| BackgroundApoptosis is a programed cell death stringently controlled by cell signaling pathways and the expression of pro and anti-apoptotic genes. Apoptosis represents an active means for multi-cellular organisms to rid of the damaged or unwanted cells under pathological condition or physiological condition during the process of tissue remodeling and embryogenesis. On the other hand, inactivation of apoptotic genes and proteins by mutation can block apoptosis, resulting in unlimited cell proliferation and cancer under certain conditions. Thus, a thorough understanding of the mechanism of apoptosis and related signaling pathways would provide novel strategies for cancer therapy.P38 MAP kinase is one of the most important members of the MAP kinase family that is critically important in mediating inflammatory responses and apoptosis under cell stress. The functional significance of P38 kinase activation in apoptosis is still unclear. Published data suggest that P38 activation can be either pro-apoptotic or anti-apoptotic in response to stress stimuli, likely determined by the type of stimuli and the state of cell proliferation and differentiation. Few studies have been reported on the role of P38 MAP kinase in cisplatin induced apoptosis. More studies are essential to further establish the role of P38 MAP kinase in genotoxin and other cell stress triggered apoptosis in cancerous cells. Esophageal carcinorma is one of the most common and debilitating cancers in China. Here,we intend to explore the role of P38 kinase in genotoxin cisplatin and UPR(unfolded protein response) inducer (DTT) dithiothreitol induced apoptosis inesophageal cancer cells. This study may provide new insight into the pathwaysleading to the apoptosis of esophageal cancer cells.Materials and methodsThis experiments are divided in two phases.1. Phase One: Phosphorylation of P38 kinase during cisplatin and DTT induced apoptosis.Ecal09 cells were cultured to 70% confluency and then treated with l0mg/ml cisplatin, 2 mM DTT, or l0ug/ml cisplatin plus 2 mM DTT for 24 hours. The untreated cells were as the control. At the end of treatment, cells were collected and used for immunohistochmestry for p-P38 or for preparing DNA for agarose gel analysis of DNA ladders. For p-P38 immunohistochemisrtry, cells were fixed onto glass slide. P-P38 kinase was detected using anti-phosphorylated P38 antibody according to protocol of Beijing Zhongshan Biotechnology Co.,LTD..2. Phase two: Efffect of p-P38MAPK inhibitor SB203580 on cisplatin and DTT induced apoptosis.To determine the role of P38 activation in cisplatin and DTT induced apoptosis of Ecal09 cells, specific P38 kinase inhibitor SB203580 was used to inhibit P38 activity. The effect of P38 kinase inhibition on cisplatin and DTT induced apoptosis was examined. The experiment was done as in phase one except additional following experimental groups were included: cells treated with DTT or cisplatin or DTT plus cisplatin in the presence of lOug/ml SB203580 compound.Upon treatments,cells were collected for p-P38 immunohistochemistry and apoptosis assay using flow cytometry.Biostatistic analyses were done using SPSS 10.0 software package. Data from imunohistochmistry and flow cytometry were analyzed by Chi-Square and Kruskal-Wallis tests.Results1. Phase one.1.1 Detection of cisplatin and DTT induced apoptosis by DNA ladder formation. Celluar DNA from DTT, cisplatin, DTT + cisplatin treated groups and control group were extracted and subjected to agarose electrophoresis. DNA ladders were observed in all three treated groups, but not in the unteated control group, suggesting that both cisplatin and DTT were able to induce apoptosis in Ecal09 cells.1.2 Microspcopic studies:Immunohistochemistry: As detected by anti-p-P38 antibody by immunohistochemistry, The numbers of positive cells were about 50% in the treatment groups.Comparing with that of 10% in untreated group, the phosphorylation level of P38 was dramatically increased upon treatment of cispla... |
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