Anti-inflammatory mechanisms of cannabinoids: an immunometabolic perspective.

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.

At the core of survival: autophagy delays the onset of both apoptotic and necrotic cell death in a model of ischemic cell injury.

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.

Cancer and Covid-19: Collectively catastrophic.

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-1ß, 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.

Dietary red palm oil protects the heart against the cytotoxic effects of anthracycline.

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.

Molecular regulation of autophagy in a pro-inflammatory tumour microenvironment: New insight into the role of serum amyloid A.

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.

Fatty acids: Adiposity and breast cancer chemotherapy, a bad synergy?

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.

Ischaemic preconditioning and TNF-alpha-mediated preconditioning is associated with a differential cPLA2 translocation pattern in early ischaemia.

Both the cytokine tumour necrosis factor-alpha (TNF-alpha) and the enzyme cytosolic phospholipase A2 (cPLA2) have been implicated in ischaemic injury. Apart from the induction of apoptosis, TNF-alpha also mediates cytoprotection when present in low concentrations. However, the relationship between TNF-alpha and cPLA2 activities during cytoprotection is poorly understood. Therefore, we examined the role of cPLA2 in TNF-alpha-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.

Proanthocyanidin from grape seeds inactivates the PI3-kinase/PKB pathway and induces apoptosis in a colon cancer cell line.

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 microg/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 Ser(473) phosphorylation. GSPE might thus exert its beneficial effects by means of increased apoptosis and suppression of the important PI3-kinase survival-related pathway.

Apoptosis is mediated by cytosolic phospholipase A2 during simulated ischaemia/reperfusion-induced injury in neonatal cardiac myocytes.

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.

Ex vivo study of MAPK profiles correlated with parameters of apoptosis during cervical carcinogenesis.

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 NH(2)-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.

The role of mTOR during cisplatin treatment in an in vitro and ex vivo model of cervical cancer.

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.

Comparison of the fatty acid compositions in intraepithelial and infiltrating lesions of the cervix: part I, total fatty acid profiles.

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.

Comparison of the fatty acid compositions in intraepithelial and infiltrating lesions of the cervix: part II, free fatty acid profiles.

In the second part of this study, the emphasis is on the free fatty acids during cervical carginogenesis, since they may reflect active cell metabolism during this disease process. Lipids were extracted from biopsies of normal epithelial tissue (N) (n=36), cervical intraepithelial lesions (CIL) (n=47), and infiltrating lesions (Ca) (n=47) of the cervix. Samples, from which the free fatty acid compositions were determined, were saponified, methylated and analysed by GLC. In accordance with results obtained on total fatty acid compositions, 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). As previously mentioned by us in part I of this study, with regard to possible disturbances in metabolic pathways based on the total fatty acid profiles during stages of cervical cancer, EFAD is prevalent during cervical carcinogenesis. This EFAD in cancer cells may result in many defective cell mechanisms, since fatty acids are associated with biochemical events such as lipid peroxidation, signal transduction and immune responses. The high level of oleic acid in cancer cells is known to activate PKC and thus contribute to the continous growth stimulus thought to exist in malignant cells. From a therapeutic viewpoint, substantial changes in the fatty acid composition of the membranes can be produced in cancer cells by selective fatty acid supplementation strategies. At present, modifications of the fatty acid compositions of cell membranes represent an experimental model that has promoted increased understanding of lipid transportation, membrane remodelling, and the relationship between membrane lipids and membrane function. By addressing factors responsible for insufficient essential fatty acid levels, carginogenesis may be prevented or treated. The clinical feasibility of using modification of fatty acids in tumours or cancer by diet or perfusion as an adjunct to standard therapies should be tested.

Comparison of the fatty acid compositions in intraepithelial and infiltrating lesions of the cervix: part III, saturated and unsaturated fatty acid profiles.

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.

AHNAK: the giant jack of all trades.

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.

Phosphatidylinositol-3-kinase (PI3K) activity decreases in C2C12 myotubes during acute simulated ischemia at a cost to their survival.

AIMS: 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. MAIN METHODS: Cells are grown in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS), and incubated under 5% CO(2) 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. KEY FINDINGS: 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. SIGNIFICANCE: An ischemic microenvironment leads to a reduction in PI3K activity with subsequent induction of apoptosis.

Autophagy in heart disease: a strong hypothesis for an untouched metabolic reserve.

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.