红藤提取物联合5-氟尿嘧啶抑制肝癌细胞生长作用及机制研究
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摘要
目的研究红藤提取物联合5-氟尿嘧啶对人肝癌HepG2细胞生长的抑制作用,并初步探讨其作用机制。方法体外培养HepG2细胞,以不同质量浓度的红藤提取物作用于细胞,采用MTT法检测其对细胞生长的影响,选择后续实验浓度。将HepG2细胞分为对照组、红藤提取物(40 mg/L)组、5-氟尿嘧啶(10μmol/L)组及联合用药(红藤提取物40 mg/L+5-氟尿嘧啶10μmol/L)组,采用MTT法检测细胞增殖抑制率,流式细胞仪检测细胞周期,Western blotting法检测细胞中增殖细胞核抗原(PCNA)、细胞周期蛋白D1(Cyclin D1)及细胞周期依赖性蛋白激酶4(CDK4)表达水平。结果 MTT检测结果显示,红藤提取物呈质量浓度依赖性地抑制HepG2细胞增殖,选择其质量浓度为40mg/L用于后续实验。与对照组比较,红藤提取物组和5-氟尿嘧啶组细胞增殖抑制率显著升高,G_0/G_1期细胞比例显著升高,S期和G2/M期细胞比例显著降低,细胞中PCNA、Cyclin D1、CDK4蛋白的表达显著降低(P<0.05)。与5-氟尿嘧啶组比较,联合用药能显著抑制HepG2细胞增殖,阻滞细胞周期,抑制细胞中PCNA、Cyclin D1、CDK4蛋白表达(P<0.05)。结论红藤提取物联合5-氟尿嘧啶能够增强对HepG2细胞生长的抑制作用,其作用机制可能与抑制细胞中PCNA、Cyclin D1、CDK4蛋白表达水平有关。
Objective To investigate the effect of Sargentodoxa cuneata extracts combined with 5-fluorouracil on the growth inhibition of human hepatoma HepG2 cells, and explore its mechanism. Methods HepG2 cells were cultured in vitro and treated with different concentrations of S. cuneata extracts. The effect of S. cuneata extracts on cell growth inhibition was detected by MTT assay,and the subsequent experimental concentration was selected. HepG2 cells were randomly divided into four groups: control group, S.cuneata extracts group(40 mg/L), 5-fluorouracil(10 μmol/L) group, and combination group(S. cuneata extracts 40 mg/L +5-fluorouracil 10 μmol/L). Cell proliferation inhibition rate was detected by MTT assay and cell cycle was detected by flow cytometry.The expression levels of nuclear antigen PCNA, Cyclin D1, and cell cycle-dependent protein kinase CDK4 were detected by Western blotting. Results The results of MTT assay showed that S. cuneata extracts inhibited the proliferation of HepG2 cells in a concentration-dependent manner(P < 0.05), and S. cuneata extracts with a concentration of 40 mg/L was selected for subsequent experiments. Compared with the control group, the cell proliferation inhibition rate of the S. cuneata extracts group and the5-fluorouracil group was significantly increased, the proportion of G0/G1 phase cells was significantly increased, and the proportion of cells in the S phase and G2/M phase was significantly decreased(P < 0.05). The protein expression levels of PCNA, Cyclin D1, and CDK4 in the cells were significantly decreased(P < 0.05). Compared with the 5-fluorouracil group, the combination group significantly inhibited the proliferation of HepG2 cells, blocked the cell cycle, and inhibited the protein expression of PCNA, Cyclin D1, and CDK4 in the cells(P < 0.05). Conclusion S. cuneata extracts combined with 5-fluorouracil enhances the growth inhibition of hepatocellular carcinoma HepG2 cells, and its mechanism may be related to the inhibition of the protein expression of PCNA, Cyclin D1, and CDK4 related to the expression levels of PCNA, Cyclin D1, and CDK4 in the inhibited cells.
Objective To investigate the effect of Sargentodoxa cuneata extracts combined with 5-fluorouracil on the growth inhibition of human hepatoma HepG2 cells, and explore its mechanism. Methods HepG2 cells were cultured in vitro and treated with different concentrations of S. cuneata extracts. The effect of S. cuneata extracts on cell growth inhibition was detected by MTT assay,and the subsequent experimental concentration was selected. HepG2 cells were randomly divided into four groups: control group, S.cuneata extracts group(40 mg/L), 5-fluorouracil(10 μmol/L) group, and combination group(S. cuneata extracts 40 mg/L +5-fluorouracil 10 μmol/L). Cell proliferation inhibition rate was detected by MTT assay and cell cycle was detected by flow cytometry.The expression levels of nuclear antigen PCNA, Cyclin D1, and cell cycle-dependent protein kinase CDK4 were detected by Western blotting. Results The results of MTT assay showed that S. cuneata extracts inhibited the proliferation of HepG2 cells in a concentration-dependent manner(P < 0.05), and S. cuneata extracts with a concentration of 40 mg/L was selected for subsequent experiments. Compared with the control group, the cell proliferation inhibition rate of the S. cuneata extracts group and the5-fluorouracil group was significantly increased, the proportion of G0/G1 phase cells was significantly increased, and the proportion of cells in the S phase and G2/M phase was significantly decreased(P < 0.05). The protein expression levels of PCNA, Cyclin D1, and CDK4 in the cells were significantly decreased(P < 0.05). Compared with the 5-fluorouracil group, the combination group significantly inhibited the proliferation of HepG2 cells, blocked the cell cycle, and inhibited the protein expression of PCNA, Cyclin D1, and CDK4 in the cells(P < 0.05). Conclusion S. cuneata extracts combined with 5-fluorouracil enhances the growth inhibition of hepatocellular carcinoma HepG2 cells, and its mechanism may be related to the inhibition of the protein expression of PCNA, Cyclin D1, and CDK4 related to the expression levels of PCNA, Cyclin D1, and CDK4 in the inhibited cells.
