乳源六肽结合EEF1D逆转卵巢癌细胞耐药性及其分子机制

Ovarian cancer is one of the most serious malignant gynecological tumors. Due to primary and secondary development of multidrug resistance, the researches on decreasing chemoresistance, elucidating the molecular mechanism of resistance and improving chemotherapy sensitivity have important significance for the treatment of ovarian cancer. PGPIPN is a multifunctional hexapeptide derived from beta casein in bovine milk. Previous studies have found that PGPIPN significantly sensitized the chemotherapy of ovarian cancer cell line SKOV3, target protein of which is eukaryotic translation elongation factor 1 delta (EEF1D). This project will study that the effects of PGPIPN on the reversing chemoresistance of ovarian cancer cell, and what influence exerted by PGPIPN on genes associated multidrug resistance (MDR) and signaling pathways by binding and inactivating target protein-EEF1D. In this study, we will utilize resistant and sensitive ovarian cancer cell lines to drugs, clinical ovarian cancers insensitive to chemotherapy (progressive disease/stable disease, PD/SD) and sensitive to chemotherapy (complete response/partial response, CR/PR) in vitro, and the xenograft SCID mice model in vivo. We will study that PGPIPN contributes to reversing drug resistance of ovarian cancer mediated by EEF1D in the cellular level. By small interfering RNA (siRNA), two-dimensional electrophoresis, liquid chromatography-mass spectrometry (LC-MS), cDNA chip, real time PCR, immunohistochemistry (IHC) and Western Blot, bioinformatics (gene set enrichment, pathway, etc.) and other methods, we will probe EEF1D function as target of PGPIPN, genes related chemoresistance and signaling pathways，to clarify the reversal mechanism of ovarian cancer cell chemoresistance in the molecular level. These are validated through real-time quantitative PCR, western blot and luciferase reporter gene system. By the xenograft tumor SCID mice model, the about effects will be verified in the overall. By studying these mechanisms of PGPIPN reversing chemoresistance of ovarian cancer mediated by EEF1D, the project will provide new ideas, targets, methods and drugs for the therapy of ovarian cancer.

卵巢癌由于原发性与继发性耐药,是妇科治疗最为困难的恶性肿瘤。探索化疗耐药的分子机制, 提高化疗敏感性，对卵巢癌的治疗有重要意义。PGPIPN是一具有多种功能的乳源六肽。前期研究发现，PGPIPN显著增敏卵巢癌细胞株SKOV3的化疗，其结合靶蛋白是翻译延长因子1δ（EEF1D）。本项目利用耐药和敏感卵巢细胞株、化疗不敏感和敏感临床卵巢癌组织、SCID鼠荷瘤模型，研究PGPIPN通过结合和失活EEF1D逆转卵巢癌的药物抗性及其对耐药相关基因和信号途径影响，以阐明PGPIPN逆转卵巢癌细胞抗药及其机制。通过siRNA、2D电泳、LC-MS、IHC和cDNA芯片等，从分子水平研究EEF1D功能、耐药相关基因及信号途径，以明确耐药作用相关机制；用裸鼠种植瘤实验，从整体验证PGPIPN对EEF1D介导的耐药和相关机制。本项目通过探索乳源六肽逆转卵巢癌抗药机制，为卵巢癌治疗提供新思路、靶点、手段和药物。

卵巢癌由于原发性与继发性耐药，是妇科治疗最为困难的恶性肿瘤。探索化疗耐药的分子机制, 提高化疗敏感性，对卵巢癌的治疗有重要意义。本项目通过探索乳源六肽逆转卵巢癌抗药机制，为卵巢癌治疗提供新思路、靶点、手段和药物。PGPIPN是一种有多种功能的乳源六肽，其结合靶蛋白之一是翻译延长因子1δ（EEF1D），PGPIPN通过直接或间接结合EEF1D，降低其活性。本项目利用敏感和耐药卵巢癌细胞株、化疗不敏感和敏感临床卵巢癌组织、BALB/c荷瘤鼠模型，研究PGPIPN逆转卵巢癌的药物抗性及其机制。结果表明，乳源六肽PGPIPN显著增加卵巢癌细胞株（SKOV3/DDP、COC1/DDP）、原代卵巢癌细胞对DDP的敏感性，但对敏感细胞株（SKOV3、COC1）不敏感，作用包括增加DDP对细胞增殖的抑制、增加DDP对细胞凋亡的诱导、增加细胞周期的阻滞（阻滞在G2/M期）、增加DDP对癌细胞侵袭和迁移的抑制、增加DDP对贴壁癌细胞克隆形成的抑制。利用siRNA建立沉默EEF1D基因的稳定SKOV3/DDP细胞模型，利用CRISP/Cas9建立EEF1D基因敲除的稳定SKOV3/DDP细胞模型，结果表明敲减EEF1D基因或敲除EEF1D基因明显加强DDP对卵巢癌的增殖抑制（敲减和敲除EEF1D基因最高可分别提高19.31%和50.62%的抑制率）。实验结果也表明，在DDP作用卵巢癌细胞48h后其自噬显著增加，PGPIPN通过抑制卵巢癌细胞自噬降低其耐药性，从而促进DDP诱导细胞凋亡和增殖降低，并引起细胞周期G2/M期阻滞。PGPIPN通过akt1-PI3K-ERCC1/MDR1、HSF1-HSP70-ERCC1/MDR1和siah-1-PSMA-β-catenin等多条信号途径降低卵巢癌的耐药作用。动物实验表明DDP+PGPIPN和DDP+ EEF1D siRNA能显著加强对荷瘤生长的抑制，差别具有显著性，其中相对于对照组和DDP组，DDP+PGPIPN组抑瘤率最大可提高42.17%和29.57%，而 EEF1D siRNA组抑瘤率最大，分别达到65.59%和50.19%。DDP+PGPIPN和DDP+ EEF1D siRNA组瘤组织凋亡指数显著增加，其中DDP+shEEF1D组裸鼠瘤组织细胞凋亡指数高达61.71%。同时PGPIPN能显著改善荷瘤动物的体质和增加体重。