Ⅰ.The Antiapoptotic Effect And Its Mechanism Of Glucocorticoids In Human Ovarian Cancer Cell HO-8910 Ⅱ.The Up-regulation Of RhoB By Glucocorticoids And 1,25(OH)₂ D₃ In Human Osteosarcoma Cell Line HOS-8603 And Its Biological

Glucocorticoids play an important role in regulating cells proliferation, differentiation and apoptosis. However, these effects vary among different cells. For instance, GCs are used to suppress immunity and treat lymphoid leukemia because they can induce apoptosis in thymocyte, leukemia cells and lymphocyte. On the contrary, GCs can protect some cells such as breast cancer cell and lung carcinoma cell from apoptosis induced by irradiation or chemotherapy drugs. The mechanisms of GCs inducing apoptosis have been studied deeply so far, but the antiapoptotic mechanisms are still unclear. The first part of this study is about the antiapoptotic mechanisms of GCs in the human ovarian cancer cell line HO-8910. The second part is based on the work of our laboratory that Dex and 1,25(OH)₂D₃ could inhibit the proliferation of human osteosarcoma cell line HOS-8603 and the small G protein RhoB was involved in the inhibitory effect of Dex. We want to find out the relationship between RhoB and the inhibition on cell growth in HOS-8603 by Dex and 1,25(OH)₂ D₃ to understand whether the up-regulation of RhoB is limited in ovarian cancer cell.I The antiapoptotic effect and its mechanism of glucocorticoids in human ovarian cancer cell HO-8910As a chemotherapy drug, Cisplatin performs apoptic effect in a lot of tumor cells including ovarian cancer cell. GCs are often used in chemotherapy for their antiemetic effect. Besides, they are proved to have anti-proliferation function in different types of ovarian carcinoma clls. So we wondered that if Dex could cooperate with Cisplatin or antagonize Cisplatin in ovarian cancer therapy.We found that Dex had a strong antiapoptotic effect in certain cell and prevented Cisplatin-induced apoptosis. We presumed that Dex was able to antagonize the effect of Cisplatin.First of all, we observed the effect of Dex on cisplatin-induced apoptosis by PI staining and Annexin V-FITC/PI staining. Cells were treated for 48h with cisplatin in the presence or absence of Dex of therapeutic doses. The apoptosis rate of cisplatin alone treatment was 13.2 ± 1.34% whereas decreased to 4.68±0.84%(P<0.01) when combined with Dex treatment. Dex treatment alone had no effect on apoptosis of cells. This finding indicated that Dex inhibited cisplatin-apoptosis in HO-8910. We found RU486 restored the apoptosis sensitivity of the Dex-cotreated cells which suggested that GR mediated protection of ovarian cells by Dex.Bcl-2 expression were analyzed by semi-quantitive RT-PCR and Western Blot. Compared with the levels in control, up-regulation of Bcl-X_L and down-regulation of Bax by Dex were found in a time- and dose- dependent way. But Dex had no effect on the expression of Bcl-2 and Bad. In addition, we found that Dex decreased activity of Caspase-9 and Caspase-3 obviously. Bax and Bcl-X_L are located in mitochondrial membrane and Caspase-9 is cleaved through mitochondrial pathway, so we considered that the mitochondrial pathway was involved in the anti-apoptotic effect of Dex.Dex elevated epidermal growth factor receptor (EGFR) expression in a dose-and time- dependent way in HO-8910 cell and the maximum effect was at 24h by treated with 100nM Dex in protein level. The PI-3K-PKB signaling pathway can be activated after the epidermal growth factor binding with EGFR, then perform a prosurvival/enti-apoptotic action in HO-8910 cell. We detected the phosphorylationof PKB/Akt with phosphorylated antibody of PKB by Western Blot. The result showed that Dex could activate PKB through inducing phosphorylation of Akt. Further more, we blocked PI-3K with its inhibitor, LY294002, and the anti-apoptotic effect of Dex was suppressed by it. We considered that PI-3K/PKB signaling pathway was involved in anti-apoptotic action of Dex.Serum and glucocorticoid regulated protein kinase(SGK) is a Akt-like Ser/Thr protein kinase. With semi- quantitive RT-PCR, we found that SGK mRNA was up-regulated by Dex in HO-8910 cell.Our study proved that Dex could confer apoptosis resistance of human ovarian?carcinoma cell HO-8910 toward apoptosis induced by Cisplatin. This was partly due to the regulation of key molecules in the mitochondrial apoptosis pathway such as BcI-Xl and Bax by Dex, resulting in a blockade of caspase activity. We also found that the PI-3K-PKB signaling pathway participated in the anti-apoptotic action of Dex. In addition, the rapidly induced expression of SGK by Dex might be also involved in.II The up-regulation of RhoB by glucocorticoids and 1,25(OH)2 D3 in humanosteosarcoma cell line HOS-8603 and its biological significanceGCs and l,25(OH)2D3play an important role in regulating osteocytes proliferation, differentiation and apoptosis. We have reported that the two steroids can both inhibit proliferation and induce differentiation of human osteosarcoma cells. The growth inhibition and proapoptotic effects of GCs in osteoblasts appear to be one cause of GCs-related osteoporosis, but the molecular mechanism remains unknown. HOS-8603 is an osteoblast-like osteosarcoma cell line and we have reported that Dex and 1,25(OH)2 D3 could suppress its proliferation. However, the target genes regulated by these two steroids and if there are some genes regulated by both steroids are still unclear. The small G protein RhoB has a negative-modulatory effect on proliferation in many malignant cells and we have proved that Dex inhibited growth of human ovarian cancer cell HO-8910 by regulating RhoB expression. Based on these, we studied the regulation of RhoB expression by glucocorticoids and 1,25(OH)2D3 in human osteosarcoma cell line HOS-8603 and its biological significance.Our study proved that Dex could up-regulated the expression of RhoB in human...