OncoTargets and Therapy (May 2020)
Bufalin Induces Glioma Cell Death by Apoptosis or Necroptosis
Abstract
Hai Rui LingHu,1,* Hui Luo,2,* Lin Gang2,* 1Department of Neurosurgery and Neurocritical Care, Beijing Chaoyang Integrative Medicine Emergency Medical Center, Beijing 100022, People’s Republic of China; 2Characteristic Medical Center of Chinese People’s Armed Police Force (PAP), Tianjin 300162, People’s Republic of China*These authors contributed equally to this workCorrespondence: Hai Rui LingHuDepartment of Neurosurgery and Neurocritical Care, Beijing Chaoyang Integrative Medicine Emergency Medical Center, Chaoyang District, Beijing 100022, People’s Republic of ChinaTel +8613622098542Email [email protected]: 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.Materials and Methods: 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.Results: 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.Discussion: 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.Keywords: glioma, bufalin, RIPK1, apoptosis, necroptosis
