Background： The suillin isoform iso-suillin is a natural substance isolated from a petroleum ether extract of the fruiting bodies of the mushroom Suillus.flavus. Previous studies have found its inhibition effect on s...Background： The suillin isoform iso-suillin is a natural substance isolated from a petroleum ether extract of the fruiting bodies of the mushroom Suillus.flavus. Previous studies have found its inhibition effect on some cancer cells, and we aimed to study its effects on human small cell lung cancer H446 cell line. Methods： Cell viability was measured by 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide assay. Cellular morphological changes （apoptosis and necrosis） were evaluated using an electron microscope and Hoechst 33258 staining detected by the inverted microscope. Flow cytometry was used to detect cell apoptosis, cell cycle distribution, and mitochondrial membrane potential. Protein expression was determined by Western blotting analysis. Results： Here, we describe the ability of iso-suillin to inhibit the growth of H446 cells in time- and dose-dependent way. Iso-suillin had no obvious impact on normal human lymphocyte proliferation at low concentrations （9.09, 18.17, or 36.35 μmol/L） but promoted lymphocyte proliferation at a high concentration （72.70 μmol/L）. After treatment of different concentrations of iso-suillin （6.82, 13.63, or 20.45 μmol/L）, the apoptosis rate of H446 cells increased with increasing concentrations of iso-suillin （16.70%, 35.54%, and 49.20%, respectively, all P 〈 0.05 compared with the control）, and the expression of related apoptotic proteins in the mitochondrial pathway including cytochrome c and caspase-9 were up-regulated compared with the control （all P 〈 0.05）. On the contrary, Bcl-2/Bax ratio was down-regulated compared with the control. Besides, the expression of pro-apoptotic proteins in the death receptor apoptosis pathway, including Fas-associating protein with a novel death domain and caspase-8, and the expression of caspase-3, a downstream regulatory protein of apoptosis, were also increased compared with the control （all P 〈 0.05）. lnhibitors of caspase-9 and caspase-8 reversed the apoptosis process in H446 展开更多
目的探讨淫羊藿苷对人小细胞肺癌细胞株NCI-H446的作用及其机制。方法将对数生长期NCI-H446细胞分成对照组和淫羊藿苷组。对照组细胞常规培养,淫羊藿苷组细胞培养体系中加入淫羊藿苷(8μmol/L)。各组细胞培养48 h后,利用噻唑蓝检测细胞...目的探讨淫羊藿苷对人小细胞肺癌细胞株NCI-H446的作用及其机制。方法将对数生长期NCI-H446细胞分成对照组和淫羊藿苷组。对照组细胞常规培养,淫羊藿苷组细胞培养体系中加入淫羊藿苷(8μmol/L)。各组细胞培养48 h后,利用噻唑蓝检测细胞增殖;流式细胞术检测细胞凋亡;实时定量聚合酶链式反应检测细胞Janus激酶2(JAK2)和信号转导子与转录激活子3(STAT3)基因表达;蛋白质印迹法检测细胞中JAK2、STAT3、磷酸化JAK2(p-JAK2)、磷酸化STAT3(p-STAT3)、Bax和BCL-2蛋白的变化。结果与对照组相比,淫羊藿苷组NCI-H446细胞增殖率明显下降(P<0.05),凋亡率明显升高,NCI-H446细胞JAK2 m RNA与STAT3 m RNA无明显差异。蛋白质印迹法检测显示,淫羊藿苷组NCI-H446细胞JAK2、STAT3总蛋白无明显变化(P>0.05);但p-JAK2、p-STAT3和Bax蛋白明显增加,而BCL-2明显减少(P<0.05)。结论淫羊藿苷能抑制NCI-H446细胞增殖,促进NCI-H446细胞凋亡,其作用可能是通过影响JAK2/STAT3信号传导通路实现的。展开更多
基金Financial support and sponsorship This study was supported by a grant from the Natural Science Foundation of Hebei Province, China （No. H2015206214）.
文摘Background： The suillin isoform iso-suillin is a natural substance isolated from a petroleum ether extract of the fruiting bodies of the mushroom Suillus.flavus. Previous studies have found its inhibition effect on some cancer cells, and we aimed to study its effects on human small cell lung cancer H446 cell line. Methods： Cell viability was measured by 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide assay. Cellular morphological changes （apoptosis and necrosis） were evaluated using an electron microscope and Hoechst 33258 staining detected by the inverted microscope. Flow cytometry was used to detect cell apoptosis, cell cycle distribution, and mitochondrial membrane potential. Protein expression was determined by Western blotting analysis. Results： Here, we describe the ability of iso-suillin to inhibit the growth of H446 cells in time- and dose-dependent way. Iso-suillin had no obvious impact on normal human lymphocyte proliferation at low concentrations （9.09, 18.17, or 36.35 μmol/L） but promoted lymphocyte proliferation at a high concentration （72.70 μmol/L）. After treatment of different concentrations of iso-suillin （6.82, 13.63, or 20.45 μmol/L）, the apoptosis rate of H446 cells increased with increasing concentrations of iso-suillin （16.70%, 35.54%, and 49.20%, respectively, all P 〈 0.05 compared with the control）, and the expression of related apoptotic proteins in the mitochondrial pathway including cytochrome c and caspase-9 were up-regulated compared with the control （all P 〈 0.05）. On the contrary, Bcl-2/Bax ratio was down-regulated compared with the control. Besides, the expression of pro-apoptotic proteins in the death receptor apoptosis pathway, including Fas-associating protein with a novel death domain and caspase-8, and the expression of caspase-3, a downstream regulatory protein of apoptosis, were also increased compared with the control （all P 〈 0.05）. lnhibitors of caspase-9 and caspase-8 reversed the apoptosis process in H446
文摘目的探讨淫羊藿苷对人小细胞肺癌细胞株NCI-H446的作用及其机制。方法将对数生长期NCI-H446细胞分成对照组和淫羊藿苷组。对照组细胞常规培养,淫羊藿苷组细胞培养体系中加入淫羊藿苷(8μmol/L)。各组细胞培养48 h后,利用噻唑蓝检测细胞增殖;流式细胞术检测细胞凋亡;实时定量聚合酶链式反应检测细胞Janus激酶2(JAK2)和信号转导子与转录激活子3(STAT3)基因表达;蛋白质印迹法检测细胞中JAK2、STAT3、磷酸化JAK2(p-JAK2)、磷酸化STAT3(p-STAT3)、Bax和BCL-2蛋白的变化。结果与对照组相比,淫羊藿苷组NCI-H446细胞增殖率明显下降(P<0.05),凋亡率明显升高,NCI-H446细胞JAK2 m RNA与STAT3 m RNA无明显差异。蛋白质印迹法检测显示,淫羊藿苷组NCI-H446细胞JAK2、STAT3总蛋白无明显变化(P>0.05);但p-JAK2、p-STAT3和Bax蛋白明显增加,而BCL-2明显减少(P<0.05)。结论淫羊藿苷能抑制NCI-H446细胞增殖,促进NCI-H446细胞凋亡,其作用可能是通过影响JAK2/STAT3信号传导通路实现的。
