青藤碱衍生物ZW016抑制STAT3活化进而诱导骨髓瘤细胞凋亡的机制研究

Signal transducer and activator of transcription 3 (STAT3) is a bi-functional protein which coupled with tyrosine phosphorylation signaling pathway. Overexpressed or highly activated STAT3 is closely associated with abnormal proliferation and apoptosis of multiple myeloma (MM) cells as well as chemoresistance to standard therapies. In our previous series of in vitro experiments, it was shown that ZW016, one of sinomenine derivatives from traditional Chinese medicine, could inhibit intracellular STAT3 activation and induces cell growth arrest and apoptosis in MM cells. Based on these results, we proposed that ZW016 could display potency in the treatment of myeloma through target modulating STAT3. To prove this hypothesis，this project intends to address the followings: ① Determine the relationship between expression levels of STAT3 and p-STAT3 and prognosis of MM patients. ② Assess biological effects of ZW016 in abrogating STAT3 activation, inhibiting cell proliferation and inducing apoptosis in MM cell lines. Furthermore, by using cell free analysis, validate the interaction of ZW016 and STAT3 as well as its precise binding cites. ③ Validate biological effects of ZW016 in distinct MM mice models. ④ Explore combined effects of ZW016 and first line therapeutic agents in MM. This study would provide insights for the development of anti-MM drugs and the theoretic instructions for drug targets identification.

信号传导与转录激活因子3（STAT3）是存在于细胞浆与酪氨酸磷酸化信号通路偶联的双功能蛋白，STAT3高表达和活化与多发性骨髓瘤（MM）细胞异常增殖和凋亡以及耐药密切相关。课题组前期研究显示，青藤碱衍生物ZW016能够与MM细胞内STAT3结合并抑制其活化进而诱导MM细胞凋亡。鉴于此, 课题组提出如下假说：ZW016通过直接靶向STAT3而发挥抗MM效应。为证明此假说，课题组拟从以下方面进行研究：①临床水平验证MM患者骨髓瘤细胞内STAT3和磷酸化STAT3表达水平与其生存预后的相关性; ②在细胞水平检测ZW016抑制MM细胞内STAT3活化并诱导MM细胞增殖阻滞和凋亡的生物学效应以及ZW016与STAT3结合方式和位点; ③应用MM动物模型进一步检测ZW016抗MM生物学效应; ④探究ZW016与MM临床治疗常用药物的联合效应。本实验完成将为MM新型治疗药物研发提供理论基础和潜在靶标。

传统中药青风藤主要成分青藤碱作为一种异喹啉生物碱，具有抗炎和免疫调节等多种药理作用。前期研究显示，青藤碱衍生物YL064（原代号ZW016）可以调变多发性骨髓瘤（MM）细胞内信号传导与转录激活因子3（STAT3）通路。鉴于此，本课题以MM细胞株和原代细胞为模型，观察YL064对MM细胞及其胞内STAT3的生物学效应，进一步探究YL064和STAT3相互作用方式及结合位点，并采用小鼠MM模型进行验证，同时拓展研究YL064联合二甲双胍对MM细胞生物学行为的影响及其可能机制。结果发现，YL064能够选择性诱导MM细胞株和原代细胞凋亡，并对与骨髓基质细胞共培养MM细胞也具有凋亡诱导效应；同时，YL064可以抑制MM细胞内STAT3持续性和IL-6诱导激活，进而阻碍STAT3核内转位及其调控基因表达，进一步分析研究显示，YL064能够直接靶向结合MM细胞内STAT3的SH2 结构域进而干扰其二聚体化；小鼠MM模型验证实验提示体内YL064能够抑制MM细胞内STAT3活化进而抑制其生长；此外，YL064联合二甲双胍能够协同诱导MM细胞增殖阻滞并进一步诱导其凋亡，其机制可能与两药协同促进细胞G2/M期阻滞以及上调SQSTM1/P62表达和抑制自噬通路相关。拓展研究还发现，YL064能够下调MM细胞内CXC亚家族趋化因子受体3（CXCR3）表达并抑制其下游信号通路进而诱导其凋亡。本课题所揭示的青藤碱衍生物对MM细胞及其胞内STAT3的调变效应为构想MM新型治疗方案和药物靶标提供实验和理论基础。