Shile Huang

Curcumin (diferuloylmethane), an orange-yellow component of turmeric or curry powder, is a polyphenol natural product isolated from the rhizome of the plant Curcuma longa. For centuries, curcumin has been used in some medicinal preparation or used as a food-coloring agent. In recent years, extensive in vitro and in vivo studies suggested curcumin has anticancer, antiviral, antiarthritic, anti-amyloid, antioxidant, and anti-inflammatory properties. The underlying mechanisms of these effects are diverse and appear to involve the regulation of various molecular targets, including transcription factors (such as nuclear factor-κB), growth factors (such as vascular endothelial cell growth factor), inflammatory cytokines (such as tumor necrosis factor, interleukin 1 and interleukin 6), protein kinases (such as mammalian target of rapamycin, mitogen-activated protein kinases, and Akt) and other enzymes (such as cyclooxygenase 2 and 5 lipoxygenase). Thus, due to its efficacy and regulation of multiple targets, as well as its safety for human use, curcumin has received considerable interest as a potential therapeutic agent for the prevention and/or treatment of various malignant diseases, arthritis, allergies, Alzheimers disease, and other inflammatory illnesses. This review summarizes various in vitro and in vivo pharmacological aspects of curcumin as well as the underlying action mechanisms. The recently identified molecular targets and signaling pathways modulated by curcumin are also discussed here. Keywords: Curcumin, molecular targets, transcription factors, growth factors, inflammatory cytokines, protein kinases, enzymes, diferuloylmethane, orange-yellow component, turmeric, polyphenol natural product, rhizome, anticancer, Alzheimer's disease, inflammatory illnesses, Ayurvedic medicine, pharmacological aspects, adhesion kinase, thioredoxin reductase, electrophile response element, preoliferator-activated receptor-gamma

The mammalian target of rapamycin (mTOR), an atypical serine/threonine kinase, plays a central role in the regulation of cell proliferation, growth, differentiation, migration and survival. Dysregulation of mTOR signaling occurs in diverse human tumours, and can confer higher susceptibility to inhibitors of mTOR. Rapamycin and its derivatives, CCI-779 and RAD001 (designated rapamycins), specifically inhibit the function of mTOR, leading to inactivation of ribosomal S6K1 and inhibition of cap-dependent translation initiation through the 4E-BP1/eIF4E pathway. The overall effect is an accumulation of cells in the G1 phase of the cell-cycle, and potential apoptosis. Preclinical studies indicate that rapamycins are potent inhibitors of the proliferation of numerous tumour cell lines in culture and of murine syngeneic tumour models or human xenografts. RAD001 and CCI-779 are in phase I and II trials, respectively, as anti-cancer agents. These trials have demonstrated promising anti-cancer activity and relatively mild side effects of CCI-779. Emerging results suggest that inhibition of mTOR signaling can be exploited as a potential tumour-selective therapeutic strategy.

Influenza A viruses (IAVs) are contagious pathogens responsible for severe respiratory infection in humans and animals worldwide. Upon detection of IAV infection, host immune system aims to defend against and clear the viral infection. Innate immune system is comprised of physical barriers (mucus and collectins), various phagocytic cells, group of cytokines, interferons (IFNs), and IFN-stimulated genes, which provide first line of defense against IAV infection. The adaptive immunity is mediated by B cells and T cells, characterized with antigen-specific memory cells, capturing and neutralizing the pathogen. The humoral immune response functions through hemagglutinin-specific circulating antibodies to neutralize IAV. In addition, antibodies can bind to the surface of infected cells and induce antibody-dependent cell-mediated cytotoxicity or complement activation. Although there are neutralizing antibodies against the virus, cellular immunity also plays a crucial role in the fight against IAVs. On the other hand, IAVs have developed multiple strategies to escape from host immune surveillance for successful replication. In this review, we discuss how immune system, especially innate immune system and critical molecules are involved in the antiviral defense against IAVs. In addition, we highlight how IAVs antagonize different immune responses to achieve a successful infection.

Tumor cell migration and invasion play fundamental roles in cancer metastasis. The mammalian target of rapamycin (mTOR), a highly conserved and ubiquitously expressed serine/threonine (Ser/Thr) kinase, is a central regulator of cell growth, proliferation, differentiation and survival. Recent studies have shown that mTOR also plays a critical role in the regulation of tumor cell motility, invasion and cancer metastasis. Current knowledge indicates that mTOR functions as two distinct complexes, mTORC1 and mTORC2. mTORC1 phosphorylates p70 S6 kinase (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), and regulates cell growth, proliferation, survival and motility. mTORC2 phosphorylates Akt, protein kinase C α (PKCα) and the focal adhesion proteins, and controls the activities of the small GTPases (RhoA, Cdc42 and Rac1), and regulates cell survival and the actin cytoskeleton. Here we briefly review recent knowledge of mTOR complexes and the role of mTOR signaling in tumor cell migration and invasion. We also discuss recent efforts about the mechanism by which rapamycin, a specific inhibitor of mTOR, inhibits cell migration, invasion and cancer metastasis.

Cadmium (Cd), a highly toxic environmental pollutant, induces neurodegenerative diseases. Recently we have demonstrated that Cd induces neuronal apoptosis in part through activation of the mammalian target of rapamycin (mTOR) pathway. However, the underlying mechanism is unknown. Here we show that Cd induces the generation of reactive oxygen species (ROS) by upregulating the expression of NADPH oxidase 2 and its regulatory proteins (p22(phox), p67(phox), p40(phox), p47(phox), and Rac1) in PC12 and SH-SY5Y cells. Cd induction of ROS contributed to the activation of mTOR signaling, as pretreatment with N-acetyl-l-cysteine (NAC), a ROS scavenger, prevented this event. Further studies reveal that Cd induction of ROS increased phosphorylation of the type I insulin-like growth factor receptor (IGFR) β subunit, which was abrogated by NAC. Wortmannin, a phosphoinositide 3'-kinase (PI3K) inhibitor, partially attenuated Cd-induced phosphorylation of Akt, p70 S6 kinase 1, and eukaryotic initiation factor 4E-binding protein 1, as well as apoptosis of the neuronal cells. In addition, overexpression of wild-type phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or pretreatment with aminoimidazole carboxamide ribonucleotide, an AMP-activated protein kinase (AMPK) activator, partially prevented Cd-induced ROS and activation of the mTOR pathway, as well as cell death. The results indicate that Cd induction of ROS activates mTOR signaling, leading to neuronal cell death, in part by activating the positive regulators IGFR/PI3K and by inhibiting the negative regulators PTEN/AMPK. The findings suggest that inhibitors of PI3K and mTOR, activators of AMPK, or antioxidants may be exploited for the prevention of Cd-induced neurodegenerative diseases.

The mammalian or mechanistic target of rapamycin (mTOR) and associated phosphatidyl-inositiol 3-kinase (PI3K)/protein kinase B (Akt) pathways regulate cell growth, differentiation, migration, and survival, as well as angiogenesis and metabolism. Dysregulation of these pathways is frequently associated with genetic/epigenetic alterations and predicts poor treatment outcomes in a variety of human cancers including cutaneous malignancies like melanoma and non-melanoma skin cancers. Recently, the enhanced understanding of the molecular and genetic basis of skin dysfunction in patients with skin cancers has provided a strong basis for the development of novel therapeutic strategies for these obdurate groups of skin cancers. This review summarizes recent advances in the roles of PI3K/Akt/mTOR and their targets in the development and progression of a broad spectrum of cutaneous cancers and discusses the current progress in preclinical and clinical studies for the development of PI3K/Akt/mTOR targeted therapies with nutraceuticals and synthetic small molecule inhibitors.

Rapamycins are macrocyclic lactones that possess immunosuppressive, antifungal and antitumor properties. The parent compound, rapamycin, is approved as an immunosup-pressive agent for preventing rejection in patients receiving organ transplantation. Two analogues, CCI-779 and RAD001 are currently being investigated as anticancer agents. Rapamycins first bind a cyclophilin FKBP12, and this complex binds and inhibits the function of mTOR (mammalian target of rapamycin) a serine/threonine (Ser/Thr) kinase with homology to phosphatidylinositol 3' kinase. Currently, as mTOR is the only identified target, this places rapamycins in a unique position of being the most selective kinase inhibitor known. Consequently these agents have been powerful tools in elucidating the role of mTOR in cellular growth, proliferation, survival and tumorigenesis. Increasing evidence suggests that mTOR acts as a central controller sensing cellular environment (nutritional status or mitogenic stimulation) and regulating translation initiation through the eukaryotic initiation factor 4E, and ribosomal p70 S6 kinase pathways. Here we review the conserved TOR signaling pathways, conceptual basis for tumor selectivity, and the mechanisms of resistance to this class of antitumor agent.

Cadmium (Cd), a highly toxic environmental pollutant, induces neurodegenerative diseases. Recently we have demonstrated that Cd may induce neuronal apoptosis in part through activation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (Erk1/2) pathways. However, the underlying mechanism remains enigmatic. Here we show that Cd induced generation of reactive oxygen species (ROS), leading to apoptosis of PC12 and SH-SY5Y cells. Pretreatment with N-acetyl-L-cysteine (NAC) scavenged Cd-induced ROS, and prevented cell death, suggesting that Cd-induced apoptosis is attributed to its induction of ROS. Furthermore, we found that Cd-induced ROS inhibited serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5), leading to activation of Erk1/2 and JNK, which was abrogated by NAC. Overexpression of PP2A or PP5 partially prevented Cd-induced activation of Erk1/2 and JNK, as well as cell death. Cd-induced ROS was also linked to the activation of caspase-3. Pretreatment with inhibitors of JNK (SP600125) and Erk1/2 (U0126) partially blocked Cd-induced cleavage of caspase-3 and prevented cell death. However, zVAD-fmk, a pan caspase inhibitor, only partially prevented Cd-induced apoptosis. The results indicate that Cd induction of ROS inhibits PP2A and PP5, leading to activation of JNK and Erk1/2 pathways, and consequently resulting in caspase-dependent and -independent apoptosis of neuronal cells. The findings strongly suggest that the inhibitors of JNK, Erk1/2, or antioxidants may be exploited for prevention of Cd-induced neurodegenerative diseases.

Abstract Curcumin (diferuloylmethane), a polyphenol natural product of the plant Curcuma longa , is undergoing early clinical trials as a novel anticancer agent. However, the anticancer mechanism of curcumin remains to be elucidated. Here we show that curcumin inhibited growth of rhabdomyosarcoma cells (Rh1 and Rh30) (IC 50 = 2–5 μM) and arrested cells in G 1 phase of the cell cycle. Curcumin also induced apoptosis and inhibited the basal or type I insulin‐like growth factor‐induced motility of the cells. At physiological concentrations (˜2.5 μM), curcumin rapidly inhibited phosphorylation of the mammalian target of rapamycin (mTOR) and its downstream effector molecules, p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E‐BP1), in a panel of cell lines (Rh1, Rh30, DU145, MCF‐7 and Hela). Curcumin also inhibited phosphorylation of Akt in the cells, but only at high concentrations (>40 μM). The data suggest that curcumin may execute its anticancer activity primarily by blocking mTOR‐mediated signaling pathways in the tumor cells. © 2006 Wiley‐Liss, Inc.

Under serum-free conditions, rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), induces apoptosis of cells lacking functional p53. Cells expressing wild-type p53 or p21Cip1arrest in G1 and remain viable. In cells lacking functional p53, rapamycin or amino acid deprivation induces rapid and sustained activation of apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase, and elevation of phosphorylated c-Jun that results in apoptosis. This stress response depends on expression of eukaryotic initiation factor 4E binding protein 1 and is suppressed by p21Cip1 independent of cell cycle arrest. Rapamycin induces p21Cip1 binding to ASK1, suppressing kinase activity and attenuating cellular stress. These results suggest that inhibition of mTOR triggers a potentially lethal response that is prevented only in cells expressing p21Cip1.

Oxidative stress-induced neuronal apoptosis is a prominent feature found in neurodegenerative disorders. However, how oxidative stress induces neuronal apoptosis is not well understood. To address this question, undifferentiated and differentiated neuronal cell lines (PC12 and SH-SY5Y) were exposed to hydrogen peroxide (H2O2), a major oxidant generated when oxidative stress occurs. We observed that H2O2 induced generation of reactive oxygen species (ROS), leading to apoptosis of the cells in a concentration- and time-dependent manner. H2O2 rapidly activated the mitogen-activated protein kinases (MAPK) including extracellular signal-regulated kinase 1/2 (Erk1/2), c-Jun N-terminal kinase (JNK) and p38. Inhibition of Erk1/2, JNK or p38 with kinase inhibitors (U0126, SP600125 or PD169316, respectively), downregulation of Erk1/2 or p38 using RNA interference, or expression of dominant negative c-Jun partially prevented H2O2-induced apoptosis. Pretreatment with N-acetyl-l-cysteine (NAC) scavenged H2O2-induced ROS, blocking activation of MAPKs and cell death. Furthermore, we found that H2O2-induced ROS inhibited serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5), which was abrogated by NAC. Overexpression of PP2A or PP5 partially prevented H2O2-activation of Erk/12, JNK and p38, as well as cell death. Similar results were observed in primary murine neurons as well. The results suggest that H2O2-induction of ROS inhibit PP2A and PP5, leading to activation of Erk1/2, JNK and p38 pathways thereby resulting in neuronal apoptosis. Our findings suggest that inhibitors of MAPKs (JNK, Erk1/2 and p38), activators of phosphatases (PP2A and PP5) or antioxidants may have potentials to prevent and treat oxidative stress-induced neurodegenerative diseases.

Oxidative stress results in apoptosis of neuronal cells, leading to neurodegenerative disorders. However, the underlying molecular mechanism remains to be elucidated. Here, we show that hydrogen peroxide (H(2)O(2)), a major oxidant generated when oxidative stress occurs, induced apoptosis of neuronal cells (PC12 cells and primary murine neurons), by inhibiting the mammalian target of rapamycin (mTOR)-mediated phosphorylation of ribosomal p70 S6 kinase (S6K1) and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). N-acetyl-L-cysteine (NAC), a scavenger of reactive oxygen species (ROS), blocked H(2)O(2) inhibition of mTOR signaling. Ectopic expression of wild-type (wt) mTOR, constitutively active S6K1 or downregulation of 4E-BP1 partially prevented H(2)O(2) induction of apoptosis. Furthermore, we identified that H(2)O(2) induction of ROS inhibited the upstream kinases, Akt and phosphoinositide-dependent kinase 1 (PDK1), but not the type I insulin-like growth factor receptor (IGFR), and activated the negative regulator, AMP-activated protein kinase alpha (AMPKalpha), but not the phosphatase and tensin homolog (PTEN) in the cells. Expression of a dominant negative AMPKalpha or downregulation of AMPKalpha1 conferred partial resistance to H(2)O(2) inhibition of phosphorylation of S6K1 and 4E-BP1, as well as cell viability, indicating that H(2)O(2) inhibition of mTOR signaling is at least in part through activation of AMPK. Our findings suggest that AMPK inhibitors may be exploited for prevention of H(2)O(2)-induced neurodegenerative diseases.

Curcumin (diferuloylmethane), a polyphenol natural product of the plant Curcuma longa, is undergoing early clinical trials as a novel anticancer agent. However, the anticancer mechanism of curcumin remains to be elucidated. Recently, we have shown that curcumin inhibits phosphorylation of p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), two downstream effector molecules of the mammalian target of rapamycin complex 1 (mTORC1) in numerous cancer cell lines. This study was designed to elucidate the underlying mechanism. We observed that curcumin inhibited mTORC1 signaling not by inhibition of the upstream kinases, such as insulin-like growth factor 1 receptor (IGF-IR) and phosphoinositide-dependent kinase 1 (PDK1). Further, we found that curcumin inhibited mTORC1 signaling independently of protein phosphatase 2A (PP2A) or AMP-activated protein kinase AMPK-tuberous sclerosis complex (TSC). This is evidenced by the findings that curcumin was able to inhibit phosphorylation of S6K1 and 4E-BP1 in the cells pretreated with PP2A inhibitor (okadaic acid) or AMPK inhibitor (compound C), or in the cells expressing dominant-negative (dn) PP2A, shRNA to PP2A-A subunit, or dn-AMPKalpha. Curcumin did not alter the TSC1/2 interaction. Knockout of TSC2 did not affect curcumin inhibition of mTOR signaling. Finally, we identified that curcumin was able to dissociate raptor from mTOR, leading to inhibition of mTORC1 activity. Therefore, our data indicate that curcumin may represent a new class of mTOR inhibitor.

Cadmium (Cd), a toxic environmental contaminant, induces oxidative stress, leading to neurodegenerative disorders. Recently we have demonstrated that Cd induces neuronal apoptosis in part by activation of the mitogen-activated protein kineses (MAPK) and mammalian target of rapamycin (mTOR) pathways. However, the underlying mechanism remains elusive. Here we show that Cd elevated intracellular calcium ion ([Ca2+]i) level in PC12, SH-SY5Y cells and primary murine neurons. BAPTA/AM, an intracellular Ca2+ chelator, abolished Cd-induced [Ca2+]i elevation, and blocked Cd activation of MAKPs including extracellular signal-regulated kinase 1/2 (Erk1/2), c-Jun N-terminal kinase (JNK) and p38, and mTOR-mediated signaling pathways, as well as cell death. Pretreatment with the extracellular Ca2+ chelator EGTA also prevented Cd-induced [Ca2+]i elevation, MAPK/mTOR activation, as well as cell death, suggesting that Cd-induced extracellular Ca2+ influx plays a critical role in contributing to neuronal apoptosis. In addition, calmodulin (CaM) antagonist trifluoperazine (TFP) or silencing CaM attenuated the effects of Cd on MAPK/mTOR activation and cell death. Furthermore, Cd-induced [Ca2+]i elevation or CaM activation resulted in induction of reactive oxygen species (ROS). Pretreatment with BAPTA/AM, EGTA or TFP attenuated Cd-induced ROS and cleavage of caspase-3 in the neuronal cells. Our findings indicate that Cd elevates [Ca2+]i, which induces ROS and activates MAPK and mTOR pathways, leading to neuronal apoptosis. The results suggest that regulation of Cd-disrupted [Ca2+]i homeostasis may be a new strategy for prevention of Cd-induced neurodegenerative diseases.

Mammalian target of rapamycin (mTOR) is a central controller of cell growth, proliferation, metabolism and angiogenesis. mTOR signaling is often dysregulated in various human diseases and thus attracts great interest in developing drugs that target mTOR. Currently it is known that mTOR functions as two complexes, mTOR complex 1/2 (mTORC1/2). Rapamycin and its analogs (all termed rapalogs) first form a complex with the intracellular receptor FK506 binding protein 12 (FKBP12) and then bind a domain separated from the catalytic site of mTOR, blocking mTOR function. Rapalogs are selective for mTORC1 and effective as anticancer agents in various preclinical models. In clinical trials, rapalogs have demonstrated efficacy against certain types of cancer. Recently, a new generation of mTOR inhibitors, which compete with ATP in the catalytic site of mTOR and inhibit both mTORC1 and mTORC2 with a high degree of selectivity, have been developed. Besides, some natural products, such as epigallocatechin gallate (EGCG), caffeine, curcumin and resveratrol, have been found to inhibit mTOR as well. Here, we summarize the current findings regarding mTOR signaling pathway and review the updated data about mTOR inhibitors as anticancer agents.

