| Ovarian cancer is one of the most common life-threatening malignant tumors among women and it is the first leading cause of death from gynecologic cancers. Due to the lack of effective screening strategies and the absence of symptoms in early-stage of disease, majority of cases have progressed to an advanced stage at the time of primary diagnosis. Although most patients will undergo an attempt at surgical debulking followed by 6 cycles of chemotherapy with a platinum-based regimen, the 5-year survival remains 25-30% and 85-90% patients will develope tumor recurrence or metastasis. Cisplatin as the critical component in the chemotherapy regimens, its usage has been limited because of its cumulative toxicities, especially the toxicity to the kidney and the intrinsic or acquired resistance to cisplatin in some patients. In recent years,the interests of combining cisplatin and new active chemotherapeutic agents to treat ovarian cancer have arisen for promoting the effectiveness and reducing the side effect of cisplatin.Akt, also named protein kinase B, represents a subfamily of the serine/threonine kinase.Three members, AKT1, AKT2, and AKT3,have been identified in this subfamily. Akt is activated by extracellular stimuli in a phosphatidylino sitol 3-kinase (PI3k)-dependent manner. Full activation of Akt requires phosphorylation of threonine 308 in the activation loop and serine 473 in the COOH terminal activation domain. In addition,amplification, overexpression, and/or activation of Akt have been detected in a number of human malignancies . Ectopic expression of Akt,especially constitutively active Akt, induces cell survival and malignant transformation, whereas inhibition of Akt activity stimulates apoptosis in a range of mammalian cells. Furthermore,activation of Akt has been shown to associate with tumor invasiveness and chemoresistance. Therefore, development of potent inhibitors that block AKT pathway is an attractive therapeutic strategy for treating ovarian carcinoma.The Y-box-binding protein 1 (YB-1), which is a DNA/RNA-binding protein also known as dbpB, regulates transcription, translation, DNA damage repair and other biological processes in both the nucleus and cytoplasm. In the cytoplasm, YB-1 regulates mRNA stability and translational regulation, while in the nucleus, it plays a pivotal role in transcriptional regulation through specific recognition of the Y-box promoter element. However, as a nucleocytoplasmic shuttling protein, it is important to understand which signalling molecules are involved in the translocation of YB-1 into the nucleus.Akt activation regulates the nuclear translocation of YB-1, affecting the expression of drug-resistance genes and other genes associated with the malignant characteristics in ovarian cancer cells. Therefore, the Akt pathway could be a novel target of disrupting the nuclear translocation of YB-1 that has important implications for further development of therapeutic strategy against ovarian cancers.Objective:1. To investigate whether the expression of AKT was associated with the expression of P-gp in human ovarian cancer tissues.2.To detect whether PI3K/AKT/YB-1 cell signal pathway take part in the formation of cisplatin chemoresistance of ovarian cancer cells.3.To investigate whether PI3K/AKT cell signal pathway regulate the expression of P-gp by YB-1 nuclear translocation in human ovarian cancer cell line SKOV3/DDP.Methods1.we detected the expression of p-AKT and P-gp in epithelial ovarian cancer and normal ovarian tissue by immunohistochemistry.The relevant assay and survival curve assay were carried out about p-AKT and P-gp.The relationship between chemotherapy effect and coexpression of p-AKT and P-gp was assessed according to clinical data.2.To parental ovarian cancer cell line SKOV-3 and its corresponding cisplatin-resistant cell line SKOV-3/DDP for subjects, first of all to verify the resistance index of SKOV-3/DDP by MTT method, followed by immunocytochemical detected the expression of AKT isoform, followed by extraction of cell protein detected total AKT, pAkt (Thr 308 and Ser 473) in SKOV-3/DDP and SKOV-3 