Dong‐Hyung Cho

Mitochondria continuously undergo two opposing processes, fission and fusion. The disruption of this dynamic equilibrium may herald cell injury or death and may contribute to developmental and neurodegenerative disorders. Nitric oxide functions as a signaling molecule, but in excess it mediates neuronal injury, in part via mitochondrial fission or fragmentation. However, the underlying mechanism for nitric oxide–induced pathological fission remains unclear. We found that nitric oxide produced in response to β-amyloid protein, thought to be a key mediator of Alzheimer's disease, triggered mitochondrial fission, synaptic loss, and neuronal damage, in part via S-nitrosylation of dynamin-related protein 1 (forming SNO-Drp1). Preventing nitrosylation of Drp1 by cysteine mutation abrogated these neurotoxic events. SNO-Drp1 is increased in brains of human Alzheimer's disease patients and may thus contribute to the pathogenesis of neurodegeneration.

Excessive mitochondrial fission is a prominent early event and contributes to mitochondrial dysfunction, synaptic failure, and neuronal cell death in the progression of Alzheimer's disease (AD). However, it remains to be determined whether inhibition of excessive mitochondrial fission is beneficial in mammal models of AD. To determine whether dynamin-related protein 1 (Drp1), a key regulator of mitochondrial fragmentation, can be a disease-modifying therapeutic target for AD, we examined the effects of Drp1 inhibitor on mitochondrial and synaptic dysfunctions induced by oligomeric amyloid-β (Aβ) in neurons and neuropathology and cognitive functions in Aβ precursor protein/presenilin 1 double-transgenic AD mice. Inhibition of Drp1 alleviates mitochondrial fragmentation, loss of mitochondrial membrane potential, reactive oxygen species production, ATP reduction, and synaptic depression in Aβ-treated neurons. Furthermore, Drp1 inhibition significantly improves learning and memory and prevents mitochondrial fragmentation, lipid peroxidation, BACE1 expression, and Aβ deposition in the brain in the AD model. These results provide evidence that Drp1 plays an important role in Aβ-mediated and AD-related neuropathology and in cognitive decline in an AD animal model. Therefore, inhibiting excessive Drp1-mediated mitochondrial fission may be an efficient therapeutic avenue for AD. SIGNIFICANCE STATEMENT Mitochondrial fission relies on the evolutionary conserved dynamin-related protein 1 (Drp1). Drp1 activity and mitochondria fragmentation are significantly elevated in the brains of sporadic Alzheimer's disease (AD) cases. In the present study, we first demonstrated that the inhibition of Drp1 restored amyloid-β (Aβ)-mediated mitochondrial dysfunctions and synaptic depression in neurons and significantly reduced lipid peroxidation, BACE1 expression, and Aβ deposition in the brain of AD mice. As a result, memory deficits in AD mice were rescued by Drp1 inhibition. These results suggest that neuropathology and combined cognitive decline can be attributed to hyperactivation of Drp1 in the pathogenesis of AD. Therefore, inhibitors of excessive mitochondrial fission, such as Drp1 inhibitors, may be a new strategy for AD.

Macroautophagy, hereafter referred to as autophagy, is a catabolic process that results in the lysosomal degradation of cytoplasmic contents ranging from abnormal proteins to damaged cell organelles. It is activated under diverse conditions, including nutrient deprivation and hypoxia. During autophagy, members of the core autophagy-related (ATG) family of proteins mediate membrane rearrangements, which lead to the engulfment and degradation of cytoplasmic cargo. Recently, the nuclear regulation of autophagy, especially by transcription factors and histone modifiers, has gained increased attention. These factors are not only involved in rapid responses to autophagic stimuli, but also regulate the long-term outcome of autophagy. Now there are more than 20 transcription factors that have been shown to be linked to the autophagic process. However, their interplay and timing appear enigmatic as several have been individually shown to act as major regulators of autophagy. This Cell Science at a Glance article and the accompanying poster highlights the main cellular regulators of transcription involved in mammalian autophagy and their target genes.

Peroxisomes are rapidly degraded during amino acid and oxygen deprivation by a type of selective autophagy called pexophagy. However, how damaged peroxisomes are detected and removed from the cell is poorly understood. Recent studies suggest that the peroxisomal matrix protein import machinery may serve double duty as a quality control machinery, where they are directly involved in activating pexophagy. Here, we explored whether any matrix import factors are required to prevent pexophagy, such that their loss designates peroxisomes for degradation. Using gene editing and quantitative fluorescence microscopy on culture cells and a zebrafish model system, we found that PEX13, a component of the peroxisomal matrix import system, is required to prevent the degradation of otherwise healthy peroxisomes. The loss of PEX13 caused an accumulation of ubiquitinated PEX5 on peroxisomes and an increase in peroxisome-dependent reactive oxygen species that coalesce to induce pexophagy. We also found that PEX13 protein level is downregulated to aid in the induction of pexophagy during amino acid starvation. Together, our study points to PEX13 as a novel pexophagy regulator that is modulated to maintain peroxisome homeostasis.Abbreviations: AAA ATPases: ATPases associated with diverse cellular activities; ABCD3: ATP binding cassette subfamily D member; 3ACOX1: acyl-CoA oxidase; 1ACTA1: actin alpha 1, skeletal muscle; ACTB: actin beta; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG12: autophagy related 12; ATG16L1: autophagy related 16 like 1; CAT: catalase; CQ: chloroquine; Dpf: days post fertilization: FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; H2O2: hydrogen peroxide; HA – human influenza hemagglutinin; HBSS: Hanks’ Balanced Salt Solution; HCQ; hydroxychloroquine; KANL: lysine alanine asparagine leucine; KO: knockout; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; MTORC2: mechanistic target of rapamycin kinase complex 2; MYC: MYC proto-oncogene, bHLH transcription factor; MZ: maternal and zygotic; NAC: N-acetyl cysteine; NBR1 – NBR1 autophagy cargo receptor; PBD: peroxisome biogenesis disorder; PBS: phosphate-buffered saline; PEX: peroxisomal biogenesis factor; PTS1: peroxisome targeting sequence 1; RFP: red fluorescent protein; ROS: reactive oxygen speciess; iRNA: short interfering RNA; SKL: serine lysine leucine; SLC25A17/PMP34: solute carrier family 25 member 17; Ub: ubiquitin; USP30: ubiquitin specific peptidase 30.

Peroxiredoxins (Prx), a family of peroxidases that reduce intracellular peroxides with the thioredoxin system as the electron donor, are highly expressed in various cellular compartments. Among the antioxidant Prx enzymes, Prx2 is the most abundant in mammalian neurons, making it a prime candidate to defend against oxidative stress. Here we report that Prx2 is S-nitrosylated (forming SNO-Prx2) by reaction with nitric oxide at two critical cysteine residues (C51 and C172), preventing its reaction with peroxides. We observed increased SNO-Prx2 in human Parkinson's disease (PD) brains, and S-nitrosylation of Prx2 inhibited both its enzymatic activity and protective function from oxidative stress. Dopaminergic neurons, which are lost in PD, become particularly vulnerable. Thus, our data provide a direct link between nitrosative/oxidative stress and neurodegenerative disorders such as PD.

Autophagy is a catabolic process involved in cell death and in cell protective mechanism. Autophagic cell death is differentiated from apoptosis by the presence of double or multiple-membrane enclosed vesicles, and the ATG proteins are essential for the formation of these autophagic vesicles. Here, we show that ATG6/Beclin-1 is a novel caspase substrate. ATG6 is directly cleaved by caspases in a process inhibited by the pan caspase inhibitor, zVAD. Ectopic expression of ATG6 suppresses cell death while reduction of ATG6 levels by siRNA sensitizes cells to TRAIL-induced cell death. Also, the inhibition of caspases leads to an increase in autophagy. These results suggest that caspase-mediated cleavage of ATG6 links the apoptotic and autophagic signaling pathways.

Colorectal cancer (CRC) patients frequently experience disease recurrence and distant metastasis. This study aimed to identify prognostic indicators, including individual responses to chemotherapy, in CRC patients. RNA‐seq data was generated using 54 samples (normal colon, primary CRC, and liver metastases) from 18 CRC patients and genes associated with CRC aggressiveness were identified. A risk score based on these genes was developed and validated in four independent CRC patient cohorts ( n = 1063). Diverse statistical methods were applied to validate the risk scoring system, including a generalized linear model likelihood ratio test, Kaplan–Meier curves, a log‐rank test, and the Cox model. TREM1 and CTGF were identified as two activated regulators associated with CRC aggressiveness. A risk score based on 19 genes regulated by TREM1 or CTGF activation (TCA19) was a significant prognostic indicator. In multivariate and subset analyses based on pathological staging, TCA19 was an independent risk factor (HR = 1.894, 95% CI = 1.227–2.809, P = 0.002). Subset stratification in stage III patients revealed that TCA19 had prognostic potential and identified patients who would benefit from adjuvant chemotherapy, regardless of age. The TCA19 predictor represents a novel diagnostic tool for identifying high‐risk CRC patients and possibly predicting the response to adjuvant chemotherapy.

We screened a chemical library in MCF-7 cells stably expressing green fluorescent protein (GFP)-conjugated microtubule-associated protein 1 light chain 3 (LC3) (GFP-LC3-MCF-7) using cell-based assay, and identified BIX-01294 (BIX), a selective inhibitor of euchromatic histone-lysine N-methyltransferase 2 (EHMT2), as a strong autophagy inducer. BIX enhanced formation of GFP-LC3 puncta, LC3-II, and free GFP, signifying autophagic activation. Inhibition of these phenomena with chloroquine and increasement in punctate dKeima ratio (550/438) signal indicated that BIX activated autophagic flux. BIX-induced cell death was suppressed by the autophagy inhibitor, 3-methyladenine, or siRNA against BECN1 (VPS30/ATG6), ATG5, and ATG7, but not by caspase inhibitors. Moreover, EHMT2 siRNA augmented GFP-LC3 puncta, LC3-II, free GFP, and cell death, implying that inhibition of EHMT2 caused autophagy-mediated cell death. Treatment with EHMT2 siRNA and BIX accumulated intracellular reactive oxygen species (ROS). BIX augmented mitochondrial superoxide via NADPH oxidase activation. In addition, BIX increased hydrogen peroxide and glutathione redox potential in both cytosol and mitochondria. Treatment with N-acetyl-L-cysteine (NAC) or diphenyleneiodonium chloride (DPI) decreased BIX-induced LC3-II, GFP-LC3 puncta, and cell death, indicating that ROS instigated autophagy-dependent cell death triggered by BIX. We observed that BIX potentiated autophagy-dependent and caspase-independent cell death in estrogen receptor (ESR)-negative SKBr3 and ESR-positive MCF-7 breast cancer cells, HCT116 colon cancer cells, and importantly, in primary human breast and colon cancer cells. Together, the results suggest that BIX induces autophagy-dependent cell death via EHMT2 dysfunction and intracellular ROS accumulation in breast and colon cancer cells, therefore EHMT2 inhibition can be an effective therapeutic strategy for cancer treatment.

Neurons are known to use large amounts of energy for their normal function and activity. In order to meet this demand, mitochondrial fission, fusion, and movement events (mitochondrial dynamics) control mitochondrial morphology, facilitating biogenesis and proper distribution of mitochondria within neurons. In contrast, dysfunction in mitochondrial dynamics results in reduced cell bioenergetics and thus contributes to neuronal injury and death in many neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease, and Huntington's disease. We recently reported that amyloid-beta peptide, thought to be a key mediator of AD pathogenesis, engenders S-nitrosylation and thus hyperactivation of the mitochondrial fission protein Drp1. This activation leads to excessive mitochondrial fragmentation, bioenergetic compromise, and synaptic damage in models of AD. Here, we provide an extended commentary on our findings of nitric oxide-mediated abnormal mitochondrial dynamics.

The loss or injury of neurons associated with oxidative and nitrosative redox stress plays an important role in the onset of various neurodegenerative diseases. Specifically, nitric oxide (NO), can affect neuronal survival through a process called S-nitrosylation, by which the NO group undergoes a redox reaction with specific protein thiols. This in turn can lead to the accumulation of misfolded proteins, which generally form aggregates in Alzheimer's, Parkinson's, and other neurodegenerative diseases. Evidence suggests that S-nitrosylation can also impair mitochondrial function and lead to excessive fission of mitochondria and consequent bioenergetic compromise via effects on the activity of the fission protein dynamin-related protein 1 (Drp1). This insult leads to synaptic dysfunction and loss. Additionally, high levels of NO can S-nitrosylate a number of aberrant targets involved in neuronal survival pathways, including the antiapoptotic protein XIAP, inhibiting its ability to prevent apoptosis.

The vicinal dithiol motif is widely present in proteins, and is critical for proteins’ structures and functions. In recent years, a variety of fluorescent probes with high specificity and outstanding optical properties for sensing protein vicinal dithiols have been developed. In this review, we summarized the fluorescent probes of protein vicinal dithiols in literature. These probes are classified into four types based on their acceptor sites, i.e., biarsenical probes, monoarsenical probes, dimaleimide probes and diacrylate probes. Through analyzing the properties of different probes, we expect that this review would help readers further understand the structural factors of these probes and provide the design strategy for novel fluorescent probes with improved properties.

