Modulations of cell cycle checkpoints during HCV associated disease

Background Impaired proliferation of hepatocytes has been reported in chronic Hepatitis C virus infection. Considering the fundamental role played by cell cycle proteins in controlling cell proliferation, altered regulation of these proteins could significantly contribute to HCV disease progression and subsequent hepatocellular carcinoma (HCC). This study aimed to identify the alterations in cell cycle genes expression with respect to early and advanced disease of chronic HCV infection. Methods Using freshly frozen liver biopsies, mRNA levels of 84 cell cycle genes in pooled RNA samples from patients with early or advanced fibrosis of chronic HCV infection were studied. To associate mRNA levels with respective protein levels, four genes (p27, p15, KNTC1 and MAD2L1) with significant changes in mRNA levels (\u3e 2-fold, p-value \u3c 0.05) were selected, and their protein expressions were examined in the liver biopsies of 38 chronic hepatitis C patients. Results In the early fibrosis group, increased mRNA levels of cell proliferation genes as well as cell cycle inhibitor genes were observed. In the advanced fibrosis group, DNA damage response genes were up-regulated while those associated with chromosomal stability were down-regulated. Increased expression of CDK inhibitor protein p27 was consistent with its mRNA level detected in early group while the same was found to be negatively associated with liver fibrosis. CDK inhibitor protein p15 was highly expressed in both early and advanced group, but showed no correlation with fibrosis. Among the mitotic checkpoint regulators, expression of KNTC1 was significantly reduced in advanced group while MAD2L1 showed a non-significant decrease. Conclusion Collectively these results are suggestive of a disrupted cell cycle regulation in HCV-infected liver. The information presented here highlights the potential of identified proteins as predictive factors to identify patients with high risk of cell transformation and HCC development

Molecular pathways involved in the synergistic interaction of the PKCβ inhibitor enzastaurin with the antifolate pemetrexed in non-small cell lung cancer cells

Conventional regimens have limited impact against non-small cell lung cancer (NSCLC). Current research is focusing on multiple pathways as potential targets, and this study investigated molecular mechanisms underlying the combination of the PKCβ inhibitor enzastaurin with the multitargeted antifolate pemetrexed in the NSCLC cells SW1573 and A549. Pharmacologic interaction was studied using the combination-index method, while cell cycle, apoptosis induction, VEGF secretion and ERK1/2 and Akt phosphorylation were studied by flow cytometry and ELISAs. Reverse transcription–PCR, western blot and activity assays were performed to assess whether enzastaurin influenced thymidylate synthase (TS) and the expression of multiple targets involved in cancer signaling and cell cycle distribution. Enzastaurin-pemetrexed combination was highly synergistic and significantly increased apoptosis. Enzastaurin reduced both phosphoCdc25C, resulting in G2/M checkpoint abrogation and apoptosis induction in pemetrexed-damaged cells, and GSK3β and Akt phosphorylation, which was additionally reduced by drug combination (−58% in A549). Enzastaurin also significantly reduced pemetrexed-induced upregulation of TS expression, possibly through E2F-1 reduction, whereas the combination decreased TS in situ activity (>50% in both cell lines) and VEGF secretion. The effects of enzastaurin on signaling pathways involved in cell cycle control, apoptosis and angiogenesis, as well as on the expression of genes involved in pemetrexed activity provide a strong experimental basis to their evaluation as pharmacodynamic markers in clinical trials of enzastaurin-pemetrexed combination in NSCLC patients

Chemical–Genetic Profiling of Imidazo[1,2-a]pyridines and -Pyrimidines Reveals Target Pathways Conserved between Yeast and Human Cells

Small molecules have been shown to be potent and selective probes to understand cell physiology. Here, we show that imidazo[1,2-a]pyridines and imidazo[1,2-a]pyrimidines compose a class of compounds that target essential, conserved cellular processes. Using validated chemogenomic assays in Saccharomyces cerevisiae, we discovered that two closely related compounds, an imidazo[1,2-a]pyridine and -pyrimidine that differ by a single atom, have distinctly different mechanisms of action in vivo. 2-phenyl-3-nitroso-imidazo[1,2-a]pyridine was toxic to yeast strains with defects in electron transport and mitochondrial functions and caused mitochondrial fragmentation, suggesting that compound 13 acts by disrupting mitochondria. By contrast, 2-phenyl-3-nitroso-imidazo[1,2-a]pyrimidine acted as a DNA poison, causing damage to the nuclear DNA and inducing mutagenesis. We compared compound 15 to known chemotherapeutics and found resistance required intact DNA repair pathways. Thus, subtle changes in the structure of imidazo-pyridines and -pyrimidines dramatically alter both the intracellular targeting of these compounds and their effects in vivo. Of particular interest, these different modes of action were evident in experiments on human cells, suggesting that chemical–genetic profiles obtained in yeast are recapitulated in cultured cells, indicating that our observations in yeast can: (1) be leveraged to determine mechanism of action in mammalian cells and (2) suggest novel structure–activity relationships

