华蟾酥毒基通过FAK/PI3K/Akt通路抑制食管癌Kyse-520细胞迁移和侵袭
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摘要
目的探讨华蟾酥毒基抑制食管癌Kyse-520细胞迁移和侵袭的相关机制。方法 CCK-8法检测Kyse-520细胞经不同浓度华蟾酥毒基作用12、24、48 h后的增殖活性;划痕愈合实验和Transwell小室法检测细胞迁移和侵袭过程;实时荧光定量PCR(RT-qPCR)分析Kyse-520细胞中FAK、Akt、PTEN、VEGF-A、MMP-2、MMP-9 mRNA的表达;Western blot检测FAK、p-FAK(Tyr397)、Akt、p-Akt(Ser473)、PTEN、VEGF-A、MMP-2、MMP-9蛋白表达水平。结果 CCK-8结果显示,华蟾酥毒基可以抑制Kyse-520细胞增殖,呈现时间和浓度依赖性,划痕实验和Transwell小室实验结果表明,华蟾酥毒基可以抑制食管癌细胞迁移和侵袭;RT-qPCR和Western blot结果显示,华蟾酥毒基对FAK、Akt mRNA及总蛋白无明显影响,但可以下调p-FAK、p-Akt、VEGF-A、MMP-2、MMP-9表达,上调PTEN的表达。结论华蟾酥毒基通过诱导PTEN表达,下调FAK/PI3K/Akt信号通路,抑制食管癌Kyse-520细胞的迁移和侵袭。
Aim To investigate the effect of cinobufagin on the migration and invasion of esophageal cancer Kyse-520 cells, and to explore the underlying molecular mechanism.Methods The rates of inhibition after treated with different concentrations of cinobufagin 12, 24, 48 h were detected by CCK-8 method, and the changes of cell migration and invasion were observed with wound healing and transwell assay. The mRNA expressions of FAK, Akt, PTEN, VEGF-A, MMP-2 and MMP-9 were detected by quantitative real-time polymerase chain reaction(RT-qPCR). The protein expressions of FAK, p-FAK(Tyr397), Akt, p-Akt(Ser473), VEGF-A, PTEN, MMP-2 and MMP-9 were measured by Western blot.Results The results of CCK-8 showed that cinobufagin could inhibit the proliferation of Kyse-520 cells in a time-and concentration-dependent manner, and cinobufagin significantly inhibited the cell invasion and migration. Meanwhile, data from RT-qPCR and Western blot suggested that cinobufagin had no significant effect on mRNA and total protein of FAK and Akt, but it reduced the expression of p-FAK(Tyr397), p-Akt(Ser473), VEGF-A, MMP-2 and MMP-9, and increased the PTEN expression.Conclusions Cinobufagin significantly inhibits the invasion and migration of esophageal cancer Kyse-520 cells through inducing PTEN expression and down-regulating FAK/PI3 K/Akt pathway.
Aim To investigate the effect of cinobufagin on the migration and invasion of esophageal cancer Kyse-520 cells, and to explore the underlying molecular mechanism.Methods The rates of inhibition after treated with different concentrations of cinobufagin 12, 24, 48 h were detected by CCK-8 method, and the changes of cell migration and invasion were observed with wound healing and transwell assay. The mRNA expressions of FAK, Akt, PTEN, VEGF-A, MMP-2 and MMP-9 were detected by quantitative real-time polymerase chain reaction(RT-qPCR). The protein expressions of FAK, p-FAK(Tyr397), Akt, p-Akt(Ser473), VEGF-A, PTEN, MMP-2 and MMP-9 were measured by Western blot.Results The results of CCK-8 showed that cinobufagin could inhibit the proliferation of Kyse-520 cells in a time-and concentration-dependent manner, and cinobufagin significantly inhibited the cell invasion and migration. Meanwhile, data from RT-qPCR and Western blot suggested that cinobufagin had no significant effect on mRNA and total protein of FAK and Akt, but it reduced the expression of p-FAK(Tyr397), p-Akt(Ser473), VEGF-A, MMP-2 and MMP-9, and increased the PTEN expression.Conclusions Cinobufagin significantly inhibits the invasion and migration of esophageal cancer Kyse-520 cells through inducing PTEN expression and down-regulating FAK/PI3 K/Akt pathway.
引文
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[11] 陈璐瑶, 杨洋, 安输, 等. G蛋白偶联受体与酪氨酸激酶受体之间的信号交流在肿瘤治疗中的作用[J]. 中国药理学通报, 2017, 33(4):454-60.
[11] Chen L Y, Yang Y, An S, et al. Cross-talk of GPCRs and RTKs and its effects on oncotherapy[J]. Chin Pharmacol Bull, 2017, 33(4):454-60.
[12] 王毛毛, 周琴, 胡叶,等. 蟾蜍及其制品寒热药性理论及在肿瘤治疗中的应用[J]. 中国医药导报, 2016, 13(19):72-5.
