Effect of Hsp90 Inhibitor Ganetespib Combined with Pemetrexed on Lung Adenocarcinoma Cells
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摘要:
目的 探索Ganetespib和培美曲塞体外对人肺腺癌细胞增殖、凋亡和相关信号通路的影响。 方法 人肺腺癌细胞HCC827、H1975、A549分组(Ganetespib组、培美曲塞组、2药联用组),同时设置对照组(DMSO 0.2%),培养48 h后,Cell Titer-Glo(CTG)法体外检测抑制细胞增殖能力,Western blot检测细胞中Akt、p-Akt、Bcl-2蛋白表达水平,细胞凋亡实验检测细胞凋亡率,RNA-seq检测2药物对于HCC827细胞转录组水平的影响。 结果 抑制细胞增殖效果Ganetespib>培美曲塞(P < 0.001),2药联用时抑制效果较单药增强(P < 0.0001);细胞凋亡率Ganetespib>培美曲塞(P < 0.05),2药联用时HCC827细胞凋亡率较单药升高;Ganetespib组Akt蛋白磷酸化水平降低(P < 0.05),培美曲塞组Akt蛋白磷酸化水平升高(P < 0.05),2药联用组结果与Ganetespib组无明显差别,Bcl-2蛋白仅在A549细胞中下调(P < 0.01);2药物对HCC827细胞株的基因转录水平有影响,Ganetespib组上调的差异表达基因为102个,下调有27个,培美曲塞组的上调差异表达基因为53个,下调有8个。 结论 Ganetespib联用培美曲塞可明显抑制肺腺癌细胞HCC827、H1975和A549的增殖,诱导细胞凋亡,其分子机制可能是通过PI3K/AKT信号通路的蛋白质磷酸化来抑制肺腺癌细胞增殖。联合用药可一定程度增强抑制肺腺癌细胞增殖作用,具体机制需进一步研究。 -
关键词:
- 肺腺癌 /
- Ganetespib /
- 培美曲塞
Abstract:Objective To investigate the effects of Ganetespib and Pemetrexed on the proliferation, apoptosis and related signaling pathways of human lung adenocarcinoma cells in vitro. Methods Human lung adenocarcinoma cells HCC827, H1975, and A549 were divided into groups (Ganetespib group, pemetrexed group, combined treatment group), and a control group (DMSO 0.2%) was set up. After culturing for 48 hours, the Cell Titer-Glo (CTG) method was used to detect the inhibitory effects of cell proliferation in vitro, and Western blot was used to detect the expression levels of Akt, p-Akt, and Bcl-2 proteins in cells. The apoptosis experiment was used to detect the apoptosis rate, and RNA-seq was used to detect the effects of the two drugs on the transcription level of the HCC827 cell line. Results The inhibitory effects of cell proliferation were as follows: Ganetespib > pemetrexed (P < 0.001), and the inhibitory effect was enhanced when the two drugs were combined (P < 0.0001); the apoptosis rate was as follows: Ganetespib > pemetrexed (P < 0.05), and the apoptosis rate of HCC827 cells increased when the two drugs were combined; the phosphorylation level of Akt protein was lower in the Ganetespib group (P < 0.05), and the phosphorylation level of Akt protein was higher in the pemetrexed group (P < 0.05), and the results of the combined treatment group were similar to those of the Ganetespib group, and Bcl-2 protein was only downregulated in A549 cells (P < 0.01); the two drugs had an effect on the gene transcription level of the HCC827 cell line, with 102 upregulated differentially expressed genes and 27 downregulated genes in the Ganetespib group, 53 upregulated differentially expressed genes and 8 downregulated genes in the pemetrexed group. Conclusion The combination of Ganetespib and pemetrexed can significantly inhibit the proliferation of lung adenocarcinoma cells HCC827, H1975, and A549, induce cell apoptosis. The molecular mechanism may be through the inhibition of lung adenocarcinoma cell proliferation by protein phosphorylation in the PI3K/AKT signaling pathway. The transcriptome of HCC827 cells was affected by the combination therapy. The combined treatment can enhance the inhibition of lung adenocarcinoma cell proliferation to some extent, and the specific mechanism needs further research. -
Key words:
- Lung adenocarcinoma /
- Ganetespib /
- Pemetrexed
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图 2 Western blot检测细胞中Akt、p-Akt和Bcl-2的表达情况
A:HCC827蛋白表达;B:H1975蛋白表达;C:A549蛋白表达;D:HCC827磷酸化Akt统计学分析;E:H1975磷酸化Akt统计学分析;F:A549磷酸化Akt统计学分析;G:HCC827 Bcl-2统计学分析;H:H1975 Bcl-2统计学分析;I:A549 Bcl-2统计学分析。*P < 0.05,**P < 0.01, ***P < 0.001 ,****P < 0.0001。
Figure 2. The expression levels of Akt,p-Akt and Bcl-2 in the cells were detected by Western blot
表 1 参考基因比对结果及基因表达个数
Table 1. Reference gene comparison results and gene expression numbers
样品名 测序序列 基因占比(%) 总基因数(个) 已知基因数(个) 新基因数(个) HCC827-1 60599120 60.12 15432 15432 0 HCC827-2 84059840 63.87 15899 15899 0 HCC827-3 64289534 58.25 16287 16287 0 HCC827-4 65960004 62.49 15423 15423 0 HCC827-5 66589904 62.06 15786 15786 0 HCC827-6 62855002 59.33 16303 16303 0 HCC827-7 61549178 61.29 15467 15467 0 HCC827-8 60140870 60.83 15614 15614 0 HCC827-9 66496334 56.70 16318 16318 0 表 2 KEGG PATHWAY数据库信息分类
Table 2. Information classification of KEGG pathway database
Metabolism 新陈代谢 12个子类 Genetic Information processing 遗传信息加工 4个子类 Environmental Information processing 环境信息加工 3个子类 Cellular processes 细胞过程 5个子类 Organismal Systems 生物体系统 10个子类 Human diseases 人类疾病 12个子类 Drug development 药物开发 11个子类 -
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