Anna‐Mart Engelbrecht

Doxorubicin is currently the most effective chemotherapeutic drug used to treat breast cancer. It has, however, been shown that doxorubicin can induce drug resistance resulting in poor patient prognosis and survival. Studies reported that the interaction between signalling pathways can promote drug resistance through the induction of proliferation, cell cycle progression and prevention of apoptosis. The aim of this study was therefore to determine the effects of doxorubicin on apoptosis signalling, autophagy, the mitogen-activated protein kinase (MAPK)- and phosphoinositide 3-kinase (PI3K)/Akt signalling pathway, cell cycle control, and regulators of the epithelial-mesenchymal transition (EMT) process in murine breast cancer tumours. A tumour-bearing mouse model was established by injecting murine E0771 breast cancer cells, suspended in Hank’s Balances Salt Solution and Corning® Matrigel® Basement Membrane Matrix, into female C57BL/6 mice. Fourty-seven mice were randomly divided into three groups, namely tumour control (received Hank’s Balances Salt Solution), low dose doxorubicin (received total of 6 mg/ml doxorubicin) and high dose doxorubicin (received total of 15 mg/ml doxorubicin) groups. A higher tumour growth rate was, however, observed in doxorubicin-treated mice compared to the untreated controls. We therefore compared the expression levels of markers involved in cell death and survival signalling pathways, by means of western blotting and fluorescence-based immunohistochemistry. Doxorubicin failed to induce cell death, by means of apoptosis or autophagy, and cell cycle arrest, indicating the occurrence of drug resistance and uncontrolled proliferation. Activation of the MAPK/ extracellular-signal-regulated kinase (ERK) pathway contributed to the resistance observed in treated mice, while no significant changes were found with the PI3K/Akt pathway and other MAPK pathways. Significant changes were also observed in cell cycle p21 and DNA replication minichromosome maintenance 2 proteins. No significant changes in EMT markers were observed after doxorubicin treatment. Our results suggest that doxorubicin-induced drug resistance and tumour growth can occur through the adaptive role of the MAPK/ERK pathway in an effort to protect tumour cells. Previous studies have shown that the efficacy of doxorubicin can be improved by inhibition of the ERK signalling pathway and thereby treatment failure can be overcome.

Anthracyclines, such as doxorubicin, are among the most valuable treatments for various cancers, but their clinical use is limited due to detrimental side effects such as cardiotoxicity. Doxorubicin-induced cardiotoxicity is emerging as a critical issue among cancer survivors and is an area of much significance to the field of cardio-oncology. Abnormalities in mitochondrial functions such as defects in the respiratory chain, decreased adenosine triphosphate production, mitochondrial DNA damage, modulation of mitochondrial sirtuin activity and free radical formation have all been suggested as the primary causative factors in the pathogenesis of doxorubicin-induced cardiotoxicity. Melatonin is a potent antioxidant, is nontoxic, and has been shown to influence mitochondrial homeostasis and function. Although a number of studies support the mitochondrial protective role of melatonin, the exact mechanisms by which melatonin confers mitochondrial protection in the context of doxorubicin-induced cardiotoxicity remain to be elucidated. This review focuses on the role of melatonin on doxorubicin-induced bioenergetic failure, free radical generation, and cell death. A further aim is to highlight other mitochondrial parameters such as mitophagy, autophagy, mitochondrial fission and fusion, and mitochondrial sirtuin activity, which lack evidence to support the role of melatonin in the context of cardiotoxicity.

Impaired autophagic machinery is implicated in a number of diseases such as heart disease, neurodegeneration and cancer. A common denominator in these pathologies is a dysregulation of autophagy that has been linked to a change in susceptibility to cell death. Although we have progressed in understanding the molecular machinery and regulation of the autophagic pathway, many unanswered questions remain. How does the metabolic contribution of autophagy connect with the cell's history and how does its current autophagic flux affect metabolic status and susceptibility to undergo cell death? How does autophagic flux operate to switch metabolic direction and what are the underlying mechanisms in metabolite and energetic sensing, metabolite substrate provision and metabolic integration during the cellular stress response? In this article we focus on unresolved questions that address issues around the role of autophagy in sensing the energetic environment and its role in actively generating metabolite substrates. We attempt to provide answers by explaining how and when a change in autophagic pathway activity such as primary stress response is able to affect cell viability and when not. By addressing the dynamic metabolic relationship between autophagy, apoptosis and necrosis we provide a new perspective on the parameters that connect autophagic activity, severity of injury and cellular history in a logical manner. Last, by evaluating the cell's condition and autophagic activity in a clear context of regulatory parameters in the intra- and extracellular environment, this review provides new concepts that set autophagy into an energetic feedback loop, that may assist in our understanding of autophagy in maintaining healthy cells or when it controls the threshold between cell death and cell survival.

Pro-inflammatory and stress-activated signalling pathways are important role players in the pathogenesis of obesity and insulin resistance. Obesity and type II diabetes are associated with chronic, low-grade inflammation and elevated tumour necrosis factor-α (TNF-α) levels. There is increasing evidence that TNF-α may play a critical role in skeletal muscle atrophy. However, the effects of obesity-induced insulin resistance on these signalling pathways are poorly understood in skeletal muscle. Therefore, the present study addressed the effects of obesity-induced insulin resistance on the activity of the ubiquitin ligases, nuclear factor-B, p38 MAPK and phosphoinositide 3-kinase signalling pathways in the gastrocnemius muscle and compared these with muscle of standard chow-fed control rats. Male Wistar rats were randomly allocated to a control diet group (standard commercial chow; 60% carbohydrates, 30% protein and 10% fat) or a cafeteria diet group (65% carbohydrates, 19% protein and 16% fat) for 16 weeks. Blood analysis was conducted to determine the impact of the model of obesity on circulating insulin, glucose, free fatty acids, TNF-α and angiotensin II concentrations. The experimental animals were 18% heavier and had 68% greater visceral fat mass than their control counterparts and were dyslipidaemic. Significant increases in the ubiquitin ligase and MuRF-1, as well as in caspase-3 and poly-ADP-ribose polymerase cleavage were observed in the muscle of obese animals compared with the control rats. We propose that dyslipidaemia may be a mechanism for the activation of inflammatory/stress-activated signalling pathways in obesity and type II diabetes, which will lead to apoptosis and atrophy in skeletal muscle.

This study evaluated whether the manipulation of autophagy could attenuate the cardiotoxic effects of doxorubicin (DXR) in vitro as well as in a tumour-bearing mouse model of acute doxorubicin-induced cardiotoxicity. We examined the effect of an increase or inhibition of autophagy in combination with DXR on apoptosis, reactive oxygen species (ROS) production and mitochondrial function. H9C2 rat cardiac myoblasts were pre-treated with bafilomycin A1 (autophagy inhibitor, 10 nM) or rapamycin (autophagy inducer, 50 μM) followed by DXR treatment (3 μM). The augmentation of autophagy with rapamycin in the presence of DXR substantially ameliorated the detrimental effects induced by DXR. This combination treatment demonstrated improved cell viability, decreased apoptosis and ROS production and enhanced mitochondrial function. To corroborate these findings, GFP-LC3 mice were inoculated with a mouse breast cancer cell line (EO771). Following the appearance of tumours, animals were either treated with one injection of rapamycin (4 mg/kg) followed by two injections of DXR (10 mg/kg). Mice were then sacrificed and their hearts rapidly excised and utilized for biochemical and histological analyses. The combination treatment, rather than the combinants alone, conferred a cardioprotective effect. These hearts expressed down-regulation of the pro-apoptotic protein caspase-3 and cardiomyocyte cross-sectional area was preserved. These results strongly indicate that the co-treatment strategy with rapamycin can attenuate the cardiotoxic effects of DXR in a tumour-bearing mouse model.

The nucleoprotein AHNAK is an unusual and somewhat mysterious scaffolding protein characterised by its large size of approximately 700 kDa. Several aspects of this protein remain uncertain, including its exact molecular function and regulation on both the gene and protein levels. Various studies have attempted to annotate AHNAK and, notably, protein interaction and expression analyses have contributed greatly to our current understanding of the protein. The implicated biological processes are, however, very diverse, ranging from a role in the formation of the blood–brain barrier, cell architecture and migration, to the regulation of cardiac calcium channels and muscle membrane repair. In addition, recent evidence suggests that AHNAK might be yet another accomplice in the development of tumour metastasis. This review will discuss the different functional roles of AHNAK, highlighting recent advancements that have added foundation to the proposed roles while identifying ties between them. Implications for related fields of research are noted and suggestions for future research that will assist in unravelling the function of AHNAK are offered.

Complex associations exist between inflammation and thrombosis, with the inflammatory state tending to promote coagulation. Fibrinogen, an acute phase protein, has been shown to interact with the amyloidogenic ß-amyloid protein of Alzheimer's disease. However, little is known about the association between fibrinogen and serum amyloid A (SAA), a highly fibrillogenic protein that is one of the most dramatically changing acute phase reactants in the circulation. To study the role of SAA in coagulation and thrombosis, in vitro experiments were performed where purified human SAA, in concentrations resembling a modest acute phase response, was added to platelet-poor plasma (PPP) and whole blood (WB), as well as purified and fluorescently labelled fibrinogen. Results from thromboelastography (TEG) suggest that SAA causes atypical coagulation with a fibrin(ogen)-mediated increase in coagulation, but a decreased platelet/fibrin(ogen) interaction. In WB scanning electron microscopy analysis, SAA mediated red blood cell (RBC) agglutination, platelet activation and clumping, but not platelet spreading. Following clot formation in PPP, the presence of SAA increased amyloid formation of fibrin(ogen) as determined both with auto-fluorescence and with fluorogenic amyloid markers, under confocal microcopy. SAA also binds to fibrinogen, as determined with a fluorescent-labelled SAA antibody and correlative light electron microscopy (CLEM). The data presented here indicate that SAA can affect coagulation by inducing amyloid formation in fibrin(ogen), as well as by propelling platelets to a more prothrombotic state. The discovery of these multiple and complex effects of SAA on coagulation invite further mechanistic analyses.

Insulin plays an indispensable role in the management of hyperglycaemia that arises in a variety of settings, including Type I and II diabetes, gestational diabetes, as well as is in hyperglycaemia following a severe inflammatory insult. However, insulin receptors are also expressed on a range of cells that are not canonically implicated in glucose homeostasis. This includes immune cells, where the anti-inflammatory effects of insulin have been repeatedly reported. However, recent findings have also implicated a more involved role for insulin in shaping the immune response during an infection. This includes the ability of insulin to modulate immune cell differentiation and polarisation as well as the modulation of effector functions such as biocidal ROS production. Finally, inflammatory mediators can through both direct and indirect mechanisms also regulate serum insulin levels, suggesting that insulin may be co-opted by the immune system during an infection to direct immunological operations. Collectively, these observations implicate insulin as a bona fide immune-modulating hormone and suggest that a better understanding of insulin’s immunological function may aid in optimising insulin therapy in a range of clinical settings.

The incidence of breast cancer is often associated with geographic variation which indicates that a person's surrounding environment can be an important etiological factor in cancer development. Environmental risk factors can include exposure to sewage- or wastewater, which consist of a complex mixture of pathogens, mutagens and carcinogens. Wastewater contains primarily carbonaceous, nitrogenous and phosphorus compounds, however it can also contain trace amounts of chemical pollutants including toxic metal cations, hydrocarbons and pesticides. More importantly, the contamination of drinking water by wastewater is a potential source of exposure to mammary carcinogens and endocrine disrupting compounds. Organic solvents and other pollutants often found in wastewater have been detected in various tissues, including breast and adipose tissues. Furthermore, these pollutants such as phenolic compounds in some detergents and plastics, as well as parabens and pesticides can mimic estrogen. High estrogen levels are a well-established risk factor for estrogen-receptor (ER) positive breast cancer. Therefore, exposure to wastewater is a risk factor for the initiation, progression and metastasis of breast cancer. Carcinogens present in wastewater can promote tumourigenesis through various mechanisms, including the formation of DNA adducts, gene mutations and oxidative stress. Lastly, the presence of endocrine disrupting compounds in wastewater can have negative implications for ER-positive breast cancers, where these molecules can activate ERα to promote cell proliferation, survival and metastasis. As such, strategies should be implemented to limit exposure, such as providing funding into treatment technologies and implementation of regulations that limit the production and use of these potentially harmful chemicals.

The aim of this investigation was to evaluate the chemopreventative/antiproliferative potential of a grape seed proanthocyanidin extract (GSPE) against colon cancer cells (CaCo2 cells) and to investigate its mechanism of action. GSPE (10–100 μg/ml) significantly inhibited cell viability and increased apoptosis in CaCo2 cells, but did not alter viability in the normal colon cell line (NCM460). The increased apoptosis observed in GSPE-treated CaCo2 cells correlated with an attenuation of PI3-kinase (p110 and p85 subunits) and decreased PKB Ser473 phosphorylation. GSPE might thus exert its beneficial effects by means of increased apoptosis and suppression of the important PI3-kinase survival-related pathway.

Ischemic cell injury leads to cell death. Three main morphologies have been described: apoptosis, cell death with autophagy and necrosis. Their inherent dynamic nature, a point of no return (PONR) and molecular overlap have been stressed. The relationship between a defined cell death type and the severity of injury remains unclear. The functional role of autophagy and its effects on cell death onset is largely unknown. In this study we report a differential induction of cell death, which is dependent on the severity and duration of an ischemic insult. We show that mild ischemia leads to the induction of autophagy and apoptosis, while moderate or severe ischemia induces both apoptotic and necrotic cell death without increased autophagy. The autophagic response during mild injury was associated with an ATP surge. Real-time imaging and Fluorescence Resonance Energy Transfer (FRET) revealed that increased autophagy delays the PONR of both apoptosis and necrosis significantly. Blocking autophagy shifted PONR to an earlier point in time. Our results suggest that autophagic activity directly alters intracellular metabolic parameters, responsible for maintaining mitochondrial membrane potential and cellular membrane integrity. A similar treatment also improved functional recovery in the perfused rat heart. Taken together, we demonstrate a novel finding: autophagy is implicated only in mild injury and positions the PONR in cell death.

Muscle atrophy poses a serious concern to patients inflicted with inflammatory diseases. An increasing body of evidence implies that TNF-α plays a critical role in muscle atrophy in a number of these clinical settings. The mechanisms mediating its effects are not completely understood and conflicting data regarding its anabolic and catabolic actions exists. To examine the functional significance and detailed morphological characteristics of TNF-α-induced muscle proteolysis, differentiated L6 myotubes were subjected to increasing concentrations of recombinant TNF-α for 24 and 48 h. Data analysis of cell death showed that TNF-α induced a combination of apoptosis and necrosis in high concentrations. TNF-R1, rather than TNF-R2, was significantly upregulated. In addition, the transcription factors, NF-κB and FKHR were rapidly activated thus leading to increased expression of ubiquitin ligases, MuRF-1 and MAFbx. Muscle fiber diameter decreased with increasing TNF-α concentrations and was associated with attenuation of the PI3-K/Akt pathway as well as significant reductions in differentiation markers. Furthermore, treatment of L6 myotubes with exogenous TNF-α strongly potentiates its proteolytic effects through certain MAPKs that are activated. These observations suggest that TNF-α induces muscle proteolysis in a dose-dependent manner via various signal transduction pathways.

