Role and regulation of GnRH and receptors in Cisplatin resistant ovarian cancer

描述（申请人提供）：卵巢癌（OVCA）是最致命的妇科恶性肿瘤，化疗耐药性是成功治疗的主要障碍。参与化学敏感性调节的机制尚不完全清楚。促性腺激素释放激素（GnRHs）及其受体（gnrrs）在人卵巢表面上皮中的存在，提示OVCA中可能存在自分泌调节环，该系统参与顺铂（CDDP）诱导的细胞凋亡。我们假设GnRHs和gnrrs在控制化疗敏感性方面很重要，并且它们的失调可能导致OVCA的化疗耐药。我们的研究目的是研究GnRH及其受体在OVCA中CDDP敏感性调控中的表达和作用，使用具有相同遗传背景的对化疗敏感和化疗耐药的人OVCA细胞系，并建立p53和Akt状态。将使用sIRNA、显性阴性（DN）和sense cDNA。在Aim 1中，CDDP对化疗敏感细胞（OV2008和A2780）和化疗耐药细胞（CI 3*和A2780cp） GnRH和GnRHR表达的调节将通过RIA、免疫细胞化学、Western blotting和qRT-PCR检测。使用p53突变细胞系将揭示p53是否参与cddp诱导的gnrh介导的细胞凋亡。在Aim 2中，内源性GnRHs和gnrrs的功能将通过确定(a)在化学敏感的OVCA细胞中敲低GnRH及其受体是否赋予这些细胞对CDDP的抗性（减少凋亡）来评估；(b) GnRH受体的强制表达和/或外源性GnRH的添加会使化疗耐药的OVCA细胞对CDDP敏感（增加凋亡）。在Aim 3中，我们将通过研究CDDP治疗下敏感和耐药OVCA细胞的Ca++信号和凋亡反应，确定GnRH系统在化疗耐药中的失调机制。我们将在野生型和突变型p53、p53基因沉默和重组、组成型激活以及DN-Akt表达的OVCA细胞系中确定p53和Akt在这种调控中的作用。本研究将提高对卵巢癌化疗耐药分子机制的认识，并为卵巢癌化疗耐药的新治疗策略的开发提供新的见解。

DESCRIPTION (provided by applicant): Ovarian cancer (OVCA) is the most lethal of the gynecological malignancies and chemoresistance presents a major hurdle for successful treatment. The mechanism involved in the regulation of chemo- sensitivity is not completely known. The presence of gonadotropin releasing hormones (GnRHs) and their receptors (GnRHRs) in human ovarian surface epithelium raises the possible existence of an autocrine regulatory loop in OVCA and that this system is involved in cisplatin (CDDP)-induced apoptosis. We hypothesize that GnRHs and GnRHRs are important in the control of chemosensitivity and that their dysregulation may confer chemoresistance in OVCA. The objective of our study is to examine the expression and the role of GnRH and its receptor in the regulation of CDDP sensitivity in OVCA, using pairs of chemo-sensitive and chemoresistant human OVCA cell lines with same genetic background and established p53 and Akt status. sIRNA, dominant negatives (DN) and sense cDNA will be used. In Aim 1, regulation of GnRH and GnRHR expression in chemosensitlve (OV2008 and A2780) and chemoresistant (CI 3* and A2780cp) cells treated with CDDP will be determined by RIA, immunocytochemistry, Western blotting and qRT-PCR. Use of p53 mutant cell line will delineate if p53 is involved in CDDP-induced GnRH-mediated apoptosis. In Aim 2, the functionality of endogenous GnRHs and GnRHRs will be assessed by determining if (a) knockdown of GnRH and their receptors in chemosensitlve OVCA cells confer resistance in these cells to CDDP (decreased apoptosis); (b) forced expression of GnRH receptors and/or addition of exogenous GnRH would sensitize chemoresistant OVCA cells to CDDP (increased apoptosis). In Aim 3, we will determine the mechanism of dysregulation of the GnRH system in chemoresistance, by investigating Ca++ signaling and apoptotic response of both sensitive and resistant OVCA cells to CDDP with/without GnRH treatment. We will determine the role of p53 and Akt in this regulation in OVCA cell lines with wild type and mutant p53, p53 gene silencing and reconstitution, expression of constitutively activated as well as DN-Akt. This study will improve the understanding of the molecular mechanism of chemoresistance in ovarian cancer and offer new insight in the development of novel therapeutic strategies for chemoresistant ovarian cancer. PUBLICH HEALTH RELEVANCE: Chemoresistance is a major hurdle for successful ovarian cancer treatment. This study will improve the understanding of chemoresistance in ovarian cancer and offer new insight in the development of novel therapeutic strategies for ovarian cancer.