[12] Wang M M, Zhou Q, Hu Y, et al. Toad and its products′ cold and hot medicinal properties theory and its application in the treatment of tumor[J]. China Med Herald, 2016, 13(19):72-5.
[13] Dai G, Yu L, Yang J, et al. The synergistic antitumor effect of cinobufagin and cisplatin in human osteosarcoma cell line in vitro and in vivo[J].Oncotarget, 2017, 8(49):85150-68.
[14] He Y, Ge Y, Jiang M, et al. MiR-592 promotes gastric cancer proliferation, migration, and invasion through the PI3K/AKT and MAPK/ERK signaling pathways by targeting Spry2[J]. Cell Physiol Biochem, 2018, 47(4):1465-81.
[15] Blanco-Aparicio C, Renner O, Leal J F, Carnero A. PTEN, more than the AKT pathway[J]. Carcinogenesis, 2007, 28(7):1379-86.
[2] 韩鹏黎, 孙蕾, 吕朋举, 等. 靶向ESCCAL1基因的siRNA纳米复合物的制备及体外对食管癌EC-9706细胞的抑制作用[J]. 中国药理学通报, 2017, 33(12):1749-53.
[2] Han P L, Sun L, Lyu P J, et al. Preparation and in vitro study of ESCCAL1-targeted siRNA gene delivery of nanocomposite for treating esophageal cancer EC-9706[J]. Chin Pharmacol Bull, 2017, 33(12):1749-53.
[3] Li C, Hashimi S M, Cao S, et al. Chansu inhibits the expression of cortactin in colon cancer cell lines in vitro and in vivo[J]. BMC Complement Altern Med, 2015, 15(1):1-8.
[4] 王志美, 徐忠伟, 王佳宝, 等. 蟾酥活性成分协调索拉非尼通过下调Akt/NF-κB信号途径抑制肝癌HepG2细胞生长[J]. 中国药理学通报, 2017, 33(11):1510-6.
[4] Wang Z M, Xu Z W, Wang J B, et al. Synergy with the active ingredients of toad venom and sorafenib suppresses hepatocellular carcinoma HepG2 cells proliferation through down regulating Akt/NF-κB signaling pathways[J]. Chin Pharmacol Bull, 2017, 33(11):1510-6.
[5] Okano J, Snyder L, Rustgi A K. Genetic alterations in esophageal cancer[J]. Methods Mol Biol, 2003, 222:131-45.
[6] Zhao X, Guan J L. Focal adhesion kinase and its signaling pathways in cell migration and angiogenesis[J]. Adv Drug Deliv Rev, 2011, 63(8):610-5.
[7] You D J, Mander S, Park C R, et al. NME1L Negatively regulates IGF1-dependent proliferation of brease cancer cells[J]. J Cell Biochem,2016,117(6):1454-63.
[8] Yang Y H, Li M J, Yi Y J, et al. The root transcriptome of Achyranthes bidentata and the identification of the genes involved in the replanting benefit[J]. Plant Cell Rep, 2018, 37(4): 611-25.
[9] Yue G G, Lee J K, Li L, et al. Andrographis paniculata elicits anti-invasion activities by suppressing TM4SF3 gene expression and by anoikis-sensitization in esophageal cancer cells[J]. Am J Cancer Res, 2015, 5(12):3570-87.
[10] Liang G Z, Ding M L, Lu H T, et al. Metformin upregulates E-cadherin and inhibits B16F10 cell motility, invasion and migration[J]. Oncol Lett, 2015, 10(3):1527-32.
[11] 陈璐瑶, 杨洋, 安输, 等. G蛋白偶联受体与酪氨酸激酶受体之间的信号交流在肿瘤治疗中的作用[J]. 中国药理学通报, 2017, 33(4):454-60.
[11] Chen L Y, Yang Y, An S, et al. Cross-talk of GPCRs and RTKs and its effects on oncotherapy[J]. Chin Pharmacol Bull, 2017, 33(4):454-60.
[12] 王毛毛, 周琴, 胡叶,等. 蟾蜍及其制品寒热药性理论及在肿瘤治疗中的应用[J]. 中国医药导报, 2016, 13(19):72-5.
[12] Wang M M, Zhou Q, Hu Y, et al. Toad and its products′ cold and hot medicinal properties theory and its application in the treatment of tumor[J]. China Med Herald, 2016, 13(19):72-5.
[13] Dai G, Yu L, Yang J, et al. The synergistic antitumor effect of cinobufagin and cisplatin in human osteosarcoma cell line in vitro and in vivo[J].Oncotarget, 2017, 8(49):85150-68.
[14] He Y, Ge Y, Jiang M, et al. MiR-592 promotes gastric cancer proliferation, migration, and invasion through the PI3K/AKT and MAPK/ERK signaling pathways by targeting Spry2[J]. Cell Physiol Biochem, 2018, 47(4):1465-81.
[15] Blanco-Aparicio C, Renner O, Leal J F, Carnero A. PTEN, more than the AKT pathway[J]. Carcinogenesis, 2007, 28(7):1379-86.