cells by western blot.3.We detect the change of cisplatin chemoresistance of SKOV-3/DDP and SKOV-3 cells pretreated with API-2 by MTT. 4.SKOV-3/DDP respectively, and SKOV-3 cells for the following groups, namely control group, treated with cisplatin alone, API-2 pre-treatment 1 hour by adding cisplatin intervention group, all sub-guarantee of cisplatin on the tumor cells for more than 24h, and the same time. Grouping of different SKOV-3/DDP and SKOV-3 cell extracts were detected the inhibition of Akt activity by western blot analysis.The confocal microscopy and Annexin V/PI staining flow cytometry were carried out to investigate the effect of API-2 on apoptosis in ovarian cancer cell line SKOV-3/DDP and SKOV3.5.we detected total protein and its phosphoralation isform expression of YB-1 in SKOV-3/DDP and SKOV-3 cells by western blot and immunofluorescence.6.Respectively, for the SKOV-3, SKOV-3/DDP cells following division, that is, the control group, treated with cisplatin alone, API-2 pre-treatment 1 hour by adding cisplatin intervention group, all sub-guarantee of cisplatin on the tumor cells for more than 24h, and the same time. After inhibiting AKT activity, YB-1 expression in positioning the situation by immune cell fluorescence in confocal microscopy . Semi-quantitative Western blot analysis further verified YB-1 expression in nuclear with the immunofluorescence results.7.We detected the expression of P-gp in SKOV-3/DDP and SKOV-3 cells by western blot and immunofluorescence.8.Western blot was carried out to investigate the expression change of P-gp in SKOV-3/DDP and SKOV-3 cells after the activity of AKT was inhibited.Reults1. The expression of p-AKT and P-gp in epithelial ovarian cancer tissues were associated, Pearson contingency coefficient was r = 0.3089. P-gp and p-AKT expression on total positive of resistance was 86.67% predictive value, P-gp and p-AKT expression in a total negative response to chemotherapy was 72% predictive value. Follow-up data showed that 63 cases of epithelial ovarian cancer, seven cases of survival 5 years, the cumulative survival rate was 11.11%, the average survival time was 31 months. The average survival time of p-AKT-positive group was shorter than the negative group (30 months vs 44 months), the Log Rank test, two groups were statistically significant differences (P = 0.0003). The average survival time of P-gp positive group was shorter than the negative group (28 months vs 42 months), the Log Rank test, two groups were statistically significant differences (P = 0.0015). 2. MTT method was used to verify the resistance index of SKOV-3/DDP = 3.44. Drug-resistant cells that still maintain a good resistance characteristics, to meet the requirements of the follow-up experiments.3. The expression level of AKT1 and AKT2 of drug-resistant ovarian cancer cell line were higher than their parental cell line, may be involved in chemotherapy resistance. There was no significant difference between SKOV-3/DDP and SKOV-3 cells in the total amount of AKT protein expression ; The active pAkt (Thr 308 and Ser 473) expression of SKOV-3/DDP cells was significantly higher than SKOV-3 cells.The results suggest, the difference of pAkt (Thr 308 and Ser 473) expression between SKOV-3/DDP and SKOV-3 cells may be related to the resistance of the chemotherapy.4.AKT activity inhibitor API-2 in vitro study reversed cisplatin-resistant ovarian cancer, western blot results showed that, API-2 is indeed to achieve the purpose of inhibiting Akt activity, with the control group SKOV-3/DDP and SKOV-3 cells than, API-2 pretreatment of cells half inhibitory concentration IC50 value decreased significantly.The results of Annexinâ…¤/ PI staining by flow cytometry and Annexinâ…¤/ PI staining under confocal microscope coincides, API-2 pretreatment of cells had a higher rate of apoptosis, ovarian cancer of cisplatin resistance was improved in vitro.5.Immunofluorescence detection of YB-1 protein in SKOV-3, SKOV-3/DDP cells, results showed that YB-1 protein expression in cisplatin-resistant ovarian cancer cell lines SKOV-3/DDP was strongly positive ;while the YB-1 