健脾利湿化瘀方对人前列腺癌C4-2细胞雄激素非依赖性生长的影响
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摘要
目的探究健脾利湿化瘀方治疗去势抵抗性前列腺癌的可能作用机制。方法将健脾利湿化瘀方根据药物功效拆分为扶正药、解毒药、化瘀药。实验分为全方组、扶正组、解毒组、化瘀组,每组设4个药物浓度,分别为10、20、100、1000 mg/ml,另设对照组(含培养液和细胞)。在96孔板加入人前列腺癌C4-2细胞悬液100μl (细胞数约1. 5×10~4个),培养24 h后根据实验分组加入不同浓度药物继续培养48 h。采用MTT实验计算各组细胞抑制率。根据结果再将全方和细胞抑制率最高的拆方组中单味药物作用于C4-2细胞,筛选细胞抑制率最高的单味药物。观察健脾利湿化瘀方全方和最佳单味药物分别对C4-2细胞在含与不含雄激素的培养基的细胞增殖情况,计算细胞抑制率。结果各组药物对人前列腺癌C4-2细胞的抑制率呈浓度依赖关系(P <0. 05),药物浓度越高,抑制率越高,在不同浓度药物干预下组间比较,抑制率由高到低分别为全方组>化瘀组>解毒组>扶正组,组间两两比较差异有统计学意义(P <0. 05),拆方中化瘀组是最佳药物组。在最佳单味药物细胞抑制率筛选中,细胞抑制率由高到低依次为王不留行组>全方组>大黄组>姜黄组(P <0. 05),提示王不留行是抑制人前列腺癌C4-2细胞增殖作用最佳的中药单药。全方组不含雄激素细胞抑制率与含雄激素差异无统计学意义(P> 0. 05),而王不留行组含雄激素细胞抑制率低于不含雄激素(P <0. 05),并且王不留行组含和不含雄激素细胞抑制率均高于相应的全方组(P <0. 05)。结论健脾利湿化瘀方对人前列腺癌C4-2细胞具有很好的抑制作用,且呈剂量依赖性。其中王不留行是最佳的单味中药,王不留行增加了人前列腺癌C4-2细胞对雄激素的敏感性,可能对人前列腺癌C4-2细胞的雄激素非依赖性生长具有逆转作用。
Objective To investigate the possible mechanism of Jianpi Lishi Huayu Recipe(健脾利湿化瘀方) in the treatment of castration-resistant prostate cancer. Methods The Jianpi Lishi Huayu Recipe was divided into reinforcing healthy qi medicine,detoxification medicine and dissipating blood stasis medicine according to the actions of the drug. The experiment was divided into a whole formula group,a reinforcing healthy qi group,a detoxification group and a dissipating blood stasis group. Each group was given 4 drug concentrations,10,20,100,1000 mg/ml,and a control group containing culture medium and cells was set up. Human prostate cancer C4-2 cell suspension100 μl(1. 5 × 10~4 cells) was added to a 96-well plate. After 24 hours of culture,cells were continued to culture for 48 h by adding different concentrations drugs according to the experimental grouping. The inhibition rate of each group was calculated by MTT assay. According to the results,the single drug in the whole formula group and the single drug in the split formula group with the highest total inhibition rate were applied to C4-2 cells,and the single drug with the highest cell inhibition rate was selected. The cell proliferation of Jianpi Lishi Huayu Recipe whole formula and best single drug on C4-2 cells in androgen-containing and androgen-free medium were observed,and the cell inhibition rate was calculated. Results The inhibition rate of human prostate cancer C4-2 cells in each group was concentration-dependent(P < 0. 05). The higher the drug concentration was,the higher the inhibition rate was. Under different concentrations of drug intervention,the inhibition rate from high to low was listed as whole formula group,dissipating blood stasis group,detoxification group,reinforcing healthy qi group. The difference between the every 2 groups was statistically significant(P < 0. 05). In the split formula,the dissipating blood stasis group was the best drug group. In the best single-drug screening,the cell inhibition rate from high to low was Semen Vaccariae group,the whole formula group,Radix et Rhizoma Rhei group,Rhizoma Curcumae Longae group(P < 0. 05),suggesting that Semen Vaccariae was the best Chinese medicine single drug inhibitor of human prostate cancer C4-2 cell proliferation. There was no significant difference in the inhibition rate between androgen-containing and androgen-free in the whole formula group(P > 0. 05),while the inhibition rate of androgen-containing in Semen Vaccariae group was lower than that of androgen-free(P < 0. 05). The inhibition rate of androgen-containing and androgen-free in Semen Vaccariae group was higher than that in the corresponding whole formula group(P < 0. 05). Conclusion Jianpi Lishi Huayu Recipe has a good inhibitory effect on human prostate cancer C4-2 cells in a dose-dependent manner. Among them,Semen Vaccariae is the best single-flavor Chinese medicine. Semen Vaccariae has increased the sensitivity of human prostate cancer C4-2 cells to androgen,which may reverse the androgen-independent growth of human prostate cancer C4-2 cells.
