摘要
目的考察毛蚶水提物体外抗肿瘤活性,研究其抗肿瘤作用机制。方法MTT法检测毛蚶水提物对9种人肿瘤细胞系生长的影响,并通过Giemsa染色光学显微镜下观察肿瘤细胞核的变化;流式细胞术及TUNEL染色检测毛蚶水提物对HT-29细胞周期的影响和凋亡情况;Western blotting检测凋亡相关蛋白Caspaes-3的表达情况。结果毛蚶水提物对人结直肠癌HT-29、HCT-116细胞,肝癌HepG-2、BEL-7402细胞及肺癌A549细胞具有较强的抑制作用,抑制作用呈作用时间和剂量依赖性。流式细胞术结合细胞Giemsa染色结果显示,毛蚶水提物能引起人结直肠癌HT-29细胞G2-M期阻滞,多核细胞增加。TUNEL染色显示毛蚶水提物有一定诱导细胞凋亡的作用。400μg.mL-1毛蚶水提物对HT-29细胞Caspase-3蛋白的表达具有下调作用。结论毛蚶水提物体外具有广谱抗癌活性;毛蚶水提物抑制人结直肠癌HT-29细胞生长的主要机制为诱导细胞凋亡及引起肿瘤细胞G2-M期阻滞,进而形成多核细胞,最终导致肿瘤细胞死亡。
Objects To investigate the antitumor activities of the water extract of Arca subcrenata Lischke in vitro and disclose its antitumor mechanisms. Methods The effect of the water extract of Arca subcre- nata Lischke on the growth of 9 different kinds of human tumor cell lines were detected by MTT assay, so the sensitive cell lines were picked up and their antitumor effects of dependence on dose and time were studied. Growth state and morphological changes of human colon rectal carcinoma HT-29 cells treated with the water extract of Arca subcrenata Lischke were observed under the inverted phase con- trast microscope, the nucleus feature was analyzed by Giemsa staining. Flow cytometry and TUNEL assay were used to detect the cell cycle and apoptosis of HT-29 cells treated with the water extract. Ap- optosis related casepase-3 protein expression was analyzed by Western blotting. Results Cell viability was significantly decreased in the water extract of Arca subcrenata Lischke treated groups in human co- lon rectal carcinoma HT-29 and HCT-116 cells, human hepatocarcinoma HepG-2 and BEL-7402 cells and human lung cancer A549 cells in a time-and dose-dependent manner in vitro. The ICs0 of 72 h was 263.92~6.96, 399.38~9.17, 243.78＋10. 29, 439.45~7.93 and 392. 80___7.03/,g ~ mL-1 respective- ly. Under the inverted phase contrast microscope, the human colon rectal carcinoma HT-29 cells showed significant growth inhibition in the water extract treated group. Morphological changes includ- ed cell shrinking and rounding, separating from surrounding cells. A part of cells showed internal vacu- olization. Some cell pieces appeared in the culture medium after the water extract treated for 48h, and more in 72h. Flow cytometry analysis and Giemsa stain assay indicated that G2-M phase cells accumula- ted and multinuclear cells increased in the water extract treated group of HT-29 cells. TUNEL stain re- vealed the water extract of Arca subcrenata Lischke can induce apoptosis in HT-29 cells. Flow cytome- try analysis showed that apoptosis and necrosis cells were all increased in HT-29 cells treated with the water extract at 200~g ~ mL-1 for 48h. Apotosis related casepase-3 protein was decreased in HT-29 cells which were cultured with the water extract at 400/~g ~ mL-1 for 48h. Conclusions Water extract of Arca subcrenata Lischke has broad-spectrum antitumor effect. The mechanisms of inhibiting the growth of human colon rectal carcinoma HT-29 cells include inducing cell apoptosis and cell cycle arrest in G2- M phase, which may give rise to multinuclear cells and ultimately result in cell death.
出处
《中国海洋药物》
CAS
CSCD
北大核心
2013年第3期40-48,共9页
Chinese Journal of Marine Drugs
基金
山东省科技发展计划项目(2011YD19007)资助
关键词
毛蚶水提物
人肿瘤细胞
HT-29细胞
凋亡
细胞周期阻滞
多核细胞
water extract of Arca subcrenata Lischke~ human tumor cells~ HT-29 cells~ apoptosis~ cellcycle arrest~ multinuclear cells
