巴西苏木素对舌癌Tca8113细胞凋亡和自噬的影响及分子机制
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摘要
目的探讨巴西苏木素对人舌鳞状细胞癌Tca8113细胞增殖、凋亡和自噬的影响及其分子机制,为临床用药提供理论依据。方法用MTT法检测巴西苏木素对Tca8113细胞的增殖作用;Hoechst33342染色法观察细胞形态变化;Annexin V/PI双染色检测巴西苏木素对Tca8113细胞凋亡程度的影响;Western blot法检测凋亡相关蛋白bax、bcl-2、cleaved caspase3及自噬相关蛋白p-AMPK、p-mTOR、LC3B、p62的表达;使用AMPK通路抑制剂与巴西苏木素共同作用于Tca8113细胞,并检测通路相关蛋白p-AMPK、p-mTOR、LC3B的表达。结果 MTT结果表明,巴西苏木素能明显抑制Tca8113细胞的增殖,24 h时IC50为31.17μmol/L;Hoechst33342染色结果显示,巴西苏木素能使Tca8113细胞发生凋亡形态学改变,且与浓度正相关;Annexin V/PI染色结果显示,不同浓度巴西苏木素作用24 h后,细胞凋亡数和凋亡率均高于空白对照组;Western blot检测相关蛋白表达结果表明,巴西苏木素可抑制抗凋亡蛋白bcl-2,促进凋亡关键蛋白bax和cleaved-caspase3的表达,同时增加LC3B和p-AMPK的表达,降低p62和p-mTOR表达。在与抑制剂合用后,p-AMPK与LC3B表达量虽仍略高于空白对照组,但与单纯使用巴西苏木素相比均明显降低,而p-mTOR的表达较巴西苏木素组则明显升高(P<0.05)。结论巴西苏木素能抑制Tca8113细胞增殖并促进其凋亡,同时可通过AMPK/mTOR通路诱导细胞发生自噬。
Objective To investigate the effects of brazilin on the proliferation, apoptosis and autophagy of human tongue squamous cell carcinoma Tca8113 cells in vitro and explore its molecular mechanism. Methods The changes in the proliferation, morphology and apoptosis of Tca8113 cells in response to brazilin treatment were detected using MTT assay,Hoechst33342 staining, and Annexin V/PI double staining, respectively. The expressions of apoptosis-related protein Bax, Bcl-2,cleaved caspase-3 and autophagy-related proteins p-AMPK, p-mTOR, LC3 B, and p62 in the treated cells were detected using Western blotting. The effect of treatment with both the AMPK pathway inhibitor and brazilin on the expressions of the pathway-related proteins p-AMPK, p-mTOR, and LC3 B was assessed. Results MTT assay showed that brazilin significantly inhibited the proliferation of Tca8113 cells with an IC50 of 31.17 μmol/L at 24 h. Hoechst33342 staining showed that brazilin induced apoptotic morphological changes in Tca8113 cells in a concentration-dependent manner. Treatment with different concentrations of brazilin resulted in increased apoptosis in the cells. Brazilin obviously inhibited the expression of Bcl-2, p62 and p-mTOR and enhanced the expressions of Bax, cleaved caspase-3, LC3 B and p-AMPK. The AMPK pathway inhibitor significantly inhibited the increase in p-AMPK and LC3 B expressions and the decrease in p-mTOR expression induced by brazilin. Conclusion Brazilin can inhibit the proliferation and promote apoptosis in Tca8113 cells and at the same time induces autophagy in the cells through the AMPK/mTOR pathway.
Objective To investigate the effects of brazilin on the proliferation, apoptosis and autophagy of human tongue squamous cell carcinoma Tca8113 cells in vitro and explore its molecular mechanism. Methods The changes in the proliferation, morphology and apoptosis of Tca8113 cells in response to brazilin treatment were detected using MTT assay,Hoechst33342 staining, and Annexin V/PI double staining, respectively. The expressions of apoptosis-related protein Bax, Bcl-2,cleaved caspase-3 and autophagy-related proteins p-AMPK, p-mTOR, LC3 B, and p62 in the treated cells were detected using Western blotting. The effect of treatment with both the AMPK pathway inhibitor and brazilin on the expressions of the pathway-related proteins p-AMPK, p-mTOR, and LC3 B was assessed. Results MTT assay showed that brazilin significantly inhibited the proliferation of Tca8113 cells with an IC50 of 31.17 μmol/L at 24 h. Hoechst33342 staining showed that brazilin induced apoptotic morphological changes in Tca8113 cells in a concentration-dependent manner. Treatment with different concentrations of brazilin resulted in increased apoptosis in the cells. Brazilin obviously inhibited the expression of Bcl-2, p62 and p-mTOR and enhanced the expressions of Bax, cleaved caspase-3, LC3 B and p-AMPK. The AMPK pathway inhibitor significantly inhibited the increase in p-AMPK and LC3 B expressions and the decrease in p-mTOR expression induced by brazilin. Conclusion Brazilin can inhibit the proliferation and promote apoptosis in Tca8113 cells and at the same time induces autophagy in the cells through the AMPK/mTOR pathway.