Numerous studies have shown that long-chain polyunsaturated fatty acids can kill cancer cells in vitro as well as in vivo, while normal cells remain unaffected. Unfortunately, the cellular and molecular mechanisms responsible for this phenomenon are still poorly understood. The aim of this study was to investigate the potential chemopreventative/antiproliferative potential of docosahexaenoic acid (DHA) in an adenocarcinoma cell line (CaCo2 cells) and to evaluate the signalling pathways modulated by it. DHA (5-50 μM) significantly inhibited cell viability in a dose-dependent manner in CaCo2 cells, while the viability of normal colon cells (NCM460 cells) was not compromised. DHA also induced apoptosis in CaCo2 cells, as indicated by increases in caspase-3 activation and poly-ADP-ribose polymerase cleavage. Signalling proteins, which include extracellular signal-regulated kinase, p38 mitogen-activated protein kinase (MAPK), Akt and p53 were analysed by Western blotting using phosphospecific and total antibodies. The protein inhibitors wortmannin (phosphoinositide 3 kinase inhibitor), PD 98059 (MEK inhibitor) and SB 203580 (p38 inhibitor) as well as silencing RNA [small interfering RNA (siRNA)] of the p38 MAPK protein, were used to investigate cross-talk between signalling pathways. DHA supplementation significantly suppressed Akt phosphorylation, which also correlated with decreased cell viability and increased apoptosis in CaCo2 cells. Furthermore, siRNA experiments suggested a possible role for p38 MAPK in the phosphorylation of p53 at Ser15, a site which is associated with DNA damage. DHA might thus exert its beneficial effects by means of increased apoptosis and suppression of the important survival-related kinase, Akt.

Autophagy, apoptosis and necrosis have previously been described as distinct static processes that induce and execute cell death. Due to an increased use of novel techniques in mapping cellular death-techniques which allow for reporting of real-time data-the existence of "grey zones" between cell death modes and the existence of the "point of no return" within these have been revealed. This revelation demands the integration of new concepts in describing the cellular death process. Furthermore, since the contribution of autophagy in cell death or cell survival is still poorly understood, it is important to accurately describe its function within the dynamic framework of cell death. In this review cell death is viewed as a dynamic and integrative cellular response to ensure the highest likelihood of self-preservation. Suggestions are offered for conceptualizing cell death modes and their morphological features, both individually and in relation to one another. It addresses the need for distinguishing between dying cells and dead cells so as to better locate and control the onset of cell death. Most importantly, the fundamental role of autophagy, autophagic flux, and the effects of the intracellular metabolic environment on the kinetics of the cell death modes are stressed. It also contextualizes the kinetic dimension of cell death as a process and aims to contribute towards a better understanding of autophagy as a key mechanism within this process. Understanding the dynamic nature of the cell death process and autophagy's central role can reveal new insight for therapeutic intervention in preventing cell death.

The cancer stem cell (CSC) model has emerged as a prominent paradigm for explaining tumour heterogeneity. CSCs in tumour recurrence and drug resistance have also been implicated in a number of studies. In fact, CSCs are often identified by their expression of drug‐efflux proteins which are also highly expressed in normal stem cells. Similarly, pro‐survival or proliferation signalling often exhibited by stem cells is regularly reported as being upregulated by CSC. Here we review evidence suggesting that many aspects of CSCs are more readily described by clonal evolution. As an example, cancer cells often exhibit copy number gains of genes involved in drug‐efflux proteins and pro‐survival signalling. Consequently, clonal selection for stem cell traits may result in cancer cells developing “stemness” traits which impart a fitness advantage, without strictly following a CSC model. Finally, since symmetric cell division would give rise to more cells than asymmetric division, it is expected that more advanced tumours would depart from a CSC. Collectively, these observations suggest clonal evolution may explain many aspects of the CSC.

Mitochondrial dysfunction is a central element in the development of doxorubicin (DXR)-induced cardiotoxicity. In this context, melatonin is known to influence mitochondrial homeostasis and function. This study aimed to investigate the effects of melatonin on cardiac function, tumor growth, mitochondrial fission and fusion, PGC1-α and sirtuin activity in an acute model of DXR-induced cardiotoxicity. During the in vitro study, H9c2 rat cardiomyoblasts were pre-treated with melatonin (10 μM, 24 h) followed by DXR exposure (3 μM, 24 h). Following treatment, cellular ATP levels and mitochondrial morphology were assessed. In the in vivo study, female Sprague Dawley rats (16 weeks old), were inoculated with a LA7 rat mammary tumor cell line and tumors were measure daily. Animals were injected with DXR (3 × 4 mg/kg) and/or received melatonin (6 mg/kg) for 14 days in their drinking water. Rat hearts were used to conduct isolated heart perfusions to assess cardiac function and thereafter, heart tissue was used for immunoblot analysis. DXR treatment increased cell death and mitochondrial fission which were reduced with melatonin treatment. Cardiac output increased in rats treated with DXR + melatonin compared to DXR-treated rats. Tumor volumes was significantly reduced in DXR + melatonin-treated rats on Day 8 in comparison to DXR-treated rats. Furthermore, DXR + melatonin treatment increased cellular ATP levels, PGC1-α and SIRT1 expression which was attenuated by DXR treatment. These results indicate that melatonin treatment confers a dual cardio-protective and oncostatic effect by improving mitochondrial function and cardiac function whilst simultaneously retarding tumor growth during DXR-induced cardiotoxicity.

Autophagy plays a major role in the adaptive metabolic response of cancer cells during adverse conditions such as nutrient deprivation. However, specific data that assess metabolite profiles in context with adenosine triphosphate (ATP) availability and cell death susceptibility remain limited. Human breast cancer cells, MDAMB231, and normal breast epithelial cells, MCF12A, were subjected to short‐term amino acid starvation and the cellular apoptotic and autophagic responses assessed. The role of autophagy in the control of cellular amino acid, ATP, free fatty acid, and glucose levels during amino acid starvation were compared. We demonstrate that breast cancer cells have an increased metabolic demand contributing to significant amino acid and ATP depletion in a nutrient‐poor environment. Upregulation of autophagy was important for the generation of amino acids and free fatty acids and maintenance of cellular ATP levels. In contrast to normal cells, breast cancer cells were unable to maintain the response after 12 hours of amino acid starvation. Regulation of autophagic activity in these environments had indirect consequences on cell death susceptibility. Overall, our data provide support for autophagy as an important survival mechanism capable of providing metabolic substrates when cancer cells are faced with nutrient‐deprived environments. Significance of study The results obtained in this study helps to expand our current knowledge on how cells respond to environmental changes; the biochemical and metabolic consequences and the physiological processes activated in response. The environmental stress applied in this study is relevant to tumour physiology, and results can be translated to cancer therapeutic and clinical research areas, ultimately assisting in the specific targeting of cancer cells while avoiding harm to normal cells.

Excess adipose tissue is a hallmark of an overweight and/or obese state as well as a primary risk factor for breast cancer development and progression. In an overweight/obese state adipose tissue becomes dysfunctional due to rapid hypertrophy, hyperplasia, and immune cell infiltration which is associated with sustained low-grade inflammation originating from dysfunctional adipokine synthesis. Evidence also supports the role of excess adipose tissue (overweight/obesity) as a casual factor for the development of chemotherapeutic drug resistance. Obesity-mediated effects/modifications may contribute to chemotherapeutic drug resistance by altering drug pharmacokinetics, inducing chronic inflammation, as well as altering tumor-associated adipocyte adipokine secretion. Adipocytes in the breast tumor microenvironment enhance breast tumor cell survival and decrease the efficacy of chemotherapeutic agents, resulting in chemotherapeutic resistance. A well-know chemotherapeutic agent, doxorubicin, has shown to negatively impact adipose tissue homeostasis, affecting adipose tissue/adipocyte functionality and storage. Here, it is implied that doxorubicin disrupts adipose tissue homeostasis affecting the functionality of adipose tissue/adipocytes. Although evidence on the effects of doxorubicin on adipose tissue/adipocytes under obesogenic conditions are lacking, this narrative review explores the potential role of obesity in breast cancer progression and treatment resistance with inflammation as an underlying mechanism.

Despite remaining one of the most widely abused drugs worldwide, Cannabis sativa exhibits remarkable medicinal properties. The phytocannabinoids, cannabidiol and Δ-9-tetrahydrocannabinol, reduce nausea and vomiting, particularly during chemotherapy. This is attributed to their ability to reduce the release of serotonin from enterochromaffin cells in the small intestine, which would otherwise orchestrate the vomiting reflex. Although there are many preclinical and clinical studies on the effects of Δ-9-tetrahydrocannabinol during nausea and vomiting, little is known about the role that cannabidiol plays in this scenario. Since cannabidiol does not induce psychotropic effects, in contrast to other cannabinoids, its use as an anti-emetic is of great interest. This review aims to summarize the available literature on cannabinoid use, with a specific focus on the nonpsychotropic drug cannabidiol, as well as the roles that cannabinoids play in preventing several other adverse side effects of chemotherapy including organ toxicity, pain and loss of appetite.

Identifying new approaches to lessen inflammation, as well as the associated malignant consequences, remains crucial to improving the lives and prognosis of patients diagnosed with inflammatory bowel diseases. Although it previously has been suggested as a suitable biomarker for monitoring disease activity in patients diagnosed with Crohn's disease, the role of the acute-phase protein serum amyloid A (SAA) in inflammatory bowel disease remains unclear. In this study, we aimed to assess the role of SAA in colitis-associated cancer.We established a model of colitis-associated cancer in wild-type and SAA double-knockout (Saa1/2-/-) mice by following the azoxymethane/dextran sulfate sodium protocol. Disease activity was monitored throughout the study while colon and tumor tissues were harvested for subsequent use in cytokine analyses, Western blot, and immunohistochemistry +experiments.We observed attenuated disease activity in mice deficient for Saa1/2 as evidenced by decreased weight loss, increased stool consistency, decreased rectal bleeding, and decreased colitis-associated tissue damage. Macrophage infiltration, including CD206+ M2-like macrophages, also was attenuated in SAA knockout mice, while levels of interleukin 4, interleukin 10, and tumor necrosis factor-ɑ were decreased in the distal colon. Mice deficient for SAA also showed a decreased tumor burden, and tumors were found to have increased apoptotic activity coupled with decreased expression for markers of proliferation.Based on these findings, we conclude that SAA has an active role in inflammatory bowel disease and that it could serve as a therapeutic target aimed at decreasing chronic inflammation and the associated risk of developing colitis-associated cancer.

Breast cancer is the most frequently diagnosed cancer in women globally. Although there have been many significant advances made in the diagnosis and treatment of breast cancer, numerous unresolved challenges remain, which include prevention, early diagnosis, metastasis and recurrence. The role of inflammation in cancer development is well established and is believed to be one of the leading hallmarks of cancer progression. Recently, the role of the inflammasome, a cytosolic multiprotein complex, has received attention in different cancers. By contributing to the activation of inflammatory cytokines the inflammasome intensifies the inflammatory cascade. The inflammasome can be activated through several pathways, which include the binding of pattern associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs) to toll-like receptors (TLRs). Serum amyloid A (SAA), a non-specific acute-phase protein, can function as an endogenous DAMP by binding to pattern recognition receptors like TLRs on both breast cancer cells and cancer associated fibroblasts (CAFs). SAA can thus stimulate the production of IL-1β, thereby creating a favourable inflammatory environment to support tumour growth. The aim of this review is to highlight the possible role of SAA as an endogenous DAMP in the tumour microenvironment (TME) thereby promoting breast cancer growth through the activation of the NLRP3 inflammasome.

The Covid-19 pandemic has spread rapidly across the globe, resulting in more than 3 million deaths worldwide. The symptoms of Covid-19 are usually mild and non-specific, however in some cases patients may develop acute respiratory distress syndrome (ARDS) and systemic inflammation. Individuals with inflammatory or immunocompromising illnesses, such as cancer, are more susceptible to develop ARDS and have higher rates of mortality. This is mediated through an initial hyperstimulated immune response which results in elevated levels of pro-inflammatory cytokines and a subsequent cytokine storm. This potentiates positive feedback loops which are unable to be balanced by anti-inflammatory mediators. Therefore, elevated levels of IL-1β, as a result of NLRP3 inflammasome activation, as well as IL-6 and TNF-α amongst many others, contribute to the progression of various cancer types. Furthermore, Covid-19 progression is associated with the depletion of CD8+ and CD4+ T cells, B cell and natural killer cell numbers. Collectively, a Covid-19-dependent pro-inflammatory profile and immune suppression promotes the optimal microenvironment for tumourigenesis, initiation and immune evasion of malignant cells, tumour progression and metastasis as well as cancer recurrence. There are, however, therapeutic windows of opportunity that may combat both Covid-19 and cancer to improve patient outcomes.

Anthracycline-induced cardiotoxicity is a clinically complex syndrome that leads to substantial morbidity and mortality for cancer survivors. Despite several years of research, the underlying molecular mechanisms remain largely undefined and thus effective therapies to manage this condition are currently non-existent. This study therefore aimed to determine the contribution of the ubiquitin–proteasome pathway (UPP) and endoplasmic reticulum (ER)-stress within this context. Cardiotoxicity was induced with the use of doxorubicin (DXR) in H9C2 rat cardiomyoblasts (3 μM) for 24 h, whereas the tumour-bearing GFP-LC3 mouse model was treated with a cumulative dose of 20 mg/kg. Markers for proteasome-specific protein degradation were significantly upregulated in both models following DXR treatment, however proteasome activity was lost. Moreover, ER-stress as assessed by increased ER load was considerably augmented (in vitro) with modest binding of DXR with ER. These results suggest that DXR induces intrinsic activation of the UPP and ER stress which ultimately contributes to dysfunction of the myocardium during this phenomenon.

Cisplatin is the main chemotherapeutic drug for the treatment of cervical cancers, however resistance to cisplatin is increasingly common and therefore has limited the efficacy and use of this drug in the clinic. Dose-dependent toxicity poses an additional challenge since patients suffer long-term and often permanent side-effects after treatment. Bcl-2 up-regulation has been implicated in the resistance to cisplatin in a variety of cancer cell lines, however its role in cervical cancer is confounding.A low, non-cytotoxic concentration of cisplatin was used in the treatment of HeLa and CaSki cells. Bcl-2 expression was determined through Western blotting and immunocytochemistry before and after treatment with cisplatin. To assess the reliance of the cervical cancer cells on Bcl-2 in the presence of cisplatin, Bcl-2 knock-down was achieved through RNA interference, where after apoptosis was assessed through PARP cleavage (Western blotting), Caspase activity (Caspase-Glo(©)) and PI inclusion analysis (Flow cytometry). Finally, pre-malignant and malignant cervical tissue was analysed for the presence of Bcl-2 through Western blotting and immunofluorescence.Cervical cancer cells upregulate Bcl-2 when treated with a non-cytotoxic concentration of cisplatin, which when silenced, effectively enhanced cisplatin sensitivity, and therefore significantly induced apoptosis. Analysis of the expression profile of Bcl-2 in cervical tissue revealed its up-regulation in cervical carcinoma, which agrees with results obtained from the in vitro data.Our data strongly suggest that utilising a lower dose of cisplatin is feasible when combined with Bcl-2 silencing as an adjuvant treatment, thereby improving both the dose-dependent toxicity, as well as cervical cancer resistance.