Cell division cycle 25 A (Cdc25A), a dual-specificity protein phosphatase, is one of the most crucial cell cycle regulators, which removes the inhibitory phosphorylation in cyclin-dependent kinases (CDKs), such as CDK2, CDK4, and CDK6, and positively regulates the activities of CDKs that lead to cell cycle progression. In addition, Cdc25A also acts as a regulator of apoptosis. Overexpression of Cdc25A promotes tumorigenesis, and is frequently observed in various types of cancer. Here we briefly summarize current understanding of the role of Cdc25A in cell proliferation and apoptosis, as well as the impact of overexpression of Cdc25A on tumorigenesis.

An early event of cell migration is characterized as the rapid reorganization of the actin cytoskeleton. Recently, we have demonstrated that rapamycin inhibits tumor cell motility. To understand the underlying mechanism, this study was set to determine whether rapamycin inhibition of cell motility is related to its prevention of F-actin reorganization. We found that rapamycin prevented type I insulin-like growth factor (IGF-I)-stimulated F-actin reorganization in human rhabdomyosarcoma (Rh30), Ewing sarcoma (Rh1), glioblastoma (U-373) and prostate carcinoma (PC-3) cells, and concurrently inhibited phosphorylation of focal adhesion proteins, including focal adhesion kinase (FAK), paxillin and p130(Cas) in the cells. The effect of rapamycin was blocked by expression of a rapamycin-resistant mutant of mTOR (mTORrr), but not a kinase-dead mTORrr. Downregulation of raptor mimicked the effect of rapamycin. Cells infected with a recombinant adenovirus expressing constitutively active and rapamycin-resistant mutant of p70 S6 kinase 1 (S6K1) conferred to resistance to rapamycin. Further, IGF-I failed to stimulate F-actin reorganization and phosphorylation of the focal adhesion proteins in the S6K1-downregulated cells. Expression of constitutively hypophosphorylated eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1-5A) inhibited IGF-I-stimulated F-actin reorganization, but did not alter the cellular protein or phosphorylation levels of the focal adhesion proteins. The results suggest that rapamycin inhibits IGF-I-induced F-actin reorganization and phosphorylation of the focal adhesion proteins by disruption of mTOR-raptor complex. Both S6K1 and 4E-BP1 pathways, mediated by the mTOR-raptor complex, are involved in the regulation of IGF-I-stimulated F-actin reorganization, but only the former controls IGF-I-stimulated phosphorylation of the focal adhesion proteins.

Abstract Cadmium (Cd) may be accumulated in human body through long‐term exposure to Cd‐polluted environment, resulting in neurodegeneration and other diseases. To study the mechanism of Cd‐induced neurodegeneration, PC12 and SH‐SY5Y cells were exposed to Cd. We observed that Cd‐induced apoptosis in the cells in a time‐ and concentration‐dependent manner. Cd rapidly activated the mitogen‐activated protein kinases (MAPK) including extracellular signal‐regulated kinase 1/2 (Erk1/2), c ‐Jun N‐terminal kinase (JNK) and p38. Inhibition of Erk1/2 and JNK, but not p38, partially protected the cells from Cd‐induced apoptosis. Consistently, over‐expression of dominant negative c‐ Jun or down‐regulation of Erk1/2, but not p38 MAPK, partially prevented Cd‐induced apoptosis. To our surprise, Cd also activated mammalian target of rapamycin (mTOR)‐mediated signaling pathways. Treatment with rapamycin, an mTOR inhibitor, blocked Cd‐induced phosphorylation of S6K1 and eukaryotic initiation factor 4E binding protein 1, and markedly inhibited Cd‐induced apoptosis. Down‐regulation of mTOR by RNA interference also in part, rescued cells from Cd‐induced death. These findings indicate that activation of the signaling network of MAPK and mTOR is associated with Cd‐induced neuronal apoptosis. Our results strongly suggest that inhibitors of MAPK and mTOR may have a potential for prevention of Cd‐induced neurodegeneration.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons. Dysregulation of mammalian target of rapamycin (mTOR) has been implicated in the pathogenesis of PD. However, the underlying mechanism is incompletely elucidated. Here, we show that PD mimetics (6-hydroxydopamine, N-methyl-4-phenylpyridine or rotenone) suppressed phosphorylation of mTOR, S6K1 and 4E-BP1, reduced cell viability, and activated caspase-3 and PARP in PC12 cells and primary neurons. Overexpression of wild-type mTOR or constitutively active S6K1, or downregulation of 4E-BP1 in PC12 cells partially prevented cell death in response to the PD toxins, revealing that mTOR-mediated S6K1 and 4E-BP1 pathways due to the PD toxins were inhibited, leading to neuronal cell death. Furthermore, we found that the inhibition of mTOR signaling contributing to neuronal cell death was attributed to suppression of Akt and activation of AMPK. This is supported by the findings that ectopic expression of constitutively active Akt or dominant negative AMPKα, or inhibition of AMPKα with compound C partially attenuated inhibition of phosphorylation of mTOR, S6K1 and 4E-BP1, activation of caspase-3, and neuronal cell death triggered by the PD toxins. The results indicate that PD stresses activate AMPK and inactivate Akt, causing neuronal cell death via inhibiting mTOR-mediated S6K1 and 4E-BP1 pathways. Our findings suggest that proper co-manipulation of AMPK/Akt/mTOR signaling may be a potential strategy for prevention and treatment of PD.

The mushroom Ganoderma lucidum (G. lucidum) has been used for centuries in Asian countries to treat various diseases and to promote health and longevity. Clinical studies have shown beneficial effects of G. lucidum as an alternative adjuvant therapy in cancer patients without obvious toxicity. G. lucidum polysaccharides (GLP) is the main bioactive component in the water soluble extracts of this mushroom. Evidence from in vitro and in vivo studies has demonstrated that GLP possesses potential anticancer activity through immunomodulatory, anti-proliferative, pro-apoptotic, anti-metastatic and anti-angiogenic effects. Here, we briefly summarize these anticancer effects of GLP and the underlying mechanisms.

The mammalian target of rapamycin (mTOR) functions in cells at least as two complexes, mTORC1 and mTORC2. Intensive studies have focused on the roles of mTOR in the regulation of cell proliferation, growth, and survival. Recently we found that rapamycin inhibits type I insulin-like growth factor (IGF-1)-stimulated lamellipodia formation and cell motility, indicating involvement of mTOR in regulating cell motility. This study was set to further elucidate the underlying mechanism. Here we show that rapamycin inhibited protein synthesis and activities of small GTPases (RhoA, Cdc42, and Rac1), crucial regulatory proteins for cell migration. Disruption of mTORC1 or mTORC2 by down-regulation of raptor or rictor, respectively, inhibited the activities of these proteins. However, only disruption of mTORC1 mimicked the effect of rapamycin, inhibiting their protein expression. Ectopic expression of rapamycin-resistant and constitutively active S6K1 partially prevented rapamycin inhibition of RhoA, Rac1, and Cdc42 expression, whereas expression of constitutively hypophosphorylated 4E-BP1 (4EBP1-5A) or down-regulation of S6K1 by RNA interference suppressed expression of the GTPases, suggesting that both mTORC1-mediated S6K1 and 4E-BP1 pathways are involved in protein synthesis of the GTPases. Expression of constitutively active RhoA, but not Cdc42 and Rac1, conferred resistance to rapamycin inhibition of IGF-1-stimulated lamellipodia formation and cell migration. The results suggest that rapamycin inhibits cell motility at least in part by down-regulation of RhoA protein expression and activity through mTORC1-mediated S6K1 and 4E-BP1-signaling pathways.

The mammalian target of rapamycin (mTOR) has attracted substantial attention because of its involvement in a variety of diseases, such as cancer, cardiac hypertrophy, diabetes and obesity. Current knowledge indicates that mTOR functions as two distinct multiprotein complexes, mTORC1 and mTORC2. mTORC1 phosphorylates p70 S6 kinase (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), and regulates cell growth, proliferation, and survival by integrating hormones, growth factors, nutrients, stressors and energy signals. In contrast, mTORC2 is insensitive to nutrients or energy conditions. However, in response to hormones or growth factors, mTORC2 phosphorylates Akt, and regulates actin cytoskeleton and cell survival. These findings not only reveal the crucial role of mTOR in physiology and pathology, but also reflect the complexity of the mTOR signaling network. In this review, we discuss the advances in studies of the mTOR complexes, including the interacting proteins, the upstream regulators and the downstream effectors of mTOR complexes, as well as their implication in certain human diseases.

This study explores the neuroprotective effects and mechanisms of N-acetyl-L-cysteine (NAC) in mice exposed to cadmium (Cd).NAC (150 mg/kg) was intraperitoneally administered to mice exposed to Cd (10-50 mg/L) in drinking water for 6 weeks. The changes of cell damage and death, reactive oxygen species (ROS), antioxidant enzymes, as well as Akt/mammalian target of rapamycin (mTOR) signalling pathway in brain neurones were assessed. To verify the role of mTOR activation in Cd-induced neurotoxicity, mice also received a subacute regimen of intraperitoneally administered Cd (1 mg/kg) with/without rapamycin (7.5 mg/kg) for 11 days.Chronic exposure of mice to Cd induced brain damage or neuronal cell death, due to ROS induction. Co-administration of NAC significantly reduced Cd levels in the plasma and brain of the animals. NAC prevented Cd-induced ROS and significantly attenuated Cd-induced brain damage or neuronal cell death. The protective effect of NAC was mediated, at least partially, by elevating the activities of Cu/Zn-superoxide dismutase, catalase and glutathione peroxidase, as well as the level of glutathione in the brain. Furthermore, Cd-induced activation of Akt/mTOR pathway in the brain was also inhibited by NAC. Rapamycin in vitro and in vivo protected against Cd-induced neurotoxicity.NAC protects against Cd-induced neuronal apoptosis in mouse brain partially by inhibiting ROS-dependent activation of Akt/mTOR pathway. The findings highlight that NAC may be exploited for prevention and treatment of Cd-induced neurodegenerative diseases.

Rotenone, a common pesticide and inhibitor of mitochondrial complex I, induces loss of dopaminergic neurons and consequential aspects of Parkinson's disease (PD). However, the exact mechanism of rotenone neurotoxicity is not fully elucidated. Here, we show that rotenone induced reactive oxygen species (ROS), leading to apoptotic cell death in PC12 cells and primary neurons. Pretreatment with catalase (CAT), a hydrogen peroxide-scavenging enzyme, attenuated rotenone-induced ROS and neuronal apoptosis, implying hydrogen peroxide (H₂O₂) involved, which was further verified by imaging intracellular H₂O₂ using a peroxide-selective probe H2DCFDA. Using thenoyltrifluoroacetone (TTFA), antimycin A, or Mito-TEMPO, we further demonstrated rotenone-induced mitochondrial H₂O₂-dependent neuronal apoptosis. Rotenone dramatically inhibited mTOR-mediated phosphorylation of S6K1 and 4E-BP1, which was also attenuated by CAT in the neuronal cells. Of interest, ectopic expression of wild-type mTOR or constitutively active S6K1, or downregulation of 4E-BP1 partially prevented rotenone-induced H₂O₂ and cell apoptosis. Furthermore, we noticed that rotenone-induced H₂O₂ was linked to the activation of caspase-3 pathway. This was evidenced by the finding that pretreatment with CAT partially blocked rotenone-induced cleavages of caspase-3 and poly (ADP-ribose) polymerase. Of note, zVAD-fmk, a pan caspase inhibitor, only partially prevented rotenone-induced apoptosis in PC12 cells and primary neurons. Expression of mTOR-wt, S6K1-ca, or silencing 4E-BP1 potentiated zVAD-fmk protection against rotenone-induced apoptosis in the cells. The results indicate that rotenone induction of H₂O₂ inhibits mTOR-mediated S6K1 and 4E-BP1/eIF4E pathways, resulting in caspase-dependent and -independent apoptosis in neuronal cells. Our findings suggest that rotenone-induced neuronal loss in PD may be prevented by activating mTOR signaling and/or administering antioxidants.

The dysregulated phosphatidylinositol-3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling pathway has been implicated in various immune-mediated inflammatory and hyperproliferative dermatoses such as acne, atopic dermatitis, alopecia, psoriasis, wounds, and vitiligo, and is associated with poor treatment outcomes. Improved comprehension of the consequences of the dysregulated PI3K/Akt/mTOR pathway in patients with inflammatory dermatoses has resulted in the development of novel therapeutic approaches. Nonetheless, more studies are necessary to validate the regulatory role of this pathway and to create more effective preventive and treatment methods for a wide range of inflammatory skin diseases. Several studies have revealed that certain natural products and synthetic compounds can obstruct the expression/activity of PI3K/Akt/mTOR, underscoring their potential in managing common and persistent skin inflammatory disorders. This review summarizes recent advances in understanding the role of the activated PI3K/Akt/mTOR pathway and associated components in immune-mediated inflammatory dermatoses and discusses the potential of bioactive natural products, synthetic scaffolds, and biologic agents in their prevention and treatment. However, further research is necessary to validate the regulatory role of this pathway and develop more effective therapies for inflammatory skin disorders.

Rapamycins represent a novel family of anticancer agents, currently including rapamycin and its derivatives, CCI-779 and RAD001. Rapamycins inhibit the function of the mammalian target of rapamycin (mTOR), and potently suppress tumor cell growth by arresting cells in G1 phase or potentially inducing apoptosis of cells, in culture or in xenograft tumor models. However, recent data indicate that genetic mutations or compensatory changes in tumor cells influence the sensitivity of rapamycins. First, mutations of mTOR or FKBP12 prevent rapamycin from binding to mTOR, conferring rapamycin resistance. Second, mutations or defects of mTOR-regulated proteins, including S6K1, 4E-BP1, PP2A-related phosphatases, and p27(Kip1) also render rapamycin insensitivity. In addition, the status of ATM, p53, PTEN/Akt and 14-3-3 are also associated with rapamycin sensitivity. To better explore the role of rapamycins against tumors, this review will summarize the current knowledge of the mechanism of action of rapamycins, and progress in understanding mechanisms of acquired or intrinsic resistance.

Abstract Ciclopirox olamine (CPX) is a synthetic antifungal agent clinically used to treat mycoses of the skin and nails. Here, we show that CPX inhibited tumor growth in human breast cancer MDA‐MB‐231 xenografts. To unveil the underlying mechanism, we further studied the antitumor activity of CPX in cell culture. The results indicate that CPX inhibited cell proliferation and induced apoptosis in human rhabdomyosarcoma (Rh30), breast carcinoma (MDA‐MB231) and colon adenocarcinoma (HT‐29) cells in a concentration‐dependent manner. By cell cycle analysis, CPX induced accumulation of cells in G 1 /G 0 phase of the cell cycle. Concurrently, CPX downregulated cellular protein expression of cyclins (A, B1, D1 and E) and cyclin‐dependent kinases (CDK2 and CDK4) and upregulated expression of the CDK inhibitor p21 Cip1 , leading to hypophosphorylation of retinoblastoma protein. CPX also downregulated protein expression of Bcl‐xL and survivin and enhanced cleavages of Bcl‐2. Z‐VAD‐FMK, a pan‐caspase inhibitor, partially prevented CPX‐induced cell death, suggesting that CPX‐induced apoptosis of cancer cells is mediated at least in part through caspase‐dependent mechanism. The results indicate that CPX is a potential antitumor agent.

Abstract Cryptotanshinone (CPT), a natural compound isolated from the plant Salvia miltiorrhiza Bunge, is a potential anticancer agent. However, little is known about its anticancer mechanism. Here, we show that CPT inhibited cancer cell proliferation by arresting cells in G1-G0 phase of the cell cycle. This is associated with the inhibition of cyclin D1 expression and retinoblastoma (Rb) protein phosphorylation. Furthermore, we found that CPT inhibited the signaling pathway of the mammalian target of rapamycin (mTOR), a central regulator of cell proliferation. This is evidenced by the findings that CPT inhibited type I insulin-like growth factor I– or 10% fetal bovine serum–stimulated phosphorylation of mTOR, p70 S6 kinase 1, and eukaryotic initiation factor 4E binding protein 1 in a concentration- and time-dependent manner. Expression of constitutively active mTOR conferred resistance to CPT inhibition of cyclin D1 expression and Rb phosphorylation, as well as cell growth. The results suggest that CPT is a novel antiproliferative agent. Cancer Prev Res; 3(8); 1015–25. ©2010 AACR.

J. Neurochem. (2011) 119 , 1108–1118. Abstract Cadmium (Cd), a toxic environmental contaminant, induces neurodegenerative diseases. Recently, we have shown that Cd elevates intracellular free calcium ion ([Ca 2+ ] i ) level, leading to neuronal apoptosis partly by activating mitogen‐activated protein kinases (MAPK) and mammalian target of rapamycin (mTOR) pathways. However, the underlying mechanism remains to be elucidated. In this study, we show that the effects of Cd‐elevated [Ca 2+ ] i on MAPK and mTOR network as well as neuronal cell death are through stimulating phosphorylation of calcium/calmodulin‐dependent protein kinase II (CaMKII). This is supported by the findings that chelating intracellular Ca 2+ with 1,2‐bis( o ‐aminophenoxy) ethane‐ N , N , N ′, N ′‐tetraacetic acid tetra(acetoxymethyl) ester or preventing Cd‐induced [Ca 2+ ] i elevation using 2‐aminoethoxydiphenyl borate blocked Cd activation of CaMKII. Inhibiting CaMKII with KN93 or silencing CaMKII attenuated Cd activation of MAPK/mTOR pathways and cell death. Furthermore, inhibitors of mTOR (rapamycin), c‐Jun N‐terminal kinase (SP600125) and extracellular signal‐regulated kinase 1/2 (U0126), but not of p38 (PD169316), prevented Cd‐induced neuronal cell death in part through inhibition of [Ca 2+ ] i elevation and CaMKII phosphorylation. The results indicate that Cd activates MAPK/mTOR network triggering neuronal cell death, by stimulating CaMKII. Our findings underscore a central role of CaMKII in the neurotoxicology of Cd, and suggest that manipulation of intracellular Ca 2+ level or CaMKII activity may be exploited for prevention of Cd‐induced neurodegenerative disorders.

Cryptotanshinone is one of the major tanshinones isolated from the roots of the plant Salvia miltiorrhiza Bunge (Danshen). Danshen has been widely used in traditional Chinese medicine for treatment of a variety of diseases, including coronary artery disease, acute ischemic stroke, hyperlipidemia, chronic renal failure, chronic hepatitis, and Alzheimer's disease, showing no serious adverse effects. Recent studies have shown that cryptotanshinone not only possesses the potential for treatment and prevention of the above-mentioned diseases, but also is a potent anticancer agent. Here we briefly summarize the physical and chemical properties and the pharmacokinetic profiles of cryptotanshinone, and then comprehensively review its anticancer activities as well as the underlying mechanisms.

The mammalian target of rapamycin (mTOR), a serine/threonine protein kinase, acts as a "master switch" for cellular anabolic and catabolic processes, regulating the rate of cell growth and proliferation. Dysregulation of the mTOR signaling pathway occurs frequently in a variety of human tumors, and thus, mTOR has emerged as an important target for the design of anticancer agents. mTOR is found in two distinct multiprotein complexes within cells, mTORC1 and mTORC2. These two complexes consist of unique mTOR-interacting proteins and are regulated by different mechanisms. Enormous advances have been made in the development of drugs known as mTOR inhibitors. Rapamycin, the first defined inhibitor of mTOR, showed effectiveness as an anticancer agent in various preclinical models. Rapamycin analogues (rapalogs) with better pharmacologic properties have been developed. However, the clinical success of rapalogs has been limited to a few types of cancer. The discovery that mTORC2 directly phosphorylates Akt, an important survival kinase, adds new insight into the role of mTORC2 in cancer. This novel finding prompted efforts to develop the second generation of mTOR inhibitors that are able to target both mTORC1 and mTORC2. Here, we review the recent advances in the mTOR field and focus specifically on the current development of the second generation of mTOR inhibitors as anticancer agents.

