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. 2024 May 9;25(10):5146.
doi: 10.3390/ijms25105146.

Quercetin Induces Mitochondrial Apoptosis and Downregulates Ganglioside GD3 Expression in Melanoma Cells

Sang Young Seo  1   2 , Won Seok Ju  1   3 , Kyongtae Kim  1 , Juhwan Kim  1 , Jin Ok Yu  1 , Jae-Sung Ryu  4 , Ji-Su Kim  5 , Hyun-A Lee  6 , Deog-Bon Koo  7 , Young-Kug Choo  1   8
Affiliations

Quercetin Induces Mitochondrial Apoptosis and Downregulates Ganglioside GD3 Expression in Melanoma Cells

Sang Young Seo et al. Int J Mol Sci. .

Abstract

Malignant melanoma represents a form of skin cancer characterized by a bleak prognosis and heightened resistance to traditional therapies. Quercetin has demonstrated notable anti-carcinogenic, anti-inflammatory, anti-oxidant, and pharmacological effects across various cancer types. However, the intricate relationship between quercetin's anti-cancer properties and ganglioside expression in melanoma remains incompletely understood. In this study, quercetin manifests specific anti-proliferative, anti-migratory, and cell-cycle arrest effects, inducing mitochondrial dysfunction and apoptosis in two melanoma cancer cell lines. This positions quercetin as a promising candidate for treating malignant melanoma. Moreover, our investigation indicates that quercetin significantly reduces the expression levels of ganglioside GD3 and its synthetic enzyme. Notably, this reduction is achieved through the inhibition of the FAK/paxillin/Akt signaling pathway, which plays a crucial role in cancer development. Taken together, our findings suggest that quercetin may be a potent anti-cancer drug candidate for the treatment of malignant melanoma.

