姜黄素对胰腺癌SW1990细胞耐吉西他滨的逆转作用及机制研究
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摘要
目的:研究姜黄素对胰腺癌SW1990细胞耐吉西他滨(GEM)的逆转作用及机制。方法:采用CCK8法检测不同浓度(50、100、150、200、250μmol/L)GEM对SW1990细胞和耐GEM SW1990细胞(SW1990/GEM耐药细胞)存活率的影响,计算半数抑制浓度(IC_(50))和耐药倍数;采用CCK8法检测不同浓度(1、5、10、20、40μmol/L)姜黄素对SW1990/GEM耐药细胞存活率的影响,计算IC_(50);采用CCK8法检测2.41μmol/L姜黄素与不同浓度(25、50、75、100、125μmol/L)GEM联用对SW1990细胞和SW1990/GEM耐药细胞存活率的影响,计算GEM的IC_(50)和耐药逆转倍数。采用流式细胞仪检测以GEM的IC_(50)为给药浓度,GEM单用、姜黄素(2.41μmol/L)与GEM联用处理SW1990细胞和SW1990/GEM耐药细胞后的细胞凋亡率和细胞周期分布,并采用Western blot法检测细胞中脂肪酸合成酶(FAS)、蛋白激酶B(AKT)、磷酸化AKT(p-AKT)、磷脂酰肌醇激酶(PI3K)、磷酸化PI3K(p-PI3K)、胱天蛋白酶3(Caspase-3)、B淋巴细胞瘤2(Bcl-2)及其相关X蛋白(Bax)的蛋白表达,反转录聚合酶链式反应(RT-PCR)检测mRNA表达。结果:GEM对SW1990细胞的IC_(50)为92μmol/L,对SW1990/GEM耐药细胞的IC_(50)为216μmol/L,SW1990细胞对GEM的耐药倍数为2.35;姜黄素对SW1990/GEM耐药细胞的IC_(50)为9.2μmol/L;2.41μmol/L姜黄素下,GEM对SW1990细胞的IC_(50)为75μmol/L,对SW1990/GEM耐药细胞的IC_(50)为98μmol/L,SW1990/GEM耐药细胞对GEM的耐药逆转倍数为2.2。与GEM单用比较,姜黄素与GEM联用时SW1990细胞和SW1990/GEM耐药细胞的凋亡率均明显升高(P<0.05),主要阻滞在G0/G1期,细胞中FAS、p-AKT、p-PI3K、Bcl-2蛋白表达水平和FAS、Bcl-2 mRNA表达水平均明显降低(P<0.05),Bax、Cascapse-3蛋白表达水平和mRNA表达水平均明显升高(P<0.05)。结论:姜黄素可逆转SW1990细胞对GEM的耐药性,其机制可能与PI3K/AKT通路有关。
OBJECTIVE:To study the effects of curcumin on gemcitabine(GEM)-resistant pancreatic cancer SW1990 cells and its mechanism. METHODS:CCK8 assay was used to detect the effects of different concentrations of GEM(50,100,150,200,250 μmol/L)on the survival rate of SW1990 cells and GEM-resistant SW1990 cells(SW1991/GEM resistant cells);half inhibitory concentration(IC_(50))and drug resistance multiple were calculated. CCK8 assay was performed to detect the effects of different concentrations of curcumin(1,5,10,20,40 μ mol/L)on survival rate of SW1990/GEM resistant cells,and IC_(50) was calculated. CCK8 assay was used to detect the effects of curcumin 2.41 μ mol/L combined with different concentrations of GEM(25,50,75,100,125 μ mol/L) on the survival rate of SW1990 cells and SW1990/GEM resistant cells,and IC_(50) and drug resistance reversal fold of GEM were calculated. Flow cytometry was carried out to detect the cell cycle distribution and apoptosis rate of SW1990 after treated with GEM alone or curcumin(2.41 μ mol/L) combined with GEM using IC_(50) of GEM as drug concentration. Western blot assay was used to the protein expression of FAS,AKT,p-AKT,PI3 K,p-PI3 K,Caspase-3,Bcl-2 and related X protein(Bax). RT-PCR was used to detect m RNA expression. RESULTS:IC_(50) of GEM to SW1990 cells was 92 μmol/L.IC_(50) of GEM to SW1990/GEM resistant cells was 216 μmol/L,and drug resistance multiple SW1990 cells to GEM was 2.35. IC_(50) of curcumin to SW1990/GEM resistant cells was 9.2 μmol/L. Under 2.41 μmol/L curcumin,IC_(50) of GEM to SW1990 cells was 75μ mol/L,and IC_(50) of GEM to SW1990/GEM resistant cells was 98 μ mol/L;drug resistance reversal multiple of SW1990/GEM resistant