Study of the induction of immunogenic cell death by chrysin and chrysin-loaded nano-carriers on B16 murine melanoma cells

Abstract

Introduction: Chrysin, a flavonoid with potential in cancer treatment, shows promise as a strategy to overcome the limitations of cancer immunotherapy by inducing immunogenic cell death (ICD) and therefor enhancing the effectiveness of chemotherapy. However, poor water solubility of chrysin results in low bioavailability and therapeutic efficacy of this compound. In the present study, the potency of chrysin and chrysin-loaded micelles in inducing of ICD in the B16 melanoma cell line were assessed. Objective: Studying the inhibitory effect of chrysin and chrysin-loaded micelles on B16 melanoma cells.Methods: The growth inhibitory effects of chrysin and chrysin-loaded micelles were evaluated using MTT and trypan blue exclusion dye assays. Cell apoptosis was studied through an Annexin V/PI assay conducted with flow cytometry. Flow cytometry was also utilized to assess the surface exposure of Calreticulin (CRT). The levels of heat shock protein 90 (HSP90) and Annexin A1 were measured using an enzyme-linked immunosorbent assay (ELISA). Western blotting was performed to examine the levels of GRP78 (Glucose related protein 78) and p-PERK (the activated form of PERK). Results: Evaluation of the cytotoxicity and apoptosis induced by chrysin and chrysin-loaded micelles showed a significant difference between them, favoring the nano formulation. The presence of damage-associated molecular patterns (DAMPs), including calreticulin, HSP90 and Annexin A1, along with higher levels of activated PERK, indicate that chrysin and chrysin-loaded micelles are able to induce ICD. Lower levels of GRP78 after treatment suggest a possible pathway of action for the apoptotic effects of chrysin. Conclusion: The Findings of this study show that chrysin and nano-chrysin can induce ER stress and, consequently, ICD in B16 cells.