Activation Of Extrinsic Apoptotic Pathway Research Articles

Abstract P2077: Caspase8ap2 M6a Mrna Methylation Enhances Cardiomyocyte Apoptosis Following Myocardial Ischemia

Objective: Heart failure is the leading cause of morbidity and mortality in the United States. Cardiomyocyte death is one of the critical mediators of cardiac dysfunction following ischemic injury. However, the role of posttranscriptional regulation on cell-death-associated genes is not well explored during ischemic heart failure. N6-methyladenosine (m6A) is the most abundant and conserved chemical modification found in eukaryotic mRNA and is associated with mRNA synthesis and metabolism. We have recently shown that m6A RNA methylation regulates the angiogenic potential of endothelial cells following ischemic injury. However, the role of m6A mRNA methylation in cardiac cell death following ischemic injury is not well defined. Methods: Cardiac function (using echocardiography) was measured at baseline and four weeks post-MI in mice. In the end, mice were euthanized, and hearts were excised for biochemical and histological analysis. Cardiac cell death was assessed in H9C2 cells and cardiomyocytes using FACs (Annexin V and PI) and TUNNEL assay. The Casp8AP2 stability were measured using actinomycin D assay. To demonstrate the molecular mechanism, METTL3 was inhibited using siRNA or shRNA. Results: The cardiac function was significantly reduced following MI. Enhanced m6A mRNA methylation was observed in mice hearts following MI. Histological (TUNEL assay) and biochemical (WB: Caspase 8, cleaved cas8, caspase3, cleaved caspase3, Casp8AP2) analysis showed increased myocytes apoptosis in mice following MI. At the molecular level, hypoxic stress (nutrient depiviation+1%O2) induces global m6A mRNA methylation, which leads to Cas8AP2 gene stability and activation of extrinsic apoptotic pathways in cardiomyocytes in vitro. In contrast, METTL3 inhibition significantly reduced Cas8AP2 mRNA methylation, enhanced its degradation, and ultimately reduced cardiomyocyte cell death. Conclusion: In conclusion, we have shown that increased Casp8AP2 m6A RNA methylation enhances its stability and activates caspase-3 cleavage, which eventually leads to cardiomyocyte death following myocardial ischemia. The reduction of the RNA methylation through METTL3 inhibition (methyltransferase) reduced hypoxia-induced apoptosis of cardiomyocytes.

Regulatory T cells are less sensitive to glucocorticoid hormone induced apoptosis than CD4+ T cells

Earlier we have reported that thymic regulatory T cells (Treg) are resistant to in vivo glucocorticoid hormone (GC)-induced apoptosis, while the most GC-sensitive DP thymocytes died through the activation of mitochondrial apoptotic pathway. Here we analyzed the apoptosis-inducing effect of high dose (10–6 M) in vitro dexamethasone (DX) treatment in mouse thymic- and splenic Tregs and CD4+ T cells. Activation of both extrinsic and intrinsic apoptotic pathways started after 2 h of DX treatment in CD4 SP thymocytes and was 3 × higher than in CD4+ splenocytes, while in Treg cells, weak activation of the extrinsic apoptotic pathway started only after 3 h. We also investigated the expression of 21 apoptosis-related molecules using a protein array and found higher level of both pro-and anti-apoptotic molecules in Tregs compared to CD4+ T cells. 4 h in vitro DX treatment induced upregulation of most apoptosis-related molecules both in Tregs and CD4+ T cells, except for the decrease of Bcl-2 expression in CD4+ T cells. We found high basal cytosolic Ca2+ levels in untreated Treg cells, which further increased after DX treatment, while the specific TCR-induced Ca2+ signal was lower in Tregs than in CD4+ T cells. Our results suggest that in the background of the relative apoptosis resistance of Treg cells to GCs might be their high basal cytosolic Ca2+ level and upregulated Bcl-2 expression. In contrast, downregulation of Bcl-2 expression in CD4+ T cells can explain their higher, DX-induced apoptosis sensitivity.