引文
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[14]Fan J, Li R, Zhang R, et al. Effect of Bcl-2 and Bax on survival of side population cells from hepatocellular carcinoma cells[J]. World J Gastroenterol, 2007, 13(45):6053-9.
[15]Derakhshan A, Chen Z, Van Waes C. Therapeutic small molecules target inhibitor of apoptosis proteins in cancers with deregulation of extrinsic and intrinsic cell death pathways[J]. Clin Cancer Res,2017, 23(6):1379-87.
[16]Liu LS, Bai XQ, Gao Y, et al. PCSK9 promotes oxLDL-Induced PC12 cell apoptosis through the Bcl-2/Bax-Caspase 9/3 signaling pathway[J]. J Alzheimers Dis, 2017, 57(3):723-34.
[17] Fulda S. Targeting apoptosis for anticancer therapy[J]. Semin Cancer Biol, 2015, 31:84-8.
[18]Kuribayashi K, Mayes PA, El-Deiry WS. What are caspases 3 and 7doing upstream of the mitochondria[J]? Cancer Biol Ther, 2006, 5(7):763-5.
[19] Sun L, Liu N, Liu SS, et al. Beclin-1-independent autophagy mediates programmed cancer cell death through interplays with endoplasmic reticulum and/or mitochondria in colbat chlorideinduced hypoxia[J]. Am J Cancer Res, 2015, 5(9):2626-42.
[20]Rabinowitz JE. Autophagy and metabolism[J]. Science, 2010, 330(6009):1344-8.
[21]Zhou XT, Pu ZJ, Liu LX, et al. Inhibition of autophagy enhances adenosineinduced apoptosis in human hepatoblastoma HepG2 cells[J]. Oncol Rep, 2019, 41:829-38.
[22]Pankiv S, Clausen TH, Lamark T, et al. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy[J]. J Biol Chem, 2007, 282(33):24131-45.
[23]Velazquez AF, Jackson WT. So many roads:the multifaceted regulation of autophagy induction[J]. Mol Cell Biol, 2018, 38(21):303-18.
[24]郑庆委,高淑娴,吕杰,等.虫草素对人舌癌细胞凋亡,自噬的影响及其分子机制[J].南方医科大学学报, 2018, 38(4):390-4.
[25]Kim J, Guan KL. Regulation of the autophagy initiating kinase ULK1 by nutrients:roles of mTORC1 and AMPK[J]. Cell Cycle,2011, 10(9):1337-8.
[26]Xu L, Brink M. mTOR, cardiomyocytes and inflammation in cardiac hypertrophy[J]. Biochim Biophys Acta, 2016, 1863(7, B, SI):1894-903.
[27]Jung CH, Ro SH, Cao J, et al. mTOR regulation of autophagy[J].FEBS Lett, 2010, 584(7):1287-95.
[28]Hsiao YT, Kuo CL, Chueh FH, et al. Curcuminoids induce reactive oxygen species and autophagy to enhance apoptosis in human oral cancer cells[J]. Am J Chin Med, 2018, 46(5):1145-68.
[29]Lin CY, Hsu SH, Lee HS, et al. Enhanced expression of glucose transporter-1 in vascular smooth muscle cells via the Akt/tuberous sclerosis complex subunit 2(TSC2)/mammalian target of rapamycin(mTOR)/ribosomal S6 protein kinase(S6K)pathway in experimental renal failure[J]. J Vasc Surg, 2013, 57(2):475-85.
[2] Parkin DM, Bray F, Ferlay J, et al. Global cancer statistics, 2002[J].CA Cancer J Clin, 2005, 55(2):74-108.
[3] Sano D, Myers JN. Metastasis of squamous cell carcinoma of the oral tongue[J]. Cancer Metastas Rev, 2007, 26(3/4):645-62.
[4] Lee DY, Lee MK, Kim GS, et al. Brazilin inhibits growth and induces apoptosis in human[J]. Molecules, 2013, 18(2):2449-57.
[5] Kang K, Won M, Yuk JM, al. LinQ attenuates systemic inflammatory responses via selectively impairing the Myddosome complex formation upon TLR4 ligation[J].Biochem Pharmacol, 2016, 121:52-66.
[6] Zhang TT, Fan XP, Song LL, et al. c-Fos is involved in inhibition of human bladder carcinoma T24 cells by brazilin[J]. IUBMB Life,2015, 67(3):175-81.