Abstract In recent decades, the role of the peroxisome in physiology and disease conditions has become increasingly important. Together with the mitochondria and other cellular organelles, peroxisomes support key metabolic platforms for the oxidation of various fatty acids and regulate redox conditions. In addition, peroxisomes contribute to the biosynthesis of essential lipid molecules, such as bile acid, cholesterol, docosahexaenoic acid, and plasmalogen. Therefore, the quality control mechanisms that regulate peroxisome biogenesis and degradation are important for cellular homeostasis. Current evidence indicates that peroxisomal function is often reduced or dysregulated in various human disease conditions, such as neurodegenerative diseases. Here, we review the recent progress that has been made toward understanding the quality control systems that regulate peroxisomes and their pathological implications.

Mitochondria have several quality control mechanisms by which they maintain cellular homeostasis and ensure that the molecular machinery is protected from stress. Mitophagy, selective autophagy of mitochondria, promotes mitochondrial quality control by inducing clearance of damaged mitochondria via the autophagic machinery. Accumulating evidence suggests that mitophagy is modulated by various microbial components in an attempt to affect the innate immune response to infection. In addition, mitophagy plays a key role in the regulation of inflammatory signaling, and mitochondrial danger signals such as mitochondrial DNA translocated into the cytosol can lead to exaggerated inflammatory responses. In this review, we present current knowledge on the functional aspects of mitophagy and its crosstalk with innate immune signaling during infection. A deeper understanding of the role of mitophagy could facilitate the development of more effective therapeutic strategies against various infections.

Autophagy has paradoxical and complex functions in cancer development, and autophagy-related genes (ATG) are key regulators in autophagy. Until now, more than 30 different ATG proteins have been identified in yeast, and their mammalian counterparts also have been reported. Although the roles of a few ATG proteins in cancer have been characterized, the role of ATG10 is almost completely unknown.To investigate the clinicopathological role of ATG10 in colorectal cancer, we analyzed ATG10 expression in colorectal cancer tissues and cell lines. Protein expression analysis showed that ATG10 is highly increased in colorectal cancer (tissue - 18/37 cases, 48%; cell line -8/12 cell lines, 66%). Immunohistochemical analysis with clinicopathological features indicated a strong association of the up-regulation of ATG10 with tumor lymph node metastasis (p = 0.005) and invasion (p<0.001). Moreover, both 5-year disease free survival and overall survival rates of patients bearing tumors that did not express ATG10 were significantly higher than those of patients bearing ATG10-expressing tumors (p = 0.012).Increased expression of ATG10 in colorectal cancer is associated with lymphovascular invasion and lymph node metastasis indicating that ATG10 may be a potential prognostic maker in colorectal cancer.

Mitochondrial dynamics greatly influence the biogenesis and morphology of mitochondria. Mitochondria are particularly important in neurons, which have a high demand for energy. Therefore, mitochondrial dysfunction is strongly associated with neurodegenerative diseases. Until now various post-translational modifications for mitochondrial dynamic proteins and several regulatory proteins have explained complex mitochondrial dynamics. However, the precise mechanism that coordinates these complex processes remains unclear. To further understand the regulatory machinery of mitochondrial dynamics, we screened a mitochondrial siRNA library and identified mortalin as a potential regulatory protein. Both genetic and chemical inhibition of mortalin strongly induced mitochondrial fragmentation and synergistically increased Aβ-mediated cytotoxicity as well as mitochondrial dysfunction. Importantly we determined that the expression of mortalin in Alzheimer disease (AD) patients and in the triple transgenic-AD mouse model was considerably decreased. In contrast, overexpression of mortalin significantly suppressed Aβ-mediated mitochondrial fragmentation and cell death. Taken together, our results suggest that down-regulation of mortalin may potentiate Aβ-mediated mitochondrial fragmentation and dysfunction in AD. Mitochondrial dynamics greatly influence the biogenesis and morphology of mitochondria. Mitochondria are particularly important in neurons, which have a high demand for energy. Therefore, mitochondrial dysfunction is strongly associated with neurodegenerative diseases. Until now various post-translational modifications for mitochondrial dynamic proteins and several regulatory proteins have explained complex mitochondrial dynamics. However, the precise mechanism that coordinates these complex processes remains unclear. To further understand the regulatory machinery of mitochondrial dynamics, we screened a mitochondrial siRNA library and identified mortalin as a potential regulatory protein. Both genetic and chemical inhibition of mortalin strongly induced mitochondrial fragmentation and synergistically increased Aβ-mediated cytotoxicity as well as mitochondrial dysfunction. Importantly we determined that the expression of mortalin in Alzheimer disease (AD) patients and in the triple transgenic-AD mouse model was considerably decreased. In contrast, overexpression of mortalin significantly suppressed Aβ-mediated mitochondrial fragmentation and cell death. Taken together, our results suggest that down-regulation of mortalin may potentiate Aβ-mediated mitochondrial fragmentation and dysfunction in AD.

Autophagy is an intracellular degradation pathway for large protein aggregates and damaged organelles. Recent studies have indicated that autophagy targets cargoes through a selective degradation pathway called selective autophagy. Peroxisomes are dynamic organelles that are crucial for health and development. Pexophagy is selective autophagy that targets peroxisomes and is essential for the maintenance of homeostasis of peroxisomes, which is necessary in the prevention of various peroxisome-related disorders. However, the mechanisms by which pexophagy is regulated and the key players that induce and modulate pexophagy are largely unknown. In this review, we focus on our current understanding of how pexophagy is induced and regulated, and the selective adaptors involved in mediating pexophagy. Furthermore, we discuss current findings on the roles of pexophagy in physiological and pathological responses, which provide insight into the clinical relevance of pexophagy regulation. Understanding how pexophagy interacts with various biological functions will provide fundamental insights into the function of pexophagy and facilitate the development of novel therapeutics against peroxisomal dysfunction-related diseases.

Peroxisomes are dynamic and multifunctional organelles involved in various cellular metabolic processes, and their numbers are tightly regulated by pexophagy, a selective degradation of peroxisomes through autophagy to maintain peroxisome homeostasis in cells. Catalase, a major peroxisome protein, plays a critical role in removing peroxisome-generated reactive oxygen species (ROS) produced by peroxisome enzymes, but the contribution of catalase to pexophagy has not been reported. Here, we investigated the role of catalase in peroxisome degradation during nutrient deprivation. Both short interfering RNA-mediated silencing of catalase and pharmacological inhibition by 3-aminotriazole (3AT) decreased the number of peroxisomes and resulted in the downregulation of peroxisomal proteins, such as PMP70 and PEX14 under serum starvation. In addition, treatment with 3AT induced NBR1-dependent autophagy and PEX5 ubiquitination in the absence of serum, which was accompanied by accumulation of ROS. Co-treatment with antioxidant agent N-acetyl-l-cysteine (NAC) prevented ROS accumulation and pexophagy by modulating peroxisome protein levels and the association of NBR1, a pexophagy receptor with peroxisomes. Taken together, these findings demonstrate that catalase plays an important role in pexophagy during nutrient deprivation.

Abstract A primary cilium is an antenna-like structure on the cell surface that plays a crucial role in sensory perception and signal transduction. Mitochondria, the ‘powerhouse’ of the cell, control cell survival, and death. The cellular ability to remove dysfunctional mitochondria through mitophagy is important for cell survival. We show here that mitochondrial stress, caused by respiratory complex inhibitors and excessive fission, robustly stimulates ciliogenesis in different types of cells including neuronal cells. Mitochondrial stress-induced ciliogenesis is mediated by mitochondrial reactive oxygen species generation, subsequent activation of AMP-activated protein kinase and autophagy. Conversely, abrogation of ciliogenesis compromises mitochondrial stress-induced autophagy, leading to enhanced cell death. In mice, treatment with mitochondrial toxin, MPTP elicits ciliary elongation and autophagy in the substantia nigra dopamine neurons. Blockade of cilia formation in these neurons attenuates MPTP-induced autophagy but facilitates dopamine neuronal loss and motor disability. Our findings demonstrate the important role of primary cilia in cellular pro-survival responses during mitochondrial stress.

Quality control of peroxisomes is essential for cellular homeostasis. However, the mechanism underlying pexophagy is largely unknown. In this study, we identified HSPA9 as a novel pexophagy regulator. Downregulation of HSPA9 increased macroautophagy/autophagy but decreased the number of peroxisomes in vitro and in vivo. The loss of peroxisomes by HSPA9 depletion was attenuated in SQSTM1-deficient cells. In HSPA9-deficient cells, the level of peroxisomal reactive oxygen species (ROS) increased, while inhibition of ROS blocked pexophagy in HeLa and SH-SY5Y cells. Importantly, reconstitution of HSPA9 mutants found in Parkinson disease failed to rescue the loss of peroxisomes, whereas reconstitution with wild type inhibited pexophagy in HSPA9-depleted cells. Knockdown of Hsc70-5 decreased peroxisomes in Drosophila, and the HSPA9 mutants failed to rescue the loss of peroxisomes in Hsc70-5-depleted flies. Taken together, our findings suggest that the loss of HSPA9 enhances peroxisomal degradation by pexophagy.

Mitochondrial dynamics not only involves mitochondrial morphology but also mitochondrial biogenesis, mitochondrial distribution, and cell death. To identify specific regulators to mitochondria dynamics, we screened a chemical library and identified niclosamide as a potent inducer of mitochondria fission. Niclosamide promoted mitochondrial fragmentation but this was blocked by down-regulation of Drp1. Niclosamide treatment resulted in the disruption of mitochondria membrane potential and reduction of ATP levels. Moreover, niclosamide led to apoptotic cell death by caspase-3 activation. Interestingly, niclosamide also increased autophagic activity. Inhibition of autophagy suppressed niclosamide-induced cell death. Therefore, our findings suggest that niclosamide induces mitochondria fragmentation and may contribute to apoptotic and autophagic cell death.

To evaluate the association of microsatellite instability (MSI) with clinicopathologic features and oncologic outcomes in patients with poorly differentiated colorectal cancer (PD).Study patients were divided into well-differentiated colorectal cancer (WD) and PD, which were compared according to histologic differentiation and MSI status.Among 1,941 patients, PD was more frequent among microsatellite-unstable tumors (23.6%) than among microsatellite-stable (MSS) tumors (4.2%, P < .001). Patients with PD had worse 4-year overall survival rates than patients with WD (78.6% vs 88.2%, P = 0.010). Compared with MSS-PD tumors, MSI-PD tumors were characterized by right-colon predilection, larger size, and infrequent lymph node metastasis (P < .001 to P = .007).The clinicopathologic characteristics of PD were closely associated with those of MSI. The outcomes of MSI-PD tumors were better than those of MSS-PD tumors, but this finding did not reach statistical significance.

Mitochondrial dynamics and mitophagy are thought to be important events for the quality control of mitochondria and mitochondria-associated diseases. To identify novel mitophagy modulators, we developed a cell-based screening system and selected 1,10-phenanthroline (Phen) as a target molecule. Phen treatment highly induced mitochondrial fragmentation and mitochondrial dysfunctions in a Drp1 dependent manner. Phen treatment also increased autophagy. Moreover, prolonged exposure of Phen increased mitochondria clearance through mitophagy. Phen-mediated loss of mitochondrial mass was more reduced in ATG5 deficient cells than in wild type cells. In addition, down-regulation of Drp1 decreased autophagy activation, suggesting that mitochondrial fission is involved in Phen-mediated mitophagy. Thus, our results demonstrate that the disruption of mitochondrial dynamics and mitochondrial dysfunctions provokes mitophagy in Phen-treated cells.

Summary Mitochondrial dynamics control mitochondrial functions as well as their morphology. However, the role of mitochondrial dynamics in melanogenesis is largely unknown. Here, we show that mitochondrial dynamics regulate melanogenesis by modulating the ROS ‐ ERK signaling pathway. Genetic and chemical inhibition of D rp1, a mitochondrial fission protein, increased melanin production and mitochondrial elongation in melanocytes and melanoma cells. In contrast, down‐regulation of OPA1, a mitochondria fusion regulator, suppressed melanogensis but induced massive mitochondrial fragmentation in hyperpigmented cells. Consistently, treatment with CCCP , a mitochondrial fission chemical inducer, also efficiently repressed melanogenesis. Furthermore, we found that ROS production and ERK phosphorylation were increased in cells with fragmented mitochondria. And inhibition of ROS or ERK suppressed the antimelanogenic effect of mitochondrial fission in α‐ MSH ‐treated cells. In addition, the activation of ROS ‐ ERK pathway by mitochondrial fission induced phosphorylation of serine73 on MITF accelerating its proteasomal degradation. In conclusion, mitochondrial dynamics may regulate melanogenesis by modulating ROS ‐ ERK signaling pathway.

Although targeting radioresistant tumor cells is essential for enhancing the efficacy of radiotherapy, the signals activated in resistant tumors are still unclear.This study shows that ERp57 contributes to radioresistance of laryngeal cancer by activating STAT3.Increased ERp57 was associated with the radioresistant phenotype of laryngeal cancer cells.Interestingly, increased interaction between ERp57 and STAT3 was observed in radioresistant cells, compared to the control cells.This physical complex is required for the activation of STAT3 in the radioresistant cells.Among STAT3-regulatory genes, Mcl-1 was predominantly regulated by ERp57.Inhibition of STAT3 activity with a chemical inhibitor or siRNA-mediated depletion of Mcl-1 sensitized radioresistant cells to irradiation, suggesting that the ERp57-STAT3-Mcl-1 axis regulates radioresistance of laryngeal cancer cells.Furthermore, we observed a positive correlation between ERp57 and phosphorylated STAT3 or Mcl-1 and in vivo interactions between ERp57 and STAT3 in human laryngeal cancer.Importantly, we also found that increased ERp57-STAT3 complex was associated with poor prognosis in human laryngeal cancer, indicating the prognostic role of ERp57-STAT3 regulation.Overall, our data suggest that ERp57-STAT3 regulation functions in radioresistance of laryngeal cancer, and targeting the ERp57-STAT3 pathway might be important for enhancing the efficacy of radiotherapy in human laryngeal cancer.

