Browsing by Subject "caspase-3"
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Item Involvement of p70S6K in Cisplatin-Induced Cell Death(2008-05-01) Dhar, Rohini; Basu, Alakananda; Singh, Meharvan; Vishwanatha, JamboorRohini Dhar, Involvement of p70S6K in cisplatin-induced cell death. Doctor of Philosophy (Biochemistry and Molecular Biology) May 2008, 151 pp., 23 illustration, 3 tables, 153 references. Cisplatin is used for the treatment of solid tumors; however its success is often compromised due to relapse and chemoresistance. The purpose of this dissertation is to delineate the role of p70S6K in cisplatin-induced apoptosis. A comparison of p70S6K levels in H69 cells that acquired resistance to cisplatin (H69/CP) compared to parental H69 cells revealed that levels of phosphorylated p70S6K and S6 were elevated. Cisplatin treatment resulted in the activation of p70S6K and downregulation of total p70S6K. Inhibition of the phosphoinositide 3-kinase (P13K) pathway by itself augmented cisplatin-induced PARP cleavage and also blocked the phosphorylation of p70S6K. Inhibition of extracellular signal-regulated kinase (ERK) pathway however attenuated cisplatin-induced PARP cleavage. These results reveal that phosphorylation of p70S6K is associated with increased cisplatin resistance, and inhibition of P13K/ p70S6K pathway could reverse cisplatin resistance. We have found that cisplatin caused a time- and concentration-dependent downregulation of p70S6K. While the calpain and proteasome inhibitors had no effect on the downregulation of p70S6K, the broad specificity caspase inhibitor z-VAD-fmk (z-VAD) reversed p70S6K downregulation by cisplatin. Furthermore, the caspase-3 inhibitor and knockdown of caspase-3 prevented cisplatin-induced proteolytic cleavage of p70S6K. While, cisplatin failed to induce cleavage of p70S6K in MCF-7 cells that lack functional caspase-3, overexpression of caspase-3 in these cells resulted in cisplatin-induced cleavage of p70S6K. Thus, these results demonstrate that p70S6K is a novel substrate for caspase-3. Examination of the role of p70S6K in cisplatin-induced death shows that rapamycin in a pharmacological inhibitor of p70S6K, enhanced cisplatin-induced apoptosis in A549 cells. However, knockdown of p70S6K by siRNA resulted in a decrease in cisplatin-iduced apoptosis. In addition, caspase-3 mediated cleavage of p70S6K at the aspartic acid residue at the 393 position and site-directed mutagenesis of Asp393 to Ala resulted in protection against cisplating-mediated apoptosis. Interestingly, introduction of the N-terminal cleaved fragment [∆(394-525)] resulted in potentiation of cisplatin-induced apoptosis. These results suggest that the proteolytic cleavage of p70S6K by caspase-3 is important for cisplatin-induced apoptosis.Item Neuroprotection of Cyperus esculentus L. orientin against cerebral ischemia/reperfusion induced brain injury(Wolters Kluwer - Medknow, 2020-03) Jing, Si-Qun; Wang, Sai-Sai; Zhong, Rui-Min; Zhang, Jun-Yan; Wu, Jin-Zi; Tu, Yi-Xian; Pu, Yan; Yan, Liang-JunOrientin is a flavonoid monomer. In recent years, its importance as a source of pharmacological active substance is growing rapidly due to its properties such as anti-myocardial ischemia, anti-apoptosis, anti-radiation, anti-tumor, and anti-aging. However, the neuroprotective effects of Orientin on stroke injury have not been comprehensively evaluated. The aim of the present study was thus to investigate the neuroprotective capacity and the potential mechanisms of Cyperus esculentus L. orientin (CLO) from Cyperus esculentus L. leaves against ischemia/reperfusion (I/R) injury using standard orientin as control. For in vitro studies, we treated HT22 cells with CoCl2 as an in vitro ischemic injury model. HT22 cells in the control group were treated with CoCl2. For in vivo studies, we used rat models of middle cerebral artery occlusion, and animals that received sham surgery were used as controls. We found that CLO protected CoCl2-induced HT22 cells against ischemia/reperfusion injury by lowering lipid peroxidation and reactive oxygen species formation as well as decreasing protein oxidation. However, CLO did not reduce the release of lactate dehydrogenase nor increase the activity of superoxide dismutase. Results showed that CLO could decrease neurological deficit score, attenuate brain water content, and reduce cerebral infarct volume, leading to neuroprotection during cerebral ischemia-reperfusion injury. Our studies indicate that CLO flavonoids can be taken as a natural antioxidant and bacteriostastic substance in food and pharmaceutical industry. The molecular mechanisms of CLO could be at least partially attributed to the antioxidant properties and subsequently inhibiting activation of casepase-3. All experimental procedures and protocols were approved on May 16, 2016 by the Experimental Animal Ethics Committee of Xinjiang Medical University of China (approval No. IACUC20160516-57).