连翘提取物通过PI3K-AKT-mTOR信号通路逆转结肠癌氟尿嘧啶耐药细胞株的研究
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摘要
目的:通过体外实验观察中药连翘提取物对结肠癌氟尿嘧啶(5-FU)耐药细胞株的作用及机制。方法:构建结肠癌5-FU耐药细胞株SW480R,并运用MTT、流式细胞术、Western Blot检测不同浓度连翘提取物对SW480R的抑制增殖、诱导凋亡以及蛋白磷脂酰肌醇3-激酶(Phosphatidylinositide 3-kinases,PI3K)、磷酸化蛋白激酶B (Phosphorylated-protein kinase B,PAKT)、蛋白激酶B (Protein kinase B,AKT)、磷酸化雷帕霉素靶蛋白(Phosphorylated-mammalian target of rapamycin,P-mTOR)、雷帕霉素靶蛋白(Mammalian target of rapamycin,mTOR)、磷酸化真核翻译起始因子4E结合蛋白1 (Phosphorylated-eukaryotic translation initiation factor 4E-binding protein 1,P-4EBP1)、真核翻译起始因子4E结合蛋白1 (Eukaryotic translation initiation factor4E-binding protein 1,4EBP1)的表达。结果:经过5-FU长期诱导,构建培养而得到结肠癌5-FU耐药细胞株SW480R的半抑制浓度(The half maximal inhibitory concentration,IC50)为62.68μmol/L,而正常结肠癌细胞SW480的IC50为13.54μmol/L。与对照组比较,不同浓度连翘提取物对SW480R有抑制增殖作用(P <0.05),且呈剂量依赖性,其IC50剂量为13.63μmol/L。与对照组比较,不同浓度连翘提取物能使活化型半胱天冬酶3 (Cleaved-Caspase 3)、活化型半胱天冬酶9 (Cleaved-Caspase 9)的表达增加,原型Caspase 3、Caspase 9的表达下降,同时显著下调信号通路蛋白PI3K、P-AKT、P-mTOR、P-4EBP1的表达(P <0.05)。结论:连翘提取物能从体外明显抑制结肠癌5-FU耐药细胞株SW480R的生长,诱导凋亡,其作用机制可能通过抑制PI3K-AKTmTOR信号通路,最终起到抑制肿瘤生长的作用。
Objective: To observe the effect and mechanism of Chinese herbal Forsythia suspensa extract on fluorouracil(5-FU)resistant cell line in colon cancer by experiments in vitro. Methods:Made a 5-FU drug-resistant cell line called SW480 R in colon cancer,and applied MTT, flow cytometry and Western blotto detect the proliferation inhibition and apoptosisinduction of different concentrations of Forsythia extract for SW480 R and the expressions of phosphatidylinositide 3-kinases(PI3 K), phosphorylated-protein kinase B(P-AKT),protein kinase B(AKT),phosphorylated-mammalian target of rapamycin(P-mTOR),mammalian target of rapamycin(mT OR),phosphorylated eukaryotic translation initiation factor 4 E-binding protein 1(P-4 EBP1),eukaryotic translation initiation factor4 E-binding protein 1(4 EBP1). Results: After long-term induction of 5-FU, the half-immunity inhibitory concentration(IC50)of5-FU-resistant cell line SW480 R created by construction and culture in colon cancerwas 62.68 μmol/L, while the IC50 of SW480 in normal colon cancer cells was 13.54 μmol/L. Compared with that in the control group,different concentrations of Forsythia suspensa extract inhibited proliferation inhibitionof SW480 R(P < 0.05); there was a dose-dependent manner, with its IC50 dose being 13.63 μmol/L.Compared with that in the control group, different concentrations of Forsythia suspensa extract increased the expressions of Cleaved-Caspase 3 and Cleaved-Caspase 9 and decreasedthe expressions of prototype Caspase 3 and Caspase 9; at the same time it significantlydown-regulatedthe expressions of PI3 K, P-AKT, P-mT OR and P-4 EBP1(P < 0.05). Conclusion: The application ofForsythiasuspensa extract can significantly inhibit the growth of 5-FU resistant cell line SW480 R in colon cancer in vitro and induce the apoptosis.The mechanism of action may ultimately beachieved in that Forsythia suspensa extract inhibits the growth of tumor by inhibiting the PI3 K-AKT-mTOR signaling pathway.
