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该文研究异鼠李素(isorhamnetin,ISO)对人结肠癌细胞(LOVO)增殖和凋亡的影响,并初步探讨其分子机制。采用MTT法检测不同浓度异鼠李素作用于LOVO 24、48、72 h后的细胞增殖;采用PI染色法检测不同浓度异鼠李素处理LOVO 48 h后的贴壁细胞数;采用碘化丙啶(propidium iodide,PI)染色法和DNA电泳的梯状条带(DNA ladder)法检测不同浓度异鼠李素处理LOVO 48 h后细胞形态和DNA片段;采用Western blot法检测磷酸化的表皮生长因子受体(epidermal growth factor receptor phosphorylation,p EGFR)、Bax(Bcl-2 associated X protein)、Bcl-2(B-cell lymphoma-2)、半胱氨酸蛋白酶-3(caspase-3)和caspase-3的剪切体(cleaved caspase-3)的蛋白质水平。结果发现,异鼠李素能有效抑制LOVO的增殖,且具有浓度和时间依赖性;处理48 h后,细胞出现了典型的凋亡形态,且出现180~200 bp倍数的DNA片段。进一步研究发现,异鼠李素通过抑制EGFR磷酸化、增加Bax蛋白质水平、减少Bcl-2蛋白质水平进而活化caspase-3,实现诱导细胞凋亡并抑制细胞增殖。因此,异鼠李素通过细胞凋亡抑制结肠癌细胞的增殖,具有开发为人结肠癌临床治疗用药或辅助用药的潜力。
This study was designed to investigate the effects of isorhamnetin (ISO) on the proliferation and apoptosis of human colon carcinoma (LOVO) cells and to explore its molecular mechanism. MTT assay was used to determine the effect of isorhamnetin on proliferation of LOVO cells at 24, 48 and 72 h. PI staining was used to determine the number of adherent cells after treated with isorhamnetin for 48 h. Adopting propidium iodide Propidium iodide (PI) staining and DNA ladder were used to detect cell morphology and DNA fragments after isorhamnetin-treated LOVO at different concentrations for 48 h. Western blot was used to detect the phosphorylated epidermal growth (EGFR), Bcl-2 associated X protein, Bcl-2, caspase-3 and caspase -3 cleaved (cleaved caspase-3) protein levels. The results showed that isorhamnetin can effectively inhibit the proliferation of LOVO in a concentration- and time-dependent manner. After 48 h of treatment, typical apoptotic cells were observed and DNA fragments with multiples of 180-200 bp appeared. Further studies showed that isorhamnetin can induce apoptosis and inhibit cell proliferation by inhibiting EGFR phosphorylation, increasing Bax protein level, decreasing Bcl-2 protein level and activating caspase-3. Therefore, isorhamnetin inhibits the proliferation of colon cancer cells through apoptosis, and has the potential to be developed into a clinical therapeutic or adjuvant drug for human colon cancer.