Curcumin can induce p53-independent apoptosis. However, the underlying mechanism remains to be defined. Here, we show that curcumin-induced apoptosis in a panel of tumor cells with mutant p53. Curcumin rapidly induced activation of the mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase 1/2 (Erk1/2) and c-Jun N-terminal kinase (JNK). Inhibition of JNK (with SP600125) or Erk1/2 (with U0126) partially prevented curcumin-induced cell death in the cells. Similarly, expression of dominant negative c-Jun or downregulation of Erk1/2 in part attenuated curcumin-induced cell death. It appears that curcumin-induced activation of MAPKs and apoptosis was due to induction of reactive oxygen species (ROS), as pretreatment with N-acetyl-L-cysteine, a ROS scavenger, blocked these events. Furthermore, we found that curcumin-induced activation of MAPK pathways was related to inhibition of the serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5). Overexpression of PP2A or PP5 partially prevented curcumin-induced activation of JNK and Erk1/2 phosphorylation as well as cell death. The results suggest that curcumin induction of ROS activates MAPKs, at least partially by inhibiting PP2A and PP5, thereby leading to p53-independent apoptosis in tumor cells.

Influenza virus neuraminidase (NA) is transported to the virus assembly site at the plasma membrane and is a major viral envelope component that plays a critical role in the release of progeny virions and in determination of host range restriction. However, little is known about the host factors that are involved in regulating the intracellular and cell surface transport of NA. Here we identified the Cdc42-specific GAP, ARHGAP21 differentially expressed in host cells infected with influenza A virus using cDNA microarray analysis. Furthermore, we have investigated the involvement of Rho family GTPases in NA transport to the cell surface. We found that expression of constitutively active or inactive mutants of RhoA or Rac1 did not significantly affect the amount of NA that reached the cell surface. However, expression of constitutively active Cdc42 or depletion of ARHGAP21 promoted the transport of NA to the plasma membranes. By contrast, cells expressing shRNA targeting Cdc42 or overexpressing ARHGAP21 exhibited a significant decrease in the amount of cell surface-localized NA. Importantly, silencing Cdc42 reduced influenza A virus replication, whereas silencing ARHGAP21 increased the virus replication. Together, our results reveal that ARHGAP21- and Cdc42-based signaling regulates the NA transport and thereby impacts virus replication.

Cryptotanshinone (CPT), a natural compound isolated from the plant Salvia miltiorrhiza Bunge, is a potential anticancer agent. However, the underlying mechanism is not well understood. Here, we show that CPT induced caspase-independent cell death in human tumor cells (Rh30, DU145, and MCF-7). Besides downregulating antiapoptotic protein expression of survivin and Mcl-1, CPT increased phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase (JNK), and inhibited phosphorylation of extracellular signal-regulated kinases 1/2 (Erk1/2). Inhibition of p38 with SB202190 or JNK with SP600125 attenuated CPT-induced cell death. Similarly, silencing p38 or c-Jun also in part prevented CPT-induced cell death. In contrast, expression of constitutively active mitogen-activated protein kinase kinase 1 (MKK1) conferred resistance to CPT inhibition of Erk1/2 phosphorylation and induction of cell death. Furthermore, we found that all of these were attributed to CPT induction of reactive oxygen species (ROS). This is evidenced by the findings that CPT induced ROS in a concentration- and time-dependent manner; CPT induction of ROS was inhibited by N-acetyl-L-cysteine (NAC), a ROS scavenger; and NAC attenuated CPT activation of p38/JNK, inhibition of Erk1/2, and induction of cell death. The results suggested that CPT induction of ROS activates p38/JNK and inhibits Erk1/2, leading to caspase-independent cell death in tumor cells.

Although alteration in host cellular translation machinery occurs in virus-infected cells, the role of such alteration and the precise pathogenic processes are not well understood. Influenza A virus (IAV) infection shuts off host cell gene expression at transcriptional and translational levels. Here, we found that the protein level of eukaryotic translation initiation factor 4B (eIF4B), an integral component of the translation initiation apparatus, was dramatically reduced in A549 cells as well as in the lung, spleen, and thymus of mice infected with IAV. The decrease in eIF4B level was attributed to lysosomal degradation of eIF4B, which was induced by viral NS1 protein. Silencing eIF4B expression in A549 cells significantly promoted IAV replication, and conversely, overexpression of eIF4B markedly inhibited the viral replication. Importantly, we observed that eIF4B knockdown transgenic mice were more susceptible to IAV infection, exhibiting faster weight loss, shorter survival time, and more-severe organ damage. Furthermore, we demonstrated that eIF4B regulated the expression of interferon-induced transmembrane protein 3 (IFITM3), a critical protein involved in immune defense against a variety of RNA viruses, including influenza virus. Taken together, our findings reveal that eIF4B plays an important role in host defense against IAV infection at least by regulating the expression of IFITM3, which restricts viral entry and thereby blocks early stages of viral production. These data also indicate that influenza virus has evolved a strategy to overcome host innate immunity by downregulating eIF4B protein.Influenza A virus (IAV) infection stimulates the host innate immune system, in part, by inducing interferons (IFNs). Secreted IFNs activate the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, leading to elevated transcription of a large group of IFN-stimulated genes that have antiviral function. To circumvent the host innate immune response, influenza virus has evolved multiple strategies for suppressing the production of IFNs. Here, we show that IAV infection induces lysosomal degradation of eIF4B protein; and eIF4B inhibits IAV replication by upregulating expression of interferon-induced transmembrane protein 3 (IFITM3), a key protein that protects the host from virus infection. Our finding illustrates a critical role of eIF4B in the host innate immune response and provides novel insights into the complex mechanisms by which influenza virus interacts with its host.

Protein kinase R (PKR) is a vital component of host innate immunity against viral infection. However, the mechanism underlying inactivation of PKR by influenza A virus (IAV) remains elusive. Here, we found that vault RNAs (vtRNAs) were greatly induced in A549 cells and mouse lungs after infection with IAV. The viral NS1 protein was shown to be the inducer triggering the upregulation of vtRNAs. Importantly, silencing vtRNA in A549 cells significantly inhibited IAV replication, whereas overexpression of vtRNAs markedly promoted the viral replication. Furthermore, in vivo studies showed that disrupting vtRNA expression in mice significantly decreased IAV replication in infected lungs. The vtRNA knockdown animals exhibited significantly enhanced resistance to IAV infection, as evidenced by attenuated acute lung injury and spleen atrophy and consequently increased survival rates. Interestingly, vtRNAs promoted viral replication through repressing the activation of PKR and the subsequent antiviral interferon response. In addition, increased expression of vtRNAs was required for efficient suppression of PKR by NS1 during IAV infection. Moreover, vtRNAs were also significantly upregulated by infections of several other viruses and involved in the inactivation of PKR signaling by these viruses. These results reveal a novel mechanism by which some viruses circumvent PKR-mediated innate immunity.

Since leukemic stem cells (LSCs) or cancer stem cells (CSCs) were found in acute myeloid leukemia (AML) in 1997, extensive studies have been contributed to identification and characterization of such cell populations in various tissues. LSCs are now generally recognized as a heterogeneous cell population that possesses the capacities of self-renewal, proliferation and differentiation. It has been shown that LSCs are regulated by critical surface antigens, microenvironment, intrinsic signaling pathways, and novel molecules such as some ncRNAs. To date, significant progress has been made in understanding of LSCs, leading to the development of numerous LSCs-targeted therapies. Moreover, various novel therapeutic agents targeting LSCs are undergoing clinical trials. Here, we review current knowledge of LSCs, and discuss the potential therapies and their challenges that are being tested in clinical trials for evaluation of their effects on leukemias.

Cadmium (Cd) is a highly toxic metal that affects the central nervous system. Recently we have demonstrated that inhibition of mTOR by rapamycin rescues neuronal cells from Cd-poisoning. Here we show that rapamycin inhibited Cd-induced mitochondrial ROS-dependent neuronal apoptosis. Intriguingly, rapamycin remarkably blocked phosphorylation of JNK, Erk1/2 and p38 in neuronal cells induced by Cd, which was strengthened by co-treatment with Mito-TEMPO. Inhibition of JNK and Erk1/2 by SP600125 and U0126, respectively, potentiated rapamycin's prevention from Cd-induced apoptosis. Consistently, over-expression of dominant negative c-Jun or MKK1 also potently improved the inhibitory effect of rapamycin on Cd neurotoxicity. Furthermore, pretreatment with SP600125 or U0126, or expression of dominant negative c-Jun or MKK1 enhanced the inhibitory effects of rapamycin or Mito-TEMPO on Cd-induced ROS. Further investigation found that co-treatment with Mito-TEMPO/rapamycin more effectively rescued cells by preventing Cd inactivation of PP2A than treatment with rapamycin or Mito-TEMPO alone. Over-expression of wild-type PP2A reinforced rapamycin or Mito-TEMPO suppression of activated JNK and Erk1/2 pathways, as well as ROS production and apoptosis in neuronal cells in response to Cd. The findings indicate that rapamycin ameliorates Cd-evoked neuronal apoptosis by preventing mitochondrial ROS inactivation of PP2A, thereby suppressing activation of JNK and Erk1/2 pathways. Our results underline that rapamycin may have a potential in preventing Cd-induced oxidative stress and neurodegenerative diseases.

Psoriasis is a chronic immune-mediated skin disease that involves the interaction of immune and skin cells, and is characterized by cytokine-driven epidermal hyperplasia, deviant differentiation, inflammation, and angiogenesis. Because the available treatments for psoriasis have significant limitations, dietary products are potential natural sources of therapeutic molecules, which can repair the molecular defects associated with psoriasis and could possibly be developed for its management. Fisetin (3,7,3′,4′-tetrahydroxyflavone), a phytochemical naturally found in pigmented fruits and vegetables, has demonstrated proapoptotic and antioxidant effects in several malignancies. This study utilized biochemical, cellular, pharmacological, and tissue engineering tools to characterize the effects of fisetin on normal human epidermal keratinocytes (NHEKs), peripheral blood mononuclear cells (PBMC), and CD4+ T lymphocytes in 2D and 3D psoriasis-like disease models. Fisetin treatment of NHEKs dose- and time-dependently induced differentiation and inhibited interleukin-22-induced proliferation, as well as activation of the PI3K/Akt/mTOR pathway. Fisetin treatment of TNF-α stimulated NHEKs also significantly inhibited the activation of p38 and JNK, but had enhanced effect on ERK1/2 (MAPK). In addition, fisetin treatment significantly decreased the secretion of Th1/Th-17 pro-inflammatory cytokines, particularly IFN-γ and IL-17A by 12-O-tetradecanolylphorbol 13-acetate (TPA)-stimulated NHEKs and anti-CD3/CD28-activated human PBMCs. Furthermore, we established the in vivo relevance of fisetin functions, using a 3D full-thickness human skin model of psoriasis (FTRHSP) that closely mimics in vivo human psoriatic skin lesions. Herein, fisetin significantly ameliorated psoriasis-like disease features, and decreased the production of IL-17 by CD4+ T lymphocytes co-cultured with FTRHSP. Collectively, our data identify the prodifferentiative, antiproliferative, and anti-inflammatory effects of fisetin, via modulation of the PI3K-Akt-mTOR and p38/JNK pathways and the production of cytokines in 2D and 3D human skin models of psoriasis. These results suggest that fisetin has a great potential to be developed as an effective and inexpensive agent for the treatment of psoriasis and other related inflammatory skin disorders.

Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATL), a rapidly progressing clonal malignancy of CD4+ T lymphocytes. Exploring the host-HTLV-1 interactions and the molecular mechanisms underlying HTLV-1-mediated tumorigenesis is critical for developing efficient therapies against the viral infection and associated leukemia/lymphoma. It has been demonstrated to date that several HTLV-1 proteins play key roles in the cellular transformation and immortalization of infected T lymphocytes. Of note, the HTLV-1 oncoprotein Tax inhibits the innate IFN response through interaction with MAVS, STING and RIP1, causing the suppression of TBK1-mediated phosphorylation of IRF3/IRF7. The HTLV-1 protein HBZ disrupts genomic integrity and inhibits apoptosis and autophagy of the target cells. Furthermore, it is revealed that HBZ enhances the proliferation of ATL cells and facilitates evasion of the infected cells from immunosurveillance. These studies provide insights into the molecular mechanisms by which HTLV-1 mediates the formation of cancer as well as useful strategies for the development of new therapeutic interventions against ATL. In this article, we review the recent advances in the understanding of the pathogenesis, the underlying mechanisms, clinical diagnosis and treatment of the disease caused by HTLV-1 infection. In addition, we discuss the future direction for targeting HTLV-1-associated cancers and strategies against HTLV-1.

Environmental exposure to cadmium (Cd) links to neurodegenerative disorders. Autophagy plays an important role in controlling cell survival/death. However, how autophagy contributes to Cd's neurotoxicity remains enigmatic. Here, we show that Cd induced significant increases in autophagosomes with a concomitant elevation of LC3-II and p62 in PC12 cells and primary neurons. Using autophagy inhibitor 3-MA, we demonstrated that Cd-increased autophagosomes contributed to neuronal apoptosis. Impairment of Cd on autophagic flux was evidenced by co-localization of mCherry and GFP tandem-tagged LC3 puncta in the cells. This is further supported by the findings that administration of chloroquine (CQ) potentiated the basic and Cd-elevated LC3-II and p62 levels, autophagosome accumulation and cell apoptosis, whereas rapamycin relieved the effects in the cells in response to Cd. Subsequently, we noticed that Cd evoked the phosphorylation of Akt and BECN1. Silencing BECN1 and especially expression of mutant BECN1 (Ser295A) attenuated Cd-increased autophagosomes and cell death. Of note, inhibition of Akt with Akt inhibitor X, or ectopic expression of dominant negative Akt (dn-Akt), in the presence or absence of 3-MA, significantly alleviated Cd-triggered phosphorylation of Akt and BECN1, autophagosomes, and apoptosis. Importantly, we found that Cd activation of Akt functioned in impairing autophagic flux. Collectively, these results indicate that Cd results in accumulation of autophagosomes-dependent apoptosis through activating Akt-impaired autophagic flux in neuronal cells. Our findings underscore that inhibition of Akt to improve autophagic flux is a promising strategy against Cd-induced neurotoxicity and neurodegeneration.

The mechanistic/mammalian target of rapamycin (mTOR), a serine/threonine kinase, is a central regulator for human physiological activity. Deregulated mTOR signaling is implicated in a variety of disorders, such as cancer, obesity, diabetes, and neurodegenerative diseases. The papers published in this special issue summarize the current understanding of the mTOR pathway and its role in the regulation of tissue regeneration, regulatory T cell differentiation and function, and different types of cancer including hematologic malignancies, skin, prostate, breast, and head and neck cancer. The findings highlight that targeting the mTOR pathway is a promising strategy to fight against certain human diseases.

Psoriasis is a chronic autoimmune inflammatory skin disorder characterized by epidermal hyperplasia and aberrant immune response. In addition to aberrant cytokine production, psoriasis is associated with activation of the Akt/mTOR pathway. mTOR/S6K1 regulates T-lymphocyte activation and migration, keratinocytes proliferation and is upregulated in psoriatic lesions. Several drugs that target Th1/Th17 cytokines or their receptors have been approved for treating psoriasis in humans with variable results necessitating improved therapies. Fisetin, a natural dietary polyphenol with anti-oxidant and anti-proliferative properties, covalently binds mTOR/S6K1. The effects of fisetin on psoriasis and its underlying mechanisms have not been clearly defined. Here, we evaluated the immunomodulatory effects of fisetin on Th1/Th17-cytokine-activated adult human epidermal keratinocytes (HEKa) and anti-CD3/CD28-stimulated inflammatory CD4+ T cells and compared these activities with those of rapamycin (an mTOR inhibitor). Transcriptomic analysis of HEKa revealed 12,713 differentially expressed genes (DEGs) in the fisetin-treated group compared to 7,374 DEGs in the rapamycin-treated group, both individually compared to a cytokine treated group. Gene ontology analysis revealed enriched functional groups related to PI3K/Akt/mTOR signaling pathways, psoriasis, and epidermal development. Using in silico molecular modeling, we observed a high binding affinity of fisetin to IL-17A. In vitro, fisetin significantly inhibited mTOR activity, increased the expression of autophagy markers LC3A/B and Atg5 in HEKa cells and suppressed the secretion of IL-17A by activated CD4+ T lymphocytes or T lymphocytes co-cultured with HEKa. Topical administration of fisetin in an imiquimod (IMQ)-induced mouse psoriasis model exhibited a better effect than rapamycin in reducing psoriasis-like inflammation and Akt/mTOR phosphorylation and promoting keratinocyte differentiation and autophagy in mice skin lesions. Fisetin also significantly inhibited T-lymphocytes and F4/80+ macrophage infiltration into skin. We conclude that fisetin potently inhibits IL-17A and the Akt/mTOR pathway and promotes keratinocyte differentiation and autophagy to alleviate IMQ-induced psoriasis-like disease in mice. Altogether, our findings suggest fisetin as a potential treatment for psoriasis and possibly other inflammatory skin diseases.

