抑制Shh信号通路增加胰腺癌细胞对紫杉醇敏感性的实验研究
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摘要
目的研究抑制音猬因子(Shh)信号通路对胰腺癌细胞紫杉醇敏感性的影响。方法给予人胰腺癌SW1990细胞0、10 nmol/L、20 nmol/L、40 nmol/L、80 nmol/L、160 nmol/L的紫杉醇刺激,MTT测定细胞增殖情况,计算其半数抑制浓度约为50 nmol/L。用Western blot方法测定0、50 nmol/L紫杉醇处理后细胞中Shh信号通路蛋白Shh、锌指转录因子1(Gli1)、补丁蛋白1(Ptch 1)的表达水平。分别用紫杉醇、Shh信号通路抑制剂cyclopamine处理SW1990细胞,用紫杉醇和cyclopamine共同处理SW1990细胞,MTT测定增殖,克隆实验测定细胞克隆能力,流式细胞术测定细胞凋亡,Western blot测定细胞中剪切的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)、Bcl-2相关X蛋白(Bax)、Shh、Gli1、Ptch 1蛋白水平。结果 10 nmol/L、20 nmol/L、40 nmol/L、80 nmol/L、160 nmol/L的紫杉醇抑制SW1990细胞的增殖,50 nmol/L的紫杉醇可以抑制细胞中Shh、Gli1、Ptch 1蛋白表达。紫杉醇和cyclopamine单独处理后的细胞增殖能力和克隆形成能力降低,细胞凋亡增多,细胞中Cleaved Caspase-3、Bax蛋白水平升高。与紫杉醇和cyclopamine单独处理的细胞比较,紫杉醇和cyclopamine共同处理后的细胞增殖和克隆能力下降更多,细胞凋亡率更高,细胞中Cleaved Caspase-3、Bax蛋白水平更高,细胞中Shh、Gli1、Ptch 1蛋白水平更低。结论抑制Shh信号通路能够增加胰腺癌细胞对紫杉醇的敏感性。
Objective Study on the inhibitory effect of Shh signaling pathway on paclitaxel sensitivity in pancreatic cancer cells. Methods SW1990 cells were stimulated by 0 nmol/L,10 nmol/L,20 nmol/L,40 nmol/L,80 nmol/Land 160 nmol/L of paclitaxel,the proliferation of cells was measured by MTT,it was calculated that the median inhibitory concentration was about 50 nmol/L. The expression level of Shh signaling pathway protein Shh,Gli1 and Ptch 1 in 0 and 50 nmol/L paclitaxel treated cells was detected by Western blot. Paclitaxel and Shh signaling pathway inhibitor cyclopamine were used to treat cells respectively. Combined paclitaxel and cyclopamine co processed SW1990 cells,MTT to determine proliferation,cloning assay was used to determine the cell clone ability,cell apoptosis was measured by flow cytometry,Western blot was used to measure the levels of Cleaved Caspase-3,Bax,Shh,Gli1 and Ptch 1 protein in cells. Results 10 nmol/L,20 nmol/L,40 nmol/L,80 nmol/L,160 nmol/L paclitaxel inhibited the proliferation of SW1990 cells,paclitaxel from 50 nmol/L inhibited the expression of Shh,Gli1 and Ptch 1 protein in cells. Paclitaxel and cyclopamine alone reduced cell proliferation and colony forming ability,cell apoptosis increased,and the levels of Cleaved Caspase-3 and Bax protein increased,compared with paclitaxel and cyclopamine alone treated cells,paclitaxel combined with cyclopamine could decrease cell proliferation and clone ability more,the rate of cell apoptosis was higher,the level of Cleaved Caspase-3 and Bax protein was higher,the levels of Shh,Gli1 and Ptch 1 protein were lower in the cells. Conclusion Inhibition of Shh signaling pathway increases the sensitivity of pancreatic cancer to paclitaxel.
