7,8-Dihydroxyflavone induces mitochondrial apoptosis and down-regulates the expression of ganglioside GD3 in malignant melanoma cells

Malignant melanoma is a skin cancer with poor prognosis and high resistance to conventional treatment. 7,8-dihydroxyflavone (7,8-DHF) has shown anti-carcinogenic, anti-inflammatory, anti-oxidant, and pharmacological effects in several types of cancer. However, the relationship between ganglioside expression and the anti-cancer effects of 7,8-DHF in melanoma is not fully understood. In the present study, 7,8-DHF exhibits specific anti-proliferation, anti-migration, and G2/M phase cell-cycle arrest effects on both melanoma cancer cell lines, and induces mitochondrial dysfunction and apoptosis, making it a potent candidate for anti-melanoma treatment. Furthermore, we confirmed that 7,8-DHF significantly reduces the expression levels of ganglioside GD3 and its synthase, which are known to be closely involved in carcinogenesis. Taken together, our findings suggest that 7,8-DHF may be a potent anti-cancer drug candidate for the treatment of malignant melanoma. Supplementary Information The online version contains supplementary material available at 10.1007/s12672-023-00643-0.


Introduction
Malignant melanoma worldwide is a highly perilous form of representative skin cancers including non-melanomas (basal cell carcinoma and squamous cell carcinoma), which leads to poor prognosis with limited survival rates and unsuccessful therapy approaches due to their substantial side effects and significant toxicities for patients [1][2][3][4]. Despite the advancements in cancer therapy with the addition of immunotherapy and targeted drugs, the response rate remains 1 3

Nuclear staining with DAPI
The levels of nuclear condensation and fragmentation in malignant melanoma cells were confirmed by using nucleic acid staining and DAPI staining. Melanoma cell line (SK-MEL-2 and G-361) and HaCaT cells treated with 7,8-DHF at different concentration (0, 100, 200, and 300 µM for 24 h) were harvested by trypsinization, and fixed in 4% paraformaldehyde (Sigma, St. Louis, MO, USA) in Dulbecco's modified phosphate-buffered saline (DPBS; Welgene, Gyeongsan, Gyeongbuk, Republic of Korea) at room temperature for 30 min. The cells were spread on slides, stained with DAPI solution (2 µg/ mL), and analyzed under a fluorescence microscope (Carl Zeiss, Ulm, Germany).

Cell cycle analysis
For facile and quantitative measurements of the percentage of cells in a variety of cell cycles, both melanoma SK-MEL-2 (1.0 × 10 5 cells/well) and G-361 cells (1.0 × 10 5 cells/well) were seeded into 6-well plates (Thermo Fisher Scientific lnc., Waltham, MA, USA) and maintained for 24 h. After exposure to 7,8-DHF at different concentrations (100, 200, and 300 µM) for 24 h, the cells were detached by trypsinization and washed twice with DPBS. Detached samples were fixed in 70% ethanol at -20 ℃ until staining. Fixed samples were then washed with DPBS, collected by centrifugation at 300 × g for 5 min, and mixed with 150 µL Muse ™ Cell Cycle Reagent (Luminex Corp., TX, USA). After suspension, the mixed samples were incubated at room temperature for 30 min and protected from light. Cells were analyzed by flow cytometry using a Muse ™ Cell Analyzer (Merck Millipore, MA, USA).

Annexin v & dead cell assay
For the quantitative analysis of live cells, early and late apoptosis, and cell death in melanoma cells, both SK-MEL-2 (3.1 × 10 5 cells/well) and G-361 (4.3 × 10 5 cells/well) cells were seeded on 6-well plates (Thermo Fisher Scientific lnc., Waltham, MA, USA) and cultured for 24 h. After treatment with different concentrations (100, 150, 200, 250, and 300 µM) of 7,8-DHF for 24 h, the cells were stained with Muse ™ Annexin V & Dead Cell kit (Luminex Corp., TX, USA) for 20 min at room temperature in the dark. Stained samples were analyzed by using a Muse ™ Cell Analyzer (Merck Millipore, MA, USA).

Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from melanoma cell lines (SK-MEL-2 and G-361) treated with 7,8-DHF at different concentrations (0 and 200 µM for 24 h) by using TRIzol ™ Reagent (ThermoFisher Scientific lnc., Waltham, MA, USA), and 1.5 µg of RNA was used to synthesize cDNA using the AccuPower® RT PreMix kit (Bioneer Corp., Daejeon, Korea). The cDNA for RT-PCR was obtained by the amplification using specific primers such as β-actin, ST3GAL5, and ST8SIA1. For quantitative PCR, 2 µL of cDNA, 10 pmol of the forward primer, 10 pmol of the reverse primer, and 5 µL of 20X premix buffer were added with distilled water to a final volume of 20 µL. The mixture was under the following conditions: 94 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, 62 °C for 30 s, and 72 °C for 30 s, with a final extension at 72 °C for 10 min. Primer information and the specific PCR conditions used in this study are provided in Additional file 1: Table S1. first cultured and then treated with 7,8-DHF at different concentrations (100-200 µM) for 24 h. Cells were washed twice with DPBS, added 5 µM JC-1 in 1 × dilution buffer, and then incubated at 37 ℃ for 15 min in the dark. The samples were washed twice with DPBS. MMPs of the mitochondria in the cancer cells were observed using a fluorescence microscope (Carl Zeiss, Ulm, Germany). The aggregated form of JC-1 yielded a red to orange colored emission (590 ± 17.5 nm), whereas the monomeric form yielded a green colored emission of 530 ± 15 nm.

Isolation of mitochondria and protein fractionation
Mitochondrial and cytosolic fractions were isolated using a Mitochondria and Cytosol Fractionation Kit (Abcam Inc., Cambridge CB2, UK), according to the manufacturer's instructions [46]. After treatment with 7,8-DHF for up to 24 h, both melanoma SK-MEL-2 and G-361 cells were harvested and then mixed with 1 mL of 1 × cytosol extraction buffer mix containing DTT and protease inhibitors. After incubation on ice for 10 min, the cell samples were homogenized in an ice-cold Dounce glass tissue grinder (DWK Life Science, St. Modwen Park, Lincoln LN6 9BJ, UK) for 40-60 passes. The samples were then centrifuged at 700 × g at 4 ℃ for 10 min, the supernatant was collected, and the pellet was discarded. The supernatants were centrifuged at 10,000 × g at 4 ℃ for 30 min to separate the mitochondrial (pellets) and cytosolic (supernatants) fractions. Finally, mitochondrial pellets were suspended in 150 µL of mitochondrial extraction buffer mix containing DTT and protease inhibitors. All isolated samples were stored at − 80 ℃ until use.

Western blot analysis
SK-MEL-2 and G-361 cells were treated with various concentrations of 7,8-DHF for different incubation times (100, 200, and 300 µM, for 12 and 24 h) and washed twice with DPBS. Cells were homogenized well with a protein extraction solution (RIPA) (ELPIS Biotech Inc., Daejeon, Korea). The extracted proteins were quantified using the Pierce ® BCA Protein Assay kit (Thermo Fisher Scientific lnc., Waltham, MA, USA). Equal amounts of protein samples were separated by 8%, 10%, and 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) along with protein markers and then transferred to polyvinylidene difluoride (PVDF) blotting membranes. The membranes were blocked at room temperature for one hour with 5% non-fat dried milk in 1 × TBS (Biosesang, Seongnam, Gyeonggi, Korea) containing 0.1% Tween-20 (Bio-Rad Inc., CA, USA) and then incubated overnight at 4 ℃ with specific primary antibodies. After incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature for two hours, immunoreactive bands were visualized using a SuperSignal West Pico PLUS chemiluminescent substrate (ThermoFisher Scientific lnc., Waltham, MA, USA).

Wound healing assay
Confluent SK-MEL-2 (5.0 × 10 5 cells/well) and G-361 cells (5.0 × 10 5 cells/well), grown in 60 mm culture dishes (Thermo Fisher Scientific lnc., Waltham, MA, USA), were sharply scratched using a sterile tip, and each well was washed twice with DPBS to remove cell debris. After scratching, the cells were treated with 200 µM 7,8-DHF and then cultured for 24, 48, and 72 h to observe the perimeter of the area with a central cell-free zone under an inverted phase-contrast microscope (Carl Zeiss, Ulm, Germany).

Ganglioside extraction and purification
Gangliosides from melanoma cell lines (SK-MEL-2 and G-361) and HaCaT cells treated with 7,8-DHF at different concentration (0 and 200 µM for 24 h) was prepared as previously described [35]. Briefly, total lipids were extracted using chloroform/methanol (1:1, v/v). Next, neutral lipids were filtered off with 10 mL chloroform/methanol/H 2 O (15:30:4, v/v/v) by applying a DEAE Sephadex A25 column (Sigma, St. Louis, MO, USA). Acidic lipids were then extracted with 5 mL chloroform/methanol/0.8 M sodium acetate (15:30:4, v/v/v). The eluted samples were dried with N 2 gas at 30 ℃, dissolved in chloroform/methanol (1:1, v/v), and incubated for neutralization with 12N NH 4 OH overnight at room temperature. After the neutralized samples were dried with N 2 gas, they were dissolved in distilled water and the salt was removed using a Sep-Pak C18 cartridge (Merck Millipore, MA, USA) to obtain gangliosides. The eluted gangliosides were dried under N 2 gas at 30 °C. Finally, the dried samples were stored at − 80 ℃ until further experiments.

