| Objective: Androgens and estrogens have been associated with the risk for epithelial OVCA. Both IL-6 and IL-8 are also likely involved in the progression of OVCA. In order to discover the underline molecular mechanism, we investigated the modulation of these two steroid hormones and two cytokines in the growth of epithelial OVCA.On the other hand, Raf kinase inhibitor protein (RKIP) levels were found to be reduced or lost in prostate cancer, breast cancer, melanoma cells, and insulinomas. Studies in cell cultures and animal models have suggested a role of RKIP in suppressing the metastatic spread of prostate cancer, breast cancer, and melanoma cells. However, the expression and role of RKIP in OVCA has not been previously reported. Therefore, we investigated the expression of RKIP in human ovarian epithelial tumors and five OVCA cell lines, and the potential role of RKIP in the metastasis and development of epithelial OVCA.Methods: In these studies, the expression of androgen receptor (AR), estrogen receptor (ER) a, ERp, IL-6 receptor (IL-6R α and gp130) and IL-8 receptor (IL-8RA and IL-8RB) in five epithelial OVCA cell lines was detected by Western blot analysis and Reverse transcription polymerase chain reaction (RT-PCR). OVCA cells expressing AR and/or ER, IL-6 and IL-8 receptors were selected to study related molecular mechanism. MTT assay was performed to investigated the effect of 5 α -dihydrotestosterone (DHT) , 17 β -estradiol (E2), IL-6 and IL-8 on the proliferation of the above cells.Furthermore, the regulatory effect of DHT and E2 on the expression of IL-6, IL-8 and their respective receptors was investigated, reciprocally, the effect of IL-6 andIL-8 on AR, ERa, and ERp expression was also analyzed by Enzyme-linked immnosorbent assay (ELISA), RT-PCR, Western blot analysis, and Luciferase reporter assay.At the same time, to examine RKIP expression and distribution in vivo and in vitro, clinical samples of human ovarian epithelial tumors, including 10 samples of benign ovarian epithelial tumor, 8 samples of borderline OVCA, and 22 samples of epithelial OVCA, as well as high-metastatic human OVCA HO-8910PM cell line and the parental low-metastatic HO-8910 cell line were immunostained with RKIP antibody. Furthermore, in order to determine the potential effect of RKIP on the invasive ability of OVCA cells, RT-PCR and Western blot analysis were performed to investigate the expression of RKIP in five epithelial OVCA cell lines, Transwell Chamber system were performed to analyze the in vitro invasion ability of different correlated OVCA cells.Moreover, stable cell lines overexpressing or deleting of RKIP were cloned to investigate the function of RKIP in OVCA cells. The recombinant plasmids expressing sense (ss) or antisense (as) RKIP cDNA or empty vector was transfected into human OVCA cell line SKOV-3 by lipofectamine, the positive cell clone was selected by G418. The expression level of RKIP protein and the effect of RKIP on MAP kinase activity in OVCA cells were detected by Western blot analysis. Assays of cell proliferation, soft-agar colony formation, in vitro cell adhesion, and in vitro cell invasion were used to examine the malignant phenotypes of the transfected cells. Flow cytometric analysis was performed to observe the effect of RKIP on cell cycle distribution, and ELISA and RT-PCR were performed to examine variant expression of IL-6 and IL-8 before and after transfection.Results: In this study, we found that DHT and E2 markedly promoted OVCA cellproliferation, and enhanced IL-6 and IL-8 secretion, meanwhile, regulated respective expression of IL-6 and IL-8 receptor. Flutamide (Flu), an AR antagonist, or tamoxifen (Txf), an ER antagonist, completely abolished DHT- or E2-stimulated cell growth and the expression of IL-6 and IL-8. On the other hand, IL-6 and IL-8 also significantly increased OVCA cell proliferation, which was completely blocked by their specific neutralizing antibodies. Moreover, IL-6 or IL-8 antibody partially inhibited DHT- or E2-induced cell growth.In the absence of androgen, both cytokines enhanced AR expression and AR promoter activation, which was completely blocked by Flu. However, Flu failed to reduce IL-6- or IL-8-induced cell growth. Similarly, in the absence of estrogen, both cytokines activated estrogen-responsive promoter, which was completely blocked by Txf, up-regulated the expression of functional ER a expression, and down-regulated the expression of ER P protein. Txf also reduced IL-6- or IL-8-induced cell growth. Pretreatment of OVCA cells with p38 MAPK, MEK1/2, and ErbB2 MAPK inhibitors, respectively, resulted in the blockage of IL-6-mediated enhancement of AR transcription and activation of estrogen-responsive promoter, while Src inhibitor blocked IL-8 induced AR transcription and estrogen-responsive promoter activation.In this study, the expression levels of RKIP protein in epithelial OVCA samples from patients were found to be reduced markedly than those with benign ovarian epithelial tumor and borderline OVCA. Furthermore, the expression levels of RKIP protein in poorly differentiated OVCA were significantly decreased than in well-differentiated or moderately differentiated OVCA. We also discovered that RKIP expression was inversely associated with the invasiveness of five OVCA cell lines in vitro.SKOV-3 clones stably expressing full-length recombinant pcDNA3.1(+)-ssRKIP, pcDNA3.1(-)-asRKIP, and their respective empty vector were obtained. RKIP wasable to block basal levels of MEK and ERK activation by Raf-1 in OVCA cells. Abilities of cell proliferation, anchorage-independent growth, cell adhesion, cell invasion, and IL-8 expression in the ssRKIP vector-transfected cells were decreased compared with the empty vector pcDNA3.1 (+)-transfected and/or non-transfected cells, whereas those in the asRKIP vector-transfected cells were increased compared with the empty vector pcDNA3.1(-)-transfected and/or non-transfected cells. Compared with the empty vector-transfected and non-transfected cells, ssRKIP-expressing cells had a significant increase in the Gl phase and decrease in the G2 + S phase, while asRKIP-expressing cells had a significant decrease in the G1 phase and increase in the G2 + S phase, suggesting RKIP inhibited OVCA cell proliferation by altering cell cycle distribution, rather than by promoting apoptosis.Conclusion: Our present study provides a novel mechanism that androgens, estrogens, IL-6 and IL-8 may form a common amplifying signaling cascade to modulate OVCA growth. It provides the insight into the mechanisms underlying the connection of androgen/estrogen and IL-6/IL-8 in OVCA proliferation.Metastasis suppressor genes have been shown to suppress the growth of metastases without affecting the growth of the primary tumor. These genes differ from tumor suppressor genes, which suppress the growth of primary tumors. In our studies, RKIP not only inhibits the metastasis, but also the growth of OVCA cells. Thus, we hypothesize that RIKP appears to be a tumor suppressor gene rather than metastasis suppressor genes in epithelial OVCA.Our data show an inverse correlation tendency between RKIP protein expression and malignant degree of ovarian epithelial tumors, suggesting that RKIP could be used as a marker in estimating the malignant degree of ovarian epithelial tumors, evaluating the malignant potential of borderline OVCA, and in differential diagnosisof borderline OVCAand OVCA.
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