Hydroxychavicol sensitizes imatinib-resistant chronic myelogenous leukemia cells to TRAIL-induced apoptosis by ROS-mediated IAP downregulation.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of cytokine superfamily, induces apoptosis in a number of tumor cells through the activation of extrinsic apoptotic pathway but shows little or no cytotoxicity toward normal cells. However some tumor cells are inherently resistant to TRAIL-mediated apoptosis, which needs to be addressed to establish TRAIL as a potential chemotherapeutic drug. In this study, our aim was to manipulate TRAIL-apoptosis pathway by hydroxychavicol (HCH), a polyphenol from Piper betel leaf, for the induction of apoptosis in TRAIL resistant chronic myeloid leukemia cell. When imatinib-resistant K562 cells were treated with HCH, it made these K562 cells sensitive to TRAIL. It was observed that HCH downregulated antiapoptotic proteins XIAP and FLIP, whereas the expression of TRAIL receptors, DR4 and DR5, remains unchanged. Moreover, we observed that reactive oxygen species or ROS played a crucial role in the downregulation of FLIP and XIAP because ROS scavenger significantly reversed the decrease of XIAP, and FLIP. Ubiquitin-proteasome pathway was observed to play a crucial role in the downregulation of XIAP and FLIP, as proteasomal inhibitor MG132 significantly reversed the downregulation of XIAP and FLIP. In conclusion, this study demonstrates the combinatorial treatment of TRAIL and HCH as promising alternative therapeutic approach to treat the imatinib-resistant leukemia, which are also resistant to TRAIL.

Abstract 5841: Ceramide synthase-6 binding to CD95/Fas confers resistance to chemotherapy in T-cell acute lymphoblastic leukemia cells

Abstract Ceramide synthase (CERS) produces ceramides, key intermediators in the biosynthesis of complex sphingolipids and play an important role in cell growth, differentiation, apoptosis, and senescence. Six isoforms of ceramide synthases (CERS1-6) with varying substrate specificity generate ceramides of diverse chain lengths. In normal tissues, CERS2, synthesizing ceramides with C24 acyl chain (C24-Cer), is highly expressed in most of the tissues while CerS6 generates C16-Cer with low and tissue-specific distribution. Our data showed that CERS6 and C16-Cer levels were significantly higher in acute lymphoblastic leukemia (ALL) cells in comparison to peripheral blood mononuclear cells and T-Lymphocytes derived from healthy human volunteers. The purpose of this study was to investigate the role of CERS6 in chemo resistance in T-ALL models. Stable knockdown/overexpression of CERS6 was achieved by lentiviral vector transduction. Ceramide levels were measured by LC/MS. Cytotoxicity of conventional and investigational anti-leukemia agents was evaluated in T- ALL cells with altered CERS6 levels using DIMSCAN assay. CERS6 knockdown significantly decreased C16-Cer by 4-fold (p<0.01) while CERS6 overexpression increased C16-Cer by 2-fold (p<0.05). CERS6 knockdown in CCRF-CEM cells increased their sensitivity to a pan BCL-2 inhibitor, ABT-737 as well as a glucocorticoid, dexamethasone (DXM). The percent survival of CCRF-CEM cells with CERS6 knockdown treated with ABT-737 (100nM) or DXM (100nM) was 5% and 2% at 72h while cells transduced with non-targeting shRNA showed survival of 87% and 33% (p<0.001). In CCRF-CEM cells with CERS6 overexpression, the percent survival, when treated with ABT-737 (300nM) or DXM (300nM) for 72h, was significantly higher relative to the cells transduced with vector control: 39% vs 2% for ABT-737 and 33% vs 0.4% for DXM, p<0.01. Higher cleavage of PARP, Caspase 3 and Caspase 8 was seen in ABT-737 or DXM treated cells with CERS6 knockdown, which was reversed by the overexpression of CERS6. Sensitivity to ABT-737 in CERS6 knockdown cells was significantly reduced by a Caspase 8 inhibitor, suggesting that CERS6 alters ALL cell sensitivity to chemotherapy via extrinsic pathway of apoptosis. Pull down of CERS6 in CCRF-CEM cells identified CD95/Fas, a death receptor leading to activation of extrinsic apoptotic pathway, as a CERS6 binding partner. In CERS6 knockdown cells, a higher level of FADD was detected in Fas pull-down samples when treated with ABT-737 compared with cells transduced with non-targeting shRNA, indicating CERS6 interferes Fas/FADD assembly in inducing apoptosis. Taken together, CERS6 modulation affected the sensitivity of T-ALL cells to chemotherapy by interfering with Fas/FADD assembly in extrinsic apoptotic pathway. CERS6 may serve as a biomarker in determining the efficacy of anticancer drugs acting via extrinsic apoptotic pathway in T-ALL. Citation Format: Dattesh U. Verlekar, Hwangeui Cho, Sung-Jen Wei, Min H. Kang. Ceramide synthase-6 binding to CD95/Fas confers resistance to chemotherapy in T-cell acute lymphoblastic leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5841.

