胰蛋白酶抑制剂BBI通过RIP3激活线粒体途径诱导肿瘤细胞凋亡的机制研究

Bowman-birk trypsin inbitor BBI has been shown to have anticarcinogenic effects on many different cell lines for its induction of apoptosis, but the biochemical and molecular bases for this mechanism of action need to be further elucidated. The capacity of BBI for inducing RIP3 expression, increasing the transcription of Bax, decreasing the level of Bcl-2 mRNA and little influencing on the Fas/FasL mRNA has also been demonstrated in our previous study. We applied Western-blot assay to further detect the role of Mung bean BBI on tumor cell apoptosis, and the result confirmed that Mung bean BBI enhanced the releasing of cytochrome C from mitochondria as well as markedly induced the activation of caspase3, Caspase9 and caspase8. Our previous result suggested that the apoptosis induced by Mung bean BBI was correlated with mitochondrial dysfunction. Mung bean BBI has also been evidenced by inhibiting the growth of tumor in tumor bearing mice model in our animal experiment. So，we proposed that the possible mechanism of mung bean BBI anti-cancer properties is via mitochondrial apoptosis signal path: 1, BBI may activate the transcription and expression of tumor RIP3 gene, subsequently RIP3 directly triggers signals of mitochondrial apoptosis pathway; 2, the expression of RIP3 in tumor cells may negatively regulate PI3K-AKT/PI3K-NF-кB pathway which can normally inhibit apoptosis. We will collate the expression and activation of RIP3, the changes of mitochondrial signaling molecules in the mitochondrial pathway with the result of animal model and chip to find the details of mung bean BBI's anti-tumor function. This study will clarify that the roles of BBI's in anti-tumor is based on the activation of RIP3 by inducing mitochondrial apoptosis pathway，which will also provide novel therapeutics target, new prognostic indicators, and novel idea for drug development.

胰蛋白酶抑制BBI可通过诱导肿瘤细胞凋亡发挥其抗肿瘤作用，对正常组织和细胞毒害作用不明显，其机理尚未明确。RIP3广泛表达于正常组织，未见在肿瘤中表达。我们前期研究发现绿豆BBI能诱导肿瘤细胞表达RIP3，抑制Bcl-2 转录，促进Bax转录，促使cyt-c释放到细胞质中，激活caspase3、caspase9，但不影响Fas/Fas配体转录，不能活化caspase8，结果提示绿豆BBI可能通过RIP3介导激活细胞凋亡的线粒体途径导致肿瘤细胞凋亡；动物实验还证实绿豆BBI能抑制小鼠的肿瘤生长。本研究拟通过分析绿豆BBI处理后肿瘤细胞中RIP3及细胞凋亡线粒体途径中信号分子的表达、激活等情况，结合动物模型和基因芯片找出BBI激活RIP3表达对肿瘤细胞凋亡通路的影响，阐明RIP3调控肿瘤细胞凋亡的线粒体途径和BBI抗肿瘤机制，为临床提供新的预后指标，为抗肿瘤药物研发提供新的治疗靶点和新思路。

丝氨酸蛋白酶在生物体内参与了许多的生理过程，如消化，凝血，纤溶，免疫反应，细胞周期进程和细胞凋亡。某些丝氨酸蛋白酶功能或活性异常与癌症、血管生成、类风湿性关节炎，神经退行性疾病、心血管等疾病密切相关。植物丝氨酸蛋白酶抑制剂在人类多种的疾病防治中，包括癌症、神经退行性疾病、心血管疾病、肌肉萎缩和炎症占据重要地位。丝氨酸蛋白酶抑制剂Bowman Birk（BBI）在临床上可有效抑制前列腺增生症、口腔白斑、溃疡性结肠炎和结肠癌。绿豆BBI是本室研制的丝氨酸蛋白酶抑制剂，前期的研究发现：该蛋白能特异性的诱发肿瘤细胞凋亡和抑制肿瘤生长。因此，本项目在前期的基础上研究绿豆BBI诱导肿瘤细胞凋亡的机制，旨在找到BBI抗肿瘤药物靶点和治疗肿瘤机理。研究发现BBI能抑制多种肿瘤细胞增殖和诱导凋亡，并激活RIP3表达。敲低RIP3表达可有效抵抗BBI诱发的肿瘤细胞死亡。BBI通过激活RIP3来诱发肿瘤细胞死亡，并在小鼠实体瘤上得以验证。本项目共投入80万元，结余167226.47万元（尚有测试费用和试剂费用14左右的款项未结），各项支出基本与预算相符，剩余经费计划用于本项目研究后续支出。项目资助号资助的文章共6篇，其中sci三篇，中文论文三篇。代发相关文章sci三篇，正在申请专利1项，投稿中中文核心3篇。