金莲花总黄酮诱导人HT-29结肠癌细胞凋亡机制的研究

Apoptotic Mechanism in Human HT-29 Colon Cancer Cells Induced by Total Flavones from Trollius chinensis Bunge

摘要:

本文研究了金莲花总黄酮乙醇提取物（TFETC）诱导人HT-29结肠癌细胞凋亡的作用机制。MTT法测定TFETC对HT-29细胞增殖的影响，200 μg/mL浓度TFETC对癌细胞的抑制率达到81%。通过4, 6-diamidino-2-phenylindole（DAPI）法和流式细胞术检测到金莲花总黄酮乙醇提取物可以诱导癌细胞凋亡，200 μg/mL浓度TFETC处理的癌细胞通过显微镜观察出现凋亡，亚G1期DNA含量达到28.9%。逆转录聚合酶链式反应(RT-PCR)法测定TFETC对HT-29细胞内Bcl-2，Bcl-xL，Bax，caspase-9，caspase-3和COX-2等基因表达的影响。TFETC在mRNA水准上下调抗凋亡基因Bcl-2和Bcl-xL，上调促凋亡基因Bax，caspase-9和caspase-3的表达。此外，TFETC还可抑制HT-29细胞内COX-2基因的表达，并呈剂量效应关系。本研究结果显示金莲花总黄酮乙醇提取物可以通过内源性线粒体途径诱导人HT-29结肠癌细胞的凋亡，同时金莲花总黄酮乙醇提取物还可通过下调COX-2基因的表达抑制HT-29细胞的增殖。

Abstract:

The mechanism of the ethanol extract of the total flavones from Trollius chinensis Bunge (TFETC) inducing apoptosis in HT-29 human colon cancer cells was investigated. MTT assay, which is used to determine the anti-proliferation effect to cancer cells, showed that TFETC inhibited the proliferation of HT-29 cells in a dose-dependent manner, and the inhibitory rate was 81% when treated with 200 μg/mL TFETC. 4,6-diamidino-2-phenylindole (DAPI) staining. Flow cytometry analysis showed that 200 μg/mL TFETC induced apoptosis of HT-29 cells and the sub-G1 DNA content was 28.9%. Reverse transcription polymerase chain reaction (RT-PCR) was applied to evaluate the impact of TFETC on mRNA expression levels of Bcl-2, Bcl-xL, Bax, caspase-9, caspase-3 and COX-2 in HT-29 cells. The results showed that TFETC reduced the mRNA expression levels of anti-apoptosis genes Bcl-2 and Bcl-xL, and up-regulated the mRNA expression of pro-apoptosis genes Bax, caspase-9, and caspase-3 in HT-29 cells. Moreover, TFETC down-regulated the mRNA expression of COX-2 in HT-29 cells in a dose-dependent manner. These results indicated that TFETC could induce the apoptosis through an intrinsic mitochondria pathway in HT-29 cells. Meanwhile, TFETC could reduce cell proliferation through down-regulating the gene expression of COX-2.