cells to GEM was 2.2. Compared with GEM alone,the apoptosis rate of SW1990 cells and SW1990/GEM resistant cells were increased significantly after curcumin combined with GEM(P<0.05), blocking at G_0/G_1 phase; the protein expression of FAS,p-AKT,p-PI3K and Bcl-2 and mRNA expression of FAS and Bcl-2 were decreased significantly(P<0.05); the protein and mRNA expression of Bax and Caspase-3 were increased significantly(P<0.05).CONCLUSIONS:Curcumin can reverse drug resistance of SW1990 cells to GEM,the mechanism of which may be associated with PI3K/AKT pathway.
OBJECTIVE:To study the effects of curcumin on gemcitabine(GEM)-resistant pancreatic cancer SW1990 cells and its mechanism. METHODS:CCK8 assay was used to detect the effects of different concentrations of GEM(50,100,150,200,250 μmol/L)on the survival rate of SW1990 cells and GEM-resistant SW1990 cells(SW1991/GEM resistant cells);half inhibitory concentration(IC_(50))and drug resistance multiple were calculated. CCK8 assay was performed to detect the effects of different concentrations of curcumin(1,5,10,20,40 μ mol/L)on survival rate of SW1990/GEM resistant cells,and IC_(50) was calculated. CCK8 assay was used to detect the effects of curcumin 2.41 μ mol/L combined with different concentrations of GEM(25,50,75,100,125 μ mol/L) on the survival rate of SW1990 cells and SW1990/GEM resistant cells,and IC_(50) and drug resistance reversal fold of GEM were calculated. Flow cytometry was carried out to detect the cell cycle distribution and apoptosis rate of SW1990 after treated with GEM alone or curcumin(2.41 μ mol/L) combined with GEM using IC_(50) of GEM as drug concentration. Western blot assay was used to the protein expression of FAS,AKT,p-AKT,PI3 K,p-PI3 K,Caspase-3,Bcl-2 and related X protein(Bax). RT-PCR was used to detect m RNA expression. RESULTS:IC_(50) of GEM to SW1990 cells was 92 μmol/L.IC_(50) of GEM to SW1990/GEM resistant cells was 216 μmol/L,and drug resistance multiple SW1990 cells to GEM was 2.35. IC_(50) of curcumin to SW1990/GEM resistant cells was 9.2 μmol/L. Under 2.41 μmol/L curcumin,IC_(50) of GEM to SW1990 cells was 75μ mol/L,and IC_(50) of GEM to SW1990/GEM resistant cells was 98 μ mol/L;drug resistance reversal multiple of SW1990/GEM resistant cells to GEM was 2.2. Compared with GEM alone,the apoptosis rate of SW1990 cells and SW1990/GEM resistant cells were increased significantly after curcumin combined with GEM(P<0.05), blocking at G_0/G_1 phase; the protein expression of FAS,p-AKT,p-PI3K and Bcl-2 and mRNA expression of FAS and Bcl-2 were decreased significantly(P<0.05); the protein and mRNA expression of Bax and Caspase-3 were increased significantly(P<0.05).CONCLUSIONS:Curcumin can reverse drug resistance of SW1990 cells to GEM,the mechanism of which may be associated with PI3K/AKT pathway.
