蟾毒灵干预CaMKKβ/CaMKIV轴调控肝内胆管癌细胞增殖与凋亡的机制研究

The prognosis of intrahepatic cholangiocarcinoma is dismal. Thus, exploration and identification of new drugs and targets are of vital importance in practice. Our previous studies have found that venenum Bufonis, a potent monomer of Chinese toad, inhibited the proliferation and promoted the apoptosis of intrahepatic cholangiocarcinoma cells. By detecting the proteome and genomic microarrays, bufalin could directly bind to CaMKKβ protein, and the gene expression of CaMKIV was positively correlated to CaMKKβ. Combined with relevant aticle reports, we preliminary confirmed that bufalin can affect the downstream mTOR/S6K signaling pathway. Thus, it can be assumed that bufalin inhibits CaMKIV by directly inhibiting the activity of CaMKKβ, thereby affecting its downstream mTOR/S6K signaling pathway to exert its anti-tumor effect. This research intends to study in vitro and in vivo perspectives that bufalin regulates the CaMKKβ/CaMKIV axis to inhibite mTOR/S6K signaling pathway to exert its anti-tumor mechanism. This study will explain the anti-tumor mechanism of bufalin which might suggest the new drug and target for ICC therapy.

肝内胆管细胞癌恶性程度高，预后差，已有治疗药物疗效差，因此寻找新的治疗药物和作用靶点具有重要意义。我们前期研究发现中药蟾酥的有效单体蟾毒灵能抑制肝内胆管癌细胞增殖并促进其凋亡，通过蛋白质组和基因组芯片检测发现蟾毒灵能直接结合CaMKKβ蛋白，且基因CaMKIV的表达与CaMKKβ的表达呈正相关。结合已有文献报道，我们初步证实蟾毒灵可影响下游mTOR/S6K信号通路。由此可假设：蟾毒灵通过直接抑制CaMKKβ的活性抑制CaMKIV进而影响下游mTOR/S6K信号通路发挥其抗肿瘤作用。本课题拟从体外、体内角度研究蟾毒灵调控CaMKKβ/CaMKIV轴影响mTOR/S6K信号通路发挥其抗肿瘤机制，此研究成果将解释蟾毒灵在肝内胆管癌中发挥作用的具体机制，为肝内胆管细胞癌的有效治疗提供新药物和新靶点。

肝内胆管癌是胆道系统最常见的恶性肿瘤之一，恶性程度高，危害大。手术切除是其早期治疗的最主要方式，对于不可行手术切除患者5年生存率＜5%。目前对于不能通过手术的进展期肝内胆管癌患者，仍没有一致认同的标准化疗方案。在过去的20年里，肿瘤专家通过提高化疗剂量和变换不同的细胞毒性药物，都不能改善肝内胆管癌患者整体预后。中医药治疗具有独特的优势，蟾酥具有祛瘀攻毒散实邪之功效，临床上可配伍应用于进展期肿瘤的治疗。蟾毒灵是蟾酥的有效单体，我们研究发现蟾毒灵具有良好的抗肝内胆管癌效果，包括抑制其增殖、侵袭迁移，促其凋亡；主要抗肿瘤机制可能是蟾毒灵通过靶向抑制CaMKKβ从而抑制mTOR/S6K通路发挥作用；蟾毒灵在肝内胆管癌皮下移植瘤动物模型中同样具有良好的治疗效果；CaMKKβ在肝内胆管癌组织中明显上调，在早期组织中即可检测到，并与肝内胆管癌患者的临床预后显著相关，可能称为肝内胆管癌诊断和预后判断的潜在生物标志物。由此可见，蟾毒灵有望开发成为肝内胆管癌治疗的新型药物，本项目为肝内胆管癌的新型药物开发提供了新策略和新思路。