The Effects of Combination Treatment of Silibinin and Chrysin on the Induction of Immunogenic Cell Death in B16 cells (Melanoma Cell Line)

Abstract

Background: Induction of immunogenic cell death (ICD), a type of apoptosis, is a promising strategy in cancer immunotherapy. Silibinin and chrysin, belonging to flavonoids, can induce ICD in monotherapy trials. Also, their anticancer effect is already known.Aim: The present study intended to examine whether silibinin-chrysin combination can induce a synergistic effect in induction of ICD in the B16F10 cancer cell line.Material and Methods: The growth inhibitory effects of the combination of silibinin and chrysin were determined using MTT assay and trypan blue exclusion dye assays. Flow cytometry was used to study cell apoptosis and examine calreticulin (CRT) surface exposure. The levels of heat shock protein 90 (HSP90) have been measured by ELISA. The Protein level of elf-2α and p-elf-2α was examined by western blotting analysis.Results: Treatment of B16F10 cells with the mentioned combination caused synergistic anticancer effects, with a combination index (CI) of 0.48869. Combination therapy promoted key markers of ICD compared to monotherapy. CRT expression was 44.75±4.2% in silibinin-treated (80µM) and 59.72± 3.3 % in chrysin-treated cells (40µM). A significant increase was observed (94.2±5.01%) in combination therapy with silibinin (80µM) and chrysin (40µM) compared to the monotherapies (p<0.001). Combining silibinin and chrysin significantly increased apoptosis to 80.59± 4.9% from 47.26 ±6.05 and 39.42 ±5.7 for silibinin and chrysin alone. Also, HSP90 levels were 5.69±0.38 in the silibinin-treated cells and 8.44 ±0.29 in the chrysin-treated cells. The combination therapy potentiates HSP90 level to 14.36 ±0.53 compared to monotherapies (p<0.001). Compared to the control group, p-eIF2α levels were enhanced in the cells exposed to monotherapies (p<0.001), and this elevation was intensified in combination therapy compared with monotherapies (p<0.01 and p<0.001).Conclusion: Based on our research combination therapy with silibinin and chrysin induces a synergistic effect in the induction of ICD in B16F10 melanoma cells.