Capsaicin Induces Immunogenic Cell Death In Human Osteosarcoma Cells

Background:Osteosarcoma, which is the most common primary malignant bone tumor, occurs most frequently in adolescent. The incidence of adolescent is 8-11 cases/million population/year. Prior to the 1980s, the relative 5-year survival rate of patients with osteosarcoma was approximately 20%. With the introduction of chemotherapy into the multi-modal treatment for osteosarcoma, the survival rate is greatly improved to approximately 60%?80%. However, improvements in osteosarcoma survival during the last decade have been limited and the side effect of osteosarcoma chemotherapy drug is very serious. Clearly, the development of new osteosarcoma chemotherapy drugs has very important significance.Immunogenic cell death (ICD) of tumor cells means a cell death modality, which is not only able to cause cell apoptosis but also stimulate response against homologous tumor cells. ICD is characterized by a series of alterations that usually do not occur in the context of apoptosis: (i) the pre-apoptotic exposure of calreticulin (CRT) on the cell surface, (ii) release of ATP during the blebbing phase of apoptosis, and (iii) post-apoptotic exodus of the chromatinbinding protein high mobility group B1 (HMGB1). Up to now only a few kinds of anti-cancer chemicals were found to induce ICD, so it has important clinical significance to explore the new chemicals that can induce ICD of osteosarcoma.Objective:The purpose of this research is to study the growth inhibition and apoptosis effect of capsaicin and cisplatin on osteosarcoma cells, and then study the mechanism of actions, further investigate whether capsaicin and cisplatin can induce osteosarcoma cells immunogenic cell death or not. This lay a good foundation for the development of new osteosarcoma chemotherapy drugs.Methods:Part ?:In this study, MTT assay was used to examine the growth inhibiting effects of MG-63 cells when they were treated by capsaicin or cisplatin. DNA ladder, Mitochondrial membrane potential (MMP) and western blot analysis were used to investigate the capsaicin- and cisplatin- induced apoptotosis.Part ?:Capsaicin and cisplatin acted on the MG-63 cells 12 or 24 hours later, flow cytometry was used to detect the expression of CRT on the cell membrane, fluorescein enzymatic method was used to detect the release of ATP, and ELISA was used to detect the secretion of HMGB1. After co-incubation of capsaicin or cisplatin treated MG-63 cells with DC cells, the phagocytic efficiency of the phagocytes on MG-63 cells was detected and induced T cell's ability in secreting IL-4 and IFN-y was analyzed. These data can used to identify whether capsaicin induce ICD in human osteosarcoma cells or not.Results:Part ?:MTT results showed that 200?M capsaicin and 32?g/ml cisplatin can prevent 50% of MG-63 cells proliferation. DNA ladder, Mitochondrial membrane potential (MMP) and western blot analysis were showed that capsaicin and cisplatin can induce MG-63 cells apoptotosis and by Bcl-2 pathway.Part ?:In early apoptosis, capsaicin induced translocation of CRT from endoplasmic reticulum to the cell surface. In late apoptosis, capsaicin induced secretion of extracellular ATP and HMGB1. Capsaicin-treatment increased phagocytosis of MG-63 cells by dendritic cells (DCs) and these MG-63-loaded DCs could efficiently upregulate the secretion of IFN-y in the lymphocytes.Conclusion: These data demonstrate that capsaicin can induce apoptosis and immunogenic cell death in osteosarcoma cells.