芪术方诱导人肝癌细胞SMMC-7721凋亡机制研究
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摘要
目的:探讨芪术方(QZP)诱导人肝癌细胞SMMC-7721凋亡的分子机制。方法:以不同浓度的QZP干预人肝癌细胞SMMC-7721,流式细胞仪检测细胞凋亡及周期分布情况,RT-PCR法检测AKT1、AKT2、Caspase-9mRNA的表达;蛋白质印迹法检测p-AKT、PTEN蛋白表达。结果:QZP能阻滞人肝癌细胞SMMC-7721从G0/G1期进入S期,并诱发细胞凋亡;能抑制AKT1、AKT2表达,提高Caspase-9表达,且跟浓度呈正比例关系。结论:QZP能诱导人肝癌细胞SMMC-7721凋亡和阻滞细胞周期,其机制可能为促进人肝癌细胞SMMC-7721 PTEN表达的增加,使得PI3K/AKT信号通路激活受阻,同时抑制p-AKT、AKT1、AKT2的表达,激活下游Caspase-9促凋亡分子的表达,从而为PI3K/AKT信号通路介导细胞凋亡这一设想提供了理论依据。
Objective: To discuss molecular mechanism of QiZhu prescription(QZP) inducing the apoptosis of human liver cancer cells SMMC-7721. Methods: Different concentrations of QZP were used to intervene human liver cancer cells SMMC-7721, flow cytometry was used to detect cellular apoptosis and cycle distribution conditions,RT-PCR was adopted to detect the expressions of AKT1, AKT2 and Caspase-9 mRNA, Western blotting was used to detect the expressions of p-AKT and PTEN protein. Results: QZP could arrest human liver cancer cells SMMC-7721 transiting from G0/G1 phase to S phase, induce cellular apoptosis; it could inhibit the expressions of AKT1 and AKT2, raise the expression of caspase-9, and the effects of QZP varies directly with the concentrations. Conclusion:QZP could induce the apoptosis of human liver cancer cell SMMC-7221 and arrest cellular cycle, and it might increase the expressions of human liver cancer cells SMMC-7721 PTEN, block the activation of PI3 K/AKT signal channel, inhibit the expressions of p-AKT, AKT1 and AKT2, and activate the expressions of caspase-9, therefore to provide theoretical evidence for PI3 K/AKT signal channel mediating cellular apoptosis.
Objective: To discuss molecular mechanism of QiZhu prescription(QZP) inducing the apoptosis of human liver cancer cells SMMC-7721. Methods: Different concentrations of QZP were used to intervene human liver cancer cells SMMC-7721, flow cytometry was used to detect cellular apoptosis and cycle distribution conditions,RT-PCR was adopted to detect the expressions of AKT1, AKT2 and Caspase-9 mRNA, Western blotting was used to detect the expressions of p-AKT and PTEN protein. Results: QZP could arrest human liver cancer cells SMMC-7721 transiting from G0/G1 phase to S phase, induce cellular apoptosis; it could inhibit the expressions of AKT1 and AKT2, raise the expression of caspase-9, and the effects of QZP varies directly with the concentrations. Conclusion:QZP could induce the apoptosis of human liver cancer cell SMMC-7221 and arrest cellular cycle, and it might increase the expressions of human liver cancer cells SMMC-7721 PTEN, block the activation of PI3 K/AKT signal channel, inhibit the expressions of p-AKT, AKT1 and AKT2, and activate the expressions of caspase-9, therefore to provide theoretical evidence for PI3 K/AKT signal channel mediating cellular apoptosis.
引文
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[5]Low SW,Lin AW.Apoptosis in Cancer[J].Carcinogenesis,2000,21(3):485-495.
[6]Ngeow J,Tan IB,Choo SP.Targeted therapies in the treatment of gastric cancer[J].Asia Pac J Clin Oncol,2011,7(3):224-235.
[7]温文胜,张哲,谢莹,等.PI3K-AKT抑制剂诱导人鼻咽癌细胞CNE2凋亡的作用[J].临床耳鼻咽喉头颈外科杂志,2010,24(8):370-372.
[8]Xiong JB,Li ZR,Yang Z,et al.PRL-3 promotes the peritoneal metastasis of gastric cancer through the PI3K/AKT signaling pathway by regulating PTEN[J].Oncology Reports,2016,36(4):1819-1828.
[9]周炳刚,魏昌晟,张颂,等.苦参碱逆转人乳腺癌MCF-7/ADR细胞株耐药性及对PI3K/AKT信号通路下游因子的影响[J].现代肿瘤医学,2017,25(1):9-13.
[10]Altomare DA,Testa JR.Perturbations of the AKT signaling pathway in human cancer[J].Oncogene,2005,24(50):7455-7472.
[11]Masure S,Haefner B,Wesselink JJ,et al.Molecular cloning,expression and characterization of the human serine/threonine kinase AKT-3[J].European Journal of Biochemistry,1999,265(1):353-360.
[12]Konishi H,Kuroda S,Tanaka M,et al.Molecular cloning and c haracterization of a new member of the RACprotein kinase family:association of the pleckstrin homology domain of 3 types of RAC protein kinase with protein kinase C subspecies andβγsubunits of G proteins[J].Biochemical&Biophysical Research Communications,1995,216(2):526-534.
[13]Kennedy SG,Wagner AJ,Conzen SD,et al.The PI 3-kinase/AKT signaling pathway delivers an anti-apoptotic signal[J].Genes Dev,1997,11(6):701-713.
[14]Liu AX,Testa JR,Hamilton TC,et al.AKT2,a member of the protein kinase B family,is activated by growth factors,v-Ha-ras,and v-src through phosphatidylinositol 3-kinase in human ovarian epithelial cancer cells[J].Cancer Research,1998,58(14):2973-2977.
[15]Dobbin ZC,Landen CN.The importance of the PI3K/AKT/MTORpathway in the progression of ovarian cancer[J].International Journal of Molecular Sciences,2013,14(4):8213-8227.
[16]Yang M,Wang HY,Zhou M,et al.The natural compound sulforaphene,as a novel anticancer reagent,targeting PI3K-AKT signaling pathway in lung cancer[J].Oncotarget,2016,7(47):76656-76666.
[17]Banumathy G,Cairns P.Signaling pathways in renal cell carcinoma[J].Cancer Biology&Therapy,2010,10(7):658-664.
[18]Alnemri ES,Livingston DJ,Nicholson DW,et al.Human ICE/CED-3 protease nomenclature[J].Cell,1996,87(2):171-183.
[19]Mackenzie SH,Clark AC.Targeting cell death in tumors by activating caspases[J].Current Cancer Drug Targets,2008,8(2):98-109.
[20]Choi BH,Kim W,Wang QC,et al.Kinetin riboside preferentially induces apoptosis by modulating Bcl-2family proteins and caspase-3 in cancer cells[J].Cancer Letters,2008,261(1):37-45.
[21]Allan LA,Clarke PR.Apoptosis and autophagy:regulation of caspase-9 by phosphorylation[J].Febs Journal,2009,276(21):6063-6073.