华蟾毒精抑制人肝癌细胞株增殖、促凋亡作用及机制探讨
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摘要
目的:观察华蟾毒精对人肝癌细胞株增殖抑制及促凋亡作用,并探讨其机制。方法:选取人肝癌细胞HepG2进行培养,取对数生长期HepG2细胞分为实验组1(浓度为25μmol/L华蟾毒精处理)、实验组2(浓度为50μmol/L华蟾毒精处理)、实验组3(浓度为100μmol/L华蟾毒精处理)和对照组(加等体积的生理盐水),培养24h、48h后采用MTT法和流式细胞术检测对照组和实验组肝癌细胞HepG2增殖抑制率和凋亡率。培养48h后荧光定量PCR检测AKT、mTOR、Caspase3、Bax和Bcl-2 m RNA表达量,免疫印迹法检测Caspase3、Bax和Bcl-2蛋白表达量。结果:培养24h、48h,实验组1、实验组2和实验组3细胞增殖抑制率均显著高于对照组(P<0.05),且实验组间两两比较差异均有统计学意义(P<0.05);培养24h、48h实验组1、实验组2和实验组3细胞凋亡率均显著高于对照组(P<0.05),实验组间两两比较差异均有统计学意义(P<0.05);培养48h,实验组1、实验组2和实验组3细胞AKT、mTOR、Bcl-2 m RNA表达量与对照组相比均显著降低(P<0.05),Bax和Caspase-3 m RNA表达量均显著增加(P<0.05),且实验组Caspase3、Bax和Bcl-2蛋白表达与m RNA表达结果趋势一致。结论:华蟾毒精能够抑制肝癌细胞HepG2增殖,并促进其凋亡,作用机制可能与通过下调Bcl-2蛋白和上调Bax、Caspase-3蛋白的表达,调控AKT/mTOR信号通路有关。
Objective:. Methods: To observe the effect and mechanism of cinobufagin on proliferation and pro-apoptotic of human hepatoma cell line. Results: Human hepatoma cell line HepG2 was cultured,and the HepG2 cells were divided into the experimental group 1( added 25 μmol/L cinobufagin),the experimental group 2( added 50 μmol/L cinobufagin),the experimental group 3( added 100 μmol/L cinobufagin) and the control group( added equal volume of saline). The proliferation inhibition rates were measured by MTT assay and the apoptotic rates were determined by flow cytometry in the control group and the experimental group after 24 h and 48 h of culture. The m RNA expressions of AKT,mTOR,Caspase3,Bax and Bcl-2 were detected by Real time Quantitative PCR and the protein expressions of Caspase3,Bax and Bcl-2 were detected by Western blotting. Results: After 24 h and 48 h of culture,the cell proliferation inhibition rates of the three experimental groups were significantly higher than those of the control group( P < 0. 05),and the differences between each two experimental groups were statistically significant( P < 0. 05). The apoptosis rates of the three experimental groups were significantly higher than those of the control group( P < 0. 05),and the differences between each two experimental groups were statistically significant( P < 0. 05). Compared with the control group,the expressions of AKT,mTOR and Bcl-2 m RNA of the three experimental groups were significantly decreased( P < 0. 05),and the expressions of Bax and Caspase-3 m RNA were increased significantly( P < 0. 05) in the three experimental groups at 48 h,of which the expressions of the proteins of Caspase3,Bax and Bcl-2 were consistent with the m RNA expressions. Conclusion: cinobufagin can inhibit proliferation and promote apoptosis of hepatoma cell line,and it suggests that the down-regulation of Bcl-2 and up-regulation of Bax and Caspase-3 can inhibit the proliferation and promote apoptosis of HCC cells,which is related to AKT/mTOR signaling pathway.
