大蒜素对5-氟尿嘧啶治疗肝癌的增效作用及机制
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摘要
目的研究大蒜素增强肝癌细胞SMMC-7721对5-氟尿嘧啶(5-FU)敏感性作用及机制。方法用不同浓度的大蒜素和亚叶酸钙(CF)同时联合5-FU处理肝癌细胞株SMMC-7721。将对数生长的SMMC-7721细胞株随机分为空白对照组、5-FU(100 mg/L)组、低浓度大蒜素(25 mg/L)+5-FU(100 mg/L)组、中浓度大蒜素(50 mg/L)+5-FU(100 mg/L)组、高浓度大蒜素(100 mg/L)+5-FU (100 mg/L)组、低浓度CF(5 mg/L)+5-FU(100 mg/L)组、中浓度CF(10 mg/L)+5-FU(100 mg/L)组、高浓度CF(20 mg/L)+5-FU(100 mg/L)组。采用CCK-8检测细胞增殖抑制率,选择同类药物中抑制率最高的组完成后续实验。采用流式细胞术检测细胞周期。用Hoechst 33258染色观察细胞凋亡。采用Western Blot法检测与耐药相关的P糖蛋白(Pgp)和多药耐药相关蛋白-1(MRP-1)表达。结果与5-FU组比较,低、中、高浓度大蒜素均可协同5-FU抑制SMMC-7721生长,而高浓度大蒜素+5-FU组的增殖抑制作用最明显(P<0.05);选择效果较好的高浓度大蒜素+5-FU组和中浓度CF+5-FU组继续完成后续实验。各给药组均能将细胞阻断在S期,高浓度大蒜素+5-FU组的S期阻滞作用较其他组更为明显,并且还具有明显的G2/M期细胞阻滞作用(P<0.05)。高浓度大蒜素+5-FU组能更明显地诱导肝癌细胞凋亡,降低Pgp和MRP-1蛋白表达水平(P<0.05)。结论大蒜素能够增强5-FU的抗肿瘤效果。其机制可能与大蒜素降低肝癌细胞内Pgp和MRP-1的表达,逆转耐药有关。大蒜素具有G2/M期细胞阻滞作用,可能与5-FU发生协同作用。
Objective To investigate the effect of allicin on enhancing the sensitivity of hepatocellular carcinoma cell line SMMC-7721 to 5-FU and its possible mechanism. Methods Allicin and Calcium folinate(CF) at different concentration were used as chemotherapeutic sensitizers of 5-FU to intervene human HCC SMMC-7721 cells. Human HCC SMMC-7721 cells at the logarithmic phase were randomly divided into the blank control group, 5-FU(100 mg/L)group, low concentration allicin(25 mg/L) + 5-FU(100 mg/L)group, middle concentration allicin(50 mg/L) + 5-FU(100 mg/L)group, high concentration allicin(100 mg/L) + 5-FU(100 mg/L)group, low concentration CF(5 mg/L)+ 5-FU(100 mg/L)group, middle concentration CF(10 mg/L)+ 5-FU(100 mg/L)group, and high concentration CF(20 mg/L)+ 5-FU(100 mg/L)group. CCK-8 was used to invest the cell proliferation ability of each group, and the inhibitory rates were calculated. According to the results of CCK-8, we chose the best concentration to complete the study. Flow cytometry(FCM) was used to detect the cell cycle in each group, and to observe the cell cycle arrest effect. Hoechst 33258 staining was conducted to testify the cell apoptosis, and the apoptosis rates were concluded by cell counting. The expression of proteins which were closely related to the inducement of multidrug resistance like P-glycoprotein(Pgp) and multidrug resistance-associated protein-1(MRP-1) were determined by Western Blot. Results Compared with 5-FU group, the allicin in low, medium and high concentration all had the synergistic effect in inhibiting the growth of SMMC-7721,while the high concentration of allicin combined with 5-FU group had the most obvious inhibitory effect on the cell growth(P<0.05). We chose high concentration allicin + 5-FU group and middle concentration CF+ 5-FU group to complete study. All the groups could block the cells in S phase, but the high concentration allicin + 5-FU group was most obvious, and also showed the obvious G2/M phase cell block effect(P<0.05). The high concentration allicin+5-FU group showed the more obvious effect on inducing the cell apoptosis, and could reduce the expression level of Pgp and MRP-1(P<0.05). Conclusions Allicin can enhance the sensitivity of hepatoma cell SMMC-7721 to 5-FU. The mechanism may be related to allicin lowering the expression of Pgp and MRP-1 of SMMC-7721 and reversing drug resistance. Allicin has G2/M phase blocking effect and may play a synergistic role with 5-FU.
