姜黄素调控自噬在抑制HPV阳性宫颈癌细胞的体外研究
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摘要
目的研究姜黄素在体外应用于诱导HPV阳性宫颈癌细胞凋亡与自噬的影响。方法采用MTT检测法和流式细胞仪检测姜黄素(10,20,30,40μmol/L)对HPV阳性宫颈癌SiHa细胞增殖和凋亡的影响;采用自噬抑制剂3-MA与姜黄素联用,观察自噬对姜黄素抑制HPV阳性宫颈癌细胞增殖和诱导细胞凋亡的影响;分光光度法检测SiHa细胞Caspase-3活性;应用ELISA检测姜黄素和(或)自噬抑制剂3-MA对宫颈癌SiHa细胞内Bcl-2、Bax和Beclin-1表达的影响。结果姜黄素对HPV阳性的宫颈癌SiHa细胞的生长具有抑制作用,而且这种抑制作用呈浓度依赖性和时间依赖性;而姜黄素与3-MA联用后,对HPV阳性宫颈癌SiHa细胞诱导凋亡的作用明显下降;与对照组相比,姜黄素组细胞Caspase-3活性明显提高(P<0.01)、Bax和Beclin-1的表达均明显升高(P<0.01),而Bcl-2的表达明显降低(P<0.01);与单独应用姜黄素组相比,姜黄素与3-MA联用组细胞Caspase-3活性和Bax表达均明显下降(P<0.01),而Bcl-2的表达明显增加(P<0.01)。结论姜黄素能有效抑制HPV阳性宫颈癌SiHa细胞的增殖,可以提高细胞Caspase-3活性、促进促凋亡因子Bax的表达升高和抑制凋亡因子Bcl-2表达下调,同时促进自噬相关蛋白Beclin 1的表达上调,从而诱导HPV阳性宫颈癌SiHa细胞发生凋亡和自噬性细胞死亡。
Objective To study the effect of curcumin on the apoptosis and autophagy of HPV-positive cervical cancer cells in vitro.Methods The effects of curcumin(10,20,30,40 μmol/L)on the proliferation and apoptosis of HPV-positive cervical cancer SiHa cells were detected by MTT assay and flow cytometry;autophagy inhibitor 3-MA was used in combination with curcumin to observe the effect of autophagy on curcumin inhibition of HPV positive cervical cancer cell proliferation and induction of apoptosis;the expression of Caspase-3 in SiHa cells was detected by spectrophotometry,and the effects of curcumin and/or autophagy inhibitors(3-MA)on the expression of Bcl-2 and Bax in cervical cancer SiHa cells were detected by ELISA.Results Curcumin inhibited the growth of HPV-positive cervical cancer SiHa cells,and the inhibitory effect was in a dose-dependent and time-dependent manner.After curcumin combined with 3-MA,the effect of curcumin on the HPV-positive cervical cancer SiHa The effect of cell apoptosis was significantly decreased.Compared with the control group,the expression of Caspase-3,Bax and Beclin-1 in the curcumin group was significantly higher than that in the control group(P<0.01),while the expression of Bcl-2 was significantly decreased(P<0.01)(P<0.01),and the expression of Bcl-2 was significantly higher in the combination of curcumin and 3-MA group(P<0.01),and the expression of Caspase-3,Bax and Beclin-1 increased(P<0.01).Conclusion Curcumin can effectively inhibit the proliferation of HPV-positive cervical cancer SiHa cells,can promote the expression of pro-apoptotic factors Bax,Caspase-3 and inhibit the down-regulation of apoptosis factor Bcl-2 expression,while promoting the expression of autophagy-related protein Beclin 1 Upregulation,which induced HPV-positive cervical cancer SiHa cells apoptosis and autophagic cell death.