引文
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[24]Liu Y,Wang X,Wang Y,et al.Combination of SNX-2112with 5-FU exhibits antagonistic effect in esophageal cancer cells[J].Int J Oncol,2015,46(1):299-307.
[2]Jiang E,Shangguan A J,Chen S,et al.The progress and prospects of routine prophylactic antiviral treatment in hepatitis B-related hepatocellular carcinoma[J].Cancer Lett,2016,379(2):262-267.
[3]梁淑影,王峰,王珺,等.5-氟尿嘧啶血药浓度对替吉奥联合紫杉醇治疗晚期胃癌疗效的评估价值[J].郑州大学学报:医学版,2017,52(3):339-343.
[4]程康文,李琪,詹勇强,等.5-氟尿嘧啶对肝癌细胞株PLC/RAF/5生物学特性及耐药机制的影响[J].临床肝胆病杂志,2015,31(9):1458-1463.
[5]武超,许杜娟,杨翠,等.黄芪皂苷II增加5-氟尿嘧啶对人肝癌细胞株HepG2增殖抑制作用[J].安徽医科大学学报,2016,51(1):78-82.
[6]Jimeno A,Machiels J P,Wirth L,et al.Phase Ib study of duligotuzumab(MEHD7945A)plus cisplatin/5-fluorouracil or carboplatin/paclitaxel for first-line treatment of recurrent/metastatic squamous cell carcinoma of the head and neck[J].Cancer,2016,122(24):3803-3811.
[7]陈兆榕,程晓平,褚剑锋,等.红藤水提物活化Nrf2细胞信号通路促进抗氧化基因HO-1表达的研究[J].康复学报,2017,27(4):27-32.
[8]Tang J,Qian Y,Li Q Q,et al.Cinnamic acid derivatives from the ethyl acetate fraction of Sargentodoxa cuneata[J].Chem Nat Comp,2012,48(1):118-119.
[9]韩磊,韩涛,赵冲,等.我国原发性肝癌治疗理念的转变[J].中国临床实用医学,2017,8(4):1-2.
[10]Li M,Bu W,Ren J,et al.Enhanced synergism of thermo-chemotherapy for liver cancer with magnetothermally responsive nanocarriers[J].Theranostics,2018,8(3):693-709.
[11]Falkson G,Moertel C G,Lavin P,et al.Chemotherapy studies in primary liver cancer:A prospective randomized clinical trial[J].Cancer,2015,42(5):2149-2156.
[12]李银英,陈成群,张振中.2-甲氧基雌二醇联合冬凌草甲素对胃癌SGC-7901细胞增殖和凋亡的影响[J].郑州大学学报:医学版,2016,51(5):602-606.
[13]Liao Y H,Lin C C,Li T C,et al.Utilization pattern of traditional Chinese medicine for liver cancer patients in Taiwan[J].Bmc Complement Altern Med,2012,12(1):146-146.
[14]毕丽华,武刚,田敬荣,等.红藤养胃汤对胃癌患者血清MG7-Ag、TPS及OPN水平的影响[J].现代生物医学进展,2018,18(5):943-946.
[15]鲁平,张小峰,赵阳.红藤诱发胃腺癌细胞MCGS03凋亡的研究[J].医学信息旬刊,2011,24(7):219-220.
[16]刘涛,王执民,韦建学.红藤脂酸钠对人肝癌细胞系SMMC-7721体外抑瘤作用的实验研究[J].介入放射学杂志,2005,14(4):401-403.
[17]王明华.红藤四萜大环内酯酸钠对肝癌的药效学及作用机制研究[D].西安:第四军医大学,2011.
[18]Peng Y C,Lu S D,Zhong J H,et al.Combination of5-fluorouracil and 2-morphilino-8-phenyl-4H-chromen-4-one may inhibit liver cancer stem cell activity[J].Tumor Biol,2016,37(8):10943-10958.
[19]Cheng M,Gao X,Wang Y,et al.Synthesis of glycyrrhetinic acid-modified chitosan 5-fluorouracil nanoparticles and its inhibition of liver cancer characteristics in vitro and in vivo[J].Mar Drugs,2013,11(9):3517-3536.
[20]张继新,庞志刚,刘超,等.阿霉素、5-氟尿嘧啶联合热疗对SMMC-7721细胞增殖及凋亡的影响[J].郑州大学学报:医学版,2014,49(3):365-367.
[21]Zhao H,Ho P C,Lo Y H,et al.Interaction of proliferation cell nuclear antigen(PCNA)with c-Abl in cell proliferation and response to DNA damages in breast cancer[J].PLoS One,2012,7(1):e29416.
[22]Tao Y,Lin F,Li R,et al.Prolyl hydroxylase-2 inhibits liver tumor cell proliferation and cyclin D1 expression in a hydroxylase-dependent manner[J].Int J Biochem Cell Biol,2016,77(Pt A):129-140.
[23]李莉,贾慧,李保平,等.上皮性卵巢癌中Let-7a及其靶基因HMGA2、Cyclin D1的表达[J].郑州大学学报:医学版,2017,52(4):398-402.
[24]Liu Y,Wang X,Wang Y,et al.Combination of SNX-2112with 5-FU exhibits antagonistic effect in esophageal cancer cells[J].Int J Oncol,2015,46(1):299-307.