Under serum-free conditions, rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), induces a cellular stress response characterized by rapid and sustained activation of the apoptosis signal-regulating kinase 1 (ASK1) signaling pathway and selective apoptosis of cells lacking functional p53. Here we have investigated how mTOR regulates ASK1 signaling using p53-mutant rhabdomyosarcoma cells. In Rh30 cells, ASK1 was found to physically interact with protein phosphatase 5 (PP5), previously identified as a negative regulator of ASK1. Rapamycin did not affect either protein level of PP5 or association of PP5 with ASK1. Instead, rapamycin caused rapid dissociation of the PP2A-B″ regulatory subunit (PR72) from the PP5-ASK1 complex, which was associated with reduced phosphatase activity of PP5. This effect was dependent on expression of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Down-regulation of PP5 activity by rapamycin coordinately activated ASK1, leading to elevated phosphorylation of c-Jun. Amino acid deprivation, which like rapamycin inhibits mTOR signaling, also inhibited PP5 activity, caused rapid dissociation of PR72, and activated ASK1 signaling. Overexpression of PP5, but not the PP2A catalytic subunit, blocked rapamycin-induced phosphorylation of c-Jun, and protected cells from rapamycin-induced apoptosis. The results suggest that PP5 is downstream of mTOR, and positively regulated by the mTOR pathway. The findings suggest that in the absence of serum factors, mTOR signaling suppresses apoptosis through positive regulation of PP5 activity and suppression of cellular stress. Under serum-free conditions, rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), induces a cellular stress response characterized by rapid and sustained activation of the apoptosis signal-regulating kinase 1 (ASK1) signaling pathway and selective apoptosis of cells lacking functional p53. Here we have investigated how mTOR regulates ASK1 signaling using p53-mutant rhabdomyosarcoma cells. In Rh30 cells, ASK1 was found to physically interact with protein phosphatase 5 (PP5), previously identified as a negative regulator of ASK1. Rapamycin did not affect either protein level of PP5 or association of PP5 with ASK1. Instead, rapamycin caused rapid dissociation of the PP2A-B″ regulatory subunit (PR72) from the PP5-ASK1 complex, which was associated with reduced phosphatase activity of PP5. This effect was dependent on expression of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Down-regulation of PP5 activity by rapamycin coordinately activated ASK1, leading to elevated phosphorylation of c-Jun. Amino acid deprivation, which like rapamycin inhibits mTOR signaling, also inhibited PP5 activity, caused rapid dissociation of PR72, and activated ASK1 signaling. Overexpression of PP5, but not the PP2A catalytic subunit, blocked rapamycin-induced phosphorylation of c-Jun, and protected cells from rapamycin-induced apoptosis. The results suggest that PP5 is downstream of mTOR, and positively regulated by the mTOR pathway. The findings suggest that in the absence of serum factors, mTOR signaling suppresses apoptosis through positive regulation of PP5 activity and suppression of cellular stress. Rapamycin, and its analogues CCI-779, RAD001, and AP23573 now in clinical trials as anticancer agents potently inhibit tumor cell proliferation (1Huang S. Houghton P.J. Curr. Opin. Pharmacol. 2003; 3: 371-377Crossref PubMed Scopus (407) Google Scholar). These agents inhibit the function of the mammalian target of rapamycin, mTOR 1The abbreviations used are: mTOR, mammalian target of rapamycin; ASK1, apoptosis signal-regulating kinase 1; PP2A, protein phosphatase 2A; PP5, protein phosphatase 5; PR72, PP2A-B″ regulatory subunit; 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; S6K1, p70S6 kinase; TPR, tetratricopeptide repeat; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; PBS, phosphate-buffered saline; FACS, fluorescent-activated cell signaling; TOP, 5′-terminal oligopyrimidine tracts; FITC, fluorescein isothiocyanate. (also designated FRAP, RAFT1, or RAPT1), a 289 kDa Ser/Thr kinase, which lies downstream of phosphatidylinositol 3′-kinase, and senses mitogenic stimuli, nutrient conditions (2Hardwick J.S. Kuruvilla F.G. Tong J.K. Shamji A.F. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14866-14870Crossref PubMed Scopus (469) Google Scholar, 3Kim D.H. Sarbassov D.D. Ali S.M. King J.E. Latek R.R. ErdjumentBromage H. Tempst P. Sabatini D.M. Cell. 2002; 110: 163-175Abstract Full Text Full Text PDF PubMed Scopus (2387) Google Scholar, 4Hara K. Maruki Y. Long X. Yoshino K. Oshiro N. Hidayat S. Tokunaga C. Avruch J. Yonezawa K. Cell. 2002; 110: 177-189Abstract Full Text Full Text PDF PubMed Scopus (1454) Google Scholar), and ATP (5Dennis P.B. Jaeschke A. Saitoh M. Fowler B. Kozma S.C. Thomas G. Science. 2001; 294: 1102-1105Crossref PubMed Scopus (807) Google Scholar), and regulates multiple cellular events required for cellular growth (size) and proliferation (6Huang S. Houghton P.J. Curr. Opin. Investig. Drugs. 2002; 3: 295-304PubMed Google Scholar), differentiation (7Coolican S.A. Samuel D.S. Ewton D.Z. McWade F.J. Florini J.R. J. Biol. Chem. 1997; 272: 6653-6662Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar, 8Erbay E. Chen J. J. Biol. Chem. 2001; 276: 36079-36082Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 9Shu L. Zhang X. Houghton P.J. J. Biol. Chem. 2002; 277: 16726-16732Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), migration (10Poon M. Marx S.O. Gallo R. Badimon J.J. Taubman M.B. Marks A.R. J. Clin. Investig. 1996; 98: 2277-2283Crossref PubMed Scopus (464) Google Scholar, 11Sun J. Marx S.O. Chen H.J. Poon M. Marks A.R. Rabbani L.E. Circulation. 2001; 103: 2967-2972Crossref PubMed Scopus (178) Google Scholar), and survival (12Hosoi H. Dilling M.B. Shikata T. Liu L.N. Shu L. Ashmun R.A. Germain G.S. Abraham R.T. Houghton P.J. Cancer Res. 1999; 59: 886-894PubMed Google Scholar, 13Huang S. Liu L.N. Hosoi H. Dilling M.B. Shikata T. Houghton P.J. Cancer Res. 2001; 61: 3373-3381PubMed Google Scholar, 14Huang S. Shu L. Dilling M.B. Easton J. Harwood F.C. Ichijo H. Houghton P.J. Mol. Cell. 2003; 11: 1491-1501Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Rapamycin first forms a complex with a 12 kDa cytosolic protein designated FK-506-binding protein 12 (FKBP-12) and this complex binds mTOR and inhibits its function. Subsequently, eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) becomes hypophosphorylated and associates with eIF4E (15Brunn G.J. Hudson C.C. Sekulic A. Williams J.M. Hosoi H. Houghton P.J. Lawrence J.C. Abraham R.T. Science. 1997; 277: 99-104Crossref PubMed Scopus (813) Google Scholar, 16Gingras A.C. Raught B. Gygi S.P. Niedzwiecka A. Miron M. Burley S.K. Polakiewicz R.D. Wyslouch-Cieszynska A. Aebersold R. Sonenberg N. Genes Dev. 2001; 15: 2852-2864Crossref PubMed Scopus (1126) Google Scholar). This association prevents binding of eIF4E to eIF4G and formation of eIF4F initiation complex, thereby inhibiting cap-dependent translation of mRNA. Inhibition of mTOR by rapamycin also directly or indirectly inactivates ribosomal p70S6 kinase (S6K1), thereby blocking translation of mRNA species containing 5′-terminal oligopyrimidine tracts (TOP) (17Dennis P.B. Pullen N. Kozma S.C. Thomas G. Mol. Cell. Biol. 1996; 16: 6242-6451Crossref PubMed Scopus (224) Google Scholar, 18Peterson R.T. Desai B.N. Hardwick J.S. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4438-4442Crossref PubMed Scopus (429) Google Scholar). However, the requirement for S6K1 activity in translation of TOP containing mRNAs has been recently challenged. Importantly, complete inhibition of mTOR by rapamycin had only a slight repressive effect on translation of TOP mRNAs leading to the conclusion that regulation by growth factors and mitogens is primarily through the PI3K pathway with a minor role for mTOR in regulation of TOP mRNA translation (19Tang H. Hornstein E. Stolovich M. Levy G. Livingstone M. Templeton D. Avruch J. Meyuhas O. Mol. Cell. Biol. 2001; 21: 8671-8683Crossref PubMed Scopus (258) Google Scholar, 20Stolovich M. Tang H. Hornstein E. Levy G. Cohen R. Bae S.S. Birnbaum M.J. Meyuhas O. Mol. Cell. Biol. 2002; 22: 8101-8113Crossref PubMed Scopus (198) Google Scholar). In many cell lines, exposure to rapamycin reduces overall protein synthesis only ∼15–20% but results in a specific G1 accumulation. We previously found that proliferation of human rhabdomyosarcoma cells was inhibited by low concentrations of rapamycin (21Dilling M.B. Dias P. Shapiro D.N. Germain G.S. Johnson R.K. Houghton P.J. Cancer Res. 1994; 54: 903-907PubMed Google Scholar, 22Hosoi H. Dilling M.B. Liu L.N. Danks M.K. Shikata T. Sekulic A. Abraham R.T. Lawrence Jr, J.C. Houghton P.J. Mol. Pharmacol. 1998; 54: 815-824Crossref PubMed Scopus (165) Google Scholar). Under serum-free culture conditions rapamycin treatment induces apoptosis in these and other cells lacking functional p53 (12Hosoi H. Dilling M.B. Shikata T. Liu L.N. Shu L. Ashmun R.A. Germain G.S. Abraham R.T. Houghton P.J. Cancer Res. 1999; 59: 886-894PubMed Google Scholar, 13Huang S. Liu L.N. Hosoi H. Dilling M.B. Shikata T. Houghton P.J. Cancer Res. 2001; 61: 3373-3381PubMed Google Scholar). Ectopic expression of p53 or p21Cip1 protects cells from apoptosis (13Huang S. Liu L.N. Hosoi H. Dilling M.B. Shikata T. Houghton P.J. Cancer Res. 2001; 61: 3373-3381PubMed Google Scholar). Rapamycin inhibition of mTOR induces a cellular stress response characterized by rapid and sustained activation of apoptosis signal-regulating kinase 1 (ASK1) signaling in p53-mutant cells. In contrast only transient activation of ASK1 signaling occurs in cells expressing functional p53/p21Cip1 (14Huang S. Shu L. Dilling M.B. Easton J. Harwood F.C. Ichijo H. Houghton P.J. Mol. Cell. 2003; 11: 1491-1501Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). However, little is known about the mechanism by which inhibition of mTOR causes activation of ASK1 signaling. Protein phosphatase 5 (PP5, also named PPT) is a novel member of Ser/Thr phosphatase family, which also includes PP1, PP2A, PP2B (also termed PP3 or calcineurin A), PP2C, PP4 (also termed PPX), PP6 (also named PPV in Drosophila, or Sit4 in yeast), and PP7 (reviewed in Ref. 23Chinkers M. Trends Endocrinol. Metab. 2001; 12: 28-32Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). PP5 differs from the other Ser/Thr phosphatases in that it uniquely contains three tetratricopeptide repeat (TPR) motifs at its N terminus (24Chen M.X. McPartlin A.E. Brown L. Chen Y.H. Barker H.M. Cohen P.T. EMBO J. 1994; 13: 4278-4290Crossref PubMed Scopus (257) Google Scholar, 25Becker W. Kentrup H. Klumpp S. Schultz J.E. Joost H.G. J. Biol. Chem. 1994; 269: 22586-22592Abstract Full Text PDF PubMed Google Scholar) that serve as protein-protein interaction domains (26Blatch G.L. Lassle M. Bioessays. 1999; 21: 932-939Crossref PubMed Scopus (967) Google Scholar). Removal of the TPR domains by limited proteolysis results in a substantial increase in phosphatase activity suggesting that the TPR domains also function as autoinhibitory domains (27Chen M.X. Cohen P.T. FEBS Lett. 1997; 400: 136-140Crossref PubMed Scopus (119) Google Scholar, 28Sinclair C. Borchers C. Parker C. Tomer K. Charbonneau H. Rossie S. J. Biol. Chem. 1999; 274: 23666-23672Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). PP5 has been shown to interact with a number of proteins through TPR motifs (29Chinkers M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11075-11079Crossref PubMed Scopus (137) Google Scholar, 30Chen M.S. Silverstein A.M. Pratt W.B. Chinkers M. J. Biol. Chem. 1996; 271: 32315-32320Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 31Silverstein A.M. Galigniana M.D. Chen M.S. Owens-Grillo J.K. Chinkers M. Pratt W.B. J. Biol. Chem. 1997; 272: 16224-16230Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 32Russell L.C. Whitt S.R. Chen M.S. Chinkers M. J. Biol. Chem. 1999; 274: 20060-20063Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 33Zhao S. Sancar A. Photochem. Photobiol. 1997; 66: 727-731Crossref PubMed Scopus (80) Google Scholar, 34Ollendorff V. Donoghue D.J. J. Biol. Chem. 1997; 272: 32011-32018Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 35Kono Y. Maeda K. Kuwahara K. Yamamoto H. Miyamoto E. Yonezawa K. Takagi K. Sakaguchi N. Genes Cells. 2002; 7: 821-834Crossref PubMed Scopus (32) Google Scholar, 36Shao J. Hartson S.D. Matts R.L. Biochemistry. 2002; 41: 6770-6779Crossref PubMed Scopus (60) Google Scholar, 37Yamaguchi Y. Katoh H. Mori K. Negishi M. Curr. Biol. 2002; 12: 1353-1358Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 38Galigniana M.D. Harrell J.M. Murphy P.J. Chinkers M. Radanyi C. Renoir J.M. Zhang M. Pratt W.B. Biochemistry. 2002; 41: 13602-13610Crossref PubMed Scopus (93) Google Scholar). Importantly, PP5 associates with ASK1 (39Morita K. Saitoh M. Tobiume K. Matsuura H. Enomoto S. Nishitoh H. Ichijo H. EMBO J. 2001; 20: 6028-6036Crossref PubMed Scopus (254) Google Scholar), and other protein phosphatase subunits such as PP2A-A and -B subunits (40Lubert E.J. Hong Y. Sarge K.D. J. Biol. Chem. 2001; 276: 38582-38587Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). As in yeast, mTOR appears to regulate several protein phosphatases through binding to the Ig receptor-binding protein α4, the mammalian homologue of yeast Tap42. Association of α4 with PP2A, PP4, and PP6 has been linked to rapamycin sensitivity (41Murata K. Wu J. Brautigan D.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10624-10629Crossref PubMed Scopus (195) Google Scholar, 42Inui S. Sanjo H. Maeda K. Yamamoto H. Miyamoto E. Sakaguchi N. Blood. 1998; 92: 539-546Crossref PubMed Google Scholar), although it is considered that PP2A is negatively regulated by mTOR (18Peterson R.T. Desai B.N. Hardwick J.S. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4438-4442Crossref PubMed Scopus (429) Google Scholar, 41Murata K. Wu J. Brautigan D.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10624-10629Crossref PubMed Scopus (195) Google Scholar, 43Gingras A.C. Raught B. Sonenberg N. Genes Dev. 2001; 15: 807-826Crossref PubMed Scopus (1187) Google Scholar, 44Jacinto E. Hall M.N. Nat. Rev. Mol. Cell. Biol. 2003; 4: 117-126Crossref PubMed Scopus (515) Google Scholar), this remains controversial. In yeast Tap42 may redirect PP2A activity (61Di Como C.J. Arndt K.T. Genes Dev. 1996; 10: 1904-1916Crossref PubMed Scopus (442) Google Scholar). Very recent results in yeast indicate that Tap42 can both inhibit and activate protein phosphatases (47Duvel K. Santhanam A. Garrett S. Schneper L. Broach J.R. Mol. Cell. 2003; 11: 1467-1478Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), suggesting even greater complexity of Tor signaling. At this time it is unknown whether the mTOR pathway regulates PP5. As in yeast, contradictory information on the consequences of α4 dissociation from PP2A or PP6 has been reported. For instance, rapamycin treatment inhibits cell proliferation by decreasing PP2A activity through dissociating α4 from PP2A catalytic subunit (42Inui S. Sanjo H. Maeda K. Yamamoto H. Miyamoto E. Sakaguchi N. Blood. 1998; 92: 539-546Crossref PubMed Google Scholar). Other studies (45Chen J. Peterson R.T. Schreiber S.L. Biochem. Biophys. Res. Commun. 1998; 247: 827-832Crossref PubMed Scopus (159) Google Scholar, 46Kloeker S. Reed R. McConnell J.L. Chang D. Tran K. Westphal R.S. Law B.K. Colbran R.J. Kamoun M. Campbell K.S. Wadzinski B.E. Protein Expr. Purif. 2003; 31: 19-33Crossref PubMed Scopus (59) Google Scholar) do not demonstrate rapamycin-induced dissociation of α4 from PP2A or PP6. ASK1, a recently identified mitogen-activated protein kinase (MAPK) kinase kinase, activates the c-Jun N-terminal kinase (JNK) and p38 MAPK pathways in response to a variety of cytotoxic stimuli, including oxidative stress, Fas ligand, tumor necrosis factor, nutrient depletion and antitumor agents (14Huang S. Shu L. Dilling M.B. Easton J. Harwood F.C. Ichijo H. Houghton P.J. Mol. Cell. 2003; 11: 1491-1501Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 39Morita K. Saitoh M. Tobiume K. Matsuura H. Enomoto S. Nishitoh H. Ichijo H. EMBO J. 2001; 20: 6028-6036Crossref PubMed Scopus (254) Google Scholar, 48Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (2035) Google Scholar, 49Tobiume K. Matsuzawa A. Takahashi T. Nishitoh H. Morita K. Takeda K. Minowa O. Miyazono K. Noda T. Ichijo H. EMBO Rep. 2001; 2: 222-228Crossref PubMed Scopus (1015) Google Scholar, 50Chang H.Y. Nishitoh H. Yang X. Ichijo H. Baltimore D. Science. 1998; 281: 1860-1863Crossref PubMed Scopus (534) Google Scholar, 51Gotoh Y. Cooper J.A. J. Biol. Chem. 1998; 273: 17477-17482Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 52Nishitoh H. Saitoh M. Mochida Y. Takeda K. Nakano H. Rothe M. Miyazono K. Ichijo H. Mol. Cell. 1998; 2: 389-395Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar, 53Saitoh M. Nishitoh H. Fujii M. Takeda K. Tobiume K. Sawada Y. Kawabata M. Miyazono K. Ichijo H. EMBO J. 1998; 17: 2596-2606Crossref PubMed Scopus (2089) Google Scholar, 54Wang T.H. Wang H.S. Ichijo H. Giannakakou P. Foster J.S. Fojo T. Wimalasena J. J. Biol. Chem. 1998; 273: 4928-4936Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 55Wang T.H. Popp D.M. Wang H.S. Saitoh M. Mural J.G. Henley D.C. Ichijo H. Wimalasena J. J. Biol. Chem. 1999; 274: 8208-8216Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 56Song J.J. Rhee J.G. Suntharalingam M. Walsh S.A. Spitz D.R. Lee Y.J. J. Biol. Chem. 2002; 277: 46566-46575Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). Numerous protein phosphatases can dephosphorylate and inactivate JNK and p38 MAPK (57Tamura S. Hanada M. Ohnishi M. Katsura K. Sasaki M. Kobayashi T. Eur. J. Biochem. 2002; 269: 1060-1066Crossref PubMed Scopus (92) Google Scholar). However, PP5 is the only protein phosphatase that has so far been identified to physically interact with, and negatively regulate ASK1 (39Morita K. Saitoh M. Tobiume K. Matsuura H. Enomoto S. Nishitoh H. Ichijo H. EMBO J. 2001; 20: 6028-6036Crossref PubMed Scopus (254) Google Scholar). Here we show that inhibition of mTOR by rapamycin or amino acid deprivation causes rapid dissociation of PP2A-B″ subunit (designated PR72) from PP5, that is associated with decreased phosphatase activity of PP5, and concurrently activating ASK1. Furthermore, down-regulation of PP5 activity by inhibition of mTOR is dependent on expression of 4E-BP1. Our findings suggest that PP5 is downstream in the mTOR pathway, is positively regulated by mTOR and that mTOR regulates ASK1 activity via PP5. Cell Lines and Growth Conditions—Human rhabdomyosarcoma cell line Rh30 that expresses a mutant p53 (Arg273 → Cys), and Rh30/mTOR-rr expressing a rapamycin-resistant mutant mTOR (Ser2035 → Ile) (12Hosoi H. Dilling M.B. Shikata T. Liu L.N. Shu L. Ashmun R.A. Germain G.S. Abraham R.T. Houghton P.J. Cancer Res. 1999; 59: 886-894PubMed Google Scholar) were grown in antibiotic-free RPMI 1640 medium supplemented with 10% fetal bovine serum and 2 mm l-glutamine at 37° and 5% CO2. For experiments where cells were deprived of serum overnight, cell monolayers were washed with RPMI 1640 containing 2 mm l-glutamine, and incubated in the same medium. For prolonged serum-free conditions, Rh30 cells were cultured in modified N2E medium (MN2E; DMEM/F12 supplemented with 1 μg/ml human transferrin, 30 nm sodium selenate, 20 nm progesterone, 100 μm putrescine, 30 nm vitamin E phosphate, 50 μm ethanolamine, and 1 mg/ml bovine serum albumin). For amino acid deprivation, cells were cultured in a specific RPMI 1640 medium (R-7130, without l-glutamine, l-leucine, l-lysine, and l-methionine) (Sigma). Plasmids and Transfection of Cells—Rh30 cells (5 × 105 cells) in 6-well plates were transfected with plasmid expression vectors using FuGENE 6 transfection reagent (Roche Applied Science) as directed by the manufacturer. Plasmids encoding N-terminal FLAG-tagged wild-type rat PP2Aα catalytic subunit (a gift from Dr. Hitoshi Nakagama, Hokkaido University, Sapporo, Japan) and human PP5 were as described previously (39Morita K. Saitoh M. Tobiume K. Matsuura H. Enomoto S. Nishitoh H. Ichijo H. EMBO J. 2001; 20: 6028-6036Crossref PubMed Scopus (254) Google Scholar). Wild-type 4E-BP1 (pcDNA3-PHAS-I) was from Robert T. Abraham (Burnham Institute, La Jolla, CA). After transfection for 48 h, cells were transferred to 100-mm dishes and cultured for 2 weeks at 37°, 5% CO2 in growth medium containing 500 μg/ml geneticin (G418 sulfate; Invitrogen, Carlsbad, CA). Drug-resistant cells were cloned and expanded in growth medium containing G418 (500 μg/ml). Clones were screened by Western blot analysis for expression of the appropriate protein. 4E-BP1 was probed with an antibody to 4E-BP1 (Zymed Laboratories Inc., South San Francisco, CA), and FLAG-PP2Aα catalytic subunit and FLAG-PP5 were detected with an antibody to FLAG (M2, Sigma). A short hairpin-activated gene silencing (SHAG) strategy was used for siRNA to down-regulate 4E-BP1, as described previously (14Huang S. Shu L. Dilling M.B. Easton J. Harwood F.C. Ichijo H. Houghton P.J. Mol. Cell. 2003; 11: 1491-1501Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). To suppress 4E-BP1 expression Rh30 cells were transfected with 1 μg of plasmid (piNEO-4E-BP1 or control piNEO) using 3 μl of FuGENE 6 per the manufacturer's instructions. Three days post-transfection the cells were replated in a 10-cm dish with 10% fetal bovine serum/RPMI 1640 medium containing 500 μg/ml G418 for selection. Ten days after initial selection the piNEO and piNEO-4E-BP1 cells were analyzed for expression of 4E-BP1 protein by Western blot. Empty vector piNEO did not affect 4E-BP1 expression, whereas piNEO-4E-BP1 reduced 4E-BP1 expression by about 80%. Both piNEO and piNEO-4E-BP1 cells were expanded and used for experiments. Immunoprecipitation and Western Blot Analysis—Cells were briefly washed with cold PBS. On ice, cells were lysed in radioimmune precipitation assay buffer (50 mm Tris, pH 7.2, 150 mm NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Triton-X 100, 10 mm NaF, 1 mm Na3VO4, protease inhibitor mixture (1:1000, Sigma)) for direct Western blot analysis and in lysis buffer (20 mm Tris, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm β-glycerol phosphate, 1 mm Na3VO4, 1 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride) for immunoprecipitation. Lysates were sonicated for 10 s and centrifuged at 14,000 rpm for 15 min at 4 °C. Protein concentration was determined by bicinchoninic acid assay with bovine serum albumin as standard (Pierce). Immune complexes were immunoprecipitated from 1 mg of cleared cell lysate with corresponding antibody and protein A/G-agarose (Santa Cruz Biotechnology) and washed four times with the lysis buffer. For Western blot analysis, equivalent amounts of protein were separated on 7.5–12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were incubated with PBS containing 0.05% Tween 20 and 5% nonfat dry milk to block nonspecific binding and were incubated with primary antibodies, then with secondary antibodies conjugated to horseradish peroxidase. We used primary antibodies to phospho-c-Jun (Ser63) and c-Jun (Santa Cruz Biotechnology), 4E-BP1, S6K1 (Cell Signaling, Beverly, MA), α4 protein (a gift from D. Brautigan, University of Virginia and Upstate Biotechnology, Lake Placid, NY), FLAG (M2, Sigma), mTOR (58Zhang X. Shu L. Hosoi H. Murti K.G. Houghton P.J. J. Biol. Chem. 2002; 277: 28127-28134Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), PP2Ac, and PP5 (Transduction Laboratories, Lexington, KY), PP2A-A and -B subunits (Upstate Biotechnology), and ASK1 (48Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (2035) Google Scholar). Immunoreactive bands were visualized by using Renaissance chemiluminescence reagent (PerkinElmer Life Sciences, Boston, MA). To check the amount of protein loaded, the immunoblots were treated with stripping solution (62.5 mm Tris buffer, pH 6.7, containing 2% SDS and 100 mm β-mercaptoethanol) for 30 min at 50 °C and incubated with mouse monoclonal anti-β-tubulin antibody (Sigma) followed by horseradish peroxidase-coupled goat anti-mouse IgG (Pierce). In Vitro Kinase Assay—Cells were briefly washed with cold PBS, and disrupted in lysis buffer (20 mm Tris, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm β-glycerol phosphate, 1 mm Na3VO4, 1 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride), followed by immunoprecipitation with rabbit polyclonal antibodies to ASK1 (48Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (2035) Google Scholar) and protein A/G-agarose. The kinase activity of ASK1 in the immunocomplexes was analyzed using recombinant GST-MKK6 as a substrate, as described previously (39Morita K. Saitoh M. Tobiume K. Matsuura H. Enomoto S. Nishitoh H. Ichijo H. EMBO J. 2001; 20: 6028-6036Crossref PubMed Scopus (254) Google Scholar). After detection of phosphorylation of GST-MKK6, polyvinylidene difluoride membranes were immunoblotted with ASK1 antibody. In Vitro Phosphatase Assay—Cells were lysed in 50 mm Tris-HCl buffer, pH 7.0, containing 1% Nonidet P-40, 2 mm EDTA, and protease inhibitor mixture (Sigma, 1:1000). PP2Ac or PP5 was immunoprecipitated with antibodies to PP2A catalytic subunit (rabbit polyclonal, Upstate Biotechnology) or PP5 (mouse monoclonal), respectively, and protein A/G-agarose. Subsequently, the beads were washed three times with the above lysis buffer, and twice with the phosphatase assay buffer (50 mm Tris-HCl, pH 7.0, 0.1 mm CaCl2). The phosphatase activity of immunoprecipitated PP2A or PP5 was assayed with a Ser/Thr Phosphatase Assay kit 1 using KRpTIRR as the substrate peptide (Upstate Biotechnology) following the manufacturer's instructions. Growth Inhibition Assay—Cells were seeded on 6-well plates (5 × 104 cells/well) in triplicate. Rapamycin (0–10,000 ng/ml) was added the next day. After incubation for 7 days, cells were enumerated by counting the nuclei as described previously (21Dilling M.B. Dias P. Shapiro D.N. Germain G.S. Johnson R.K. Houghton P.J. Cancer Res. 1994; 54: 903-907PubMed Google Scholar). FACS Analysis of Apoptosis—Cells (1.7 × 106 per 162-cm2 flask) were grown overnight in MN2E. Rapamycin (100 ng/ml) was added the next day, and cells were exposed for up to 6 days. Parallel control cells were not treated with rapamycin. Cells were treated with trypsin, washed with PBS, resuspended in 200 μl of binding buffer (Clontech Laboratories), and incubated with 10 μl annexin V-FITC (final concentration, 1 μg/ml; Clontech Laboratories) and 500 ng propidium iodide (final volume, 410 μl). Cells were incubated at room temperature without light for 10 min before flow cytometric analysis (FACSCalibur) (12Hosoi H. Dilling M.B. Shikata T. Liu L.N. Shu L. Ashmun R.A. Germain G.S. Abraham R.T. Houghton P.J. Cancer Res. 1999; 59: 886-894PubMed Google Scholar). Rapamycin Inhibits PP5 Activity in an mTOR-dependent Manner—We previously found that under conditions of serum-free growth rapamycin induced apoptosis of p53-mutant/null cells. Although detection of apoptosis was relatively delayed, early events were characterized by rapid and sustained activation of the ASK1 signaling pathway (12Hosoi H. Dilling M.B. Shikata T. Liu L.N. Shu L. Ashmun R.A. Germain G.S. Abraham R.T. Houghton P.J. Cancer Res. 1999; 59: 886-894PubMed Google Scholar, 13Huang S. Liu L.N. Hosoi H. Dilling M.B. Shikata T. Houghton P.J. Cancer Res. 2001; 61: 3373-3381PubMed Google Scholar, 14Huang S. Shu L. Dilling M.B. Easton J. Harwood F.C. Ichijo H. Houghton P.J. Mol. Cell. 2003; 11: 1491-1501Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Rapamycin did not affect the level of ASK1, but increased the kinase activity of ASK1 (14Huang S. Shu L. Dilling M.B. Easton J. Harwood F.C. Ichijo H. Houghton P.J. Mol. Cell. 2003; 11: 1491-1501Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Recently, PP5 has been shown to interact with ASK1 and identified as a negative regulator of ASK1 in HeLa cells (39Morita K. Saitoh M. Tobiume K. Matsuura H. Enomoto S. Nishitoh H. Ichijo H. EMBO J. 2001; 20: 6028-6036Crossref PubMed Scopus (254) Google Scholar). Activation of ASK1 signaling could be attributed to either down-regulation of PP5 protein level, decreased association of PP5 with ASK1, or decreased phosphatase activity of PP5. To answer these questions, serum-starved Rh30 cells with mutant p53 (Arg273 → Cys) were exposed to rapamycin (100 ng/ml) for up to 24 h, followed by either direct Western blot with antibodies to ASK1, PP5, and β-tubulin (loading control), or immunoprecipitation with antibodies to PP5, and then immunoblotting with antibodies to ASK1 and PP5, respectively, or in vitro phosphatase assay for PP5. As seen previously (14Huang S. Shu L. Dilling M.B. Easton J. Harwood F.C. Ichijo H. Houghton P.J. Mol. Cell. 2003; 11: 1491-1501Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar), rapamycin did increase the kinase activity