Arachidonic acid activates extrinsic apoptotic pathway to enhance tumoricidal action of bleomycin against IMR-32 cells

Bleomycin is a commonly used anti-cancer drug in the management of a variety of cancers. Previously, we showed that arachidonic acid (AA) augmented the growth inhibitory action of bleomycin on IMR-32 (human neuroblastoma cells) in vitro by enhancing oxidative stress. Despite these results, the exact molecular mechanism of cytotoxic action of bleomycin and its augmentation by AA is not known. Our current study revealed that a combination of bleomycin and AA significantly enhanced the expression of FAS gene, which is involved in programmed cell death (apoptosis) and caspases 3 and 8 compared to either bleomycin or AA alone implying that activation of extrinsic apoptotic pathway plays a major role in their (bleomycin + AA) tumoricidal action. It is interesting to note that AA by itself enhanced the expression of FAS and caspases 3 and 8 compared to control. Since caspases have a role in inflammation, cell proliferation, tumour suppression, cell differentiation, neural development and axon guidance and ageing, this may explain pleiotropic actions of AA and their metabolites.

Executioner caspases and CAD are essential for mutagenesis induced by TRAIL or vincristine

Chemotherapy drugs interfere with cellular processes to generate genotoxic lesions that activate cell death pathways. Sustained DNA damage induced by these drugs can provoke mutations in surviving non-cancerous cells, potentially increasing the risk of therapy-related cancers. Ligation of death receptors by ligands such as TRAIL, and subsequent activation of extrinsic apoptotic pathways, also provokes mutations. In this study, we show that executioner caspase activation of the apoptotic nuclease CAD/DFF40 is essential for TRAIL-induced mutations in surviving cells. As exposure to chemotherapy drugs also activates apoptotic caspases and presumably CAD, we hypothesized that these pathways may also contribute to the mutagenesis induced by conventional chemotherapy drugs, perhaps augmenting the mutations that arise from direct DNA damage provoked by these agents. Interestingly, vincristine-mediated mutations were caspase and CAD dependent. Executioner caspases accounted for some of the mutations caused by the topoisomerase poisons doxorubicin and SN38, but were dispensable for mutagenesis following treatment with cisplatin or temozolomide. These data highlight a non-apoptotic role of caspases in mutagenesis mediated by death receptor agonists, microtubule poisons and topoisomerase inhibitors, and provide further evidence for a potential carcinogenic consequence of sublethal apoptotic signaling stimulated by anticancer therapies.

Oxidative imbalance and kidney damage in spontaneously hypertensive rats: activation of extrinsic apoptotic pathways.

In both humans and animals, essential hypertension acts as a risk factor for subclinical kidney damage and precedes renal dysfunction. Several lines of evidence indicate that hypertension and oxidative stress are closely related. The increase in vascular oxidative stress plays a key role in the pathophysiological consequences of hypertension, including kidney disease. Our study examined this issue in spontaneously hypertensive rat (SHR), a reliable model of essential hypertension. We used SHR 20 weeks old when hypertension is stably developed, vascular remodeling started, but kidney function is preserved. We examined plasmatic pro-oxidant and antioxidant status showing a significant alteration in oxidative balance in SHR. As index of oxidative damage, we evaluated lipid peroxidation in kidney, liver, and skeletal muscle, detecting a significant rise in lipid peroxidation levels in all SHR tissues, particularly relevant in kidney. In addition, we analyzed the expression of cytoplasmic antioxidant enzymes, superoxide dismutase 1 (SOD1) and glutatione S-tranferasi P1 (GSTP1). In SHR liver, SOD1 expression slight increased while we have not detected any variation in other tissues. Concerning GSTP1, SHR renal tissues did not display variations in enzyme expression, while in the other tissues, we observed a significant increase in both monomeric and pro-apoptotic dimeric form of the enzyme. By analyzing apoptotic signal, we founded c-Jun N-terminal kinase (JNK) activation in all SHR tissues, but only kidney presented extrinsic apoptotic pathway activation. Our results suggest that, in hypertensive animals with preserved renal function, despite the remarkable oxidative damage of renal tissues, only the extrinsic apoptotic pathway is activated.

