二氢杨梅素靶向Wnt和NF-κB信号通路杀灭骨肉瘤干细胞的机制研究

A considerable number of osteosarcoma patients either are not sensitive to chemotherapy or develop drug resistance to current chemotherapy regimens. Therefore, it is necessary to develop several potentially useful therapeutic agents. The previous study examined the anti-tumor activity both in vitro and in vivo without noticeable side effects and the underlying mechanism of action of dihydromyricetin (DMY) in osteosarcoma cells. In this study, we investigated DMY induced increased p21 expression and G2/M cell cycle arrest, caused DNA damage, activated ATM/CHK2/H2AX signaling pathways, and induced apoptosis in osteosarcoma cells. Mechanistic analysis showed that the anti-tumor potential of DMY may be due to the activation of AMPKα and p38MAPK, as the activating AMPKα led to the inactivation of GSK-3β in osteosarcoma cells. Moreover, GSK-3β deletion or GSK-3β inhibition by LiCl treatment resulted in increased p21 expression and reduced Sox2 expression in osteosarcoma cells. Taken together, our results strongly indicate that the ant-tumor potential of DMY is correlated with P38 MAPK and AMPKα/GSK-3β/Sox2 signaling pathway. We also demonstrated that DMY significantly reduced the sphere formation capability by downregulating Sox2 expression. Sox2 plays a key role in stem cell maintenance characteristics, the possibleinhibition of stem cell osteosarcoma. The Wnt and NF- κ B pathways were activated in osteosarcoma stem cells, they play a key role in maintaining stem cellcharacteristics, and GSK-3 β play an important role in regulatoring Wnt pathway and regulation of NF- κ B. Ultimately,the topic aims at providing theorem for DMY preventing osteosarcoma,and laying the foundation for exploitation of drugs targeted osteosarcoma stem cell.

由于化疗耐药的原因，有必要开发新药物。我们研究表明二氢杨梅素具有良好的体内外抗骨肉瘤细胞和干细胞活性且无明显的副作用；发现其能诱导p21表达增加，G2/ M期阻滞，引起DNA损伤，激活ATM/Chk2/H2AX的信号转导途径，并诱导骨肉瘤细胞凋亡。深入机制研究结果表明，二氢杨梅素抗肿瘤可能通过p38MAPK和AMPKα/GSK-3β/Sox2的信号通路，Sox2在干细胞维持特性中起关键作用，部分说明了其抑制干细胞的作用。而骨肉瘤干细胞中Wnt和NF-κB通路活性增强，二者对维持其特性起关键作用， GSK-3β是Wnt的调节因子且调控NF-κB发挥重要作用，提示：二氢杨梅素是否靶向Wnt和NF-κB杀灭骨肉瘤干细胞？本研究拟通过研究二氢杨梅素作用于Wnt和NF-κB信号通路的情况，明确其拮抗骨肉瘤干细胞的关键蛋白靶点，探讨作用机制，在体内实验证实，为开发针对骨肉瘤干细胞的治疗药物打下基础。

由于化疗耐药的原因，有必要开发新药物。研究从藤茶中提取出来的黄酮类化合物-二氢杨梅素抗骨肉瘤效果及相应机制。发现二氢杨梅素对骨肉瘤细胞U2OS、MG63、Saos2、HOS、143B、ZOS、ZOS-M 及耐药性骨肉瘤细胞U2OS/MTX300具有良好的体内外抗骨肉瘤细胞和干细胞活性且无明显的副作用，二氢杨梅素能诱导p21蛋白表达增加，G2/ M期阻滞，引起DNA损伤，激活ATM/Chk2/H2AX的信号转导途径，并诱导骨肉瘤细胞凋亡。深入机制研究结果表明，二氢杨梅素抗骨肉瘤作用与p38MAPK和AMPKα/GSK-3β/Sox2的信号通路相关，Sox2在干细胞维持特性中起关键作用，部分说明了其抑制干细胞的作用。而骨肉瘤干细胞中Wnt和NF-κB通路活性增强，二者对维持其特性起关键作用，GSK-3β是Wnt的调节因子且调控NF-κB发挥重要作用。研究还发现二氢杨梅素在发挥体内抑瘤作用的同时，对裸鼠的心、肝、肾等重要器官无明显毒副作用。二氢杨梅素与传统化疗药物阿霉素联合应用后，不但能起到协同体内抑瘤作用，而且能降低阿霉素对体重的影响和对机体重要脏器的损伤。项目研究结果，为开发新型骨肉瘤治疗的药物打下了基础，应用前景广阔。