Combination of Vildagliptin and Ischemic Postconditioning in Diabetic Hearts as a Working Strategy to Reduce Myocardial Reperfusion Injury by Restoring Mitochondrial Function and Autophagic Activity

Purpose: Diabetic hearts are resistant to cardioprotection by ischemic-postconditioning (IPostC). Protection of diabetic hearts and finding related interfering mechanisms would have clinical benefits. This study investigated the combination effects of vildagliptin (Vilda) and IPostC on cardioprotection and the levels of autophagy and mitochondrial function following myocardial ischemia/reperfusion (I/R) injury in type-II diabetic rats. Methods: Diabetes was established by high fat diet/low dose of streptozotocin and lasted for 12 weeks. The diabetic rats received Vilda (6 mg/kg/day, orally) for one month before I/R. Myocardial regional ischemia was induced through the ligation of left coronary artery, and IPostC was applied immediately at the onset of reperfusion. The infarct size was assessed by a computerised planimetry and left ventricles samples were harvested for cardiac mitochondrial function studies (ROS production, membrane potential and staining) and western blotting was used for determination of autophagy markers. Results: None of Vilda or IPostC but combination of them could significantly reduce the infarct size of diabetic hearts, comparing to control (P<0.001). IPostC could not significantly affect p62 expression level in diabetic hearts, but pre-treatment with Vilda alone (p<0.05) and in combination with IPostC (p<0.01) more significantly decreased p62 expression in comparison with corresponding control group. The expression of LC3B-II and LC3BII/LC3BI as well as mitochondrial ROS production were decreased significantly in treatment groups (p<0.001). Mitochondrial membrane depolarization was significantly higher and mitochondrial density was lower in untreated diabetic I/R hearts than treated groups (p<0.001). IPostC in combination with vildagliptin prevented the mitochondrial membrane depolarization and increased the mitochondrial content more potent than IPostC alone in diabetic hearts. Conclusion: Combination of vildagliptin and IPostC in diabetic hearts was a well-working strategy to reduce myocardial I/R damages by restoring mitochondrial membrane potential and ROS production and modulating the autophagic activity in I/R hearts

Modulation of lipolysis and glycolysis pathways in cancer stem cells changed multipotentiality and differentiation capacity toward endothelial lineage

Abstract Cancer stem cells obtain energy demand through the activation of glycolysis and lipolysis. It seems that the use of approached targeting glycolysis and lipolysis could be an effective strategy for the inhibition of cancer stem cells. In the current experiment, we studied the potential effect of glycolysis and lipolysis inhibition on cancer stem cells differentiation and mesenchymal–epithelial-transition capacity. Cancer stem cells were enriched from human ovarian cells namely SKOV3 by using MACS technique. Cells were exposed to Lonidamine, an inhibitor of glycolysis, and TOFA, a potent inhibitor of lipolysis for 7 days in endothelial differentiation medium; EGM-2 and cell viability was studied by MTT assay. At the respective time point, the transcription level of genes participating in EMT such as Zeb-1, -2, Vimentin, Snail-1, -2 and VE-cadherin were measured by real-time PCR analysis. Our data noted that the inhibition of lipolysis and glycolysis could decrease cell viability compared to the control of cancer stem cells. The inhibition of glycolysis prohibited the expression of Zeb-1, Snails, and Vimentin while increased endothelial differentiation rate indicated by the expression of VE-cadherin. In contrast, the inhibition of lipolysis increased EMT associated genes and reduced endothelial differentiation rate by suppressing the transcription of VE-cadherin. Notably, the simultaneous inhibition of glycolysis and lipolysis had moderate effects on the transcription of EMT genes. We concluded that the modulation of the metabolic pathway of glycolysis in ovarian CSCs is more effective than the inhibition of lipolysis in the control of angiogenesis potential and stemness feature

Promoter Methylation Status of Survival-Related Genes in MOLT- 4 Cells Co-Cultured with Bone Marrow Mesenchymal Stem Cells under Hypoxic Conditions

Objective DNA methylation is a well-studied epigenetic mechanism that is a potent arm of the gene expression controlling machinery. Since the hypoxic situation and the various cells of bone marrow microenvironment, e.g. mesenchymal stem cells, play a role in the in vivo and in vitro biology of leukemic cells, we decided to study the effects of hypoxia and mesenchymal stem cells (MSCs) on the promoter methylation pattern of BAX and BCL2 genes. Materials and Methods In this experimental study, the co-culture of MOLT-4 cells with MSCs and treatment with CoCl2 was done during 6, 12, and 24 hour periods. Total DNA was extracted using commercial DNA extraction kits, and sodium bisulfite (SBS) treatment was performed on the extracted DNA. Methylation specific polymerase chain reaction (MSP) was used to evaluate the methylation status of the selected genes’ promoter regions. Results The BAX and BCL2 promoters of untreated MOLT-4 cells were in partial methylated and fully unmethylated states, respectively. After incubating the cancer cells with CoCl2and MSCs, the MSP results after 6, 12, and 24 hours were the same as untreated MOLT-4 cells. In other words, the exposure of MOLT-4 cells to the hypoxia-mimicry agent and MSCs in various modes and different time frames showed that these factors have exerted no change on the methylation signature of the studied fragments from the promoter region of the mentioned genes. Conclusion Hypoxia and MSCs actually have no notable effect on the methylation status of the promoters of BAX and BCL2 in the specifically studied regions. DNA methylation is probably not the main process by which MSCs and CoCl2 induced hypoxia regulate the expression of these genes. Finally, we are still far from discovering the exact functional mechanisms of gene expression directors, but these investigations can provide new insights into this field for upcoming studies

Treatment of cancer stem cells from human colon adenocarcinoma cell line HT-29 with resveratrol and sulindac induced mesenchymal-endothelial transition rate

WOS: 000468934500006PubMed ID: 30758710In the current experiment, the combined regime of resveratrol and a Wnt-3a inhibitor, sulindac, were examined on the angiogenic potential of cancer stem cells from human colon adenocarcinoma cell line HT-29 during 7days. Cancer stem cells were enriched via a magnetic-activated cell sorter technique and cultured in endothelial induction medium containing sulindac and resveratrol. Expression of endothelial markers such as the von Willebrand factor (vWF) and vascular endothelial cadherin (VE-cadherin) and genes participating in mesenchymal-to-epithelial transition was studied by real-time PCR assay. Protein levels of Wnt-3a and angiogenic factor YKL-40 were examined by western blotting. ELISA was used to determine the level of N-acetylgalactosaminyltransferase 11 (GALNT11) during mesenchymal-endothelial transition. Autophagy status was monitored by PCR array under treatment with the resveratrol plus sulindac. Results showed that resveratrol and sulindac had the potential to decrease the cell survival of HT-29 cancer cells and the clonogenic capacity of cancer stem cells compared with the control (p<0.05). The expression of VE-cadherin and vWF was induced in cancer stem cells incubated with endothelial differentiation medium enriched with resveratrol (p<0.05). Interestingly, the Wnt-3a level was increased in the presence of resveratrol and sulindac (p<0.05). YKL-40 was reduced after cell exposure to sulindac and resveratrol. The intracellular content of resistance factor GALNT11 was diminished after treatment with resveratrol (p<0.05). Resveratrol had the potential to induce the transcription of autophagy signaling genes in cancer stem cells during endothelial differentiation (p<0.05). These data show that resveratrol could increase cancer stem cell trans-differentiation toward endothelial lineage while decrease cell resistance by modulation of autophagy signaling and GALNT11 synthesis.Tabriz University of Medical SciencesThis study was supported by a grant from the Tabriz University of Medical Sciences