Data analysis
For statistical analysis, all data were analyzed for statistical significance with One-way ANOVA, post hoc Tukey's multiple range test by using the Statistical Package for the Social Science statistics software (IBM SPSS Corp., Armonk, NY, USA) (ver. 25). Other data are represented as mean ± standard deviation (SD). A p value (p < 0.05) in this study was considered statistically significant.  (Fig. 1B). The inhibition of viability by 7,8-DHF was significant in both melanoma cell lines when the concentration ranged from 200 to 250 µM. In HaCaT cells, no significant change was observed while HaCaT cells were exposed up to 300 µM. To analyze morphological changes, SK-MEL-2 and G-361 cells were treated with 7,8-DHF (100-300 µM, for 24 h). The number of cells was markedly decreased, and the cell size also decreased and became more rounded (Fig. 1C). In contrast, HaCaT cells showed no distinctive morphological changes after treatment with 300 µM 7,8-DHF.

7,8-DHF induced apoptosis in melanoma cells
The effects of 7,8-DHF on cellular apoptosis in SK-MEL-2, G-361, and HaCaT cells were determined by nuclear morphology using DAPI staining and Annexin-V/7-ADD staining analysis, and these results were verified by the images of the morphological changes. It was confirmed that 7,8-DHF (100, 200, and 300 µM for 24 h) induced nuclear condensation and perinuclear apoptotic bodies in melanoma cells in a concentration-dependent manner. Morphological and nuclear changes were also induced in HaCaT cells by 7,8-DHF (200 µM), although to a lower degree ( Fig. 2A). The percentage of cells with nuclear fragmentation in 7,8-DHF-treated cells vs. non-treated cells is shown in Fig. 2B. Additionally, the apoptotic effect of 7,8-DHF on SK-MEL-2 and G-361 melanoma cells was evaluated after 24 h using the Annexin-V/7-ADD assay (Fig. 2C). For the upper panel in Fig. 2C, the average percentage of early apoptotic cells in the SK-MEL-2 group was 4.14, 13.
To verify this change of cell-cycle, the levels of G2/M phase-related proteins (p53, p21, cyclin B, CDK1) were analyzed. As shown in Fig. 3C, The expression levels of p53 and p21 were increased, whereas the expression levels of CDK1 and cyclin B were decreased compared to those in the control. Also, the expression levels of Mcl-1 and Survivin were slightly decreased in dose-dependent manner (Fig. 3C).
To further investigate the relationship between the changes of cell-cycle and cell apoptosis, the apoptosis-related proteins, including Bid, Caspase 3 (CASP3), and poly (ADP-ribose) polymerase (PARP), were analyzed; they were truncated dramatically. Specifically, the expression levels of Bax, cleaved-CASP3, and cleaved-PARP were highly increased, whereas the expression of the anti-apoptotic protein BCL-xL was decreased as shown in Fig. 3D.

7,8-DHF changed the Mitochondrial membrane potential (MMP) and induced the release of cytochrome c from the intermembrane space in melanoma cells
To investigate whether 7,8-DHF-mediated apoptosis in SK-MEL-2 and G-361 cells through the mitochondrial pathway, we measured the MMP using JC-1 staining, and examined the level of Bcl-2 family proteins and cytochrome c. As shown in Fig. 4A, JC-1 monomer (green) was dose-dependently increased, indicating depolarization of the MMP in melanoma cells. Next, we investigated the expression levels of fractionations into mitochondrial and cytosolic proteins related to the mitochondrial apoptotic pathway in both cells. As shown in Fig. 4 B, 7,8-DHF induced mitochondrial cytochrome c release into the cytosol in SK-MEL-2 and G-361 cells in a time-dependent manner, which was followed by an increase in mitochondrial Bax. In addition, the expression levels of cleaved-CASP3 and cleaved-PARP were dramatically increased, whereas the expression levels of pro-apoptotic proteins including Bid and Caspase-9 and the antiapoptotic protein Bcl-xL were highly decreased.

