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近年来,我国的癌症发病率和死亡率不断在增加,癌症已成为我国居民的主要死亡原因,肺癌的发病率和死亡率居各类恶性肿瘤之首,其恶性程度高、进展快[1]。目前临床上针对肺癌的治疗手段主要以手术、化疗及放疗为主,但大部分肺癌病人确诊时已是晚期,失去了手术的最佳时机,而传统化疗方案疗效有限,亦存在耐药现象,放疗不良反应较大。因此需探索新的肺癌治疗方式。近年来随着中药提纯技术的提高,应用中药及其有效成分进行综合治疗已成为肿瘤领域的研究热点。华蟾素为干蟾皮经科学方法提取加工制成的水化萃取物,具有清热解毒、利水消肿等作用,对多种肿瘤细胞具有抑制作用,其抗肿瘤的机制尚未完全明确。本研究以肺癌细胞株A549为研究对象,探讨华蟾素抗肺癌的分子机制。现作报道。
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CCK8法检测结果示,与对照组比较,0.2 μg/mL华蟾素作用A549细胞株48 h后显示明显抑制作用(P < 0.01),0.8 μg/mL华蟾素处理12 h后均具有不同程度抑制作用(P < 0.05~P < 0.01)(见表 1)。进一步应用EdU及Ki67免疫荧光法检测检测华蟾素对A549细胞株增殖的影响(见图 1),结果显示,与对照组比较,0.2、0.8 μg/mL华蟾素作用72 h后,A549细胞增殖均受到抑制(P < 0.05~P < 0.01)(见表 2)。
华蟾素浓度/(μg/mL) n 6 h 12 h 24 h 48 h 72 h 0.0 3 0.325±0.020 0.515±0.054 0.612±0.081 0.871±0.099 1.045±0.126 0.2 3 0.287±0.049 0.403±0.051 0.528±0.068 0.626±0.078** 0.791±0.086** 0.8 3 0.305±0.041 0.367±0.051* 0.393±0.055** 0.486±0.062** 0.623±0.066** F — 0.74 6.57 7.71 17.25 14.28 P — >0.05 < 0.05 < 0.05 < 0.01 < 0.01 MS组内 — 0.001 0.018 0.037 0.114 0.133 与对照组比较*P < 0.05,**P < 0.01 表 1 CCK8法检测对照组和华蟾素处理组不同时间点细胞增殖情况(x±s)
华蟾素浓度/(μg/mL) n Ki67 EdU Ki67/EdU 0.0 3 39.333±5.163 28.364±5.164 14.182±3.954 0.2 3 28.125±3.842* 14.287±3.606** 7.379±2.518* 0.8 3 11.369±2.054** 8.333±2.517** 2.337±1.082** F — 39.06 20.70 13.74 P — < 0.01 < 0.01 < 0.01 MS组内 — 594.235 317.412 106.000 与对照组比较*P < 0.05,**P < 0.01 表 2 Ki67、EdU检测对照组和华蟾素处理组细胞增殖情况(x±s)
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PTEN/AKT/mTOR通路活性0.2、0.8 μg/mL华蟾素作用于细胞72 h后,p-PI3K、p-AKT、p-mTOR、p-S6、p-PTEN表达量均较对照组下降(P < 0.05~P < 0.01),而PI3K、AKT、mTOR、S6及PTEN表达量与对照组差异无统计学意义(P < 0.05)(见图 2、表 3)。
华蟾素浓度/(μg/mL) p-PI3K PI3K p-AKT AKT p-mTOR mTOR p-S6 S6 p-PTEN PTEN 0.0 0.261±0.056 0.816±0.082 0.624±0.075 0.959±0.181 0.478±0.056 0.480±0.057 0.754±0.086 0.685±0.077 0.841±0.081 0.576±0.082 0.2 0.137±0.024* 0.804±0.079 0.375±0.059** 0.926 ±0.175 0.286±0.032** 0.522±0.058 0.481±0.059** 0.596±0.084 0.537±0.052* 0.560±0.079 0.8 0.102±0.016** 0.789±0.086 0.224±0.050** 0.915±0.189 0.217±0.036** 0.477±0.064 0.328±0.052** 0.612±0.079 0.414±0.049** 0.619±0.086 F 15.83 0.081 31.57 0.135 27.65 0.415 30.86 0.792 37.27 0.412 P < 0.01 >0.05 < 0.01 >0.05 < 0.01 >0.05 < 0.01 >0.05 < 0.01 >0.05 MS组内 0.021 0.000 0.122 0.004 0.055 0.002 0.140 0.120 0.145 0.003 与对照组比较*P < 0.05,**P < 0.01 表 3 对照组和华蟾素处理组AKT/mTOR信号通路蛋白表达量比较(ni=3;x±s)
华蟾素对非小细胞肺癌细胞株A549细胞增殖及PTEN/AKT/mTOR信号通路表达的影响
Effect of cinobufacin on the proliferation and expression of PTEN/AKT/mTOR signaling pathway in non-small cell lung cancer cell line A549
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摘要:
目的观察华蟾素对肺癌细胞A549及PTEN/AKT/mTOR信号通路蛋白表达的影响。 方法在培养肺癌细胞株NCI-A549中分别加入不同浓度的华蟾素,应用细胞计数试剂盒-8检测24、48、72 h后细胞增殖活力,Ki67及EdU检测72 h细胞增殖活力,免疫印迹法检测磷脂酰肌醇-3激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白以及磷酸化第10号染色体缺失的磷酸酶和张力蛋白的同源基因(phosphorylated-phosphatase and tensin homolog deleted on chromosome ten,p-PTEN)蛋白表达。 结果0.2 μg/mL华蟾素作用于A549细胞株48 h后显示明显抑制增殖作用(P < 0.01),0.8 μg/mL华蟾素处理12 h后均具有不同程度抑制增殖作用(P < 0.05~P < 0.01)。不同浓度华蟾素作用于细胞72 h后,p-PI3K、p-AKT、p-mTOR、p-S6、p-PTEN表达量均较对照组下降(P < 0.05~P < 0.01),而PI3K、AKT、mTOR、S6及PTEN表达量与对照组差异无统计学意义(P>0.05)。 结论华蟾素可能通过抑制PI3K/AKT/mTOR信号通路蛋白磷酸化,抑制肺癌细胞A549的增殖。 -
