靶向抑制突变体p53激活p63/p73－蟾毒灵及其衍生物CNJ-2治疗淋巴瘤的机制研究

Discovering new drug from traditional Chinese medicine is a very important and valuable road. We found that Cinobufacin has certain curative effects for the treatment of advanced lymphomain in our previous clinical studies. Bufalin is the main antitumor component of cinobufotalin. We found bufalin can induce apoptosis of Raji lymphoma cells and significantly inhibit the mutant p53 (mutp53) expression, increase p63/p73 expression. We hypothesized that bufalin targeted inhibition mutp53 and activate p63 / p73 promotes tumor apoptosis. We design overexpression and shRNA mutp53 in Raji cells, followed by Bufalin treatment in vitro and in vivo to clarify its molecular mechanism. CNJ-2, the novel bufalin derivative with high efficiency and low toxicity were screened by microbial cell suspensions system. We next to reveal CNJ-2 has a same molecular mechanism with bufalin through the in vitro and in vivo experiment. By patient-derived xenograft lymphoma biopsy transplant NCG mouse establish PDTX model to further validate the efficacy of CNJ-2 treatment of lymphoma in vivo and its relationship with mutp53. Thereby clarify the molecular targets and mechanisms of CNJ-2 for treatment of lymphoma. This work may helpful for development of new targeting drugs to treatment of lymphoma.

从中医药宝库中发掘治疗淋巴瘤的新药是非常重要而宝贵的途径。我们发现华蟾素单药治疗中晚期淋巴瘤具有肯定的疗效，初步实验证实，华蟾素抗肿瘤主要成分蟾毒灵可诱导淋巴瘤细胞株凋亡，mRNAseq分析及western提示其抑制mutp53蛋白，RT-PCR提示p63/p73上调及survivin下调，我们推测蟾毒灵靶向抑制mutp53激活p63/p73促进肿瘤凋亡。本研究拟通过在Raji细胞中过表达及下调mutp53，其后体内外予蟾毒灵干预明确其作用的分子机制。利用微生物细胞悬浮体系筛选出高效低毒的蟾毒灵衍生物CNJ-2，通过体内外实验研究，明确CNJ-2具有与蟾毒灵相同的分子机制。通过患者来源的淋巴瘤活检组织异种移植NCG小鼠建立PDTX模型，进一步验证CNJ-2治疗淋巴瘤的体内疗效及与mutp53的关系, 从而阐明CNJ-2治疗淋巴瘤的分子靶点和作用机制，为开发淋巴瘤的新型靶向药物打下基础。

目的背景：我们前期发现华蟾素单药治疗中晚期淋巴瘤具有肯定的疗效，拟通过实验研究进一步揭示其分子机制。.主要研究内容和关键数据: 实验证实，华蟾素抗肿瘤主要成分蟾毒灵可诱导淋巴瘤细胞株凋亡，mRNAseq分析及western提示其抑制mutp53蛋白，涉及的信号通路是由miR-21/TP63通路激活介导的，蟾毒灵抑制miR-21的表达，其后上调TP63的水平，TP63促进凋亡相关蛋白FAS、Puma、P21的表达，最后通过caspase9和caspase3蛋白的激活抑制淋巴瘤细胞增殖和促进细胞凋亡。此外本研究通过利用微生物细胞悬浮体系筛选出高效低毒的蟾毒灵衍生物CNJ-2/BF211，通过体内外实验研究明确CNJ-2/BF211针对B细胞来源的浆细胞肿瘤具有明显的杀伤作用，IL-6能够诱导浆细胞肿瘤JAK2/STAT3信号通路的激活，进而引起相关磷酸化分子p-JAK2, p-STAT3蛋白表达的上调，而磷酸化分子p-JAK2, p-STAT3蛋白的上调能够被BF211以浓度梯度的方式逆转。进一步通过患者来源的淋巴瘤活检组织异种移植NCG小鼠，成功建立人淋巴瘤的PDTX模型，为开发淋巴瘤的新型靶向药物打下基础。进一步的拓展实验中我们发现，中药蟾毒灵具有多靶点效应，对淋巴瘤细胞生长过程中富集的髓系免疫抑制细胞（MDSC）具有抑制作用，为此课题组建立了一套体外培养MDSC的创新实验体系，为开展下一步研究打下基础。.科学意义：课题通过体内体外实验揭示了蟾毒灵及其衍生物治疗B细胞来源肿瘤的分子机理，建立了个体化评估药效的淋巴瘤PDTX模型，并且拓展性地研究了蟾毒灵对淋巴瘤相关MDSC的影响。全方位多角度的研究了蟾毒灵及其衍生物的抗肿瘤活性及机制。