Objective Study on the inhibitory effect of Shh signaling pathway on paclitaxel sensitivity in pancreatic cancer cells. Methods SW1990 cells were stimulated by 0 nmol/L,10 nmol/L,20 nmol/L,40 nmol/L,80 nmol/Land 160 nmol/L of paclitaxel,the proliferation of cells was measured by MTT,it was calculated that the median inhibitory concentration was about 50 nmol/L. The expression level of Shh signaling pathway protein Shh,Gli1 and Ptch 1 in 0 and 50 nmol/L paclitaxel treated cells was detected by Western blot. Paclitaxel and Shh signaling pathway inhibitor cyclopamine were used to treat cells respectively. Combined paclitaxel and cyclopamine co processed SW1990 cells,MTT to determine proliferation,cloning assay was used to determine the cell clone ability,cell apoptosis was measured by flow cytometry,Western blot was used to measure the levels of Cleaved Caspase-3,Bax,Shh,Gli1 and Ptch 1 protein in cells. Results 10 nmol/L,20 nmol/L,40 nmol/L,80 nmol/L,160 nmol/L paclitaxel inhibited the proliferation of SW1990 cells,paclitaxel from 50 nmol/L inhibited the expression of Shh,Gli1 and Ptch 1 protein in cells. Paclitaxel and cyclopamine alone reduced cell proliferation and colony forming ability,cell apoptosis increased,and the levels of Cleaved Caspase-3 and Bax protein increased,compared with paclitaxel and cyclopamine alone treated cells,paclitaxel combined with cyclopamine could decrease cell proliferation and clone ability more,the rate of cell apoptosis was higher,the level of Cleaved Caspase-3 and Bax protein was higher,the levels of Shh,Gli1 and Ptch 1 protein were lower in the cells. Conclusion Inhibition of Shh signaling pathway increases the sensitivity of pancreatic cancer to paclitaxel.
引文
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[20]Lu Y,Wu D,Wang J,et al.miR-320a regulates cell proliferation and apoptosis in multiple myeloma by targeting pre-B-cell leukemia transcription factor 3[J].Biochem Biophys Res Commun,2016,473(4):1315-1320.
[21]Huang F,Jin Y,Wei Y.MicroRNA-187 induces diffuse large B-cell lymphoma cell apoptosis via targeting BCL6[J].Oncol Lett,2016,11(4):2845-2850.
[22]Chung H,Vilaysane A,Lau A,et al.NLRP3 regulates a non-canonical platform for caspase-8 activation during epithelial cell apoptosis[J].Cell Death Differ,2016,23(8):1331-1346.
[23]Zhang X,Yu H.Matrine inhibits diethylnitrosamine-induced HCC proliferation in rats through inducing apoptosis via p53,Bax-dependent caspase-3 activation pathway and down-regulating MLCK overexpression[J].Iran J Pharm Res,2016,15(2):491-499.
[24]钱小英,胡春宏.吉西他滨对乳腺癌细胞凋亡作用及其对Bcl-2、Bax蛋白表达的影响[J].临床和实验医学杂志,2016,15(14):1394
[25]Yeo JK,Cha SD,Cho CH,et al.Se-methylselenocysteine induces apoptosis through caspase activation and Bax cleavage mediated by calpain in SKOV-3 ovarian cancer cells[J].Cancer Lett,2002,182(1):83-92.
[26]Song Y,Song Y,Cao W,et al.F10,a novel hydatidiform mole-associated gene,inhibits the paclitaxel sensitivity of A549 lung cancer cells by downregulating BAX and caspase-3[J].Oncol Lett,2017,13(4):2563-2568.
[2]杨梅,朱春富.表柔比星联合多西他赛或紫杉醇治疗乳腺癌的效果分析[J].实用临床医药杂志,2016,20(21):151-151.
[3]Ai B,Bie Z,Zhang S,et al.Paclitaxel targets VEGF-mediated angiogenesis in ovarian cancer treatment[J].Am J Cancer Res,2016,6(8):1624-1635.
[4]Frese KK,Neesse A,Cook N,et al.nab-Paclitaxel potentiates gemcitabine activity by reducing cytidine deaminase levels in a mouse model of pancreatic cancer[J].Cancer Discov,2012,2(3):260-269.
[5]Suyama K,Onishi H,Imaizumi A,et al.CD24 suppresses malignant phenotype by downregulation of SHH transcription through STAT1 inhibition in breast cancer cells[J].Cancer Lett,2016,374(1):44-53.
[6]张志波,胡婷嫣,何峰,等.胆道闭锁肝脏组织中SHH信号与上皮间充质转化关系的研究[J].中国医科大学学报,2010,39(4):305-307.
[7]Liang H,Ma J,Duan W,et al.Pancreatic stellate cells contribute pancreatic cancer pain via activation of s HH signaling pathway[J].Oncotarget,2016,7(14):18146-18158.