Objective:. Methods: To observe the effect and mechanism of cinobufagin on proliferation and pro-apoptotic of human hepatoma cell line. Results: Human hepatoma cell line HepG2 was cultured,and the HepG2 cells were divided into the experimental group 1( added 25 μmol/L cinobufagin),the experimental group 2( added 50 μmol/L cinobufagin),the experimental group 3( added 100 μmol/L cinobufagin) and the control group( added equal volume of saline). The proliferation inhibition rates were measured by MTT assay and the apoptotic rates were determined by flow cytometry in the control group and the experimental group after 24 h and 48 h of culture. The m RNA expressions of AKT,mTOR,Caspase3,Bax and Bcl-2 were detected by Real time Quantitative PCR and the protein expressions of Caspase3,Bax and Bcl-2 were detected by Western blotting. Results: After 24 h and 48 h of culture,the cell proliferation inhibition rates of the three experimental groups were significantly higher than those of the control group( P < 0. 05),and the differences between each two experimental groups were statistically significant( P < 0. 05). The apoptosis rates of the three experimental groups were significantly higher than those of the control group( P < 0. 05),and the differences between each two experimental groups were statistically significant( P < 0. 05). Compared with the control group,the expressions of AKT,mTOR and Bcl-2 m RNA of the three experimental groups were significantly decreased( P < 0. 05),and the expressions of Bax and Caspase-3 m RNA were increased significantly( P < 0. 05) in the three experimental groups at 48 h,of which the expressions of the proteins of Caspase3,Bax and Bcl-2 were consistent with the m RNA expressions. Conclusion: cinobufagin can inhibit proliferation and promote apoptosis of hepatoma cell line,and it suggests that the down-regulation of Bcl-2 and up-regulation of Bax and Caspase-3 can inhibit the proliferation and promote apoptosis of HCC cells,which is related to AKT/mTOR signaling pathway.
引文
[1]王新青,卜阳,于松宁,等.精准肝切除治疗原发性肝癌近期疗效的Meta分析[J].中国普通外科杂志,2016,25(1):57-67.
[2]Aliberti C,Carandina R,Sarti D,et al.Hepatic Arterial Infusion of Polyethylene Glycol Drug-eluting Beads for Primary and Metastatic Liver Cancer Therapy.[J].Anticancer Research,2016,36(7):3515-3521.
[3]谢闪闪.华蟾毒精对先天免疫细胞功能的调节作用研究[D].吉林大学,2016.
[4]舒波,游川,刘福.华蟾素胶囊对中晚期癌症癌痛患者疼痛及生存质量的影响[J].中医药导报,2017,23(15):48-50.
[5]杨斯文,孙涛.吉西他滨±顺铂联合华蟾素治疗老年晚期非小细胞肺癌对肿瘤周期影响的观察研究[J].临床心身疾病杂志,2016,22(1):106-108.
[6]Weiler E,Farbman AI.Supporting cell proliferation in the olfactory epithelium decreases postnatally[J].Glia,2015,22(4):315-328.
[7]丁宇霜.华蟾毒精、土木香内酯诱导人结肠癌细胞凋亡及其机制的研究[D].吉林大学,2017.
[8]Benarye E,Samuels N,Goldstein LH,et al.Potential risks associated with traditional herbal medicine use in cancer care:A study of Middle Eastern oncology health care professionals.[J].Cancer,2016,122(4):598-610.
[9]李娜,贠可力.自拟清热解毒、活血化瘀方联合华蟾素治疗湿热瘀毒型宫颈癌临床观察[J].世界中医药,2016,11(11):2324-2326.
[10]Seydi E,Rasekh H R,Salimi A,et al.Myricetin Selectively InducesApoptosis on Cancerous Hepatocytes by directly Targeting their Mitochondria[J].Basic&Clinical Pharmacology&Toxicology,2016,119(3):249-258.
[11]孟显华.华蟾毒精对淋巴细胞和巨噬细胞免疫调节活性的研究[D].锦州医科大学,2016.
[12]张伟珍,徐凤秋,王小芬,等.华蟾素胶囊联合TP方案治疗晚期宫颈癌临床观察[J].浙江中西医结合杂志,2015,25(2):145-147.