Objective To study the effect of curcumin on the apoptosis and autophagy of HPV-positive cervical cancer cells in vitro.Methods The effects of curcumin(10,20,30,40 μmol/L)on the proliferation and apoptosis of HPV-positive cervical cancer SiHa cells were detected by MTT assay and flow cytometry;autophagy inhibitor 3-MA was used in combination with curcumin to observe the effect of autophagy on curcumin inhibition of HPV positive cervical cancer cell proliferation and induction of apoptosis;the expression of Caspase-3 in SiHa cells was detected by spectrophotometry,and the effects of curcumin and/or autophagy inhibitors(3-MA)on the expression of Bcl-2 and Bax in cervical cancer SiHa cells were detected by ELISA.Results Curcumin inhibited the growth of HPV-positive cervical cancer SiHa cells,and the inhibitory effect was in a dose-dependent and time-dependent manner.After curcumin combined with 3-MA,the effect of curcumin on the HPV-positive cervical cancer SiHa The effect of cell apoptosis was significantly decreased.Compared with the control group,the expression of Caspase-3,Bax and Beclin-1 in the curcumin group was significantly higher than that in the control group(P<0.01),while the expression of Bcl-2 was significantly decreased(P<0.01)(P<0.01),and the expression of Bcl-2 was significantly higher in the combination of curcumin and 3-MA group(P<0.01),and the expression of Caspase-3,Bax and Beclin-1 increased(P<0.01).Conclusion Curcumin can effectively inhibit the proliferation of HPV-positive cervical cancer SiHa cells,can promote the expression of pro-apoptotic factors Bax,Caspase-3 and inhibit the down-regulation of apoptosis factor Bcl-2 expression,while promoting the expression of autophagy-related protein Beclin 1 Upregulation,which induced HPV-positive cervical cancer SiHa cells apoptosis and autophagic cell death.
引文
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[2]Munger K,Baldwin A,Edwards KM,et al.Mechanisms of human papillomavirus-induced oncogenesis[J].J Virol,2004,78(21):11451.
[3]曾新,韩一栩,吴丽香.HPV在宫颈炎、宫颈癌前病变、宫颈癌中的检测意义分析[J].中国实验诊断学,2014,18(01):127.
[4]Lim CB,Ky N,Ng HM,et al.Curcuma wenyujin extract induces apoptosis and inhibits proliferation of human cervical cancer cells in vitro and in vivo[J].Integr Cancer Ther,2010,9(1):36.
[5]Singh AK,Misra K.Human papilloma virus 16e6protein as a target for curcuminoids,curcumin conjugates and congeners for chemoprevention of oral and cervical cancers[J].Interdiscip Sci,2013,5(2):112.
[6]Paulraj F,Abas F,Lajis NH,et al.The curcumin analogue 1,5-bis(2-hydroxyphenyl)21,4-pentadiene-3-one induces apoptosis and downregulates E6and E7oncogene expression in HPV16and HPV18-infected cervical cancer cells[J].Molecules,2015,20(7),11830.
[7]Sahebkar A,Cicero AF,Simental-Mendia LE,et al.Curcumin downregulates human tumor necrosis factoralpha levels:a systematic review and meta-analysis ofrandomized controlled trials[J].Pharmacol Res,2016,107,234.
[8]狄建彬,顾振纶,赵笑东,等.姜黄素的抗氧化和抗炎作用研究进展[J].中草药,2010,41(05):854.
[9]Panahi Y,Hosseini MS,Khalili N,et al.Antioxidant and anti-inflammatory effects of curcuminoid-piperine combination in subjects with metabolic syndrome:A randomized controlled trial and an updated meta-analysis[J].Clin Nutr,2015,34(6):1101.
[10]Panahi Y,Ghanei M,Hajhashemi A,et al.Effects of curcuminoids-piperine combination on systemic oxidative stress,clinical symptoms and quality of life in subjects with chronic pulmonary complications due to sulfur mustard:a randomized controlled trial[J].J Diet Suppl,2016,13(1):93.
[11]孙永,彭明利.姜黄素及其衍生物在肝脏相关疾病中防治作用的研究进展[J].药学学报,2014,49(11):1483.
[12]Panahi Y,Rahimnia AR,Sharafi M,et al.Curcuminoid treatment for knee osteoarthritis:a randomized double-blind placebocontrolled trial[J].Phytother Res,2014,28(11):1625.
[13]Momtazi,A A,Shahabipour F,Khatibi S,et al.Curcumin as a MicroRNA regulator in cancer:a review[J].Rev Physiol Biochem Pharmacol,2016,171:1.
[14]Deretic V,Saitoh T,Akira S.Autophagy in infection,inflammation and immunity[J].Nat Rev Immunol,2013,13(10):722.
[15]Janku F,McConkey DJ,Hong DS,et al.Autophagy as a target for anticancer therapy[J].Nat Rev Clin Oncol.2011,8(9):528.
[16]Miracco C,Cevenini G,Franchi A,et al.Beclin 1and LC3autophagic gene expression in cutaneous melanocytic lesions[J].Hum Pathol 41(4):503,2010.
[17]高小胜,吕应年,吴科锋,等.半边旗提取物5F诱发结直肠癌细胞生长抑制及细胞凋亡[J].药学研究,2016,35(04):193,211.
[18]Stankov MV,El KM,Kumar TB,et al.Histone deacetylase inhibitors induce apoptosis in myeloid leukemia by suppressing autophagy[J].Leukemia,2014,28(3):577.