水飞蓟宾联合阿法替尼抑制卵巢癌细胞增殖和侵袭的机制研究

Study on Inhibitory Effects of Silibinin Combined with Afatinib on Proliferation and Invasion of Ovarian Cancer Cells and Its Mechanism

关键词:  水飞蓟宾|阿法替尼|卵巢癌|黏着斑激酶|细胞骨架

Key words:  silibinin|afatinib|ovarian cancer|focal adhesion kinase|cytoskeleton

摘要:

目的 探讨水飞蓟宾联合阿法替尼对卵巢癌的治疗作用及其可能机制。方法 将不同浓度阿法替尼和水飞蓟宾单独或联合作用于卵巢癌SKOV3和CP70细胞48 h，采用CCK-8法检测水飞蓟宾和阿法替尼单独或联用对细胞增殖的影响，并计算半数抑制浓度(IC₅₀)；采用集落形成试验检测药物对细胞增殖的影响；采用Transwell试验检测药物对细胞侵袭的影响；采用免疫荧光和鬼笔环肽染色检测黏着斑激酶(focal adhesion kinase，FAK)和细胞骨架的影响；采用Western blotting检测药物对FAK及其磷酸化的影响；通过数据库分析FAK在卵巢癌中表达水平以及表达水平与预后之间的关联。结果 水飞蓟宾和阿法替尼均能抑制SKOV3和CP70细胞的生长，且抑制作用呈浓度依赖性，水飞蓟宾和阿法替尼对SKOV3的IC₅₀分别为81.2，1.9 μmol·L^-1，对CP70的IC₅₀分别为154.1，4.1 μmol·L^-1。两药联合应用能显著增加生长抑制率；两药均能抑制SKOV3细胞的侵袭能力，且两药联合作用能显著增加对SKOV3细胞侵袭的抑制作用；两药联用能抑制FAK表达水平及磷酸化。水飞蓟宾和阿法替尼均能够显著破坏细胞骨架的完整性，联用时对细胞骨架的破坏更强；数据库分析表明FAK在卵巢癌中的高表达，且FAK的高表达与卵巢癌预后呈负相关。结论 初步证明水飞蓟宾联合阿法替尼用药能显著抑制SKOV3和CP70的增殖，其作用机制可能是通过破坏细胞骨架和减少FAK的形成来抑制SKOV3细胞的侵袭能力。

Abstract:

OBJECTIVE To explore the inhibitory effect and possible mechanism of silibinin combined with afatinib against ovarian cancer. METHODS Different concentrations of afatinib and silibinin were treated on ovarian cancer cell lines, SKOV3 or CP70 cells, alone or in combination for 48 h. The inhibitory effects of silibinin and afatinib alone or in combination on cell proliferation were detected by CCK-8 assay. The half inhibitory concentration(IC₅₀) was calculated. Transwell test was applied to detect the effect of drugs on cell proliferation. Transwell test was used to detect the effect of drugs on cell invasion. Immunofluorescence and phalloidin staining were applied to detect focal adhesion kinase(FAK) and cytoskeleton. Western blotting was used to evaluate the protein levels and phosphorylation of FAK. Finally, the expression of FAK in ovarian cancer and the correlation between expression and prognosis were analyzed using database. RESULTS Both silibinin and afatinib could inhibit the growth of SKOV3 and CP70 cells via a dose-dependent manner. The IC₅₀ values of silibinin and afatinib against SKOV3 were 81.2, 1.9 μmol·L^-1, respectively; the IC₅₀ values of silibinin and afatinib against CP70 was 154.1, 4.1 μmol·L^-1, respectively. The combination of the two drugs could significantly increase the survival inhibitory rate. In addition, both drugs inhibited the invasion of SKOV3 cells, and the combination of the two drugs significantly increased the inhibitory rate of invasion in SKOV3 cells. The combination of the two drugs inhibited the expression and phosphorylation of FAK. Furthermore, silibinin and afatinib significantly reduced the integrity of cytoskeleton, and the destruction of cytoskeleton was stronger when silibinin and afatinib were combined. Finally, FAK was highly expressed in ovarian cancer through the database, which was negatively correlated with the ovarian cancer prognosis. CONCLUSION It is preliminarily proved that the combination of silibinin and afatinib can significantly inhibit the proliferation of SKOV3 and CP70 cells, and may inhibit the invasion of SKOV3 cells by reducing the formation of cytoskeleton and suppressing the FAK signaling pathway.