基于GSK3β及其相关自噬信号通路的槐定酸类新化合物IMB-08B抗肝癌作用机理研究

Hepatocellular cancer (HCC) is the fifth most common malignancy and a major health burden worldwide. Therefore, a thorough understanding of the molecular carcinogenic mechanisms and development of efficacious therapeutics of HCC is a global scientific challenge. Overwhelming evidence implicates that increased Glycogen synthase kinase3β (GSK3β) activity and reduced autophagy may promote the development of HCC pathogenesis, thus therapeutic targeting of the GSK3β and autophagic pathways may be an effective way to enhance the therapeutic activity of anticancer drugs against HCC. During our screening program for natural anticancer drugs against HCC, we have recently discovered that IMB-08B, a novel sophoridinic acid derivative, has potent antihepatocarcinoma activity. Our primary results demonstrated that IMB-08B inhibited hepatoma cell survival by suppressing the activation of GSK3β and increasing the autophagy level. Meanwhile, we also found that GSK3β regulated the autophagy level in hepatoma cell by interacting with TAB2 as well as controlling the expression of c-FLIP. Since TAB2 and c-FLIP inhibition of autophagy through interaction with Beclin1 and Atg3, respectively, we hypothesize that IMB-08B regulates hepatoma cell survival through GSK3β-TAB2-Beclin1 and GSK3β-c-FLIP-Atg3 autophagic pathways. This proposal is novel, as it will demonstrate the effect of autophagy on the antihepatocarcinoma activity of IMB-08B, delineate the molecular mechanism by which IMB-08B regulates autophagic cell death through GSK3β-TAB2-Beclin1 signaling pathway, to elucidate the mechanism of IMB-08B control the autophagic cell death by GSK3-c-FLIP-Atg3 pathway in hepatoma cell with the use of multiple cellular and molecular biotechnologies. This study will clarify a new role of IMB-08B suppressing hepatoma cell survival via GSK3β and its related autohaogic pathways, and provide a theoretical basis for the mechanism study of sophoridinic acid derivatives against HCC.

诱导自噬性细胞死亡是抗肿瘤药物研发的新策略。糖原合成酶激酶3β（GSK3β）及其相关自噬信号通路活性与肝癌发生密切相关，抑制GSK3β活性、提高自噬水平具有抗肝癌作用。前期研究发现我们自主创新、具有全新骨架结构的槐定酸类化合物IMB-08B具有明确的抗肝癌作用，可抑制肝癌细胞中GSK3β的活性，诱导自噬性细胞死亡的发生，并具有较好的成药性特征。 本项目拟在此原创性工作基础上，以GSK3β是TAB2的新作用分子及GSK3β调控c-FLIP表达为依据，深入研究：（1）自噬对IMB-08B抗肝癌作用的影响；（2）IMB-08B通过GSK3β调控TAB2-Beclin1自噬通路的确切机制；（3）IMB-08B通过GSK3β调控c-FLIP-Atg3自噬途径的机理，力求阐明IMB-08B通过GSK3β及相关自噬信号通路抑制肝癌生长的分子机制，为槐定酸类化合物抗肿瘤的信号通路机制研究提供新思路。

诱导自噬性细胞死亡是抗肿瘤药物研发的新策略。糖原合成酶激酶3β（GSK-3β）及相关自噬信号通路活性与肿瘤发生密切相关，抑制GSK-3β活性、提高自噬水平具有抗肿瘤作用。我们前期研究证实槐定酸衍生物具有显著的抗肿瘤活性。本研究从多个角度系统阐明了槐定酸类新化合物IMB-08B（后命名为IMB-6G）的抗肿瘤作用及分子机制。我们发现IMB-6G可通过激活肝癌细胞中IRE1α-ASK1和 PERK-CHOP两条内质网应激信号通路，分别引起JNK1磷酸化和Bax转移到线粒体内膜从而诱发内源性细胞凋亡。此外，GSK-3β高表达于肝癌等恶性肿瘤中， IMB-6G可抑制GSK-3β的活性，阻断胰腺癌细胞中的自噬流，促进自噬小体的聚集进而引起溶酶体膜通透性的增加，使Cathepsin B和D从溶酶体转位到细胞质中，诱导胰腺癌细胞凋亡的发生。同时，IMB-6G抑制GSK-3β的活性后，也可通过激活TFEB的转录活性，促进肿瘤细胞中溶酶体的生物合成与活性，从而降解c-FLIP蛋白，诱导自噬性细胞死亡的发生。构效分析结果表明，在槐定酸的N端引入亚芳基可大大提高其抗肿瘤活性，机制是引起肿瘤细胞中溶酶体的pH值升高，阻断自噬流，进而诱发细胞凋亡。本研究阐明了IMB-6G通过GSK-3β及相关自噬信号通路抑制肿瘤生长的分子机制，为槐定酸类化合物发展成为一类新型抗肿瘤药物奠定了基础。