Effects of Dioscin on the Growth of Tamoxifen-resistant Breast Cancer Cells

doi:10.12114/j.issn.1007-9572.2019.00.270

Abstract

Abstract: Background　Tamoxifen is a main drug used for endocrine treatment of breast cancer.It is difficult to treat breast cancer resistant to tamoxifen，and new treatments are needed urgently.Diosgenin（Dio） can inhibit the growth of cancer to some extent.Studying the effect of Dio on the growth of tamoxifen-resistant（TAM-R） breast cancer cells can provide a reference for clinical treatment.Objective　To study the effects of Dio on the growth of TAM-R breast cancer cells.Methods　From January 2017 to June 2018，TAM-R breast cancer cells were cultured.Corresponding experiments were carried out on cell growth and apoptosis，autophagy and apoptotic markers，and anti-cancer proteins.The details are as follows：automated cell counters were used to count the number of TAM-R breast cancer cells in six groups（control，Dio 0.625 μg/ml，Dio 0.800 μg/ml，Dio 1.000 μg/ml，Dio 1.250 μg/ml and Dio 2.500 μg/ml） after being treated by different doses of Dio for five days，and to measure the apoptosis of TAM-R breast cancer cells in 6 groups（control，TAM 10-7 mol/L，Dio 1 μg/ml，Dio 2 μg/ml，TAM 10-7 mol/L+Dio 1 μg/ml，and TAM 10-7 mol/L+Dio 2 μg/ml） treated with different doses of TAM and Dio for three days.Immunoblotting was used to detect the effects of different doses of Dio on TAM-R autophagy markers LC3 and Beclin-1，and apoptotic markers Bax，Bim，pAMPK and p53 proteins in four groups（control，Dio 0.80 μg/ml，Dio 1.25 μg/ml，and Dio 2.50 μg/ml）．Autophagy of TAM-R breast cancer cells treated with Dio and its inhibitors and inducers in six groups（control，Rapamycin 20 nmol/L，Chloroquine 10 μmol/L，TAM 10-7 mol/L，Dio 1 μg/ml，and TAM 10-7 mol/L+Dio 1 μg/ml） was observed by autophagic fluorescence microscopy.Results　The number of TAM-R breast cancer cells differed significantly across different groups （P<0.05）．To be specific，the control group had much less TAM-R breast cancer cells than Dio groups（P<0.05）．The number of apoptotic TAM-R breast cancer cells varied obviously across different groups（P<0.05）．Specifically，the control group showed much less apoptotic TAM-R breast cancer cells than other five groups（P<0.05）．Moreover，TAM 10-7 mol/L+Dio 2 μg/ml group showed significantly less TAM-R breast cancer cells than Dio 2 μg/ml group（P<0.05）．The expressions of LC3，Beclin-1，Bax，Bim，pAMPK and p53 varied significantly across the groups （P<0.05）．Fluorescence microscopy showed that autophagic activity level of TAM 10-7 mol/L + Dio 1 μg/ml group was higher than that of Dio 1 μg/ml group and TAM 10-7 mol/L group.Conclusion　Dio could inhibit the growth of TAM-R breast cancer cells，and strengthen the growth inhibition and apoptosis enhancement effects of TAM on such cells.Moreover，both autophagy and apoptotic marker proteins were also influenced.