The serine/threonine kinase, mTOR (mammalian Target of Rapamycin) has become a focus for cancer drug development. Rapamycins are highly specific inhibitors of mTOR and potently suppress tumour cell growth by retarding cells in G1 phase or potentially inducing apoptosis. Currently, both rapamycin and several analogues are being evaluated as anticancer agents in clinical trials. Results indicate that many human cancers have intrinsic resistance and tumours initially sensitive to rapamycins become refractory, demonstrating acquired resistance. Here, we consider mechanisms of resistance to inhibitors of mTOR.

Mammalian target of rapamycin (mTOR) controls lymphangiogenesis. However, the underlying mechanism is not clear. Here we show that rapamycin suppressed insulin-like growth factor 1 (IGF-1)- or fetal bovine serum (FBS)-stimulated lymphatic endothelial cell (LEC) tube formation, an in vitro model of lymphangiogenesis. Expression of a rapamycin-resistant and kinase-active mTOR (S2035T, mTOR-T), but not a rapamycin-resistant and kinase-dead mTOR (S2035T/D2357E, mTOR-TE), conferred resistance to rapamycin inhibition of LEC tube formation, suggesting that rapamycin inhibition of LEC tube formation is mTOR kinase activity dependent. Also, rapamycin inhibited proliferation and motility in the LECs. Furthermore, we found that rapamycin inhibited protein expression of VEGF receptor 3 (VEGFR-3) by inhibiting protein synthesis and promoting protein degradation of VEGFR-3 in the cells. Down-regulation of VEGFR-3 mimicked the effect of rapamycin, inhibiting IGF-1- or FBS-stimulated tube formation, whereas over-expression of VEGFR-3 conferred high resistance to rapamycin inhibition of LEC tube formation. The results indicate that rapamycin inhibits LEC tube formation at least in part by downregulating VEGFR-3 protein expression.

Abstract Alterations in translation occur in cancer cells, but the precise pathogenic processes and mechanistic underpinnings are not well understood. In this study, we report that interactions between Pim family kinases and the translation initiation factor eIF4B are critical for Abl oncogenicity. Pim kinases, Pim-1 and Pim-2, both directly phosphorylated eIF4B on Ser406 and Ser422. Phosphorylation of eIF4B on Ser422 was highly sensitive to pharmacologic or RNA interference-mediated inhibition of Pim kinases. Expression and phosphorylation of eIF4B relied upon Abl kinase activity in both v-Abl- and Bcr-Abl–expressing leukemic cells based on their blockade by the Abl kinase inhibitor imatinib. Ectopic expression of phosphomimetic mutants of eIF4B conferred resistance to apoptosis by the Pim kinase inhibitor SMI-4a in Abl-transformed cells. In contrast, silencing eIF4B sensitized Abl-transformed cells to imatinib-induced apoptosis and also inhibited their growth as engrafted tumors in nude mice. Extending these observations, we found that primary bone marrow cells derived from eIF4B-knockdown transgenic mice were less susceptible to Abl transformation, relative to cells from wild-type mice. Taken together, our results identify eIF4B as a critical substrate of Pim kinases in mediating the activity of Abl oncogenes, and they highlight eIF4B as a candidate therapeutic target for treatment of Abl-induced cancers. Cancer Res; 73(15); 4898–908. ©2013 AACR.

Pyruvate kinase isozyme type M2 (PKM2) was first found in hepatocellular carcinoma (HCC), and its expression has been thought to correlate with prognosis. A large number of studies have demonstrated that epithelial-mesenchymal transition (EMT) is a crucial event in hepatocellular carcinoma (HCC) and associated metastasis, resulting in enhanced malignancy of HCC. However, the roles of PKM2 in HCC EMT and metastasis remain largely unknown. The present study aimed to determine the effects of PKM2 in EGF-induced HCC EMT and elucidate the molecular mechanisms in vitro. Our results showed that EGF promoted EMT in HCC cell lines as evidenced by altered morphology, expression of EMT-associated markers, and enhanced invasion capacity. Furthermore, the present study also revealed that nuclear translocation of PKM2, which is regulated by the ERK pathway, regulated <TEX>${\beta}$</TEX>-catenin-TCF/LEF-1 transcriptional activity and associated EMT in HCC cell lines. These discoveries provide evidence of novel roles of PKM2 in the progression of HCC and potential therapeutic target for advanced cases.

Background: Ciclopirox (CPX) has been used as an antifungal agent in various formulations to treat superficial fungal infection for decades. Its effectiveness and safety in treatments have been demonstrated by multiple studies. Methods: Here we briefly summarize the pharmacological and toxicological properties of CPX as an antifungal agent, the new medical uses of CPX, as well as the correspondent molecular mechanisms. Results: Increasing evidence has demonstrated that CPX is able to inhibit tumor growth, ameliorate diabetes and its complications, prevent human immunodeficiency virus (HIV) infection, and improve age-associated cardiovascular defects. Interestingly, its antifungal activity and all those newly observed effects are more or less related to its capability of chelating iron and interfering with the related signaling pathways. Mechanistically, CPX is capable of modulating the activities of certain enzymes or signaling pathways, such as ribonucleotide reductase (RR), deoxyhypusine hydroxylase (DOHH)/eukaryotic translation initiation factor 5A (eIF5A), Wnt/ β-catenin, hypoxia-inducible factor-1α (HIF-1 α)/vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 3 (VEGFR-3)/extracellular signal-regulated protein kinases 1/2, mammalian target of rapamycin, and cyclin dependent kinases (CDKs). Most of these activities are related to its chelation of iron. Conclusion: CPX, as an antifungal agent, may be repositioned for treatment of cancer and other human diseases. Keywords: Ciclopirox, iron chelator, fungicide, cancer, diabetes, HIV.

Long noncoding RNAs (lncRNAs), defined as the transcripts longer than 200 nt without protein-coding capacity, have been found to be aberrantly expressed in diverse human diseases including cancer. A reciprocal translocation between chromosome 9 and 22 generates the chimeric Bcr-Abl oncogene, which is associated with several hematological malignancies. However, the functional relevance between aberrantly expressed lncRNAs and Bcr-Abl-mediated leukemia remains obscure.LncRNA cDNA microarray was used to identify novel lncRNAs involved in Bcr-Abl-mediated cellular transformation. To study the functional relevance of novel imatinib-upregulated lncRNA (IUR) family in Abl-induced tumorigenesis, Abl-transformed cell survival and xenografted tumor growth in mice was evaluated. Primary bone marrow transformation and in vivo leukemia transplant using lncRNA-IUR knockdown (KD) transgenic mice were further conducted to corroborate the role of lncRNA-IUR in Abl-induced tumorigenesis. Transcriptome RNA-seq, Western blot, RNA pull down and RNA Immunoprecipitation (RIP) were employed to determine the mechanisms by which lncRNA-IUR-5 regulates Bcr-Abl-mediated tumorigenesis.We identified a conserved lncRNA-IUR family as a key negative regulator of Bcr-Abl-induced tumorigenesis. Increased expression of lncRNA-IUR was detected in both human and mouse Abl-transformed cells upon imatinib treatment. In contrast, reduced expression of lncRNA-IUR was observed in the peripheral blood lymphocytes derived from Bcr-Abl-positive acute lymphoblastic leukemia (ALL) patients compared to normal subjects. Knockdown of lncRNA-IUR remarkably promoted Abl-transformed leukemic cell survival and xenografted tumor growth in mice, whereas overexpression of lncRNA-IUR had opposite effects. Also, silencing murine lncRNA-IUR promoted Bcr-Abl-mediated primary bone marrow transformation and Abl-transformed leukemia cell survival in vivo. Besides, knockdown of murine lncRNA-IUR in transgenic mice provided a favorable microenvironment for development of Abl-mediated leukemia. Finally, we demonstrated that lncRNA-IUR-5 suppressed Bcr-Abl-mediated tumorigenesis by negatively regulating STAT5-mediated expression of CD71.The results suggest that lncRNA-IUR may act as a critical tumor suppressor in Bcr-Abl-mediated tumorigenesis by suppressing the STAT5-CD71 pathway. This study provides new insights into functional involvement of lncRNAs in leukemogenesis.

Porcine epidemic diarrhea virus (PEDV) infection causes immunosuppression and clinical symptoms such as vomiting, watery diarrhea, dehydration, and even death in piglets. TRIM28, an E3 ubiquitin ligase, is involved in the regulation of autophagy. However, the role of TRIM28 in PEDV infection is unknown. This study aimed to determine whether TRIM28 acts as a host factor for PEDV immune escape. We found that depletion of TRIM28 inhibited PEDV replication, whereas overexpression of TRIM28 promoted the viral replication in host cells. Furthermore, knockdown of TRIM28 reversed PEDV-induced downregulation of the JAK/STAT1 pathway. Treatment with the mitophagic activator carbonyl cyanide 3-chlorophenylhydrazone (CCCP) attenuated the activating effect of TRIM28 depletion on the expression of the STAT1 pathway-related proteins. Treatment with CCCP also reduced the nuclear translocation of pSTAT1. Moreover, TRIM28, via its RING domain, interacted with PEDV N. Overexpression of TRIM28 induced mitophagy, which could be enhanced by co-expression with PEDV N. The results indicate that PEDV infection upregulates the expression of TRIM28, which induces mitophagy, leading to inhibition of the JAK-STAT1 pathway. This research unveils a new mechanism by which PEDV can hijack host cellular TRIM28 to promote its own replication.

Dihydroartemisinin (DHA), an antimalarial drug, has previously unrecognized anticancer activity, and is in clinical trials as a new anticancer agent for skin, lung, colon and breast cancer treatment. However, the anticancer mechanism is not well understood. Here, we show that DHA inhibited proliferation and induced apoptosis in rhabdomyosarcoma (Rh30 and RD) cells, and concurrently inhibited the signaling pathways mediated by the mammalian target of rapamycin (mTOR), a central controller for cell proliferation and survival, at concentrations (<3 μM) that are pharmacologically achievable. Of interest, in contrast to the effects of conventional mTOR inhibitors (rapalogs), DHA potently inhibited mTORC1-mediated phosphorylation of p70 S6 kinase 1 and eukaryotic initiation factor 4E binding protein 1 but did not obviously affect mTORC2-mediated phosphorylation of Akt. The results suggest that DHA may represent a novel class of mTORC1 inhibitor and may execute its anticancer activity primarily by blocking mTORC1-mediated signaling pathways in the tumor cells.

