| Ovarian carcinoma is a leading cause of death from gynecological cancer in China and many other countries. The major reason for the mortality of this cancer is the high metastatic ability and the resistance to chemotherapy. The response of the primary tumor to taxane and platinum-based chemotherapy is high, but about 20% of patients never achieve a clinical response and the majority of the patients will relapse and eventually die of drug-resistant disease. Although great improvement has been made in standard therapy for ovarian cancer, the mechanism of the drug-resistant and relapses is not clear yet. Identifying the molecules that differentially expresses in the drug-resistant cells is a hot field for exploring the target for new drugs.Glycogen synthase kinase-3 beta (GSK-3β), a serine/threonine kinase active in resting cells, is now recognized as a key component of a surprisingly large number of cellular processes, such as cellular structure, growth ,motility and apoptosis. Dysregulation of the expression and activity of GSK-3βhas been linked to several prevalent pathological conditions, including tumorigenesis.It has been reported that the level of expression of GSK-3βis significantly high in ovarian carcinoma tissue.Because of the importance of GSK-3βin apoptotic procedure, it is plausible that the pharmacological inhibiton of GSK-3βshould cure ovarian carcinoma. And the latest report also indicated that GSK-3βpositively regulates the proliferation of ovarian carcinoma cell line SKOV3, via the upregulation of cyclin D1, which can be inhibited by lithium chloride, an approved GSK-3βinhibitor(12). However, in this context, we offered the contrary evidence that the inhibiton of GSK-3βby lithium chloride offered the chemoresistance of ovarian carcinoma cells to cisplatin, and the molecular mechanisms responsible for the chemoresistance focused on the induction of p53, p21 and GADD45.AIM:Detect the role of GSK-3βin the cisplatin chemoresistance of ovarian cancer cells.METHODS:Our research work was divided into two parts. The first, we detected total protein and its phosphoralation isform expression of GSK-3βin A2780 and CP70 cells by western blot and immunofluorescence. Then we detect the change of cisplatin chemoresistance of A2780 and CP70 cells pretreated with LiCl by MTT, DAPI staining and flow cytometry.The second, after transient transfection of S9A mutant GSK-3βvector and GSK-3βsiRNA in CP70 cells, the change of cisplatin chemoresistance and the ability of DNA repairment were detected by MTT and DAPI staining, flow cytometry and atomic absorption spectroscopy. And then, the possible molecular mechanism was detected by western blot.At last, the data was analyzed with SPSS 13.0 statistical software.RESULTS:First, we detected the expression and localization of total GSK-3βand its inhibitive phosphorylated isoforms, pGSK-3βser9 in A2780 and CP70 cells. Western blot analysis showed that both A2780 and CP70 expressed GSK-3β, and the levels of total GSK-3βin two cell lines were almost aequalis (1:1.08±0.13,P>0.05), however, the level of pGSK-3βser9 isoforms in A2780 was lower than in CP70 (1:2.34±0.21,P<0.01). As we know, the function of GSK-3βis closely correlated with its localization. So the localization of GSK-3βwas detected by immunofluorescence. We found that GSK-3βalmost localized in cytoplasmic in A2780, but in cytoplasmic and nucleus in CP70. After treatment of cisplatin, GSK-3βtranslocated obviously into the nucleus of the two cell lines ,and pGSK-3βser9 is the main composition of GSK-3βin the nucleus.To confirm whether the increase of pGSK-3βser9 contributes to the cisplatin-resistant of A2780, CP70 cell lines, we examined the inhibitory concentration 50% (IC50) of cisplatin to A2780 and CP70 pretreated with lithium chloride. IC50 of A2780 and A2780/CP70 respectively significantly increased from 0.51±0.13μm, 5.43±0.15μm to 3.04±0.28μm , 13.28±0.34μm after lithium pretreatment. The results of DAPI and flow cytometry confirmed these consequence.To further verify that the effect of lithium was due to inhibition of GSK-3β, CP70 cells were transiently transfected with pcDNA3-GSK-3β-S9A and GSK-3βshort interfering RNA (siRNA), respectively. We observed that overexpression of GSK-3β-S9A resulted in the depression of cisplatin chemoresistance in CP70 cell, on the contrary, inhibition of GSK-3βexpression by siRNA led to the increase of cisplatin chemoresistance.It has been demonstrated that the cytotoxicity of cisplatin is thought to be due to the formation of intrastrand and interstrand cross-links in the DNA and cisplatin-DNA adducts, which may induce cell cycle arrest or apoptosis. So platinum cotent was measured by atomic absorption spectroscopy to determine the ability of removal of platinum-DNA adducts. The results of atomic absorption spectroscopy indicated that overexpression of GSK-3β-S9A resulted in the reduction of DNA repairment ability.As we know, in A2780 and CP70 cells, the resistance to cisplatin correlates with prolonged p53 protein stabilization and accumulation. Therefor, we examined the expression of p53 protein in CP70 cells, in which GSK-3βS9A mutant overexpressed or GSK-3βexpression was inhibited by siRNA. The results demonstrated that the inhibition of GSK-3βexpression resulted in an obvious increase of p53 expression, compared with those transfected with pcDNA3-GSK-3β-S9A, and this increase was associated with up-regulation of p21 and GADD45, two target genes of p53, which were correlated with cell cycle arrest and DNA repair. Furthermore, we found the expression of cleaved caspase-3 was reduced when p53 was induced by the inhibition of GSK-3β.CONCLUSIONS:Our results shows high level expression and nuclear location of pGSK-3βser9 in CP70 cells is associated with its resistance to cisplatin and lithium is able to promote the chemoresistance to cisplatin through induction of inactive phosphoralation of GSK-3βat serine 9 and p53. Therefore, the administration of lithium to ovarian cancer patients should be scrupulous.
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