华蟾毒配基联合索拉非尼通过AURKA/Ras/Raf/ERK信号通路对肝癌Huh7细胞增殖与凋亡的影响
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摘要
目的研究华蟾毒配基联合索拉非尼对肝癌Huh7细胞增殖与凋亡的影响及作用机制。方法 MTT法检测Huh7细胞增殖;Hoechst33342/PI荧光双染法检测Huh7细胞凋亡形态变化;流式细胞仪检测细胞周期;免疫细胞化学法检测Ki67蛋白表达;Western blotting法检测Bax、Bcl-2、Caspase-8、极光激酶A(AURKA)、Ras、Raf、细胞外调节蛋白激酶(ERK)和p-ERK蛋白的表达变化。结果华蟾毒配基、索拉非尼及联合用药对Huh7细胞的增殖均有抑制作用,联合用药组的增殖抑制作用更明显,具有协同效应;荧光染色可见细胞凋亡形态变化;索拉非尼引起Huh7细胞周期S期阻滞,华蟾毒配基和联合用药引起Huh7细胞周期G2/M期阻滞,联合用药组G2/M期阻滞较单药组更明显;华蟾毒配基和索拉非尼均能减弱Ki67表达,联合用药组的作用更为显著;华蟾毒配基、索拉非尼及联合用药上调Bax和Caspase-8蛋白表达,下调Bcl-2蛋白表达,上调Bax/Bcl-2比例,对ERK蛋白表达无明显影响,显著下调AURKA、Ras、Raf和p-ERK蛋白表达,联合用药组的作用更为显著(P<0.05)。结论华蟾毒配基联合索拉非尼通过AURKA/Ras/Raf/ERK信号通路抑制肝癌Huh7细胞增殖,诱导其凋亡。
Objective To investigate the effect and mechanism of cinobufagin combined with Sorafenib on the proliferation and apoptosis of hepatocellular carcinoma Huh7 cells. Methods The proliferation of Huh7 cells was measured using MTT assay; The apoptosis morphological changes of Huh7 cells were detected using Hoechst33342/PI fluorescence staining; The cells cycle was detected by flow cytometry; The expression of Ki67 protein was detected by immunocytochemistry; The expressions of Bax, Bcl-2, Caspase-8, AURKA, Ras, Raf, ERK, and p-ERK proteins were measured using Western blotting. Results Cinobufagin, Sorafenib, and combination therapy inhibited the proliferation of Huh7 cells, and the inhibitory effect of the combination group was more obvious with synergistic effect. Fluorescence staining showed morphological changes of apoptosis. Sorafenib induced the cell cycle S phase arrest, cinobufagin and combination therapy induced the cell cycle G2/M phase arrest, combination group had more obvious cell cycle arrest in G2/M phase than single drug groups. Both cinobufagin and Sorafenib attenuated the expression of Ki67, and the effect of combination group was more significant. Cinobufagin, Sorafenib, and combination therapy up-regulated the expression of Bax and Caspase-8 proteins; down-regulated the expression of Bcl-2 protein; up-regulated the ratio of Bax/Bcl-2; had no obvious effect on the expression of ERK protein; significantly down-regulated the expression of AURKA, Ras, Raf, and p-ERK proteins; And the effect of combination group was more significant(P < 0.05). Conclusion Cinobufagin combined with Sorafenib could inhibit the proliferation and induce the apoptosis of hepatocellular carcinoma Huh7 cells through AURKA/Ras/Raf/ERK signaling pathway.
Objective To investigate the effect and mechanism of cinobufagin combined with Sorafenib on the proliferation and apoptosis of hepatocellular carcinoma Huh7 cells. Methods The proliferation of Huh7 cells was measured using MTT assay; The apoptosis morphological changes of Huh7 cells were detected using Hoechst33342/PI fluorescence staining; The cells cycle was detected by flow cytometry; The expression of Ki67 protein was detected by immunocytochemistry; The expressions of Bax, Bcl-2, Caspase-8, AURKA, Ras, Raf, ERK, and p-ERK proteins were measured using Western blotting. Results Cinobufagin, Sorafenib, and combination therapy inhibited the proliferation of Huh7 cells, and the inhibitory effect of the combination group was more obvious with synergistic effect. Fluorescence staining showed morphological changes of apoptosis. Sorafenib induced the cell cycle S phase arrest, cinobufagin and combination therapy induced the cell cycle G2/M phase arrest, combination group had more obvious cell cycle arrest in G2/M phase than single drug groups. Both cinobufagin and Sorafenib attenuated the expression of Ki67, and the effect of combination group was more significant. Cinobufagin, Sorafenib, and combination therapy up-regulated the expression of Bax and Caspase-8 proteins; down-regulated the expression of Bcl-2 protein; up-regulated the ratio of Bax/Bcl-2; had no obvious effect on the expression of ERK protein; significantly down-regulated the expression of AURKA, Ras, Raf, and p-ERK proteins; And the effect of combination group was more significant(P < 0.05). Conclusion Cinobufagin combined with Sorafenib could inhibit the proliferation and induce the apoptosis of hepatocellular carcinoma Huh7 cells through AURKA/Ras/Raf/ERK signaling pathway.
引文
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[14]Yu P,Ye L,Wang H,et al.NSK-01105 inhibits proliferation and induces apoptosis of prostate cancer cells by blocking the Raf/MEK/ERK and PI3K/Akt/m TOR signal pathways[J].Tumour Biol,2015,36(3):2143-2153.
