青蒿琥酯联合TRAIL对前列腺癌PC-3细胞系作用的实验研究
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摘要
目的:联合使用青蒿琥酯(Artesunate,ART)和肿瘤坏死因子相关的凋亡诱导配体(TNF Related Apoptosis Inducing Ligand,TRAIL),探讨两种药物联合使用对人前列腺癌PC-3细胞增殖、周期、凋亡及细胞培养液上清中前列腺特异性抗原(Prostate specific antigen, PSA)浓度等方面的影响及可能机制,寻求对前列腺癌更为有效的治疗方法。
方法: (1)根据文献资料和课题组前期研究基础,确定青蒿琥酯四个浓度和TRAIL三个浓度进行配对分组,另设单独使用青蒿琥酯组、单独使用TRAIL组和阴性对照组共15个组,药物处理对数生长期PC-3细胞48小时后,MTT法检测两种药物联合使用的增殖抑制率,进行统计学分析。(2)根据MTT结果,分别选取青蒿琥酯三个浓度和TRAIL两个浓度配对分组,另设阴性对照组共7组,药物处理48小时后流式细胞术进行细胞周期分析。(3)按以上分组,药物处理48小时后运用Annexin V-FITC/PI双染色法在流式细胞仪上检测细胞凋亡率。(4)按以上分组,药物处理48小时后,ELISA法检测PC-3细胞培养液上清中PSA的浓度。
结果: (1)MTT结果显示,青蒿琥酯和TRAIL联合应用能够有效抑制前列腺癌PC-3细胞增殖,经过析因方差分析,增殖抑制率的差异与青蒿琥酯和TRAIL均有较好的剂量依赖关系。(2)流式细胞仪细胞周期分析结果显示,G2/M期细胞比例随两种药物浓度的增高而增高,差异有统计学意义,说明联合运用青蒿琥酯和TRAIL能够有效的将PC-3细胞阻滞在G2/M期,但药物处理后G0/G1和S期的细胞比例的变化缺乏统计学意义。(3) Annexin V-FITC/PI双染色法在流式细胞议上检测细胞凋亡率发现,联合用药能有效诱导PC-3细胞凋亡,凋亡率随着两种药物浓度的增高而增高,统计学分析与两种药物均呈良好的剂量依赖关系。(4)ELISA法检测PC-3细胞培养液上清中的PSA浓度发现,联合用药能有效降低培养液上清中的PSA浓度,PSA随着两种药物浓度的增高而降低,统计学分析与两种药物均呈良好的剂量依赖关系。
结论:青蒿琥酯联合应用TRAIL,能够有效抑制人前列腺癌PC-3增殖,导致PC-3细胞G2/M期周期阻滞,有效诱导PC-3细胞凋亡,降低PC-3细胞培养液上清PSA等,且两种药物均呈良好的剂量依赖关系,联合用药作用强于单独用药,两者具有协同作用。中药青蒿琥酯联合应用TRAIL有望成为临床治疗前列腺癌新的治疗方案。
Objectives: To investigate the anti-proliferation, cell cycle arrest, induceing apoptosis and down regulate PSA effects and mechanism of Artesunate with TNF related apoptosis inducing ligand (TRAIL) on prostate carcinoma cell lines PC-3 in vitro, which may provide some clues to clinical application.
Methods: (1)According to references and the results we studied before, we combinate four concentrations of Artesunate with three concentrations of TRAIL on human prostate carcinoma cell lines PC-3, the inhibition ratio of proliferation were investigated by MTT assay. (2)According to the results of MTT, three concentrations of Artesunate were conbimated with two concentrations of TRAIL on PC-3 cells, after treated 48hours, cell cycle were analyzed by flow cytometry(FCM) assay. (3)Three concentrations of Artesunate were conbimated with two concentrations of TRAIL on PC-3 cells, after treated 48hours, cell apoptosis ratio were investigated on flow cytometer by Annexin V-FITC/PI double staining method. (4)Three concentrations of Artesunate were conbimated with two concentrations of TRAIL on PC-3 cells, after treated 48hours, PSA of cell culture fluid were detected by ELISA method.
Results: (1)MTT results showed that combination of Artesunate with TRAIL can effectively inhibit proliferation of human prostate carcinoma cell lines PC-3. Factorial analysis variance showed that Artesunate and TRAIL could inhibit proliferation of PC-3cells in dose dependent manner. (2)Cell cycle analysis results of FCM showed that after treated by combination of Artesunate with TRAIL 48hours, a concentration dependent increase of cells in G2/M phase could be detected, but no obvious change of cell count was found in G0 /G1 phase and S phase. (3)Cell apoptosis ratio were assayed on flow cytometer by Annexin V-FITC/PI double staining method, the results showed that combination of Artesunate with TRAIL can effectively induce PC-3 cells apoptosis, both of two has dose dependent manner. (4)ELISA results showed that a concentration dependent decrease of PSA of cell culture fluid were detected.
Conclusions: Combination of chinese medicine Artesunate with TRAIL can actively inhibit proliferation of prostate carcinoma cell lines PC-3, induce cell cycle arrest at G2/M, effectively induce cell apoptosis of PC-3, and decrease the PSA level of cell culture fluid. All of these effects have well does dependent manner with Artesunate and TRAIL, factorial analysis variance showed that the function of simultaneous two agents was higher than only one agent applying. Chinese medicine Artesunate combinate with TRAIL will be a new therapeutic regimen in clinic therapy of prostate carcinoma.