Objective To investigate the possible mechanism of Jianpi Lishi Huayu Recipe(健脾利湿化瘀方) in the treatment of castration-resistant prostate cancer. Methods The Jianpi Lishi Huayu Recipe was divided into reinforcing healthy qi medicine,detoxification medicine and dissipating blood stasis medicine according to the actions of the drug. The experiment was divided into a whole formula group,a reinforcing healthy qi group,a detoxification group and a dissipating blood stasis group. Each group was given 4 drug concentrations,10,20,100,1000 mg/ml,and a control group containing culture medium and cells was set up. Human prostate cancer C4-2 cell suspension100 μl(1. 5 × 10~4 cells) was added to a 96-well plate. After 24 hours of culture,cells were continued to culture for 48 h by adding different concentrations drugs according to the experimental grouping. The inhibition rate of each group was calculated by MTT assay. According to the results,the single drug in the whole formula group and the single drug in the split formula group with the highest total inhibition rate were applied to C4-2 cells,and the single drug with the highest cell inhibition rate was selected. The cell proliferation of Jianpi Lishi Huayu Recipe whole formula and best single drug on C4-2 cells in androgen-containing and androgen-free medium were observed,and the cell inhibition rate was calculated. Results The inhibition rate of human prostate cancer C4-2 cells in each group was concentration-dependent(P < 0. 05). The higher the drug concentration was,the higher the inhibition rate was. Under different concentrations of drug intervention,the inhibition rate from high to low was listed as whole formula group,dissipating blood stasis group,detoxification group,reinforcing healthy qi group. The difference between the every 2 groups was statistically significant(P < 0. 05). In the split formula,the dissipating blood stasis group was the best drug group. In the best single-drug screening,the cell inhibition rate from high to low was Semen Vaccariae group,the whole formula group,Radix et Rhizoma Rhei group,Rhizoma Curcumae Longae group(P < 0. 05),suggesting that Semen Vaccariae was the best Chinese medicine single drug inhibitor of human prostate cancer C4-2 cell proliferation. There was no significant difference in the inhibition rate between androgen-containing and androgen-free in the whole formula group(P > 0. 05),while the inhibition rate of androgen-containing in Semen Vaccariae group was lower than that of androgen-free(P < 0. 05). The inhibition rate of androgen-containing and androgen-free in Semen Vaccariae group was higher than that in the corresponding whole formula group(P < 0. 05). Conclusion Jianpi Lishi Huayu Recipe has a good inhibitory effect on human prostate cancer C4-2 cells in a dose-dependent manner. Among them,Semen Vaccariae is the best single-flavor Chinese medicine. Semen Vaccariae has increased the sensitivity of human prostate cancer C4-2 cells to androgen,which may reverse the androgen-independent growth of human prostate cancer C4-2 cells.
引文
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[11]涂玥,孙伟,顾刘宝,等.大黄酸调控m TOR信号通路活性抑制肾小管上皮细胞自噬蛋白表达的分子机制[J].中国中药杂志,2014,39(21):4090.
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[13]花慧,冯磊,金坚,等.王不留行中抑制血管生成的活性物质研究[J].时珍国医国药,2009,20(3):698-670.
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[15]MELLADO B,CODONY J,RIBAL MJ,et al.Molecular biology of androgen-independent prostate cancer:the role of the androgen receptor pathway[J].Clin Transl Oncol,2009,11(1):5-10.
