Effect of Hsp90 Inhibitor Ganetespib Combined with Pemetrexed on Lung Adenocarcinoma Cells
-
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
-
图 1 Ganetespib、培美曲塞及联用对肺腺癌细胞增殖能力的影响
A：HCC827细胞增殖情况；B：H1975细胞增殖情况；C：A549细胞增殖情况。**P < 0.05，***P < 0.001，****P < 0.0001。
Figure 1. Effects of Ganetespib，pemetrexed and combined treatment on the proliferation of lung adenocarcinoma cells
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
图 3 Ganetespib和pemetrexed对细胞凋亡率的影响
A：HCC827；B：H1975；C：A549。****P < 0.0001。
Figure 3. Effect of Ganetespib and pemetrexed on apoptosis rate
图 4 差异基因表达数目
图中红色表示上调基因数量，蓝色表示下调基因数量。
Figure 4. Number of differential gene expression
图 5 PCA分析结果
X轴表示第1主成份贡献率，Y轴表示第2主成份贡献率。不同的点代表不同的样品，相同的颜色表示为同1个样品组。
Figure 5. PCA analysis results
图 6 差异基因表达情况火山图
A：Ganetespib-VS-对照组；B：培美曲塞-VS-对照组。x轴是某个具体基因因子数量上的差额（log2），y轴是该种生物因素的变化程度和其相应的负向逻辑指数计算结果。红点代表上调的基因，蓝点代表下调的基因。
Figure 6. Volcano map of differential gene expression
图 7 基因差异表达的MA图
A：Ganetespib-VS-对照组；B：培美曲塞-VS-对照组。观察到的是MA图像上的显著区别标志着各种生物体内的独特蛋白质序列的存在。该轴是A参数，它反映了FPKM的均方根变化率的变化情况。而M是另1个重要指标，用于衡量 log2 （ FC ） 对样本间蛋白水平差别的度量标准，这被用来评估这些数据是否存在统计学意义和实际效应的大小。红色圆圈标识出那些呈现上升趋势的关键变种，蓝色则是下降模式的表现形式，黑色符号显示没有明显的差别现象发生的情况。
Figure 7. MA map of gene differential expression
图 8 差异表达基因的聚类热图结果
A：Ganetespib、培美曲塞和对照组组间交集差异基因分析；B：Ganetespib、培美曲塞和对照组组间并集差异基因分析；C：Ganetespib和对照组组间差异基因分析。横轴展示了每个样本和其对应的分类结果，而纵轴则是每1个有显著差别的基因及它们各自的分类成果。
Figure 8. Cluster heat map results of differentially expressed genes
图 9 差异基因GO功能分类图
A：Ganetespib处理后GO功能分类；B：培美曲塞处理后GO功能分类。X轴：差异表达基因的数量，Y轴：不同的大类用不同的颜色表示；每个大类中单独的柱子表示其中的1个子类。
Figure 9. GO functional classification map of differential genes
图 10 差异基因的KEGG富集散点图
A：Ganetespib处理后KEGG功能分类；B：培美曲塞处理后KEGG功能分类。横轴代表Richfactor，其值越大，富集程度越高；纵轴则是Pathway的名称。在这个pathway中，差异性基因的数量由相应点的大小来标识，而qvalue范围则与对应点的颜色深浅进行匹配。
Figure 10. KEGG distribution point diagram of differential genes
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 
下载: 导出CSV
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
下载: 导出CSV
-
[1] Lee E,Kazerooni E A. Lung cancer screening[J]. Seminars in Respiratory and Critical Care Medicine,2022,43(6):839-850. doi: 10.1055/s-0042-1757885 [2] Schabath M B, Cote M L. Cancer progress and priorities: Lung cancer [J]. Cancer Epidemiology, Biomarkers & Prevention, 2019, 28（10）: 1563-1579.Schabath M B,Cote M L. Cancer progress and priorities: Lung cancer [J]. Cancer Epidemiology,Biomarkers & Prevention,2019,28(10): 1563-1579. [3] Sung H,Ferlay J,Siegel R L,et al. Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer Journal for Clinicians,2021,71(3):209-249. doi: 10.3322/caac.21660 [4] Goss P E,Strasser-weippl K,Lee-bychkovsky B L,et al. Challenges to effective cancer control in China,India,and Russia[J]. Lancet Oncology,2014,15(5):489-538. doi: 10.1016/S1470-2045(14)70029-4 [5] Ruiz-cordero R,Devine W P. Targeted therapy and checkpoint immunotherapy in lung cancer[J]. Surgical Pathology Clinics,2020,13(1):17-33. doi: 10.1016/j.path.2019.11.002 [6] Chaft J E,Shyr Y,Sepesi B,et al. Preoperative and postoperative systemic therapy for operable non-small-cell lung cancer[J]. Journal of Clinical Oncology,2022,40(6):546-555. doi: 10.1200/JCO.21.01589 [7] Yousefi M,Bahrami T,Salmaninejad A,et al. Lung cancer-associated brain metastasis: Molecular mechanisms and therapeutic options[J]. Cellular Oncology (Dordrecht),2017,40(5):419-441. doi: 10.1007/s13402-017-0345-5 [8] Yousefi M E,Cavalu S,Hasan A M,et al. Role of ganetespib,An HSP90 inhibitor,in