Remote Limb Preconditioning Generates a Neuroprotective Effect by Modulating the Extrinsic Apoptotic Pathway and TRAIL-Receptors Expression.

As remote limb preconditioning (RPC) ameliorates brain damage after ischemic cerebral stroke (ICS), the purpose of the present study was to explore the molecular mechanisms in the course of RPC. Results of TUNEL staining and cleaved caspase-3 expression showed that ischemia-induced neuronal apoptosis was inhibited by RPC. The expression changes in cleaved caspase-8, cFLIP, Bid itself, and its truncated form represented that RPC suppressed the activation of extrinsic apoptotic pathway during ICS. Then, the level of cytoplasmic cytochrome c was also decreased by RPC. In addition, RPC might partially suppress TNF-related apoptosis-inducing ligand (TRAIL)-induced extrinsic apoptosis through downregulation of TRAIL death receptors and upregulation of TRAIL decoy receptors. As a counterproof, immunoneutralization of TRAIL in dMCAO rats resulted in significant restraint of tissue damage and in a marked functional recovery. Our data complemented the knowledge of RPC neuroprotective mechanism and provided new evidence for its clinical application.

The secreted protein acidic and rich in cysteine is a critical mediator of cell death program induced by WIN/TRAIL combined treatment in osteosarcoma cells.

Secreted protein acidic and rich in cysteine (SPARC) is a multi-functional protein which modulates cell-cell and cell-matrix interactions. In cancer cells, SPARC behaves as a tumor promoter in a number of tumors, but it can also act as a tumor suppressor factor. Our previous results showed that the synthetic cannabinoid WIN55,212-2 (WIN), a potent cannabinoid receptor agonist, is able to sensitize osteosarcoma MG63 cells to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis which is accompanied with endoplasmic reticulum (ER)-stress induction and the increase in autophagic markers. In the present investigation, we studied the role of SPARC in WIN/TRAIL-induced apoptosis demonstrating that WIN increased the level of SPARC protein and mRNA in a time-dependent manner. This event was functional to WIN/TRAIL-dependent apoptosis as demonstrated by RNA interfering analysis which indicated that SPARC-silenced cells were less sensitive to cytotoxic effects induced by the combined treatment. Our experiments also demonstrate that SPARC interacts with caspase-8 thus probably favoring its translocation to plasma membrane and the activation of extrinsic apoptotic pathway. In conclusion, to the best of our knowledge, our results are the first to show that WIN-dependent increase in the level of SPARC plays a critical role in sensitizing osteosarcoma cells to TRAIL action.