Key words: Breast neoplasms, Dioscin, Tamoxifen, Apoptosis, Autophagy

摘要： 背景　三苯氧胺（TAM）是乳腺癌内分泌治疗的主要药物，当前乳腺癌耐药TAM（TAM-R）细胞治疗困难，迫切需要探索新的治疗方法。薯蓣皂苷（Dio）对肿瘤有一定的抑制作用，设想研究Dio对乳腺癌TAM-R细胞生长的影响，期望为治疗提供参考。目的　研究Dio对乳腺癌TAM-R细胞生长的影响。方法　2017年1月—2018年6月，培养TAM-R细胞，分别针对细胞生长与凋亡，自噬、凋亡、代谢标志物以及自噬情况等多项指标进行相应实验。具体为细胞计数器测定六组（对照组、Dio 0.625 μg/ml组、Dio 0.800 μg/ml组、Dio 1.000 μg/ml组、Dio 1.250 μg/ml组、Dio 2.500 μg/ml组）不同剂量的Dio处理TAM-R细胞5 d细胞生长情况；测定六组（对照组、TAM 10-7 mol/L组、Dio 1 μg/ml组、Dio 2 μg/ml组、TAM 10-7 mol/L+Dio 1 μg/ml组、TAM 10-7 mol/L+Dio 2 μg/ml组）不同剂量TAM和Dio处理TAM-R细胞3 d细胞凋亡情况；Western blotting法检测四组（对照组、Dio 0.80 μg/ml组、Dio 1.25 μg/ml组、Dio 2.50 μg/ml组）不同剂量的Dio对TAM-R自噬标志物LC3、Beclin-1，凋亡标志物Bax、Bim以及pAMPK、p53的影响；采用荧光显微术观察六组（对照组、Rapamycin 20 nmol/L组、Chloroquine 10 μmol/L组、TAM 10-7 mol/L组、Dio 1 μg/ml组、TAM 10-7 mol/L+Dio 1 μg/ml组）Dio及其抑制剂、诱导剂处理的TAM-R细胞的自噬情况。结果　六组TAM-R细胞数量比较，差异有统计学意义（P<0.05）；其中各Dio浓度组TAM-R细胞数量低于对照组（P<0.05）。六组TAM-R细胞凋亡数量比较，差异有统计学意义（P<0.05）；其中TAM 10-7 mol/L组、Dio 1 μg/ml组、Dio 2 μg/ml组、TAM 10-7 mol/L+Dio 1 μg/ml组、TAM 10-7 mol/L+Dio 2 μg/ml组TAM-R细胞凋亡数量高于对照组，TAM 10-7 mol/L+Dio 2 μg/ml组TAM-R细胞凋亡数量高于Dio 2 μg/ml组（P<0.05）。各组LC3、Beclin-1、Bax、Bim、pAMPK、p53表达水平比较，差异均有统计学意义（P<0.05）。荧光显微术检测显示，TAM 10-7 mol/L+Dio 1 μg/ml组自噬活动高于Dio 1 μg/ml组和TAM 10-7 mol/L组。结论　Dio对TAM-R细胞生长有抑制作用，且能增强TAM对TAM-R细胞的生长抑制和凋亡作用，对自噬、凋亡标志物蛋白均有影响。

关键词: 乳腺肿瘤, 薯蓣皂苷, 三苯氧胺, 细胞凋亡, 自噬

CHEN Haiyan,DING Xiaoqing. Effects of Dioscin on the Growth of Tamoxifen-resistant Breast Cancer Cells　[J]. Chinese General Practice, 2019, 22(29): 3570-3575. DOI: 10.12114/j.issn.1007-9572.2019.00.270.
谌海燕,丁晓庆. 薯蓣皂苷对耐三苯氧胺乳腺癌细胞生长的影响研究[J]. 中国全科医学, 2019, 22(29): 3570-3575. DOI: 10.12114/j.issn.1007-9572.2019.00.270.