Activation of the immune system is metabolically costly, yet a hallmark of an infection is a reduction in appetite with a subsequent reduction in metabolite provision. What is the functional value of decreasing nutrient intake when an infection imposes large demands on metabolic parameters? Here, we propose that sickness-associated anorexia (SAA) upregulates the ancient process of autophagy systemically, thereby profoundly controlling not only immune- but also nonimmune-competent cells. This allows an advanced impact on the resolution of an infection through direct pathogen killing, enhancement of epitope presentation and the contribution toward the clearance of noxious factors. By rendering a ‘free meal,’ autophagy is thus most fundamentally harnessed during an anorexic response in order to promote both host tolerance and resistance. These findings strongly suggest a reassessment of numerous SAA-related clinical applications and a re-evaluation of current efforts in patient care.

There is an ongoing debate regarding the efficacy of glycaemic control in critically ill patients. Here we briefly highlight the key function of elevated glucose in critically ill patients, namely, to enable elevation of aerobic glycolysis in rapidly dividing cells. In particular, aerobic glycolysis provides metabolic intermediates necessary for expansion of biomass in immune cells and promotion of tissue repair. Furthermore, we emphasise that insulin may inhibit autophagy, a cell survival process used in the bulk degradation of cellular debris and damaged organelles. These observations provide a rational basis for tolerating elevated glucose levels in certain critically ill patients.

The majority of human tumours are comprised of cancerous epithelial cells that coexist with a multitude of different cell types and extracellular matrix components creating the cancer microenvironment. Cancer-associated fibroblasts (CAFs) are the most abundant mesenchymal cell types present within most human carcinomas. Recent evidence suggests that nutrient deprived epithelial cancer cells are able to survive these conditions, as a result of their ability to undergo extensive metabolic reprogramming and exploit the metabolic capacities of surrounding CAFs. Although several studies support the role of CAFs in tumour progression and metastasis, the molecular mechanisms underlying this pro-tumourigenic interaction remains to be elucidated. This review will discuss the complex metabolic interaction that exists between epithelial cancer cells and CAF's: focussing primarily on their functional role in tumour progression, metastasis and chemotherapeutic resistance. Attempts are made at delineating the molecular mechanisms underlying this pro-tumourigenic interaction, and potential CAF-based targets are suggested.

Activation of the immune system is associated with an increase in the breakdown of various peripheral tissues, including bone. Despite the widely appreciated role of inflammatory mediators in promoting bone resorption, the functional value behind this process is not completely understood. Recent advances in the field of immunometabolism have highlighted the metabolic reprogramming that takes place in activated immune cells. It is now believed that the breakdown of peripheral tissue provides metabolic substrates to fuel metabolic anabolism in activated immune cells. We argue that phosphate, liberated by bone resorption, plays an indispensable role in sustaining immune cell metabolism. The liberated phosphate is then incorporated into macromolecules such as nucleotides and phospholipids, and is also used for the phosphorylation of metabolites (e.g. glycolytic intermediates). In addition, magnesium, also liberated during the breakdown of bone, is an essential cofactor required by various metabolic enzymes which are upregulated in activated immune cells. Finally, calcium activates various additional molecules involved in immune cell migration. Taken together, these factors suggest a key role for bone resorption during infection.

Breast cancer cells modulate lipid and fatty acid metabolism to sustain proliferation. The role of adipocytes in cancer treatment efficacy remains, however, to be fully elucidated. We investigated whether diet-induced obesity (DIO) affects the efficacy of doxorubicin treatment in a breast tumour-bearing mouse model. Female C57BL6 mice were fed a high fat or low fat diet for the full duration of the study (12 weeks). After 8 weeks, mice were inoculated with E0771 triple-negative breast cancer cells in the fourth mammary gland to develop breast tumour allographs. Tumour-bearing mice received either vehicle (Hank’s balanced salt solution) or doxorubicin (chemotherapy). Plasma inflammatory markers, tumour and mammary adipose tissue fatty acid composition, as well as protein expression of lipid metabolism markers were determined. The HFD attenuated the treatment efficacy of doxorubicin. Both leptin and resistin concentrations were significantly increased in the HFD group treated with doxorubicin. Suppressed lipogenesis (decreased stearoyl CoA-desaturase-1) and lipolysis (decreased hormone-sensitive lipase) were observed in mammary adipose tissue of the DIO animals, whereas increased expression was observed in the tumour tissue of doxorubicin treated HFD mice. Obesogenic conditions induced altered tissue fatty acid (FA) compositions, which reduced doxorubicin’s treatment efficacy. In mammary adipose tissue breast cancer cells suppressed the storage of FAs, thereby increasing the availability of free FAs and favoured inflammation under obesogenic conditions.

Numerous studies have reported the protective properties of carotenoid supplementation against skin and eye associated diseases. However, conflicting data concerning the efficacy of beta-carotene in the pathogenesis of cancers and cardiovascular disease exist. It has been shown that beta-carotene is an effective antioxidant on its own or in combination with other antioxidants. Red palm oil (RPO) is a potent anti-oxidant rich oil which consists of carotenoids, tocopherols, tocotrienols and lycopenes as well as lipid fractions such as squalene, saturated and unsaturated fatty acids (which maximize absorption of these anti-oxidants) and Co-enzyme Q10. alpha and beta-carotene account for more than 90% of the total carotene in RPO. It is known that ischaemia/reperfusion-induced injury causes an imbalance in oxygen supply which can lead to oxidative stress in the heart. It has been shown that the mitogen-activated protein kinases (MAPKs), PKB/Akt and the NO-cGMP all play vital roles in ischaemia/reperfusion injury in the heart. Therefore, our review mainly focuses on the signaling pathways involved in functional recovery induced by a natural carotenoid oil after ischaemia/reperfusion injury.

Cisplatin is used as a cytotoxic agent for the management of cervical cancer. However, the severity of the side-effects limits the use of this drug, particularly at high doses. Resistance to cisplatin is often attributed to a disruption in the normal apoptotic response via aberrant activation of pathways such as the mTOR pathway. Here we assess the role of mTOR and its effect on cell death sensitization and autophagy in response to a low concentration of cisplatin in cervical cancer cells. Additionally we measured the expression profile of mTOR in normal, low- and high-grade squamous intraepithelial (LSIL and HSIL) lesions and cancerous tissue. An in vitro model of cervical cancer was established using HeLa and CaSki cells. mTOR protein expression as well as autophagy-related proteins were evaluated through Western blotting. Inhibition of mTOR was achieved with the use of rapamycin and RNA silencing. A low concentration of cisplatin administered as a single agent induces autophagy, but not apoptosis. Cisplatin cytotoxicity was greatly enhanced in cancer cells when mTOR had been inhibited prior to cisplatin treatment which was likely due to autophagy being increased above cisplatin-induced levels, thereby inducing apoptosis. Cervical tissue samples revealed an increase in mTOR protein expression in LSIL and carcinoma tissue which suggests a change in autophagy control. Our data suggest that utilising a lower dose of cisplatin combined with mTOR inhibition is a viable treatment option and addresses the challenge of cisplatin dose-dependent toxicity, however future studies are required to confirm this in a clinical setting.

Preclinical studies suggest that fasting prior to chemotherapy may be an effective strategy to protect patients against the adverse effects of chemo-toxicity. Fasting may also sensitize cancer cells to chemotherapy. It is further suggested that fasting may similarly augment the efficacy of oncolytic viral therapy. The primary mechanism mediating these beneficial effects is thought to relate to the fact that fasting results in a decrease of circulating growth factors. In turn, such fasting cues would prompt normal cells to redirect energy towards cell maintenance and repair processes, rather than growth and proliferation. However, fasting is also known to up-regulate autophagy, an evolutionarily conserved catabolic process that is up-regulated in response to various cell stressors. Here we review a number of mechanisms by which fasting-induced autophagy may have an impact on both chemo-tolerance and chemo-sensitization. Firstly, fasting may exert a protective effect by mobilizing autophagic components prior to chemo-induction. In turn, the autophagic apparatus can be repurposed for removing cellular components damaged by chemotherapy. Autophagy also plays a key role in epitope expression as well as in modulating inflammation. Chemo-sensitization resulting from fasting may in fact be an effect of enhanced immune surveillance as a result of better autophagy-dependent epitope processing. Finally, autophagy is involved in host defense against viruses, and aspects of the autophagic process are also often targets for viral subversion. Consequently, altering autophagic flux by fasting may alter viral infectivity. These observations suggest that fasting-induced autophagy may have an impact on therapeutic efficacy in various oncological contexts.

Understanding the response of cancer cells to anti-cancer therapies is crucial to unraveling and preventing the development of therapeutic resistance. The human AHNAK protein is a giant scaffold protein implicated in several diverse cellular functions. The role of AHNAK in cancer is however unclear as the protein has previously been described as a tumor suppressor, as well as being essential for tumor metastasis and invasion, while also being implicated in selected chemotherapeutic responses. To clarify the role of AHNAK in cancer, we investigated the effect of doxorubicin treatment on AHNAK in doxorubicin-sensitive MCF-7 and doxorubicin-resistant MDA-MB-231 breast cancer cell lines, as well as in a tumor-bearing mouse model. The role of AHNAK in the cellular response of breast cancer cells to doxorubicin was also investigated. We report here, for the first time, an association between AHNAK and resistance to doxorubicin. While treatment with doxorubicin modulated AHNAK protein expression both in vitro and in vivo in a dose-dependent manner, no changes in its cellular localization were observed. AHNAK knockdown prevented doxorubicin-induced modulation of cleaved caspase 7 protein expression and cell cycle arrest, while its overexpression decreased cleaved caspase 7 and cleaved PARP levels and induced S-phase arrest, changes that were comparable to the effects of doxorubicin. This novel association was restricted to doxorubicin-resistant cells, implicating the protein in therapeutic resistance. These findings confirm that AHNAK does indeed function in the chemotherapeutic response of breast cancer cells while also emphasizing the need for further investigation into potential implications for AHNAK in terms of predicting and modulating treatment response.

Inflammatory mediators have a well-established role in mediating metabolic disturbances. Chronic low-grade inflammation is implicated in the pathogenesis of obesity and the development of metabolic syndrome. This phenomenon is even more pronounced in severe inflammatory states such as in critically ill patients where hyperglycaemia invariably manifests. Similarly, though inflammatory mediators have a well-established role in promoting bone resorption, the adaptive function of this process remains unknown. Here we review emerging evidence from the field of immunometabolism suggesting that these two processes serve a common goal, namely, to sustain the rapid proliferation of immune cells during an infection. Activated immune cells exhibit an increased demand for glucose which not only provides energy, but also glycolytic intermediates which are fluxed into biosynthetic processes. Similarly, phosphate liberated from bone is consumed during the phosphorylation of glycolytic intermediates, which plays a critical role in the synthesis of nucleotides and phospholipids. Taken together, these considerations suggest that metabolic alterations induced by inflammatory mediators do not manifest as an inability to maintain homeostatic levels of metabolites but represent an adaptive shift in the homeostatic set point during an infection.

A number of studies have implicated cannabinoids as potent anti-inflammatory mediators. However, the exact mechanism by which cannabinoids exert these effects remains to be fully explained. The recent resurgence in interest regarding the metabolic adaptations undergone by activated immune cells has highlighted the intricate connection between metabolism and an inflammatory phenotype. In this regard, evidence suggests that cannabinoids may alter cell metabolism by increasing AMPK activity. In turn, emerging evidence suggests that the activation of AMPK by cannabinoids may mediate an anti-inflammatory effect through a range of processes. First, AMPK may promote oxidative metabolism, which have been shown to play a central role in immune cell polarisation towards a tolerogenic phenotype. AMPK activation may also attenuate anabolic processes which in turn may antagonise immune cell function. Furthermore, AMPK activity promotes the induction of autophagy, which in turn may promote anti-inflammatory effects through various well-described processes. Taken together, these observations implicate cannabinoids to mediate part of their anti-inflammatory effects through alterations in immune cell metabolism and the induction of autophagy.

Globally, breast cancer continues to be a major concern in women's health. Lifestyle related risk factors, specifically excess adipose tissue (adiposity) has reached epidemic proportions and has been identified as a major risk factor in the development of breast cancer. Dysfunctional adipose tissue has evoked research focusing on its association with metabolic-related conditions, breast cancer risk and progression. Adipose dysfunction in coordination with immune cells and inflammation, are responsible for accelerated cell growth and survival of cancer cells. Recently, evidence also implicates adiposity as a potential risk factor for chemotherapy resistance. Chemotherapeutic agents have been shown to negatively impact adipose tissue. Since adipose tissue is a major storage site for fatty acids, it is not unlikely that these negative effects may disrupt adipose tissue homeostasis. It is therefore argued that fatty acid composition may be altered due to the chemotherapeutic pharmacokinetics, which in turn could have severe health related outcomes. The underlying molecular mechanisms elucidating the effects of fatty acid composition in adiposity-linked drug resistance are still unclear and under explored. This review focuses on the potential role of adiposity in breast cancer and specifically emphasizes the role of fatty acids in cancer progression and treatment resistance.

Abstract Despite sound basis to suspect that aggressive and early administration of nutritional support may hold therapeutic benefits during sepsis, recommendations for nutritional support have been somewhat underwhelming. Current guidelines (ESPEN and ASPEN) recognise a lack of clear evidence demonstrating the beneficial effect of nutritional support during sepsis, raising the question: why, given the perceived low efficacy of nutritionals support, are there no high-quality clinical trials on the efficacy of permissive underfeeding in sepsis? Here, we review clinically relevant beneficial effects of permissive underfeeding, motivating the urgent need to investigate the clinical benefits of delaying nutritional support during sepsis.

Breast cancer is a leading cause of death worldwide and a better understanding of this disease is needed to improve treatment outcomes. Recent evidence indicates that bacterial dysbiosis is associated with breast cancer, but the bacteria involved remain poorly characterised. Furthermore, an association between periodontal disease, characterised by oral dysbiosis, and breast cancer have also been discovered, but the mechanisms responsible for this association remains to be elucidated. The oral bacterium involved in periodontal disease, Fusobacterium nucleatum, have recently been detected in human breast tumour tissue and it promoted tumour growth and metastatic progression in a mouse model. The mechanisms of how F. nucleatum might colonise breast tissue and how it might promote tumour progression has not been fully elucidated yet. Here we discuss the breast tumour microbiota, its colonisation by F. nucleatum, possible mechanisms by which F. nucleatum might promote breast cancer progression and how this might impact breast cancer treatment. Literature indicates that F. nucleatum might promote breast cancer progression through activating the Toll-like receptor 4 pathway and by supressing the immune system. This results in cell growth and treatment resistance through autophagy as well as immune evasion. Targeted treatment directed at F. nucleatum combined with immunotherapy and autophagy inhibitors might therefore be a feasible treatment strategy for breast cancer patients.