Raloxifene is a selective estrogen receptor modulator (SERM) that binds to the estrogen receptor (ER), and exhibits potent anti-tumor and autophagy-inducing effects in breast cancer cells. However, the mechanism of raloxifene-induced cell death and autophagy is not well-established. So, we analyzed mechanism underlying death and autophagy induced by raloxifene in MCF-7 breast cancer cells. Treatment with raloxifene significantly induced death in MCF-7 cells. Raloxifene accumulated GFP-LC3 puncta and increased the level of autophagic marker proteins, such as LC3-II, BECN1, and ATG12-ATG5 conjugates, indicating activated autophagy. Raloxifene also increased autophagic flux indicators, the cleavage of GFP from GFP-LC3 and only red fluorescence-positive puncta in mRFP-GFP-LC3-expressing cells. An autophagy inhibitor, 3-methyladenine (3-MA), suppressed the level of LC3-II and blocked the formation of GFP-LC3 puncta. Moreover, siRNA targeting BECN1 markedly reversed cell death and the level of LC3-II increased by raloxifene. Besides, raloxifene-induced cell death was not related to cleavage of caspases-7, -9, and PARP. These results indicate that raloxifene activates autophagy-dependent cell death but not apoptosis. Interestingly, raloxifene decreased the level of intracellular adenosine triphosphate (ATP) and activated the AMPK/ULK1 pathway. However it was not suppressed the AKT/mTOR pathway. Addition of ATP decreased the phosphorylation of AMPK as well as the accumulation of LC3-II, finally attenuating raloxifene-induced cell death. Our current study demonstrates that raloxifene induces autophagy via the activation of AMPK by sensing decreases in ATP, and that the overactivation of autophagy promotes cell death and thereby mediates the anti-cancer effects of raloxifene in breast cancer cells.

Autophagy plays complex roles in tumor initiation and development, and the expression of autophagy-related genes (ATGs) is differentially regulated in various cancer cells, depending on their environment. In this study, we analyzed the expressional relationship between polypyrimidine tract-binding protein 1 (PTBP1) and ATG10 in metastatic colorectal cancer. PTBP1 is associated with tumor metastasis in primary colorectal tumors and colorectal cancer liver metastasis (CLM) tissues. In addition, PTPB1 directly interacts with mRNA of ATG10, and regulates ATG10 expression level in colorectal cancer cells. Ectopic expression of PTBP1 decreased ATG10 expression, whereas down-regulation of PTBP1 increased ATG10 level. In contrast to PTBP1, expression of ATG10 was decreased in CLM tissues. Knock down of ATG10 promoted cell migration and invasion of colorectal cancer cells. Moreover, depletion of ATG10 modulated epithelial-mesenchymal transition-associated proteins in colorectal cancer cells: N-cadherin, TCF-8/ZEB1, and CD44 were up-regulated, whereas E-cadherin was down-regulated. Taken together, our findings suggest that expression of ATG10 negatively regulated by PTBP1 is associated with metastasis of colorectal cancer cells.

To characterize the mutation profiles of colorectal cancer (CRC) primary tumors (PTs) and liver metastases (CLMs), we performed both whole-exome and RNA sequencing. Ten significantly mutated genes, including BMI1, CARD11, and NRG1, were found in 34 CRCs with CLMs. We defined three mutation classes (Class 1 to 3) based on the absence or presence of mutations during liver metastasis. Most mutations were classified into Class 1 (shared between PTs and CLMs), suggesting the common clonal origin of PTs and CLMs. Class 1 was more strongly associated with the clinical characteristics of advanced cancer and was more frequently superimposed with chromosomal deletions in CLMs than Class 2 (PT-specific). The integration of exome and RNA sequencing revealed that variant-allele frequencies (VAFs) of mutations in the transcriptome tended to have stronger functional implications than those in the exome. For instance, VAFs of the TP53 and APC mutations in the transcriptome significantly correlated with the expression level of their target genes. Additionally, mutations with high functional impact were enriched with high VAFs in the CLM transcriptomes. We identified 11 mutation-associated splicing events in the CRC transcriptomes. Thus, the integration of the exome and the transcriptome may elucidate the underlying molecular events responsible for CLMs.

We previously reported that BIX-01294 (BIX), a small molecular inhibitor of euchromatic histone-lysine N-methyltransferase 2 (EHMT2/G9a), induces reactive oxygen species (ROS)-dependent autophagy in MCF-7 cells.Herein, we analyzed the epigenetic mechanism that regulates the transcription of Beclin-1, a tumor suppressor and an autophagy-related gene (ATG).Inhibition of EHMT2 reduced dimethylation of lysine 9 on histone H3 (H3K9me2) and dissociated EHMT2 and H3K9me2 from the promoter of Beclin-1.To this promoter, RNA polymerase II and nuclear factor kappa B (NF-κB) were recruited in a ROS-dependent manner, resulting in transcriptional activation.Moreover, treatment with BIX reversed the suppression of Beclin-1 by the cooperative action of EHMT2 and DNA methyltransferase 1 (DNMT1).Accordingly, a combination treatment with BIX and 5-Aza-2'-deoxycytidine (5-Aza-Cd), a DNMT1 inhibitor, exerted a synergistic effect on Beclin-1 expression.Importantly, high levels of EHMT2 expression showed a significant association with low levels of Beclin-1 expression, which was related to a poor prognosis.These findings suggest that EHMT2 can directly repress Beclin-1 and that the inhibition of EHMT2 may be a useful therapeutic approach for cancer prevention by activating autophagy.

The key step for faithful chromosome segregation during meiosis is kinetochore assembly. Defects in this process result in aneuploidy, leading to miscarriages, infertility and various birth defects. However, the roles of kinetochores in homologous chromosome segregation during meiosis are ill-defined. Here we found that Zwint-1 is required for homologous chromosome segregation during meiosis. Knockdown of Zwint-1 accelerated the first meiosis by abrogating the kinetochore recruitment of Mad2, leading to chromosome misalignment and a high incidence of aneuploidy. Although Zwint-1 knockdown did not affect Aurora C kinase activity, the meiotic defects following Zwint-1 knockdown were similar to those observed with ZM447439 treatment. Importantly, the chromosome misalignment following Aurora C kinase inhibition was not restored after removing the inhibitor in Zwint-1-knockdown oocytes, whereas the defect was rescued after the inhibitor washout in the control oocytes. These results suggest that Aurora C kinase-mediated correction of erroneous kinetochore-microtubule attachment is primarily regulated by Zwint-1. Our results provide the first evidence that Zwint-1 is required to correct erroneous kinetochore-microtubule attachment and regulate spindle checkpoint function during meiosis.

Mitochondrial quality control maintains mitochondrial function by regulating mitochondrial dynamics and mitophagy. Despite the identification of mitochondrial quality control factors, little is known about the crucial regulators coordinating both mitochondrial fission and mitophagy. Through a cell-based functional screening assay, FK506 binding protein 8 (FKBP8) was identified to target microtubule-associated protein 1 light chain 3 (LC3) to the mitochondria and to change mitochondrial morphology. Microscopy analysis revealed that the formation of tubular and enlarged mitochondria was observed in FKBP8 knockdown HeLa cells and the cortex of Fkbp8 heterozygote-knockout mouse embryos. Under iron depletion-induced stress, FKBP8 was recruited to the site of mitochondrial division through budding and colocalized with LC3. FKBP8 was also found to be required for mitochondrial fragmentation and mitophagy under hypoxic stress. Conversely, FKBP8 overexpression induced mitochondrial fragmentation in HeLa cells, human fibroblasts and mouse embryo fibroblasts (MEFs), and this fragmentation occurred in Drp1 knockout MEF cells, FIP200 knockout HeLa cells and BNIP3/NIX double knockout HeLa cells, but not in Opa1 knockout MEFs. Interestingly, we found an LIR motif-like sequence (LIRL), as well as an LIR motif, at the N-terminus of FKBP8 and LIRL was essential for both inducing mitochondrial fragmentation and binding of FKBP8 to OPA1. Together, we suggest that FKBP8 plays an essential role in mitochondrial fragmentation through LIRL during mitophagy and this activity of FKBP8 together with LIR is required for mitophagy under stress conditions.

Cysteine (Cys) plays a pivotal role in many physiological and pathological processes, including detoxification and protein synthesis. The abnormal levels of Cys are linked to many diseases. In this study, a novel red-emitting off-on fluorescent probe Cys-TCF was masterly constructed for discriminative detection of Cys. After a series of experimental assessment, Cys-TCF displayed higher selectivity and sensitivity for Cys over other biothilols with a low detection limit (0.04 μmol/L). More notably, the probe was also successfully applied to image Cys in live cells and live zebrafishes with low cytotoxicity.

Abstract As a dynamic organelle, mitochondria continuously fuse and divide with adjacent mitochondria. Imbalance in mitochondria dynamics leads to their dysfunction, which implicated in neurodegenerative diseases. However, how mitochondria alteration and glucose defect contribute to pathogenesis of Alzheimer’s disease (AD) is still largely unknown. Dynamin‐related protein 1 (Drp1) is an essential regulator for mitochondria fission. Among various posttranslational modifications, O -GlcNAcylation plays a role as a sensor for nutrient and oxidative stress. In this study, we identified that Drp1 is regulated by O -GlcNAcylation in AD models. Treatment of Aβ as well as PugNAc resulted in mitochondrial fragmentation in neuronal cells. Moreover, we found that AD mice brain exhibits an upregulated Drp1 O -GlcNAcylation. However, depletion of OGT inhibited Drp1 O -GlcNAcylation in Aβ-treated cells. In addition, overexpression of O -GlcNAc defective Drp1 mutant (T585A and T586A) decreased Drp1 O -GlcNAcylation and Aβ-induced mitochondria fragmentation. Taken together, these finding suggest that Aβ regulates mitochondrial fission by increasing O -GlcNAcylation of Drp1.

Autophagy plays a key role in eliminating and recycling cellular components in response to stress, including starvation. Dysregulation of autophagy is observed in various diseases, including neurodegenerative diseases, cancer, and diabetes. Autophagy is tightly regulated by autophagy-related (ATG) proteins. Autophagy-related 4 (ATG4) is the sole cysteine protease, and four homologs (ATG4A-D) have been identified in mammals. These proteins have two domains: catalytic and short fingers. ATG4 facilitates autophagy by promoting autophagosome maturation through reversible lipidation and delipidation of seven autophagy-related 8 (ATG8) homologs, including microtubule-associated protein 1-light chain 3 (LC3) and GABA type A receptor-associated protein (GABARAP). Each ATG4 homolog shows a preference for a specific ATG8 homolog. Post-translational modifications of ATG4, including phosphorylation/dephosphorylation, O-GlcNAcylation, oxidation, S-nitrosylation, ubiquitination, and proteolytic cleavage, regulate its activity and ATG8 processing, thus modulating its autophagic activity. We reviewed recent advances in our understanding of the effect of post-translational modification on the regulation, activity, and function of ATG4, the main protease that controls autophagy.

Perfluorooctanesulfonate (PFOS) is a ubiquitous environmental pollutant associated with increasing health concerns and environmental hazards. Toxicological analyses of PFOS exposure are hampered by large interspecies variations and limited studies on the mechanistic details of PFOS-induced toxicity. We investigated the effects of PFOS exposure on Xenopus laevis embryos based on the reported developmental effects in zebrafish. X. laevis was selected to further our understanding of interspecies variation in response to PFOS, and we built upon previous studies by including transcriptomics and an assessment of ciliogenic effects. Midblastula-stage X. laevis embryos were exposed to PFOS using the frog embryo teratogenesis assay Xenopus (FETAX). Results showed teratogenic effects of PFOS in a time- and dose-dependent manner. The morphological abnormalities of skeleton deformities, a small head, and a miscoiled gut were associated with changes in gene expression evidenced by whole-mount in situ hybridization and transcriptomics. The transcriptomic profile of PFOS-exposed embryos indicated the perturbation in the expression of genes associated with cell death, and downregulation in adenosine triphosphate (ATP) biosynthesis. Moreover, we observed the effects of PFOS exposure on cilia development as a reduction in the number of multiciliated cells and changes in the directionality and velocity of the cilia-driven flow. Collectively, these data broaden the molecular understanding of PFOS-induced developmental effects, whereby ciliary dysfunction and disrupted ATP synthesis are implicated as the probable modes of action of embryotoxicity. Furthermore, our findings present a new challenge to understand the links between PFOS-induced developmental toxicity and vital biological processes.

The role of autophagy in tumor development is paradoxical. Although some genetic evidence has indicated that autophagy has as a tumor suppressor function, it also provides some advantages to tumors under metabolic stress conditions. Autophagy is regulated by several autophagy-related gene (ATG) proteins. In mammals, 16 different ATG genes have been identified. To investigate the clinicopathological role of ATG5 in colorectal cancer, we firstly investigated its expression in patients with sporadic colorectal cancer. Expression analysis revealed ATG5 to be strongly down-regulated in colorectal cancer (38/40 patients). Interestingly, immunohistochemical analysis of colorectal cancer tissues indicated that increased ATG5 expression is associated with lymphovascular invasion (p=0.035). The findings in our limited clinical cohort indicate that ATG5 could be a potential prognostic or diagnostic biomarker.