Cadmium (Cd), a toxic environmental contaminant, induces neurodegenerative diseases. Celastrol, a plant-derived triterpene, has shown neuroprotective effects in various disease models. However, little is known regarding the effect of celastrol on Cd-induced neurotoxicity. Here, we show that celastrol protected against Cd-induced apoptotic cell death in neuronal cells. This is supported by the findings that celastrol strikingly attenuated Cd-induced viability reduction, morphological change, nuclear fragmentation, and condensation, as well as activation of caspase-3 in neuronal cells. Concurrently, celastrol remarkably blocked Cd-induced phosphorylation of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinases 1/2 and p38, in neuronal cells. Inhibition of JNK by SP600125 or over-expression of dominant negative c-Jun potentiated celastrol protection against Cd-induced cell death. Furthermore, pre-treatment with celastrol prevented Cd down-regulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and activation of phosphoinositide 3'-kinase/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling in neuronal cells. Over-expression of wild-type PTEN enhanced celastrol inhibition of Cd-activated Akt/mTOR signaling and cell death in neuronal cells. The findings indicate that celastrol prevents Cd-induced neuronal cell death via targeting JNK and PTEN-Akt/mTOR network. Our results strongly suggest that celastrol may be exploited for the prevention of Cd-induced neurodegenerative disorders. Celastrol, a plant-derived triterpene, has shown neuroprotective effects. However, little is known regarding the effect of celastrol on cadmium (Cd) neurotoxicity. This study underscores that celastrol prevents Cd-induced neuronal apoptosis via inhibiting activation of JNK (c-Jun N-terminal kinase) and Akt/mTOR network. Celastrol suppresses Cd-activated Akt/mTOR pathway by elevating PTEN (phosphatase and tensin homolog). The findings suggest that celastrol may be exploited for the prevention of Cd-induced neurodegenerative disorders.

The polyphenol natural product curcumin possesses a plethora of biological and pharmacological properties. For years, much interest has been placed in the development and use of curcumin and its derivatives for the prevention and treatment of cardiovascular, diabetic, and neurodegenerative diseases, as well as cancer. Increasing evidence suggests that curcumin displays amazing molecular versatility, and the number of its proposed cellular targets grows as the research continues. The mammalian target of rapamycin (mTOR) is a master kinase, regulating cell growth/proliferation, survival, and motility. Dysregulated mTOR signaling occurs frequently in cancer, and targeting mTOR signaling is a promising strategy for cancer therapy. Recent studies have identified mTOR as a novel target of curcumin. Here we focus on reviewing current knowledge regarding the effects of curcumin on mTOR signaling for better understanding the anticancer mechanism of curcumin. The emerging studies of mTOR signaling and clinical studies on curcumin with cancer patients are also discussed here. Keywords: Curcumin, mTOR, Akt, Cancer, Cell proliferation, Cell death.

Avermectin (AVM) is a macrocyclic lactone agent widely used as a nematicide, acaricide and insecticide in veterinary medicine and plant protection. P-glycoprotein (P-gp) is an ATP-dependent drug efflux pump for xenobiotic compounds, and is involved in multidrug resistance. To understand the development of AVM resistance in invertebrates, we investigated the mechanisms by which AVM affected P-gp expression in Drosophila S2 cells. We found that AVM induced upregulation of P-gp protein expression, increased P-gp ATPase activity and enhanced cellular efflux of the P-gp substrate rhodamine 123 from cells. Furthermore, we observed that AVM-induced expression of P-gp was due to elevation of intracellular calcium concentration ([Ca2+]i). This occurred both directly, by activating calcium ion channels, and indirectly, by activating chloride ion channels. These results are supported by our observations that verapamil, a Ca2+ channel blocker, and niflumic acid, a chloride channel antagonist, significantly attenuated AVM-induced [Ca2+]i elevation, thereby reducing P-gp expression. Inhibition of P-gp with anti-P-gp antibody or cyclosporine A (a P-gp inhibitor) reduced the AVM-induced elevation of [Ca2+]i, implying that P-gp and [Ca2+]i regulate each other. Finally, we found that trifluoperazine, a calmodulin inhibitor, and pyrrolidine dithiocarbamic acid, an NF-κB inhibitor, attenuated the AVM-induced expression of P-gp, suggesting that AVM induces P-gp protein expression via the calmodulin/Relish (NF-κB) signaling pathway.

Background and Purpose Increasing evidence has suggested cadmium (Cd), as an inducer of ROS, is a potential pathogenic factor in human neurodegenerative diseases. Thus, it is important to find effective interventions for Cd‐induced oxidative stress in the CNS. Here, we have studied the effects of celastrol, a plant‐derived triterpene, on ROS production and cell death in neuronal cells, induced by Cd. Experimental Approach PC12, SH‐SY5Y cells and primary murine neurons were used to study celastrol neuroprotection against Cd‐poisoning. Cd‐induced changes in cell viability, apoptosis, ROS and AMP‐activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway in the cells were analysed by Trypan blue exclusion, DAPI and TUNEL staining, ROS imaging, immunofluorescence staining and Western blotting. Pharmacological and genetic approaches were employed to investigate the mechanisms underlying Cd neurotoxicity. Results Celastrol attenuated Cd‐induced apoptosis by suppressing Cd activation of mTOR, which was attributed to preventing Cd inactivation of AMPK. Inhibition of AMPK with compound C or expression of dominant negative AMPKα prevented celastrol from hindering Cd‐induced dephosphorylation of AMPKα, activation of mTOR and apoptosis. Inhibition of mTOR with rapamycin or knockdown of mTOR potentiated prevention by celastrol, of Cd‐induced phosphorylation of p70 S6 kinase 1/eukaryotic initiation factor 4E binding protein 1 and apoptosis. Celastrol attenuated Cd‐induced cell death by suppressing induction of mitochondrial ROS. Conclusions and Implications Celastrol prevented the inactivation of AMPK by mitochondrial ROS, thus attenuating Cd‐induced mTOR activation and neuronal apoptosis. Celastrol may be a promising agent for prevention of Cd‐induced oxidative stress and neurodegenerative diseases.

Cadmium (Cd), an environmental and industrial pollutant, affects the nervous system and consequential neurodegenerative disorders. Recently, we have shown that celastrol prevents Cd-induced neuronal cell death partially by suppressing Akt/mTOR pathway. However, the underlying mechanism remains to be elucidated. Here, we show that celastrol attenuated Cd-elevated intracellular-free calcium ([Ca2+ ]i ) level and apoptosis in neuronal cells. Celastrol prevented Cd-induced neuronal apoptosis by inhibiting Akt-mediated mTOR pathway, as inhibition of Akt with Akt inhibitor X or ectopic expression of dominant negative Akt reinforced celastrol's prevention of Cd-induced phosphorylation of S6K1/4E-BP1 and cell apoptosis. Furthermore, chelating intracellular Ca2+ with BAPTA/AM or preventing [Ca2+ ]i elevation using EGTA potentiated celastrol's repression of Cd-induced [Ca2+ ]i elevation and consequential activation of Akt/mTOR pathway and cell apoptosis. Moreover, celastrol blocked Cd-elicited phosphorylation of CaMKII, and pretreatment with BAPTA/AM or EGTA enhanced celastrol's suppression of Cd-increased phosphorylation of CaMKII in neuronal cells, implying that celastrol hinders [Ca2+ ]i -mediated CaMKII phosphorylation. Inhibiting CaMKII with KN93 or silencing CaMKII attenuated Cd activation of Akt/mTOR pathway and cell apoptosis, and this was strengthened by celastrol. Taken together, these data demonstrate that celastrol attenuates Cd-induced neuronal apoptosis via inhibiting Ca2+ -CaMKII-dependent Akt/mTOR pathway. Our findings underscore that celastrol may act as a neuroprotective agent for the prevention of Cd-induced neurodegenerative disorders. J. Cell. Physiol. 232: 2145-2157, 2017. © 2016 Wiley Periodicals, Inc.

Abstract Celastrol, a plant‐derived triterpene, has neuroprotective benefit in the models of neurodegenerative disorders that are characterized by overproduction of reactive oxygen species ( ROS ). Recently, we have reported that cadmium (Cd) activates c‐Jun N‐terminal kinase ( JNK ) pathway leading to neuronal cell death by inducing ROS inactivation of protein phosphatase 5 ( PP 5), and celastrol prevents Cd‐activated JNK pathway against neuronal apoptosis. Therefore, we hypothesized that celastrol could hinder Cd induction of ROS ‐dependent PP 5‐ JNK signaling pathway from apoptosis in neuronal cells. Here, we show that celastrol attenuated Cd‐induced expression of NADPH oxidase 2 ( NOX 2) and its regulatory proteins (p22 phox , p40 phox , p47 phox , p67 phox , and Rac1), as well as the generation of ROS in PC 12 cells and primary neurons. Also, N ‐acetyl‐ l ‐cysteine, a ROS scavenger, potentiated celastrol's inhibition of the events in the cells triggered by Cd, implying neuroprotection by celastrol via blocking Cd‐evoked NOX 2‐derived ROS . Further research revealed that celastrol was involved in the regulation of PP 5 inactivation and JNK /c‐Jun activation induced by Cd, as celastrol prevented Cd from reducing PP 5 expression, and over‐expression of wild‐type PP 5 or dominant negative c‐Jun strengthened celastrol's inhibition of Cd‐induced phosphorylation of JNK and/or c‐Jun, as well as apoptosis in neuronal cells. Of importance, inhibiting NOX 2 with apocynin or silencing NOX 2 by RNA interference enhanced the inhibitory effects of celastrol on Cd‐induced inactivation of PP 5, activation of JNK /c‐Jun, ROS , and apoptosis in the cells. Furthermore, we noticed that expression of wild‐type PP 5 or dominant negative c‐Jun, or pretreatment with JNK inhibitor SP 600125 reinforced celastrol's suppression of Cd‐induced NOX 2 and its regulatory proteins, and consequential ROS in neuronal cells. These findings indicate that celastrol ameliorates Cd‐induced neuronal apoptosis via targeting NOX 2‐derived ROS ‐dependent PP 5‐ JNK signaling pathway. Our data highlight a beneficial role of celastrol in the prevention of Cd‐induced oxidative stress and neurodegenerative diseases. image

Cadmium (Cd), a heavy metal pollutant, contributes to neurodegenerative disorders. Recently, we have demonstrated that Cd induction of reactive oxygen species (ROS) causes apoptosis in neuronal cells. Whether X-linked inhibitor of apoptosis protein (XIAP) is involved in Cd-induced ROS-dependent neuronal apoptosis remains unclear. Here, we show that Cd-induced ROS reduced the expression of XIAP, which resulted in up-regulation of murine double minute 2 homolog (MDM2) and down-regulation of p53, leading to apoptosis in PC12 cells and primary neurons. Inhibition of MDM2 with Nutlin-3a reversed Cd-induced reduction of p53 and substantially rescued cells from excess ROS-dependent death. Overexpression of XIAP protected against Cd induction of ROS-dependent neuronal apoptosis. Inhibition of XIAP by Embelin strengthened Cd-induced ROS and apoptosis in the cells. Furthermore, we found that Cd inactivation of XIAP pathway was attributed to Cd induction of mitochondrial ROS, as evidenced by using a mitochondrial superoxide indicator MitoSOX and a mitochondria-targeted antioxidant Mito-TEMPO. Taken together, these results indicate that Cd induces mitochondrial ROS inactivation of XIAP-MDM2-p53 pathway leading to apoptosis in neuronal cells. Our findings suggest that activators of XIAP or modulation of XIAP-MDM2-p53 pathway by antioxidants may be exploited for the prevention of Cd-induced oxidative stress and neurodegenerative diseases.

The cyclin-dependent kinase inhibitor p21(Cip1) regulates multiple cellular functions and protects cells from genotoxic and other cellular stresses. Activation of apoptosis signal-regulating kinase 1 (ASK1) induced by inhibition of mTOR signaling leads to sustained phospho-c-Jun that is suppressed in cells with functional p53 or by forced expression of p21(Cip1). Here we show that small deletions of p21(Cip1) around S98 abrogate its association with ASK1 but do not affect binding to Cdk1, hence distinguishing between the cell cycle-regulating functions of p21(Cip1) and its ability to suppress activation of the ASK1/Jun N-terminal protein kinase (JNK) pathway. p21(Cip1) is phosphorylated in vitro by both ASK1 and JNK1 at S98. In vivo phosphorylation of p21(Cip1), predominantly carried out by ASK1, is associated with binding to ASK1 and inactivation of ASK1 kinase function. Binding of p21(Cip1) to ASK1 requires ASK1 kinase function and may involve phosphorylation of S98.

Parkinson disease (PD) results from the slow, progressive loss of dopaminergic neurons in the substantia nigra. Alterations in α-synuclein (aSyn), such as mutations or multiplications of the gene, are thought to trigger this degeneration. Here, we show that aSyn disrupts mitogen-activated protein kinase (MAPK)-controlled stress signaling in yeast and human cells, which results in inefficient cell protective responses and cell death. aSyn is a substrate of the yeast (and human) polo-like kinase Cdc5 (Plk2), and elevated levels of aSyn prevent Cdc5 from maintaining a normal level of GTP-bound Rho1, which is an essential GTPase that regulates stress signaling. The nine N-terminal amino acids of aSyn are essential for the interaction with polo-like kinases. The results support a unique mechanism of PD pathology.

Cadmium (Cd), a toxic environmental contaminant, induces neurodegenerative disorders. Resveratrol, a natural product, has been found to exert neuroprotective effects. However, little is known regarding the effect of resveratrol on Cd-evoked neurotoxicity. Here, we show that resveratrol effectively reversed Cd-elicited cell viability reduction, morphological change, nuclear fragmentation and condensation, as well as activation of caspase-3 in neuronal cells, implying neuroprotection against Cd-poisoning by resveratrol. Further research revealed that both c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinases 1/2 (Erk1/2) were involved in the inhibitory effect of resveratrol on Cd-induced cell death, as selective inhibitors of Erk1/2 (U0126) and JNK (SP600125), or over-expression of dominant negative mitogen-activated protein kinase kinase 1 (MKK1) or dominant negative c-Jun potentiated resveratrol's prevention of Cd-induced phosphorylation of JNK and Erk1/2, as well as cell death in neuronal cells. Interestingly, resveratrol potently rescued the cells from Cd-induced suppression of protein phosphatases 2A (PP2A) and 5 (PP5) activity. Over-expression of PP2A or PP5 strengthened the inhibitory effects of resveratrol on Cd-induced activation of Erk1/2 and/or JNK, as well as cell death. The results indicate that resveratrol prevents Cd-induced activation of Erk1/2 and JNK pathways and neuronal cell death in part via activating PP2A and PP5. Our findings strongly support the notion that resveratrol may serve as a potential therapeutic agent in the prevention of Cd-induced neurodegenerative diseases.

Ciclopirox olamine (CPX), an off-patent fungicide, has recently been identifiedas a novel anticancer agent.However, the molecular mechanism underlying its anticancer action remains to be elucidated.Here we show that CPX inhibits cell proliferation in part by downregulating the protein level of Cdc25A in tumor cells. Our studies revealed that CPX did not significantly reduceCdc25A mRNA level or Cdc25A protein synthesis, but remarkably promoted Cdc25A protein degradation.This resulted in inhibition of G 1 -cyclin dependent kinases (CDKs), as evidenced by increased inhibitory phosphorylation of G 1 -CDKs.Since Cdc25A degradation is tightly related to its phosphorylation status, we further examined whether CPX alters Cdc25A phosphorylation.The results showed that CPX treatment increased the phosphorylation of Cdc25A (S76 and S82), but only Cdc25A-S82A mutant was resistant to CPX-induced degradation.Furthermore, ectopic expression of Cdc25A-S82A partially conferred resistance to CPX inhibition of cell proliferation.Therefore, our findings indicate that CPX inhibits cell proliferation at least in part by promoting Cdc25A degradation.

Ganoderma lucidum (G. lucidum) extracts, as dietary supplements, have been found to exert potent anticancer activity, which is attributed to the presence of polysaccharides and triterpenes. However, the molecular mechanism underlying the anticancer action of G. lucidum extracts remains to be investigated. Here, we show that ReishiMax GLp, containing G. lucidum polysaccharides and triterpenes (GLPT), inhibited cell proliferation and induced cell death in human lung cancer cells (A549 and A427) and simultaneously suppressed the signaling pathways of mammalian target of rapamycin complexes 1 and 2 (mTORC1 and mTORC2), respectively. Mechanistically, GLPT downregulated the phosphorylation and protein levels of insulin-like growth factor 1 receptor (IGFR) and phosphoinositide 3-kinase (PI3K) as well as the protein level of RAS homolog enriched in brain (Rheb). In addition, GLPT also activated the AMP-activated protein kinase (AMPK) network. This was evidenced by observations that GLPT increased the phosphorylation of AMPKα (T172) and its substrates tuberous sclerosis complex 2 (TSC2, S1387) and regulatory-associated protein of mTOR (raptor, S792). Ectopic expression of dominant-negative AMPKα partially mitigated the inhibitory effect of GLPT on mTORC1, indicating that GLPT inhibits mTORC1 partly by activating AMPK. The results suggest that G. lucidum extracts exert anticancer action at least partly by suppressing mTORC1/2 signaling via activation of AMPK and inhibition of IGFR/PI3K/Rheb in tumor cells.

Serine/threonine (Ser/Thr) protein phosphatase 2A (PP2A) has been implicated as a novel component of the mammalian target of rapamycin (mTOR) signaling pathway. Recently we have demonstrated that mTOR regulates cell motility in part through p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) pathways. Little is known about the role of PP2A in the mTOR-mediated cell motility. Here we show that rapamycin inhibited the basal or insulin-like growth factor 1 (IGF-1)-induced motility of human Ewing sarcoma (Rh1) and rhabdomyosarcoma (Rh30) cells. Treatment of the cells with rapamycin activated PP2A activity, and concurrently inhibited IGF-1 stimulated phosphorylation of Erk1/2. Inhibition of Erk1/2 with PD98059 did not significantly affect the basal mobility of the cells, but dramatically inhibited IGF-1-induced cell motility. Furthermore, inhibition of PP2A with okadaic acid significantly attenuated the inhibitory effect of rapamycin on IGF-1-stimulated phosphorylation of Erk1/2 as well as cell motility. Consistently, expression of dominant negative PP2A conferred resistance to IGF-1-stimulated phosphorylation of Erk1/2 and cell motility. Expression of constitutively active MKK1 also attenuated rapamycin inhibition of IGF-1-stimulated phosphorylation of Erk1/2 and cell motility. The results suggest that rapamycin inhibits cell motility, in part by targeting PP2A-Erk1/2 pathway.

Ciclopirox olamine (CPX), an off-patent antifungal agent used to treat mycoses of skin and nails, has recently been demonstrated to be a potential anticancer agent. However, the underlying mechanism is not well understood. Here, for the first time, we show that CPX inhibited lymphangiogenesis in an in vitro model (tube formation). This effect was, in part, associated with inhibition of vascular endothelial growth factor receptor-3 (VEGFR-3) expression, as overexpression of VEGFR-3 conferred partial resistance to CPX inhibitory effect on tube formation in lymphatic endothelial cells (LECs), whereas downregulation of VEGFR-3 mimicked the effect of CPX, blocking the tube formation. Further study revealed that CPX did not alter mRNA level, but inhibited protein synthesis and promoted protein degradation of VEGFR-3. In addition, we found that CPX inhibited phosphorylation of the extracellular signal-related kinase 1/2 (ERK1/2), a downstream effector of VEGFR-3. Overexpression of VEGFR-3 attenuated CPX inhibition of ERK1/2 phosphorylation, whereas downregulation of VEGFR-3 inhibited ERK1/2 phosphorylation in LECs. Ectopic expression of constitutively active mitogen-activated protein kinase kinase 1 (MKK1) resulted in activation of ERK1/2 and partially prevented CPX inhibition of LEC tube formation. The results suggest that CPX inhibits LEC tube formation at least, in part, through inhibiting VEGFR-3-mediated ERK signaling pathway.