The mechanisms by which long-chain dietary polyunsaturated fatty acids (PUFAs) protect against cardiovascular disease are largely unknown. The present study determines the effects of eicosapentaenoic acid (EPA) and arachidonic acid (ARA) on the response of neonatal rat cardiomyocytes to simulated ischaemia (SI) and reperfusion (R). Myocytes isolated from 1-2 day old Wistar rat hearts were cultured with or without EPA or ARA and exposed to 1 h SI followed by 30 minutes reperfusion. Apoptosis was evaluated by caspase-3 activation, poly-(ADP-ribose) polymerase (PARP) cleavage and nuclear condensation. EPA (20microM) and ARA (20microM) significantly inhibited caspase-3 activation and PARP-cleavage and reduced the apoptotic index during reperfusion. Both fatty acids significantly increased ERK phosphorylation and decreased p38 phosphorylation during reperfusion. The mechanism of action of ARA on the MAPKs was further investigated with okadaic acid (to inhibit serine-threonine phosphatases) and orthovanadate (to inhibit tyrosine phosphatases). Vanadate, but not okadaic acid, significantly reduced ARA-induced inhibition of p38 phosphorylation, suggesting the involvement a tyrosine phosphatase during SI/R. Mitogen-activated protein kinase phosphatase-1 (MKP-1), a dual-specificity phosphatase, was targeted and a significant induction of MKP-1 by ARA and EPA was observed. It was demonstrated for the first time that EPA and ARA protect neonatal cardiac myocytes from ischaemia/reperfusion-induced apoptosis through activation of ERK as well as induction of a dual-specific phosphatase, causing dephosphorylation of the pro-apoptotic kinase, p38. The cardioprotective effects of EPA and ARA could also be demonstrated on the functional recovery of isolated perfused hearts subjected to global ischemia.

It has been shown that dietary red palm oil (RPO) supplementation improves reperfusion function. However, no exact protective cellular mechanisms have been established. To determine a potential mechanism for functional improvement, we examined the regulation of both mitogen-activated protein kinases (MAPKs) and PKB/Akt in the presence and absence of dietary RPO supplementation in ischemia/reperfusion-induced injury. Wistar rats were fed a control diet or control diet plus 7 g RPO/kg diet for 6 weeks. Hearts were excised and mounted on an isolated working heart perfusion apparatus. Cardiac function was measured before and after hearts were subjected to 25 min of total global ischemia. Hearts subjected to the same conditions were freeze clamped and used to characterize the degree of phosphorylation of extracellular signal-regulated kinase, p38, c-Jun NH(2)-terminal protein kinase (JNK) and PKB/Akt. Dietary RPO supplementation significantly improved aortic output recovery (72.1 +/- 3.2% vs. 54.0 +/- 3.2%, P < .05). This improved aortic output recovery was associated with significant increases in p38 and PKB/Akt phosphorylation during reperfusion when compared with control hearts. Furthermore, a significant decrease in JNK phosphorylation and attenuation of poly(ADP-ribose) polymerase cleavage occurred in the RPO-supplemented group during reperfusion. Our results suggest that dietary RPO supplementation caused differential phosphorylation of the MAPKs and PKB/Akt during ischemia/reperfusion-induced injury. These changes in phosphorylation were associated with improved functional recovery and reduced cleavage of an apoptotic marker, arguing that dietary RPO supplementation may confer protection via the MAPK and PKB/Akt signaling pathways during ischemia/reperfusion-induced injury.

We have previously shown that dietary red palm oil (RPO) supplementation improves functional recovery in hearts subjected to ischaemia-reperfusion. However, little knowledge exists concerning the effects of RPO supplementation of a high-cholesterol diet on ischaemia-reperfusion injury. The signalling mechanisms responsible for RPO's effects in the presence of cholesterol also remain to be elucidated. Therefore, the aim of the present study was to examine the effects of RPO, given with a high-cholesterol diet, on mitogen-activated protein kinase (MAPK) phosphorylation and apoptosis. Long-Evans rats were fed a control diet, a control diet containing 2% cholesterol, or a control diet containing 2% cholesterol and 7 g RPO per kg (CRPO) for 5 weeks. Hearts were excised and mounted on an isolated working heart perfusion apparatus. Cardiac function was measured after which hearts were freeze-clamped and used to assess MAPK phosphorylation and to evaluate apoptosis. Cholesterol supplementation caused a poor aortic output (AO) recovery compared with the control group (35.5 (sem 6.2) v. 55.4 (sem 2.5) %), but when RPO was added, the percentage AO increased significantly. The cholesterol group's poor AO was associated with a significant increase in p38-MAPK phosphorylation, whereas the CRPO-supplemented group showed as significant reduction in p38-MAPK phosphorylation when compared with the cholesterol-supplemented group. This significant reduction in p38-MAPK was also associated with reduced apoptosis as indicated by significant reductions in caspase-3 and poly(ADP-ribose) polymerase cleavage.

During an infection, expansion of immune cells, assembly of antibodies, and the induction of a febrile response collectively place continual metabolic strain on the host. These considerations also provide a rationale for nutritional support in critically ill patients. Yet, results from clinical and preclinical studies indicate that aggressive nutritional support does not always benefit patients and may occasionally be detrimental. Moreover, both vertebrates and invertebrates exhibit a decrease in appetite during an infection, indicating that such sickness-associated anorexia (SAA) is evolutionarily conserved. It also suggests that SAA performs a vital function during an infection. We review evidence signifying that SAA may present a mechanism by which autophagic flux is upregulated systemically. A decrease in serum amino acids during an infection promotes autophagy not only in immune cells, but also in nonimmune cells. Similarly, bile acids reabsorbed postprandially inhibit hepatic autophagy by binding to farnesoid X receptors, indicating that SAA may be an attempt to conserve autophagy. In addition, augmented autophagic responses may play a critical role in clearing pathogens (xenophagy), in the presentation of epitopes in nonprovisional antigen presenting cells and the removal of damaged proteins and organelles. Collectively, these observations suggest that some patients might benefit from permissive underfeeding.

Over-nutrition and a sedentary lifestyle are the driving forces behind the development of metabolic diseases. Conversely, caloric restriction and exercise have proven to be the most effective strategies in combating metabolic diseases. Interestingly, exercise and caloric restriction share a common feature: both represent a potent mechanism for upregulating autophagy. Autophagy is rapidly induced by nutrient deprivation, and conversely, inactivated by amino acids as well as growth factors (e.g. insulin). Here, we review evidence demonstrating that autophagy may indeed be attenuated in metabolic tissue such as liver, muscle, and adipose, in the context of obesity. We also highlight the mechanistic basis by which defective autophagy may contribute to the manifestation of metabolic diseases. This includes a compromised ability of the cell to perform quality control on the mitochondrial matrix, since autophagy plays a pivotal role in the degradation of defective mitochondria. Similarly, autophagy also plays an indispensable role in the clearance of protein aggregates and redundant large protein platforms such as inflammasomes. Autophagy might also play a key role in the metabolism of endotoxins, implicating the importance of autophagy in the pathogenesis of metabolic endotoxemia. These observations underpin an unprecedented role of autophagy in the manifestation of obesity-induced metabolic derangement.

Skeletal muscle protein loss, known as atrophy, occurs during inactivity, disease, and aging. Atrophy may be the result of increased catabolic factors, e.g. glucocorticoids, or reduced influence of anabolic factors, e.g. insulin. The purpose of this study was to investigate atrophy, signaling mechanisms, and apoptosis in a rat model of restraint stress in 40 adult male Wistar rats. Due to the anxiolytic effects of Sutherlandia frutescens, we also determined if any of the molecular events in gastrocnemius muscle would be affected by daily treatment with S. frutescens. Rats were randomly assigned to four experimental groups: control placebo (CP); control Sutherlandia (CS) treatment; Restraint Placebo (RP) and Restraint Sutherlandia (RS) treatment. Restraint resulted in a significant increase in myostatin which was significantly reduced with Sutherlandia treatment. In addition, MyoD expression was significantly attenuated in RP and this effect was also counteracted by Sutherlandia treatment. Restraint also resulted in a significant attenuation of the PI3-Kinase/Akt signaling pathway and increased apoptosis which was reversed with Sutherlandia treatment. This study demonstrates for the first time that psychological stress elevates markers of muscle atrophy and apoptosis, whilst a herbal remedy, Sutherlandia, inhibits apoptosis, and signaling pathways associated with muscle atrophy.

Increasing resistance to cisplatin as well as the severity of the adverse effects limit the use of this drug, particularly at high doses. Evidence has implicated the importance of autophagy in cancer resistance as well as the fact that various chemotherapy agents induce autophagy in cancer cells. We therefore aimed to first assess the role of autophagy in cisplatin treatment and second to assess whether a nontoxic concentration of cisplatin, together with autophagy inhibition, is able to maintain its cancer-specific cytotoxic action.Three human cervical cell lines were used: a noncancerous ectocervical epithelial cell line (Ect1/E6E7) and 2 cancerous cervical cell lines (HeLa and CaSki). Autophagy was monitored through the presence of the classical protein markers LC-3 II and p62 under basal and treatment conditions, and inhibited using bafilomycin and autophagy protein 5 (ATG5) siRNA under treatment conditions. Cell death was analyzed through examination of the apoptotic markers PARP and caspase-3 through Western blotting, as well as the Caspase-Glo assay to confirm caspase-3/7 activity. Cervical biopsies were analyzed for the presence of LC-3 using Western blotting and immunofluorescence to determine if a correlation between autophagic levels and the progression of the disease exists.Cervical cancer cells exhibit increased basal autophagic levels in comparison to the noncancerous counterparts. Cisplatin treatment enhanced autophagic activity in all 3 cell lines. Inhibition of this autophagic response together with cisplatin treatment leads to significant increases in cancer cell death. Expression profiles of LC-3 in normal, premalignant (low-grade and high-grade squamous intraepithelial lesion), and cancerous cervical tissue revealed that autophagy is significantly up-regulated in HSILs and carcinoma cervical tissue, which emphasized the role of autophagy in the progression of the disease.The inhibition of autophagy improves the cytotoxicity of a nontoxic concentration of cisplatin and provides a promising new avenue for the future treatment of cervical cancer.

Inflammatory mediators have an established role in inducing insulin resistance and promoting hyperglycemia. In turn, hyperglycemia has been argued to drive immune cell dysfunction as a result of mitochondrial dysfunction. Here, the authors review the evidence challenging this view. First, it is pointed out that inflammatory mediators are known to induce altered mitochondrial function. In this regard, critical care patients suffer both an elevated inflammatory tone as well as hyperglycemia, rendering it difficult to distinguish between the effects of inflammation and hyperglycemia. Second, emerging evidence indicates that a decrease in mitochondrial respiration and an increase in reactive oxygen species (ROS) production are not necessarily manifestations of pathology, but adaptations taking shape as the mitochondria is abdicating its adenosine triphosphate (ATP)‐producing function (which is taken over by glycolysis) and instead becomes “retooled” for an immunological role. Collectively, these observations challenge the commonly held belief that acute hyperglycemia induces mitochondrial damage leading to immune cell dysfunction.

Chronic inflammation, systemic or local, plays a vital role in tumour progression and metastasis. Dysregulation of key physiological processes such as autophagy elicit unfavourable immune responses to induce chronic inflammation. Cytokines, growth factors and acute phase proteins present in the tumour microenvironment regulate inflammatory responses and alter crosstalk between various signalling pathways involved in the progression of cancer. Serum amyloid A (SAA) is a key acute phase protein secreted by the liver during the acute phase response (APR) following infection or injury. However, cancer and cancer-associated cells produce SAA, which when present in high levels in the tumour microenvironment contributes to cancer initiation, progression and metastasis. SAA can activate several signalling pathways such as the PI3K and MAPK pathways, which are also known modulators of the intracellular degradation process, autophagy. Autophagy can be regarded as having a double edged sword effect in cancer. Its dysregulation can induce malignant transformation through metabolic stress which manifests as oxidative stress, endoplasmic reticulum (ER) stress and DNA damage. On the other hand, autophagy can promote cancer survival during metabolic stress, hypoxia and senescence. Autophagy has been utilised to promote the efficiency of chemotherapeutic agents and can either be inhibited or induced to improve treatment outcomes. This review aims to address the known mechanisms that regulate autophagy as well as illustrating the role of SAA in modulating these pathways and its clinical implications for cancer therapy.

A number of mechanisms have been proposed to explain the well-established link between diabetic status and an increased susceptibility to infection. Notably, diabetes has been shown to be one of the strongest factors influencing healthcare outcome in COVID-19 infections. Though it has long been noted that lymphocytes upregulate insulin receptors following immune activation, until recently, this observation has received little attention. Here, we point out key findings implicating dysregulated insulin signalling in immune cells as a possible contributing factor in the immune pathology associated with diabetes. Mechanistically, insulin, by activating the PI3K/Akt/mTOR pathway, regulates various aspects of both myeloid cells and lymphocytes, such as cell survival, metabolic reprogramming and the polarization and differentiation of immune cells. PI3K signalling is also supressed by immune checkpoint proteins, suggesting that insulin signalling may antagonize peripheral tolerance. Remarkably, it has also recently been shown that, following insulin binding, the insulin receptor translocates to the nucleus where it plays a key role in regulating the transcription of various immune-related genes, including pathways involved in viral infections. Taken together, these observations suggest that dysregulated insulin signalling may directly contribute to a defective immune response during COVID-19 infections.

Breast cancer is the most commonly diagnosed cancer and the second most common cause of cancer death in women. The anthracycline, doxorubicin, is a well-known and highly effective treatment for breast cancer patients; however, many patients present with resistance to chemotherapeutic drugs, which ultimately results in treatment failure and contributes to high mortality rates. It is well established that the mitogen-activated protein kinase phosphatase 1 (MKP-1) mediates the response to chemotherapy, where upregulated MKP-1 is associated with chemoresistance. We investigated whether MKP-1 inhibition or silencing can sensitise triple-negative MDA-MB-231 breast cancer cells to doxorubicin therapy. We found that MKP-1 inhibition and silencing sensitises breast cancer cells to doxorubicin-induced apoptosis. Additionally, the inhibition of MKP-1 in combination with doxorubicin treatment promotes autophagy induction, while doxorubicin and not MKP-1 modulation increased lysosomal acidic compartments. As such, this study demonstrated that MKP-1 inhibition has a potential therapeutic benefit for breast cancer patients by increasing the efficacy of conventional chemotherapy. Therefore, MKP-1 inhibition should be developed as a clinically relevant adjuvant therapy, which could provide a novel avenue for therapeutic intervention in combination with chemotherapy in breast cancer patients.