Inositol polyphosphate 4-phosphatase type II (INPP4B) was recently identified as a tumor resistance factor in laryngeal cancer cells. Herein, we show that INPP4B-mediated resistance is associated with increased glycolytic phenotype. INPP4B expression was induced by hypoxia and irradiation. Intriguingly, overexpression of INPP4B enhanced aerobic glycolysis. Of the glycolysis-regulatory genes, hexokinase 2 (HK2) was mainly regulated by INPP4B and this regulation was mediated through the Akt-mTOR pathway. Notably, codepletion of INPP4B and HK2 markedly sensitized radioresistant laryngeal cancer cells to irradiation or anticancer drug. Moreover, INPP4B was significantly associated with HK2 in human laryngeal cancer tissues. Therefore, these results suggest that INPP4B modulates aerobic glycolysis via HK2 regulation in radioresistant laryngeal cancer cells.

Abstract Previous studies have shown that mesenchymal stem cell (MSC)-based therapies have varying efficacies for the treatment of various diseases, including cartilage defects. In this study, we demonstrated that the chondrogenic differentiation potential of human umbilical cord blood-derived MSCs (hUCB-MSCs) obtained from different individual donors varies, and we investigated the molecular basis for this variation. Microarray gene expression analysis identified thrombospondin-2 (TSP2) as a candidate gene underlying the interindividual variation in the chondrogenic differentiation potential of hUCB-MSCs. To assess the association between TSP-2 and the differentiation potential, we evaluated chondrogenic differentiation of hUCB-MSCs treated with TSP2 siRNA. In addition, we studied the effect of supplementing exogenous recombinant TSP-2 on TSP2 siRNA-treated hUCB-MSCs. We found that TSP-2 autocrinally promoted chondrogenic differentiation of hUCB-MSCs via the Notch signaling pathway, which was confirmed in MSCs from other sources such as bone marrow and adipose tissue. Interestingly, we observed that TSP-2 attenuated hypertrophy, which inevitably occurs during chondrogenic differentiation of hUCB-MSCs. Our findings indicated that the variable chondrogenic differentiation potential of MSCs obtained from different donors is influenced by the TSP-2 level in the differentiating cells. Thus, the TSP-2 level can be used as a marker to select MSCs with superior chondrogenic differentiation potential for use in cartilage regeneration therapy. Stem Cells 2015;33:3291–3303

Autophagy degrades cellular components and organelles through a cooperative process involving autophagosomes and lysosomes. Although autophagy is known to mainly regulate the turnover of cellular components, the role of autophagy in melanogenesis has not been well addressed. Here, we show that inhibition of autophagy suppresses the antimelanogenesis activity of resveratrol (RSV), a well-known antimelanogenic agent. RSV strongly increased autophagy in melanocytes. However, the depletion of ATG5 significantly suppressed RSV-mediated antimelanogenesis as well as RSV-induced autophagy in melanocytes. Moreover, suppression of ATG5 retrieved the RSV-mediated downregulation of tyrosinase and TRP1 in α-MSH-treated cells. Most importantly, electron microscopy analysis revealed that autophagosomes engulfed melanin or melanosomes after combined treatment of α-MSH and RSV. Taken together, these results suggest that RSV-mediated autophagy regulates melanogenesis.

Skin pigmentation involves multiple processes, including melanin synthesis, transport, and melanosome release. Melanin content determines skin color and protects against UV radiation-induced damage. Autophagy is a cooperative process between autophagosomes and lysosomes that degrades cellular components and organelles. In the present study, B16F1 cells were treated with Rhizoma Arisaematis extract (RA) and assessed for pigmentation and autophagy regulation. RA treatment suppressed the α-MSH-stimulated increase of melanogenesis and down-regulated the expression of tyrosinase and TRP1 proteins in B16F1 cells. In addition, autophagy was activated in RA-treated cells. Inhibition of autophagy reduced the anti-melanogenic activity of RA in α-MSH-treated B16F1 cells. We identified schaftoside as an effector molecule by LC-MS analysis of RA. Consistently, treatment of schaftoside showed anti-melanogenic effect and induced autophagy activation in B16F1 cells. Inhibition of autophagy by 3 MA treatment reduced the anti-melanogenic effect of the schaftoside and recovered expression level of melanogenesis regulators in α-MSH-treated B16F1 cells. Taken together, our results suggest that schaftoside from RA inhibits skin pigmentation through modulation of autophagy.

The present study aimed to identify molecules associated with lymphovascular invasion (LVI) and perineural invasion (PNI) and to examine their biological behavior in colorectal cancer (CRC). LVI- and PNI-associated molecules were identified and verified using sequential processes including (1) identification of 117 recurrence-associated genes differentially expressed on RNA-seq analysis using primary cancer tissues from 130 CRC patients with and without systemic recurrence; (2) analysis of molecules associated with LVI and PNI; (3) assessment of biological properties by measuring proliferation, anoikis, invasion/migration, epithelial-mesenchymal transition and autophagy flux; and (4) verification of disease-free survival using public datasets. Gelsolin (GSN) and 2'-5'-oligoadenylate synthetase 2 (OAS2) were associated with PNI and LVI, respectively. Invasion potential was >2-fold greater in GSN-overexpressing LoVo cells than in control cells (p<0.001–0.005), whereas OAS2-overexpressing RKO cells showed reduced invasion (p<0.001–0.005). GSN downregulated E-cadherin, β-catenin, claudin-1 and snail, and upregulated N-cadherin and ZEB1, whereas OAS2 overexpression had the opposite effects. Several autophagy-related proteins including ATG5-12, ATG6/BECN1, ATG7 and ATG101 were downregulated in GSN-overexpressing LoVo cells, whereas the opposite pattern was observed in OAS2-overexpressing RKO cells. Patients with low GSN expression had significantly higher 5-year recurrence-free survival (RFS) rates than those with GSN overexpression (73.6% vs. 64.7%, p = 0.038), whereas RFS was longer in patients with OAS2 overexpression than in those with underexpression (73.4% vs. 63.7%, p = 0.01). In conclusion, GSN and OAS2 were positively and negatively associated with recurrence, respectively, suggesting their potential value as predictors of recurrence or therapeutic targets in CRC patients.

A smart fluorescent probe DPAS-Cys has been rationally designed based on a typical AIEgen DPAS and an acrylate moiety. The probe DPAS-Cys not only can be used for the detection of cysteine (Cys) selectively with large Stokes shift (200 nm) and relatively low detection limit (2.4 μM), but also shows lipid droplets (LDs) targeting property. The response mechanism for Cys was carefully verified. Importantly, due to the aggregation-induced emission characteristic, the introduction of considerable percentage of traditional organic solvent is avoidable, which makes it suitable for bioimaging in physiological systems. In addition, the confocal fluorescence imaging demonstrates that DPAS-Cys is able to detect Cys in LDs of different cell lines with universality. Our study opens a new avenue to understand the importance of LDs in biosystem, for which the gap between the essential biothiol Cys and the energy storage organelle LDs was bridged for the first time.

The effect of glucose-dependent insulinotropic polypeptide (GIP) on cells under oxidative stress induced by glutamate, a neurotransmitter, and the underlying molecular mechanisms were assessed in the present study. We found that in the pre-treatment of HT-22 cells with glutamate in a dose-dependent manner, intracellular ROS were excessively generated, and additional cell damage occurred in the form of lipid peroxidation. The neurotoxicity caused by excessive glutamate was found to be ferroptosis and not apoptosis. Other factors (GPx-4, Nrf2, Nox1 and Hspb1) involved in ferroptosis were also identified. In other words, it was confirmed that GIP increased the activity of sub-signalling molecules in the process of suppressing ferroptosis as an antioxidant and maintained a stable cell cycle even under glutamate-induced neurotoxicity. At the same time, in HT-22 cells exposed to ferroptosis as a result of excessive glutamate accumulation, GIP sustained cell viability by activating the mitogen-activated protein kinase (MAPK) signalling pathway. These results suggest that the overexpression of the GIP gene increases cell viability by regulating mechanisms related to cytotoxicity and reactive oxygen species production in hippocampal neuronal cell lines.

We describe the isolation and characterization of a new apaf-1-interacting protein (APIP) as a negative regulator of ischemic injury. APIP is highly expressed in skeletal muscle and heart and binds to the CARD of Apaf-1 in competition with caspase-9. Exogenous APIP inhibits cytochrome <i>c</i>-induced activation of caspase-3 and caspase-9, and suppresses cell death triggered by mitochondrial apoptotic stimuli through inhibiting the downstream activity of cytochrome <i>c</i> released from mitochondria. Conversely, reduction of APIP expression potentiates mitochondrial apoptosis. APIP expression is highly induced in mouse muscle affected by ischemia produced by interruption of the artery in the hindlimb and in C2C12 myotubes created by hypoxia <i>in vitro</i>, and the blockade of APIP up-regulation results in TUNEL-positive ischemic damage. Furthermore, forced expression of APIP suppresses ischemia/hypoxia-induced death of skeletal muscle cells. Taken together, these results suggest that APIP functions to inhibit muscle ischemic damage by binding to Apaf-1 in the Apaf-1/caspase-9 apoptosis pathway.

Autophagy is a catabolic cellular process involving self-digestion and turnover of macromolecules and entire organelles. Autophagy is primarily a protective process in response to cellular stress, but it can be associated with cell death. Genetic evidence also supports autophagy function as a tumor suppressor mechanism. To identify specific regulators to autophagy, we screened the Lopac 1280 and the Prestwick chemical libraries using a cell-based screening system with autophagy marker (green fluorescence protein conjugated LC3 protein (GFP-LC3)). We identified ARP101, a selective matrix metalloproteinase-2 (MMP-2) inhibitor as one of the most potent inducer of autophagy. ARP101 treatment was highly effective in inducing the formation of autophagosome and conversion of LC3I into LC3II. Moreover, ARP101-induced autophagy was completely blocked in mouse embryo fibroblasts that lacked autophagy related gene 5 (ATG5−/− MEF). Interestingly, cell death induced by ARP101 was not inhibited by zVAD, a pan caspase inhibitor, whereas, it was efficiently suppressed by addition of 3-methyladenine, an autophagy inhibitor. These results suggest that the selective MMP-2 inhibitor, ARP101, induces autophagy and autophagy-associated cell death.

Context: Cinnamon bark is a very popular herb used in traditional medicine to treat various disorders such as chronic gastric symptoms, arthritis, and the common cold.Objective: The immunomodulatory effect of water extract of cinnamon bark (CWE) on cytokine secretion and involvement of intracellular signaling molecules in activated T cells have been examined.Materials and methods: Mice were orally administered CWE for 7 days. Serum was obtained 90 min after intravenous injection of anti-CD3 antibody (Ab). Splenocytes were cultured with anti-CD3 Ab and CWE for cytokine expression, cell cycle, apoptotic/necrotic changes, and viability. IκBα, p38, JNK, ERK1/2, STAT4, and STAT6 were analyzed using western blotting.Results: Administration of CWE decreased systemic levels of IFN-γ, but not the levels of IL-4 or IL-2. In vitro, CWE inhibited anti-CD3 Ab-stimulated IFN-γ and IL-4 at the mRNA and secreted protein levels. Despite its inhibition of IL-2 transcript, CWE enhanced IL-2 secretion. CWE treatment caused a reduction in the sub-G1 phase, accompanied by an increased ratio of apoptotic cells to necrotic cells. The increased IL-2 secretion by CWE was not mediated by its direct effect on CD4 T cells. CWE inhibited the activation of p38, JNK, ERK1/2, and STAT4, but not IκBα degradation or STAT6.Discussion and conclusions: These observations provided evidence that CWE was able to down-regulate IFN-γ expression in activated T cells without altering IL-2 production, involving inhibition of p38, JNK, ERK1/2, and STAT4. Our results contribute to a better understanding of the immunomodulatory action of cinnamon bark for the application of inflammatory disorders.

Autophagy is associated with cell survival and cell death. Autophagy is implicated in the pathophysiology of various human diseases. In order to identify autophagy regulatory molecules, we screened a chemical drug library in SH-SY5Y cells and selected Sertindole as a potent autophagy inducer. Sertindole was developed as an antipsychotic drug for Schizophrenia. Sertindole treatment highly induced the formation of autophagosomes as well as LC3 conversion. Subsequently, Sertindole-induced autophagy was efficiently suppressed by down regulation of ATG5. Sertindole also increased reactive oxygen species (ROS) production, which contributes to autophagy-associated cell death in neuroblastoma cells. ROS scavengers such as N-acetylcysteine and Trolox suppressed not only ROS generation but also autophagy activation by Sertindole. These results suggest Sertindole induces autophagy and autophagy-associated cell death by ROS production in neuroblastoma cells.

Mitochondrial morphology is dynamically regulated by forming small, fragmented units or interconnected networks, and this is a pivotal process that is used to maintain mitochondrial homeostasis. Although dysregulation of mitochondrial dynamics is related to the pathogenesis of several human diseases, its molecular mechanism is not fully elucidated. In this study, we demonstrate the potential role of miR-27 in the regulation of mitochondrial dynamics. Mitochondrial fission factor (MFF) mRNA is a direct target of miR-27, whose ectopic expression decreases MFF expression through binding to its 3'-untranslated region. Expression of miR-27 results in the elongation of mitochondria as well as an increased mitochondrial membrane potential and mitochondrial ATP level. Our results suggest that miR-27 is a novel regulator affecting morphological mitochondrial changes by targeting MFF.

