Quercetin

Quercetin, a bioactive plant-derived flavonoid abundant in fruits and vegetables, has shown remarkable promise in fighting melanoma, a deadly skin cancer. Research has revealed that quercetin can inhibit tumor growth without toxicity, boosting antitumor immunity by modulating the tumor immune microenvironment. This natural compound has been found to increase the percentages of immune cells, suppress cell proliferation and migration, and promote phagocytosis, making it a potential therapeutic agent for enhancing the efficacy of existing melanoma treatments. With its anti-inflammatory, antioxidant, and anticancer properties, quercetin holds great potential as a complementary therapy for melanoma, offering new hope for patients seeking effective and natural solutions.

Fig.1. Chemical structure of Quercetin

Quercetin's Anti-Tumor Effects in Live Models: Research demonstrated quercetin's ability to combat melanoma in vivo. Mice with subcutaneously implanted tumors received daily quercetin treatment for 18 days, resulting in significantly slowed melanoma development, suppressed tumor growth, and reduced tumor weight (Fig. 2A-2D). These findings confirm quercetin's potential to inhibit tumor growth in live models, offering promising implications for melanoma treatment.

Fig.2. Quercetin inhibited tumor growth in vivo. (A) Tumor-free rates of quercetin group and control group. (B) Tumor volume was measured with Vernier calipers every 2 days from the day of administration; the tumor volume changes are shown. (C) Representative tumor images are shown. (D) Tumor weights of quercetin group and control group (n ¼ 6).

Quercetin Boosts Melanoma Immunity: Quercetin enhances anti-tumor immunity by modulating the immune microenvironment in melanoma. Flow cytometry analysis revealed that quercetin treatment significantly increased macrophages, M1 macrophages, CD4+ T cells, and CD8+ T cells in tumors and spleens of tumor-bearing mice (Figs. 3A-2L). Additionally, quercetin stimulated CD8+ T cells to secrete higher levels of IL-2 and IFN-γ cytokines, crucial for fighting cancer. These findings demonstrate quercetin's ability to strengthen immune responses against melanoma.

Fig.3. Quercetin enhanced antitumor immunity. (A) The frequencies of macrophages in the tumors. (B) The frequencies of macrophages in the spleens. (C) The frequencies of M1 macrophages in the tumors. (D) The frequencies of M1 macrophages in the spleens. (E) The frequencies of CD4þ T cells in the tumors. (F) The frequencies of CD4þ T cells in the spleens. (G) The frequencies of CD8þ T cells in the tumors. (H) The frequencies of CD8þ T cells in the spleens. (I) The frequencies of IL-2 secreted by CD8þ T cells in the tumors. (J) The frequencies of IL-2 secreted by CD8þ T cells in the spleens. (K) The frequencies of IFN-γ secreted by CD8þ T cells in the tumors. (L) The frequencies of IFN-γ secreted by CD8þ T cells in the spleens.

Quercetin Suppresses Melanoma Cell Growth and Migration: Quercetin demonstrated potent anti-proliferative and anti-migratory effects on B16 melanoma cells. The CCK-8 assay revealed that quercetin significantly reduced B16 cell viability, with IC50 values of 454.77 μM (24h), 274.3 μM (48h), and 157.27 μM (72h) (Fig. 4A). CFSE staining and flow cytometry analysis showed increased mean fluorescence intensity (MFI) with rising quercetin concentrations, indicating inhibited cell proliferation (Fig. 4B). Wound healing assays (Fig. 4C) and transwell assays (Fig. 4D) further confirmed quercetin's ability to suppress B16 cell migration and invasion.

Fig.4. Quercetin inhibited the proliferation and migration of B16 cells. (A) Changes in cell viability of B16 cells after 24 h, 48 h, and 72 h treatment with quercetin. (B) The effects of quercetin on proliferation of B16 cells stained with CFSE. (C) Effects and quantification of quercetin’s effects on the migration capacity of B16 cells (scale bar 250 μm). (D) Effects and quantification of quercetin’s effects on the invasion capacity of B16 cells .

Uncovering Quercetin's Targets in Melanoma Inhibition: Network pharmacological analysis revealed potential targets of quercetin in melanoma treatment. This analysis identified 43 common targets (Fig.5A) and enriched GO terms (Fig. 5B), which were visualized as an intersection network highlighting quercetin, melanoma, and their targets (Fig. 5C). Notably, CD47 emerged as a key target in regulating cell proliferation (p=1.71E-05). To further investigate, RNA-Seq analysis was performed on quercetin-treated B16 cells, identifying 110 differentially expressed genes (DEGs), with 102 up-regulated and 8 down-regulated genes (Figs. 5D-5F). Phosphoinositide-dependent kinase 1 (PDK1) was identified as a crucial gene correlated with CD47. Previous studies have shown that quercetin inhibits PDK1 activation and CD47 expression decreases when PDK1 is down-regulated.

