The anti-melanoma roles and mechanisms of tricholoma isoflavone derivative CA028

Abstract

As a form of skin cancer, melanoma's incidence rate is continuing to rise globally. Therefore, there is an urgent need to find new agents to improve survival in melanoma patients. Isoflavones, a class of phytoestrogens, are primarily found in soy and other legumes. Cumulating evidence demonstrates that isoflavones exhibits significant anti-tumor properties and is beneficial for the prevention and treatment of melanoma. In the present study, we aim to investigate the anti-melanoma role of tricholoma isoflavone derivative CA028. By using in vitro melanoma cell line models, A375 and A2058 and in vivo xenograft mouse model, our results indicate that melanoma proliferation, migration, and invasion are attenuated following CA028 treatment. In addition, the treatment of CA028 induced cell apoptosis of melanoma. Finally, we addressed the mechanism of CA028 against melanoma by comparative transcriptomic analysis. The results of gene ontology highlighted the involvement of CA028's targets in the cell proliferation, cell apoptosis, and migration ability of melanoma cells. Furthermore, Ingenuity Pathway Analysis constructed the network involved in the apoptotic roles of CA028 through targeting p53 signaling and death receptor signaling. For the first time, our data suggested the possible use of modified isoflavone for therapeutic applications against melanoma.

Fig. 1. CA028 inhibited the proliferation of melanoma cells.

A CCK8 proliferation assay showed the inhibition of proliferation of melanoma cells A375 and A2058 caused by CA028 treatment. B Colony formation showed the reduced colony formation ability of melanoma cells A375 and A2058 caused by CA028 treatment. C Annexin V/PI staining followed by flow cytometry analysis showed the induction of melanoma cell apoptosis caused by CA028 treatment. D Hoechst33258 staining showed that the treatment of CA028 induced the % of apoptotic melanoma cells.

Fig. 2. CA028 inhibited migration and invasion ability of melanoma cells.

A Wound haling assay showed the reduced migration ability of melanoma cell caused by CA028 treatment. B Migration assay showed the reduced migration ability of melanoma cell caused by CA028 treatment. C Invasion assay showed the reduced invasiveness of of melanoma cell caused by CA028 treatment.

Fig. 3. CA028 regulated the genes involved in proliferation, apoptosis, and migration of melanoma cells.

A Comparative transcriptome sequencing demonstrated the differential gene expression in melanoma cell A375 under CA028 treatment. Blue dots represented downregulated genes; red dots represented upregulated genes. Cutoff: 1<log2 fold change < –1 and log10 q-value > 1.3. Gene ontology (GO) enrichment analysis highlighted the roles of CA028’ target genes in (B) DNA damage and cell cycle and C cell death and apoptosis. D Cricos plot showed the involvement of genes in the selected GO terms. GO enrichment analysis highlighted the roles of CA028’ target genes in (E) cell growth and proliferation and F wound healing and cell migration. The size of bubble represented the number of genes involved in the biological processes. The color of bubble represented the significance of the biological processes.

Fig. 4. CA028 regulated the cell signaling involved in carcinogenicity of melanoma.

KEGG pathway enrichment analysis showed the involvement of CA028’s targets in A carcinogenicity, B cancer signaling, and C apoptosis and metabolisms. The size of bubble represented the number of genes involved in the pathways. The color of bubble represented the significance of the pathways. D Ingenuity Pathway Analysis highlighted the importance of CA028’s targets in cell death and DNA damage response. E Gene network showed the anti-melanoma roles of CA028.

Fig. 5. Validation of transcriptome sequencing’s result.

A qPCR analysis was used to determine the expression of anti-proliferation genes (BAK1 and P21) and anti-migration and -invasion genes (SUFU and BMP4). B immunohistochemical staining was used to determine the change of protein expression of SUFU and P21 in melanoma after CA028 treatment.

Fig. 6. CA028 reduced the growth of melanoma in the nude mice model.

A The use of CA028 significantly reduced the tumor growth of melanoma raised by A2058 in nude mice as compared with control group. B The use of CA028 attenuated the reduced body weight in nude mice inoculated with A2058 melanoma cells.