Excessive mitochondrial fission is associated with the pathogenesis of neurodegenerative diseases. Dynamin-related protein 1 (Drp1) possesses specific fission activity in the mitochondria and peroxisomes. Various post-translational modifications of Drp1 are known to modulate complex mitochondrial dynamics. However, the post-transcriptional regulation of Drp1 remains poorly understood. Here, we show that the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) regulates Drp1 expression at the post-transcriptional level. hnRNP A1 directly interacts with Drp1 mRNA at its 3'UTR region, and enhances translation potential without affecting mRNA stability. Down-regulation of hnRNP A1 induces mitochondrial elongation by reducing Drp1 expression. Moreover, depletion of hnRNP A1 suppresses 3-NP-mediated mitochondrial fission and dysfunction. In contrast, over-expression of hnRNP A1 promotes mitochondrial fragmentation by increasing Drp1 expression. Additionally, hnRNP A1 significantly exacerbates 3-NP-induced mitochondrial dysfunction and cell death in neuroblastoma cells. Interestingly, treatment with 3-NP induces subcellular translocation of hnRNP A1 from the nucleus to the cytoplasm, which accelerates the increase in Drp1 expression in hnRNP A1 over-expressing cells. Collectively, our findings suggest that hnRNP A1 controls mitochondrial dynamics by post-transcriptional regulation of Drp1.

Autophagy is a catabolic degradation process and maintains cellular homeostasis.And autophagy is activated in response to various stress conditions.Although O-GlcNAcylation functions a sensor for nutrient and stress, the relationship between O-GlcNAcylation and autophagy is largely unknown.Here, we identified that ATG4B is novel target for O-GlcNAcylation under metabolic stress condition.Treatment with PugNAc, an O-GlcNAcase inhibitor increased activation of autophagy in SH-SY5Y cells.Both bimolecular fluorescence complementation and immunoprecipitation assay indicated that OGT directly interacts with ATG4B in SH-SY5Y cells.We also found that the O-GlcNAcylated ATG4B was increased in autophagy activation conditions, and down-regulation of OGT reduces O-GlcNAcylation of ATG4B under low glucose condition.Furthermore, the proteolytic activity of ATG4B for LC3 cleavage was enhanced in PugNAc-treated cells.Taken together, these results imply that O-GlcNAcylation of ATG4B regulates autophagy activation by increasing its proteolytic activity under metabolic stress condition.

Several studies have shown that dysfunction of macroautophagy/autophagy is associated with many human diseases, including neurodegenerative disease and cancer. To explore the molecular mechanisms of autophagy, we performed a cell-based functional screening with SH-SY5Y cells stably expressing GFP-LC3, using an siRNA library and identified TMED10 (transmembrane p24 trafficking protein 10), previously known as the γ-secretase-modulating protein, as a novel regulator of autophagy. Further investigations revealed that depletion of TMED10 induced the activation of autophagy. Interestingly, protein-protein interaction assays showed that TMED10 directly binds to ATG4B (autophagy related gene 4B cysteine peptidase), and the interaction is diminished under autophagy activation conditions such as rapamycin treatment and serum deprivation. In addition, inhibition of TMED10 significantly enhanced the proteolytic activity of ATG4B for LC3 cleavage. Importantly, the expression of TMED10 in AD (Alzheimer disease) patients was considerably decreased, and downregulation of TMED10 increased amyloid-β (Aβ) production. Treatment with Aβ increased ATG4B proteolytic activity as well as dissociation of TMED10 and ATG4B. Taken together, our results suggest that the AD-associated protein TMED10 negatively regulates autophagy by inhibiting ATG4B activity.Abbreviations: Aβ: amyloid-β; AD: Alzheimer disease; ATG: autophagy related; BECN1: beclin 1; BiFC: bimolecular fluorescence complementation; CD: cytosolic domain; GFP: green fluorescent protein; GLUC: Gaussia luciferase; IP: immunoprecipitation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LD: luminal domain; PD: Parkinson disease; ROS: reactive oxygen species; siRNA: small interfering RNA; SNP: single-nucleotide polymorphisms; TD: transmembrane domain; TMED10: transmembrane p24 trafficking protein 10; VC: C terminus of Venus fluorescent protein; VN: N terminus of Venus fluorescent protein.

The stimulation of autophagy or lysosomes has been considered therapeutic for neurodegenerative disorders because the accumulation of misfolded proteins is commonly observed in the brains of individuals with these diseases. Although zinc is known to play critical roles in the functions of lysosomes and autophagy, the mechanism behind this regulatory relationship remains unclear. Therefore, in this study, we examined which mechanism is involved in zinc-mediated activation of autophagy and lysosome. Exposure to zinc at a sub-lethal concentration activated autophagy in a concentration-dependent manner in mRFP-GFP-LC3-expressing H4 glioma cells. Zinc also rescued the blocking of autophagic flux arrested by pharmaceutical de-acidification. Co-treatment with zinc attenuated the chloroquine (CQ)-induced increase in the number and size of mRFP-GFP-LC3 puncta in H4 cells and accumulation of p62 by CQ or ammonium chloride in both H4 and mouse cerebrocortical cultures. Zinc rapidly induced the expression of cathepsin B (CTSB) and cathepsin D (CTSD), representative lysosomal proteases in neurons, which appeared likely to be mediated by transcription factor EB (TFEB). We observed the translocation of TFEB from neurite to nucleus and the dephosphorylation of TFEB by zinc. The addition of cycloheximide, a chemical inhibitor of protein synthesis, inhibited the activity of CTSB and CTSD at 8 h after zinc exposure but not at 1 h, indicating that only late lysosomal activation was dependent on the synthesis of CTSB and CTSD proteins. At the very early time point, the activation of cathepsins was mediated by an increased assembly of V-ATPase on lysosomes and resultant lysosomal acidification. Finally, considering that P301L mutation in tau protein causes frontotemporal dementia through aggressive tau accumulation, we investigated whether zinc reduces the accumulation of protein aggregates in SK-N-BE(2)-C neuroblastoma cells expressing wild-type tau or mutant P301L-tau. Zinc markedly attenuated the levels of phosphorylated tau and total tau as well as p62 in both wild-type and mutant tau-overexpressing cells. We also observed that zinc was more effective than rapamycin at inducing TFEB-dependent CTSB and CTSD expression and V-ATPase-dependent lysosomal acidification and CTSB/CTSD activation. These results suggest that the regulation of zinc homeostasis could be a new approach for developing treatments for neurodegenerative diseases, including Alzheimer's and Parkinson's.

Abstract The Arg/N-degron pathway, which is involved in the degradation of proteins bearing an N-terminal signal peptide, is connected to p62/SQSTM1-mediated autophagy. However, the impact of the molecular link between the N-degron and autophagy pathways is largely unknown in the context of systemic inflammation. Here, we show that chemical mimetics of the N-degron Nt-Arg pathway (p62 ligands) decreased mortality in sepsis and inhibited pathological inflammation by activating mitophagy and immunometabolic remodeling. The p62 ligands alleviated systemic inflammation in a mouse model of lipopolysaccharide (LPS)-induced septic shock and in the cecal ligation and puncture model of sepsis. In macrophages, the p62 ligand attenuated the production of proinflammatory cytokines and chemokines in response to various innate immune stimuli. Mechanistically, the p62 ligand augmented LPS-induced mitophagy and inhibited the production of mitochondrial reactive oxygen species in macrophages. The p62 ligand-mediated anti-inflammatory, antioxidative, and mitophagy-activating effects depended on p62. In parallel, the p62 ligand significantly downregulated the LPS-induced upregulation of aerobic glycolysis and lactate production. Together, our findings demonstrate that p62 ligands play a critical role in the regulation of inflammatory responses by orchestrating mitophagy and immunometabolic remodeling.

Abstract The primary cilium, an antenna-like structure on the cell surface, acts as a mechanical and chemical sensory organelle. Primary cilia play critical roles in sensing the extracellular environment to coordinate various developmental and homeostatic signaling pathways. Here, we showed that the depletion of heat shock protein family A member 9 (HSPA9)/mortalin stimulates primary ciliogenesis in SH-SY5Y cells. The downregulation of HSPA9 enhances mitochondrial stress by increasing mitochondrial fragmentation and mitochondrial reactive oxygen species (mtROS) generation. Notably, the inhibition of either mtROS production or mitochondrial fission significantly suppressed the increase in primary ciliogenesis in HSPA9-depleted cells. In addition, enhanced primary ciliogenesis contributed to cell survival by activating AKT in SH-SY5Y cells. The abrogation of ciliogenesis through the depletion of IFT88 potentiated neurotoxicity in HSPA9 -knockdown cells. Furthermore, both caspase-3 activation and cell death were increased by MK-2206, an AKT inhibitor, in HSPA9-depleted cells. Taken together, our results suggest that enhanced primary ciliogenesis plays an important role in preventing neurotoxicity caused by the loss of HSPA9 in SH-SY5Y cells.

Apoptosis repressor with CARD (ARC) possesses the ability not only to block activation of caspase 8 but to modulate caspase-independent mitochondrial events associated with cell death. However, it is not known how ARC modulates both caspase-dependent and caspase-independent cell death. Here, we report that ARC is a Ca(2+)-dependent regulator of caspase 8 and cell death. We found that in Ca(2+) overlay and Stains-all assays, ARC protein bound to Ca(2+) through the C-terminal proline/glutamate-rich (P/E-rich) domain. ARC expression reduced not only cytosolic Ca(2+) transients but also cytotoxic effects of thapsigargin, A23187, and ionomycin, for which the Ca(2+)-binding domain of ARC was indispensable. Conversely, direct interference of endogenous ARC synthesis by targeting ARC enhanced such Ca(2+)-mediated cell death. In addition, binding and immunoprecipitation analyses revealed that the protein-protein interaction between ARC and caspase 8 was decreased by the increase of Ca(2+) concentration in vitro and by the treatment of HEK293 cells with thapsigargin in vivo. Caspase 8 activation was also required for the thapsigargin-induced cell death and suppressed by the ectopic expression of ARC. These results suggest that calcium binding mediates regulation of caspase 8 and cell death by ARC.

Hereditary colorectal cancer develops through a series of well-defined genetic and histological changes. However, elucidation of the canonical pathway based on hereditary colorectal cancer has not provided a clear explanation of the molecular mechanisms of sporadic colorectal cancer. To identify the alterative pathways involved in sporadic colorectal tumorigenesis, we performed gene expression analysis in patients with sporadic colorectal tumors. A comparison analysis of gene expression profiles revealed a pattern of upregulation of small proline rich repeat protein 3 (SPRR3) in tumor samples. SPRR3 has previously been reported to be downregulated in esophageal cancer. However, in the present study, we observed that SPRR3 was strongly upregulated in 31 of 35 samples of sporadic colorectal tumors (88%). We also determined that overexpression of SPRR3 not only accelerates colorectal cancer cell proliferation but also is associated with lymphovascular invasion in colorectal cancer. Moreover, AKT was activated and p53 levels were decreased in cells that overexpressed SPRR3. In contrast to the pattern seen in esophageal cancer, these results suggest that increased expression of SPRR3 is involved in colorectal tumorigenesis.

Abstract South East Asia has some of the highest prevalence rates of hepatitis B virus (HBV) infection (≥8%) in the world, and the emergence of hepatitis B surface antigen (HBsAg) mutant strains is a growing problem. Assays with the highest levels of sensitivity, including mutant detection, should be used for routine HBsAg screening. In this large multicenter study, the clinical and technical performance of the fully automated Elecsys HBsAg II assay was compared with the Architect, AxSYM, and Advia Centaur HBsAg assays for HBsAg screening. Nine laboratories (three each from Thailand, Korea, and Singapore) compared the Elecsys HBsAg II assay with their routine HBsAg screening assay against a range of stored and routine clinical samples, including recombinant mutants. The Elecsys HBsAg II assay demonstrated equivalent sensitivity and specificity to the Architect HBsAg assay. However, the Elecsys HBsAg II assay recognized a native mutant sample (L94S, L97V, L98V, T123A) that the Architect HBsAg assay failed to detect. The AxSYM and Advia Centaur HBsAg assays appeared less sensitive for the detection of early HBV infection and also failed to detect some of the recombinant mutant strains. There was almost complete agreement between the Elecsys HBsAg II assay and comparator assays with respect to routine serum samples. The results of this study demonstrate that the Elecsys HBsAg II assay is a highly sensitive and specific screening assay for HBsAg and detects reliably the most important and clinically relevant HBV mutants and genotypes. It is suitable for routine HBsAg screening in Asia. J. Med. Virol. 82: 755–762, 2010. © 2010 Wiley‐Liss, Inc.

Autophagy is a cellular degradation process for cellular aggregates and unneeded cellular compartments including damaged mitochondria, ER, and peroxisomes. Melanosome is cellular organelle that is the cellular site of generation, storage and transports of melanin in melanocytes. Despite potential importance of autophagy, the role of autophagy in melanogenesis and melanosome autophagy are largely unknown. In here, we identified 3'-hydroxydaidzein (3'-ODI) as an autophagy inducer from a phytochemical library screening. Treatment with 3'-ODI significantly reduced α-MSH-mediated melanogenesis but efficiently increased autophagy both in melanoma cells and melanocytes. Furthermore, inhibition of autophagy significantly reduced the anti-melanogenic effects of 3'-ODI in α-MSH-stimulated melanoma cells. Taken together, these results suggest that autophagy mediates anti-melanogenic activity of 3'-ODI.

