丹参新酮调控AP4-Lgr5通路对胃癌化疗增敏的作用和机制研究

Multidrug resistance (MDR) strictly restricts the effect of chemotherapy in patients with advanced gastric cancer (GC). Traditional Chinese medicine is of great power to increase the sensitivity of chemotherapy and to improve patients’ living quality. Our previous study observed that miltirone, which was extracted from traditional Chinese medicine Salvia miltiorrhiza Bunge, could effectively increase the sensitivity to chemotherapy of gastric cancer cells, and down-regulate the expression of transcription factor AP4. In addition, AP4 modulated GC cells stem cell-like phenotype by regulating Lgr5 expression, and was important target for therapy of advanced gastric cancer. Therefore, we hypothesized that miltirone might increase chemotherapy sensitization by inhibiting generation of tumor cell stem cell-like phenotype through regulation of AP4-Lgr5 pathway. This project aims ① to investigate the efficiency of enhancing chemotherapy sensitization by miltirone through regulating AP4 in GC, ② to evaluate the effect on chemotherapy sensitization of miltirone through regulation of AP4-Lgr5 pathway in GC in vivo, ③ to elucidate the roles of AP4 on generation of stem cell-like phenotype and the control of AP4 on Lgr5 transcription in GC. This project will clarify the mechanism of miltirone on chemotherapy sensitization in gastric cancer, and transcriptional control of AP4 on Lgr5 gene, extend theoretical basis for anti-cancer study of traditional Chinese herbal medicine, and provide new targets and ideas for the treatment of advanced gastric cancer.

肿瘤多药耐药是晚期胃癌化疗疗效不佳的关键原因。中药在增加化疗敏感性和改善治疗效果提高患者生活质量方面具有独特效果。课题组前期研究发现中药丹参提取物丹参新酮能有效增加胃癌细胞对化疗药物敏感性，并下调转录因子AP4的表达；胃癌中AP4调控Lgr5的表达，促进胃癌细胞干细胞样，是化疗增敏在内的癌症治疗的重要靶点。基于此，我们提出假设，丹参新酮通过调控AP4-Lgr5通路抑制胃癌细胞干细胞样表型达到化疗增敏效应。本项目拟进行以下研究：①丹参新酮调控AP4对胃癌细胞化疗增敏作用；②体内评估丹参新酮调控AP4-Lgr5通路对胃癌细胞化疗增敏的作用；③转录因子AP4调控胃癌细胞干细胞样作用及对Lgr5基因转录调控机制。本项目的实施有助于阐明丹参新酮对胃癌细胞的化疗增敏机制，明确AP4对Lgr5的转录调控机制，为传统中草药的抗肿瘤研究提供新理论基础，为晚期胃癌的治疗提供新的靶点和治疗思路。

我国是胃癌高发地区。目前，多药耐药的产生依然严重制约胃癌临床治疗效果。本项目研究发现，中药丹参提取物丹参新酮可以抑制胃癌细胞增殖、成球和转移，诱导胃癌细胞凋亡；下调STAT3-c-Myc-AP4-Lgr5表达水平；过表达AP4促进胃癌细胞迁移侵袭。丹参新酮与顺铂联用对胃癌细胞的抑制率均显著高于顺铂单药组。裸鼠移植瘤实验结果显示顺铂、丹参新酮、丹参新酮联合顺铂均可抑制肿瘤生长，联合用药组效果最好。转录组测序结果显示丹参新酮作用于胃癌细胞后，差异表达基因中存在167个基因表达上升，230个基因表达下调。涉及PI3K-Akt signaling pathway、FoxO signaling pathway、transcriptional misregulation in cancers等多条通路，为后续实验展开提供了方向。IHC检测胃癌组织芯片显示转录因子AP4在胃癌组织中阳性率为69.4%，与胃癌无病生存期（DFS）有关，高表达的AP4提示胃癌预后不良。LGR5在胃癌组织中阳性率为29.3%，与胃癌无病生存期（DFS）有关，高表达的LGR5提示胃癌预后不良。低表达的LGR5与胞质AP4表达水平正相关。本项目的实施，阐明丹参新酮对胃癌细胞的化疗增敏机制，为传统中草药的抗肿瘤研究提供新理论基础，为晚期胃癌的治疗提供新的靶点和治疗思路。