Objective To investigate the effect of allicin on enhancing the sensitivity of hepatocellular carcinoma cell line SMMC-7721 to 5-FU and its possible mechanism. Methods Allicin and Calcium folinate(CF) at different concentration were used as chemotherapeutic sensitizers of 5-FU to intervene human HCC SMMC-7721 cells. Human HCC SMMC-7721 cells at the logarithmic phase were randomly divided into the blank control group, 5-FU(100 mg/L)group, low concentration allicin(25 mg/L) + 5-FU(100 mg/L)group, middle concentration allicin(50 mg/L) + 5-FU(100 mg/L)group, high concentration allicin(100 mg/L) + 5-FU(100 mg/L)group, low concentration CF(5 mg/L)+ 5-FU(100 mg/L)group, middle concentration CF(10 mg/L)+ 5-FU(100 mg/L)group, and high concentration CF(20 mg/L)+ 5-FU(100 mg/L)group. CCK-8 was used to invest the cell proliferation ability of each group, and the inhibitory rates were calculated. According to the results of CCK-8, we chose the best concentration to complete the study. Flow cytometry(FCM) was used to detect the cell cycle in each group, and to observe the cell cycle arrest effect. Hoechst 33258 staining was conducted to testify the cell apoptosis, and the apoptosis rates were concluded by cell counting. The expression of proteins which were closely related to the inducement of multidrug resistance like P-glycoprotein(Pgp) and multidrug resistance-associated protein-1(MRP-1) were determined by Western Blot. Results Compared with 5-FU group, the allicin in low, medium and high concentration all had the synergistic effect in inhibiting the growth of SMMC-7721,while the high concentration of allicin combined with 5-FU group had the most obvious inhibitory effect on the cell growth(P<0.05). We chose high concentration allicin + 5-FU group and middle concentration CF+ 5-FU group to complete study. All the groups could block the cells in S phase, but the high concentration allicin + 5-FU group was most obvious, and also showed the obvious G2/M phase cell block effect(P<0.05). The high concentration allicin+5-FU group showed the more obvious effect on inducing the cell apoptosis, and could reduce the expression level of Pgp and MRP-1(P<0.05). Conclusions Allicin can enhance the sensitivity of hepatoma cell SMMC-7721 to 5-FU. The mechanism may be related to allicin lowering the expression of Pgp and MRP-1 of SMMC-7721 and reversing drug resistance. Allicin has G2/M phase blocking effect and may play a synergistic role with 5-FU.
引文
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[8] Johnson Z L, Chen J. Structural basis of substrate recognition by the multidrug resistance protein MRP1[J]. Cell, 2017, 168(6): 1075-1085.
[9] Samarin J, Laketa V, Malz M, et al. PI3K/AKT/mTOR-dependent stabilization of oncogenic far-upstream element binding proteins in hepatocellular carcinoma cells[J]. Hepatology, 2016, 63(3): 813-826.
[10] Fletcher JI, Williams RT, Henderson MJ, et al. ABC transporters as mediators of drug resistance and contributors to cancer cell biology[J]. Drug Resist Updat, 2016, 26: 1-9.
[11] Lee JM, Lee MS, Koh D, et al. A new synthetic2'-hydroxy-2, 4, 6-trimethoxy-5', 6'-naphthochalcone induces G2/M cell cycle arrest and apoptosis by disrupting the microtubular network of human colon cancer cells[J]. Cancer Lett, 2014, 354(2): 348-354.
[2] 孔春芳, 丁江华. 大蒜素抗癌作用与信号传导通路[J]. 重庆医学, 2013, 42(10): 1175-1177.
[3] 马锐, 何红梅, 袁媛. 大蒜素与细胞周期特异性化疗药联合应用抗肿瘤的实验研究[J]. 中国肿瘤临床, 2005, 32(19): 1129-1132.
[4] Lee S, Lee M, Kim JB, et al. 17β-estradiol exerts anticancer effects in anoikis-resistant hepatocellular carcinoma cell lines by targeting IL-6/STAT3 signaling[J]. Biochem Biophys Res Commun, 2016, 473(4): 1247-1254.
[5] Gao K, Liang Q, Zhao ZH, et al. Synergistic anticancer properties of docosahexaenoic acid and 5-fluorouracil through interference with energy metabolism and cell cycle arrest in human gastric cancer cell line AGS cells[J]. World J Gastroenterol, 2016, 22(10): 2971-2980.
[6] Chen F, Li H, Wang Y, et al. Inhibition of allicin in Eca109 and EC9706 cells via G2/M phase arrest and mitochondrial apoptosis pathway[J]. J Funct Foods, 2016, 25: 523-536.
[7] Han GZ, Zhang CZ, Sun YB, et al. Effect of allicin on spleen and cell cycle of splenocytes in mice[J]. China Tropical Med, 2016, 16(9): 883-885.
[8] Johnson Z L, Chen J. Structural basis of substrate recognition by the multidrug resistance protein MRP1[J]. Cell, 2017, 168(6): 1075-1085.
[9] Samarin J, Laketa V, Malz M, et al. PI3K/AKT/mTOR-dependent stabilization of oncogenic far-upstream element binding proteins in hepatocellular carcinoma cells[J]. Hepatology, 2016, 63(3): 813-826.
[10] Fletcher JI, Williams RT, Henderson MJ, et al. ABC transporters as mediators of drug resistance and contributors to cancer cell biology[J]. Drug Resist Updat, 2016, 26: 1-9.
[11] Lee JM, Lee MS, Koh D, et al. A new synthetic2'-hydroxy-2, 4, 6-trimethoxy-5', 6'-naphthochalcone induces G2/M cell cycle arrest and apoptosis by disrupting the microtubular network of human colon cancer cells[J]. Cancer Lett, 2014, 354(2): 348-354.