References

［1］郑莹，吴春晓，张敏璐.乳腺癌在中国的流行状况和疾病特征［J］．中国癌症杂志，2013，23（8）：561-569．DOI：10.3969/j.issn.1007-3969.2013.08.001.
ZHENG Y，WU C X，ZHANG M L.The epidemic and characteristics of female breast cancer in China［J］．China Oncology，2013，23（8）：561-569．DOI：10.3969/j.issn.1007-3969.2013.08.001.
［2］李军，黄梅，石新蕾.三七总皂苷联合三苯氧胺对人乳腺癌细胞的抑制作用及对PI3K-AKT-mTOR信号通路的影响［J］．安徽医药，2017，21（10）：1780-1784．DOI：10.3969/j.issn.1009-6469.2017.10.007.
LI J，HUANG M，SHI X L.The inhibitory effect of panax notoginseng saponins in combination with tamoxifen on human breast cancer cells and the effect on PI3K-AKT-mTOR signaling pathway［J］．Anhui Medical and Pharmaceutical Journal，2017，21（10）：1780-1784．DOI：10.3969/j.issn.1009-6469.2017.10.007.
［3］陆清，薛晓红.“乳癌术后方”拮抗乳腺癌三苯氧胺治疗耐药的体外实验研究［J］．江苏中医药，2018，50（1）：79-82．DOI：10.3969/j.issn.1672-397X.2018.01.031.
LU Q，XUE X H.In vitro experimental study of “postoperative prescription for breast cancer” on anti-drug resistance to tamoxifen in breast cancer［J］．Jiangsu Journal of Traditional Chinese Medicine，2018，50（1）：79-82．DOI：10.3969/j.issn.1672-397X.2018.01.031.
［4］王淑荣，凌霜，张庆光，等．薯蓣皂苷现代药理学研究进展［J］．中国药理学通报，2017，33（2）：161-166．DOI：10.3969/j.issn.1001-1978.2017.02.004.
WANG S R，LING S，ZHANG Q G，et al．Advance on the pharmacological research of dioscin［J］．Chinese Pharmacological Bulletin，2017，33（2）：161-166．DOI：10.3969/j.issn.1001-1978.2017.02.004.
［5］王弥，李卫，郭亚春，等．薯蓣皂苷元抗肿瘤作用机制研究进展［J］．承德医学院学报，2017，34（6）：511-513.
WANG M，LI W，GUO Y C，et al．Research progress on anti-tumor mechanisms of diosgenin［J］．Journal of Chengde Medical College，2017，34（6）：511-513.
［6］邓运宗，孙向东，郭寒冰，等．薯蓣皂苷元对三阴乳腺癌细胞系HCC1937增殖及凋亡的影响［J］．中华实用诊断与治疗杂志，2017，31（7）：657-659．DOI：10.13507/j.issn.1674-3474.2017.07.009.
DENG Y Z，SUN X D，GUO H B，et al．Influence of diosgenin on the proliferation and apoptosis of triple-negative breast cancer cell line HCC1937［J］．Journal of Chinese Practical Diagnosis and Therapy，2017，31（7）：657-659．DOI：10.13507/j.issn.1674-3474.2017.07.009.
［7］方瑞，赵晶晶，盛飞凤，等．薯蓣皂苷激活p38MAPK/FOXO3a信号抑制三阴性乳腺癌细胞上皮-间质转化及侵袭［J］．中国细胞生物学学报，2018，40（5）：640-647．DOI：10.11844/cjcb.2018.05.0313.
FANG R，ZHAO J J，SHENG F F，et al．Dioscin inhibits EMT and invasion of triple negative breast cancer cells via activation of p38MAPK/FOXO3a signaling［J］．Chinese Journal of Cell Biology，2018，40（5）：640-647．DOI：10.11844/cjcb.2018.05.0313.
［8］阚月一，王娅杰，李琦，等．肿瘤发展过程中自噬与凋亡的相互作用［J］．中国比较医学杂志，2018，28（2）：106-112．DOI：10.3969.j.issn.1671-7856.2018.02.019.
KAN Y Y，WANG Y J，LI Q，et al．Interaction between autophagy and apoptosis during tumor development［J］．Chinese Journal of Comparative Medicine，2018，28（2）：106-112．DOI：10.3969.j.issn.1671-7856.2018.02.019.
［9］李程豪，骆亚莉，刘永琦，等．肿瘤微环境与中性粒细胞相互作用关系的研究进展［J］．中国肿瘤，2018，27（11）：847-850．DOI：10.11735/j.issn.1004-0242.2018.11.A006.
LI C H，LUO Y L，LIU Y Q，et al．Research progress on the interaction between tumor microenvironment and neutrophil［J］．China Cancer，2018，27（11）：847-850．DOI：10.11735/j.issn.1004-0242.2018.11.A006.
［10］梁炜，潘磊，付敏，等．LncRNA在肿瘤及微环境中的作用研究进展［J］．临床检验杂志，2016，34（5）：329-331．DOI：10.13602/j.cnki.jcls.2016.05.03.
