基于Nrf2介导的细胞代谢重编程探讨隐丹参酮逆转NSCLC顺铂获得性耐药的作用机制

The nuclear factor erythroid-2–related factor 2 (Nrf2) contributes to cellular metabolic reprogramming and plays an important role in cancer chemotherapy resistance. In our previous study, it was found that cryptotanshinone (CTS), an active component of traditional Chinese medicine Salvia miltiorrhiza Bge., could enhance the sensitivity of cisplatin-resistant non-small cell lung cancer (NSCLC) cells to cisplatin, and we also found that the high protein level of Nrf2 in cisplatin-resistant NSCLC cells was significantly decreased by CTS, however, the molecular mechanisms underlying this effects remain unclear. This project will explore the mechanisms underlying the function of CTS in vitro and in vivo from a new perspective: Nrf2 mediated reprogramming of cellular metabolism. The project will conduct the following studies: (1) investigate the characteristics of the reversal effect of CTS on cisplatin resistant NSCLC in cells and in nude mice model; (2) reveal the molecular mechanisms of the inhibitory effect of CTS on Nrf2 expression in cisplatin-resistant NSCLC cells from gene expression, Keap1 mediated ubiquitination, and regulation of Nrf2 phosphorylation et al.; (3) investigate the dose- and time-dependent characteristics of CTS on Nrf2 mediated cellular metabolic reprogramming from several levels including redox banlance, energy metabolism and the gene expression of key metabolism enzymes; (4) elucidate the intrinsic relation and mechanism underlying the reversal effect of CTS on cisplatin-resistant NSCLC by investigating the change of the reversal effect of CTS and its effect on Nrf2 mediated metabolic reprogramming before and after intervention of Nrf2 and its key target metabolic genes. The implementation of this project will supply scientific basis for the application of CTS in the cisplatin-resistant NSCLC chemotherapy, and provide a new insight in the mechanistic study of the reversal effect of traditional Chinese medicine on cancer chemotherapy resistance.

Nrf2可以重编程细胞代谢且在肿瘤耐药中扮演着重要角色。本课题组前期研究发现中药丹参中活性成分隐丹参酮能增强非小细胞肺癌（NSCLC）获得性耐药细胞对顺铂的敏感性，且明显抑制耐药细胞Nrf2表达，但具体机制尚不明确。本课题拟从Nrf2介导细胞代谢重编程的新视角，在分子、细胞和动物水平，考察其逆转NSCLC顺铂耐药的作用特点；从对Nrf2表达、泛素化、磷酸化等环节的影响，揭示其抑制Nrf2的分子机理；从Nrf2调控的细胞氧化还原平衡、物质和能量代谢、关键代谢酶基因表达等多环节探讨其对代谢重编程影响的量效-时效特征；对Nrf2及细胞代谢关键环节的基因抑制或过表达，观察隐丹参酮逆转耐药作用特征和细胞代谢重编程的变化，阐明影响Nrf2介导细胞代谢重编程与其逆转NSCLC顺铂获得性耐药的内在机理。本研究将为隐丹参酮用于逆转NSCLC顺铂耐药提供科学依据，为中药逆转化疗耐药的机制研究提供新思路。

中药在临床辅助肿瘤化疗中一直发挥着十分重要的作用。近年来，大量研究证实多种中药活性成分可以通过多种方式实现对肿瘤耐药性的逆转。因此，从中药中寻找高效、低毒、多靶点的肿瘤耐药逆转剂或增敏剂具有广阔的前景。本研究探讨了中药丹参的主要活性成分隐丹参酮对非小细胞肺癌（NSCLC）顺铂耐药的逆转作用，并从Nrf2介导细胞代谢角度，探讨了其作用的分子机制，旨在为为隐丹参酮用于逆转NSCLC顺铂耐药提供科学依据，为中药丹参逆转化疗耐药的机制研究提供新思路。.本项目选择以NSCLC细胞A549和顺铂耐药A549/DDP细胞分别构建相应的细胞和裸鼠移植瘤动物模型，研究隐丹参酮逆转NSCLC顺铂化疗耐药的作用特征及机制，首先，通过体外和体内研究确证顺铂和隐丹参酮联用可明显抑制耐顺铂的A549/DDP细胞增殖，促进凋亡，增加化疗药顺铂对A549/DDP的杀伤作用，且两者联用在抑制肿瘤增殖的同时对动物无明显毒性。然后，考察了隐丹参酮对Nrf2及其调控的下游代谢靶基因的影响及调控机制，结果发现隐丹参酮可以抑制顺铂耐药A549/DDP细胞中高表达的Nrf2和p-Nrf2及其下游代谢靶基因的蛋白表达，作用呈浓度和时间依赖性。同时发现，隐丹参酮对Nrf2的下调作用与下调p-JNK, p-ERK, p-p38, p-Akt和p-STAT3的磷酸化表达密切相关。siNrf2沉默耐药细胞A549/DDP内源性Nrf2基因后，与siNrf2对照处理组相比，隐丹参酮对Nrf2及其下游主要靶基因的表达无显著改变，同时，顺铂对siNrf2和隐丹参酮联用组的IC50值未见明显变化，确证了Nrf2信号通路在隐丹参酮逆转NSCLC细胞对顺铂的耐药性中发挥重要作用。最后，采用细胞代谢组学结合分子生物学技术，发现顺铂耐药细胞代谢表型与敏感细胞具有显著性改变，确证了Nrf2介导的磷酸戊糖途径及其主要代谢酶G6PD在A549细胞顺铂耐药中的重要作用，同时，研究发现隐丹参酮可显著影响耐药肿瘤细胞的代谢表型及其磷酸途径主要代谢酶G6PD和TKT表达，进而发挥其逆转耐药作用。综上，本研究从Nrf2介导细胞代谢调控的角度揭示了隐丹参酮逆转NSCLC顺铂耐药机制，为中药丹参活性成分隐丹参酮用于逆转NSCLC顺铂耐药提供理论和实验依据。