柴胡皂苷D对肿瘤化疗耐药细胞的肿瘤干细胞特征影响及机制研究

In addition to possessing characteristic of cross drug resistance, acquired chemoresistant cancer cells also possess aberrant metastatic ability. Currently, investigations of combating acquired chemoresistance are mostly focused on either drug resistance or metastasis, instead of both on drug resistance and metastasis. It has been shown that cancer stem cells possess chemoresistant property and aberrant metastatic ability. On the other hand, our published works together with reports from other laboratories demonstrate that acquired chemoresistant cancer cells possess properties of cancer stem cells, and hedgehog (Hh) signaling pathway is essential for maintaining these properties of acquired chemoresistant cancer cells. These reports suggest that disrupting the cancer stem cell-like properties of acquired chemoresistant cancer cells may simultaneously circumvent the drug resistance and suppress the metastasis ability of acquired chemoresistant cancer cells. Our pilot studies for this proposal indicate that the saikosaponin D， the most effective component of Radix bupleuri, a famous drug from Traditional Chinese Medicine, may disrupt many cancer stem cell-like properties of acquired chemoresistant cancer cells K562/A02 and KB/VCR, reverse the resistance of KB/VCR to VCR, and inhibit its migration ability. Meanwhile, we observed that saikosaponin D may inhibit the Gli luciferase activity provoked by shh, suggesting that saikosaponin D may suppress the Hh pathway acitivity. Hence, this proposal is aimed to elucidate: 1). whether saikosaponin D may disrupt the cancer stem cell-like properties of acquired chemoresistant cancer cells; 2). whether saikosaponin D may simultaneously circumvent the drug resistance and suppress the metastatic ability of acquired chemoresistant cancer cells; and 3). determining the effect of saikosaponin D on the Hh pathway and mapping its molecular target. This study will provide a bench foundation for using saikosaponin D to combat acquired chemoresistance including both drug resistance and metastasis ability.

获得性耐药肿瘤细胞除了具有交叉耐药点的特征外，还具有极强的转移能力。目前针对获得性耐药的研究多仅聚焦于耐药或转移的一方面，同时干预耐药和转移的研究尚不多见。研究发现肿瘤干细胞具有耐药及高转移的特性。我们已发表的工作和其他实验室的工作发现获得性耐药肿瘤细胞具有肿瘤干细胞特征，而Hedgehog（Hh）信号通路的活化对维持其干细胞特征具有重要作用。这些研究提示通过干预获得性耐药肿瘤细胞的肿瘤干细胞特征或许能同时逆转耐药及抑制其转移能力。我们前期工作发现具有和解表里、升阳举陷功效的中药柴胡的有效成分柴胡皂苷D能干扰耐药细胞的部分肿瘤干细胞特征，逆转耐药细胞KB/VCR的耐药及降低其迁移能力，并抑制Hh信号通路转录因子Gli的转录活性。本项目拟在此基础上深入探讨柴胡皂苷D能否通过干扰获得性耐药肿瘤细胞的肿瘤干细胞特征，从而逆转其耐药及抑制其转移能力，并从Hh信号通路角度阐明其作用机制。

Hedgehog信号通路异常活化与肿瘤及脏器纤维化有关，寻找Hh信号通路抑制剂具有广泛的应用前景。柴胡是传统药用植物，本项目系统研究了三种柴胡皂苷B1、B2和D对Hedgehog信号通路的抑制作用、作用机制及相关用途。结果显示，三种皂苷均可以剂量依赖性抑制Hh信号通路关键分子Gli1的转录活性，IC50分别为539.2nM，490nM及156.8nM，并下调Gli下游靶基因Gli1和Ptch1的基因表达；三种皂苷对于TNF-α/ NF-κB和PGE2 / TCF / LEF信号通路活性无明显抑制作用，提示其对Hh信号通路的抑制作用具有选择性。进一步研究发现，三种皂苷对于Smo突变体、敲减通路抑制分子Sufu以及Gli2ΔN异常激活的Hh通路无抑制作用，揭示了其分子靶点位于膜受体Smo，是三种Smo的靶向拮抗剂。应用研究，选取Hh通路异常激活的髓母细胞瘤进行体内抑瘤试验，B1和D显示出明显的抗肿瘤效应，降低了Gli的基因表达水平，且毒性较低；对于传统的UUO肾脏纤维化动物模型及Shh诱导的体外纤维化模型，B1及B2可以减轻纤维化的病理改变，减少细胞外基质的沉积，缓解纤维化的进程，且抗纤维化作用与其抑制Hh信号通路相关。综上，我们的研究为开发柴胡皂苷用于抗肿瘤、缓解脏器纤维化提供了重要的实验依据。