Activity of ABT-199 and Acquired Resistance in Follicular Lymphoma Cells

ABT-199, a platelet-sparing pro-apoptotic BH3 mimetic shows promise in the treatment of B-cell malignancy such as chronic lymphocytic leukemia (CLL). Given the central role of BCL2 expression in follicular lymphoma (FL), we studied the effects of ABT-199 on proliferation, induction of apoptosis, and signaling pathways in the t(14;18)+ FL cell lines WSU-FSCCL and FC-TxFL2 and in primary FL cells. We also developed an ABT-199-resistant FC-TxFL2 cell line by continuous treatment with ABT-199.The growth of tested cell lines and primary FL cells was significantly inhibited with nanomolar concentrations of ABT-199. FC-TxFL2 cells (IC50 = 7 nM) were more sensitive to ABT-199 treatment than WSU-FSCCL cells (IC50 = 110 nM). The increased sensitivity was associated with higher levels of BIM pro-apoptotic protein. ABT-199 induced rapid phosphorylation of JNK kinase and substantial decrease of mitochondrial potential in FC-TxFL2 cells. Inhibition of JNK activation further augmented the apoptosis induced with ABT-199. All other tested anti-apoptotic (such as BCL-2, BCL-xL, MCL-1) and pro-apoptotic proteins (such as Bax, Puma, Bak, Bad, Noxa, Bid, Bok) were similarly expressed in both cell lines and did not change upon ABT-199 treatment. Surprisingly, cleavage of Bid was observed, which points also to activation of extrinsic apoptotic pathway. In cells with acquired resistance after continuous ABT-199 exposure, we identified increased levels of MCL-1, and elevated phosphorylation of BCL-2 on T56 and of AKT on S473, but also evidence for increased autophagy, as assessed by LC3BI/II expression, compared to parental cells. These cells remained resistant several weeks after omitting ABT-199 from culture medium.In a FL murine xenograft model, ABT-199 showed anti-tumor activity. Treatment significantly decreased tumors volumes compared to untreated mice. Subsequently isolated xenograft FC-TxFL2 cells previously exposed to ABT-199 showed increased resistance to ABT-199. However, unlike in vitro exposed cells, the resistance quickly diminished after cultivation without ABT-199. Next, the ability to overcome ABT-199 resistance with epigenetic modifiers such as decitabine and vorinostat was tested. A strong synergistic effect in potentiation of apoptosis with these therapeutics was observed.Despite the theoretic rationale for BH3 mimetic activity in FL, activity in patients with FL has not been as dramatic as has been observed for CLL. Relatively easily acquired resistance observed in our model may explain this failure. Therefore, combinations that can prevent resistance acquisition should be intensively studied and moved quickly to clinical trials. DisclosuresSmith:Abbvie: Research Funding.

Effects of gastrokine‑2 expression on gastric cancer cell apoptosis by activation of extrinsic apoptotic pathways.

Gastrokine-2 is a putative gastric cancer-specific tumor suppressor gene, the loss of which is known to be involved in the development and progression of gastric cancer, and restoration of gastrokine-2 expression inhibits growth of gastric cancer cells in vitro. However, the underlying mechanism of these effects requires elucidation. In the present study, expression patterns of gastrokine-2 protein were examined in gastric cancer tissues and cell lines. Expression of gastrokine-2 was restored in gastric cancer cells in order to assess its effect on cell viability, apoptosis and gene expression. A total of 76 gastric cancer tissues with corresponding normal mucosae samples, and two gastric cancer cell lines (SGC-7901 and AGS) were subjected to western blot analysis of gastrokine-2 expression. SGC-7901 cells were transiently transfected with gastrokine-2 cDNA and then treated with anti-CD95 and/or anti-Fas antibodies prior to analysis of cell viability, apoptosis and gene expression levels. Expression of gastrokine-2 protein was reduced or absent in gastric cancer tissues and gastric cancer cell lines. Following restoration of gastrokine-2 expression, the protein expression level of Fas was significantly increased, but no marked change was observed in the levels of bcl-2 and Bax proteins. Expression of gastrokine-2 protein reduced gastric cancer cell viability and induced apoptosis. Activity of caspase-3 and caspase-8 was increased, but caspase-9 activity remained unchanged in the SGC-7901 cells. Reduction or knockout of gastrokine-2 protein expression may contribute to gastric cancer development or progression, as the current study demonstrated that restoration of gastrokine-2 expression induces apoptosis of gastric cancer cells through the extrinsic apoptosis pathway.

Activation of Extrinsic Apoptotic Pathway From Bladder Biopsy in Patients With Interstitial Cystitis/Painful Bladder Syndrome