A pathology-supported genetic testing (PSGT) framework was established in South Africa to improve access to precision medicine for patients with breast carcinomas. Nevertheless, the frequent identification of variants of uncertain significance (VUSs) with the use of genome-scale next-generation sequencing has created a bottleneck in the return of results to patients. This review highlights the importance of incorporating functional genomics into the PSGT framework as a proposed initiative. Here, we explore various model systems and experimental methods available for conducting functional studies in South Africa to enhance both variant classification and clinical interpretation. We emphasize the distinct advantages of using in vitro, in vivo, and translational ex vivo models to improve the effectiveness of precision oncology. Moreover, we highlight the relevance of methodologies such as protein modelling and structural bioinformatics, multi-omics, metabolic activity assays, flow cytometry, cell migration and invasion assays, tube-formation assays, multiplex assays of variant effect, and database mining and machine learning models. The selection of the appropriate experimental approach largely depends on the molecular mechanism of the gene under investigation and the predicted functional effect of the VUS. However, before making final decisions regarding the pathogenicity of VUSs, it is essential to assess the functional evidence and clinical outcomes under current variant interpretation guidelines. The inclusion of a functional genomics infrastructure within the PSGT framework will significantly advance the reclassification of VUSs and enhance the precision medicine pipeline for patients with breast carcinomas in South Africa.

Daunorubicin (DNR) and doxorubicin (DOX) are two of the most effective anthracycline drugs known for the treatment of systemic neoplasms and solid tumors. However, their clinical use is hampered due to profound cardiotoxicity. The mechanism by which DNR injures the heart remains to be fully elucidated. Recent reports have indicated that DOX activates ubiquitin proteasome-mediated degradation of specific transcription factors; however, no reports exist on the effect of DNR on the E3 ubiquitin ligases, MURF-1 (muscle ring finger 1) and MAFbx (muscle atrophy F-box). The aim of this study was to investigate the effect of DNR treatment on the protein and organelle degradation systems in the heart and to elucidate some of the signalling mechanisms involved. Adult rats were divided into two groups where one group received six intraperitoneal injections of 2 mg/kg DNR on alternate days and the other group received saline injections as control. Hearts were excised and perfused on a working heart system the day after the last injection and freeze-clamped for biochemical analysis. DNR treatment significantly attenuated cardiac function and increased apoptosis in the heart. DNR-induced cardiac cytotoxicity was associated with upregulation of the E3 ligases, MURF-1 and MAFbx and also caused significant increases in two markers of autophagy, beclin-1 and LC3. These changes observed in the heart were also associated with attenuation of the phosphoinositide 3-kinase/Akt signalling pathway.

Mammalian circadian rhythms form an integral physiological system allowing for the synchronisation of all metabolic processes to daily light/dark cycles, thereby optimising their efficacy. Circadian disruptions have been implicated in the onset and progression of various cancers, including those arising in the breast. Several links between the circadian protein Per2 and DNA damage responses exist. Aberrant Per2 expression results in potent downstream effects on both cell cycle and apoptotic targets, suggestive of a tumour suppressive role for Per2. Due to the severe dose limiting side effects associated with current chemotherapeutic strategies, including the use of doxorubicin, a need for more effective adjuvant therapies to increase cancer cell susceptibility has arisen. This study was therefore aimed at characterizing the role of Per2 in normal breast epithelia (MCF-12A) and in ER − breast cancer cells (MDA-MB-231) and also at determining the role of Per2 in doxorubicin-induced cell death. In both cell lines Per2 protein expression displayed a 24-hour circadian rhythm in both cell lines. Per2 was located predominantly in the cytoplasm, with nuclear localization observed with lower cytoplasmic fluorescent intensities. Our results show that Per2 silencing effectively sensitizes the chemoresistant MDA-MB-231 breast cancer cells to the cytotoxic effects of doxorubicin.

Autophagy is a conserved catabolic process for long-lived proteins and organelles and is primarily responsible for nonspecific degradation of redundant or faulty cell components. Although autophagy has been described as the cell’s major adaptive strategy in response to metabolic challenges, its influence on the cell’s energy profile is poorly understood. In the myocardium, autophagy is active at basal levels and is crucial for maintaining its contractile function. Defects in the autophagic machinery cause cardiac dysfunction and heart failure. In this paper we propose that (1) autophagy contributes significantly to the metabolic balance sheet of the heart. (2) Increased autophagy contributes to an improved myocardial energy profile through changing the cardiac substrate preference. (3) Substrates generated through autophagy give rise to an alternative for ATP production with an oxygen-sparing effect. These elements identify autophagy in a new context of myocardial metabolic interregulation, which we discuss in the settings of myocardial infarction, heart failure and the diabetic heart. It is hoped that the hypothesis presented can lead to new insights aimed at exploiting autophagy to improve existing metabolic-based therapy in heart disease.

Sutherlandia frutescens is a plant that is endemic to South Africa and is traditionally used for the treatment of cancer and many other ailments. Although previous studies have shown S. frutescens to exert anti-proliferative and apoptotic effects in human mammary adenocarcinoma- and leukemia cell lines, its potential cytotoxic effects on colon cancer cells have not yet been established. Moreover, limited information is available elucidating the major signaling pathways involved upon exposure to the herbal extract. The aim of this study was therefore to assess the cytotoxic effect of S. frutescens on the colon cancer cell line CaCo2, to investigate molecular role players of apoptosis and to assess the involvement of the PI3-K survival pathway. CaCo2 cells exposed to ethanolic extracts of S. frutescens showed significant decreased cell viability, indicated by reduced MTT reductive capacity, increased pyknosis as well as loss in cellular membrane integrity. Western blot analysis revealed a down-regulation in the PI3-K and Akt phosphorylation, a decrease in forkhead transcription factor (FKHR) phosphorylation as well as a concomitant induction of apoptosis. This study demonstrates that Sutherlandia treatment attenuates proliferation of colon cancer cells in vitro by the disruption of the key molecules in the PI-3K pathway thereby inducing apoptosis.

Future OncologyVol. 11, No. 14 EditorialMelatonin: a protective role against doxorubicin-induced cardiotoxicityJenelle Govender, Ben Loos & Anna-Mart EngelbrechtJenelle Govender Department of Physiological Sciences, Stellenbosch University, Stellenbosch, 7600, South AfricaSearch for more papers by this author, Ben Loos Department of Physiological Sciences, Stellenbosch University, Stellenbosch, 7600, South AfricaSearch for more papers by this author & Anna-Mart Engelbrecht*Author for correspondence: E-mail Address: ame@sun.ac.za Department of Physiological Sciences, Stellenbosch University, Stellenbosch, 7600, South AfricaSearch for more papers by this authorPublished Online:22 Jul 2015https://doi.org/10.2217/fon.15.48AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit View articleKeywords: cardio-oncologydoxorubicin-induced cardiotoxicityheartmelatoninmitochondriaPapers of special note have been highlighted as: • of interest; •• of considerable interestReferences1 Weiss RB. 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Sci. 7, 444–458 (2000).Crossref, Medline, CAS, Google Scholar18 Galano A, Tan DX, Reiter RJ. On the free radical scavenging activities of melatonin's metabolites, AFMK and AMK. J. Pineal. Res. 54, 245–257 (2013).• This review extensively discusses how melatonin and its metabolites provide continuous protection via the generation of free radical scavenging cascades.Crossref, Medline, CAS, Google Scholar19 Gustafsson AB, Gottlieb RA. Heart mitochondria: gates of life and death. Cardiovasc. Res. 77, 334–343 (2008).• The role of mitochondria in the control of cell death in cardiac myocytes is extensively reviewed.Crossref, Medline, CAS, Google Scholar20 Andrabi SA, Sayeed I, Siemen D et al. Direct inhibition of the mitochondrial permeability transition pore: a possible mechanism responsible for anti-apoptotic effects of melatonin. FASEB J. 18, 869–871 (2004).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByHsp90 protected chicken primary myocardial cells from heat-stress injury by inhibiting oxidative stress and calcium overload in mitochondriaBiochemical Pharmacology, Vol. 209Pharmacological Activation of Rev-erbα Attenuates Doxorubicin-Induced Cardiotoxicity by PGC-1α Signaling PathwayCardiovascular Therapeutics, Vol. 2023Melatonin attenuates branch chain fatty acid induced apoptosis mediated neurodegeneration21 November 2020 | Environmental Toxicology, Vol. 36, No. 4Sirtuins, melatonin, and the relevance of circadian oscillatorsMelatonin prevents doxorubicin-induced cardiotoxicity through suppression of AMPKα2-dependent mitochondrial damage18 December 2020 | Experimental & Molecular Medicine, Vol. 52, No. 12Heat shock protein 90 relieves heat stress damage of myocardial cells by regulating Akt and PKM2 signaling in�vivo31 March 2020 | International Journal of Molecular MedicineAMPK/PGC1α activation by melatonin attenuates acute doxorubicin cardiotoxicity via alleviating mitochondrial oxidative damage and apoptosisFree Radical Biology and Medicine, Vol. 129Cardio-oncology: a special focus issue from Future OncologyRobin L Jones & Maria Teresa Sandri22 July 2015 | Future Oncology, Vol. 11, No. 14 Vol. 11, No. 14 eToC Sign up Follow us on social media for the latest updates Metrics Downloaded 117 times History Published online 22 July 2015 Published in print July 2015 Information© Future Medicine LtdKeywordscardio-oncologydoxorubicin-induced cardiotoxicityheartmelatoninmitochondriaFinancial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.PDF download

Jawless vertebrates utilize a form of adaptive immunity that is functionally based on molecular effectors that are completely different from those of vertebrates. This observation raises an intriguing question: why did vertebrates, representing only 5% of all animals, twice evolve a system as complex as adaptive immunity? Theories aimed at identifying a selective pressure that would 'drive' the development of an adaptive immune system (AIS) fail to explain why invertebrates would not similarly develop an AIS. We argue that an AIS can only be implemented in a certain physiological context, i.e., that an AIS represents an unevolvable trait for invertebrates. The immune system is functionally integrated with other systems; therefore a preexisting physiological innovation unique to vertebrates may have acted as the prerequisite infrastructure that allowed the development of an AIS. We propose that future efforts should be directed toward identifying the evolutionary release that allowed the development of an adaptive immune system in vertebrates. In particular, the advent of specialized adipocytes might have expanded the metabolic scope of vertebrates, allowing the opportunistic incorporation of an AIS. However, physiological innovations, unique to (or more developed in) vertebrates, support the implementation of an AIS. Thus, understanding the interaction between systems (e.g. neural-immune-adipose connection) may illuminate our understanding regarding the perplexing immunological dimorphism within the animal kingdom.

Abstract We have previously shown that dietary red palm oil (RPO) supplementation improves functional recovery in hearts subjected to ischaemia/reperfusion-induced injury. Unfortunately, the cellular and molecular mechanisms responsible for this phenomenon are still poorly understood and no knowledge exists regarding the effects of RPO supplementation on the phosphoinositide 3-kinase (PI3-K) signaling pathway and apoptosis during ischaemia/reperfusion injury. Therefore, the aims of the present study were three fold: (i) to establish the effect of RPO on the functional recovery of the heart after ischaemia/reperfuion injury; (ii) to determine the effect of the PI3-K pathway in RPO-induced protection with the aid of an inhibitor (wortmannin); and (iii) to evaluate apoptosis in our model. Wistar rats were fed a standard rat chow control diet or a control diet plus 7 g RPO/kg for six weeks. Hearts were excised and mounted on a Langendorff perfusion apparatus. Mechanical function was measured after a 25 min period of total global ischaemia followed by 30 minutes of reperfusion. Hearts subjected to the same conditions were freeze-clamped for biochemical analysis at 10 min during reperfusion to determine the involvement of the PI3-Kinase signaling pathway and apoptosis in our model. Dietary RPO supplementation significantly increased % rate pressure product recovery during reperfusion (71.0 ± 6.3% in control vs 92.36 ± 4.489% in RPO; p &lt; 0.05). The % rate pressure product recovery was significantly reduced when wortmannin was added during perfusion (92.36 ± 4.489% in the RPO group vs 75.21 ± 5.26% in RPO + Wm). RPO + Wm also significantly attenuated PI3-K induction compared with the RPO group (59.2 ± 2.8 pixels in RPO vs 37.9 ± 3.4 pixels in RPO + Wm). We have also demonstrated that PI3-K inhibition induced PARP cleavage (marker of apoptosis) in the hearts during ischaemia/reperfusion injury and that RPO supplementation counteracted this effect.

Strong anti-neoplastic anthracyclines like daunorubicin (DNR) and doxorubicin (DOX) have high efficacy against systemic neoplasm and solid tumours. However, clinically, they cause chronic cardiomyopathy and congestive heart failure. Red palm oil (RPO) supplementation can protect the heart against ischemic injury. We therefore hypothesize that supplementation with RPO during chemotherapy may protect the heart. Control rats received a standard diet, and the experimental group received RPO in addition for 4 weeks. Each group was subsequently injected with either saline or DNR over a 12-day period towards the end of 4 weeks. Hearts were excised and perfused on a working heart system. Functional parameters were measured. Tissue samples were collected for analysis of mRNA and protein levels. DNR + RPO increased aortic output by 25% (p < 0.05) compared with DNR only. Furthermore, DNR treatment significantly reduced tissue mRNA levels of superoxide dismutase 1 (SOD1) and nitric oxide synthase 1 (NOS1) compared with untreated controls. Protein expression of SOD1 followed the same pattern as mRNA levels. NOS1 protein levels were significantly increased in DNR treated rats when compared with untreated controls. In addition, DNR increased phosphorylation of p38 and Jun N-terminal kinase compared with untreated controls, whereas DNR + RPO completely counteracted this activation. DNR + RPO significantly up regulated the protein extracellular signal-regulated kinase 1 level compared with DNR only. In this model of DNR treatment, RPO is associated with stabilization of important antioxidant enzymes such NOS and SOD, and inhibition of the 'stress' induced mitogen-activated protein kinase pathways. Dietary RPO also maintained function, similar to control, in DNR treated hearts.

Breast cancer is frequently diagnosed in women and poses a major health problem throughout the world. Currently, the unresponsiveness of cancer cells to chemotherapeutics is a major concern. During chemotherapeutic treatment with Doxorubicin, neighbouring cells in the tumor microenvironment are also damaged. Depending on the concentration of Doxorubicin, apoptotic or senescent fibroblasts in the tumor microenvironment can then secrete a variety of bioactive molecules which promote tumor growth, metastasis and drug resistance. Mouse embryonic fibroblasts (MEFs) were treated with Doxorubicin to induce apoptosis and senescence respectively. Conditioned media was collected from the MEFs and was used to assess the paracrine effects between fibroblasts and E0771 murine breast cancer cells. Senescent fibroblasts significantly increased cell viability in E0771 cells following Doxorubicin treatment by activating Akt and ERK. Autophagy contributed to cancer cell death and not to treatment resistance in breast cancer cells. Our results highlight the complexity of the tumor microenvironment where chemotherapeutic agents such as Doxorubicin can induce significant changes fibroblasts which can affect tumor growth via the secretion of paracrine factors. Here we have demonstrated that those secreted paracrine factors enhance breast cancer growth and induce therapeutic resistance through the evasion of apoptotic cell death.