cancer therapy: From molecular mechanisms to clinical practice[J]. International Journal of Molecular Sciences,2023,24(5):.5014 doi: 10.3390/ijms24055014 [9] Birbo B,Madu E E,Madu C O,et al. Role of HSP90 in cancer[J]. International Journal of Molecular Sciences,2021,22(19):.10317 doi: 10.3390/ijms221910317 [10] Stivarou T,Patsavoudi E. Extracellular molecules involved in cancer cell invasion[J]. Cancers,2015,7(1):238-265. doi: 10.3390/cancers7010238 [11] Proia D A,Foley K P,Korbut T,et al. Multifaceted intervention by the Hsp90 inhibitor ganetespib (STA-9090) in cancer cells with activated JAK/STAT signaling[J]. PloS One,2011,6(4):e18552. doi: 10.1371/journal.pone.0018552 [12] Xiang L,Gilkes D M,Chaturvesi P,et al. Ganetespib blocks HIF-1 activity and inhibits tumor growth,vascularization,stem cell maintenance,invasion,and metastasis in orthotopic mouse models of triple-negative breast cancer[J]. Journal of Molecular Medicine (Berlin,Germany),2014,92(2):151-164. doi: 10.1007/s00109-013-1102-5 [13] Gadgeel S,Rodriguez-abreu D,Speranza G,et al. Updated analysis from KEYNOTE-189: Pembrolizumab or placebo plus pemetrexed and platinum for previously untreated metastatic nonsquamous non-small-cell lung cancer[J]. Journal of Clinical Oncology,2020,38(14):1505-1517. doi: 10.1200/JCO.19.03136 [14] Zhang J,Song C,Tian Y,et al. Single-cell RNA sequencing in lung cancer: Revealing phenotype shaping of stromal cells in the microenvironment[J]. Frontiers in Iimmunology,2021,12:802080. [15] Hirsch F R,Scagliotti G V,Mulshine J L,et al. Lung cancer: current therapies and new targeted treatments[J]. Lancet,2017,389(10066):299-311. doi: 10.1016/S0140-6736(16)30958-8 [16] Park S,Keam B,Kim S H,et al. Pemetrexed singlet versus nonpemetrexed-based platinum doublet as second-line chemotherapy after first-line epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor failure in non-small cell lung cancer patients with EGFR mutations[J]. Cancer Research and Treatment,2015,47(4):630-637. doi: 10.4143/crt.2014.244 [17] Jones G S,Baldwin D R. Recent advances in the management of lung cancer[J]. Clinical Medicine (London,England),2018,18(Suppl 2):s41-s46. [18] Yoshimura N,Okishio K,Mitsuoka S,et al. Prospective assessment of continuation of erlotinib or gefitinib in patients with acquired resistance to erlotinib or gefitinib followed by the addition of pemetrexed[J]. Journal of Thoracic Oncology,2013,8(1):96-101. doi: 10.1097/JTO.0b013e3182762bfb [19] Fuld A D,Dragnev K H,Rigas J R. Pemetrexed in advanced non-small-cell lung cancer[J]. Expert Opinion on Pharmacotherapy,2010,11(8):1387-1402. doi: 10.1517/14656566.2010.482560 [20] Wu S G,Yang C H,Yu C J,et al. Good response to pemetrexed in patients of lung adenocarcinoma with epidermal growth factor receptor (EGFR) mutations[J]. Lung Cancer,2011,72(3):333-339. doi: 10.1016/j.lungcan.2010.10.012 [21] Cui J,Zhang Y,Su D,et al. Efficacy of combined icotinib and pemetrexed in EGFR mutant lung adenocarcinoma cell line xenografts[J]. Thoracic Cancer,2018,9(9):1156-1165. doi: 10.1111/1759-7714.12818 [22] Feng X,Zhang Y,Li T,et al. Sequentially administrated of pemetrexed with icotinib/erlotinib in lung adenocarcinoma cell lines in vitro[J]. Oncotarget,2017,8(69):114292-114299. doi: 10.18632/oncotarget.23224 [23] Noronha V,Patil V M,Joshi A,et al. Gefitinib versus gefitinib plus pemetrexed and carboplatin chemotherapy in EGFR-mutated lung cancer[J]. Journal of