自噬抑制剂硫酸羟氯喹对去势抵抗性前列腺癌化疗敏感性的影响
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摘要
目的探讨自噬抑制剂硫酸羟氯喹(HCQ)干预自噬对去势抵抗性前列腺癌22RV1细胞株体内外化疗药多西他赛(DOC)敏感性的影响,及其自噬基因Bcelin-1、自噬特异性底物P62、促凋亡基因Bax mRNA的表达变化与自噬蛋白Bcelin-1、自噬特异性标记物LC3B、促凋亡蛋白Bax表达影响。方法体外培养22RV1细胞株,分别设置空白对照组(不加药物)、DOC组、HCQ(20μmol/L)+DOC组3个组,后两组DOC浓度均为10~(-6) mol/L、10~(-7) mol/L、10~(-8) mol/L,分组培养72 h后CCK-8法检测细胞增殖。于裸鼠皮下一次性注射22RV1细胞悬液,建立22RV1细胞株裸鼠移植瘤,造模成功后随机分为模型组(生理盐水)、DOC组、HCQ+DOC组3个组,每组5只,均为腹腔注射,干预4周。观察移植瘤生长体积的变化。应用实时荧光定量PCR检测22RV1细胞株和移植瘤中自噬和凋亡相关基因(Beclin-1、P62、Bax)以及Western blot检测自噬和凋亡相关蛋白(Beclin-1、LC3B、Bax)的表达水平。结果体外实验中,与空白对照组相比,DOC组和HCQ+DOC组细胞的增殖能力减弱(P<0.05);在同一DOC浓度下,与DOC组相比,HCQ+DOC组细胞的增殖被抑制(P<0.05);HCQ+DOC组DOC的半数抑制浓度较DOC组低。体内实验中,与模型组相比,各时点DOC组及HCQ+DOC组的移植瘤周体积增长值均减小;HCQ+DOC组的同时点周体积增长值均小于DOC组(P<0.05),以第4周最为明显。在体内、外实验中,与其余两组比较,HCQ+DOC组Beclin-1、P62、Bax mRNA和Beclin-1、LC3B、Bax蛋白的表达均出现上调(P<0.05)。结论 HCQ可干预去势抵抗性前列腺癌细胞的自噬,抑制其增殖,增强其对化疗药物的敏感性。
Objective To determine the effects of autophagy inhibitor hydroxychloroquine(HCQ) on chemosensitivity of castration-resistant prostate cancer 22RV1 cell line in vitro and in vivo, and changes in its mRNA expressions of autophagy gene Bcelin-1, autophagy specific substrate P62 gene, pro-apoptotic gene Bax. Methods 22RV1 cells were cultured in vitro and divided into blank control(no drug), DOC, and HCQ(20 μmol/L)+DOC groups. The concentration of DOC was set at 10~(-6) mol/L, 10~(-7) mol/L, and 10~(-8) mol/L in the tests.Cell proliferation activities were detected by CCK-8 method 72 h after drug treatments. The 22RV1 cell suspension was injected subcutaneously into the left axilla of nude mice to establish transplanted tumor. The successfully modeled mice were randomly divided into three groups(five each) treated by physiological saline, DOC and HCQ+DOC(injected intraperitoneally for 4 weeks), respectively. Changes in growth of the transplanted tumor were observed. The mRNA expressions of Beclin-1, P62, and Bax were detected by qPCR. The protein expressions of Beclin-1, LC3B, and Bax were detected by Western blot.Results In vitro: compared with the blank control, the DOC and HCQ+DOC groups showed decreased proliferation of cells(P<0.05); HCQ further lowered cell proliferation in the presence of DOC(P<0.05), resulting in reduced half maximal inhibitory concentration(IC50) of DOC. In vivo: Compared with the model mice, the DOC and HCQ+DOC groupshad decreased volume of transplanted tumor. HCQ slowed the weekly growth of tumor in the presence of DOC(P<0.05), most obvious at the 4th week. In vitro and in vivo, HCQ+DOC upregulated the mRNA and protein expressions of Beclin-1, P62 and Bax(P<0.05). Conclusion HCQ can interfere with the autophagy of castration-resistant prostate cancer cells, inhibiting its proliferation and enhancing its sensitivity to chemotherapeutic drugs.