方法: (1)根据文献资料和课题组前期研究基础,确定青蒿琥酯四个浓度和TRAIL三个浓度进行配对分组,另设单独使用青蒿琥酯组、单独使用TRAIL组和阴性对照组共15个组,药物处理对数生长期PC-3细胞48小时后,MTT法检测两种药物联合使用的增殖抑制率,进行统计学分析。(2)根据MTT结果,分别选取青蒿琥酯三个浓度和TRAIL两个浓度配对分组,另设阴性对照组共7组,药物处理48小时后流式细胞术进行细胞周期分析。(3)按以上分组,药物处理48小时后运用Annexin V-FITC/PI双染色法在流式细胞仪上检测细胞凋亡率。(4)按以上分组,药物处理48小时后,ELISA法检测PC-3细胞培养液上清中PSA的浓度。
结果: (1)MTT结果显示,青蒿琥酯和TRAIL联合应用能够有效抑制前列腺癌PC-3细胞增殖,经过析因方差分析,增殖抑制率的差异与青蒿琥酯和TRAIL均有较好的剂量依赖关系。(2)流式细胞仪细胞周期分析结果显示,G2/M期细胞比例随两种药物浓度的增高而增高,差异有统计学意义,说明联合运用青蒿琥酯和TRAIL能够有效的将PC-3细胞阻滞在G2/M期,但药物处理后G0/G1和S期的细胞比例的变化缺乏统计学意义。(3) Annexin V-FITC/PI双染色法在流式细胞议上检测细胞凋亡率发现,联合用药能有效诱导PC-3细胞凋亡,凋亡率随着两种药物浓度的增高而增高,统计学分析与两种药物均呈良好的剂量依赖关系。(4)ELISA法检测PC-3细胞培养液上清中的PSA浓度发现,联合用药能有效降低培养液上清中的PSA浓度,PSA随着两种药物浓度的增高而降低,统计学分析与两种药物均呈良好的剂量依赖关系。
结论:青蒿琥酯联合应用TRAIL,能够有效抑制人前列腺癌PC-3增殖,导致PC-3细胞G2/M期周期阻滞,有效诱导PC-3细胞凋亡,降低PC-3细胞培养液上清PSA等,且两种药物均呈良好的剂量依赖关系,联合用药作用强于单独用药,两者具有协同作用。中药青蒿琥酯联合应用TRAIL有望成为临床治疗前列腺癌新的治疗方案。
Objectives: To investigate the anti-proliferation, cell cycle arrest, induceing apoptosis and down regulate PSA effects and mechanism of Artesunate with TNF related apoptosis inducing ligand (TRAIL) on prostate carcinoma cell lines PC-3 in vitro, which may provide some clues to clinical application.
Methods: (1)According to references and the results we studied before, we combinate four concentrations of Artesunate with three concentrations of TRAIL on human prostate carcinoma cell lines PC-3, the inhibition ratio of proliferation were investigated by MTT assay. (2)According to the results of MTT, three concentrations of Artesunate were conbimated with two concentrations of TRAIL on PC-3 cells, after treated 48hours, cell cycle were analyzed by flow cytometry(FCM) assay. (3)Three concentrations of Artesunate were conbimated with two concentrations of TRAIL on PC-3 cells, after treated 48hours, cell apoptosis ratio were investigated on flow cytometer by Annexin V-FITC/PI double staining method. (4)Three concentrations of Artesunate were conbimated with two concentrations of TRAIL on PC-3 cells, after treated 48hours, PSA of cell culture fluid were detected by ELISA method.
Results: (1)MTT results showed that combination of Artesunate with TRAIL can effectively inhibit proliferation of human prostate carcinoma cell lines PC-3. Factorial analysis variance showed that Artesunate and TRAIL could inhibit proliferation of PC-3cells in dose dependent manner. (2)Cell cycle analysis results of FCM showed that after treated by combination of Artesunate with TRAIL 48hours, a concentration dependent increase of cells in G2/M phase could be detected, but no obvious change of cell count was found in G0 /G1 phase and S phase. (3)Cell apoptosis ratio were assayed on flow cytometer by Annexin V-FITC/PI double staining method, the results showed that combination of Artesunate with TRAIL can effectively induce PC-3 cells apoptosis, both of two has dose dependent manner. (4)ELISA results showed that a concentration dependent decrease of PSA of cell culture fluid were detected.
Conclusions: Combination of chinese medicine Artesunate with TRAIL can actively inhibit proliferation of prostate carcinoma cell lines PC-3, induce cell cycle arrest at G2/M, effectively induce cell apoptosis of PC-3, and decrease the PSA level of cell culture fluid. All of these effects have well does dependent manner with Artesunate and TRAIL, factorial analysis variance showed that the function of simultaneous two agents was higher than only one agent applying. Chinese medicine Artesunate combinate with TRAIL will be a new therapeutic regimen in clinic therapy of prostate carcinoma.
引文
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[23]Munshi A., TJ McDonnell, RE Meyn. Chemotherapeutic agents enhance TRAIL-induced apoptosis in prostate cancer cells[J]. Cancer Chemother Pharmacol,2002,50(1):46-52.
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[26]Kelly MM, BD Hoel, C Voelkel-Johnson. Doxorubicin pretreatment sensitizes prostate cancer cell lines to TRAIL induced apoptosis which correlates with the loss of c-FLIP expression[J].Cancer Biol Ther, 2002,1(5):520-527.
[27]Shanka S, X Chen, RK Srivastava. Effects of sequential treatments with chemotherapeutic drugs followed by TRAIL on prostate cancer in vitro and in vivo[J].Prostate, 2005,62(2):165-186.
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