Rotenone, a neurotoxic pesticide, induces loss of dopaminergic neurons related to Parkinson's disease. Previous studies have shown that rotenone induces neuronal apoptosis partly by triggering hydrogen peroxide (H2O2)-dependent suppression of mTOR pathway. However, the underlying mechanism is not fully understood. Here, we show that rotenone elevates intracellular free calcium ion ([Ca2+]i) level, and activates CaMKII, resulting in inhibition of mTOR signaling and induction of neuronal apoptosis. Chelating [Ca2+]i with BAPTA/AM, preventing extracellular Ca2+ influx using EGTA, inhibiting CaMKII with KN93, or silencing CaMKII significantly attenuated rotenone-induced H2O2 production, mTOR inhibition, and cell death. Interestingly, using TTFA, antimycin A, catalase or Mito-TEMPO, we found that rotenone-induced mitochondrial H2O2 also in turn elevated [Ca2+]i level, thereby stimulating CaMKII, leading to inhibition of mTOR pathway and induction of neuronal apoptosis. Expression of wild type mTOR or constitutively active S6K1, or silencing 4E-BP1 strengthened the inhibitory effects of catalase, Mito-TEMPO, BAPTA/AM or EGTA on rotenone-induced [Ca2+]i elevation, CaMKII phosphorylation and neuronal apoptosis. Together, the results indicate that the crosstalk between Ca2+ signaling and mitochondrial H2O2 is required for rotenone inhibition of mTOR-mediated S6K1 and 4E-BP1 pathways. Our findings suggest that how to control over-elevation of intracellular Ca2+ and overproduction of mitochondrial H2O2 may be a new approach to deal with the neurotoxicity of rotenone.

Ciclopirox olamine (CPX), an off-patent antifungal agent, has recently been identified as a potential anticancer agent. The mammalian target of rapamycin (mTOR) is a central controller of cell growth, proliferation and survival. Little is known about whether and how CPX executes its anticancer action by inhibiting mTOR. Here we show that CPX inhibited the phosphorylation of p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1), two downstream effector molecules of mTOR complex 1 (mTORC1), in a spectrum of human tumor cells, indicating that CPX inhibits mTORC1 signaling. Using rhabdomyosarcoma cells as an experimental model, we found that expression of constitutively active mTOR (E2419K) conferred resistance to CPX inhibition of cell proliferation, suggesting that CPX inhibition of mTORC1 contributed to its anticancer effect. In line with this, treatment with CPX inhibited tumor growth and concurrently suppressed mTORC1 signaling in RD xenografts. Mechanistically, CPX inhibition of mTORC1 was neither via inhibition of IGF-I receptor or phosphoinositide 3-kinase (PI3K), nor by activation of phosphatase and tensin homolog (PTEN). Instead, CPX inhibition of mTORC1 was attributed to activation of AMP-activated protein kinase (AMPK)-tuberous sclerosis complexes (TSC)/raptor pathways. This is supported by the findings that CPX activated AMPK; inhibition of AMPK with Compound C or ectopic expression of dominant negative AMPKα partially prevented CPX from inhibiting mTORC1; silencing TSC2 attenuated CPX inhibition of mTORC1; and CPX also increased AMPK-mediated phosphorylation of raptor (S792). Therefore, the results indicate that CPX exerts the anticancer effect by activating AMPK, resulting in inhibition of mTORC1 signaling.

Activation of eIF4B correlates with Abl-mediated cellular transformation, but the precise mechanisms are largely unknown. Here we show that eIF4B is a convergent substrate of JAK/STAT/Pim and PI3K/Akt/mTOR pathways in Abl transformants. Both pathways phosphorylated eIF4B in Abl-transformed cells, and such redundant regulation was responsible for the limited effect of single inhibitor on Abl oncogenicity. Persistent inhibition of one signaling pathway induced the activation of the other pathway and thereby restored the phosphorylation levels of eIF4B. Simultaneous inhibition of the two pathways impaired eIF4B phosphorylation more effectively, and synergistically induced apoptosis in Abl transformed cells and inhibited the growth of engrafted tumors in nude mice. Similarly, the survival of Abl transformants exhibited a higher sensitivity to the pharmacological inhibition, when combined with the shRNA-based silence of the other pathway. Interestingly, such synergy was dependent on the phosphorylation status of eIF4B on Ser422, as overexpression of eIF4B phosphomimetic mutant S422E in the transformants greatly attenuated the synergistic effects of these inhibitors on Abl oncogenicity. In contrast, eIF4B knockdown sensitized Abl transformants to undergo apoptosis induced by the combined blockage. Collectively, the results indicate that eIF4B integrates the signals from Pim and PI3K/Akt/mTOR pathways in Abl-expressing leukemic cells, and is a promising therapeutic target for such cancers.

Necroptosis is a programmed form of necrotic cell death, which is tightly regulated by the necroptotic signaling pathway containing receptor-interacting protein (RIP)1, RIP3, and mixed-lineage kinase domain-like (MLKL) protein. In addition to the RIP1-RIP3-MLKL axis, other factors regulating necroptosis are still largely unknown. Here a cell-based small-molecule screening led to the finding that BET inhibitors protected cells from necroptosis in the TNFα/Smac-mimetic/Z-VAD-FMK (TSZ)-induced cell necroptosis model. Mechanistic studies revealed that BET inhibitors acted by downregulating MLKL expression. Further research demonstrated that BRD4, IRF1, P-TEFb, and RNA polymerase II formed a transcription complex to regulate the expression of MLKL, and BET inhibitors interfered with the transcription complex formation. In necroptosis-related disease model, the BET inhibitor JQ-1 showed promising therapeutic effects. Collectively, our studies establish, for the first time, BRD4 as a new epigenetic factor regulating necroptosis, and highlight the potential of BET inhibitors in the treatment of necroptosis-related diseases.

The mammalian target of rapamycin (mTOR) functions as two complexes (mTORC1 and mTORC2), regulating cell growth and metabolism. Aberrant mTOR signaling occurs frequently in cancers, so mTOR has become an attractive target for cancer therapy. Iron chelators have emerged as promising anticancer agents. However, the mechanisms underlying the anticancer action of iron chelation are not fully understood. Particularly, reports on the effects of iron chelation on mTOR complexes are inconsistent or controversial. Here, we found that iron chelators consistently inhibited mTORC1 signaling, which was blocked by pretreatment with ferrous sulfate. Mechanistically, iron chelation-induced mTORC1 inhibition was not related to ROS induction, copper chelation, or PP2A activation. Instead, activation of AMPK pathway mainly and activation of both HIF-1/REDD1 and Bnip3 pathways partially contribute to iron chelation-induced mTORC1 inhibition. Our findings indicate that iron chelation inhibits mTORC1 via multiple pathways and iron is essential for mTORC1 activation.

Rabies, a highly fatal zoonotic disease, is a significant global public health threat. Currently, the pathogenic mechanism of rabies has not been fully elucidated, and no effective treatment for rabies is available. Increasing evidence shows that the tripartite-motif protein (TRIM) family of proteins participates in the host's regulation of viral replication. Studies have demonstrated the upregulated expression of tripartite-motif protein 21 (TRIM21) in the brain tissue of mice infected with the rabies virus. Related studies have shown that TRIM21 knockdown inhibits RABV replication, while overexpression of TRIM21 exerted the opposite effect. Knockdown of interferon-alpha and interferon-beta modulates the inhibition of RABV replication caused by TRIM21 knockdown and promotes the replication of the virus. Furthermore, our previous study revealed that TRIM21 regulates the secretion of type I interferon during RABV infection by targeting interferon regulatory factor 7 (IRF7). IRF7 knockdown reduced the inhibition of RABV replication caused by the knockdown of TRIM21 and promoted viral replication. TRIM21 regulates RABV replication via the IRF7-IFN axis. Our study identified TRIM21 as a novel host factor required by RABV for replication. Thus, TRIM21 is a potential target for rabies treatment or management.

Rabies remains a great threat to public health worldwide. So far, the mechanism of rabies virus (RABV) infection is not fully understood, and there is no effective treatment for rabies. Identifying more host restriction factors of RABV will spur the development of novel therapeutic interventions against rabies. Accumulating studies suggest that tripartite motif-containing (TRIM) proteins have great effects on virus replication. TRIMs control the antiviral responses through either direct interaction with viral proteins or indirect regulation of innate immune signaling molecules in the host. The role of TRIM25 in rabies virus (RABV) infection is poorly understood. Using next-generation sequencing, we found that TRIM25 is upregulated during HEP-Flury infection. Knockdown of TRIM25 enhances HEP-Flury production, while overexpression of TRIM25 suppresses HEP-Flury replication. Knockdown of interferon α and interferon β weakens the anti-RABV response induced by TRIM25 overexpression, and potentiates RABV production. Furthermore, we found that TRIM25 regulates type-I interferon response by targeting retinoic acid-inducible gene I (RIG-I) during HEP-Flury infection. Knockdown of RIG-I weakens the anti-HEP-Flury response induced by TRIM25 overexpression, indicating that TRIM25 regulates RABV production via the RIG-I-IFN axis. In addition, we observed that TRIM25 does not directly interact with HEP-Flury structural proteins, suggesting that TRIM25 regulates HEP-Flury production indirectly. Taken together, our work identifies TRIM25 as a new host factor involved in HEP-Flury infection, which may be a potential target for the development of antiviral drugs against RABV.

The target of rapamycin, mTOR, acts as a sensor for mitogenic stimuli, such as insulin-like growth factors and cellular nutritional status, regulating cellular growth and division. As many tumors are driven by autocrine or paracrine growth through the type-I insulin-like growth factor receptor, mTOR is potentially an attractive target for molecular targeted treatment. Further, a rationale for anticipating tumor-selective activity based on transforming events frequently identified in malignant disease is becoming established.

Xanthatin, a sesquiterpene lactone purified from Xanthium strumarium L., possesses prominent anticancer activity. We found that disruption of GSK3β activity was essential for xanthatin to exert its anticancer properties in non-small cell lung cancer (NSCLC), concurrent with preferable suppression of constitutive activation of STAT3. Interestingly, inactivation of the two signals are two mutually exclusive events in xanthatin-induced cell death. Moreover, we surprisingly found that exposure of xanthatin failed to trigger the presumable side effect of canonical Wnt/β-Catenin followed by GSK3β inactivation. We further observed that the downregulation of STAT3 was required for xanthatin to fine-tune the risk. Thus, the discovery of xanthatin, which has ability to simultaneously orchestrate two independent signaling cascades, may have important implications for screening promising drugs in cancer therapies.

Human albumin has recently been demonstrated to protect brain neurons from injury in rat ischemic brain. However, there is no information available about whether human albumin can prevent loss of tyrosine hydroxylase (TH) expression of dopaminergic (DA) neurons induced by 6-hydroxydopamine (6-OHDA) toxicity that is most commonly used to create a rat model of Parkinson's disease (PD). In the present study, two microliters of 1.25% human albumin were stereotaxically injected into the right striatum of rats one day before or 7 days after the 6-OHDA lesion in the same side. D-Amphetamine-induced rotational asymmetry was measured 7 days, 3 and 10 weeks after 6-OHDA lesion. We observed that intrastriatal administration of human albumin significantly reduced the degree of rotational asymmetry. The number of TH-immunoreactive neurons present in the substantia nigra was greater in 6-OHDA lesioned rats following human albumin-treatment than non-human albumin treatment. TH-immunoreactivity in the 6-OHDA-lesioned striatum was also significantly increased in the human albumin-treated rats. To examine the mechanisms underlying the effects of human albumin, we challenged PC12 cells with 6-OHDA as an in vitro model of PD. Incubation with human albumin prevented 6-OHDA-induced reduction of cell viability in PC12 cell cultures, as measured by MTT assay. Furthermore, human albumin reduced 6-OHDA-induced formation of reactive oxygen species (ROS) and apoptosis in cultured PC12 cells, as assessed by flow cytometry. Western blot analysis showed that human albumin inhibited 6-OHDA-induced activation of JNK, c-Jun, ERK, and p38 mitogen-activated protein kinases (MAPK) signaling in PC12 cultures challenged with 6-OHDA. Human albumin may protect against 6-OHDA toxicity by influencing MAPK pathway followed by anti-ROS formation and anti-apoptosis.

Maduramicin, a polyether ionophore antibiotic derived from the bacterium Actinomadura yumaensis, is currently used as a feed additive against coccidiosis in poultry worldwide. It has been clinically observed that maduramicin can cause skeletal muscle and heart cell damage, resulting in skeletal muscle degeneration, heart failure, and even death in animals and humans, if improperly used. However, the mechanism of its toxic action in myoblasts is not well understood. Using mouse myoblasts (C2C12) and human rhabdomyosarcoma (RD and Rh30) cells as an experimental model for myoblasts, here we found that maduramicin inhibited cell proliferation and induced cell death in a concentration-dependent manner. Further studies revealed that maduramicin induced accumulation of the cells at G0/G1 phase of the cell cycle, and induced apoptosis in the cells. Concurrently, maduramicin downregulated protein expression of cyclin D1, cyclin-dependent kinases (CDK4 and CDK6), and CDC25A, and upregulated expression of the CDK inhibitors (p21Cip1 and p27Kip1), resulting in decreased phosphorylation of Rb. Maduramicin also induced expression of BAK, BAD, DR4, TRADD and TRAIL, leading to activation of caspases 8, 9 and 3 as well as cleavage of poly ADP ribose polymerase (PARP). Taken together, our results suggest that maduramicin executes its toxicity in myoblasts at least by inhibiting cell proliferation and inducing apoptotic cell death.

Cadmium (Cd), a toxic environmental contaminant, contributes to neurodegeneration. Rapamycin, a macrocyclic lactone, has shown preventive effect on Cd-induced neuronal cell death. However, the underlying mechanism is not fully understood. Here, we show that rapamycin prevented Cd-induced apoptotic cell death in neuronal cells. Coincidently, rapamycin markedly blocked Cd-induced phosphorylation of Akt, S6K1 and 4E-BP1 in the cells. Expression of a rapamycin-resistant and kinase-active mTOR (S2035T, mTOR-T), but not a rapamycin-resistant and kinase-dead mTOR (S2035T/D2357E, mTOR-TE), conferred resistance to rapamycin inhibition of Cd-induced cell death, implying that the preventive effect of rapamycin on Cd-induced neurotoxicity is mTOR kinase activity-dependent. It appeared that both mTORC1 and mTORC2 were involved in the inhibitory activity of rapamycin, as silencing raptor, rictor or raptor/rictor enhanced rapamycin's blockage of Cd-induced cell death. Furthermore, downregulation of S6K1, ectopic expression of constitutively hypophosphorylated 4E-BP1 or dominant negative Akt, or co-treatment with Akt inhibitor also potentiated the rapamycin's inhibitory effect. The findings indicate that rapamycin prevents Cd-induced neuronal cell death via suppressing both mTORC1 and mTORC2 pathways. Our results highlight that rapamycin may be exploited for the prevention of Cd-induced neurodegenerative disorders.

In serum-free medium containing serum replacements but totally lacking in protein growth factors, diploid human fibroblasts remained quiescent if the extracellular Ca2+ concentration was only 0.1 mM. However, when the Ca2+ concentration in this medium was increased to 1 mM, the cells replicated as rapidly as they do in medium supplemented with protein growth factors. When quiescent cells in medium with only 0.1 mM Ca2+ were exposed to 1 or 10 mM Ca2+ or 100 ng/ml epidermal growth factor (EGF), the 42 kDa and 44 kDa forms of mitogen-activated protein kinase (MAPK) were rapidly activated, as demonstrated by a characteristic electrophoretic mobility shift of these proteins and by their enhanced ability to phosphorylate myelin basic protein (MBP). Analysis of fractions from Mono Q anion-exchange chromatography of lysates of cells exposed to 10 mM Ca2+ or 100 ng/ml EGF revealed a peak of MBP phosphorylation activity that was coeluted with p42 and p44 MAPK as shown by immunoblot analysis. Activation of MAPK by extracellular Ca2+ was dose-dependent and biphasic, with a peak of activation at 5-10 min after exposure, followed by a period of sustained activation of MAPK at a lower level. This pattern has been shown [Vouret-Craviari, Van Obberghen-Schilling, Scimeca, Van Obberghen and Pouysségur (1993) Biochem J. 289, 209-214] to correlate with the re-entry of mammalian cells into the cell cycle.

Cryptotanshinone (CPT), isolated from the plant Salvia miltiorrhiza Bunge, is a potential anticancer agent. However, the underlying mechanism remains to be defined. Here, we show that CPT inhibited lymphangiogenesis in an in vitro model (tube formation). This effect was partly attributed to inhibiting expression of VEGF receptor 3 (VEGFR-3) in murine lymphatic endothelial cells (LEC), as overexpression of VEGFR-3 conferred resistance to CPT inhibition of the tube formation, whereas downregulation of VEGFR-3 mimicked the effect of CPT, blocking the tube formation. Furthermore, CPT inhibited phosphorylation of the extracellular signal-related kinase 1/2 (ERK1/2). Overexpression of VEGFR-3 attenuated CPT inhibition of ERK1/2 phosphorylation, whereas downregulation of VEGFR-3 inhibited ERK1/2 phosphorylation in LECs. Expression of constitutively active MKK1 resulted in activation of ERK1/2 and partially prevented CPT inhibition of LEC tube formation. In addition, CPT also inhibited protein expression and activities of Rac1 and Cdc42 but not RhoA. Expression of constitutively active Rac1 and Cdc42 concurrently, but not Rac1 or Cdc42 alone, conferred resistance to CPT inhibition of LEC tube formation. Taken together, the results suggest that CPT inhibits LEC tube formation, in part, by inhibiting VEGFR-3-mediated ERK1/2 phosphorylation and, in part, by inhibiting expression of the small GTPases.

The use of herbal medicine has rapidly increased in recent decades, prompting an increase in toxicity concerns. Here we investigated whether and how essential oil of Curcuma zedoaria may induce reproductive and developmental toxicity. Whole embryo culture in rats revealed that the essential oil produced a concentration-dependent toxicity ex vivo in the embryos on gestation Day 9.5 (GD9.5). Weight loss, abnormal hematological and biochemical effects on dams and embryos were also observed in GD17 pregnant rats orally administrated with 100 mg kg−1 or 200 mg kg−1 essential oil from GD7 onward. Induction of embryotoxicity may be related to placental calcification attributed to inhibition of vascular endothelial growth factor (VEGF)-mediated angiogenesis. Analysis by gas chromatography–mass spectrometry demonstrated that the main toxic compounds in essential oil were sesquiterpenoids. Our results suggest that the reproductive toxicity of C. zedoaria may be caused by sesquiterpenoids in the essential oil blocking VEGF-mediated angiogenesis.

