粉防己碱等中药单体逆转乳腺癌MCF-7/TAM细胞耐药的自噬机制研究

Tamoxifen (TAM) is the main treatment of estrogen receptor-positive breast cancer, but long-term use can induce drug resistance. Our preliminary study found out three traditional Chinese medicine (TCM) monomers, tetrandrine, dioscin, artesunate can reverse tamoxifen resistance of breast cancer cells (MCF-7/TAM), but the mechanism of the reversal is unknown. Some study found out the drug resistance of MCF-7/TAM cells associated with autophagy, the mechanism of autophagy in TCM is the deficiency of body resistance and the integration of phlegm and stasis. The theoretical research of TCM micro-syndrome differentiation indicated that the tetrandrine has the effect of resolving phlegm, the dioscin has the effect of strengthening body resistance, and the artesunate has the effect of eliminating stasis. Based on the above study, we hypothesized that the three TCM monomers are by inhibiting the MCF-7/TAM cells autophagy to reverse the effect of drug resistance. Therefore, we are the first study focusing on the mechanism of TCM monomers reversing the drug resistance of MCF-7/TAM cells from the perspective of autophagy. We would firstly transfect Micro RNA101 to MCF-7/TAM cells to inhibit the autophagy and observe whether the resistance is reversed, then detect the formation of autophagosome, distribution and expression of LC3, and the expression levels of LC3-II/LC3-I, Beclin-1, p62 and mTOR to explore whether the TCM monomers reverse the drug resistance by inhibiting the autophagy of MCF-7/TAM cells, and compare the similarities and differences of three monomers’ targets. We expect this study could provide experimental evidences for screening efficient reversal agents and developing Chinese herbal compound.

三苯氧胺是雌激素受体阳性乳腺癌的主要治疗药物，但长期使用可产生耐药。我们前期研究发现粉防己碱、薯蓣皂苷、青蒿琥酯均可逆转耐三苯氧胺乳腺癌细胞（MCF-7/TAM）的耐药，但机制不明。研究发现MCF-7/TAM细胞耐药与自噬有关，自噬的中医机制为正虚、痰瘀互结。中医微观辨证认为粉防己碱、薯蓣皂苷、青蒿琥酯分别有化痰、扶正、化瘀作用。基于以上研究，我们推测以上中药单体通过抑制MCF-7/TAM自噬达到逆转耐药的作用。故本课题首次从自噬角度研究中药单体逆转MCF-7/TAM耐药的机制。首先转染miR-101至MCF-7/TAM抑制自噬，观察耐药是否被逆转。然后检测自噬小体的形成、LC3的分布和表达，LC3-II/LC3-I 、Beclin-1、p62和mTOR的表达水平，探索中药单体是否通过抑制自噬逆转MCF-7/TAM耐药，并比较三种单体作用靶点的异同。为筛选高效逆转剂及复方研发提供实验依据。

三苯氧胺（TAM）是雌激素受体阳性乳腺癌的主要治疗药物，但长期使用易产生耐药性，严重限制了临床疗效。我们前期研究发现粉防己碱（Tet）、薯蓣皂苷（Dio）、青蒿琥酯（Art）三种中药单体均可逆转耐三苯氧胺乳腺癌细胞（MCF-7/TAM）的耐药性，但是逆转耐药机制不明。研究发现MCF-7/TAM细胞耐药与自噬有关，自噬的中医机制为正虚、痰瘀互结。中医微观辨证理论研究发现粉防己碱有化痰作用，薯蓣皂苷有扶正作用，青蒿琥酯有化瘀作用。基于以上研究，我们推测上述三种中药单体是通过抑制MCF-7/TAM细胞自噬达到逆转耐药的作用。故本课题拟从自噬角度研究中药单体逆转MCF-7/TAM细胞耐药的机制。研究结果如下：1、MCF-7/TAM细胞与MCF-7细胞相比，自噬相关蛋白LC3、Beclin-1增加，Micro RNA101减少；凋亡相关蛋白Survivin增加，Bax减少；证实了MCF-7/TAM细胞耐药机制与自噬增强有关，同时提示可能还由凋亡减弱导致。2、Art对MCF-7/TAM细胞自噬 影响不大，主要直接引起凋亡。能轻度恢复MCF-7/TAM对tamoxifen 敏感性。 3、Dio、Tet单独作用对MCF-7/TAM细胞生长均有抑制作用，联合TAM能增强TAM对MCF-7/TAM细胞的生长抑制和凋亡作用，且能抑制自噬、增加凋亡蛋白的表达，从而起到增效，逆转耐药作用。故本实验结果表明中药单体粉防己碱、薯蓣皂苷逆转细胞耐药与抑制自噬及增强凋亡有关。为今后从中药单体及复方制剂中研发出高效的耐三苯氧胺乳腺癌的逆转剂提供前期基础。