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

描述（由申请人提供）：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。涉及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（GnRHRs）在人类卵巢表面上皮细胞的存在，提出了可能存在的自分泌调节环OVCA和该系统参与顺铂（CDDP）诱导的细胞凋亡。我们假设GnRHs和GnRHRs在控制化疗敏感性方面很重要，它们的失调可能会导致OVCA的化疗耐药性。我们的研究目的是研究GnRH及其受体在卵巢癌CDDP敏感性调节中的表达和作用，使用具有相同遗传背景和已建立的p53和Akt状态的成对的化疗敏感和化疗耐药人卵巢癌细胞系。将使用sIRNA、显性阴性（DN）和正义cDNA。目的1：通过放射免疫分析、免疫细胞化学、Western blotting和qRT-PCR检测顺铂对化疗敏感细胞（OV 2008和A2780）和化疗耐药细胞（CI 3* 和A2780 cp）GnRH和GnRHR表达的调节作用。使用p53突变细胞系将描述p53是否参与CDDP诱导的GnRH介导的细胞凋亡。在目的2中，将通过确定（a）化学敏感性OVCA细胞中GnRH及其受体的敲低是否赋予这些细胞对CDDP的抗性（细胞凋亡减少）;（B）GnRH受体的强制表达和/或外源性GnRH的添加是否会使化学抗性OVCA细胞对CDDP敏感（细胞凋亡增加）来评估内源性GnRH和GnRH受体的功能。在目标3中，我们将通过研究Ca++信号和对CDDP敏感和耐药的OVCA细胞在有/无GnRH处理的情况下的凋亡反应，确定GnRH系统在化学抗性中失调的机制。我们将在具有野生型和突变型p53的OVCA细胞系中确定p53和Akt在这种调节中的作用，p53基因沉默和重建，组成性激活的表达以及DN-Akt。本研究将提高对卵巢癌化疗耐药的分子机制的理解，并为卵巢癌化疗耐药的新治疗策略的发展提供新的见解。 公共卫生相关性：化疗耐药性是卵巢癌成功治疗的主要障碍。这项研究将提高对卵巢癌化疗耐药性的认识，并为卵巢癌新的治疗策略的发展提供新的见解。

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.