Fisetin Induces Apoptosis and Inhibits Migration of Melanoma Cells by Suppressing Glycolytic Metabolism via PKM2 Inhibition

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Melanoma is one of the most aggressive forms of skin cancer, often exhibiting resistance to conventional therapies and a high rate of metastasis¹,². The Warburg effect, a hallmark of cancer metabolism, is characterized by increased glycolysis even under normoxic conditions, facilitating rapid energy production and biosynthesis in tumor cells³. Pyruvate kinase M2 (PKM2) is a key enzyme that catalyzes the final step of glycolysis and is predominantly expressed in proliferating cancer cells, including melanoma⁴. Beyond its metabolic role, PKM2 also modulates gene expression and cell signaling pathways, contributing to tumor growth and metastasis⁴,⁵. Given this central role, PKM2 is considered a promising therapeutic target for metabolic intervention in cancer⁴,⁵.

Fisetin (3,3',4',7-tetrahydroxyflavone) is a naturally occurring flavonoid found in various fruits and vegetables with reported anti-inflammatory, antioxidant, and anticancer properties (Fig. 1A). Previous studies have demonstrated that fisetin can inhibit proliferation, induce apoptosis, and modulate signaling pathways, including PI3K/Akt and MAPK, in various cancer types⁶. However, the specific metabolic effects of fisetin, especially its impact on glycolysis and PKM2 activity in melanoma cells, remain poorly understood. In this study, we investigated the expression level of PKM2 in human melanoma tissue and explored the anticancer effects of fisetin against melanoma through PKM2-mediated glycolytic metabolism.

Fig. 1. PKM2 overexpression in human melanoma tissue, and effects of fisetin on melanoma cell survival, migration and apoptosis through glycolysis inhibition. (A) Chemical structure of fisetin. (B) Western blot analysis of PKM2 protein expression in normal skin tissue vs. melanoma tissue. Quantitative densitometric analysis is shown in the bar graph. (C) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability assay of melanoma cells treated with fisetin (0–120 μM) for 48 hours. (D) 4′,6-Diamidino-2-phenylindole nuclear staining of melanoma cells treated with fisetin (0, 10, 20, 30 μM) for 48 hours. Quantitative analysis of apoptotic cell percentage is presented in the bar graph. (E) Western blot analysis of apoptosis markers (cleaved caspase-3, caspase-3, cleaved PARP) in melanoma cells treated with fisetin (0, 10, 20, 30 μM) for 48 hours. Quantitative analysis of caspase-3/7 activity is shown in the bar graph. (F) Wound healing assay of G361 cells treated with fisetin (0, 10, 20, 30 μM) at 48 hours with quantitative analysis of wound closure percentage. (G) Western blot analysis of PKM2 expression in G361 cells treated with fisetin (0, 10, 20, 30 μM) under control siRNA (si-control) and PKM2 knockdown (si-PKM2) conditions (left panel), with quantitative analysis of cell viability (middle panel). Western blot analysis of key glycolytic enzymes (HK-II, PFKP, PDH) in G361 cells treated with fisetin (0, 10, 20, 30 μM) for 48 hours (right panel). (H) Functional metabolic assays showing fisetin-induced suppression of glycolysis. Intracellular ATP levels and HK enzymatic activity were measured in G361 cells treated with fisetin (0, 10, 20, 30 μM) for 24 hours and expressed as percentages of untreated controls. The quantitative data were presented as mean ± standard deviation.

PKM2: pyruvate kinase M2, fisetin: 3,3',4',7-tetrahydroxyflavone, PARP: poly(ADP-ribose) polymerase, HK: hexokinase, PFKP: phosphofructokinase platelet type, PDH: pyruvate dehydrogenase, si: small interfering, IC₅₀: half-maximal inhibitory concentration.

^*p<0.05 compared to untreated controls.

PKM2 expression level was analyzed in 6 malignant melanoma tissues and 6 matched normal skin tissues ex vivo. Details about the materials and methods are provided in the Supplementary Data 1. Western blot analysis revealed significantly higher expression of PKM2 proteins in all melanoma tissues vs. normal skin tissues (Fig. 1B). The relative protein expressions were statistically significant (Mann-Whitney U test, p<0.05). The median score for PKM2 in melanoma vs. normal skin was 0.865 (0.703–1.107) vs. 0.496 (0.321–0.539).