To investigate the intrinsic or extrinsic pathway of apoptosis from bladder biopsy that was performed in interstitial cystitis/painful bladder syndrome (IC/PBS). Although previous studies have reported observations of the dysfunction, denudation, and thinning of the bladder urothelium in patients associated with an increase of cell apoptosis, the molecular mechanism is unclear. The study group consisted of 32 patients with IC/PBS, and the control group consisted of 12 volunteers without any symptoms of IC. Bladder biopsies were obtained from both the groups. The expression of apoptosis-associated proteins was observed by detecting the Bcl-2/Bax ratio and the levels of cleaved caspase-9, Fas, cleaved caspase-8, and cleaved caspase-3 to differentiate intrinsic or extrinsic pathway. The data were analyzed using Mann-Whitney U test. Increased levels of cleaved caspase-3 were found in the IC/PBS group relative to the control group (P <.05). The levels of the extrinsic apoptotic pathway proteins, Fas and cleaved caspase-8, were also increased in the study group compared with the control group (P <.05). There was no significant difference in the levels of the intrinsic apoptotic pathway proteins, including cleaved caspase-9 and the Bcl-2/Bax ratio, between the control and study groups. Our findings demonstrate the activation of extrinsic apoptotic pathway from bladder biopsy in patients with IC/PBS. This study might help us to clarify the molecular changes and lead to a better understanding of this bladder disease.

Cflar Genetic Variant Resulting In Alternate Splicing Determines Isoform Specific Production Of H-Cflar Protein and Is Predictive Of Cellular Cytoxicity Of Triptolide In Hapmap Lymphoblast and AML Cell Lines

Triptolide is a therapeutic diterpenoid derived from the Chinese herb Tripterygium wilfordii Hook f. It has shown to have immunosuppressive, anti-inflammation and antineoplastic effect Triptolide also serves as a potent inhibitor against different cancer types by regulating/arresting various cell signaling mechanisms such as inducing apoptotic signals by activation of pro-apoptotic proteins, inhibiting NFkB and c-KIT pathways, suppressing the Jak2 transcription, activating MAPK8/JNK signaling and modulating the heat shock responses. Further, as many of the current drugs target p53 mediated pathway to induce growth arrest or apoptosis, dysfunction or mutation in p53 leads to resistance to chemotherapy. In such patients, triptolide provide an edge over other chemotherapeutic drugs, due to its P53 independent mode of action. Genetic subsets for pancreatic cancer cells were found to be significantly correlated with triptolide sensitivity. In recent years, Epstein-Barr-virus transformed lymphoblastoid cell lines (LCLs) that are part of Interenational HapMap project (www.hapMap.org) have been used to study drug response and genetic variation correlation. Genotype data is publically available for these HapMap LCLs, thus allowing genotype-phenotype as well as genome-wide association studies. In the present study, we used HapMap LCLs derived from 60 unrelated subjects with Caucasian ancestry to identify genetic markers that are predictive of cellular sensitivity to triptolide. Our results identified a novel QTL with SNPs on chromosome 2 with significant association with triptolide cellular sensitivity. Detailed analysis of the top 100 highly significant SNPs (observed p<0.0001), identified genes such as CFLAR, PPIl3, Caspase 8, Caspase 10, NFkB and STAT6 that are involved in cell death receptor and apoptotic pathway. As per our findings, the CFLAR topped among all genes which have highest number of SNPs that are significantly associated to triptolide activity as well as showing most promising gene expression correlation It has been reported that CFLAR is a key regulator of apoptosis and chemo resistance in variety of tumors. Hence, our study provides first line of evidence that CFLAR genetic variation may play a crucial role in variety of cancer treatments with triptolide. An interesting SNP, rs10190751 observed in the vicinity of splicing region, is predicted to play a pivotal role in cell cytotoxicity induced by triptolide as it is directly correlated to CFLAR expression (Figure 1). In general, CFLAR is expressed as long (c-FLIPL), short (c-FLIPS) and Raji (c-FLIPR), splice variants in human cells. Variation in rs10190751 (G to A) changes the expression ratio of long to short form (Figure 1), which impacts chemosensitivity of cells towards triptolide. rs10190751 A/A produces a CFLAR-R form instead of CFLAR-S form which increase the sensitivity to triptolide to a significant level. It has been reported in AML that, ratio of CFLAR-L/CFLARs is a potential biomarker for overall survival in an adult patient cohort Overall higher median expression of CFLAR-L has been reported to be significantly correlated with lower overall survival. Our results of CFLAR-knock down in THP1 AML cell line significantly increased sensitivity towards triptolide. In summary, the results of the present study indicates that activation of extrinsic apoptotic pathway and CFLAR protein could be a major signaling cascade for triptolide mediated cytotoxicity. rs10190751, SNP present in CFLAR could be a potential genetic variation marker for a dose dependent effect of triptolide. Overall our results identified novel biomarkers with immense biological significance that can predict response to triptolide and would be of high clinical relevance to patients being treated by triptolide. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Role of ceramide in diabetes mellitus: evidence and mechanisms

