Study Of The Role Of PI3K/AKT/GSK3β Cell Singnaling Pathway In Chemotherapeutics Induced Apoptosis In Ovarian Cancer

Part oneStudy of the role and mechanism of PI3K/AKT/GSK3βcellsingnaling pathway in paclitaxel induced apoptosis in ovarian cancerObjective: To investigate the effects of PI3K/AKT/GSK3βcell signal pathway on theapoptosis and cell cycle arrest induced by paclitaxel in ovarian carcinoma cell lines.Methods :1. Western Blot was used to detect the expression of p-GSK3βand the anti-apoptoticproteins Bcl-2,Bcl-xl and Mcl-1 in ovarian carcinoma cell lines treated by differentconcentration paclitaxel or by paclitaxel and LiCl.2. The apoptotic ratio test : the AnnexinV-FITC apoptosis detection kit was use to measurethe apoptotic ratio of OV20008 induced by paclitaxel and LiCl; the change of cellmorphology was identified by the Hochest33342 staining; and the Western Blot was use toevaluate the expression of Caspase 9 and Caspase 3.3. The effects of LiCl on mitochondrial membrane potential of OV2008 treated bypaclitaxel was measured by mitochondrial membrane potential detection Kit, then observedthe fluorescence under fluorescence microscope.4. Immunofluorescence and the mitochondria isolation kit were used to detect thetransposition of Bax from cytoplasm to mitochondria.5. Cell cycle were detected by flow cytometry after stained with PI in ovarian cancer cellstreated by LY294002 and LiCl.Results:1. LiCl could enhance the expression of p-GSK3β.The results of FACS showed that theapoptotic rates of cells treated with paclitaxel (26.77%,27.35%,32.28%)was higher than the cells treated with paclitaxel and LiCl (17.82%,20.17%,18.96%). Hochest33342 stainingshowed that LiCl could inhibit the apoptosis induced by pacitaxel, compared to the cellswithout inhibitor .And the Western Blot also showed that LiCl could inhibit the clip ofCaspase 9 and Caspase 3.2. The JC-1 staining showed that LiCl could inhibit the reduce of red fluorescence when thecells treated only by paclitaxel were detected stronger green fluorescence and lower redfluorescence.3. The results of immunofluorescence and Western Blot suggested that LiCl could block thetransposition of Bax from cytoplasm to mitochondria which induced by paclitaxel. TheWestern Blot also showed LiCl could suppress the down-regulation of Bcl-2,Bcl-xl andMcl-1.4. The results of FACS showed that LiCl could enhanced the G₂M arrest induced bypaclitaxel (88.13%) and in contrast LY294002 could improve the cell cycle arrest( 44.06% )5. Western Blot showed down-regulation of AKT by RNAi or by LY294002, both couldpromote the apoptosis induced by paclitaxel.Conclusions: The augment of p-GSK3βexpression could inhibit the apoptosisinduced by paclitaxel through up-regulating the function of Bax and down-regulating theanti-apoptotic proteins (Bcl-2,Bcl-xl,Mcl-1); and could also enhanced the G₂M arrest inovarian carcinoma cell lines.Part twoStudy of the effects of PI3K/AKT/GSK3βcell singnaling pathway oncisplatin induced apoptosis in ovarian cancerObjective: To investigate the effects of enhanced p-GSK3βon the apoptosis induced bypaclitaxel in ovarian carcinoma cell lines.Methods: LiCl was used to enhance the expression of p-GSK3β.The apoptosis wasdetected by FACS. The expression of p-GSK3βand GSK3βwas measured by WesternBlot. Results: The results of Western Blot showed that after treated by different concentrationcisplatin 24h, the expression of p-GSK3βwas up-regulated in the ovarian cancer cellsOV2008 and SKOV3.The results of FACS showed that the apoptotic rates of OV2008cells treated with different concentration cisplatin was 3.34 %,23.48 % and 55.17%,when pretreated with LiCl, the apoptotic rates reduced to 1.21%,16.09% and 21.73 % ;in another ovarian cancer cell SKOV3 , the apoptotic rates treated with differentconcentration cisplatin was 24.98%,53.34% and 64.93%, when pretreated with LiCl, theapoptotic rates reduced to 13.94%,20.65% and 35.75%. The results of Western Blotshowed that LiC1 could enhance the expression of p-GSK3βand the cisplatin also couldup-regulated the expression ofp-GSK3β.Conclusions: The augment ofp-GSK3βexpression could inhibit the apoptosis inducedby cisplatin in ovarian carcinoma cell lines.