异莲心碱靶向NF-κB信号通路关键转录因子p65诱导肝癌细胞凋亡的机制

Hepatocellular carcinoma (HCC) is a kind of malignant tumor which severely threatens public health. However, the clinical outcomes of anti-HCC drugs currently available are not so satisfied. Isoliensinine exists abundantly in the traditional Chinese herb "Lianxin" (lotus plumule). Till now, it remains poorly understood whether isoliensinine exhibits any anti-tumor activity. Our previous investigations revealed that isoliensinine dramatically induced HCC cell apoptosis both in vitro and in nude mice, and showed few toxic effects to untransformed hepatocytes. These observations suggested that isoliensinine has the potential to be applied in HCC treatment. Further studies revealed that isoliensinine was capable of selectively suppressing the transactivity of transcription factor p65 in HCC cells. This event downregulated NF-κB anti-apoptotic signalling and promoted HCC cell apoptosis. Based on these findings, to understand the molecular mechanisms through which isoliensinine suppresses the transactivity of p65, this proposal is planning to explore the effects of isoliensinine on the subcellular localization, post- translational modifications and functional interactions of p65 subunit, identify the p65 transactivity-regulating proteins whose expressional levels, post- translational modifications or functional interactions can be affected by isoliensinine, and then disclose the key molecular events mediating the inhibition of p65 transactivity after isoliensinine treatment, thereby unveiling the mechanisms underlying isoliensinine-induced HCC cell apoptosis. These studies will not only expand the understandings about small natural chemicals-induced cancer cell apoptosis targeting NF-κB pathway, but also provide potential theoretical guidance for considering isoliensinine as a lead compound in researching and developing novel anti-HCC drugs.

肝癌是严重威胁人类健康的恶性肿瘤，但现有药物疗效并不理想。异莲心碱是传统药材莲心的主要化学组分之一，目前对其在抗肿瘤方面的活性缺乏系统的研究。项目申请人前期工作发现，它在体外和裸鼠体内均能显著诱导肝癌细胞凋亡，对非恶性肝细胞毒性很低，有用于肝癌治疗的潜力。异莲心碱能相对选择性地抑制肝癌细胞内转录因子p65的转录激活活性，从而下调NF-κB凋亡抑制信号，促进细胞凋亡。 基于上述工作，为深入探讨异莲心碱抑制p65活性的机理，本项目拟检测异莲心碱对肝癌细胞内p65蛋白亚细胞定位、翻译后修饰以及与辅因子相互作用的影响，确定表达水平、翻译后修饰或功能性相互作用被异莲心碱改变的p65活性调控蛋白，揭示异莲心碱抑制p65活性过程中涉及到的关键性分子事件，阐明其诱导凋亡的作用机理。该工作将丰富对天然小分子靶向NF-κB诱导癌细胞凋亡的认识，并为考虑以异莲心碱为先导化合物开发抗肝癌新药提供理论依据。

课题组前期研究发现，异莲心碱抑制肝癌细胞中的NF-κB活性并诱导肝癌细胞凋亡。本项目旨在探索其中的分子机理。研究发现，异莲心碱能抑制NF-κB关键因子p65蛋白上536位丝氨酸的磷酸化。在肝癌细胞内过表达p65的磷酸化模拟突变体能抑制异莲心碱诱导的细胞凋亡。进一步研究表明，异莲心碱促进p65与PP2A的相互作用。抑制PP2A活性或敲减PP2A催化亚基的表达水平均可阻断异莲心碱诱导的p65去磷酸化。I2PP2A是PP2A的内源性抑制因子。异莲心碱直接干预PP2A与I2PP2A的相互作用。在肝癌细胞中敲减I2PP2A的表达可促进p65与PP2A的相互作用以及p65蛋白536位丝氨酸的去磷酸化。过表达I2PP2A则可以明显抑制异莲心碱诱导的p65去磷酸化与细胞凋亡。非转化肝细胞对于异莲心碱诱导的NF-κB活性抑制和细胞凋亡并不敏感。在非转化肝细胞中，基本水平的I2PP2A以及p65蛋白536位丝氨酸磷酸化均明显低于肝癌细胞。以上研究结果综合说明，在肝癌细胞中，异莲心碱可干预PP2A与I2PP2A的相互作用，促进PP2A依赖的p65去磷酸化，最终诱导NF-κB活性抑制和细胞凋亡。