Clinical Oncology,2020,38(2):124-136. doi: 10.1200/JCO.19.01154 [24] Taipale M,Jarosz D F,Lindquist S. HSP90 at the hub of protein homeostasis: Emerging mechanistic insights[J]. Nature Reviews Molecular Cell Biology,2010,11(7):515-528. doi: 10.1038/nrm2918 [25] Shimamura T,Li D,JI H,et al. Hsp90 inhibition suppresses mutant EGFR-T790M signaling and overcomes kinase inhibitor resistance[J]. Cancer Research,2008,68(14):5827-5838. doi: 10.1158/0008-5472.CAN-07-5428 [26] Acquaviva J,Smith D L,Sang J,et al. Targeting KRAS-mutant non-small cell lung cancer with the Hsp90 inhibitor ganetespib[J]. Molecular Cancer Therapeutics,2012,11(12):2633-2643. doi: 10.1158/1535-7163.MCT-12-0615 [27] Sequist L V,Gettinger S,Senzer N N,et al. Activity of IPI-504,a novel heat-shock protein 90 inhibitor,in patients with molecularly defined non-small-cell lung cancer[J]. Journal of Clinical Oncology,2010,28(33):4953-4960. doi: 10.1200/JCO.2010.30.8338 [28] Neckers L,Workman P. Hsp90 molecular chaperone inhibitors: Are we there yet?[J]. Clinical Cancer Research,2012,18(1):64-76. doi: 10.1158/1078-0432.CCR-11-1000 [29] Proia D A,Sang J,HE S,et al. Synergistic activity of the Hsp90 inhibitor ganetespib with taxanes in non-small cell lung cancer models[J]. Investigational New Drugs,2012,30(6):2201-2209. doi: 10.1007/s10637-011-9790-6 [30] Smith D L,Acquaviva J,Sequeira M,et al. The HSP90 inhibitor ganetespib potentiates the antitumor activity of EGFR tyrosine kinase inhibition in mutant and wild-type non-small cell lung cancer[J]. Targeted Oncology,2015,10(2):235-245. doi: 10.1007/s11523-014-0329-6 [31] Fennell D A,Danson S,Woll P J,et al. Ganetespib in combination with pemetrexed-platinum chemotherapy in patients with pleural mesothelioma (MESO-02): A phase Ib trial[J]. Clinical Cancer Research,2020,26(18):4748-4755. doi: 10.1158/1078-0432.CCR-20-1306 [32] Stecklein S R,Kumaraswamy E,Behbod F,et al. BRCA1 and HSP90 cooperate in homologous and non-homologous DNA double-strand-break repair and G2/M checkpoint activation[J]. Proceedings of the National Academy of Sciences of the United States of America,2012,109(34):13650-13655. [33] Abu Lila A S,Kato C,Fukshima M,et al. Downregulation of thymidylate synthase by RNAi molecules enhances the antitumor effect of pemetrexed in an orthotopic malignant mesothelioma xenograft mouse model[J]. International Journal of Oncology,2016,48(4):1399-1407. doi: 10.3892/ijo.2016.3367 [34] Zucali P A,Giovannetti E,Destro A,et al. Thymidylate synthase and excision repair cross-complementing group-1 as predictors of responsiveness in mesothelioma patients treated with pemetrexed/carboplatin[J]. Clinical Cancer Research,2011,17(8):2581-2590. doi: 10.1158/1078-0432.CCR-10-2873 [35] Deng Z,Qin Y,Liu Y,et al. Role of antiangiogenic agents combined with EGFR tyrosine kinase inhibitors in treatment-naive lung cancer: A meta-analysis[J]. Clinical Lung Cancer,2021,22(1):e70-e83. doi: 10.1016/j.cllc.2020.08.005 [36] Xue J,Li B,Wang Y,et al. Efficacy and safety of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor combination therapy as first-line treatment for patients with advanced EGFR-mutated,non-small cell lung cancer: A systematic review and bayesian network meta-analysis[J]. Cancers,2022,14(19):4894. doi: 10.3390/cancers14194894 期刊类型引用(1)
1. 李珊珊，武方翠，韩志南. 口腔正畸固定矫治青少年患儿心理痛苦现状及其影响因素分析. 心理月刊. 2024(14): 17-20 . 
百度学术其他类型引用(0)
-
点击查看大图
计量
- 文章访问数: 435
- HTML全文浏览量: 474
- PDF下载量: 7
- 被引次数: 1
