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Use of the conventional cancer chemotherapy (i.e. vincristine) is limited in tumor cells exhibiting pre-existing or acquired resistance. Here, we found that C6 ceramide (C6) dramatically sensitized vincristine's activity. In vitro, C6 and vincristine coadministration induced substantial necrosis and apoptosis in multiple human cancer cell lines, which were accompanied by a profound AMP-activated protein kinase (AMPK) activation, subsequent p53 activation, mTORC1 inactivation and Bcl-2/HIF-1α downregulation. Such synergistic effects were attenuated by AMPK inactivation through genetic mutation or short hairpin RNA silencing. Coadministration-activated p53 translocated to mitochondria, and formed a complex with cyclophilin-D, leading to mitochondrial permeability transition pore opening and cell necrosis. Disrupting p53-Cyp-D complexation through pharmacological or genetic means reduced costimulation-induced cytotoxicity. In vivo, a liposomal C6 was synthesized, which dramatically enhanced the antiproliferative activity of vincristine on HCT-116 or A2780 xenografts. Together, C6 sensitizes vincristine-induced anticancer activity in vivo and in vitro, involving activating AMPK-p53 signaling.

There is a growing body of literature suggesting the role of interactions between genes and the environment in development of type 2 diabetes mellitus (T2DM). However, the interplay between environment and genetic in developing and progressing T2MD is not fully understood. To determine the effects of high-glucose-lipid on the status of DNA methylation in beta cells, and clarify the mechanism of glucolipotoxicity on beta-cell deterioration, the DNA methylation profile was detected in beta-cells cultured with high-glucose-lipid medium. We utilized a high throughput NimbleGen RN34 CpG Island & Promoter Microarray to investigate the DNA methylation profile in beta-cells cultured with high-glucose-lipid medium. To validate the results of microarray, the immunoprecipitation (MeDIP) PCR was used to test the methylation status of some selected genes. The mRNA and protein expression of insulin and Tcf7l2 in these cells were quantified by RT-PCR and western blot, respectively. We have identified a lot of loci which experienced aberrant DNA methylation in beta-cells cultured with high-glucose-lipid medium. The results of MeDIP PCR were consistency to the microarray. An opposite regulation in transcription and translation of Tcf7l2 gene was found. Furthermore, the insulin mRNA and protein expression in beta-cells also decreased after cultured with high-glucose-lipid medium compared with the control cells. We conclude that chronic glucolipotoxicity could induce aberrant DNA methylation of some genes and may affect these genes expression in beta-cells, which might contribute to beta-cell function failure in T2DM and be helpful to explain, at least partially, the mechanism of glucolipotoxicity on beta-cells deterioration.

Summary Multidrug resistance protein 3 (MPR3), encoded by the ATP-binding cassette, subfamily C (CFTR/MRP), member 3 (ABCC3) gene, functions as an important drug efflux transporter. The ABCC3 –211C/T polymorphism is associated with decreased MRP3 mRNA expression, and low MRP3 mRNA expression is associated with increased clopidogrel response in patients. The aim of the present study was to determine whether the –211C/T polymorphism is associated with altered antiplatelet effects and clinical outcomes in clopidogrel-treated patients. A subcohort of 249 patients not carrying the CYP2C19*2, *3 or *17 variant was identified from a total of 617 consecutive clopidogrel-treated patients undergoing percutaneous coronary intervention and then categorized into three groups on the basis of their ABCC3 –211C/T genotype. Baseline data, clinical characteristics and DNA samples were collected for all patients. Light transmittance aggregometry was used to determine ADP-induced maximum platelet aggregation (MPA) in blood samples obtained from patients on Day 3 after starting daily clopidogrel maintenance doses. Genotyping of CYP2C19*2, *3 and *17 variants and the ABCC3 –211C/T polymorphism was performed using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. The primary clinical end-point was a definite stent thrombosis (ST) episode, whereas secondary end-points were other major adverse cardiovascular events within 12 months after stenting. There were no differences in MPA values according to ABCC3 –211C/T genotype. A multiple linear regression model revealed that the ABCC3 –211C/T polymorphism was not independently associated with ADP-induced MPA measurements; a multiple logistic regression model revealed that carrying the ABCC3 –211C allele was not associated with the risk of developing an ST event in clopidogrel-treated patients not harbouring CYP2C19*2, *3 and *17 variants. In conclusion, the ABCC3 –211C/T polymorphism seems not to be associated with altered antiplatelet effects and clinical outcomes in clopidogrel-treated patients.