关键词:
- 肺肿瘤 /
- 华蟾素 /
- 肺癌细胞株A549 /
- PTEN/AKT/mTOR信号通路 /
- 增殖
Abstract:ObjectiveTo observe the expression changes of PTEN/AKT/mTOR signaling pathway protein after the lung cancer cells A549 treated with cinobufacin. MethodsDifferent concentrations of cinobufacin were added to the cultured lung cancer cell line NCI-A549, and the cell proliferation activity was measured using cell counting kit-8 after 24, 48 and 72 h.Ki67 and EdU were used to detect the proliferation of cells after 72 h of treatment with cinobufacin.Western blotting was used to detect the expression levels of protein kinase B/mammalian rapamycin target protein pathway protein(AKT/mTOR) and phosphorylated-phosphatase and tensin homolog deleted on chromosome ten(P-PTEN). ResultsThe proliferation of A549 cell line inhibited after treatment with 0.2 μg/mL cinobufacin for 48 h(P < 0.01), and the proliferation of A549 cell line was inhibited after treatment with 0.8 μg/mL cinobufacin for 12 h(P < 0.05 to P < 0.01).Compared with the control group, the expression levels of p-PI3K, p-AKT, p-mTOR, p-S6 and p-PTEN in experimential group after 72 h of treatment with different concentrations of cinobufacin decreased(P < 0.05 to P < 0.01), while the differences of the expression levels of PI3K, AKT, mTOR, S6 and PTEN were not statistically significant between the control group and erperimantial group(P>0.05). ConclusionsCinobufacin may inhibit the proliferation of lung cancer cells A549 by inhibiting the phosphorylation of PI3K/AKT/mTOR signaling pathway protein. -
表 1 CCK8法检测对照组和华蟾素处理组不同时间点细胞增殖情况(x±s)
华蟾素浓度/(μg/mL) n 6 h 12 h 24 h 48 h 72 h 0.0 3 0.325±0.020 0.515±0.054 0.612±0.081 0.871±0.099 1.045±0.126 0.2 3 0.287±0.049 0.403±0.051 0.528±0.068 0.626±0.078** 0.791±0.086** 0.8 3 0.305±0.041 0.367±0.051* 0.393±0.055** 0.486±0.062** 0.623±0.066** F — 0.74 6.57 7.71 17.25 14.28 P — >0.05 < 0.05 < 0.05 < 0.01 < 0.01 MS组内 — 0.001 0.018 0.037 0.114 0.133 与对照组比较*P < 0.05,**P < 0.01 表 2 Ki67、EdU检测对照组和华蟾素处理组细胞增殖情况(x±s)
华蟾素浓度/(μg/mL) n Ki67 EdU Ki67/EdU 0.0 3 39.333±5.163 28.364±5.164 14.182±3.954 0.2 3 28.125±3.842* 14.287±3.606** 7.379±2.518* 0.8 3 11.369±2.054** 8.333±2.517** 2.337±1.082** F — 39.06 20.70 13.74 P — < 0.01 < 0.01 < 0.01 MS组内 — 594.235 317.412 106.000 与对照组比较*P < 0.05,**P < 0.01 表 3 对照组和华蟾素处理组AKT/mTOR信号通路蛋白表达量比较(ni=3;x±s)
华蟾素浓度/(μg/mL) p-PI3K PI3K p-AKT AKT p-mTOR mTOR p-S6 S6 p-PTEN PTEN 0.0 0.261±0.056 0.816±0.082 0.624±0.075 0.959±0.181 0.478±0.056 0.480±0.057 0.754±0.086 0.685±0.077 0.841±0.081 0.576±0.082 0.2 0.137±0.024* 0.804±0.079 0.375±0.059** 0.926 ±0.175 0.286±0.032** 0.522±0.058 0.481±0.059** 0.596±0.084 0.537±0.052* 0.560±0.079 0.8 0.102±0.016** 0.789±0.086 0.224±0.050** 0.915±0.189 0.217±0.036** 0.477±0.064 0.328±0.052** 0.612±0.079 0.414±0.049** 0.619±0.086 F 15.83 0.081 31.57 0.135 27.65 0.415 30.86 0.792 37.27 0.412 P < 0.01 >0.05 < 0.01 >0.05 < 0.01 >0.05 < 0.01 >0.05 < 0.01 >0.05 MS组内 0.021 0.000 0.122 0.004 0.055 0.002 0.140 0.120 0.145 0.003 与对照组比较*P < 0.05,**P < 0.01 -
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