消癌平注射液通过调控PXR/CAR增强紫杉醇抗卵巢癌的作用机制研究

Paclitaxel (PTX) is a first-line chemotherapy drug of ovarian cancer, which is metabolized and transferred by CYP2C8,CYP3A4/5 and P-gp, the downstream genes of nuclear receptors PXR and CAR. However, multidrug resistance represses the therapeutic effect of paclitaxel. The Xiaoaiping injection (XAP) combined with paclitaxel has been used for the treatment of ovarian cancer in clinic. However, the mechanism is unclear. As PXR/CAR is one of the targets of cancer multidrug resistance, in the previous experiment, we found that Xiaoaiping injection could downregulate the activity of CYP2C8 and CYP3A4 and also enhance the antitumor effect of paclitaxel in vitro and affect the expression of PXR and CAR. Therefore, it turns out that XAP may regulate PXR/CAR to affect the downstream genes CYP2C8, CYP3A4/5 and P-gp, then increase the exposure of paclitaxel to achieve synergistic effect. Hence, this research will identify an ovarian cancer cell line with high-expressed of PXR and CAR and establish its xenograft tumor model of nude mice. Plasmid transfection, co-immunoprecipitation, luciferase reporter gene, RT-PCR and LC-MS/MS assay will be used to elucidate the molecular mechanism of XAP enhanced antitumor effect of PTX on ovarian cancer, which is significant to reveal the synergistic interaction of Chinese herbal and chemical drug combination.

紫杉醇（PTX）是卵巢癌的一线化疗药物，主要通过核受体PXR和CAR的下游基因CYP2C8、CYP3A4/5和P-gp进行代谢和转运，但多药耐药影响了其有效性。消癌平注射液（XAP）在临床上已与PTX联合用于治疗卵巢癌，但机制不清。PXR和CAR是肿瘤多药耐药的靶点之一，我们前期研究发现XAP可以抑制CYP2C8和CYP3A4的活性，预实验表明XAP对PTX具有协同增效作用且能影响PXR和CAR的表达。故推测XAP能通过调控PXR/CAR的表达而影响其下游基因CYP2C8、CYP3A4/5和P-gp的活性，增加PTX的暴露量等达到协同增效作用。因此，本研究拟使用高表达PXR和CAR的卵巢癌细胞及其异位移植瘤裸鼠模型，应用质粒转染、免疫共沉淀、双荧光素酶报告基因、RT-PCR和LC-MS/MS等技术和方法，阐明消癌平注射液增强紫杉醇抗卵巢癌的作用机制，对揭示中西药联用协同增效规律有重要意义。

紫杉醇是治疗卵巢癌的一线化疗药，但在临床应用过程中易引起耐药而导致肿瘤复发和转移。中药消癌平注射液在临床上常与紫杉醇等化疗药联合用于卵巢癌的治疗，两药联合的作用及分子机制仍有待阐明。本课题从体内外两个方面开展研究，体外采用卵巢癌A2780细胞和SKOV3细胞研究消癌平注射液对紫杉醇的增效作用，采用中效原理评价发现两药之间存在协同作用；通过过表达质粒和SiRNA的干扰实验，发现消癌平注射液通过抑制核受体PXR和CAR及其下游药物代谢酶CYP2C8、CYP3A4、CYP3A5和转运体P-gp，及对凋亡相关蛋白Bax和Bcl-2的调控，发挥协同增效作用。大鼠体内药物代谢与药物动力学研究显示，消癌平注射液增加紫杉醇在体内的暴露量，通过超高效液相色谱-飞行时间质谱联用（UPLC-QTOF-MS）技术，鉴定出紫杉醇在大鼠体内的代谢产物包括3’-p-羟基紫杉醇、2-羟基紫杉醇、6α-2-二羟基紫杉醇和6α-5’’-二羟基紫杉醇。裸鼠卵巢癌异位移植瘤模型的研究显示，消癌平注射液联合紫杉醇可协同抑制肿瘤的生长，且对体外发现的作用机制进行了验证。本项目阐明消癌平注射液通过调控核受体PXR/CAR对紫杉醇抗卵巢癌的增效作用和分子机制，对揭示中西药联用协同增效规律有重要意义，同时为卵巢癌治疗的新的中西药联用方案提供基础研究数据支撑。