新型NF-κB抑制剂4-氯苯甲酰小檗胺靶向抗骨髓瘤细胞增殖分子机制的研究

Based on the molecular mechanism of the malignant proliferation of myeloma, new three-dimensional treatment strategies aim at myeloma cells, myeloma stem cells and proliferation-related signaling pathways. The traditional chemotherapy combined with multi-targeted drugs will control the recurrence, and cure the disease fundamentally. Our preliminary experiments showed that berbamine, a small chemical compound developed from traditional Chinese medicine product, is a naturally occurring NF-κB pathway inhibitor with anti-myeloma activity. After screening a series of berbamine derivatives, we identified 4-chlorobenzoyl berbamine which has stronger inhibitory effect on myeloma cell proliferation than berbamine. The mechanism of its action involves blocking NF-κB signaling pathway and eradicating myeloma stem cells. Based on our previous work, this project aims to ① gain more insight into the molecular mechanism of anti-myeloma effect conferred by 4-chlorobenzoyl berbamine, focusing on the NF-κB signaling pathway as well as other signaling network related to the malignant proliferation of myeloma cells; ② determine its potential activity of eliminating myeloma stem cells. The findings will be of both basic and clinical significance for further development of novel potent but less toxic anti-myeloma drugs with independent intellectual property rights.

针对骨髓瘤恶性增殖的分子机制，新的治疗策略更新为针对骨髓瘤细胞、骨髓瘤干细胞和肿瘤增殖相关信号通路的三维治疗，应用传统化疗药物结合多靶点攻击靶向药物的多药联合才能从根本上控制复发、达到治愈疾病的目的。我们的前期实验发现：中药单体小分子化合物小檗胺是一种天然的NF-κB抑制剂，具有抗骨髓瘤效应，经过对小檗胺的修饰改造和分离纯化，从中成功筛选出4-氯苯甲酰小檗胺，比较其母体具有更强的抑制骨髓瘤细胞恶性增殖作用，其机制涉及NF-κB信号通路的活化阻断并且可能对骨髓瘤干细胞有清除作用。本课题拟在前期工作基础上继续：① 4-氯苯甲酰小檗胺抗骨髓瘤效应详尽分子机制，涉及到NF-κB信号通路以及与骨髓瘤细胞恶性增殖密切相关的细胞信号网络；② 4-氯苯甲酰小檗胺对骨髓瘤干细胞的清除效应。研究结果对于进一步研发具有自主知识产权的新型高效低毒靶向性抗骨髓瘤药物提供科学理论数据，具有十分重要的理论和临床意义。

多发性骨髓瘤是不可治愈的B淋巴细胞恶性肿瘤，复发和耐药是限制患者长期生存的主要障碍，高效低毒的多靶点攻击的新药研发一直是骨髓瘤研究的重点。本报告研究新型的小分子化合物4-氯苯甲酰小檗胺对人骨髓瘤细胞株增殖抑制和诱导凋亡的作用并探讨其相关机制，采用MTT方法、流式细胞术细胞周期及凋亡分析、间接免疫荧法和Western blot 检测等方法。研究结果表明，4-氯苯甲酰小檗胺与其母体小檗胺比较具有更强的抗骨髓瘤细胞增殖活性， 3种骨髓瘤细胞株( KM3, RPMI8226, U266 ) 48h的IC50值分别为0.65μg/ml0、69 μg/ml 和0.72 μg/ml 。同时低剂量的4-氯苯甲酰小檗胺可以增加骨髓瘤细胞对常规化疗药物的敏感性，而在相同浓度范围内对正常造血细胞没有明显毒性。流式细胞术分析显示4-氯苯甲酰小檗胺可以引起细胞周期G1/S期阻滞，继而诱导骨髓瘤细胞凋亡。进一步研究4-氯苯甲酰小檗胺抗骨髓瘤作用的分子靶点，胞核内p65蛋白表达减少，NF-κB活化关键的上游激酶IKK和p-IκBα受抑，下游蛋白cyclin D1和survivin的表达减少，同时下调β-catenin蛋白的表达。对其它相关信号通路的研究发现，4-氯苯甲酰小檗胺可活化JNK信号通路，p-JNK表达明显增高且其下游c-Jun蛋白表达增高，在此过程中，AKT和ERK蛋白表达不受影响。本研究发现4-氯苯甲酰小檗胺比较母体小檗胺具有更强的抗骨髓瘤活性，其抗肿瘤作用可能与抑制NF-κB活性表达、抑制β-catenin信号通路和活化JNK信号通路相关，提示4-氯苯甲酰小檗胺抗骨髓瘤效应具备多靶点攻击效应，这些研究为后续新型抗骨髓瘤药物的研发奠定扎实的实验基础。