Abstract Maduramicin (Mad), a polyether ionophore antibiotic, has been reported to be toxic to animals and humans because of being used at high doses or for long time, resulting in heart failure. However, the toxic mechanism of Mad in cardiac muscle cells is not well understood. Here, we show that Mad induced cell viability reduction and apoptosis in cardiac‐derived H9c2, HL‐1 cells, primary cardiomyocytes, and murine cardiac muscles, which was because of the inhibition of extracellular‐signal‐regulated kinase 1/2 (Erk1/2). Expression of constitutively active mitogen‐activated protein kinase kinase 1 (MKK1) attenuated Mad‐induced cell death in H9c2 cells, whereas silencing Erk1/2 or ectopic expression of dominant negative MKK1 strengthened Mad‐induced cell death. Moreover, we found that both phosphatase and tensin homolog on chromosome 10 (PTEN) and protein kinase B (Akt) were implicated in the regulation of Erk1/2 inactivation and apoptosis in the cells and tissues exposed to Mad. Overexpression of dominant negative PTEN and/or constitutively active Akt, or constitutively active Akt and/or constitutively active MKK1 rescued the cells from Mad‐induced dephosphorylated‐Erk1/2 and cell death. Furthermore, Mad‐induced reactive oxygen species (ROS) activated PTEN and inactivated Akt–Erk1/2 contributing to cell death, as N‐acetyl‐ L ‐cysteine ameliorated the event. Taken together, the results disclose that Mad inhibits Erk1/2 via ROS‐dependent activation of PTEN and inactivation of Akt, leading to cell death in cardiac muscle cells. Our findings suggest that manipulation of the ROS–PTEN–Akt–Erk1/2 pathway may be a potential approach to prevent Mad‐induced cardiotoxicity.

Autophagy-related protein 7 (ATG7) is an essential autophagy effector enzyme. Although it is well known that autophagy plays crucial roles in the infections with various viruses including influenza A virus (IAV), function and underlying mechanism of ATG7 in infection and pathogenesis of IAV remain poorly understood. Here, in vitro studies showed that ATG7 had profound effects on replication of IAV. Depletion of ATG7 markedly attenuated the replication of IAV, whereas overexpression of ATG7 facilitated the viral replication. ATG7 conditional knockout mice were further employed and exhibited significantly resistant to viral infections, as evidenced by a lower degree of tissue injury, slower body weight loss, and better survival, than the wild type animals challenged with either IAV (RNA virus) or pseudorabies virus (DNA virus). Interestingly, we found that ATG7 promoted the replication of IAV in autophagy-dependent and -independent manners, as inhibition of autophagy failed to completely block the upregulation of IAV replication by ATG7. To determine the autophagy-independent mechanism, transcriptome analysis was utilized and demonstrated that ATG7 restrained the production of interferons (IFNs). Loss of ATG7 obviously enhanced the expression of type I and III IFNs in ATG7-depleted cells and mice, whereas overexpression of ATG7 impaired the interferon response to IAV infection. Consistently, our experiments demonstrated that ATG7 significantly suppressed IRF3 activation during the IAV infection. Furthermore, we identified long noncoding RNA (lncRNA) GAPLINC as a critical regulator involved in the promotion of IAV replication by ATG7. Importantly, both inactivation of IRF3 and inhibition of IFN response caused by ATG7 were mediated through control over GAPLINC expression, suggesting that GAPLINC contributes to the suppression of antiviral immunity by ATG7. Together, these results uncover an autophagy-independent mechanism by which ATG7 suppresses host innate immunity and establish a critical role for ATG7/GAPLINC/IRF3 axis in regulating IAV infection and pathogenesis.

Rabies virus (RABV) is a single-stranded negative-sense RNA virus belonging to the Rhabdoviridae family and Lyssavirus genus, which is highly neurotropic and can infect almost all warm-blooded animals, including humans. Autophagy and apoptosis are two evolutionarily conserved and genetically regulated processes that maintain cellular and organismal homeostasis, respectively. Autophagy recycles unnecessary or dysfunctional intracellular organelles and molecules in a cell, whereas apoptosis eliminates damaged or unwanted cells in an organism. Studies have shown that RABV can induce both autophagy and apoptosis in target cells. To advance our understanding of pathogenesis of rabies, this paper reviews the molecular mechanisms of autophagy and apoptosis induced by RABV and the effects of the two cellular events on RABV replication.

Porcine epidemic diarrhea (PED) virus (PEDV) is one of the main pathogens causing diarrhea in piglets and fattening pigs. The clinical signs of PED are vomiting, acute diarrhea, dehydration, and mortality resulting in significant economic losses and becoming a major challenge in the pig industry. PEDV possesses various crucial structural and functional proteins, which play important roles in viral structure, infection, replication, assembly, and release, as well as in escaping host innate immunity. Over the past few years, there has been progress in the study of PEDV pathogenesis, revealing the crucial role of the interaction between PEDV viral proteins and host cytokines in PEDV infection. At present, the main control measure against PEDV is vaccine immunization of sows, but the protective effect for emerging virus strains is still insufficient, and there is no ideal safe and efficient vaccine. Although scientists have persistently delved their research into the intricate structure and functionalities of the PEDV genome and viral proteins for years, the pathogenic mechanism of PEDV remains incompletely elucidated. Here, we focus on reviewing the research progress of PEDV structural and nonstructural proteins to facilitate the understanding of biological processes such as PEDV infection and pathogenesis.

Tripartite motif (TRIM) proteins are a multifunctional E3 ubiquitin ligase family that participates in various cellular processes. Recent studies have shown that TRIM proteins play important roles in regulating host–virus interactions through specific pathways, but their involvement in response to rabies virus (RABV) infection remains poorly understood. Here, we identified that several TRIM proteins are upregulated in mouse neuroblastoma cells (NA) after infection with the rabies virus using RNA-seq sequencing. Among them, TRIM44 was found to regulate RABV replication. This is supported by the observations that downregulation of TRIM44 inhibits RABV replication, while overexpression of TRIM44 promotes RABV replication. Mechanistically, TRIM44-induced RABV replication is brought about by activating autophagy, as inhibition of autophagy with 3-MA attenuates TRIM44-induced RABV replication. Additionally, we found that inhibition of autophagy with rapamycin reverses the TRIM44-knockdown-induced decrease in LC3B expression and autophagosome formation as well as RABV replication. The results suggest that TRIM44 promotes RABV replication by an autophagy-dependent mechanism. Our work identifies TRIM44 as a key host factor for RABV replication, and targeting TRIM44 expression may represent an effective therapeutic strategy.

O-linked-β-D-N-acetylglucosamine (O-GlcNAc) glycosylation (O-GlcNAcylation), which is dynamically regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), is a post-translational modification involved in multiple cellular processes. O-GlcNAcylation of proteins can regulate their biological functions via crosstalk with other post-translational modifications, such as phosphorylation, ubiquitination, acetylation, and methylation. Liver diseases are a major cause of death worldwide; yet, key pathological features of the disease, such as inflammation, fibrosis, steatosis, and tumorigenesis, are not fully understood. The dysregulation of O-GlcNAcylation has been shown to be involved in some severe hepatic cellular stress, viral hepatitis, liver fibrosis, nonalcoholic fatty acid liver disease (NAFLD), malignant progression, and drug resistance of hepatocellular carcinoma (HCC) through multiple molecular signaling pathways. Here, we summarize the emerging link between O-GlcNAcylation and hepatic pathological processes and provide information about the development of therapeutic strategies for liver diseases.

The central nervous system of the mollusc Helix pomatia was stimulated with NO donors sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP) or hydroxylamine, in the presence of a phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (IBMX). Radioimmunoassay revealed that all of the three NO donors significantly increased cGMP levels by 22–27-fold above basal levels. Compared with controls, strong cGMP immunoreactivity was observed in axons and cytoplasm of the stimulated neurons. About 80% of cGMP-immunoreactive neurons colocalized with NADPH-diaphorase activity. Some glial cells and giant neurons were not stained by NADPH-diaphorase histochemistry but were cGMP-immunoreactive. The results suggest the existence of a NO/cGMP pathway and indicate NO as an intra- and intercellular signaling molecule in the Helix central nervous system.

Resveratrol, a natural polyphenol compound, has been shown to possess anticancer activity. However, how resveratrol inhibits cancer cell adhesion has not been fully elucidated. Here, we show that resveratrol suppressed the basal or type I insulin‐like growth factor (IGF)‐1‐stimulated adhesion of cancer cells (Rh1, Rh30, HT29, and HeLa cells) by inhibiting the extracellular signal‐regulated kinase 1/2 (Erk1/2) pathway. Inhibition of Erk1/2 with U0126, knockdown of Erk1/2, or overexpression of dominant‐negative mitogen‐activated protein kinase kinase 1 (MKK1) strengthened resveratrol’s inhibition of the basal or IGF‐1‐stimulated of Erk1/2 phosphorylation and cell adhesion, whereas ectopic expression of constitutively active MKK1 attenuated the inhibitory effects of resveratrol. Further research revealed that both protein phosphatase 2A (PP2A) and phosphatase and tensin homolog (PTEN)–Akt were implicated in resveratrol‐inactivated Erk1/2‐dependent cell adhesion. Inhibition of PP2A with okadaic acid or overexpression of dominant‐negative PP2A rendered resistance to resveratrol’s suppression of the basal or IGF‐1‐stimulated phospho‐Erk1/2 and cell adhesion, whereas expression of wild‐type PP2A enhanced resveratrol’s inhibitory effects. Overexpression of wild‐type PTEN or dominant‐negative Akt or inhibition of Akt with Akt inhibitor X strengthened resveratrol’s inhibition of the basal or IGF‐1‐stimulated Erk1/2 phosphorylation and cell adhesion. Furthermore, inhibition of mechanistic/mammalian target of rapamycin (mTOR) with rapamycin or silencing mTOR enhanced resveratrol’s inhibitory effects on the basal and IGF‐1‐induced inhibition of PP2A–PTEN, activation of Akt–Erk1/2, and cell adhesion. The results indicate that resveratrol inhibits Erk1/2‐mediated adhesion of cancer cells via activating PP2A–PTEN signaling network. Our data highlight that resveratrol has a great potential in the prevention of cancer cell adhesion.

To develop an in vivo tool to probe brain genotoxic stress, we designed a viral proxy as a single-cell genetic sensor termed PRISM that harnesses the instability of recombinant adeno-associated virus genome processing and a hypermutable repeat sequence-dependent reporter. PRISM exploits the virus-host interaction to probe persistent neuronal DNA damage and overactive DNA damage response. A Parkinson's disease (PD)-associated environmental toxicant, paraquat (PQ), inflicted neuronal genotoxic stress sensitively detected by PRISM. The most affected cell type in PD, dopaminergic (DA) neurons in substantia nigra, was distinguished by a high level of genotoxic stress following PQ exposure. Human alpha-synuclein proteotoxicity and propagation also triggered genotoxic stress in nigral DA neurons in a transgenic mouse model. Genotoxic stress is a prominent feature in PD patient brains. Our results reveal that PD-associated etiological factors precipitated brain genotoxic stress and detail a useful tool for probing the pathogenic significance in aging and neurodegenerative disorders.

Fusarochromanone (FC101) is a small molecule fungal metabolite with a host of interesting biological functions, including very potent anti-angiogenic and direct anti-cancer activity.Herein, we report that FC101 exhibits very potent in-vitro growth inhibitory effects (IC50 ranging from 10nM-2.5 μM) against HaCat (pre-malignant skin), P9-WT (malignant skin), MCF-7 (low malignant breast), MDA-231 (malignant breast), SV-HUC (premalignant bladder), UM-UC14 (malignant bladder), and PC3 (malignant prostate) in a time-course and dose-dependent manner, with the UM-UC14 cells being the most sensitive. FC101 induces apoptosis and an increase in proportion of cells in the sub-G1 phase in both HaCat and P9-WT cell lines as evidenced by cell cycle profile analysis. In a mouse xenograft SCC tumor model, FC101 was well tolerated, non-toxic, and achieved a 30% reduction in tumor size at a dose of 8 mg/kg/day. FC101 is also a potent anti-angiogenenic agent. At nanomolar doses, FC101 inhibits the vascular endothelial growth factor-A (VEGF-A)-mediated proliferation of endothelial cells.Our data presented here indicates that FC101 is an excellent lead candidate for a small molecule anti-cancer agent that simultaneously affects angiogenesis signaling, cancer signal transduction, and apoptosis. Further understanding of the underlying FC101's molecular mechanism may lead to the design of novel targeted and selective therapeutics, both of which are pursued targets in cancer drug discovery.

Abstract Maduramicin, a polyether ionophore antibiotic, is widely used as an anticoccidial agent in the poultry industry. It has been reported that maduramicin may cause heart and skeletal muscle cell damage, resulting in heart failure, skeletal muscle degeneration and even death in animals and humans, if improperly used. However, the molecular mechanism behind its capability to cause death of cardiac cells is not known. Here, we show that maduramicin induced apoptosis and necrosis in rat myocardial cells (H9c2). Maduramicin did not apparently upregulate the expression of pro‐apoptotic proteins (e.g., BAD, BAK and BAX) or downregulate the expression of anti‐apoptotic proteins (e.g. Bcl‐2, Bcl‐xL, Mcl‐1 and survivin). Interestingly, maduramicin increased the expression of DR4 and TRAIL, activating caspases 8/3 and triggering cleavage of poly ADP ribose polymerase ( PARP ). In addition, maduramicin induced nuclear translocation of apoptosis inducing factor. Furthermore, maduramicin blocked autophagic flux, as evidenced by inducing accumulation of both LC3‐II and p62/SQSTM1. Taken together, the above results suggest that maduramicin executes its toxicity in the myocardial cells at least by inducing caspase‐dependent cell death through TRAIL/DR4‐mediated extrinsic pathway and caspase‐independent cell death by inducing apoptosis inducing factor nuclear translocation and blocking autophagic flux. Our findings provide a new insight into the molecular mechanism of maduramicin's toxicity in myocardial cells.

Ciclopirox olamine (CPX), an off-patent anti-fungal drug, has been found to inhibit the G 1 -cyclin dependent kinases partly by increasing the phosphorylation and degradation of Cdc25A.However, little is known about the molecular target(s) of CPX responsible for Cdc25A degradation.Here, we show that CPX induced the degradation of Cdc25A neither by increasing CK1α or decreasing DUB3 expression, nor via activating GSK3β, but through activating Chk1 in rhabdomyosarcoma (Rh30) and breast carcinoma (MDA-MB-231) cells.This is strongly supported by the findings that inhibition of Chk1 with TCS2312 or knockdown of Chk1 profoundly attenuated CPXinduced Cdc25A degradation in the cells.Furthermore, we observed that CPX caused DNA damage, which was independent of reactive oxygen species (ROS) induction, but related to iron chelation.CPX treatment resulted in the activation of ataxia telangiectasia mutated (ATM) and ATM-and RAD3-related (ATR) kinases.Treatment with Ku55933 (a selective ATM inhibitor) failed to prevent CPX-induced Chk1 phosphorylation and Cdc25A degradation.In contrast, knockdown of ATR conferred high resistance to CPX-induced Chk1 phosphorylation and Cdc25A degradation.Therefore, the results suggest that CPX-induced degradation of Cdc25A is attributed to the activation of ATR-Chk1 signaling pathway, a consequence of iron chelationinduced DNA damage.

Recent studies have shown that fusarochromanone (FC101), a mycotoxin, is cytotoxic in a variety of cell lines. However, the molecular mechanism underlying its cytotoxicity remains elusive. Here we found that FC101 induced cell death in COS7 and HEK293 cells in part by activating JNK pathway. This is evidenced by the findings that inhibition of JNK with SP600125 or expression of dominant negative c-Jun partially prevented FC101-induced cell death. Furthermore, we observed that FC101-activated JNK pathway was attributed to induction of reactive oxygen species (ROS). Pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger and antioxidant, suppressed FC101-induced activation of JNK and cell death. Moreover, we noticed that FC101 inhibited the serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5) in the cells, which was abrogated by NAC. Overexpression of PP2A or PP5 partially prevented FC101-induced activation of JNK and cell death. The results indicate that FC101-induced ROS inhibits PP2A and PP5, leading to activation of JNK pathway and consequently resulting in cell death.

Our previous work has shown that maduramicin, an effective coccidiostat used in the poultry production, executed its toxicity by inducing apoptosis of skeletal myoblasts. However, the underlying mechanism is not well understood. Here we show that maduramicin induced apoptosis of skeletal muscle cells by activating c-Jun N-terminal kinase (JNK) pathway in murine C2C12 and L6 myoblasts as well as skeletal muscle tissue. This is supported by the findings that inhibition of JNK with SP600125 or ectopic expression of dominant negative c-Jun attenuated maduramicin-induced apoptosis in C2C12 cells. Furthermore, we found that treatment with maduramicin reduced the cellular protein level of protein phosphatase 5 (PP5). Overexpression of PP5 substantially mitigated maduramicin-activated JNK and apoptosis. Moreover, we noticed that treatment with maduramicin elevated intracellular reactive oxygen species (ROS) level. Pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger and antioxidant, suppressed maduramicin-induced inhibition of PP5 and activation of JNK as well as apoptosis. The results indicate that maduramicin induction of ROS inhibits PP5, which results in activation of JNK cascade, leading to apoptosis of skeletal muscle cells. Our finding suggests that manipulation of ROS-PP5-JNK pathway may be a potential approach to prevent maduramicin-induced apoptotic cell death in skeletal muscle.

Rabies is caused by infection of rabies virus (RABV) and remains a serious threat to the global public health. Except for the requirement for cold chain and high cost of human rabies immune globulin, no small molecule drugs are currently available for clinical treatment of rabies. So, it is of great importance to identify novel compounds that can effectively inhibit RABV infection. Artesunate (ART) and dihydroartemisinin (DHA), two derivatives of artemisinin, are widely used for treatment of malaria in adults and children, showing high safety. In this study, we found that both ART and DHA were able to inhibit RABV replication in host cells at a low concentration (0.1 μmol/L). The antiviral effects of ART and DHA were independent of viral strains and cell lines. Pre-treatment with ART or DHA for 2 h in vitro did not affect the viral replication in host cells, implying that ART and DHA neither reduced the viability of RABV directly nor inhibited the binding and entrance of the virus to host cells. Further studies revealed that ART and DHA inhibited RABV genomic RNA synthesis and viral gene transcription. Treatment with ART or DHA (5 mg/kg) by intramuscular injection improved, to some extent, the survival rate of RABV-challenged mice. Combination treatment with derivatives of artemisinin and mannitol significantly improved the survival rate of RABV-challenged mice. The results suggest that ART and DHA have a great potential to be explored as new anti-rabies agents for treatment of rabies.

Goatpox, caused by goatpox virus (GTPV), is one of the most serious infectious diseases associated with high morbidity and mortality in goats. However, little is known about involvement of host innate immunity during the GTPV infection. For this, goats were experimentally infected with GTPV. The results showed that GTPV infection significantly induced mRNA expression of type I interferon (IFN)-α and IFN-β in peripheral blood lymphocytes, spleen and lung. In addition, GTPV infection enhanced expression of several inflammatory cytokines, including interleukin (IL)-1β, IL-6, IL-18; and tumor necrosis factor-α (TNF-α). Strikingly, infection with GTPV activated signal transducers and activators of transcription 3 (STAT3), a critical cytokine signaling molecule. Interestingly, the virus infection induced expression of suppressor of cytokine signaling (SOCS)-1. Importantly, the infection resulted in an increased expression of some critical interferon-stimulated genes, such as interferon-induced transmembrane protein (IFITM) 1, IFITM3, interferon stimulated gene (ISG) 15 and ISG20. Furthermore, we found that infection with GTPV up-regulated expression of Toll-like receptor (TLR) 2 and TLR9. These results revealed that GTPV infection activated host innate immune signaling and thereby triggered antiviral innate immunity. The findings provide novel insights into complex mechanisms underlying GTPV–host interaction and pathogenesis of GTPV.