Sex-related differences in death control of somatic cells

In 2001, The United States Institute of Medicine (IOM) Committee on Understanding the Biology of Sex and Gender Differences concluded that ‘Sex…should be considered when designing and analysing studies in all areas and at all levels of biomedical and health-related research…’ and stated an apparent paradox i.e.: ‘every cell has a sex’ 1. Sex is defined as ‘the classification of living things, generally as male or female according to their reproductive organs and functions assigned by chromosomal complement’ whereas gender is defined as ‘a person's self representation as male or female, or how that person is responded to by social institutions based on the individual's gender presentation. Gender is rooted in biology and shaped by environment and experience’ 1. It is unchallenged that there are health differences between males and females and that social and cultural factors could contribute to the observed differences. Anyway, the sex-dependent differences also have a biological base which sometimes has not been deeply investigated. Scientists studying health differences between male and female aim to both considering social/cultural environment and investigating biological/molecular mechanisms different between sexes. Some experimental studies have elucidated important differences in cell death control 2. A sex disparity, in fact, has been shown both in the propensity to apoptosis and in the activation of the autophagic pathway. In the context of cell fate control, hormones represent important regulators of both apoptosis and autophagy. In the cardiovascular system, for example, oestrogens inhibit cardiomyocyte apoptosis by decreasing reactive oxygen species production and increasing intracellular antioxidants 3. Oestrogens may also indirectly control autophagy as they up-regulate urocortin 4, a neuropeptide hormone able to inhibiting autophagy in cardiomyocytes. Conversely, increasing evidence suggests possible adverse effects of androgens on the vasculature showing that androgens, as opposed to oestrogens, may worsen vascular dysfunction in men, thus contributing to sex-based differences in cardiovascular diseases 5. However, it is currently emerging that some cell death programs are differentially controlled by sex-related hormone-independent cellular genetics. Differences in cell death sensitivity in male and female may then occur in the absence of an hormonal context. This is not an immediately obvious finding; Penaloza C et al., 6 have shown that the apoptosis amount differs between the sexes in isolated embryonic cells exposed to similar conditions and this happens at embryonal stages where there are no hormonal influences. Previous studies had reported a sexual dimorphism in embryonic neuronal signal transduction pathways and consequently differences in cell survival 7. Death pathways in XX and XY cells have been poorly investigated as most studies have been performed on established cells lines often irrespective of their male or female origin. Recently, using freshly isolated cells from male and female individuals gave important information on sex disparity in cell fate control. Such sex specificity has been in part clarified thanks to cell culture models where sex steroids can be removed from the media. Even sex-related differences in caspase activation have been found to be independent on hormone exposure. More in detail, cell death occurring in cortical neurons after ischaemia proceeds predominantly via an apoptosis-inducing factor-dependent pathway (a caspase-independent pathway) in male neurons while proceeds via a cytochrome C-dependent pathway (a process mediated by caspase activation) in female neurons 8. In this context, a sex-specific microRNA expression after ischaemia has been described in in vivo studies. In particular, it has been demonstrated that microRNA-23a, by binding the mRNA of the caspase inhibitor named XIAP, induces its translational repression in females, leading to enhanced caspase signalling in the ischaemic female brain. This effect has been shown to be independent of circulating oestrogen levels 9. Sex differences in ischaemic brain injury and cerebrovascular regulation have been observed in clinical and experimental studies and an important determinant of such differences is also represented by the integrity of endothelial cells. In fact, endothelial function is improved in women compared with men, contributing to female cellular higher resistance after ischaemic brain injury. Gupta NC et al. 10 showed that female cerebrovascular endothelial cells express lower level of soluble epoxide hydrolase and consequently have higher levels of vasoprotective epoxyeicosatrienoic acids as compared with male endothelial cells. This study therefore presents a novel additional mechanism underlying differences between male and female cells in apoptotic response after oxygen-glucose deprivation, contributing to explain higher resistance observed in females as compared with males. This study remarks again that differences between male and female cells do not necessarily depend on the hormonal context but may be inherent the cells. We believe that this apparently paradoxical concept has not been sufficiently highlighted in the scientific literature. The present ‘Letter to the Editor’ therefore aims at underlining such an important issue which deserves more attention and discussion in the researchers' community. A practical consequence of sex-dependent discrepancies in cell death control is that cellular response to any stimulus or treatment, in any physiological or pathological context, may well depend on the sex of the cell line used; journals guidelines should therefore require authors to state in any case the sex of the cell lines used in any in vitro study. In addition, at least to some extent, sex-matched or sex-unmatched cell controls may be necessary in many experimental settings. In conclusion, sex-related differences in cell death mechanism may have strong implications for experimental studies and sexual dimorphism dependent on chromosomal rather than hormonal differences have important implications for planning preclinical studies and clinical interventions

c-Flip KO fibroblasts display lipid accumulation associated with endoplasmic reticulum stress

