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.

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