LingHu Hai Rui, Luo Hui, Gang Lin
Department of Neurosurgery and Neurocritical Care, Beijing Chaoyang Integrative Medicine Emergency Medical Center, Beijing 100022, People's Republic of China.
Characteristic Medical Center of Chinese People's Armed Police Force (PAP), Tianjin 300162, People's Republic of China.
Onco Targets Ther. 2020 May 27;13:4767-4778. doi: 10.2147/OTT.S242567. eCollection 2020.
Bufalin is a component of Chinese traditional medicine, Chansu, which is reported to induce cell death among various kinds of tumors. Apoptosis evasion is a common problem of cancer treatment.
The proliferation of U-87 and U-373 treated by bufalin combined with or without apoptosis inhibitor was detected by MTT assay. The protein levels related to apoptosis and necroptosis were measured by Western blotting. Immunoprecipitation (IP) was applied for monitoring the formation of necrosome. The gene knockdown by CRISPR/Cas9 was applied to determine the roles of the proteins in apoptosis and necroptosis.
In this study, we found that bufalin could induce apoptosis or necroptosis when U-87 and U-373 escaped from apoptosis. Bufalin triggered cell death by upregulating tumor necrosis factor (TNF) -α, TNF receptor 1 (TNFR1) and receptor-interacting protein 1 (RIPK1). Antagonizing cellular inhibitor of apoptosis 1 (cIAP1) and cIAP2 were also contributory. Caspase-8 activation led to apoptosis. When caspase-8 was functionally lost, necrosome consisted of RIPK1, receptor-interacting protein 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL) formed and necroptosis happened. The knockdown of above genes or the drug treatment confirmed the mechanism of bufalin-induced cell death. Cytotoxicity of bufalin to caspase-8 knockdown cell lines made control cell lines more sensitive to bufalin in their mixture.
The cytotoxicity of bufalin to U-87 and U-373 was by inducing apoptosis or necroptosis when they were sensitive to apoptosis or not. The results indicated that seeking for treatments that could induce apoptosis and necroptosis was a good solution for the tumor evasion of apoptosis.
蟾毒灵是中药蟾酥的一种成分，据报道其可诱导多种肿瘤细胞死亡。逃避凋亡是癌症治疗中的一个常见问题。
采用MTT法检测蟾毒灵联合或不联合凋亡抑制剂处理的U - 87和U - 373细胞的增殖情况。通过蛋白质免疫印迹法检测与凋亡和坏死性凋亡相关的蛋白水平。应用免疫沉淀法监测坏死小体的形成。采用CRISPR/Cas9基因敲除技术确定这些蛋白在凋亡和坏死性凋亡中的作用。
在本研究中，我们发现当U - 87和U - 373逃避凋亡时，蟾毒灵可诱导其凋亡或坏死性凋亡。蟾毒灵通过上调肿瘤坏死因子（TNF）-α、TNF受体1（TNFR1）和受体相互作用蛋白1（RIPK1）引发细胞死亡。拮抗细胞凋亡抑制蛋白1（cIAP1）和cIAP2也有作用。半胱天冬酶 - 8的激活导致凋亡。当半胱天冬酶 - 8功能丧失时，由RIPK1、受体相互作用蛋白3（RIPK3）和混合谱系激酶结构域样蛋白（MLKL）组成的坏死小体形成，进而发生坏死性凋亡。上述基因的敲除或药物处理证实了蟾毒灵诱导细胞死亡的机制。蟾毒灵对敲除半胱天冬酶 - 8的细胞系的细胞毒性使得对照细胞系在混合体系中对蟾毒灵更敏感。
当U - 87和U - 373对凋亡敏感或不敏感时，蟾毒灵对它们的细胞毒性是通过诱导凋亡或坏死性凋亡实现的。结果表明，寻找能够诱导凋亡和坏死性凋亡的治疗方法是解决肿瘤凋亡逃避问题的一个良好方案。