protein expression in ovarian cancer cell line SKOV-3 of was weakly positive.The results of Western blot semi-quantitative analysis showed that: in SKOV-3/DDP cells, phospho-YB-1 (Ser102) and YB-1 protein expression was significantly higher than in SKOV-3 cells.6. After AKT activity was inhibited, YB-1 expression in positioning was compared between SKOV-3 and SKOV-3/DDP cells by immune cells fluorescence . In SKOV-3 cells, YB-1 of the nucleus translocate to the cytoplasm; in SKOV-3/DDP cells we have also seen significant YB-1 accumulation in the cytoplasm.Western blot analysis of semi-quantitative detection of the expression of YB-1 in the nucleus.In the nucleus, compared with SKOV-3 cells, total YB-1 protein and phospho-YB-1 (Ser102) expression increased in SKOV-3/DDP cell nucleus. After SKOV-3 and SKOV-3/DDP cells dealed with cisplatin, treated group compared with the control group, total YB-1 protein and phospho-YB-1 (Ser102) is also a slight increase in expression. API-2 pre-treatment 1 hour by adding cisplatin intervention group After inhibiting AKT activity, YB-1 phosphorylation was also inhibited expression, resulting in nuclear YB-1 expression to reduce.7. The results of immune cells fluorescence: compared with SKOV-3 cells, P-gp expression levels in SKOV-3/DDP cells was higher.8.Extraction of total cellular protein, Western blot semi-quantitative analysis showed that: compared with SKOV-3 cells, SKOV-3/DDP cells P-gp expression levels was higher.After two types of cells dealed with cisplatin, treated group compared with the control group, P-gp expression also increased slightly, API-2 pre-treatment 1 hour by adding cisplatin intervention group after suppress AKT activity, YB-1 phosphorylation expression was also inhibited, resulting in the nuclear expression of YB-1 reduced, P-gp expression was partly inhibited.Conclusions1. The relevant analysis of p-AKT and P-gp expression in epithelial ovarian cancer tissue was first carried out.2. p-AKT and P-gp expression were positive for epithelial ovarian cancer have a certain predictive value of chemotherapy resistance, p-AKT over-expression may have a positive role in promoting P-gp expression. The patients with p-AKT and P-gp-positive expression had poor prognosis.3.Sub-AKT expression in the cisplatin-resistant ovarian cancer cell lines was first detected by Immunocytochemical. The results suggest that the level of AKT1 and AKT2 in drug-resistant ovarian cancer cell lines was higher than the parental cell lines, may be involved in chemotherapy resistance.4.The first choice of AKT activity inhibitor API-2 in vitro in the reversal of cisplatin-resistant ovarian cancer, western blot results showed that, API-2 is indeed to achieve the purpose of inhibiting Akt activity, with the control group SKOV-3/DDP and SKOV-3 cells than, API-2 pretreatment of cells half inhibitory concentration IC50 value decreased significantly.The results of Annexinâ…¤/ PI staining by flow cytometry and Annexinâ…¤/ PI staining under confocal microscope coincides, API-2 pretreatment of cells had a higher rate of apoptosis, ovarian cancer of cisplatin resistance was improved in vitro. 5.One of the biological characteristics that SKOV-3/DDP cells is different from SKOV-3 cells is cells total YB-1 protein and phospho-YB-1 (Ser102) expression enhancement.6. Cisplatin can stimulate the expression of YB-1 and phospho-YB-1 (Ser102) in SKOV-3 and SKOV-3/DDP cells, and enhanced the expression of YB-1 protein in nuclear translocation.7. API-2 pretreatment of SKOV-3 and SKOV-3/DDP cells partly inhibit protein YB-1 nuclear translocation and expression of P-gp.This study confirmed the PI3K/AKT cell signaling pathway involved in cisplatin-resistant ovarian cancer, YB-1 is the AKT substrate, PI3K/AKT cell signaling pathway mediated YB-1 nuclear translocation through phosphorylation of YB-1, and regulated the P-glycoprotein expression in SKOV3/DDP resistant ovarian cancer cells. PI3K/AKT/YB-1 signaling pathway for the reversal of drug resistance in ovarian cancer therapy targeted provide new research ideas.
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