异甘草素通过Rac1-MEK1/Src信号通路抑制卵巢癌转移的作用及机制研究

High mortality rate of ovarian cancer is most likely to be caused by late diagnosis when patients are already in advanced stages. Almost all of patients will meet their demise owing to metastasis. Like other epithelial-derived tumors, EMT ( Epithelial–Mesenchymal Transition) has been demonstrated as a critical step for ovarian cancer metastasis progression, and is significantly associated with survival of ovarian cancer patients. Our previous study has revealed that Rac1-MEK1/Src signaling pathway is important for ovarian cancer metastasis through sustaining EMT process. Importantly, we demonstrated that combined use of MEK1/2 and Src inhibitors effectively suppresses development of intraperitoneal xenografts and prolongs the survival of ovarian cancer-bearing mice. Based on drug screening, we further found that isoliquiritigenin was able to inhibiting the activation of MEK1 and Src. Thus, this project is aimed to evaluated the anti-metastasis effects of isoliquiritigenin on ovarian cancer, and confirm the critical role of Rac1-MEK1/Src signaling pathway.

腹腔内播散和远处转移是导致卵巢癌患者死亡的主要因素。上皮-间充质转化（ Epithelial–Mesenchymal Transition， EMT）是卵巢癌转移过程中的关键步骤, 与腹腔转移及与卵巢癌病人的存活率十分相关。本人前期已发表的研究结果证实，Rac1-MEK/Src信号通路可通过EMT过程参与卵巢癌转移，并且MEK1、Src的小分子抑制剂联用能够减少卵巢癌腹腔转移及显著延长动物的存活期。在此基础上，我们发现异甘草素能够抑制MEK1、Src的活性。因此，本项目的是评价异甘草素是否具有逆转卵巢癌 EMT 过程从而抑制卵巢癌转移的作用，并确证其分子机制是否通过Rac1-MEK1/Src信号通路实现的。

卵巢癌是常见的妇科肿瘤之一，其死亡率居妇科恶性肿瘤之首。由于缺乏早期的临床征状和有效筛查指标，大约 75%的患者在确诊时已进入晚期，癌症已经转移到整个腹腔，存活率一般只有17-36% 。上皮-间充质转化是卵巢癌转移过程中的关键步骤，与腹腔转移及与卵巢癌病人的存活率十分相关。因此若能抑制卵巢癌EMT过程及转移，将提高卵巢癌病人的存活率，为治疗卵巢癌提供一个有效的途径。本项目主要研究了异甘草素是否能够具有逆转卵巢癌 EMT 过程从而抑制卵巢癌转移的作用，并且探讨了其具体的分子机制。首先采用MTT实验证明0-10μM低浓度的异甘草素对卵巢癌细胞生长没有明显的抑制作用。从细胞形态观察发现10uM 的异甘草素能使间质样卵巢癌细胞SKOV3和OVCAR5向上皮样细胞形态转变，同时逆转Rac1G12V持续激活诱导的卵巢癌EMT过程，也能逆转MEK1-DD和Src-Y527F共同诱导的上皮样卵巢癌细胞EMT过程。划痕，transwell和侵袭实验证明10uM 的异甘草素能明显抑制卵巢癌细胞SKOV3、OVCAR5、HEC-1A/Rac1G12V和OVCAR3/ Rac1G12V的迁移和侵袭，也能逆转MEK1-DD和Src-Y527F同时诱导的卵巢癌细胞迁移和侵袭能力。Western blot 实验证明10uM 的异甘草素逆转卵巢癌SKOV3和OVCAR5 EMT标志蛋白的表达，也能逆转Rac1诱导的或MEK1-DD和Src-Y527F同时诱导的卵巢癌EMT标志蛋白。采用EMT PCR Array和qRT-PCR筛选及证明异甘草素能降低SKOV3、OVCAR5、HEC-1A/Rac1G12V和OVCAR3/ Rac1G12V细胞中转录因子ZEB1表达。体内动物实验证明异甘草素能延长卵巢癌动物的存活及逆转卵巢癌EMT过程从而降低卵巢癌的转移。总之，异甘草素通过Rac1-MEK1/Src信号通路来逆转卵巢癌EMT过程，同时ZEB1转录因子调控该过程，从而发挥抑制卵巢癌转移的作用。因此，本项目研究为卵巢癌临床治疗提供理论和实验依据，为新药研发奠定基础，同时体现中药抗肿瘤治疗的巨大应用潜力，具有一定的临床转化意义。