番石榴叶中三萜类成分通过调控RIP3蛋白磷酸化诱导结肠癌耐药细胞发生necroptosis的作用及机制研究

Multidrug resistance (MDR) of colon carcinoma is a major problem in chemotherapy and involved in many intrinsic and extrinsic factors. Most reversal agents of MDR, regardless of their various resistance mechanisms,exhibit the low-efficacy and high-toxicity defects. In our previous work, a series of triterpenoids, including four new ones, were isolated from Psidium guajava, and their structures were characterised as ursane- and oleane-type triterpenoids. The bioactive results indicated that 12,13-dihydroxyl substituted triterpenoids could induce necroptosis obviously on MDR colon carcinoma cells.Preliminary mechanism research prompted that the biological activity were possibly associated with the phosphorylation of protein kinase RIP3 and with the activation of downstream mitogen necrosis kinase.Necroptosis could be considered as a special pathway that circumvented apoptotic resistance and transporter proteins overexpression. Our continuing research aims to the systematic seperation and structural elucidation of triterpenoids, to the evaluation of their bioactivities on MDR colon carcinoma, and to the clarification of the necroptosis mechanism, as well as the RIP3 regulation function on MDR colon carcinoma cell. This research is supposed to be a scientific basis on MDR colon carcinoma drug research.

结肠癌多药耐药呈现的多机制、多因素的复杂性，已成为影响临床治疗的重要障碍。针对不同耐药机制设计的肿瘤逆转剂普遍存在低效高毒的缺点。本课题组在前期工作中，从番石榴叶中分离并鉴定了包括4个新化合物在内的一系列乌苏烷型和齐墩果烷型三萜类成分；并首次发现具有12,13-二羟基取代的新三萜类成分可显著诱导结肠癌耐药细胞发生程序性坏死(necroptosis)；初步的机制研究提示该作用可能与RIP3蛋白被磷酸化并激活下游促坏死激酶有关。本项目拟在前期工作基础上，对番石榴叶中的三萜类成分进行进一步系统分离，评价此类成分逆转结肠癌耐药的生物活性，探讨此类成分诱导necroptosis的发生机制和对RIP3蛋白及相关信号转导通路的调控机制。我们所提出的通过necroptosis途径以绕开各种耐药机制的研究思路，为肿瘤耐药研究提供了新方向；开展RIP3的功能研究有望为新型抗结肠癌耐药药物的研发提供科学依据。

本项目开展了番石榴叶三萜提取部位的化学成分系统分离和抗结肠癌活性及作用机制研究。获得了以下实验结果：(1) 分离并鉴定40个化合物，其中15个化合物为首次从该植物中发现。(2) 新增“番石榴叶三萜部位的HPLC指纹图谱”这一实验内容。通过对三萜提取部位中的10种指标性化合物进行定量分析，对提取物的质量进行控制，为后期的药效研究提供保障。(3) 细胞活性筛选实验发现三萜提取物和受试单体成分均对结直肠癌细胞表现出显著的抑制作用，但二者诱导结肠癌细胞的死亡方式不同，单体化合物以细胞凋亡为主，而提取物表现为肿瘤细胞发生程序性坏死necroptosis。分别选择三萜活性部位和代表化合物积雪草酸进行不同的抗肿瘤作用机制研究。(4) 作用机制研究实验发现积雪草酸能够改变肿瘤细胞PI3K/AKT/mTOR信号通路上的相关蛋白的表达，并首次发现其对下游蛋白PDCD4具有调控作用。三萜活性部位能够对necroptosis相关的RIP3蛋白信号通路产生表达变化。