Using our data set (GSE50760) previously established by RNA sequencing, the present study aimed to identify upregulated genes associated with colorectal cancer (CRC) liver metastasis (CLM) and verify their biological behavior. The potential roles of candidate genes in tumors were assessed using cell proliferation and invasion assays. Tissue samples were collected from 18 CRC patients with synchronous CLM and two CRC cell lines (SW480 and SW620) were used for transfection and cloning. The roles of the genes identified in CLM were verified using immunohistochemistry in 48 nude mice after intrasplenic transplantation of CRC cells. mRNA and protein expression was determined by quantitative real-time reverse transcription polymerase chain reaction and western blot, respectively. Nine genes were initially selected according to the relevance of their molecular function and biological process and, finally, ALDH1A1 and IGFBP1 were chosen based on differential mRNA expression and a positive correlation with protein expression. The overexpression of ALDH1A1 and IGFBP1 significantly and time-dependently decreased cell proliferation (p ≤ 0.001-0.003) and suppressed invasiveness by ≥3-fold over control cells (p < 0.001) in the SW480 cell line, whereas they had a slight effect on reducing SW620 cell proliferation. The protein expression levels of E-cadherin, N-cadherin, claudin-1, and vimentin were significantly higher in CLM than in primary tumor tissues (p < 0.05). However, the cadherin switch, namely, N-cadherin overexpression with reduced E-cadherin expression, was not observed in CLM tissues and transfected CRC cells. Irrespective of reduced proliferation and invasion found on in vitro cell assays, persistent overexpression of β-catenin, vimentin, and ZO-1 in IGFBP1-overexpressing SW480 cells possibly contributed to CLM development in mice implanted with IGFBP1-overexpressing SW480 cells (CLM occurrences: SW480/IGFBP1-transfected mice vs. SW480/vector- and SW480/ALDH1A1-transfected mice, 4/8 vs. 0/10, p = 0.023). In conclusion, ALDH1A1 and IGFBP1 are differentially overexpressed in CLM and may play a dual role, functioning as both tumor suppressors and metastasis promoters in CRC.

The purpose of this study was to evaluate the prognostic significance of serum CEA (s-CEA) changes in colorectal cancer (CRC) patients with sustained elevated postoperative s-CEA levels.Between January 1999 and December 2008, 9,380 CRC patients underwent surgery. Curative resection was performed in 1,242 CRC patients with high preoperative s-CEA levels (>6 ng/mL). High s-CEA levels were normalized in 924 patients (74.4%) within 2 weeks from surgery, whereas high s-CEA levels were persistent in 318 patients (25.6%). Patients were divided into 2 groups according to their postoperative s-CEA levels: group 1 (37 patients with a 1-year postoperative s-CEA>6 ng/mL) and group 2 (281 patients with a 1-year postoperative s-CEA≤6 ng/mL).A postoperative recurrence was identified in 24 patients (64.9%) in group 1 and 65 patients (23.1%) in group 2 (P < 0.001). A curative resection after recurrence was performed in 22 patients (33.8%) from group 2, but no patients from group 1 (P = 0.001). The 5-year overall survival and time to recurrence were significantly lower in patients with recurrent cancer in group 1 (P < 0.001).Patients with persistent elevated postoperative s-CEA levels are at high risk for recurrence and a low survival rate. More intensive surveillance of patients with high postoperative s-CEA levels should be mandatory.

Approximately half of colorectal cancer (CRC) patients experience disease recurrence and metastasis, and these individuals frequently fail to respond to treatment due to their clinical and biological diversity. Here, we aimed to identify a prognostic signature consisting of a small gene group for precisely predicting CRC heterogeneity. We performed transcriptomic profiling using RNA-seq data generated from the primary tissue samples of 130 CRC patients. A prognostic index (PI) based on recurrence-associated genes was developed and validated in two larger independent CRC patient cohorts (n = 795). The association between the PI and prognosis of CRC patients was evaluated using Kaplan-Meier plots, log-rank tests, a Cox regression analysis and a RT-PCR analysis. Transcriptomic profiling in 130 CRC patients identified two distinct subtypes associated with systemic recurrence. Pathway enrichment and RT-PCR analyses revealed an eleven gene signature incorporated into the PI system, which was a significant prognostic indicator of CRC. Multivariate and subset analyses showed that PI was an independent risk factor (HR = 1.812, 95% CI = 1.342-2.448, P < 0.001) with predictive value to identify low-risk stage II patients who responded the worst to adjuvant chemotherapy. Finally, a comparative analysis with previously reported Consensus Molecular Subgroup (CMS), high-risk patients classified by the PI revealed a distinct molecular property similar to CMS4, associated with a poor prognosis. This novel PI predictor based on an eleven gene signature likely represents a surrogate diagnostic tool for identifying high-risk CRC patients and for predicting the worst responding patients for adjuvant chemotherapy.

Exposure to fine particulate matter (PM) with diameter <2.5 µm (PM2.5) causes epithelium injury and endothelial dysfunction. Primary cilia are sensory organelles that transmit extracellular signals into intracellular biochemical responses and have roles in physiology. To date, there have been no studies investigating whether PM2.5 affects primary cilia in skin. We addressed this in the present study using normal human epidermal keratinocytes (NHEKs) and retinal pigment epithelium (RPE) cells. We found that formation of primary cilium is increased in differentiated NHEKs. However, treatment with PM2.5 blocked increased ciliogenesis in NHEKs and RPE cells. Furthermore, PM2.5 transcriptionally upregulated small proline rich protein 3 (SPRR3) expression by activating c-Jun, and ectopic expression of SPRR3 inhibits suppressed the ciliogenesis. Accordingly, treatment with c-Jun activator (anisomycin) induced SPRR3 expression, whereas the inhibitor (SP600125) recovered the ciliated cells and cilium length in PM2.5-treated cells. Moreover, c-Jun inhibitor suppressed upregulation of SPRR3 in PM2.5-treated cells. Taken together, our finding suggested that PM2.5 inhibits ciliogenesis by increasing SPRR3 expression via c-Jun activation in RPE cells and keratinocytes.

Vicinal dithiol-containing proteins (VDPs) play an important role in maintaining the structures and functions of proteins mainly through the conversion between dithiols and disulfide bonds. The content of VDPs also reflects the redox status of an organism. To specifically and expediently detect VDPs, we developed a turn-on monoarsenical fluorescent probe (NEP) based on the intramolecular charge transfer mechanism. Naphthalimide was chosen as a fluorophore and linked with the receptor moiety (cyclic dithiarsolane) via carbamate segment. In the presence of VDPs, NEP displays a strong green fluorescence signal produced by the cyclic dithiarsolane cleavage and subsequent intramolecular cyclization to liberate the fluorophore. Furthermore, NEP exhibits high selectivity toward VDPs over other protein thiols and low molecular weight thiols. The favorable properties of NEP enable it readily to detect VDPs in live cells and in vivo. In addition, a remarkable decrease of VDPs in parkinsonism was disclosed for the first time, highlighting that regulating VDPs level has a therapeutic potential for parkinsonism.

Mitochondria dynamics controls not only their morphology but also functions of mitochondria. Therefore, an imbalance of the dynamics eventually leads to mitochondria disruption and cell death. To identify specific regulators of mitochondria dynamics, we screened a bioactive chemical compound library and selected Tyrphostin A9, a tyrosine kinase inhibitor, as a potent inducer of mitochondrial fission. Tyrphostin A9 treatment resulted in the formation of fragmented mitochondria filament. In addition, cellular ATP level was decreased and the mitochondrial membrane potential was collapsed in Tyr A9-treated cells. Suppression of Drp1 activity by siRNA or over-expression of a dominant negative mutant of Drp1 inhibited both mitochondrial fragmentation and cell death induced by Tyrpohotin A9. Moreover, treatment of Tyrphostin A9 also evoked mitochondrial fragmentation in other cells including the neuroblastomas. Taken together, these results suggest that Tyrphostin A9 induces Drp1-mediated mitochondrial fission and apoptotic cell death.

Mefloquine is an effective treatment drug for malaria. However, it can cause several adverse side effects, and the precise mechanism associated with the adverse neurological effects of Mefloquine is not clearly understood. In this study, we investigated the effect of Mefloquine on autophagy in neuroblastoma cells. Mefloquine treatment highly induced the formation of autophagosomes and the conversion of LC3I into LC3II. Moreover, Mefloquine-induced autophagy was efficiently suppressed by an autophagy inhibitor and by down regulation of ATG6. The autophagy was also completely blocked in ATG5 deficient mouse embryonic fibroblast cells. Moreover, suppression of autophagy significantly intensified Mefloquine-mediated cytotoxicity in SH-SY5Y cells. Our findings suggest that suppression of autophagy may exacerbate Mefloquine toxicity in neuroblastoma cells.

Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) secrete various beneficial molecules, which have anti-apoptotic activity and cell proliferation. However, the effect of hUCB-MSCs in melanogenesis is largely unclear. In this study, we show that conditioned media (CM) derived from hUCB-MSCs inhibit melanogenesis by regulating microphthalmia-associated transcription factor (MITF) expression via the ERK signalling pathway. Treatment of hUCB-MSC-CM strongly inhibited the alpha-melanocyte stimulating hormone-induced hyperpigmentation in melanoma cells as well as melanocytes. Treatment of hUCB-MSC-CM induced ERK1/2 activation in melanocytes. In addition, inhibition of ERK1/2 suppressed the anti-pigmentation activity of the hUCB-MSC-CM in melanocytes and in vitro artificial skin models. We also found that the expression of MITF was appreciably diminished while expression of phosphorylated MITF, which leads to its proteasomal degradation, was increased in cells treated with hUCB-MSC-CM. These results suggested that hUCB-MSC-CM significantly suppresses melanin synthesis via MITF degradation by the ERK pathway activation.

Cytokinesis is the final step in cell division that results in the separation of a parent cell into daughter cells. Unlike somatic cells that undergo symmetric division, meiotic division is highly asymmetric, allowing the preservation of maternal resources for embryo development. Beclin-1/BECN1, the mammalian homolog of yeast Atg6, is a key molecule of autophagy. As part of a class III phosphatidylinositol 3-kinase (PI3K-III) complex, BECN1 initiates autophagosome formation by coordinating membrane trafficking. However, emerging evidence suggests that BECN1 regulates chromosome segregation and cytokinesis during mitosis. Thus, we investigated the function of BECN1 during oocyte meiotic maturation. BECN1 was widely distributed during meiotic maturation forming small vesicles. Interestingly, BECN1 is also detected at the midbody ring during cytokinesis. Depletion of BECN1 impaired the cytokinetic abscission, perturbing the recruitment of ZFYVE26 at the midbody. Similar phenotypes were observed when PI3K-III activity was inhibited. However, inhibition of autophagy by depleting Atg14L did not disturb meiotic maturation. Therefore, our results not only demonstrate that BECN1 as a PI3K-III component is essential for cytokinesis, but also suggest that BECN1 is not associated with autophagy pathway in mouse oocytes.

To investigate alternative or subordinate pathways involved in colorectal tumorigenesis and tumor growth, possibly determining at-risk populations and predicting responses to treatment.Using microarray gene-expression analysis, we analyzed patterns of gene expression relative to canonical molecular changes and clinicopathological features in 84 sporadic colorectal cancer patients, standardized by tumor location. Subsets of differentially expressed genes were confirmed by real-time reverse-transcript polymerase chain reaction (RT-PCR).The largest number of genes identified as being differentially expressed was by tumor location, and the next largest number by lymphovascular or neural invasion of tumor cells and by mismatch repair (MMR) defects. Amongst biological processes, the immune response was significantly implicated in entire molecular changes observed during colorectal tumorigenesis (P < 0.001). Amongst 47 differentially expressed genes, seven (PISD, NIBP, BAI2, STOML1, MRPL21, MRPL16, and MKKS) were newly found to correlate with tumorigenesis and tumor growth. Most location-associated molecular changes had distinct effects on gene expression, but the effects of the latter were sometimes contradictory.We show that several differentially expressed genes were associated with canonical molecular changes in sporadic colorectal cancers, possibly constituting alternative or subordinate pathways of tumorigenesis. As tumor location was the dominant factor influencing differential gene expression, location-specific analysis may identify location-associated pathways and enhance the accuracy of class prediction.

Although autophagy regulates the quality and quantity of cellular organelles, the regulatory mechanisms of peroxisomal autophagy remain largely unknown. In this study, we developed a cell-based image screening assay, and identified 1,10-phenanthroline (Phen) as a novel pexophagy inducer from chemical library screening. Treatment with Phen induces selective loss of peroxisomes but not endoplasmic reticulum and Golgi apparatus in hepatocytes. In addition, Phen increases autophagic engulfment of peroxisomes in an ATG5 dependent manner. Interestingly, treatment of Phen excessively produces peroxisomal reactive oxygen species (ROS), and inhibition of the ROS suppresses loss of peroxisome in Phen-treated cells. Taken together, these results suggest that Phen triggers pexophagy by enhancing peroxisomal ROS.

Primary cilia are conserved cellular organelles that regulate diverse signaling pathways. Autophagy is a complex process of cellular degradation and recycling of cytoplasmic proteins and organelles, and plays an important role in cellular homeostasis. Despite its potential importance, the role of autophagy in ciliogenesis is largely unknown. In this study, we identified sertraline as a regulator of autophagy and ciliogenesis. Sertraline, a known antidepressant, induced the growth of cilia and blocked the disassembly of cilia in htRPE cells. Following treatment of sertraline, there was an increase in the number of cells with autophagic puncta and LC3 protein conversion. In addition, both a decrease of ATG5 expression and the treatment of an autophagy inhibitor resulted in the suppression of the sertraline-induced activation of autophagy in htRPE cells. Interestingly, we found that genetic and chemical inhibition of autophagy attenuated the growth of primary cilia in htRPE cells. Taken together, our results suggest that the inhibition of autophagy suppresses sertraline-induced ciliogenesis.