[8]Jia X,Min L,Zhu S,et al.Loss of sonic hedgehog gene leads to muscle development disorder and megaesophagus in mice[J].Cancer Lett,2016,374(1):44-53.
[9]Zhang H,Yan L,Carvalhobarbosa MD,et al.Dorsal root ganglion infiltration by macrophages contributes to paclitaxel chemotherapy induced peripheral neuropathy[J].J Pain,2016,17(7):775-786.
[10]Zhang W,Li C,Shen C,et al.Prodrug-based nano-drug delivery system for co-encapsulate paclitaxel and carboplatin for lung cancer treatment[J].Drug Deliv,2016,23(7):2575-2580.
[11]周晓燕,许良中,何开玲,等.紫杉醇对三株不同类型淋巴瘤细胞的体外作用[J].中华医学杂志,2000,80(1):62-65.
[12]霍小蕾,韩玲娜,裴振,等.香菇多糖联合紫杉醇对食管癌细胞生物学行为的影响[J].东南大学学报(医学版),2017,36(6):897-900.
[13]Oudejans J J,Harijadi A,Cillessen S A G M,et al.Absence of caspase 3 activation in neoplastic cells of nasopharyngeal carcinoma biopsies predicts rapid fatal outcome[J].Mod Pathol,2005,18(7):877-885.
[14]Zhang R,Lee C,Lawson LY,et al.SHH Protein Variance in the Limb Bud Is Constrained by Feedback Regulation and Correlates with Altered Digit Patterning[J].G3(Bethesda),2017,7(3):851-858.
[15]Solanki A,Lau CI,Salda1a JI,et al.The transcription factor Gli3 promotes B cell development in fetal liver through repression of Shh[J].J Exp Med,2017,214(7):2041-2058.
[16]李慧,秦苏萍,孙德旭,等.Sonic Hedgehog(SHH)促进胶原蛋白诱导关节炎大鼠滑膜成纤维细胞的增殖[J].细胞与分子免疫学杂志,2016,32(5):630-634.
[17]封彦青.Shh和Foxm1在胆囊癌中的表达和临床意义及其与肿瘤的关系[D].南昌:南昌大学,2015.
[18]胡伟国,王春友,刘涛,等.SHH、EGFR和PCNA蛋白在胰腺癌组织中的表达及其意义[J].癌症,2007,26(9):947-951.
[19]Statkiewicz M,Maryan N,Lipiec A,et al.The role of the SHH gene in prostate cancer cell resistance to paclitaxel[J].Prostate,2014,74(11):1142-1152.
[20]Lu Y,Wu D,Wang J,et al.miR-320a regulates cell proliferation and apoptosis in multiple myeloma by targeting pre-B-cell leukemia transcription factor 3[J].Biochem Biophys Res Commun,2016,473(4):1315-1320.
[21]Huang F,Jin Y,Wei Y.MicroRNA-187 induces diffuse large B-cell lymphoma cell apoptosis via targeting BCL6[J].Oncol Lett,2016,11(4):2845-2850.
[22]Chung H,Vilaysane A,Lau A,et al.NLRP3 regulates a non-canonical platform for caspase-8 activation during epithelial cell apoptosis[J].Cell Death Differ,2016,23(8):1331-1346.
[23]Zhang X,Yu H.Matrine inhibits diethylnitrosamine-induced HCC proliferation in rats through inducing apoptosis via p53,Bax-dependent caspase-3 activation pathway and down-regulating MLCK overexpression[J].Iran J Pharm Res,2016,15(2):491-499.
[24]钱小英,胡春宏.吉西他滨对乳腺癌细胞凋亡作用及其对Bcl-2、Bax蛋白表达的影响[J].临床和实验医学杂志,2016,15(14):1394
[25]Yeo JK,Cha SD,Cho CH,et al.Se-methylselenocysteine induces apoptosis through caspase activation and Bax cleavage mediated by calpain in SKOV-3 ovarian cancer cells[J].Cancer Lett,2002,182(1):83-92.
[26]Song Y,Song Y,Cao W,et al.F10,a novel hydatidiform mole-associated gene,inhibits the paclitaxel sensitivity of A549 lung cancer cells by downregulating BAX and caspase-3[J].Oncol Lett,2017,13(4):2563-2568.