c-Flip proteins are well-known apoptosis modulators. They generally contribute to tissue homeostasis maintenance by inhibiting death-receptor-mediated cell death. In the present manuscript, we showthat c-Flip knock-out (KO) mouse embryonic fibroblasts (MEFs) kept in culture under starvation conditions gradually modify their phenotype and accumulate vacuoles, becoming progressively larger according to the duration of starvation. Large vacuoles are present in KO MEFs though not in WT MEFs, and are Oil Red-O positive, which indicates that they represent lipid droplets. Western blot experiments reveal that, unlikeWTMEFs, KOMEFs express high levels of the lipogenic transcription factor PPAR-γ. Lipid droplet accumulation was found to be associated with endoplasmic reticulum (ER) stress activation and autophagic modulation valuated by means of BIP increase, LC3 lipidation and AMP-activated protein kinase (AMPK) phosphorylation, and p62 accumulation. Interestingly, XBP-1, an ER stress-induced lipogenic transcription factor, was found to preferentially localize in the nucleus rather than in the cytoplasm of KO MEFs. These data demonstrate that, upon starvation, c-Flip affects lipid accumulation, ER stress and autophagy, thereby pointing to an important role of c-Flip in the adaptive response and ER stress response programs under both normal and pathological conditions

Cancer microenvironment and endoplasmic reticulum stress response

Different stressful conditions such as hypoxia, nutrient deprivation, pH changes, or reduced vascularization, potentially able to act as growth-limiting factors for tumor cells, activate the unfolded protein response (UPR). UPR is therefore involved in tumor growth and adaptation to severe environments and is generally cytoprotective in cancer. The present review describes the molecular mechanisms underlying UPR and able to promote survival and proliferation in cancer. The critical role of UPR activation in tumor growth promotion is discussed in detail for a few paradigmatic tumors such as prostate cancer and melanoma

Lipid storage and autophagy in melanoma cancer cells

Cancer stem cells (CSC) represent a key cellular subpopulation controlling biological features such as cancer progression in all cancer types. By using melanospheres established from human melanoma patients, we compared less differentiated melanosphere-derived CSC to differentiating melanosphere-derived cells. Increased lipid uptake was found in melanosphere-derived CSC vs. differentiating melanosphere-derived cells, paralleled by strong expression of lipogenic factors Sterol Regulatory Element-Binding Protein-1 (SREBP-1) and Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ). An inverse relation between lipid-storing phenotype and autophagy was also found, since microtubule-associated protein 1A/1B-Light Chain 3 (LC3) lipidation is reduced in melanosphere-derived CSC. To investigate upstream autophagy regulators, Phospho-AMP activated Protein Kinase (P-AMPK) and Phospho-mammalian Target of Rapamycin (P-mTOR) were analyzed; lower P-AMPK and higher P-mTOR expression in melanosphere-derived CSC were found, thus explaining, at least in part, their lower autophagic activity. In addition, co-localization of LC3-stained autophagosome spots and perilipin-stained lipid droplets was demonstrated mainly in differentiating melanosphere-derived cells, further supporting the role of autophagy in lipid droplets clearance. The present manuscript demonstrates an inverse relationship between lipid-storing phenotype and melanoma stem cells differentiation, providing novel indications involving autophagy in melanoma stem cells biology

A novel role of c-FLIP protein in regulation of ER stress response

Cellular-Flice-like Inhibitory Protein (c-FLIP) is an apoptosis modulator known to inhibit the extrinsic apoptotic pathway thus blocking Caspase-8 processing in the Death Inducing Signalling Complex (DISC). We previously demonstrated that c-FLIP localizes at the Endoplasmic Reticulum (ER) and that c-FLIP-deficient Mouse Embryonic Fibroblasts (MEFs) display an enlarged ER morphology. In the present study, we have addressed the consequences of c-FLIP ablation in the ER stress response by investigating the effects of pharmacologically-induced ER stress in Wild Type (WT) and c-FLIP-/- MEFs. Surprisingly, c-FLIP-/- MEFs were found to be strikingly more resistant than WT MEFs to ER stress-mediated apoptosis. Analysis of Unfolded Protein Response (UPR) pathways revealed that Pancreatic ER Kinase (PERK) and Inositol-Requiring Enzyme 1 (IRE1) branch signalling is compromised in c-FLIP-/- cells when compared with WT cells. We found that c-FLIP modulates the PERK pathway by interfering with the activity of the serine threonine kinase AKT. Indeed, c-FLIP-/- MEFs display higher levels of active AKT than WT MEFs upon ER stress, while treatment with a specific AKT inhibitor of c-FLIP-/- MEFs subjected to ER stress restores the PERK but not the IRE1 pathway. Importantly, the AKT inhibitor or dominant negative AKT transfection sensitizes c-FLIP-/- cells to ER stress-induced cell death while the expression of a constitutively active AKT reduces WT cells sensitivity to ER stress-induced death. Thus, our results demonstrate that c-FLIP modulation of AKT activity is crucial in controlling PERK signalling and sensitivity to ER stress, and highlight c-FLIP as a novel molecular player in PERK and IRE1-mediated ER stress response