Various histone residues are post-translationally modified during the cell cycle. Among these, histone H3 phosphorylation at threonine 3 (H3T3ph) is newly characterized and has been considered to be crucial for chromosome dynamics during mitosis. However, little is known about the role of H3T3ph during mouse oocyte maturation. In the present study, we examined H3T3ph expression and localization during oocyte meiosis. Our results showed that H3T3ph was tightly associated with condensed chromosomes during meiotic maturation. H3T3ph along the chromosome arms was dissociated at anaphase/telophase I, but centromeric H3T3ph remained intact. Moreover, the inhibition of H3T3ph with the small molecule inhibitors CHR-6494 and 5-Itu impaired segregation of homologous chromosomes during meiosis. Partial inhibition of H3T3ph revealed that centromeric Aurora B/C kinase is sufficient to complete meiosis I, but Aurora B/C kinase along the chromosome arms is required to ensure accurate homologous chromosome segregation. Therefore, our results demonstrate that H3T3ph is a universal regulator of chromosome dynamics during oocyte meiosis and mitosis.

Melanosomes are specialized membrane-bound organelles that are involved in melanin synthesis. Unlike melanosome biogenesis, the melanosome degradation pathway is poorly understood. Among the cellular processes, autophagy controls degradation of intracellular components by cooperating with lysosomes. In this study, we showed that ursolic acid inhibits skin pigmentation by promoting melanosomal autophagy, or melanophagy, in melanocytes. We found that B16F1 cells treated with ursolic acid suppressed alpha-melanocyte stimulating hormone (α-MSH) stimulated increase in melanin content and activated autophagy. In addition, we found that treatment with ursolic acid promotes melanosomal degradation, and bafilomycin A1 inhibition of autophagosome–lysosome fusion blocked the removal of melanosomes in α-MSH-stimulated B16F1 cells. Furthermore, depletion of the autophagy-related gene 5 (ATG5) resulted in significant suppression of ursolic acid–mediated anti-pigmentation activity and autophagy in α-MSH-treated B16F1 cells. Taken together, our results suggest that ursolic acid inhibits skin pigmentation by increasing melanosomal degradation in melanocytes.

Primary cilia have critical roles in coordinating multiple cellular signaling pathways. Dysregulation of primary cilia is implicated in various ciliopathies. To identify specific regulators of autophagy, we screened chemical libraries and identified mefloquine, an anti-malaria medicine, as a potent regulator of primary cilia in human retinal pigmented epithelial (RPE) cells. Not only ciliated cells but also primary cilium length was increased in mefloquine-treated RPE cells. Treatment with mefloquine strongly induced the elongation of primary cilia by blocking disassembly of primary cilium. In addition, we found that autophagy was increased in mefloquine-treated cells by enhancing autophagic flux. Both chemical and genetic inhibition of autophagy suppressed ciliogenesis in mefloquine-treated RPE cells. Taken together, these results suggest that autophagy induced by mefloquine positively regulates the elongation of primary cilia in RPE cells.

The tumor necrosis factor-related apoptosis inducing ligand (TRAIL) preferentially induces apoptosis in cancer cells. However, many tumors are resistant to TRAIL-induced apoptosis, and resistance mechanisms are not fully understood. To identify novel regulatory molecules of TRAIL resistance, we screened a siRNA library targeting the human kinome, and NEK4 (NIMA-related kinase-4) was identified. Knockdown of NEK4 sensitized TRAIL-resistant cancer cells and in vivo xenografts to cell death. In contrast, over expression of NEK4 suppressed TRAIL-induced cell death in TRAIL-sensitive cancer cells. In addition, loss of NEK4 resulted in decrease of the anti-apoptotic protein survivin, but an increase in apoptotic cell death. Interestingly, NEK4 was highly upregulated in tumor tissues derived from patients with lung cancer and colon cancer. These results suggest that inhibition of NEK4 sensitizes cancer cells to TRAIL-induced apoptosis by regulation of survivin expression.

Abstract Arsenic is reportedly a biphasic inorganic compound for its toxicity and anticancer effects in humans. Recent studies have shown that certain arsenic compounds including arsenic hexoxide (AS 4 O 6 ; hereafter, AS6) induce programmed cell death and cell cycle arrest in human cancer cells and murine cancer models. However, the mechanisms by which AS6 suppresses cancer cells are incompletely understood. In this study, we report the mechanisms of AS6 through transcriptome analyses. In particular, the cytotoxicity and global gene expression regulation by AS6 were compared in human normal and cancer breast epithelial cells. Using RNA-sequencing and bioinformatics analyses, differentially expressed genes in significantly affected biological pathways in these cell types were validated by real-time quantitative polymerase chain reaction and immunoblotting assays. Our data show markedly differential effects of AS6 on cytotoxicity and gene expression in human mammary epithelial normal cells (HUMEC) and Michigan Cancer Foundation 7 (MCF7), a human mammary epithelial cancer cell line. AS6 selectively arrests cell growth and induces cell death in MCF7 cells without affecting the growth of HUMEC in a dose-dependent manner. AS6 alters the transcription of a large number of genes in MCF7 cells, but much fewer genes in HUMEC. Importantly, we found that the cell proliferation, cell cycle, and DNA repair pathways are significantly suppressed whereas cellular stress response and apoptotic pathways increase in AS6-treated MCF7 cells. Together, we provide the first evidence of differential effects of AS6 on normal and cancerous breast epithelial cells, suggesting that AS6 at moderate concentrations induces cell cycle arrest and apoptosis through modulating genome-wide gene expression, leading to compromised DNA repair and increased genome instability selectively in human breast cancer cells.

RNA polymerase II (Pol II)-dependent transcription in stimulus-inducible genes requires topoisomerase IIβ (TOP2B)-mediated DNA strand break and the activation of DNA damage response signalling in humans. Here, we report a novel function of the breast cancer 1 (BRCA1)-BRCA1-associated ring domain 1 (BARD1) complex in this process. We found that BRCA1 is phosphorylated at S1524 by the kinases ataxia-telangiectasia mutated and ATR during gene activation, and that this event is important for productive transcription. Our biochemical and genomic analyses showed that the BRCA1-BARD1 complex interacts with TOP2B in the EGR1 transcription start site and in a large number of protein-coding genes. Intriguingly, the BRCA1-BARD1 complex ubiquitinates TOP2B, which stabilizes TOP2B binding to DNA while BRCA1 phosphorylation at S1524 controls the TOP2B ubiquitination by the complex. Together, these findings suggest the novel function of the BRCA1-BARD1 complex in the regulation of TOP2B and Pol II-mediated gene expression.

Autophagy is a pivotal biological process responsible for maintaining the homeostasis of intracellular organelles. Yet the molecular intricacies of peroxisomal autophagy (pexophagy) remain largely elusive. From a ubiquitin-related chemical library for screening, we identified several inhibitors of the Von Hippel–Lindau (VHL) E3 ligase, including VH298, thereby serving as potent inducers of pexophagy. In this study, we observed that VH298 stimulates peroxisomal degradation by ATG5 dependently and escalates the ubiquitination of the peroxisomal membrane protein ABCD3. Interestingly, the ablation of NBR1 is similar to the curtailed peroxisomal degradation in VH298-treated cells. We also found that the pexophagy induced by VH298 is impeded upon the suppression of gene expression by the translation inhibitor cycloheximide. Beyond VHL inhibition, we discovered that roxadustat, a direct inhibitor of HIF-α prolyl hydroxylase, is also a potent inducer of pexophagy. Furthermore, we found that VH298-mediated pexophagy is blocked by silencing HIF-1α. In conclusion, our findings suggest that VH298 promotes pexophagy by modulating VHL-mediated HIF-α transcriptional activity.

A specialized membrane-bound organelle, named the melanosome, is central to the storage and transport of melanin as well as melanin synthesis in melanocytes. Although previous studies have linked melanosomal degradation to autophagy, the precise mechanisms remain elusive. Autophagy, a complex catabolic process involving autophagosomes and lysosomes, plays a vital role in cellular constituent degradation. In this study, the role of autophagy in melanosomal degradation was explored, employing a cell-based screening system designed to unveil key pathway regulators. We identified specific dipeptidyl peptidase-4 inhibitors, such as teneligliptin hydrobromide and retagliptin phosphate, as novel agents inducing melanophagy through a comprehensive screening of a ubiquitination-related chemical library. We found that treatment with teneligliptin hydrobromide or retagliptin phosphate not only diminishes melanin content elevated by alpha-melanocyte-stimulating hormone (α-MSH) but also triggers autophagy activation within B16F1 cells. In addition, the targeted inhibition of unc-51-like kinase (ULK1) significantly attenuated both the anti-pigmentation effects and autophagy induced by teneligliptin hydrobromide and retagliptin phosphate in α-MSH-treated cells. Collectively, our data demonstrate a new frontier in understanding melanosomal degradation, identifying teneligliptin hydrobromide and retagliptin phosphate as promising inducers of melanophagy via autophagy activation. This study contributes essential insights into cellular degradation mechanisms and offers potential therapeutic avenues in the regulation of pigmentation.

Abstract Inflammatory bowel disease (IBD) is a chronic inflammatory condition that is influenced by various factors, including environmental factors, immune responses, and genetic elements. Among the factors that influence IBD progression, macrophages play a significant role in generating inflammatory mediators, and an increase in the number of activated macrophages contributes to cellular damage, thereby exacerbating the overall inflammatory conditions. HSPA9, a member of the heat shock protein 70 family, plays a crucial role in regulating mitochondrial processes and responding to oxidative stress. HSPA9 deficiency disrupts mitochondrial dynamics, increasing mitochondrial fission and the production of reactive oxygen species. Based on the known functions of HSPA9, we considered the possibility that HSPA9 reduction may contribute to the exacerbation of colitis and investigated its relevance. In a dextran sodium sulfate-induced colitis mouse model, the downregulated HSPA9 exacerbates colitis symptoms, including increased immune cell infiltration, elevated proinflammatory cytokines, decreased tight junctions, and altered macrophage polarization. Moreover, along with the increased mitochondrial fission, we found that the reduction in HSPA9 significantly affected the superoxide dismutase 1 levels and contributed to cellular death. These findings enhance our understanding of the intricate mechanisms underlying colitis and contribute to the development of novel therapeutic approaches for this challenging condition.

Hypoxic responses have been implicated in critical pathologies, including inflammation, immunity, and tumorigenesis. Recently, efforts to identify effective natural remedies and health supplements are increasing. Previous studies have reported that the cell lysates and the cell wall-bound lipoteichoic acids of Lactiplantibacillus plantarum K8 (K8) exert anti-inflammatory and immunomodulative effects. However, the effect of K8 on cellular hypoxic responses remains unknown. In this study, we found that K8 lysates had a potent suppressive effect on gene expression under hypoxia. K8 lysates markedly downregulated hypoxia-induced HIF1α accumulation in the human bone marrow and lung cancer cell lines, SH-SY5Y and H460. Consequently, the transcription of known HIF1α target genes, such as p21, GLUT1, and ALDOC, was notably suppressed in the K8 lysate supplement and purified lipoteichoic acids of K8, upon hypoxic induction. Intriguingly, K8 lysates decreased the expression of PHD2 and VHL proteins, which are responsible for HIF1α destabilization under normoxic conditions, suggesting that K8 may regulate HIF1α stability in a non-canonical pathway. Overall, our results suggest that K8 lysates desensitize the cells to hypoxic stresses and suppress HIF1α-mediated hypoxic gene activation.

Long intergenic non-coding RNA 346 (LINC00346) has been reported to be involved in the development of atherosclerosis and specific cancers by affecting signaling pathways. However, its function in inflammation has not been thoroughly studied. Therefore, its expression pattern and function were determined in the human macrophage-like cell line THP-1. Lipopolysaccharide (LPS) treatment induced the expression of LINC00346. LPS-induced NF-κB activation and proinflammatory cytokine expression were suppressed or enhanced by the overexpression or knockdown of LINC00346, respectively. Analyses using dual luciferase assay and decoy RNAs that could block RNA-RNA interactions indicated that LINC00346 improves phosphatase and tensin homolog (PTEN) expression by sponging miR-25-3p. Subsequently, PTEN suppresses phosphoinositide-3 kinase (PI3K)-mediated conversion of phosphatidylinositol-4,5-bisphosphate (PIP2) into phosphatidylinositol-3,4,5-trisphosphate (PIP3) as well as consequent activation of protein kinase B (AKT) and NF-κB. Interestingly, database analysis revealed that the expression levels of LINC00346 and PTEN were simultaneously decreased in breast cancer tissues. Further analyses conducted using a breast cancer cell line, MDA-MB-231, confirmed the functional relationship among LINC00346, miR-25-3p, and PTEN in LPS-induced activation of NF-κB. These results indicate that miR-25-3p-sponging activity of LINC00346 affects the balance between PTEN and PI3K as well as the downstream activation of AKT/NF-κB pathway in inflammatory conditions.