To identify the anticancer effects of fisetin in melanoma, G361 melanoma cells were treated with fisetin (0–120 µM) for 24–48 hours. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed a dose-dependent decrease in cell viability, with half-maximal inhibitory concentration values within the 10–60 μM range—previously reported as biologically relevant concentrations for fisetin (Fig. 1C). Nuclear morphology analyses using 4′,6-diamidino-2-phenylindole staining showed that fisetin increased the proportion of cells with amorphous and fragmented nuclei in a concentration-dependent manner, with the most pronounced effects observed at 30 µM concentrations (Fig. 1D). Consistent with these observations, western blot analysis showed increased cleavage of poly(ADP-ribose) polymerase (PARP), with elevated levels of cleaved PARP together with cleaved caspase-3, indicating apoptosis induction (Fig. 1E). Furthermore, caspase-3/7 activity assays showed a significant concentration-dependent elevation, supporting enhanced apoptotic signaling upon fisetin exposure.

Wound healing assays confirmed that fisetin treatment markedly inhibited cell migration in a concentration-dependent manner, as evidenced by reduced wound closure over 48 hours (Fig. 1F). Quantitative analysis revealed a significant decrease in migration distance with increasing fisetin concentrations, with migration rates reduced by approximately 60%–70% at higher concentrations compared to control groups. To elucidate the metabolic mechanisms underlying fisetin’s anti-melanoma effects, we examined its impact on PKM2 and glycolytic enzymes. Fisetin dose-dependently suppressed PKM2 expression in G361 cells, an effect that remained evident even under PKM2-knockdown conditions (Fig. 1G). Notably, fisetin continued to reduce cell viability in si-PKM2 cells, indicating that fisetin may exert cytotoxic effects through PKM2-independent mechanisms. Western blot analysis further showed reduced expression of key glycolytic enzymes hexokinase (HK)-II and phosphofructokinase platelet type, while pyruvate dehydrogenase levels remained unchanged. Fisetin also functionally suppressed glycolysis, as shown by dose-dependent reductions in intracellular ATP levels and HK activity (Fig. 1H). These findings suggest that fisetin modulates melanoma cell viability through multiple pathways, with PKM2-related glycolytic regulation acting as one contributing mechanism rather than the sole determinant of its cytotoxic effects.

Previous studies have highlighted melanoma’s dependence on glycolytic metabolism², with elevated PKM2 expression playing a central role in sustaining rapid proliferation and survival⁴. Our findings confirm that PKM2 is markedly overexpressed in melanoma tissues, reinforcing its role in contributing to the metabolic phenotype. Fisetin treatment significantly reduced PKM2 levels and downregulated other key glycolytic enzymes, suggesting that fisetin contributes to the disruption of glycolytic metabolism, potentially impairing the primary energy-producing pathway in melanoma cells⁷,⁸. This metabolic inhibition was further supported by reduced ATP production and HK activity following fisetin treatment. The observed effects of fisetin—apoptosis induction, migration inhibition, and downregulation of glycolysis-related proteins—highlight its potential as a metabolic inhibitor.

Beyond inducing metabolic stress and apoptosis, fisetin effectively inhibited melanoma cell migration. Given that glycolytic intermediates and PKM2 are implicated in promoting cytoskeletal remodeling and epithelial-mesenchymal transition signaling pathways⁹, fisetin’s modulation of glycolytic pathways may help restrain metastatic potential. The disruption of glycolytic flux by fisetin may also interfere with the energy-intensive processes required for cellular invasion and metastatic colonization⁷. Considering that metastasis is a major cause of mortality in melanoma¹, these findings underscore the therapeutic relevance of targeting metabolic reprogramming to limit both tumor growth and dissemination⁷,¹⁰.

This study provides mechanistic insight into how fisetin modulates melanoma cell metabolism. Rather than establishing a direct or exclusive link between PKM2 inhibition and reduced viability, our results indicate that fisetin alters multiple cellular processes—including, but not limited to, glycolysis—to suppress melanoma cell survival and migration. Given fisetin’s natural origin and favorable safety profile, it emerges as a promising candidate for adjunctive melanoma therapy. These results highlight the potential of fisetin as a dual-action therapeutic agent that not only impairs metabolic reprogramming but also inhibits metastatic spread in melanoma. Future in vivo studies and further exploration of PKM2-targeted strategies are warranted to validate and expand upon these findings.

SUPPLEMENTARY MATERIAL

Supplementary Data 1

Supplementary Materials and Methods