Keywords: apoptosis; cell-cycle arrest; ganglioside; knockdown; malignant melanoma; quercetin.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Effects of quercetin on cell viability of malignant melanoma cells. (A) Chemical structure of quercetin. (B) Cell viability of melanoma SK-MEL-28 and G-361 and non-tumoral HaCaT cells treated with different concentrations of quercetin for 24 and 48 h (hr). Viability was detected using MTT assay kit. (C) Morphological changes in quercetin-treated SK-MEL-28, G-361, and HaCaT cells for 24 h and corresponding untreated controls. Data represent mean ± standard deviation (SD) of three independent experiments (n = 3, * p < 0.05).
Figure 2
Figure 2
Apoptosis effects induced by quercetin in malignant melanoma cells. SK-MEL-28, G-361, and HaCaT cells were cultured without or with quercetin (100, 200, and 300 µM) for 24 h. (A) Representative fluorescence images (magnification: ×200) of DAPI stained cells. White arrows indicate DNA fragmentation and nuclear condensation. (B) DNA fragmentation and nuclear condensation were quantified. Data represent mean ± SD (n = 3, * p < 0.05). (C) Quantitative detection of Annexin-V/7-ADD-positive cells using Muse Cell Analyzer. SK-MEL-28, G-361, and HaCaT cells were treated with quercetin (100, 200, and 300 µM) for 24 h. Cells stained with Annexin-V only were defined as early apoptotic. Annexin-V/7-ADD double-stained cells were defined as late apoptotic. In Annexin-V/7-ADD results, lower left quadrant represents live cells, lower right quadrant represents early apoptotic cells, upper right quadrant represents late apoptotic cells, and upper left quadrant represents dead cells. (D) Percentage of total apoptotic cell population was quantified. Data are presented as mean ± SD (n = 3, * p < 0.05).
Figure 3
Figure 3
Quercetin regulates cell-cycle G0/G1 arrest and mitochondrial apoptosis in malignant melanoma cells. Malignant melanoma SK-MEL-28 and G-361 cells were cultured without quercetin (control) or with quercetin (100, 200, and 300 µM) for 24 h. (A) Malignant melanoma SK-MEL-28 and G-361 cells were washed with DPBS, fixed in 70% ethanol overnight, stained with MuseTM Cell-Cycle reagent, and analyzed for DNA content using MuseTM Cell Analyzer. Percentage of cell-cycle phase was quantified. Data represent mean ± SD (n = 3, * p < 0.05). (B) Dose-dependent effect of quercetin on cell-cycle-related proteins, such as cyclin D, cyclin E, cdk4, and cdk6. Malignant melanoma SK-MEL-28 and G-361 cells were treated with different concentrations of quercetin for 24 h. ACTB was used as loading control. ACTB: β-actin. (C) Dose-dependent effect of quercetin on apoptosis-related proteins, such as Bax, Bid, Bcl-xL, caspase 3, cleaved caspase 3, and PARP. Malignant melanoma SK-MEL-28 and G-361 cells were treated with different concentrations of quercetin for 24 h. ACTB was used as loading control. ACTB: β-actin.
Figure 4
Figure 4
Changes in mitochondrial membrane potential of malignant melanoma cells. (A) Representative images of mitochondrial membrane depolarization (JC-1 staining) by immunofluorescence microscopy. Malignant melanoma SK-MEL-28 and G-361 cells were treated with quercetin (100, 200, and 300 μM) for 24 h. Mitochondrial membrane depolarization increased in dose-dependent manner. (B) The mitochondrial apoptotic process in malignant melanoma SK-MEL-28 and G-361 cells was determined by immunoblot analysis of mitochondrial, cytosolic, and whole-protein levels. Mitochondrial fraction was confirmed using mitochondrial marker MTCO1, and cytosolic fraction was confirmed using β-tubulin. ACTB was used as loading control in total protein. ACTB: β-actin. All data shown were representative of at least three independent experiments.
Figure 5
Figure 5
Quercetin suppressed the proliferation of proteins involved in the Raf-Akt pathway in malignant melanoma cells. (A) Protein expression levels of PCNA, survivin, p53, and phosphorylated p53 detected using immunoblotting analysis in a dose-dependent panel for 24 h. (B) Protein expression levels of Raf-1, ERK1/2, Akt1/2/3, JNK, and phosphorylated Raf-1, ERK1/2, Akt1/2/3, and JNK were detected using immunoblotting analysis in a dose-dependent panel for 24 h. ACTB was used as a loading control. ACTB: β-actin. All data shown were representative of at least three independent experiments.
Figure 6
Figure 6
Quercetin significantly inhibited the metastatic ability of malignant melanoma cells. (A) Effect of quercetin on the migration of malignant melanoma SK-MEL-28 and G-361 cells. Malignant melanoma SK-MEL-28 and G-361 cells were treated with quercetin (250 μM). Malignant melanoma cells were scratched using sterile pipette tips and incubated with or without (control group) quercetin. Samples were observed under a light microscope at 24, 48, and 72 h. (B) The area covered with cells was quantified. The blue square and red square represent SK-MEL-28 and G-361 cells, respectively. Data represent mean ± SD (n = 3, * p < 0.05).
Figure 7
Figure 7
Changes in ganglioside expression in quercetin-treated malignant melanoma cells. (A) Common ganglioside biosynthesis. G: ganglioside; M: monosialo; D: disialo; numbers denote carbohydrate sequence. Cer: ceramide; GlcCer: glucosylceramide; LacCer: lactosylceramide; GalNAc: N-acetylgalactosamine; ST3GAL5, ST3 beta-galactoside alpha-2,3-sialyltransferase 5; ST8SIA1, ST8 alpha-N-acetylneuraminide alpha-2,8-sialyltransferase 1. (B) HPTLC analysis of ganglioside in untreated SK-MEL-28 cells (L1), SK-MEL-28 cells treated with quercetin (250 μM for 24 h) (L2), untreated G-361 cells (L3), and G-361 cells treated with quercetin (250 μM for 24 h) (L4). M, marker; L, line; M1 and M2, ganglioside standard mixture marker (left panel); quantification of band intensity for GM3, GM1, and GD3 in malignant melanoma SK-MEL-28 and G-361 cells (right panel). Data represent mean ± SD (n = 3, * p < 0.05). (C) Immunofluorescence microscopy analysis of quercetin-treated malignant melanoma SK-MEL-28 and G-361 cells. Malignant melanoma SK-MEL-28 and G-361 cells were treated with different concentrations of quercetin (100, 200, and 300 μM) for 24 h, and cells were immunostained with anti-GM3 or anti-GD3 and anti-cleaved caspase 3. Signals were detected with Alexa fluor 488-conjugated anti-goat and Alexa fluor 647-conjugated anti-mouse secondary antibody. The result shown in (C) were representative of at least three independent experiments.
Figure 8
Figure 8
Effects of knockdown of ganglioside GM3 synthase or GD3 synthase siRNA on malignant melanoma cells. (A) Knockdown of ganglioside GM3 or GD3 synthase on malignant melanoma SK-MEL-28 and G-361 cells. siRNA against ganglioside GM3 or GD3 synthase was transfected with Lipofectamin®3000 reagent, and protein levels of ganglioside GM3 and GD3 synthase were examined after four days of transfection using immunoblotting (left panel). Blue and red squares represent ganglioside GM3 and GD3 synthase protein levels, respectively (right panel). ACTB was used as loading control. ACTB: β-actin. All data are presented as mean percentage levels ± SD (n = 3, * p < 0.05). (B) RT-PCR analysis of melanoma SK-MEL-28 and G-361 cells treated with ganglioside GM3 and GD3 synthase siRNA (left panel). Blue and red squares represent ganglioside GM3 and GD3 synthase protein levels, respectively (right panel). ACTB was used as loading control. ACTB: β-actin. All data are presented as mean percentage levels ± SD (n = 3, * p < 0.05).
Figure 9
Figure 9
Ganglioside siRNA inhibits relative protein levels of FAK, paxillin, and Akt in malignant melanoma cells. (A) Knockdown of ganglioside GM3 or GD3 synthase on malignant melanoma SK-MEL-28 (left panel) and G-361 (right panel) cells. Effects of knockdown of ganglioside GM3 or GD3 synthase on phosphorylation levels of paxillin and Akt in malignant melanoma SK-MEL-28 and G-361 cells. Reduction in tyrosine phosphorylation was observed, and its total protein level was not affected. ACTB was used as loading control. ACTB: β-actin. All data shown were representative of at least three independent experiments. (B) Knockdown of ganglioside GM3 or GD3 synthase treated with or without quercetin on malignant melanoma SK-MEL-28 and G-361 cells. Protein levels of FAK, paxillin, and Akt and phosphorylation levels of Akt and paxillin were examined after 24 h in untreated and quercetin-treated (250 µM) panel cells using immunoblotting analysis. ACTB was used as loading control. ACTB: β-actin. All data shown were representative of at least three independent experiments.
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