Roles Of RabJ, A Novel Small GTPase, In The Cisplatin Resistance Of Human Ovarian Carcinoma

Ovarian carcinoma is one of the common gynecological tumors. The mortality of it is the on the top of the gynecological tumors because more than 70% of ovarian carcinoma are discovered in the late stage.Chemotherapy is one of the effective therapy for the ovarian carcinoma, especially for those in the late stage. The cisplatin is one of the most effective anticancer drug and has been widely used in clinical treatment of many kinds of solid tumors. Its cytotoxicity is mainly mediated through the connection of platinum with nucleoprotein and then the formation of crosslinking between platinum and external DNA chain or internal DNA chain, resulting in the crosslinking damage of DNA and protein. The damage signal may activate apoptosis signal through mitogen-activated protein kinase (MAPK) and DNA damage-related apoptotic signaling proteins including ATR, P53 and P73 etc, and finally cause fatal damage of DNA, leading to cell apoptosis and cell cycle restrain. However, the platinum resistance of tumor cells becomes more and more stronger,which obviously affects the clinical use of platium. Previous studies have shown that the platinum resistance is resulted from multiple molecular mechanisms and is related to the unusual signal conduction inside the cell. MAPK signaling pathway is one of the signaling mechanisms that are closely correlated to drug resistance of tumors.RabJ is a representative of a novel family of small GTPase, and its biological functions are related to cell proliferation and MAPK signaling.RabJ may serve as a nuclear anchor for phosphorylated MEK1/2 and, in turn, block thenucleocytoplasmic translocation of ERK1/2, resulting in constitutive activation of ERK/12 locally in nucleus and unusual activation of the MEK/ERK signaling cascade. Based on the above background, we hypothesize that RabJ may be involved in drug resistance of human ovarian carcinoma through control of the MAPK signal pathway and blockade of RabJ expression may reverse the chemotherapy resistance of tumor cells.This research mainly focuses on the differential expression pattern of RabJ in various ovarian carcinoma cell lines and the roles of RabJ in chemoresistance of ovarian carcinoma to cisplatin. We investigated the sensitivity of ovarian cancer cells to cisplatin under conditions of stable overexpression and RNA interferencing of RabJ. Our studies may cast new light on the molecular mechanisms of cisplatin resistance of ovarian carcinoma and provide new therapeutic target for the resolve of drug resistance of tumors.Methods: 1. Extraction of the total RNA derived from various ovarian carcinoma cell lines to investigate the expression pattern of RabJ after reverse transcription and PCR amplification. 2. Overexpression of full-length RabJ in RabJ-negative HO-8910 cell lines by Lipofectamine and stable selection by neomycin. 3. Blockade of RabJ expression in SKOV-3 cells by stable selection of the expression of RabJ interferencing vectors. 4. Test the variability of different ovary cancer cells after cisplatin treatment to verify the cytotoxic sensitivity to tumor cells by using the MTT method.5. Using the method of RT-PCR to observe the variety of expression of RabJ and mRNA in different time after the cisplatin treatment of SKOV-3 cells.Results: 1. RabJ mRNA was expressed high in cisplatin-resistant ovary cancer cells such as SKOV-3 cells and CaoV-3 cells, but was not expressed in the cisplantin-sensitive ovary cancer such as HO-8910 and HO-8910pm. 2. SKOV-3 and HO-8910 have different sensitivity to cisplatin. 3. Overexpression of RabJ in HO-8910 cells induced the resistance of the tumor cells to cisplatin, and blockade ofRabJ expression in SKOV-3 cells increased the sensitivity of the tumor cells to cisplantin. 4. Cisplantin could induce the expression of RabJ on day 3 and day 4 after cisplatin stimulating of SKOV-3 cells.Conclusions: 1. Ovary cancer cells show different sensitivity to cisplatin, which is related with the expression level of RabJ. 2. RabJ can potentiate the cisplatin resistance of tumor cells. 3. RabJ-mediated enhancement of cisplantin resistance is possibly related to cisplantin-induced upregulation of RabJ and the activation of RabJ-induced NEK/ERK activation.