南蛇藤多萜靶向APOBEC3C介导的吉西他滨脱氨代谢逆转胰腺癌化疗耐药的作用研究

Supported by a series of National Natural Science Foundation of China (NSFC), we have successfully identified the total terpenoids prepared from traditional Chinese medicine Celastrus orbiculatus Thunb with significant antitumor activity. Since the standard first-line treatment by gemcitabine (GEM) in pancreatic cancer has failed to achieve better clinical outcomes due to counterproductive chemoresistance, we thus combined the total terpenoids of Celastrus orbiculatus Thunb (TTC) with GEM and found TTC could significantly increase the sensitivity of pancreatic tumor cells to GEM. However, the underlying mechanisms are still poorly understood and investigated. Previously, we found a novel role of APOBEC3C (A3C) -mediated GEM nucleotides deamination and chemoresistance as functional cytidine deaminase activity could confer GEM clearance and chemoresistance by converting GEM to inactive metabolite. Moreover, we found the potential role of TTC by targeting the process and then reversing chemoresistance of GEM. Therefore, the project is set up to establish GEM resistant pancreatic tumor patient-derived xenograft and analyze the clinical significance of A3C and GEM deamination in clinical GEM chemoresistance mixed with multiple pathological features of these patients to verify whether TTC can decrease the levels of GEM deamination and then reverse its chemoresistance. Furthermore, we will purify the recombinant as well we the deaminase active center-mutant A3C to study the chemico-biological interactions between A3C and TTC, and confirm the specific zinc-dependent enzymatic center as the critical sites for TTC inhibiting the deaminase activity. Last, we will use A3C functional rescue assays in vitro and in vivo to validate how TTC reverses chemoresistance of GEM initially by A3C deaminase-dependent suppression of the metabolic clearance of gemcitabine nucleotides in pancreatic cancer. Consequently, these studies will provide the initial underpinnings of TTC in treating pancreatic cancer as an effective GEM-sensitizing or resistance reversal agent with novel mechanisms in future clinical applications.

课题组在国家基金连续资助下，从中药南蛇藤中获得抗肿瘤活性组分南蛇藤多萜。针对胰腺癌治疗金标准吉西他滨（GEM）化疗耐药的临床困境，我们发现南蛇藤多萜能协同增强GEM抗胰腺癌活性，但机制不明。初步研究提示，胞嘧啶脱氨酶APOBEC3C（A3C）通过促进GEM脱氨代谢失活而诱导GEM耐药，南蛇藤多萜可能靶向这一过程而逆转GEM耐药。本课题首先建立临床胰腺癌GEM耐药移植瘤模型，结合病理资料分析A3C与GEM脱氨代谢对GEM耐药的临床意义，研究南蛇藤多萜能否抑制GEM脱氨代谢而逆转GEM耐药；然后制备野生及脱氨酶活性中心突变的重组A3C，研究南蛇藤多萜能否直接结合A3C锌活性中心而抑制A3C活性；最后利用A3C功能挽救的体内外实验，阐明南蛇藤多萜靶向A3C介导的GEM脱氨代谢从而逆转GEM耐药的分子机制。以上为南蛇藤多萜未来能够逆转胰腺癌GEM耐药并提高GEM临床疗效开展有价值有意义的科学探索。

DNA损伤应答（DNA damage response, DDR）的进化调控是肿瘤耐药的关键生物学机制之一。应用DDR抑制剂能显著增强一线化疗药物疗效，但临床研究结果显示，联用产生了对正常组织的剂量限制性毒性。因此，开发安全有效的组合策略以临床提高化疗药物的疗效十分重要。课题组初期发现中药南蛇藤（Celastrus orbiculatus）茎活性组分南蛇藤多萜能增强多种化疗药物的抗肿瘤敏感性，包括抗代谢药物吉西他滨。通过本项目我们进一步发现，南蛇藤多萜及其活性醌甲基三萜是天然的多位点DDR抑制剂，通过阻断DNA损伤感知（PARP1）、应答（Chk1）与修复（RAD51/53BP1）级联网络促进肿瘤基因组与染色体断裂而增强吉西他滨的抗肿瘤效应。在原位胰腺癌移植瘤模型中，证实南蛇藤多萜联合吉西他滨有效抑制了胰腺癌的原位生长与肠转移，显著减轻了化疗造成的肝毒性。此外，我们发现胞嘧啶脱氨酶APOBEC3C（A3C）可诱导吉西他滨化疗耐受，过表达或沉默A3C实验证实A3C可以促进吉西他滨对Chk1的活化。在周期调控方面，抑制Chk1可以缓解吉西他滨介导的G1/S期阻滞，而A3C促进了Chk1对S期的刹车作用。在DNA损伤效应方面，抑制Chk1可以增强吉西他滨的基因毒性，而A3C削弱了Chk1抑制剂联合吉西他滨诱导的DNA损伤标志物γH2AX与DNA双链断裂水平。因此我们认为A3C通过诱导Chk1活性促进吉西他滨耐药。综上，本项目为中药南蛇藤以及干预A3C在肿瘤DNA损伤修复及其化疗耐受中的应用提供新颖的工作机制与理论基础。