Bleeding complications and delayed healing of gastric ulcer associated with use of clopidogrel is a common clinical concern; however, the underlying mechanisms remain to be determined. This study aimed to clarify whether clopidogrel could cause the damage of the human gastric epithelial cells and to further elucidate the mechanisms involved. After human gastric epithelial cell line GES-1 had been treated with clopidogrel (0.5-2.5 mM), the cell proliferation was examined by MTT assay, apoptosis was measured with DAPI staining and flow cytometry analysis, and the barrier function of the tight junctions (TJ) was evaluated by permeability measurement and transmission electron microscopy. Moreover, expression of the TJ proteins occludin and ZO-1 and the phosphorylation of the mitogen-activated protein kinases (MAPK) p38, ERK, and JNK were examined by western blot. In addition, three MAPK inhibitors specific to p38, ERK and JNK were used, respectively, to verify the signaling pathways responsible for regulating the expression of the TJ proteins being tested. Results showed that clopidogrel significantly increased dextran permeability, induced apoptosis, suppressed GES-1 cell viability, and reduced the expression of the TJ proteins (occludin and ZO-1), acting through p38 MAPK phosphorylation. Furthermore, these observed effects were partially abolished by SB-203580 (a p38 MAPK inhibitor), rather than by either U-0126 (an ERK inhibitor) or SP-600125 (a JNK inhibitor), suggesting that clopidogrel-induced disruption in the gastric epithelial cells is mediated by the p38 MAPK pathway. It is concluded that attenuated expression of the TJ proteins occludin and ZO-1 in human gastric epithelial cells could be involved in clopidogrel-induced gastric mucosal injury through activation of the p38 MAPK pathway.

Background CD147 is a widely distributed cell surface glycoprotein that belongs to the Ig superfamily. CD147 has been implicated in numerous physiological and pathological activities. Enriched on the surface of many tumor cells, CD147 promotes tumor growth, invasion, metastasis and angiogenesis and confers resistance to some chemotherapeutic drugs. In this study, we investigated the possible role of CD147 in the progression of gastric cancer. Methods Short hairpin RNA (shRNA) expressing vectors targeting CD147 were constructed and transfected into human gastric cancer cells SGC7901 and CD147 expression was monitored by quantitative realtime RT-PCR and Western blot. Cell proliferation, the activities of MMP-2 and MMP-9, the invasive potential and chemosensitivity to cisplatin of SGC7901 cells were determined by MTT, gelatin zymography, Transwell invasion assay and MTT, respectively. Results Down-regulation of CD147 by RNAi approach led to decreased cell proliferation, MMP-2 and MMP-9 activities and invasive potential of SGC7901 cells as well as increased chemosensitivity to cisplatin. Conclusion CD147 involves in proliferation, invasion and chemosensitivity of human gastric cancer cell line SGC7901, indicating that CD147 may be a promising therapeutic target for gastric cancer.

Chronic exposure to high levels of free fatty acid (FFA) has adverse effects on the function of pancreatic beta-cell, with a consequent increase in the production of reactive oxygen species. Poly (ADP-Ribose) polymerase (PARP-1) overactivation leads to massive NAD(+) consumption and ATP depletion with induction of cellular necrosis under high reactive oxygen species. In the present study, we investigated whether inhibitors of poly(ADP-ribose) polymerase were capable of protecting beta-cells from death induced by extended exposure to FFA.RINm5F cell line was cultured in the presence of FFA (palmitate) in order to induce apoptosis, and then cells were treated with low-potency poly(ADP-ribose) polymerase inhibitor (3-aminobenzamide) or potent poly(ADP-ribose) polymerase inhibitor (PJ34). In order to explore whether poly(ADP-ribose) polymerase inhibitors could inhibit the apoptosis induced by FFA, expression of PARP-1 was measured by RT-qPCR and Western blot, while the apoptosis of RINm5F cells were analyzed by flow cytometry and Tdt-mediated dUTP Nick-End Labeling(TUNEL).Low-potency poly(ADP-ribose) polymerase inhibitor (3-aminobenzamide) significantly suppressed the impaired insulin secretion and FFA-induced apoptosis (P0.01). However, potent poly(ADP-ribose) polymerase inhibitor (PJ34) had no significant effects on FFA-induced apoptosis (P0.05). Moreover, low-potency inhibitors of PARP-1 increased PDX-1 expression down-regulated by FFA-treatment.These findings suggested that low-potency inhibitors of poly(ADP-ribose) polymerases could protect rat RINm5F cell line against free fatty acid-induced apoptosis, and it was through regulatory pathway of regulating PDX-1 expression.

To characterize the immune responses including local and systemic immunity induced by infection with H pylori, especially with CagA+ H pylori strains and the underlying immunopathogenesis.A total of 711 patients with different gastric lesions were recruited to determine the presence of H pylori infection and cytotoxin associated protein A (CagA), the presence of T helper (Th) cells and regulatory T (Treg) cells in peripheral blood mononuclear cells (PBMCs), expression of plasma cytokines, and RNA and protein expression of IFN-gamma and IL-4 in gastric biopsies and PBMCs were determined by rapid urease test, urea [(14)C] breath test, immunoblotting test, flow cytometry , real time RT-PCR and immunohistochemistry.Of the patients, 629 (88.47%) were infected with H pylori; 506 (71.16%) with CagA+ and 123 (17.30%) with CagA- strains. Among patients infected with CagA+ H pylori strains, Th1-mediated cellular immunity was associated with earlier stages of gastric carcinogenesis, while Th2-mediated humoral immunity dominated the advanced stages and was negatively associated with an abundance of Treg cells. However, there was no such tendency in Th1/Th2 polarization in patients infected with CagA- H pylori strains and those without H pylori infection.Polarization of Th cell immune responses occurs in patients with CagA+ H pylori infection, which is associated with the stage and severity of gastric pathology during the progression of gastric carcinogenesis. This finding provides further evidence for a causal role of CagA+ H pylori infection in the immunopathogenesis of gastric cancer.