[16]MONTGOMERY RB,MOSTAGHEL EA,VESSELLA R,et al.Maintenance of intratumoral androgens in metastatic prostate cancer:a mechanism for castration-resistant tumor growth[J].Cancer Res,2008,68(11):4447-4454.
[17]KOIVISTO P,KONONEN J,PALMBERG C,et al.Androgen receptor gene amplification:a possible molecular mechanism for androgen deprivation therapy failure in prostate cancer[J].Cancer Res,1997,57(2):314-319.
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[19]HAAPALA K,KUUKASJRVI T,HYYTINEN E,et al.Androgen receptor amplification is associated with increased cell proliferation in prostate cancer[J].Hum Pathol,2007,38(3):474-478.
[2]CHEN W,ZHENG R,BAADE PD.Cancer Statistics in China,2015[J].CA Cancer J Clin,2016,66(2):115-132.
[3]曾浩,种铁,贺大林,等.去势抵抗性前列腺癌最新指南解读:暨中国西部专家共识[J].现代泌尿外科杂志,2017,22(2):85-94.
[4]VAN SOEST RJ,VAN ROYEN ME,DE MORRE ES,et al.Cross-resi stance between zaxanes and new hormonal agents abiraterone and enzalutamide may affect drug sequence choices in metastatic castration-resistant prostate cancer[J].Eur J Cancer,2013,49(18):3821-3830.
[5]贾玉森,陈小均,张志杰.中医药治疗前列腺癌的临床研究概况[J].中医杂志,2012,53(24):2142-2146.
[6]贾英杰,陈军,李小江,等.前列腺癌中医证候研究的文献分析[J].辽宁中医杂志,2014,41(9):1850-1852.
[7]王金秀,李小江,陈军,等.贾英杰论前列腺癌的中医病机与治疗[J].新中医,2014,46(4):20-23.
[8]牟睿宇,李小江.“健脾利湿化瘀方”治疗晚期前列腺癌的研究[C]//中国中西医结合学会.第十五届全国中西医结合肿瘤学术大会论文集.北京:中国中西医结合学会,2017:142-143.
[9]孙彬栩,陈军.“健脾利湿化瘀方”对人前列腺癌PC-3细胞荷瘤小鼠的抑瘤作用研究[J].中华中医药学刊,2016,34(10):2468-2471.
[10]张博,叶丽红.姜黄素抗癌机制研究进展[J].中医药学报,2013,41(1):121-123.
[11]涂玥,孙伟,顾刘宝,等.大黄酸调控m TOR信号通路活性抑制肾小管上皮细胞自噬蛋白表达的分子机制[J].中国中药杂志,2014,39(21):4090.
[12]马丽萍,邱丽颖,冯磊,等.王不留行提取物抑制人微血管内皮细胞活性的研究[J].中成药,2013,35(11):2315-2319.
[13]花慧,冯磊,金坚,等.王不留行中抑制血管生成的活性物质研究[J].时珍国医国药,2009,20(3):698-670.
[14]王贵民,李延保.中医药治疗前列腺疾病用药规律的数据挖掘研究[J].西部中医药,2014,27(5):77-80.
[15]MELLADO B,CODONY J,RIBAL MJ,et al.Molecular biology of androgen-independent prostate cancer:the role of the androgen receptor pathway[J].Clin Transl Oncol,2009,11(1):5-10.
[16]MONTGOMERY RB,MOSTAGHEL EA,VESSELLA R,et al.Maintenance of intratumoral androgens in metastatic prostate cancer:a mechanism for castration-resistant tumor growth[J].Cancer Res,2008,68(11):4447-4454.
[17]KOIVISTO P,KONONEN J,PALMBERG C,et al.Androgen receptor gene amplification:a possible molecular mechanism for androgen deprivation therapy failure in prostate cancer[J].Cancer Res,1997,57(2):314-319.
[18]TAYLOR BS,SCHULTZ N,HIERONYMUS H,et al.Integrative genomic profiling of human prostate cancer[J].Cancer Cell,2010,18(1):11-22.
[19]HAAPALA K,KUUKASJRVI T,HYYTINEN E,et al.Androgen receptor amplification is associated with increased cell proliferation in prostate cancer[J].Hum Pathol,2007,38(3):474-478.