[13]刘霏,李浩然,程玺,等.蟾蜍灵对宫颈癌细胞的增殖抑制作用及其机制研究[J].中国癌症杂志,2015,25(10):780-784.
[14]Janakiraman H,House RP,Talwar S,et al.Repression of caspase-3and RNA-binding protein Hu R cleavage by cyclooxygenase-2 promotes drug resistance in oral squamous cell carcinoma[J].Oncogene,2017,36(22):3137-3148.
[15]Sidi S,Sanda T,Kennedy RD,et al.Chk1 suppresses a caspase-2 apoptotic response to DNA damage that bypasses p53,Bcl-2,and caspase-3[J].Cell,2015,133(5):864-877.
[16]陈建英,胡先全,黄三雄,等.华蟾素胶囊联合GP方案对晚期非小细胞肺癌患者免疫功能的影响[J].中国现代医生,2016,54(14):12-15.
[2]Aliberti C,Carandina R,Sarti D,et al.Hepatic Arterial Infusion of Polyethylene Glycol Drug-eluting Beads for Primary and Metastatic Liver Cancer Therapy.[J].Anticancer Research,2016,36(7):3515-3521.
[3]谢闪闪.华蟾毒精对先天免疫细胞功能的调节作用研究[D].吉林大学,2016.
[4]舒波,游川,刘福.华蟾素胶囊对中晚期癌症癌痛患者疼痛及生存质量的影响[J].中医药导报,2017,23(15):48-50.
[5]杨斯文,孙涛.吉西他滨±顺铂联合华蟾素治疗老年晚期非小细胞肺癌对肿瘤周期影响的观察研究[J].临床心身疾病杂志,2016,22(1):106-108.
[6]Weiler E,Farbman AI.Supporting cell proliferation in the olfactory epithelium decreases postnatally[J].Glia,2015,22(4):315-328.
[7]丁宇霜.华蟾毒精、土木香内酯诱导人结肠癌细胞凋亡及其机制的研究[D].吉林大学,2017.
[8]Benarye E,Samuels N,Goldstein LH,et al.Potential risks associated with traditional herbal medicine use in cancer care:A study of Middle Eastern oncology health care professionals.[J].Cancer,2016,122(4):598-610.
[9]李娜,贠可力.自拟清热解毒、活血化瘀方联合华蟾素治疗湿热瘀毒型宫颈癌临床观察[J].世界中医药,2016,11(11):2324-2326.
[10]Seydi E,Rasekh H R,Salimi A,et al.Myricetin Selectively InducesApoptosis on Cancerous Hepatocytes by directly Targeting their Mitochondria[J].Basic&Clinical Pharmacology&Toxicology,2016,119(3):249-258.
[11]孟显华.华蟾毒精对淋巴细胞和巨噬细胞免疫调节活性的研究[D].锦州医科大学,2016.
[12]张伟珍,徐凤秋,王小芬,等.华蟾素胶囊联合TP方案治疗晚期宫颈癌临床观察[J].浙江中西医结合杂志,2015,25(2):145-147.
[13]刘霏,李浩然,程玺,等.蟾蜍灵对宫颈癌细胞的增殖抑制作用及其机制研究[J].中国癌症杂志,2015,25(10):780-784.
[14]Janakiraman H,House RP,Talwar S,et al.Repression of caspase-3and RNA-binding protein Hu R cleavage by cyclooxygenase-2 promotes drug resistance in oral squamous cell carcinoma[J].Oncogene,2017,36(22):3137-3148.
[15]Sidi S,Sanda T,Kennedy RD,et al.Chk1 suppresses a caspase-2 apoptotic response to DNA damage that bypasses p53,Bcl-2,and caspase-3[J].Cell,2015,133(5):864-877.
[16]陈建英,胡先全,黄三雄,等.华蟾素胶囊联合GP方案对晚期非小细胞肺癌患者免疫功能的影响[J].中国现代医生,2016,54(14):12-15.