Diabetes mellitus is a metabolic disease with multiple complications that causes serious diseases over the years. The condition leads to severe economic consequences and is reaching pandemic level globally. Much research is being carried out to address this disease and its underlying molecular mechanism. This review focuses on the diverse role and mechanism of ceramide, a prime sphingolipid signaling molecule, in the pathogenesis of type 1 and type 2 diabetes and its complications. Studies using cultured cells, animal models, and human subjects demonstrate that ceramide is a key player in the induction of β-cell apoptosis, insulin resistance, and reduction of insulin gene expression. Ceramide induces β-cell apoptosis by multiple mechanisms namely; activation of extrinsic apoptotic pathway, increasing cytochrome c release, free radical generation, induction of endoplasmic reticulum stress and inhibition of Akt. Ceramide also modulates many of the insulin signaling intermediates such as insulin receptor substrate, Akt, Glut-4, and it causes insulin resistance. Ceramide reduces the synthesis of insulin hormone by attenuation of insulin gene expression. Better understanding of this area will increase our understanding of the contribution of ceramide to the pathogenesis of diabetes, and further help in identifying potential therapeutic targets for the management of diabetes mellitus and its complications.

Activation of Extrinsic Apoptotic Pathway Has an Important Role in Islet Amyloid-Induced Beta-Cell Death in Human Islets

Recent studies have shown that islet amyloid - previously known to be a characteristic of type 2 diabetes (T2D) - also forms in human islets during culture and following transplantation into animal models of T1D. Islet amyloid is formed by aggregation of a small beta-cell peptide named islet amyloid polypeptide (IAPP; amylin). Formation of IAPP (proto)fibrils likely contributes to beta-cell death in all three conditions. The molecular mechanism(s) by which IAPP mediates beta-cell death in primary islets has yet to be identified. The extrinsic (cell death receptor) pathway is one of the two major pathways that are primarily responsible for mediating apoptosis in most cellular systems. In the present study, we investigated the role of caspase-8 and −3, two key upstream and effector enzymes in the extrinsic pathway, in amyloid-induced beta-cell death. Freshly isolated human islets obtained from cadaveric donors were dispersed into single cells, tested for their viability (by Alamar blue), and cultured overnight in CMRL in poly-L-lysine coated plates to allow recovery. Islet cells were then treated with fibrillogenic human IAPP (hIAPP; 10 μM), non-fibrillogenic rat IAPP (rIAPP), or vehicle for up to 24 hrs. Activation of caspase-3 , -8, and apoptosis were assessed by immunocytochemistry. The number of double TUNEL/insulinpositive islet cells was significantly higher in hIAPP-treated cells compared to control cells (hIAPP: 15±3%, C: 3±2%, P<0.05, 24 h). This increase in TUNEL-positivity (apoptosis) closely correlated with the elevated number of double insulin/active caspase-8 positive cells (hIAPP: 10±3%, C: 2±1%, P<0.05, 8 h) and double insulin/active caspase-3 positive cells (hIAPP: 16±4%, C: 3±2%, P<0.05, 16 h). Treatment with non-fibrillogenic rat IAPP did not have any detectable effect on beta-cell apoptosis or activation of caspase-8 and −3. A specific peptide inhibitor of caspase-8 markedly reduced the number of TUNEL-positive beta-cells in hIAPP-treated islet cells. Interestingly, very low number of glucagon-positive (alpha cells) were TUNEL-positive in hIAPP-treated iset cells, suggesting that hIAPP-induced cell death is beta-cell specific. We conclude that the cytotoxic effects of hIAPP are mediated, at least partially, through activation of caspase-8 and its downstream enzyme caspase-3 in human islet beta-cells. Specific caspase inhibitors may protect human islets from the cytotoxic effects of amyloid formation during pretransplant culture period and following transplantation.