Objective To determine the effects of autophagy inhibitor hydroxychloroquine(HCQ) on chemosensitivity of castration-resistant prostate cancer 22RV1 cell line in vitro and in vivo, and changes in its mRNA expressions of autophagy gene Bcelin-1, autophagy specific substrate P62 gene, pro-apoptotic gene Bax. Methods 22RV1 cells were cultured in vitro and divided into blank control(no drug), DOC, and HCQ(20 μmol/L)+DOC groups. The concentration of DOC was set at 10~(-6) mol/L, 10~(-7) mol/L, and 10~(-8) mol/L in the tests.Cell proliferation activities were detected by CCK-8 method 72 h after drug treatments. The 22RV1 cell suspension was injected subcutaneously into the left axilla of nude mice to establish transplanted tumor. The successfully modeled mice were randomly divided into three groups(five each) treated by physiological saline, DOC and HCQ+DOC(injected intraperitoneally for 4 weeks), respectively. Changes in growth of the transplanted tumor were observed. The mRNA expressions of Beclin-1, P62, and Bax were detected by qPCR. The protein expressions of Beclin-1, LC3B, and Bax were detected by Western blot.Results In vitro: compared with the blank control, the DOC and HCQ+DOC groups showed decreased proliferation of cells(P<0.05); HCQ further lowered cell proliferation in the presence of DOC(P<0.05), resulting in reduced half maximal inhibitory concentration(IC50) of DOC. In vivo: Compared with the model mice, the DOC and HCQ+DOC groupshad decreased volume of transplanted tumor. HCQ slowed the weekly growth of tumor in the presence of DOC(P<0.05), most obvious at the 4th week. In vitro and in vivo, HCQ+DOC upregulated the mRNA and protein expressions of Beclin-1, P62 and Bax(P<0.05). Conclusion HCQ can interfere with the autophagy of castration-resistant prostate cancer cells, inhibiting its proliferation and enhancing its sensitivity to chemotherapeutic drugs.
引文
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[2] KURDI A,CLEENEWERCK M,VANGESTEL C,et al.ATG4B inhibitors with a benzotropolone core structure block autophagy and augment efficiency of chemotherapy in mice.Biochem Pharmacol,2017(138):150-162[2018-12-17].https://doi.org/10.1016/j.bcp.2017.06.119.
[3] 唐礼功,谢森,潘铁军,等.低氧通过诱导自噬增强小鼠前列腺癌细胞的化疗抵抗作用.华中科技大学学报,2013,42(6):651-655.
[4] PICKARD RD,SPENCER BH,MCFARLAND AJ,et al.Paradoxical effects of the autophagy inhibitor 3-methyladenine on docetaxel-induced toxicity in PC-3 and LNCaP prostate cancer cells.Naunyn Schmiedergs Arch Pharmacol,2015,388(7):793-799.
[5] HUANG K,LIU D.Targeting non-canonical autophagy overcomes erlotinib resistance in tongue cancer.Tumor Biol,2016,37(7):9625-9633.
[6] 谢昆,李密杰,蒋成砚,等.自噬相关蛋白ATG5/BECLIN-1调控细胞自噬和凋亡的分子机理研究进展.中国人兽共患病学报,2018,34(3):272-275.
[7] 王钱虎.自噬相关蛋白Beclin1、LC3-Ⅱ、Atg5在前列腺腺癌组织中的表达及意义.浙江医学,2018,40(16):1807-1810.
[8] WANG Y,ZHU WG,ZHAO Y.Autophagy substrate SQSTM1/p62 regulates chromatin ubiquitination during the DNA damage response.Autophagy,2017,13(1):212-213.
[9] MOSCAT J,KARIN M,DIAZ-MECO MT.P62 in cancer:signaling adaptor beyond autophagy.Cell,2016,167(3):606-609.
[10] ZHANG M,ZHOU YF,GONG JY,et al.Expression of autophagy-related protein LC3B,p62,and cytoplasmic p53 in human retinoblastoma tissues.Eur Rev Med Pharmacol Sci,2016,20(15):3152-3160.
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