S100A4Contributes To Cisplatin Resistance In Ovarian Cancer

BACKGROUNDOvarian cancer is one of the common gynecological malignancies, which isdifficult to be detected at the early stage due to its insidious onset, tendency tometastasis and poor prognosis. Due to the lack of specific diagnostic tools for theearly stage,70%of patients with ovarian cancer have pelvic metastases or lymphnode metastasis while they are diagnosed at the advanced stage. Chemotherapy isthe main treatment for patients at the advanced stage or patients withcytoreductive surgery. Although platinum-based combination chemotherapyfor ovarian cancer has made some progress,5-year survival rate has notsignificantly improved. Cisplatin is widely used as one of the first-linechemotherapy drugs and has significant anti-tumor activity in a variety of solidtumors. However, its effect is not satisfactory with the emergence of drugresistance. Therefore, to explore some novel molecular targets or effectivedrug-resistant reversal agents of ovarian cancer has become the most important problem.S100calcium-binding protein A4is one of the members of the family ofcalcium-binding protein S100. As a kind of calcium sensor protein, the S100family of calcium binding proteins has been shown to be involved in a variety ofphysiological functions, such as cell cycle regulation, cell proliferation anddifferentiation, cell adhesion movement, enzyme activation and apoptosis. Manystudies have confirmed that elevated levels of S100A4are closely associated withrenal cancer, colon cancer, gastric cancer, bile duct cancer, ovarian cancer andother malignancies. It is closely related to tumor development, invasion andmetastasis, chemotherapy sensitivity. Moreover, it could be regarded as one ofvaluable indicators of patients with recurrence and prognosis. Does S100A4expression have any effect on the treatment of ovarian cancer? What is therelationship between cisplatin resistance and S100A4? These issues requirefurther study.In this study, Western blot, real time RT-PCR, immunocytochemistry wereused to detect the expression of S100A4in ovarian cancer cells and to analyzethe correlation between cisplatin resistance and S100A4; then we used DNArecombinant technique to construct S100A4recombinant plasmid;the sensitivityto cisplatin was observed in cisplatin-sensitive cell line A2780transfected withS100A4recombinant plasmid; we used siRNA to downregulate S100A4geneexpression and observed the changes of the sensitivity to cisplatin in ovariancarcinoma cisplatin-resistant cell line CP70. This study provided a directexperimental evidence of the correlation beteen S100A4and cisplatin resistancein ovarian cancer. It provided a foundation for the mechanism of S100A4inovarian cancer cisplatin-resistance.OBJECTIVES 1. To observe the expression of S100A4in ovarian cancer cells.2. To observe the potential correlation between S100A4expression andcisplatin resistance in ovarian cancer cells.3. To observe the effects of S100A4recombinant plasmids and S100A4siRNAon the sensitivity to cisplatin in ovarian cancer cells, and to provide someevidences that S100A4is a molecular target to reverse cisplatin resistance inovarian cancer.METHODS AND CONTENT1. The object of study was ovarian cancer cell line SKOV3, cisplatin-sensitivecell line A2780, cisplatin resistant cell line CP70, high metastasis cell lineHO-8910PM and low metastatic cell line HO-8910. Real time PCR andWestern blot were used to observe the expression of S100A4.2. Immunocytochemistry was used to observe the expression of S100A4andthe subcellular localization in cisplatin-sensitive cell line A2780andcisplatin-resistant cell line CP70. MTT assays are performed to evaluate thedifference of cisplatin sensitivity. Western blot and Real time PCR analyzedthe changes of S100A4expression in CP70cells which are pretreated bycisplatin.3. Recombinant DNA technology is used to construct S100A4-pEGFP-N1recombinant plasmids.Real time PCR and Western blot were used to evaluatethe mRNA and protein levels of S100A4expression in A2780cells aftertransfection. MTT assays were performed to evaluate the effects of S100A4recombinant plasmids on cisplatin sensitivity of A2780cells.4. Real time PCR and Western blot were used to determine S100A4expressionrespectively in CP70cells transfected with S100A4siRNA. MTT assays were performed to evaluate the influence of S100A4gene silence oncisplatin sensitivity of CP70cells.RESULTS1. In the five kinds of ovarian cancer cell lines, the cisplatin-resistant cell lineCP70has the highest expression level of S100A4, while the sensitive cell lineA2780has the lowest expression.2. The IC50value of cisplatin in A2780and CP70cells was17.09μM and62.10μM. S100A4was mainly located in the cytoplasm of A2780and CP70cells. Western blot and Real Time PCR showed the mRNA and protein levelsof S100A4expression raised in CP70cells pretreated by cisplatin.3. A2780cells transfected with S100A4-pEGFP-N1plasmid displayed anincreased S100A4expression. Compared with empty vector group, the IC50value of S100A4-pEGFP-N1has raised from19.06μM to51.09μM. Thetolerance to cisplatin increased2.69-fold.4. CP70cells transfected with S100A4siRNA displayed an decreasedexpression of S100A4. Compared with control group, the IC50value ofS100A4siRNA has declined from64.38μM to38.12μM. The sensitivity tocisplatin enhanced1.69-fold.CONCLUSIONS1. A2780cells has low expression of S100A4, while CP70cells has highexpression of S100A4. Overexpression of S100A4significantly reducedsensitivity to cisplatin in CP70cells. Cisplatin can induce the upregulation ofS100A4in CP70cells. It occurs in the cytoplasm. It was indicated thatoverexpression of S100A4is closely associated with cisplatin-resistance inovarian cancer.2. Increased expression of S100A4strongly promotes cisplatin resistance in A2780cells. Knockdown of S100A4inhibited cisplatin resistance in CP70cells. S100A4is a potential therapeutic target for human ovarian cancer. Thissuggests that S100A4plays an important role in cisplatin resistance ofovarian cancer cell.