Intracoronary Stem Cell Infusion After Acute Myocardial Infarction A Meta-Analysis and Update on Clinical Trials

Background-Several cell-based therapies for adjunctive treatment of acute myocardial infarction have been investigated in multiple clinical trials, but the benefits still remain controversial. This meta-analysis aims to evaluate the efficacy of bone marrow-derived mononuclear cell (BMMNC) therapy in patients with acute myocardial infarction, but also explores the effect of newer generations of stem cells. Methods and Results-A random-effects meta-analysis was performed on randomized controlled trials investigating the effects of stem cell therapy in patients with acute myocardial infarction that were published between January 2002 and September 2013. The defined end points were left ventricular (LV) ejection fraction, LV end-systolic and end-diastolic volumes, infarct size, and major adverse cardiac and cerebrovascular event rates. Also, several subgroup analyses were performed on BMMNC trials. Overall, combining the results of 22 randomized controlled trials (RCTs), LV ejection fraction increased by +2.10% (95% confidence interval [CI], 0.68-3.52; P=0.004) in the BMMNC group as compared with controls, evoked by a preservation of LV end-systolic volume (-4.05 mL; 95% CI, -6.91 to -1.18; P=0.006) and a reduction in infarct size (-2.69%; 95% CI, -4.83 to -0.56; P=0.01). However, there is no effect on cardiac function, volumes, or infarct size, when only RCTs (n=9) that used MRI-derived end points were analyzed. Moreover, no beneficial effect could be detected on major adverse cardiac and cerebrovascular event rates after BMMNC infusion after a median follow-up duration of 6 months. Conclusions-Intracoronary infusion of BMMNC is safe, but does not enhance cardiac function on MRI-derived parameters, nor does it improve clinical outcome. New and possibly more potent stem cells are emerging in the field, but their clinical efficacy still needs to be defined in future trials

Final results of the HEALING IIB trial to evaluate a bio-engineered CD34 antibody coated stent (Genous™Stent) designed to promote vascular healing by capture of circulating endothelial progenitor cells in CAD patients

Objective: To assess the safety and efficacy of the Genous™ endothelial progenitor cell (EPC) capturing stent in conjunction with HmG-CoA-reductase inhibitors (statins) to stimulate EPC recruitment, in the treatment of patients with de novo coronary artery lesions. Methods and results: The HEALING IIB study was a multi-center, prospective trial, including 100 patients. The primary efficacy endpoint was late luminal loss by QCA at 6-month follow-up (FU). Although statin therapy increased relative EPC levels by 5.6-fold, the angiographic outcome at 6 month FU was not improved in patients with an overall in-stent late luminal loss of 0.76 ± 0.50 mm. The composite major adverse cardiac events (MACE) rate was 9.4%, whereas 6.3% clinically justified target lesion revascularizations (TLRs) were observed. 2 Patients died within the first 30 days after stent implantation due to angiographically verified in-stent thrombosis. At 12 month FU, MACE and TLR increased to 15.6% and 11.5% respectively and stabilized until 24 month FU. 18 Month angiographic FU showed a significant decrease in late luminal loss (0.67 ± 0.54, 11.8% reduction or 10% by matched serial analysis, P= 0.001). Conclusion: The HEALING IIB study suggests that statin therapy in combination with the EPC capture stent does not contribute to a reduction of in-stent restenosis formation for the treatment of de novo coronary artery disease. Although concomitant statin therapy was able to stimulate EPC recruitment, it did not improve the angiographic outcome of the bio-engineered EPC capture stent. Remarkably, angiographic late loss was significantly reduced between 6 and 18 months. © 2011 Elsevier Ireland Ltd