掌叶半夏脂溶性提取物PE对宫颈癌顺铂化疗的协同作用及分子机制

High recurrence rate of cervical cancer and desire for fertility-sparing of some young patients make the position of chemotherapy in cervical cancer rising. How to improve the curative effect of chmotherapy is becoming the hot spot. Anti-tumor Chinese medicine that has chemotherapy synergy can achieve better therapeutic effect within safty range. Previous clinical and experimental research on pedatisecta schott(a traditional Chinese medicine)had demonstrated its validity and safty on cervical cancer treatment. Recently, our research group isolated and purified a lipisoluble extract from pedatisecta schott, named PE. Experiments in vitro and in vivo confirmed that PE can suppress the growth of cervical cancer definitely; PE has synergistic effect with the first line chemotherapeutics cisplatin (DDP)as well. It indicates that the anti-cervical cancer effect of PE is definite, one of its effects is by suppressing proliferation of cancer cells and inducing cancer cells apoptosis, another is via elevating the reactivity of cancer cells to chemotherapeutics. Based on our previous finding that PE has the synergistic effect with the fist line chemotherapy drug DDP, this item ia aimed to carry out further reseach on the synergistic effect and associated molecular mechanisms of PE with DDP in cervical cancer chemotherapy via DDP induced DNA damage repairing signal pathway on cell and animal models. The results of this item will provide certain experiments evidence and theoretical basis for the clinical use of PE as an adjuvant therapy drug in cervical cancer.

宫颈癌的高复发率及年轻患者对保留生育功能的渴求,使得化疗在宫颈癌治疗中的地位不断提升，如何提高化疗疗效已成为关注热点。对化疗有协同作用的抗肿瘤中药可以使药物在治疗安全范围内获得更好的疗效。前期临床和实验研究证实掌叶半夏治疗宫颈癌安全、有效。近期课题组从掌叶半夏中分离纯化出一种新的脂溶性提取物（PE），体内、外实验证实PE抗宫颈癌作用确切，研究揭示PE抗宫颈癌作用之一是直接作用于癌细胞，抑制癌细胞增殖、诱导癌细胞凋亡；另一作用是提高肿瘤细胞对化疗药物的反应性。本项目拟在前期实验发现PE与一线化疗药物顺铂（DDP）有协同抗宫颈癌作用的基础上，应用细胞和动物模型，以DDP作用后可能启动的DNA损伤修复信号通路为切入点，进一步研究PE与DDP联合应用对宫颈癌的治疗作用，验证PE与顺铂的协同作用，并系统研究作用的分子机制。研究结果将为PE临床用于宫颈癌顺铂化疗的辅助治疗提供必要的实验依据和理论基础。

宫颈癌是女性最常见的恶性肿瘤之一，化疗对晚期及需要保留生育功能的患者具有重要意义。顺铂(Cis-dichlorodiammine platinum-II, CDDP)在宫颈癌化疗中的应用广泛，然而CDDP的毒副作用以及临床耐药极大的限制了化疗效果，我们希望从中西医结合的角度来探寻辅助CDDP治疗宫颈癌的药物。掌叶半夏提取物PE(Extract from Pinellia pedatisecta Schott, PE)是从掌叶半夏中分离纯化出的脂溶性物质。前期工作发现PE具有抑制宫颈癌细胞CaSki的增殖，诱导凋亡的作用。本文将对PE与CDDP联合应用对宫颈癌的治疗作用进行研究。体外实验中，与单一药物相比，PE和CDDP联合能够显著抑制宫颈癌细胞的增殖，当达到ED50的抑制作用时，联合用药指数(Combination Index, CI)SiHa为0.43，CaSki为0.68，且无论两种药物达到高细胞毒性ED90或是低细胞毒性ED25时，CI值始终小于1，意味着PE能与CDDP发挥药物协同作用。进一步实验显示与CDDP组相比，PE与CDDP联合给药对CaSki和SiHa细胞产生明显的周期阻滞效应，分别阻滞在G2/M期(12.7±1.2% vs 43.8±3.2%)(p<0.001)和G0/G1期(78.2±3.5 vs 91.1±2.3%)(p<0.01)，同时伴有S期比例的明显降低。PE+CDDP联合给药显著抑制了SiHa和CaSki细胞的侵袭能力，达88%和81%，凋亡相关cleaved-PARP及cleaved-caspase3蛋白表达亦明显增高。此外，与CDDP单药比较，PE+CDDP组ATM，CHK-1，CHK-2和γ-H2AX的磷酸化水平均得到显著提高，表明DNA损伤应答反应ATM/pChk-2信号通路的活化明显增强。而在裸鼠皮下移植瘤实验中，连续给药21天后，各组抑瘤率分别为：PE组26.3%，CDDP组50.8%，PE+CDDP组68.4%，表明PE和CDDP能够协同抑制宫颈癌的体内生长。联合用药组相对于CDDP组的中位生存时间也得到了延长。以上结果表明，PE与CDDP具有协同抑制宫颈癌发生和发展的作用，其作用机制可能是协同增强DNA损伤应答反应中ATM/pChk-2通路的激活，诱导p53介导的细胞凋亡，为研究掌叶半夏辅助治疗宫颈癌提供了重要依据。