引文
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[3]AMRUTKAR M,GLADHAUG IP.Pancreatic cancer chemoresistance to gemcitabine[J].Cancers(Basel),2017,9(11):E157-E160.
[4]仇世钦,刘经纬,孙旭杰,等.脂肪酸合成酶抑制剂浅蓝菌素对裸鼠膀胱癌移植瘤生长抑制的作用[J].中华实验外科杂志,2018,35(6):1097-1099.
[5]张嘉穗,陈惠,魏国清,等.脂肪酸合酶:抑制肿瘤的新靶点[J].华西药学杂志,2014,29(5):599-601.
[6]MENENDEZ JA,LUPU R.Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis[J].Nature Reviews Cancer,2007,7(10):763-777.
[7]MENENDEZ JA,LUPU R.Fatty acid synthase(FAS)as a therapeutic target in breast cancer[J].Expert Opin Ther Targets,2017,21(11):1001-1016.
[8]徐晓伟,王晓奎,李松.脂肪酸合成酶抑制剂的研究进展[J].国际药学研究杂志,2009,36(2):105-108,120.
[9]覃飞,王晓波,龚建平.脂肪酸合成酶在肿瘤中表达的调节机制[J].医学分子生物学杂志,2010,7(5):453-456.
[10]陈一婧,张厚德.脂肪酸合成酶抑制剂及其抗肿瘤作用研究进展[J].中国现代医生,2015,53(15):152-155.
[11]YUAN R,HOU Y,SUN W,et al.Natural products to prevent drug resistance in cancer chemotherapy:a review[J].Ann N Y Acad Sci,2017,1401(1):19-27.
[12]苗常青,许静,王军,等.脂肪酸合成酶在胰腺癌细胞凋亡过程中的作用[J].西安交通大学学报(医学版),2015.DOI:10.7652/jdyxb201506011.
[13]WU J,DU J,FU X,et al.Lciartin,a novel FASN inhibitor,exerts anti-melanoma activities through IGF-1R/STAT3signaling[J].Oncotarget,2016,7(32):51251-51269.
[14]EBRAHIMI S,HOSSEINI M,SHAHIDSALES S,et al.Targeting the Akt/PI3K signaling pathway as a potential therapeutic strategy for the treatment of pancreatic cancer[J].Currr Med Chem,2017,24(13):1321-1331.
[15]ADAMSKA A,ELASKALANI O,EMMANOUILIDI A,et al.Molecular and cellular mechanisms of chemoresistance in pancreatic cancer[J].Adv Biol Regul,2018.DOI:10.1016/j.jbior.2017.11.007.
[16]DE SOUSA CAVALCANTE L,MONTEIRO G.Gemcitabine:metabolism and molecular mechanisms of action,sensitivity and chemoresistance in pancreatic cancer[J].Eur J Pharmacol,2014.DOI:10.1016/j.ejphar.2014.07.041.
[17]GUO H,GAO M,LU Y,et al.Coordinate phosphorylation of multiple residues on single AKT1 and AKT2 molecules[J].Oncogene,2014,33(26):3463-3472.
[18]TSUKAHARAS S,YAMAMOTO S,TIN-TIN-WIN-SHWE,et al.Inhalation of low-level formaldehyde increases the bcl-2/bax expression ratio in the hippocampus of immunologically sensitized mice[J].Neuroimmunomodulation,2006,13(2):63-68.
[19]MASHIMA T,SEIMIYA H,TSURUO T.De novo fattyacid synthesis and related pathways as molecular targets for cancer therapy[J].Br J Cancer,2009,100(9):1369-1372.
[20]王祎.天然来源的脂肪酸合酶抑制剂对肿瘤细胞的作用机制[D].北京:中国科学院大学,2013.