LIANG W，PAN L，FU M，et al．Research progress on the role of LncRNA in tumors and microenvironment［J］．Chinese Journal of Clinical Laboratory Science，2016，34（5）：329-331．DOI：10.13602/j.cnki.jcls.2016.05.03.
［11］张紫薇，李滢，曹鹏，等．薯蓣皂苷的抗癌机制研究进展［J］．食品科学，2017，38（1）：297-302．DOI：10.7506/spkx1002-6630-201701049.
ZHANG Z W，LI Y，CAO P，et al．Progress in research on anticancer mechanism of dioscin［J］．Food Science，2017，38（1）：297-302．DOI：10.7506/spkx1002-6630-201701049.
［12］KIM E A，JANG J H，LEE Y H，et al．Dioscin induces caspase-independent apoptosis through activation of apoptosis-inducing factor in breast cancer cells［J］．Apoptosis，2014，19（7）：1165-1175.
［13］梁冠男，汤红平，张雁瑞，等．beclin-1、LC3及ki67在青年女性乳腺癌中的表达及临床意义［J］．临床医学，2017，37（4）：90-92．DOI：10.19528/j.issn.1003-3548.2017.04.042.
LIANG G N，TANG H P，ZHANG Y R，et al．Expression and clinical significance of beclin-1，LC3 and Ki67 in breast cancer of young women［J］．Clinical Medicine，2017，37（4）：90-92．DOI：10.19528/j.issn.1003-3548.2017.04.042.
［14］罗卓玛，胡越高，王璐红，等．薯蓣皂苷元抗肿瘤衍生物的合成及其生物活性的研究［J］．有机化学，2018，38（4）：919-925．DOI：10.6023/cjoc201711016.
LUO Z M，HU Y G，WANG L H，et al．Study on the synthesis and bioactivity of diosgenin antitumor derivatives［J］．Chinese Journal of Organic Chemistry，2018，38（4）：919-925．DOI：10.6023/cjoc201711016.
［15］蒲倩.自噬相关基因Beclin1在乳腺癌发生发展及治疗中的作用［J］．中国肿瘤生物治疗杂志，2018，25（1）：98-103．DOI：10.3872/j.issn.1007-385x.2018.01.018.
PU Q.Roles of autophagy associated gene Beclin1 in carcinogenesis，progression and therapy of breast cancer［J］．Chinese Journal of Cancer Biotherapy，2018，25（1）：98-103．DOI：10.3872/j.issn.1007-385x.2018.01.018.
［16］王晓荣.薯蓣皂苷元抑制人肝癌细胞株SMMC-7721增殖及其机制研究［D］．南京：南京中医药大学，2014.
［17］杨丽，李欣，田朗，等．柯萨奇病毒B3感染HeLa细胞后Bax和Bim的表达［J］．中国病理生理杂志，2014，30（3）：524-528．DOI：10.3969/j.issn.1000-4718.2014.03.024.
YANG L，LI X，TIAN L，et al．Expression of Bax and Bim in coxsackievirus B3-infected HeLa cells［J］．Chinese Journal of Pathophysiology，2014，30（3）：524-528．DOI：10.3969/j.issn.1000-4718.2014.03.024.
［18］UMEZAWA S，HIGURASHI T，NAKAJIMA A，et al．AMPK：therapeutic target for diabetes and cancer prevention［J］．Curr Pharm Des，2017，23（25）：3629-3644．DOI：10.2174/0929867324666170713150440.
［19］FOGARTY S，HARDIE D G.Development of protein kinase activators：AMPK as a target in metabolic disorders and cancer［J］．Biochim Biophys Acta，2010，1804（3）：581-591．DOI：10.1016/j.bbapap.2009.09.012.
［20］吴亮，王建明.乳腺癌相关基因异常甲基化与临床病理特征的关联研究［J］．医学研究杂志，2016，45（11）：82-86．DOI：10.11969/j.issn.1673-548X.2016.11.021.
WU L，WANG J M.Study on the correlation between abnormal DNA methylation and clinical features of breast cancer［J］．Journal of Medical Research，2016，45（11）：82-86．DOI：10.11969/j.issn.1673-548X.2016.11.021.
［21］王东辉，闻春艳.化疗前后乳腺癌患者血清中癌基因和抑癌基因表达水平研究［J］．中国妇幼保健，2016，31（16）：3232-3233．DOI：10.7620/zgfybj.j.issn.1001-4411.2016.16.14.
WANG D H，WEN C Y.Expression levels of serum cancer gene and tumor suppressor gene in patients with breast cancer before and after chemotherapy［J］．Maternal ＆ Child Health Care of China，2016，31（16）：3232-3233．DOI：10.7620/zgfybj.j.issn.1001-4411.2016.16.14.

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