7,8-DHF inhibited melanoma cell population and migratory capacity
In most cancer cells, the ability to invade surrounding tissues and other parts of the body is the initial step in tumor metastasis and migration. However, the inhibitory effects of 7,8-DHF on SK-MEL-2 and G-361 melanoma cells remain unclear. Therefore, we examined the effect of 7,8-DHF (200 µM) on the migratory capacity of melanoma cells using a wound healing assay. As shown in Fig. 5A, treatment with 7,8-DHF reduced the migration capacity of SK-MEL-2 and  Fig. 5A , B). Similarly, 7,8-DHF-treated G-361 cells covered 5.14%, 19.25%, and 23.43% of the scratched area at 24, 48, and 72 h after scratching, respectively (lower panel in Fig. 5A, B). In contrast, 7,8-DHF untreated melanoma cell lines showed area covered with 5%, 30.79%, and 92.03% cells at 0, 24, and 72 h (SK-MEL-2), and with 3%, 34,83%, and 81.29% at 0, 24, and 72 h (G-361) as shown in Fig. 5B. In both melanoma cells treated with 7,8-DHF, the ratio of wound closure was slightly increased, the cell population and migratory capacity were inhibited in a time-dependent manner, compared to the 7,8-DHF untreated ones, respectively.

Discussion
Malignant melanoma is an increasingly prevalent and fatal form of skin cancer. Despite being rare in the past, its incidence and mortality rates have risen significantly in recent years [3,4,36]. To avoid the harmful side effects and unexpected reactions associated with synthetic chemical, it is crucial to investigate therapeutic approaches for melanoma. Therefore, the discovery of effective anti-melanoma agents and specific biomarkers are of great importance (Additional file 2). Basically, 7,8-dihydroxyflavone (7, was suggested in many studies to provide anti-oxidant effects and anticarcinogenesis including the development and progression in different cancer types [14,37,38]. In the present study, we confirmed that 7,8-DHF effectively induces dose-and time-dependent melanoma cell death, consistent with previous studies on the anti-melanoma properties of 7,8-DHF [11,12,20,21]. However, the absolute treatment concentration setting varied due to the intra-tumor heterogeneity within melanoma caused by genetic mutations acquired irregularly [39][40][41][42][43][44]. Anti-cancer drugs are known to induce various mechanisms, such as the checkpoint in cell-cycles, in cancer cells. Specifically, we confirmed that treatment with 7,8-DHF caused G2/M phase cell-cycle arrests in both melanoma cells, which is similar to previous studies [19,45,46]. Furthermore, mitochondrial damage is followed by a decrease in mitochondrial membrane potential and the expression levels of the proteins [47]. Changes in mitochondrial function during cancer progression play an important role in the regulation and initiation of apoptosis [48]. In the present study, the decrease in mitochondrial membrane potential and changes in the expression of mitochondrial apoptosis pathway-related proteins clearly showed the anti-melanoma ability of 7,8-DHF in the present study. In squamous carcinoma cells, it has been reported that the treatment with methyl-honokiol induces mitochondrial dysfunction in the process of cancer cell death well [49]. Moreover, cancer migration and mobility are highly important for cancer progression and metastasis [50]. We confirmed 7,8-DHF effectively reduced the recovery ability and mobility in both melanoma cells line, which is also similar to previous studies [19,45]. Ganglioside is closely involved cellular metabolisms including growth, proliferation, differentiation, metastasis, and the general cell apoptosis [22][23][24][25]. Normally, ganglioside GD3 and GM3 are highly expressed in skin cancer, ganglioside GM3 in squamous carcinoma, ganglioside GM2 in pancreatic cancer, ganglioside GM3 in colorectal cancer, and ganglioside GD3 in breast cancer [54]. Especially, ganglioside GD3 is reported to be a promising target for immunotherapy in The area covered with cells was quantified. Red square and blue square represent SK-MEL-2 and G-361 cells, respectively. All data were presented as the mean ± SD (n = 3, * p < 0.05) melanoma cancers [22,23,[51][52][53][54]. In addition, ganglioside GD3 activates growth factor receptors that are combined with adhesion signals, resulting in the activation of downstream signaling molecules and in increased growth and invasion of melanoma cells [53,55] In the present study, we confirmed that the ganglioside GD3 and its synthase were highly decreased in 7,8-DHF-treated melanoma cells, compared to 7,8-DHF-untreated ones. Meanwhile, regulation of the expression of ganglioside GD3 was blocked through mitochondrial changes, decreased activation of caspase 9, and plays a crucial role in the orchestration of apoptosis signals [56], which is also similar to our findings of the present study.
Taken together, the treatment of 7,8-DHF against melanoma cells might have effectual and superior anti-cancer efficacy, indicating its potential as an anti-melanoma therapeutic agent such as a biomarker. Furthermore, ganglioside might be a double-edged sword against melanoma cells; suggesting that 7,8-DHF could be a candidate targeted anti-cancer therapy. . Data represent the mean ± SD (n = 3, * p < 0.05). C Western blot analysis and D RT-PCR analysis of the gangliosides GM3 and GD3 synthase was examined, respectively. Red square and blue square indicate SK-MEL-2 and G-361 cells, respectively. ACTB was used as a loading control. ACTB; β-actin. All data were presented as mean percentage levels ± SD (n = 3, * p < 0.05)