Cervical cancer is a leading cause of death in developing countries and is the second highest occurring cancer in women all over the world. The progression of cancer is a multistep process affecting aspects of cellular function such as proliferation, differentiation and apoptosis. Mitogen activated protein kinases (MAPKs), which include p38-MAPK, c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinases (ERKs) are closely associated with cell proliferation and apoptosis and the balance between them could determine a cell's fate. Despite the expanding research effort in vitro, little is known about MAPK activation in clinical specimens of cervical cancer. Therefore, the aim of this ex vivo study was to correlate the phosphorylation status (activity) of MAPKs (p38-MAPK, JNK and ERK), as well as poly (ADP-ribose) polymerase (PARP) and caspase-3 (two cellular markers of apoptosis), during the different stages of cervical carcinogenesis, to observe whether correlations between MAPK activities and apoptosis during the disease process exist. Decreased p38-MAPK phosphorylation was found in the carcinoma (Ca) group) compared to the normal tissues, as well when the low grade squamous intraepithelial lesion—LSIL) group and high grade squamous intraepithelial lesion—HSIL) group were compared with the Ca group. Interestingly, a significant decrease in ERK44 phosphorylation was observed in Ca when compared to LSIL and HSIL. There was also a significant decrease in JNK phosphorylation in Ca when compared with normal tissue and HSIL. As expected, caspase-3 activation and PARP cleavage was significantly lower in Ca when compared with normal tissue. Our results present the first evidence of in vivo involvement of MAPKs in cervical cancer and indicate a possible correlation between MAPK activities and apoptosis in the disease process.

Untreated menopause may have serious health implications, but treatments can have dangerous side effects. We evaluate menopausal symptoms as well as available treatments -the routes of administration and their effect on blood coagulation. Menopausal females may experience hot flushes, vulva- and vaginal atrophy and osteoporosis. Many treatments are available to relieve these symptoms such as Conjugated Equine Estrogen and bioidentical hormones. The routes of administration include oral and transdermal. Hormones that are administered orally undergo a hepatic first pass metabolism. The by-products have a lower efficacy and possibly enhanced side effects. Furthermore, hormone treatments influence the coagulation cascade through coagulation factors or their regulators. Increased coagulation poses a risk for venous thromboembolism. Currently a definite conclusion on whether the side effects from hormone treatments exceed the risk of untreated menopause cannot be made. However, a more individualised approach to hormone treatments may be the most feasible solution to this dilemma.

It has become increasingly clear that apoptosis plays a major role in ischaemia/reperfusion (I/R)-induced cell death, but the molecular basis of this process remains to be elucidated. Therefore, the aim of this study was to investigate the role of cPLA(2) in MAPK phosphorylation and apoptosis in simulated ischaemia/reperfusion (SI/R)-induced injury in neonatal cardiomyocytes. Inhibition of cPLA(2) with AACOCF(3) significantly improved cell viability during SI/R (60.17+/-1.77 to 80.17+/-1.97%, p<0.05). The increase in cell viability was associated with a significant inhibition of p38 phosphorylation (135.3+/-4.47% to 87.94+/-10.71%, p<0.001) as well as with a significant decrease in caspase-3- (320.32+/-17.32% to 146.7+/-28.69%, p<0.01) and PARP-(263.9+/-8.15% to 154.7+/-2.24%, p<0.001) cleavage during SI/R. This study provides evidence for a role for cPLA(2) during SI/R-induced injury. It appears that p38 MAPK is a central role player in the signalling pathway involved.

It has previously been demonstrated that 15-min continuous insulin infusion at immediate reperfusion affords cardioprotection. This study sought to reduce the treatment time of insulin and test if intermittent insulin infusions can mimic ischemic postconditioning.In a Langendorff perfused rat heart model of regional ischemia, hearts were at the onset of reperfusion subjected to either 5- or 1-min continuous insulin infusion or 3 × 30 s intermittent insulin infusions (InsPost); with or without inhibitors of Akt (SH-6), p70s6-kinase (rapamycin), mitochondrial ATP-sensitive potassium channels (5-hydroxydecanoic acid [5-HD]), or a scavenger of reactive oxygen species (ROS; 2-mercaptopropionyl glycine [MPG]). Infarct size is expressed as percent of area at risk and presented as mean ± standard error of the mean or s.e.m.Only InsPost was able to reduce infarct size compared with controls (InsPost 33 ± 6% vs. Ctr 52 ± 4%, p < 0.05.). This cardioprotection was abrogated by co-administering SH-6, rapamycin, 5-HD, or MPG. (InsPost + SH-6 56 ± 9%, InsPost + Rapa 55 ± 8%, InsPost + 5-HD 56 ± 7%, InsPost + MPG 60 ± 3% vs. InsPost 33 ± 6% p < 0.05). These results were corroborated by a significant increase in phosphorylated Akt and p70s6k in the InsPost group compared with controls.Short intermittent insulin infusions can mimic ischemic postconditioning and reduce myocardial infarct size via Akt/p70s6k and mKATP channels/ROS-dependent signaling.

Adipocytes in the breast tumour microenvironment promotes acquired treatment resistance. We used an in vitro adipocyte-conditioned media approach to investigate the direct paracrine effects of adipocyte secretory factors on MDA-MB-231 breast cancer cells treated with doxorubicin to clarify the underlying treatment resistance mechanisms. Cell-viability assays, and Western blots were performed to determine alterations in apoptotic, proliferation and lipid metabolism protein markers. Free fatty acids (FFA) and inflammatory markers in the collected treatment-conditioned media were also quantified. Adipocyte secretory factors increased the cell-viability of doxorubicin-treated cells (p < 0.0001), which did not correspond to apoptosis or proliferation pathways. Adipocyte secretory factors increased the protein expression of hormone-sensitive lipase (p < 0.05) in doxorubicin-treated cells. Adipocyte secretory factors increased the utilization of leptin (p < 0.05) and MCP-1 (p < 0.01) proteins and possibly inhibited release of linoleic acid by doxorubicin-treated cells (treatment-conditioned media FFA profiles). Adipocyte secretory factors induced doxorubicin treatment resistance, by increasing the utilization of inflammatory mediators and inhibiting the release of FFA by doxorubicin-treated cells. This further promotes inflammation and lipid metabolic reprogramming (lipid storage) in the tumour microenvironment, which breast cancer cells use to evade the toxic effects induced by doxorubicin and confers to acquired treatment resistance.

Both the cytokine tumour necrosis factor-α (TNF-α) and the enzyme cytosolic phospholipase A2 (cPLA2) have been implicated in ischaemic injury. Apart from the induction of apoptosis, TNF-α also mediates cytoprotection when present in low concentrations. However, the relationship between TNF-α and cPLA2 activities during cytoprotection is poorly understood. Therefore, we examined the role of cPLA2 in TNF-α-mediated (TNF-PC) and ischaemic preconditioning (IPC) in tolerance to ischaemia (SI) in C2C12 myotubes. Significant decreases in cPLA2 phosphorylation in SI compared with the preconditioned groups were observed. This was also mirrored by the p38 mitogen activated protein kinase (MAPK) phosphorylation pattern. These results correlated with fluorescence- and three-dimensional imaging, demonstrating increased translocation of phospho-cPLA2 to the nuclear region in SI compared to TNF-PC and IPC. These data suggest a p38 driven cPLA2 translocation pattern, with a possible role for cPLA2 in deciding cell fate.

Previous studies have shown that red palm oil (RPO) supplementation protected rat hearts against ischaemia-reperfusion injury. Evidence from these studies suggested that Akt may be partly responsible for the observed protection. The aim of the current study was therefore to prove or refute the involvement of Akt in the RPO-induced cardioprotection by administration of a specific Akt inhibitor (A6730). Male Wistar rats were randomly divided into 2 groups: a control group receiving standard rat chow and an experimental group receiving standard rat chow plus 2 mL RPO for six weeks. Hearts were excised and mounted on the Langendorff perfusion system. Functional recovery was documented. A different set of hearts were freeze-clamped to assess total and phosphorylation status of Akt. Another set of hearts were subjected to the same perfusion conditions with addition of A6730. Hearts from this protocol were freeze-clamped and assessed for total and phospho-Akt. RPO improved functional recovery which was associated with increased phosphorylation of Akt on Ser473 and Thr308 residues. Blockade of Akt phosphorylation caused poor functional recovery. For the first time, these results prove that Akt plays an important role in the RPO-induced cardioprotection.

The characterization of a completely novel adaptive immune system (AIS) in jawless vertebrates (hagfish and lampreys) presents an excellent opportunity for exploring similarities and differences in design principles. It also highlights a somewhat neglected question: Why did vertebrates, representing only 5 % of all animals, evolve a system as complex as an AIS twice, whereas invertebrates failed to do so? A number of theories have been presented in answer to this question. However, these theories either fail to explain why invertebrates would not similarly develop an AIS and are confounded by issues of causality, or have been challenged by more recent findings.Instead of identifying a selective pressure that would drive the development of an AIS, we hypothesise that invertebrates failed to develop an AIS because of the evolutionary constraints imposed by these animals' physiological context. In particular, we argue that a number of vascular innovations in vertebrates allowed the effective implementation of an AIS. A lower blood volume allowed for a higher antibody titer (i.e., less 'diluted' antibody concentration), rendering these immune effectors more cost-effective. In addition, both a high circulatory velocity and the ability of endothelium to coordinate immune cell trafficking promote 'epitope sampling'. Collectively, these innovations allowed the effective implementation of AIS in vertebrates.The hypothesis posits that a number of innovations to the vascular system provided the release from constraints which allowed the implementation of an AIS. However, this hypothesis would be refuted by phylogenetic analysis demonstrating that the AIS preceded these vascular innovations. The hypothesis also suggests that vascular performance would have an impact on the efficacy of an AIS, thus predicting a correlation between the vascular parameters of a species and its relative investment in AIS. The contribution of certain vascular innovations in augmenting immune functionality of an AIS can be tested by modelling the effect of different vascular parameters on AIS efficacy.The hypothesis not only explains the immunological dimorphism between vertebrates and invertebrates but also brings to attention the fact that immunity is dependent on more than just an immune system.

Scandinavian Journal of ImmunologyVolume 83, Issue 2 p. 160-161 Letter to the EditorFree Access Invertebrates: Why No Adaptive Immune System? G. van Niekerk, Corresponding Author G. van Niekerk Department of Physiological Sciences, Stellenbosch University, Stellenbosch, South AfricaCorrespondence to: G. van Niekerk, Department of Physiological Sciences, Stellenbosch University, Stellenbosch, South Africa. E-mail: gustav.v.niekerk@gmail.comSearch for more papers by this authorS. M. Hattingh, S. M. Hattingh Division of Medical Physiology, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South AfricaSearch for more papers by this authorA. M. Engelbrecht, A. M. Engelbrecht Department of Physiological Sciences, Stellenbosch University, Stellenbosch, South AfricaSearch for more papers by this author G. van Niekerk, Corresponding Author G. van Niekerk Department of Physiological Sciences, Stellenbosch University, Stellenbosch, South AfricaCorrespondence to: G. van Niekerk, Department of Physiological Sciences, Stellenbosch University, Stellenbosch, South Africa. E-mail: gustav.v.niekerk@gmail.comSearch for more papers by this authorS. M. Hattingh, S. M. Hattingh Division of Medical Physiology, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South AfricaSearch for more papers by this authorA. M. Engelbrecht, A. M. Engelbrecht Department of Physiological Sciences, Stellenbosch University, Stellenbosch, South AfricaSearch for more papers by this author First published: 26 November 2015 https://doi.org/10.1111/sji.12400Citations: 3AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat To the Editor: It is with great interest that we read the intriguing hypothesis by Corcos 1, where he proposes a novel hypothesis to explain why the adaptive immune system arises in vertebrates: to prevent cannibalism of filial gametes. This view contrasts with the 'common sense' notion that AIS must have evolved for immunological purposes. Also, the hypothesis includes the role of the ecological context in which an AIS evolves, namely, in an aquatic environment inhabited by a filter-feeding proto-vertebrate. Thus, this hypothesis provides a novel view on a very intriguing question. However, we have pointed out that any theory attempting to explain why vertebrates evolve an AIS (twice 2) should also explain why invertebrates failed to do so 3, 4. The hypothesis presented here raises the same concern: why would invertebrates representing a large spectrum of filter feeders (such as sea sponge, corals, clams) not similarly evolve an AIS to avoid cannibalism of conspecific gametes? In fact, the author claims only two constraints to the development of an AIS: 'an aquatic medium and external fertilization'. Invertebrates ostensibly qualify on both, yet have no AIS. Furthermore, Corcos quotes Hedrick 5, who argues that AIS does not represent an 'optimum solution' to the problem of parasitism. In this regard, we would like to point out that the AIS might indeed represent an immunological innovation with lasting benefits without necessarily increasing the absolute immunity of an organism, as immunological strategies are optimized in the face of competing metabolic needs. As an example, an AIS might decrease the 'total cost of ownership' associated with operating an immune system without increasing the absolute immunity of an organism. Alternatively, the addition of an AIS may provide a more flexible strategy that helps vertebrates to better allocate resources. Indeed, animals may differentially invest between innate immunity and AIS in the context of life-history events 6. A broader range of strategies may render the animals more responsive to competing metabolic demands imposed by life-history events (Fig. 1). This may be speculative, but it is worth keeping in mind that 'vertebrates are the dominant group of animals on the Earth, given their abundance, large body sizes and presence at the top of both aquatic and terrestrial food webs' 7. This may suggest that the AIS may represent an immunological strategy without necessarily increasing absolute immunity, providing emerging vertebrates with a competitive advantage (e.g. more resources to flux towards reproductive efforts) and allowing vertebrates to infiltrate ecological spaces already occupied by invertebrates. Clearly, there remain various avenues to be explored in explaining the immunological dimorphism between vertebrates and invertebrates. Figure 1Open in figure viewerPowerPoint Vector analogy demonstrating how the addition of an AIS could allow vertebrates greater scope for tailoring immune investment in context of other metabolic demands (e.g. reproduction). The graded area represents pathogen stress. In contrast to invertebrates, vertebrates could respond to developing pathogen stress not only in magnitude, but also qualitatively (i.e. investing between constitutive versus inducible immunity) as circumstances would necessitate. The immune advantage need not occur directly in terms of 'immune potency', but may represent a benefit by expanding 'operational flexibility' and allow vertebrates to optimize immune strategies in the context of competing metabolic interest. Finally, this raises the question: If an AIS provides some benefit, why did invertebrates failed to evolve an AIS? We suggest that the answer lies within the evolutionary constraints imposed by the physiology of invertebrates: a range of physiological innovations unique to vertebrates provided the evolutionary release that allowed the evolution of an AIS in both jawed and jawless vertebrates. For example, the closed vascular system would present a smaller blood volume in which antibodies (a hallmark of an AIS) are diluted 4. Similarly, although an AIS might be metabolically inexpensive to maintain, activation might impose a tremendous cost. Consequently, we have previously argued that adipocytes (a tissue type unique to vertebrates) would allow vertebrates to allocate the necessary store resources to activate the AIS 3. However, we also point out that other physiological systems might be involved. Indeed, neural crest cells (unique to vertebrates) gave rise to a number of novel tissue/anatomical innovations. We propose that strategies should aim at investigating how novel tissue types and anatomical innovations would impact on the functionality of an AIS. References 1Corcos D. Food-nonfood discrimination in ancestral vertebrates: gamete cannibalism and the origin of the adaptive immune system. Scand J Immunol 2015; 82: 409– 17. 2Boehm T, McCurley N, Sutoh Y, Schorpp M, Kasahara M, Cooper MD. VLR-based adaptive immunity. Annu Rev Immunol 2012; 30: 203– 20. 3van Niekerk G, Engelbrecht A. On the evolutionary origin of the adaptive immune system-the adipocyte hypothesis. Immunol Lett 2015; 164: 81– 7. 4van Niekerk G, Davis T, Engelbrecht A. Was the evolutionary road towards adaptive immunity paved with endothelium? Biol Direct 2015; 10: 1– 7. 5Hedrick SM. The acquired immune system: a vantage from beneath. Immunity 2004; 21: 607– 15. 6Martin LB, Weil ZM, Nelson RJ. Seasonal changes in vertebrate immune activity: mediation by physiological trade-offs. Philos Trans R Soc Lond B Biol Sci 2008; 363: 321– 39. 7Wiens JJ. Explaining large-scale patterns of vertebrate diversity. Biol Lett. DOI: 10.1098/rsbl.2015.0506. Citing Literature Volume83, Issue2February 2016Pages 160-161 FiguresReferencesRelatedInformation

