Effects of Citrus-derived Diosmetin on Melanoma: Induction of Apoptosis and Autophagy Mediated by PI3K/Akt/mTOR Pathway Inhibition

Abstract

Background: Diosmetin (DIOS) is a naturally abundant flavonoid and possesses various biological activities that hold promise as an anti-cancer agent. However, the anti-cancer activities and underlying mechanism of DIOS on cutaneous melanoma remain unclear.

Objective: This study seeks to explore the anti-tumor effect and mechanism of DIOS in cutaneous melanoma.

Methods: Here, a variety of in vitro and in vivo experiments, combined with RNA sequencing (RNA-seq), were employed to ascertain the potential anti-cutaneous melanoma capacity and mechanism of DIOS.

Results: The results demonstrated that DIOS considerably impeded cell proliferation and triggered cell apoptosis in a dose- and time-dependent manner. Concurrently, DIOS markedly elevated the expression of pro-apoptotic proteins (Cleaved caspase-3, Bax, Cleaved PARP, and Cleaved caspase-9) and downregulated the expression of Bcl-2. Additionally, DIOS markedly upregulated the protein expressions of LC3B-II and Atg5, while downregulating p62 protein expression. Notably, pre-treatment with an autophagy inhibitor significantly inhibited DIOSinduced cell apoptosis and autophagy. Mechanistically, DIOS was identified to repress the PI3K/Akt/mTOR signaling pathway by western blot analyses and RNA-seq. Finally, in vivo experiments using a syngeneic mouse model confirmed the anti-tumor effect of DIOS, which exhibited high levels of apoptosis and autophagy.

Conclusion: These findings propose that DIOS acts as a potential melanoma therapy that exerts its anti-tumor effects by triggering apoptosis and autophagy via inhibition of the PI3K/Akt/mTOR pathway.

Keywords: Diosmetin; PI3K/Akt/mTOR; anti-tumor.; apoptosis; autophagy; cutaneous melanoma.

Fig. (1)

DIOS inhibits the proliferation of A375 and SK-MEL-28 cells in vitro. (A) Chemical structure of Diosmetin (DIOS). (B) Effect of DIOS on cell viability. (C) Effect of DIOS on cell morphological change (magnification, 20×). (D) Effect of DIOS on cell clonogenesis capacity. (E) Effect of DIOS on cell migration and quantitation of cell migration area. (F) Effect of DIOS on cell proliferation detected by EdU assay. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group.

Fig. (2)

DIOS induces apoptosis in cutaneous melanoma cells in vitro. (A, B) Cell apoptosis evaluation of cell lines treated with DIOS by flow cytometry. (C) The characteristics of apoptotic nuclei in cells were observed by Calcein/PI staining. scale bar =200 μm. (D, E) The expressions of apoptotic protein. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group.

Fig. (3)

DIOS induces autophagy in melanoma cells in vitro. (A) A375 cells were exposed to 0 and 40 μM DIOS for 48 hours, and the cellular ultrastructure was examined by transmission electron microscopy (red arrow indicates autolysosome). (B) DIOS promoted autophagic flux in A375 and SK-MEL-28 cells. Cells were infected with 20 MOI for 24 h. Then the infected cells were treated with 40 μM DIOS for 48 h, followed by fluorescence microscopy observation. Bar = 50 μm. (C, D) The protein expressions of autophagy-related proteins p62, Atg5, and LC3B were measured. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group.

Fig. (4)

3-MA inhibits DIOS-induced apoptosis and autophagy. (A, B) Detection of apoptosis in melanoma cells treated with DIOS (40 μM) in the presence or absence of 3-MA (5 μM) by flow cytometry. (C, D) The protein expressions of p62, Atg 5, LC3B I, and LC3B II. (E, F) Quantitative analysis of these proteins. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group, # p < 0.05, ## p < 0.01 vs. the DIOS (40 μM) group.

Fig. (5)

DIOS induces apoptosis and autophagy via the PI3K/Akt/mTOR pathway. (A) A375 cells were exposed to 40 µM DIOS for 48 hours and then analyzed through transcriptome sequencing. The cluster analysis heat map of down-regulated differential genes. (B, C) GO and KEGG Pathway Enrichment Analysis. (D) GSEA showed that the PI3K/Akt Targets pathway was mainly enriched, and DIOS significantly inhibited this pathway. (E, F) The protein expression levels of p-PI3K, PI3K, p-Akt, Akt, p-mTOR and mTOR. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group.

Fig. (6)

DIOS exerts an anti-melanoma effect in vivo. (A) Experimental design. (B) Morphological images of subcutaneous tumor xenografts from C57BL/6 mice. (C, D) Quantitative analysis of tumor weight and tumor volume (n=5). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group. (E) HE staining of the important viscera, original magnification: 20×, scale bar =100 μm. (F) TUNEL assay, original magnification: 20×, scale bar =100 μm. (G) Immunohistochemical detection of KI67, LC3B, Cleaved caspase 3, P-Akt expression. Original magnification: 40×, scale bar: 50 μm.