[7] He ZJ, Zhu FY, Li SS, et al. Inhibiting ROS-NF-kB-dependent autophagy enhanced brazilin-induced apoptosis in head and neck squamous cell carcinoma[J]. Food Chem Toxicol, 2017, 101:55-66.
[8] Lee DY, Lee MK, Kim GS, et al. Brazilin inhibits growth and induces apoptosis in human glioblastoma cells[J]. Molecules, 2013, 18(2):2449-57.
[9]王飞.巴西苏木素联合平阳霉素对人舌癌细胞株(Tca8113)生长的影响[D].兰州:兰州大学, 2013:1-48.
[10]石璐,苏静.顺铂对人舌鳞状细胞癌细胞Tca8113细胞凋亡与自噬的影响[J].中国老年学杂志, 2013, 33(9):2088-90.
[11]Chen CF, Yang JS, Chen WK, et al. Ursolic acid elicits intrinsic apoptotic machinery by downregulating the phosphorylation of AKT/BAD signaling in human cisplatin-resistant oral cancer CAR cells[J]. Oncol Rep, 2018, 40(3):1752-60.
[12]李为,刘伟,邹君君.人参皂苷Rg5对胃癌BGC-823细胞增殖,凋亡及凋亡相关因子的影响[J].中国中医急症, 2018, 27(6):955-8.
[13]Chai J, Du C, Wu JW, et al. Structural and biochemical basis of apoptotic activation by Smac/DIABLO[J]. Nature, 2000, 406(6798):855-62.
[14]Fan J, Li R, Zhang R, et al. Effect of Bcl-2 and Bax on survival of side population cells from hepatocellular carcinoma cells[J]. World J Gastroenterol, 2007, 13(45):6053-9.
[15]Derakhshan A, Chen Z, Van Waes C. Therapeutic small molecules target inhibitor of apoptosis proteins in cancers with deregulation of extrinsic and intrinsic cell death pathways[J]. Clin Cancer Res,2017, 23(6):1379-87.
[16]Liu LS, Bai XQ, Gao Y, et al. PCSK9 promotes oxLDL-Induced PC12 cell apoptosis through the Bcl-2/Bax-Caspase 9/3 signaling pathway[J]. J Alzheimers Dis, 2017, 57(3):723-34.
[17] Fulda S. Targeting apoptosis for anticancer therapy[J]. Semin Cancer Biol, 2015, 31:84-8.
[18]Kuribayashi K, Mayes PA, El-Deiry WS. What are caspases 3 and 7doing upstream of the mitochondria[J]? Cancer Biol Ther, 2006, 5(7):763-5.
[19] Sun L, Liu N, Liu SS, et al. Beclin-1-independent autophagy mediates programmed cancer cell death through interplays with endoplasmic reticulum and/or mitochondria in colbat chlorideinduced hypoxia[J]. Am J Cancer Res, 2015, 5(9):2626-42.
[20]Rabinowitz JE. Autophagy and metabolism[J]. Science, 2010, 330(6009):1344-8.
[21]Zhou XT, Pu ZJ, Liu LX, et al. Inhibition of autophagy enhances adenosineinduced apoptosis in human hepatoblastoma HepG2 cells[J]. Oncol Rep, 2019, 41:829-38.
[22]Pankiv S, Clausen TH, Lamark T, et al. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy[J]. J Biol Chem, 2007, 282(33):24131-45.
[23]Velazquez AF, Jackson WT. So many roads:the multifaceted regulation of autophagy induction[J]. Mol Cell Biol, 2018, 38(21):303-18.
[24]郑庆委,高淑娴,吕杰,等.虫草素对人舌癌细胞凋亡,自噬的影响及其分子机制[J].南方医科大学学报, 2018, 38(4):390-4.
[25]Kim J, Guan KL. Regulation of the autophagy initiating kinase ULK1 by nutrients:roles of mTORC1 and AMPK[J]. Cell Cycle,2011, 10(9):1337-8.
[26]Xu L, Brink M. mTOR, cardiomyocytes and inflammation in cardiac hypertrophy[J]. Biochim Biophys Acta, 2016, 1863(7, B, SI):1894-903.
[27]Jung CH, Ro SH, Cao J, et al. mTOR regulation of autophagy[J].FEBS Lett, 2010, 584(7):1287-95.
[28]Hsiao YT, Kuo CL, Chueh FH, et al. Curcuminoids induce reactive oxygen species and autophagy to enhance apoptosis in human oral cancer cells[J]. Am J Chin Med, 2018, 46(5):1145-68.
[29]Lin CY, Hsu SH, Lee HS, et al. Enhanced expression of glucose transporter-1 in vascular smooth muscle cells via the Akt/tuberous sclerosis complex subunit 2(TSC2)/mammalian target of rapamycin(mTOR)/ribosomal S6 protein kinase(S6K)pathway in experimental renal failure[J]. J Vasc Surg, 2013, 57(2):475-85.