Emerging evidence has implicated insulin in regulating the phenotypes of various immune cells through canonical downstream signalling effectors of insulin, namely, the PI3K/Akt/mTOR pathway. Notably, these signalling components also exhibit crosstalk with other immune signalling pathways, such as the JAK/STAT pathway (activated by cytokines and growth factors), and, importantly, are also negatively regulated by the immune checkpoint blockers (ICBs), PD-1 and CTLA-4. Here, we point out recent findings, suggesting that insulin may promote a pro-inflammatory phenotype with potential implications on ICB therapy. As an example, the contemporary paradigm holds that, while T cell receptor recognition of distinct MHC-expressed epitopes ensures specificity, co-activation of CD28 along with signal inputs form various cytokines and insulin operates to ‘fine-tune’ the immune response via PI3K and other downstream signalling molecules. These considerations highlight the urgent need for focused investigations into the role of insulin in regulating immune cell function in the context of ICB therapies.

Abstract Background Breast cancer is a major health burden for women, worldwide. Lifestyle-related risk factors, such as obesity and being overweight, have reached epidemic proportions and contributes to the development of breast cancer. Doxorubicin (DXR) is a chemotherapeutic drug commonly used to treat breast cancer, and although effective, may cause toxicity to other organs. The mechanisms and effects of DXR on hepatic tissue, and the contributing role of obesity, in breast cancer patients are poorly understood. The aim of this study was therefore to investigate the effects of DXR on hepatic tissue in an obese tumour-bearing mouse model. Methods A diet-induced obesity (DIO) mouse model was established, where seventy-four three-week-old female C57BL6 mice were divided into two main groups, namely the high fat diet (containing 60% kcal fat) and standard diet (containing 10% kcal fat) groups. After eight weeks on their respective diets, the DIO phenotype was established, and the mice were further divided into tumour and non-tumour groups. Mice were subcutaneously inoculated with E0771 triple negative breast cancer cells in the fourth mammary gland and received three doses of 4 mg/kg DXR (cumulative dosage of 12 mg/kg) or vehicle treatments via intraperitoneal injection. The expression levels of markers involved in apoptosis and alanine aminotransferase (ALT) were compared by means of western blotting. To assess the pathology and morphology of hepatic tissue, haematoxylin and eosin staining was performed. The presence of fibrosis and lipid accumulation in hepatic tissues were assessed with Masson’s trichrome and Oil Red O staining, respectively. Results Microscopic examination of liver tissues showed significant changes in the high fat diet tumour-bearing mice treated with DXR, consisting of macrovesicular steatosis, hepatocyte ballooning and lobular inflammation, compared to the standard diet tumour-bearing mice treated with DXR and the control group (standard diet mice). These changes are the hallmarks of non-alcoholic fatty liver disease, associated with obesity. Conclusion The histopathological findings indicated that DXR caused significant hepatic parenchymal injury in the obese tumour-bearing mice. Hepatotoxicity is aggravated in obesity as an underlying co-morbidity. It has been shown that obesity is associated with poor clinical outcomes in patients receiving neo-adjuvant chemotherapy treatment regimens.

Background: Prostate cancer commonly occurs in older men. Since TNM staging excludes prostate-specific antigen (PSA) level and Gleason score, patients with prostate cancer are divided into risk groups when deciding on treatment options. This study determined the profile and risk stratification of patients with prostate cancer treated at the Department of Oncology, Universitas Annex in Bloemfontein, Free State, during 2008 to 2010.Methods: This was a cross-sectional study with retrospective data collection. Information was gathered from 497 patient files on age, race, residence, Gleason score, PSA level, TNM stage, and initial treatment. The patients’ risk group was determined from their Gleason score, PSA level, and T stage.Results: Patients were mostly (45.7%) between 65 and 75 years of age and 72.8% were in the black race group. The largest percentage of patients had a Gleason score of 8 to 10 (43.7%), PSA level > 20 ng/ml (67.9%), and a T stage ≥ T3 (62.3%). Almost half of the patients (48.7%) had stage IV disease and 38.4% received palliative hormonal therapy as initial treatment. The majority of patients (82.5%) fell into the high risk group.Conclusions: The majority of patients in each age group fell into the high risk group, which means that these patients were at a higher risk of developing metastatic prostate cancer. We recommend better education of our patient population and local clinic staff, so that people in the community can understand the prevalence of the disease, the symptoms and effect of the cancer, and that it is treatable if detected early.

Many clinical trials are beginning to assess the effectiveness of compounds known to regulate autophagy in patients receiving anti-cancer chemotherapy. However, autophagy inhibition, through exogenous inhibitors, or activation, through starvation, has revealed conflicting roles in cancer management and chemotherapeutic outcome. This study aimed to assess the effect of amino acid starvation on doxorubicin-treated breast cancer cells by assessing the roles of autophagy and apoptosis. An in vitro breast cancer model consisting of the normal breast epithelial MCF12A and the metastatic breast cancer MDAMB231 cells was used. Apoptotic and autophagic parameters were assessed following doxorubicin treatments, alone or in combination with bafilomycin, ATG5 siRNA or amino acid starvation. Inhibition of autophagy, through ATG5 siRNA or bafilomycin treatment, increased caspase activity and intracellular doxorubicin concentrations in MCF12A and MDAMB231 cells during doxorubicin treatment. While amino acid starvation increased autophagic activity and decreased caspase activity and intracellular doxorubicin concentrations in MCF12A cells, no changes in autophagic parameters or caspase activity were observed in MDAMB231 cells. Our in vivo data showed that 24 h protein starvation during high dose doxorubicin treatment resulted in increased survival of tumor-bearing GFP-LC3 mice. Results from this study suggest that short term starvation during doxorubicin chemotherapy may be a realistic avenue for adjuvant therapy, especially with regards to the protection of non-cancerous cells. More research is however, needed to fully understand the regulation of autophagic flux during starvation.

It is well known that acute ischemia resulting from several pathophysiological conditions, disturb cellular function and lead to cell and tissue damage. An increasing body of evidence implies that the phosphatidylinositol-3-kinase (PI3-K) signaling pathway plays a key role in a multitude of cellular processes which include the regulation of cell death. However, the role of the PI3-K pathway during simulated ischemia (SI) is not yet fully understood and conflicting data exists in this regard. Therefore, we aimed to determine the role of the PI3K signaling pathway during acute SI in C2C12 myotubes and analyze the related impact on cell death parameters occurring within this context. Cells are grown in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS), and incubated under 5% CO2 conditions, until reaching 90% confluency. Using DMEM supplemented with 1% horse serum, cell differentiation into myotubes was induced. Mitochondrial reductive capacity was assessed with the MTT assay. Phosphorylation of proteins was analyzed by Western blotting and immunocytochemistry was used to assess cell death. We present evidence that simulated ischemia attenuated PI3K activity which was also associated with decreased Akt-dependent phosphorylation at the level of FoxO1, FoxO4, TSC2 and mTOR. An ischemic microenvironment leads to a reduction in PI3K activity with subsequent induction of apoptosis.

GENERAL COMMENTARY article Front. Microbiol., 27 May 2015Sec. Microbial Immunology https://doi.org/10.3389/fmicb.2015.00531

The Serum Amyloid A (SAA) family of proteins is associated with various pathological conditions, including cancer. However, their role in cancer is incompletely understood. Here, we investigated the role of SAA1 in cell cycle regulation, apoptosis, survival signaling, metabolism, and metastasis in models of triple-negative breast cancer (TNBC), using RNAi. Our data show that in untransformed epithelial cells (MCF12A), the knockdown of SAA1 induces the expression of cell cycle regulators (MCM2, p53), the activation of DNA repair (PARP synthesis), and survival signaling (NFκB). In contrast, knockdown of SAA1 in the TNBC cell line (MDA-MB-231) induced the expression p16 and shifted cells in the cell cycle from the S to G2/M phase, without the activation of DNA repair. Moreover, in SAA1-deficient MDA-MB-231 and HCC70 cells, metabolism (NADH oxidation) continually increased while cell migration (% wound closure and the rate of wound closure) decreased. However, silencing of SAA1 altered epithelial and mesenchymal markers in MCF12A (E-cadherin, Laminin 1β, Vimentin) and MDA-MB-231 (α-Smooth muscle actin) cells, associated with the metastatic program of epithelial-mesenchymal transition. Nonetheless, our data provide evidence that SAA1 could potentially serve as a therapeutic target in TNBC.

In the first part of this study, the possible role of essential total fatty acids and their metabolites during cervical carcinogenesis was investigated. Since membrane lipids play a key role in cell proliferation and differentiation, disturbances in the fatty acid compositions of cell membranes and the modulation of membrane fatty acid compositions received attention in several in vitro studies. There are, however, no reported studies where the actual total and free (unesterified) fatty acid compositions have been determined during the different stages of cervical carcinogenesis. In part I of this ex vivo study, the total fatty acid compositions of normal tissue, intraepithelial and infiltrating lesions of the cervix were compared. The fatty acid profiles that were determined make it possible to speculate about the metabolic pathways followed during cervical carcinogenesis. Lipids were extracted from biopsies of normal tissue (n=36), cervical intraepithelial lesions (n=47) and infiltrating lesions (n=47). Samples, from which the total fatty acid compositions were determined, were saponified, methylated and analysed by gas liquid chromatography (GLC). Essential fatty acid deficiency (EFAD) in the intraepithelial lesions, compared with normal tissue (linoleic acid, P< 0.01), and infiltrating lesions, compared with intraepithelial lesions (linoleic acid and arachidonic acid, P< 0.01) were observed. High levels of oleic acid were also observed when infiltrating lesions were compared with normal tissue (P< 0.01). This EFAD in cancer cells may result in many defective cell mechanisms. Although there are many risk factors for cervical cancer, the human papilloma virus has emerged over the past decade as the leading candidate to be an aetiological factor. There is ample evidence that human viral infections are associated with reduced levels of linoleic acid and thus participate in the depletion of essential fatty acids in cancer cells.

Since cancer shares the same molecular machinery as the host, most therapeutic interventions that aim to target cancer would inadvertently also adversely affect the host. In addition, cancer continuously evolves, streamlining its host-derived genome for a new single-celled existence. In particular, short-term clinical success observed with most antineoplastic therapies directly relate to the fact that cancer is constantly evolving. However, the clonal evolution of cancer occasionally also render cancer cells uniquely susceptible to therapeutic interventions, as is exemplified by the clinical relevance of synthetic lethality. Synthetic lethality describes a situation where the simultaneous loss of function in two genes results in lethality, but where a loss of function in either single gene is tolerated. This observation suggests that the evolution of cancer, usually seen as a major clinical challenge, may also afford a key opportunity in lowering on-target toxicities accosted with chemotherapy. As an example, by subjecting cancer to specific selection regimes, cancer can in effect be placed on evolutionary trajectories leading to the development of "targetable" phenotypes such as synthetic lethal interactions. However, such a selection regime would have to overcome a range of obstacles such as on-target toxicity and the selection of an evolvable trait. Since the majority of cancer evolution manifests as a loss of function, we suggest that the induction of auxotrophic phenotypes (i.e., where an organism lose the ability to synthesize specific organic compounds required for growth and thus become dependent on it from dietary sources) may represent an attractive therapeutic option. As an example, animals can obtain vitamin C either by de novo synthesis or from their diet. However, since the maintenance of synthetic pathways is costly, such pathways are often lost if no longer necessary, resulting in the organism being auxotrophic toward the dietary compound. Similarly, increasing the maintenance cost of a redundant pathway in cancer cells is likely to select for clones that have lost such a redundant pathway. Inhibition of a pathway, while supporting the activity of a compensating pathway, may thus induce auxotrophism in cancer cells but not in genomic stable host cells.

Current chemotherapeutic agents have many side effects and are toxic to normal cells, providing impetus to identify agents that can effectively eliminate tumorigenic cells without damaging healthy cells. The aim of this study was to examine whether combining a novel BRD4 inhibitor, ITH-47, with the antimitotic estradiol analogue, ESE-15-ol, would have a synergistic effect on inhibiting the growth of two different breast cancer cell lines in vitro . Our docking and molecular dynamics studies showed that compared to JQ1, ITH-47 showed a similar binding mode with hydrogen bonds forming between the ligand nitrogens of the pyrazole, ASN99, and water of the BRD4 protein. Data from cell growth studies revealed that the GI 50 of ITH-47 and ESE-15-ol after 48 hours of exposure was determined to be 15 μ M and 70 nM, respectively, in metastatic MDA-MB-231 breast cancer cells. In tumorigenic MCF-7 breast cancer cells, the GI 50 of ITH-47 and ESE-15-ol was 75 μ M and 60 nM, respectively, after 48 hours of exposure. Furthermore, the combination of 7.5 μ M and 14 nM of ITH-47 and ESE-15-ol, respectively, resulted in 50% growth inhibition of MDA-MB-231 cells resulting in a synergistic combination index (CI) of 0.7. Flow cytometry studies revealed that, compared to the control, combination-treated MDA-MB-231 cells had significantly more cells present in the sub-G 1 phase and the combination treatment induced apoptosis in the MDA-MB-231 cells. Compared to vehicle-treated cells, the combination-treated cells showed decreased levels of the BRD4, as well as c-Myc protein after 48 hours of exposure. In combination, the selective BRD4 inhibitor, ITH-47, and ESE-15-ol synergistically inhibited the growth of MDA-MB-231 breast cancer cells, but not of the MCF-7 cell line. This study provides evidence that resistance to BRD4 inhibitors may be overcome by combining inhibitors with other compounds, which may have treatment potential for hormone-independent breast cancers.