[15]Zhang Z,Zhou X,Shen H,et al.Phosphorylated ERK is a potential predictor of sensitivity to sorafenib when treating hepatocellular carcinoma:Evidence from an in vitro study[J].BMC Med,2009,doi:10.1186/1741-7015-7-41.
[16]Yang S,Liu G.Targeting the Ras/Raf/MEK/ERK pathway in hepatocellular carcinoma[J].Oncol Lett,2017,13(3):1041-1047.
[17]Zhang K,Chen J,Chen D,et al.Aurora-A promotes chemoresistance in hepatocelluar carcinoma by targeting NF-kappa B/micro RNA-21/PTEN signaling pathway[J].Oncotarget,2014,5(24):12916-12935.
[18]Lassus H,Staff S,Leminen A,et al.Aurora-A overexpression and aneuploidy predict poor outcome in serous ovarian carcinoma[J].Gynecol Oncol,2011:120(1):11-17.
[19]Zeng B,Lei Y,Zhu H,et al.Aurora-A is a novel predictor of poor prognosis in patients with resected lung adenocarcinoma[J].Chin J Cancer Res,2014,26(2):166-173.
[20]Umstead M,Xiong J,Qi Q,et al.Aurora kinase A interacts with H-Ras and potentiates Ras-MAPK signaling[J].Oncotarget,2017,8(17):28359-28372.
[2]Siegel R,Ma J,Zou Z,et al.Cancer statistics,2014[J].CA Cancer J Clin,2014,64(1):9-29.
[3]Llovet J M,Ricci S,Mazzaferro V,et al.Sorafenib in advanced hepatocellular carcinoma[J].N Engl J Med,2008,359(4):378-390.
[4]Muntane J.Targeting cell death and survival receptors in hepatocellular carcinoma[J].Anticancer Agents MedChem,2011,11(6):576-584.
[5]Escudier B,Eisen T,Stadler W M,et al.Sorafenib in advanced clear-cell renal-cell carcinoma[J].N Engl J Med,2007,356(2):125-134.
[6]Xu Z,Wang F,Fan F,et al.Quantitative proteomics reveals that the inhibition of Na+/K+-ATPase activity affects S-phase progression leading to a chromosome segregation disorder by attenuating the aurora a function in hepatocellular carcinoma cells[J].J Proteome Res,2015,14(11):4594-4602.
[7]Wan X B,Long Z J,Yan M,et al.Inhibition of aurora-A suppresses epithelial-mesenchymal transition and invasion by downregulating MAPK in nasopharyngeal carcinoma cells[J].Carcinogenesis,2008,29(10):1930-1937.
[8]金正均.合并用药中的相加[J].中国药理学报,1980,1(2):70-76.
[9]Mijatovic T,Van Quaquebeke E,Delest B,et al.Cardiotonic steroids on the road to anti-cancer therapy[J].Biochim Biophys Acta,2007,1776(1):32-57.
[10]Luo Y,Ren F,Liu Y,et al.Clinicopathological and prognostic significance of high Ki-67 labeling index in hepatocellular carcinoma patients:A meta-analysis[J].IntJ Clin Exp Med,2015,8(7):10235-10247.
[11]Tomasin R,Gomes-Marcondes M C.Oral administration of Aloe vera and honey reduces Walker tumour growth by decreasing cell proliferation and increasing apoptosis in tumour tissue[J].Phytother Res,2011,25(4):619-623.
[12]Alonso E,Cano-Abad M F,Moreno-Ortega A J,et al.Nanomolar ouabain elicits apoptosis through a direct action on He La cell mitochondria[J].Steroids,2013,78(11):1110-1118.
[13]Nakamura K,Bossy-Wetzel E,Burns K,et al.Changes in endoplasmic reticulum luminal environment affect cell sensitivity to apoptosis[J].J Cell Biol,2000,150(4):731-740.
[14]Yu P,Ye L,Wang H,et al.NSK-01105 inhibits proliferation and induces apoptosis of prostate cancer cells by blocking the Raf/MEK/ERK and PI3K/Akt/m TOR signal pathways[J].Tumour Biol,2015,36(3):2143-2153.
[15]Zhang Z,Zhou X,Shen H,et al.Phosphorylated ERK is a potential predictor of sensitivity to sorafenib when treating hepatocellular carcinoma:Evidence from an in vitro study[J].BMC Med,2009,doi:10.1186/1741-7015-7-41.
[16]Yang S,Liu G.Targeting the Ras/Raf/MEK/ERK pathway in hepatocellular carcinoma[J].Oncol Lett,2017,13(3):1041-1047.
[17]Zhang K,Chen J,Chen D,et al.Aurora-A promotes chemoresistance in hepatocelluar carcinoma by targeting NF-kappa B/micro RNA-21/PTEN signaling pathway[J].Oncotarget,2014,5(24):12916-12935.
[18]Lassus H,Staff S,Leminen A,et al.Aurora-A overexpression and aneuploidy predict poor outcome in serous ovarian carcinoma[J].Gynecol Oncol,2011:120(1):11-17.
[19]Zeng B,Lei Y,Zhu H,et al.Aurora-A is a novel predictor of poor prognosis in patients with resected lung adenocarcinoma[J].Chin J Cancer Res,2014,26(2):166-173.
[20]Umstead M,Xiong J,Qi Q,et al.Aurora kinase A interacts with H-Ras and potentiates Ras-MAPK signaling[J].Oncotarget,2017,8(17):28359-28372.