盐霉素抑制骨肉瘤细胞的作用机制研究及靶向增敏

The malignant/recurrent osteoblastoma (OS) has poor prognosis, thus the search for novel and more efficient chemo-agents against this disease is urgent. Our previous studies have shown that salinomycin, the novel anti-cancer drug, induces growth inhibition, cell cycle arrest, apoptosis and autophagy in cultured OS cells, which was associated with reactive oxygen species (ROS) production, mitochondrial permeability transition pore (mPTP) opening and cytochrome c release as well as a profound AMP-activated protein kinase (AMPK) and JNK activation. Interestingly, AMPK inhibition by its inhibitor compound C, or by AMPKα RNAi prevented salinomycin-induced autophagy activation, while facilitating cancer cell death and apoptosis. Thus, we propose that salinomycin activates AMPK signaling in osteoblastoma cells, which serves as a negative regulator against cell apoptosis. AMPK inhibition might be a novel strategy to sensitize salinomycin’s effect in cancer cells. In the current proposal, by using multiple biomedical assays, we are set to test the anti-OS effect of salinomycin both in vivo and in vitro, and to study the underlying mechanism by focusing on ROS production, mPTP opening and JNK’s involvements. We will elucidate the relationship between salinomycin-induced AMPK activation and OS cell apoptosis. Experiments will also be performed to test the upstream kinase for salinomycin-induced AMPK activation, and its possible downstream targets. Thus, AMPK inhibition might be a novel strategy to sensitize salinomycin’s effect in cancer cells. This results of this study will provide theoretical and experimental evidence for using salinomycin as a novel anti-OS agent.

寻找有效的化学药物治疗复发性及耐受性骨肉瘤（OS）是目前OS研究的热点。预实验结果显示盐霉素（Salinomycin）显著抑制OS细胞存活，并诱导OS细胞凋亡和自噬。机制方面：在OS细胞中，盐霉素诱导ROS生成、线粒体通道mPTP开放、细胞色素C释放，并激活JNK和AMPK信号。而抑制AMPK则显著降低盐霉素诱导的OS细胞自噬，并增加OS细胞凋亡。因此，我们提出盐霉素诱导的AMPK活化，后者作为负反馈调节因子抑制OS细胞凋亡；而且通过干预AMPK信号通路可增加OS细胞对盐霉素的敏感性。本项目中我们将体内、外系统观察盐霉素诱导的抗OS作用，解析其作用机制，明确ROS生成、mPTP开放及JNK激活等在其中的作用；阐明盐霉素激活AMPK-自噬通路与OS细胞凋亡的关系，并通过调节AMPK-自噬通路增敏盐霉素；明确盐霉素诱导AMPK活化的分子机制及下游靶点。旨在为OS治疗学的突破进行有益探索。

骨肉瘤是最常见的原发性骨恶性肿瘤，寻找更加有效的化疗药物治疗复发性及转移性恶性骨肉瘤迫在眉睫。申请者前期研究结果证实，盐霉素能显著抑制OS细胞的存活，并诱导癌细胞周期停滞和凋亡。DNA-PKcs作为骨肉瘤细胞中盐霉素的主要抗性因子，DNA-PKcs抑制剂（NU7026，NU7441和LY294002）或DNA-PKcs shRNA敲除后，可显著增强盐霉素诱导的死亡和细胞凋亡（U2OS和MG-63骨肉瘤细胞系）。microRNA-101的强制表达下调DNA-PKcs后，可增敏盐霉素抗骨肉瘤细胞的作用；而DNA-PKcs过度表达后，则抑制盐霉素对骨肉瘤细胞的毒性作用。机制研究结果显示，DNA-PKcs是盐霉素诱导促生存自噬活动所必需的。DNA-PKcs抑制剂、DNA-PKcs shRNA干扰以及miR-101表达均可抑制盐霉素诱导的Beclin1表达和自噬发生，同时Beclin1 shRNA增敏盐霉素诱导的抗骨肉瘤细胞的作用。在体实验研究显示，盐霉素给药后，U2OS移植瘤的在体生长受到抑制，与DNA-PKcs联合用药后，其抗肿瘤活性明显增强。DNA-PKcs抑制剂或沉默DNA-PKcs后可增敏盐霉素抗骨肉瘤细胞的作用。