Fig.5. CD47 and PDK1 are potential targets of quercetin to inhibit melanoma. (A) Venn diagram of melanoma and quercetin targets. (B) GO analysis was performed on screened genes. The top 20 terms for BPs are shown. (C) The intersection network of the melanoma PPI network and quercetin PPI network was visualized using Cytos- cape. (D) Volcanic map of RNA-Seq results from control and quercetin-treated B16 cells, with 110 up-regulated DEGs and 8 down-regulated DEGs. DEGs were defined as having an FDR of less than 0.05. FC, fold change. (E) Histogram of RNA-Seq results from control and quercetin-treated B16 cells. (F) Heat map of 7 genes in up-regulated and down-regulated differentially expressed genes.

Quercetin's Mechanism of Action: Targeting PDK1/CD47 Axis in Melanoma: Research revealed that quercetin significantly decreases the protein and mRNA content of PDK1 and CD47 in melanoma cells (B16 and A375), as shown in Figures 6A-6D. To confirm PDK1's regulatory effect on CD47, siRNA experiments against PDK1 demonstrated decreased CD47 levels in B16 and A375 cells (Figures 6E-6H). Immunohistochemical staining of melanoma tissues from mice further supported this, showing reduced PDK1 and CD47 expressions in the quercetin-treated group compared to the control group (Figures 6I-6L). Additionally, rescue experiments overexpressing PDK1 in B16 cells, followed by quercetin treatment, showed increased CD47 levels reversed by quercetin (Figures 6M and 6N). These findings collectively suggest that quercetin down-regulates PDK1 to inhibit CD47 in melanoma, uncovering a critical mechanism of quercetin's anti-tumor effects.

Fig.6 Shows  the events how  Quercetin inhibited CD47 by down-regulating PDK1 in melanoma.

Quercetin Blocks CD47 Expression, Boosting Macrophage Phagocytosis: Quercetin inhibited CD47 expression in melanoma cells (B16 and A375) as shown by flow cytometry (Figs. 7A and 7B). Co-culturing quercetin-treated B16 cells with macrophages revealed enhanced phagocytosis (Figs. 7C and 7D). These findings demonstrate quercetin's ability to suppress CD47 and promote macrophage-mediated elimination of tumor cells.

Fig.7. Quercetin inhibited the expression of CD47 and enhanced phagocytosis by macrophages. (A) The MFI of CD47 in quercetin-treated B16 and control cells. (B) The MFI of CD47 in quercetin-treated A375 and control cells. (C) The percentages of CFSEþF4/80þ PMs that phagocytosed cancer cells. (D) The percentages of CFSEþF4/80þ BMDMs that phagocytosed cancer cells.

Quercetin Enhances CD8+ T Cell-Mediated Antitumor Immunity: Quercetin stimulates CD8+ T cell proliferation and cytokine secretion, boosting antitumor immunity. CD8+ T cells from quercetin-treated tumor-bearing mice showed increased IL-2 and IFN-γ secretion (Figs. 8A and 8B) and enhanced proliferation (Figs. 8C and 8D). This mechanism contributes to quercetin's antitumor effects in melanoma, illustrated in Fig. 8E.

Fig.8. Quercetin stimulated the secretion of IL-2 and IFN-γ and the proliferation of CD8þ T cells. (A and B) The changes in secreted IL-2 and IFN-γ levels from CD8þ T cells in co-culture system. (C) Quercetin promotes CD8þ T cell proliferation. (D) The MFI of CFSE in quercetin-treated CD8þ T and control cells. (E) The schematic diagram of the mechanism through which quercetin inhibits melanoma was drawn using Figdraw.

In short, this research demonstrates Quercetin's potent anti-tumor effects in melanoma by inhibiting tumor growth and enhancing antitumor immunity. Quercetin achieves this through multiple mechanisms: promoting immune cell infiltration, stimulating cytokine secretion, inhibiting tumor cell proliferation, and blocking CD47 expression via the PDK1/CD47 axis, thereby enhancing macrophage phagocytosis. Overall, Quercetin shows significant potential as a natural compound for melanoma treatment and prevention, warranting further clinical investigation.