<title>Abstract</title> NS1 binding protein (NS1-BP), a non-structural NS1-binding protein of influenza A virus, regulates viral or host RNA processing/export, cancer progression, or neurite/dendritic spine regulation. However, its precise roles in stress-induced responses without viral infection are largely unknown. Therefore, this study aims to investigate the novel roles of NS1-BP, which interact with GABARAP subfamily proteins, including LC3-interacting region-containing proteins, in regulating stress granules (SGs) during oxidative stress. NS1-BP interacts with core SG components and localizes to GABARAP-containing SGs during oxidative stress. Moreover, it associates with p62, acting as an adaptor for selective autophagy via its Kelch-motif and ubiquitin-associated domain in p62 in a stress-dependent manner. NS1-BP knockout (KO) HeLa cells demonstrated altered SG dynamics, mirroring observation in p62 KO or GABARAP triple KO cells, indicating impaired autophagic SG degradation. NS1-BP KO cells, compared to wild-type (WT) cells, showed increased p62 ubiquitination, leading to autophagic p62 degradation, while NS1-BP overexpression reduces p62 ubiquitination. In NS1-BP KO cells, overexpression of p62 WT, not p62 K420R or K435R, restored SGs size and number. Additionally, amyotrophic lateral sclerosis (ALS)-induced pluripotent stem cell-derived motor neurons showed reduced NS1-BP levels, resulting in SG morphology dysregulation. Our findings reveal the novel role of NS1-BP in negatively regulating p62 ubiquitination, influencing SG dynamics and clearance during oxidative stress. This highlights its relevance to ALS pathogenesis associated with SGs.

Primary cilia are crucial for cellular balance, serving as sensors for external conditions.Nephronophthisis and related ciliopathies, which are hereditary and degenerative, stem from genetic mutations in cilia-related genes.However, the precise mechanisms of these conditions are still not fully understood.Our research demonstrates that downregulating PDIA6, leading to cilia removal, makes cells more sensitive to ferroptotic death caused by endoplasmic reticulum (ER) stress.The reduction of PDIA6 intensifies the ER stress response, while also impairing the regulation of primary cilia in various cell types.PDIA6 loss worsens ER stress, hastening ferroptotic death in proximal tubule epithelial cells, HK2 cells.Counteracting this ER stress can mitigate PDIA6 depletion effects, restoring both the number and length of cilia.Moreover, preventing ferroptosis corrects the disrupted primary ciliogenesis due to PDIA6 depletion in HK2 cells.Our findings emphasize the role of PDIA6 in primary ciliogenesis, and suggest its absence enhances ER stress and ferroptosis.These insights offer new therapeutic avenues for treating nephronophthisis and similar ciliopathies.BMB Rep. www.bmbreports.org

The actin-based cytoskeleton is considered a fundamental driving force for cell differentiation and development. Destrin (dstn), a member of the actin depolymerizing factor family, regulates actin dynamics by treadmilling actin filaments and increasing globular actin pools. However, the specific developmental roles of dstn have yet to be fully elucidated. Here, we investigated the physiological functions of dstn during early embryonic development using Xenopus laevis as an experimental model organism. dstn is expressed in anterior neural tissue and neural plate during Xenopus embryogenesis. Depleting dstn promoted morphants with short body axes and small heads. Moreover, dstn inhibition extended the neural plate region, impairing cell migration and distribution during neurulation. In addition to the neural plate, dstn knockdown perturbed neural crest cell migration. Our data suggest new insights for understanding the roles of actin dynamics in embryonic neural development, simultaneously presenting a new challenge for studying the complex networks governing cell migration involving actin dynamics.

Chemotherapies for colon cancer have recently advanced. However, there is still a need to develop agents and identify effective regimens for better treatments of colon cancer. Histone deacetylase inhibitors (HDACIs) have shown potential as anti-cancer agents. We investigated the anti-tumor effects of CG2 (an HDACI) in combination with irinotecan, 5-FU, or oxaliplatin. Combinations of CG2 with SN38 (the active form of irinotecan), 5FU, or oxaliplatin were more effective than the agents alone when used to inhibit the growth of HCT116 cells. The protein expressions of acetyl-H3, p21, caspase-3, -8, and -9, PARP, and XIAP were affected in a time- and dose-dependent manner in HCT116 cells treated with the CG2 alone or combined CG2 and SN-38. In HCT116 xenografts, the HDACI CG2 in combination with irinotecan dramatically inhibited tumor growth without showing additive toxicity. These data indicate that CG2 together with irinotecan is a promising combination novel treatment for colon cancer.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family with apoptosis-inducing activity. Given that TRAIL selectively induces cell death in various tumors but has little or no toxicity to normal cells, TRAIL agonists have been considered as promising anti-cancer therapeutic agents. However, the resistance of many primary tumors and cancer cells to TRAIL poses a challenge. In our present study, we found that carnitine, a metabolite that transfers long-chain fatty acids into mitochondria for beta-oxidation and modulates protein kinase C activity, sensitizes TRAIL-resistant cancer cells to TRAIL. Combination of carnitine and TRAIL was found to synergistically induce apoptotic cell death through caspase activation, which was blocked by a pan caspase inhibitor, but not by an inhibitor of autophagy or an inhibitor of necrosis. The combination of carnitine and TRAIL reversed the resistance to TRAIL in lung cancer cells, colon carcinoma cells, and breast carcinoma cells. We further demonstrate that carnitine, either alone or in combination with TRAIL, enhances the expression of the pro-apoptotic Bcl-2 family protein, Bcl-2-associated X protein (Bax). The down-regulation of Bax expression by small interfering RNA reduced caspase activation when cells were treated with TRAIL, and experiments with cells from Bax knockout mice confirmed this result. Taken together, our current results suggest that carnitine can reverse the resistance of cancer cells to TRAIL by up-regulating Bax expression. Thus, a combined delivery of carnitine and TRAIL may represent a new therapeutic strategy to treat TRAIL-resistant cancer cells.

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) induces apoptosis selectively in cancer cells without affecting the majority of normal human cells. However, hepatocellular carcinoma (HCC) cells often display resistance to TRAIL-induced apoptosis. Ibulocydine (IB) is an isobutyrate ester pro-drug of a novel synthetic Cdk inhibitor that targets Cdk7 and Cdk9. In this study, we show that treatment with subtoxic doses of IB in combination with TRAIL displays potent cytotoxicity in TRAIL-resistant human HCC cells. Combination of IB and TRAIL was found to synergistically induce apoptosis through activation of caspases, which was blocked by a pan-caspase inhibitor (zVAD). Although the expression of Mcl-1 and survivin were reduced by IB plus TRAIL, overexpression of Mcl-1 and survivin did not block the cell death induced by co-treatment. Moreover, overexpression of Bcl-xL did not significantly interfere with the cell death induced by co-treatment of IB and TRAIL. Interestingly, the combination treatment induced cleavage of Bax, which was translocated to mitochondria upon induction of apoptosis. Furthermore, down-regulation of Bax by small interfering RNA effectively reduced the cell death and loss of mitochondrial membrane potential (MMP) caused by co-treatment with IB and TRAIL. Finally, pre-treatment of HCC cells with a calpain inhibitor effectively blocked IB plus TRAIL-induced cleavage of Bax and apoptosis. Collectively, our results demonstrate that IB increases the sensitivity of human HCC cells to TRAIL via mitochondria signaling pathway mediated by calpain-induced cleavage of Bax, suggesting that combined treatment with IB and TRAIL may offer an effective therapeutic strategy for human HCC.

The ultraviolent irradiation resistance-associated gene (UVRAG), a component of the Beclin 1/autophagy-related 6 complex, regulates the autophagy initiation step and functions in the DNA-damage response. UVRAG is frequently mutated in various cancer types, and mutations of UVRAG increase sensitivity to chemotherapy by impairing DNA-damage repair. In this study, we addressed the epigenetic regulation of UVRAG in colorectal cancer cells. UVRAG expression was increased in cells treated with histone deacetylase (HDAC) inhibitors, such as valproic acid and suberoylanilide hydroxamic acid. Down-regulation of HDAC1 enhanced UVRAG expression in colorectal cancer cells. In addition, both chemical and genetic inhibition of HDAC1 reduced the activation of caspase-3 and cytotoxicity in 5-fluorouracil (5FU)-treated cancer cells. In contrast, UVRAG overexpression inhibited caspase activation and cell death in 5FU-treated cells. Taken together, our findings suggest that up-regulation of UVRAG by HDAC1 inhibition potentiates DNA-damage–mediated cell death in colorectal cancer cells.

Previously, we showed that BIX-01294 treatment strongly activates autophagy. Although, the interplay between autophagy and ciliogenesis has been suggested, the role of autophagy in ciliogenesis is controversial and largely unknown. In this study, we investigated the effects of autophagy induced by BIX-01294 on the formation of primary cilia in human retinal pigmented epithelial (RPE) cells. Treatment of RPE cells with BIX-01294 caused strong elongation of the primary cilium and increased the number of ciliated cells, as well as autophagy activation. The elongated cilia in serum starved cultured cells were gradually decreased by re-feeding the cells with normal growth medium. However, the disassembly of cilia was blocked in the BIX-01294-treated cells. In addition, both genetic and chemical inhibition of autophagy suppressed BIX-01294-mediated ciliogenesis in RPE cells. Taken together, these results suggest that autophagy induced by BIX-01294 positively regulates the elongation of primary cilium.

Background and purpose Ibulocydine (IB), a novel prodrug of CDK inhibitor, has been reported to have anti-cancer effect in human hepatoma cells. In order to address its feasibility as a radiosensitizer to improve radiotherapeutic efficacy for human cancers, this study was designed. Material and methods Human cancer cells of lung and colon were treated with IB and/or radiotherapy (RT). The cellular effects were assessed by CCK-8, clonogenic, flow cytometric, and western blotting assays. In vivo radiotherapeutic efficacy was evaluated using the xenograft mouse model. Results Combined treatment of IB and RT significantly reduced viability and survival fraction of the cells. Apoptotic cell death accompanied with activation of caspases, decrease in Bcl-2/Bax expression, loss of mitochondrial membrane potential (MMP) leading to release of cytochrome c into cytosol was observed. Recovery of Bcl-2 expression level by introducing Bcl-2 expressing plasmid DNA compromised the loss of MMP and apoptosis induced by IB and RT. In vivo therapeutic efficacy of combined treatment was verified in the xenograft mouse model, in which tumor growth was markedly delayed by RT with IB. Conclusions IB demonstrated the property of sensitizing human cancer cells to RT by induction of mitochondria-mediated apoptosis, suggesting that IB deserves to be applied for chemoradiotherapy.

This article has been corrected. See ERRATUM: Prognostic Implications of Extranodal Extension in Relation to Colorectal Cancer Location.

Many non-coding RNAs (ncRNAs) serve as regulatory molecules in various physiological pathways, including gene expression in mammalian cells. Distinct from protein-coding RNA expression, ncRNA expression is regulated solely by transcription and RNA processing/stability. It is thus important to understand transcriptional regulation in ncRNA genes but is yet to be known completely. Previously, we identified that a subset of mammalian ncRNA genes is transcriptionally regulated by RNA polymerase II (Pol II) promoter-proximal pausing and in a tissue-specific manner. In this study, human ncRNA genes that are expressed in the early G1 phase, termed immediate early ncRNA genes, were monitored to assess the function of positive transcription elongation factor b (P-TEFb), a master Pol II pausing regulator for protein-coding genes, in ncRNA transcription. Our findings indicate that the expression of many ncRNA genes is induced in the G0-G1 transition and regulated by P-TEFb. Interestingly, a biphasic characteristic of P-TEFb-dependent transcription of serum responsive ncRNA genes was observed: Pol II carboxyl-terminal domain phosphorylated at serine 2 (S2) was largely increased in the transcription start site (TSS, -300 to +300) whereas overall, it was decreased in the gene body (GB, > +350) upon chemical inhibition of P-TEFb. In addition, the three representative, immediate early ncRNAs, whose expression is dependent on P-TEFb, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), nuclear enriched abundant transcript 1 (NEAT1), and X-inactive specific transcript (XIST), were further analyzed for determining P-TEFb association. Taken together, our data suggest that transcriptional activation of many human ncRNAs utilizes the pausing and releasing of Pol II, and that the regulatory mechanism of transcriptional elongation in these genes requires the function of P-TEFb. Furthermore, we propose that ncRNA and mRNA transcription are regulated by similar mechanisms while P-TEFb inhibition unexpectedly increases S2 Pol II phosphorylation in the TSSs in many ncRNA genes. One Sentence Summary: P-TEFb regulates Pol II phosphorylation for transcriptional activation in many stimulus-inducible ncRNA genes.

Colorectal cancer (CRC) is the leading cause of cancer mortality worldwide. Its poor prognosis can be ascribed primarily to high recurrence rates. Accordingly, the aim of this study was to identify novel prognostic biomarkers and therapeutic targets for management of CRC.To develop prognostic biomarkers, we performed RNA-seq analysis and real-time RT-PCR in primary cancer tissues with or without systemic recurrence. To characterize the molecular functions of the encoded proteins, CRC cells underexpressing or overexpressing the candidate genes were established and appropriate cell-based assays were applied.ITGB1 and RHOC mRNA levels were up-regulated in the recurrence group of CRC patients. Overexpression of ITGB1 or RHOC stimulated CRC cell proliferation, invasion and migration, whereas the opposite effects were observed in cells underexpressing either protein. Five-year recurrence-free survival rates were significantly higher in the ITGB1- and RHOC-underexpression groups than those in the overexpression.ITGB1 and RHOC are potential predictors of recurrence and therapeutic targets for CRC, possibly predicting a high-risk group of stage II patients.