It has been established that the acute phase protein, Serum amyloid A (SAA), which is usually synthesized by the liver, is also synthesized by cancer cells and cancer-associated cells in the tumor microenvironment. SAA also activates modulators of autophagy, such as the PI3K/Akt and MAPK signaling pathways. However, the role of SAA in autophagy in breast cancer still remains to be elucidated. The aim of this study was to investigate the role of SAA in the regulation of signaling pathways and autophagy in in vitro and in vivo models of breast cancer. The MDA-MB-231 and MCF7 cell lines were transiently transfected to overexpress SAA1. A tumor-bearing SAA1/2 knockout mouse model was also utilized in this study. SAA1 overexpression activated ERK signaling in the MDA-MB-231 cells, downregulated the PI3K pathway protein, PKB/Akt, in the MCF7 cell line, while SAA1/2 knockout also inhibited Akt. Furthermore, SAA1 overexpression in vitro downregulated autophagy, while the expression of SQSTM1/p62 was increased in the MCF7 cells, and SAA1/2 knockout induced autophagy in vivo . SAA overexpression in the MDA-MB-231 and MCF7 cells resulted in an increase in cell viability and increased the expression of the proliferation marker, MCM2, in the MCF7 cells. Furthermore, knockout of SAA1/2 resulted in an altered inflammatory profile, evident in the decrease of plasma IL-1β, IL-6 and IL-10, while increasing the plasma levels of MCP-1 and TNF-α. Lastly, SAA1/2 knockout promoted resistance to apoptosis and necrosis through the regulation of autophagy. SAA thus regulates autophagy in breast cancer cells to promote tumorigenesis.

Nutritional support continues to receive much attention as a possible intervention to prevent loss of lean tissue mass, promote recovery and re-establish proper immune function in critical care patients. Yet there remains much controversy regarding the clinical efficacy of such interventions. In addition to the direct effect of nutrition in terms of micro- and macronutrient content, nutritional formulations may exert an effect via the physiological response to feeding. Here, we highlight the key role of postprandial reabsorbed bile acids in attenuating both the inflammatory response and autophagy. These observations suggest that not all patients would benefit from aggressive nutritional support.

Immunologists have recently taken note of the fact that a host not only resists infection, but also exhibits a capacity to manage the pathology associated with such infection – a concept referred to as tolerance. Here we explore how the tolerance/resistance (T/R) framework can be implemented within an oncological context and explore a number of implications. In particular, the T/R framework distinguishes between pathology manifesting from extensive tumor burden, versus cancers intrinsically expressing a more pathogenic phenotype. Consequently, the T/R framework provides novel methodology in studying the nature of cancer pathology and for marker identification. Additionally, this framework may aid in redefining the therapeutic end point under suitable circumstances: establishing cancer as a chronic, manageable disease.

In the first part of this study, the possible role of essential total fatty acids and their metabolites during cervical carcinogenesis was investigated. Since membrane lipids play a key role in cell proliferation and differentiation, disturbances in the fatty acid compositions of cell membranes and the modulation of membrane fatty acid compositions received attention in several in vitro studies. There are, however, no reported studies where the actual total and free (unesterified) fatty acid compositions have been determined during the different stages of cervical carcinogenesis. In part I of this ex vivo study, the total fatty acid compositions of normal tissue, intraepithelial and infiltrating lesions of the cervix were compared. The fatty acid profiles that were determined make it possible to speculate about the metabolic pathways followed during cervical carcinogenesis. Lipids were extracted from biopsies of normal tissue (n = 36), cervical intraepithelial lesions (n = 47) and infiltrating lesions (n = 47). Samples, from which the total fatty acid compositions were determined, were saponified, methylated and analysed by gas liquid chromatography (GLC). Essential fatty acid deficiency (EFAD) in the intraepithelial lesions, compared with normal tissue (linoleic acid, P < 0.01), and infiltrating lesions, compared with intraepithelial lesions (linoleic acid and arachidonic acid, P < 0.01) were observed. High levels of oleic acid were also observed when infiltrating lesions were compared with normal tissue (P < 0.01). This EFAD in cancer cells may result in many defective cell mechanisms. Although there are many risk factors for cervical cancer, the human papilloma virus has emerged over the past decade as the leading candidate to be an aetiological factor. There is ample evidence that human viral infections are associated with reduced levels of linoleic acid and thus participate in the depletion of essential fatty acids in cancer cells.

Estrogen, an endogenous female sex hormone, plays a significant role in promoting the progression of estrogen receptor positive (ER+) breast cancer cells by enhancing proliferation, inhibiting apoptosis, and facilitating invasion and metastasis. Conversely, melatonin, a hormone with well-documented anti-tumourigenic properties, demonstrates the ability to counteract these cancer-promoting effects. Dysregulated estrogen and melatonin levels have been shown to increase breast cancer risk. Additionally, both hormones decline during ageing and it has been hypothesised that ageing associated decline in melatonin is a risk factor for cancer. While the individual effects of estrogen and melatonin on cancer have been extensively studied, emerging evidence suggests that these hormones exert opposing influences on various cancer hallmarks. To explore this phenomenon, we conducted a study using the MCF-7 ER+ breast adenocarcinoma cell line to investigate whether melatonin can attenuate the protumourigenic effects exerted by 17β-estradiol (E2). Our findings revealed that E2 stimulated cell proliferation and migration, whereas the addition of melatonin attenuated these effects. Moreover, the combination of melatonin with E2 demonstrated a potential to promote apoptosis in breast cancer cells. These results shed light on the capacity of melatonin to mitigate the protumourigenic effects of estrogen in ER+ breast cancer cells. Importantly, it opens new avenues for considering melatonin supplementation as a preventive and therapeutic strategy for ER+ breast cancer in menopausal individuals and those with cancer. Future studies should delve deeper into these findings to fully comprehend the underlying mechanisms and establish the clinical relevance of melatonin in breast cancer prevention and treatment.

Capacitive biosensors have shown promise in detecting surface modifications and changes in analyte as a result of biological events on a transducer surface. The development of an impedance analyser with complex capacitance analysis that has been tailored to low frequency biosensing applications using gold interdigitated electrodes, is discussed in this paper. The designed device bridges the gap between expensive and complex analysers that are used for research, and a simple device that can directly compute complex capacitance and can be used in point-of-care environments. The device was calibrated, and results validated against the PalmSens4. Impedance and capacitance spectroscopy tests were run comparing different dilutions of phosphate buffered saline. The complex capacitance computation showed an error of 1.222% and 0.656% for the real and imaginary components respectively. The designed impedance analyser successfully differentiated between changes in concentration of phosphate buffered saline, and therefore has potential for use in the detection of various proteins.

The purpose of the third part of this study is to construct a basic lipid model (this includes information regarding total and free saturated, monounsaturated and polyunsaturated fatty acid contents, as well as total and free fatty acid saturation and double bond indexes, and comparisons of total and free n-3, n-6, n-7 and n-9 fatty acids in normal epithelial tissue, and intraepithelial and infiltrating lesions of the cervix) which, together with the individual total and free fatty acid profiles given in parts I and II of this study, should provide an understanding of the turnover of total and free acids, especially essential fatty acids, during cervical carcinogenesis. Such information can serve as a sound basis for further studies in an attempt to access this disease process. We observed an increase in monounsaturated fatty acid values in cancer tissue compared with normal tissue and a decrease of saturated fatty acid values in cancer tissue compared with normal tissue. Based on our observations, we speculate that because of the depletion of polyunsaturated fatty acids, monounsaturated fatty acid are synthesized to compensate for this loss; a possible source for the monounsaturated fatty acids are the saturated fatty acids via elongation and/or desaturation. Of particular interest is the n-3 fatty acid docosahexaenoic acid, the most unsaturated lipid in the biological systems, detected in very small amounts only in cancer cells of the cervix.

Oxygen deprivation is a key hallmark within solid tumours that contributes to breast-tumour pathophysiology. Under these conditions, neoplastic cells activate several genes, regulated by the HIF-1 transcription factor, which alters the tumour microenvironment to promote survival - including resistance to cell death in therapeutic attempts such as doxorubicin (Dox) treatment.We investigated HIF-1ɑ as a therapeutic target to sensitize breast cancer cells to Dox treatment. Under both normoxic (21% O2) and hypoxic (∼0.1% O2) conditions, the HIF-1 inhibitor, 2-methoxyestradiol (2-ME), was investigated as an adjuvant for its ability to alter MCF-7 cell viability, apoptosis, autophagy and molecular pathways which are often associated with increased cell survival.Here we observed that an inverse relationship between HIF-1ɑ and apoptosis exists and that Dox promotes autophagy under hypoxic conditions. Although adjuvant therapy with 2-ME induced an antagonistic effect in breast cancer cells, upregulated HIF-1ɑ expression in a hypoxic environment promotes treatment resistance and this was attenuated once HIF-1ɑ gene expression was silenced.Therefore, highlighting the identification of possible hypoxia-targeting therapies for breast cancer patients can be beneficial by promoting more favourable treatment responses.

Pancreatic cancer has one of the highest cancer mortality rates, as it is often detected in late stages, when unresectable tumours are present.Researchers have identified a biomarker associated with the early detection of pancreatic cancer, called Carbohydrate Antigen 19-9 (CA19-9)-researchers have recommended it for pancreatic cancer screening, and for the monitoring of the efficacy of pancreatic cancer treatments.The development of a biosensor for the detection of CA19-9 is discussed in this paper.The biosensor uses capacitive spectroscopy on gold interdigitated electrodes.This electrochemical transducer mechanism was selected as appropriate due to its increased popularity in point-of-care applications.Mouse monoclonal anti-CA19-9 antibodies were covalently bound to the gold surface using cysteamine hydrochloride and glutaraldehyde, and immobilization was verified with a Zeiss AxioObserver fluorescence microscope.Next, the antigen was prepared in different concentrations, and added to the prepared electrodes.Impedance spectroscopy was carried out using the PalmSens4 Electrochemical Interface, where five different concentrations of CA19-9 were detected in this process.The concentrations ranged from 10 U/mL to 300 U/mL, which includes the threshold concentration of CA19-9 for the detection of pancreatic cancer, of 37 U/mL.This biosensor is, therefore, suited to detect the CA19-9 concentrations needed for pancreatic cancer screening.

Glucocorticoids (GC) are commonly used as anti-inflammatory and immunosuppressive therapy by approximately 1% of the total adult population. Glucocorticoid therapy has also been used in non-autoimmune and non-inflammatory conditions such as acute myocardial infarction, angina, endocarditis as well as in invasive cardiology, coronary interventions and cardiopulmonarybypass surgery. Despite ample evidence for GC’s role as a natural, physiologic regulator of the immune system, little is known about the molecular events induced by GCs during a stress response. Autophagy is a survival mechanism which is upregulated in response to stress in the cell. It has been described as the cell’s major adaptive strategy in response to a multitude of extracellular stresses, such as nutrient deprivation, mitochondrial damage, endoplasmic reticulum stress or infection. Conserved in all eukaryotes, it is mediated by a unique organelle, the autophagosome, which, under inclusion of cytoplasmic cargo, fuses with lysosomes in order to yield recyclable nutrient metabolites. Basal autophagic activity plays a vital role in maintaining homeostasis during cellular stress. Its malfunction has been implicated with human pathologies such as heart disease, neurological storage disease and cancer.

Background: Red palm oil (RPO) is known as a potent anti-oxidant rich oil. Previous results have shown that RPO can protect against the consequences of ischaemia/reperfusion. It has been shown that RPO offered protection via the NO-cGMP pathway during ischaemia (Esterhuyse et al., 2006). Hypothesis: RPO may protect against ischaemia/reperfusion injury via the PKB/Akt and MAPK signalling pathways. Methods: Male Wistar rats were fed RPO for 6 weeks. Hearts were excised and mounted on a working rat heart perfusion system. Hearts were exposed to ischaemia for 25 min and reperfused for 20 min. Functional recovery was measured and biochemical analysis was performed in a different set of freeze clamped hearts to determine the degree of kinase phosphorylation. Results: Aortic output recovery was improved (72.1±3.2% in RPO vs. 54.0±3.2% in control, p<0.05). No changes in activation of PKB/Akt and p38 could be found during ischaemia. The improved functional recovery was associated with an increased phosphorylation of PKB/Akt(Ser473) and p38 during reperfusion. During reperfusion RPO significantly decreased phosphorylation of both JNK54 and JNK46 and attenuated PARP cleavage. Discussion: The results in this study show that dietary RPO supplementation can protect against the consequences of ischaemia/reperfusion. This protection may be caused by the ability of RPO to increase phosphorylation of PKB/Akt and p38 MAPK and dephosphorylation of JNK, which might be associated with inhibition of apoptosis.

Despite major progress in the imaging of gout, it is unclear which domains these techniques can evaluate and whether imaging modalities have the potential to provide valid outcome measures. The aim of this study was to assess the use of imaging instruments in gout according to the Outcomes in Rheumatology Clinical Trials (OMERACT) filter to inform the development of imaging as an outcome measure.A systematic literature search of imaging modalities for gout was undertaken. Articles were assessed by two reviewers to identify imaging domains and summarize information according to the OMERACT filter.The search identified 78 articles (one abstract). Modalities included were conventional radiography (CR) (16 articles), ultrasound (US) (29), conventional computed tomography (CT) (11), dual energy computed tomography (DECT) (20), and magnetic resonance imaging (MRI) (16). Three domains were identified as follows: urate deposition, joint damage, and inflammation. Although sufficient data were available to assess feasibility, validity, and reliability, comprehensive assessment of discrimination was not possible due to the paucity of prospective imaging studies. CR is widely accessible, inexpensive with a validated damage scoring system. US and MRI offer radiation-free methods of evaluating urate deposition, damage and inflammation, but may be limited by accessibility. DECT provides excellent definition of urate deposition and bone damage, but has restricted availability and requires radiation.Imaging methods can detect urate deposition, damage, and inflammation in gout. More than one modality may be required depending on the domains and therapeutic agent of interest. No single imaging method currently fulfils all aspects of the OMERACT filter for any domain.

Impaired autophagic machinery is implicated in heart disease, which still remains one of the leading causes of mortality and morbidity worldwide. Although basal levels of autophagy are required for cardiomyocyte survival, dysregulation of autophagy has been linked to a change in susceptibility to cell death. Autophagy is a catabolic process that degrades long-lived proteins by dynamic rearrangements of membrane structures, sequestering proteins and damaged organelles into autophagosomes. In the diseased myocardium, the majority of cell death that manifests in the infarction occurs during ischemia, where autophagy, apoptosis, and necrosis are inextricably and dynamically linked to one another. Recent evidence suggests that the control of autophagic flux during myocardial disease pathogenesis can be utilized for therapeutic gain. Although we have progressed in understanding the molecular machinery and regulation of the autophagic pathway, many unanswered questions remain with regard to the mechanisms that determine cellular fate in the diseased myocardium. Here we provide an overview addressing protective and detrimental functions of autophagy in the heart. By highlighting the role of autophagy, and its flux and metabolism in the context of energetic sensing in the myocardium, this chapter discusses possible applications of current understanding, challenges, and unanswered questions regarding the treatment of heart disease.