Objective: To observe the effect and mechanism of Chinese herbal Forsythia suspensa extract on fluorouracil(5-FU)resistant cell line in colon cancer by experiments in vitro. Methods:Made a 5-FU drug-resistant cell line called SW480 R in colon cancer,and applied MTT, flow cytometry and Western blotto detect the proliferation inhibition and apoptosisinduction of different concentrations of Forsythia extract for SW480 R and the expressions of phosphatidylinositide 3-kinases(PI3 K), phosphorylated-protein kinase B(P-AKT),protein kinase B(AKT),phosphorylated-mammalian target of rapamycin(P-mTOR),mammalian target of rapamycin(mT OR),phosphorylated eukaryotic translation initiation factor 4 E-binding protein 1(P-4 EBP1),eukaryotic translation initiation factor4 E-binding protein 1(4 EBP1). Results: After long-term induction of 5-FU, the half-immunity inhibitory concentration(IC50)of5-FU-resistant cell line SW480 R created by construction and culture in colon cancerwas 62.68 μmol/L, while the IC50 of SW480 in normal colon cancer cells was 13.54 μmol/L. Compared with that in the control group,different concentrations of Forsythia suspensa extract inhibited proliferation inhibitionof SW480 R(P < 0.05); there was a dose-dependent manner, with its IC50 dose being 13.63 μmol/L.Compared with that in the control group, different concentrations of Forsythia suspensa extract increased the expressions of Cleaved-Caspase 3 and Cleaved-Caspase 9 and decreasedthe expressions of prototype Caspase 3 and Caspase 9; at the same time it significantlydown-regulatedthe expressions of PI3 K, P-AKT, P-mT OR and P-4 EBP1(P < 0.05). Conclusion: The application ofForsythiasuspensa extract can significantly inhibit the growth of 5-FU resistant cell line SW480 R in colon cancer in vitro and induce the apoptosis.The mechanism of action may ultimately beachieved in that Forsythia suspensa extract inhibits the growth of tumor by inhibiting the PI3 K-AKT-mTOR signaling pathway.
引文
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[10]CHEN J,SHAO R,LI L,et al.Effective inhibition of colon cancer cell growth with Mg Al-layered double hydroxide(LDH)loaded 5-FU and PI3K/m TOR dual inhibitor BEZ-235 through apoptotic pathways[J].Int JNanomed,2014,16(9):3403-3441.
[11]LAPLANTE M,SABATINI D M.m TOR signaling in growth control and disease[J].Cell,2012,149(2):274-293.
[2]ELEZ E,HENDLISZ A,DELAUNOIT T,et al.PhaseⅡstudy of necitumumab plus modified FOLFOX6 as first-line treatment in patients with locally advanced or metastatic colorectal cancer[J].Br J Cancer,2016,114(4):372-380.
[3]WANG J,WANG X,ZHAO M,et al.Potentially functional SNPs(pfSNPs)as novel genomic predictors of 5-FU response in metastatic colorectal cancer patients[J].PLo S One,2014,9(11):e111694.
[4]叶嘉诺,郑坚,朱莹杰,等.中药提取物逆转结直肠癌多药耐药的研究进展[J].吉林中医药,2017,37(4):429-432.
[5]刘广遐,王婷婷,胡文静,等.连翘醇提物对恶性胸腹水中原代肿瘤细胞的抗肿瘤作用[J].实用老年医学,2009,23(5):359-363.
[6]邢国秀,李楠,王童,等.连翘中黄酮类化学成分的研究进展[J].中国中药杂志,2003,28(7):593-597.
[7]毛威.连翘化学成分及其抗肿瘤活性的研究[D].武汉:湖北中医学院,2009.
[8]DANIELSEN S A,EIDE P W,NESBAKKEN A,et al.Portrait of the PI3K/AKT pathway in colorectal cancer[J].Biochim Biophys Acta,2015,1855(1):104-121.
[9]温彦斐,王俊,毕经旺.PI3K/AKT信号通路与结肠癌细胞5-氟尿嘧啶耐药机制的关系[J].中国肿瘤生物治疗杂志.2015,22(6):815-818.
[10]CHEN J,SHAO R,LI L,et al.Effective inhibition of colon cancer cell growth with Mg Al-layered double hydroxide(LDH)loaded 5-FU and PI3K/m TOR dual inhibitor BEZ-235 through apoptotic pathways[J].Int JNanomed,2014,16(9):3403-3441.
[11]LAPLANTE M,SABATINI D M.m TOR signaling in growth control and disease[J].Cell,2012,149(2):274-293.