Using Flavonoid Substitution Status to Predict Anticancer Effects in Human Melanoma Cancers: An In Vitro Study

Simple Summary The rising incidence of skin cancer, particularly melanoma, over the last few decades requires natural-product-based treatments with a lack of side effects. To evaluate the anticancer potential of 37 structurally diverse flavonoids, cytotoxic tests, DNA biosynthesis inhibition levels, and apoptosis pathways involved in cell death were investigated in A375 and C32 melanoma cells. Compounds 1, 6, 15, and 37 reduced the viability of cell lines via the intrinsic (caspase-9) and extrinsic (caspase-8) pathways of apoptosis, where 16 and 17 mainly influenced DNA biosynthesis in cancer cells. In our study, we performed an anticancer analysis of compound 15 (5,6-dihydroxyflavone) for the first time. Although our data provide new insights into the molecular mechanisms of flavonoid-induced toxicity, in vivo studies are needed to assess their anti-cancer activities, especially flavonoid-core-containing hydroxyl groups. Abstract Skin cancers are a dominant type of cancer that impacts millions per year. Cancer is a heterogeneous disease triggered by the irreversible impairment of cellular homeostasis and function. In this study, we investigated the activity of 37 structurally diverse flavonoids to find potentially active substances using two melanoma cell lines: C32 and A375. First, the cytotoxic potential and DNA biosynthesis inhibition of flavonoids were tested to determine the most active compounds in cancer and normal cells. Second, the molecular mechanism of the anticancer activity of flavonoids was elucidated using Western blot and immunofluorescence analyses. Compounds 1, 6, 15, and 37 reduced the viability of A375 and C32 cell lines via the intrinsic and extrinsic pathways of apoptosis, whereas 16 and 17 acted in a higher degree via the inhibition of DNA biosynthesis. In our experiment, we demonstrated the anticancer activity of compound 15 (5,6-dihydroxyflavone) for the first time. The in vitro studies pointed out the importance of the flavonoid core in hydroxyl groups in the search for potential drugs for amelanotic melanoma.

The MTT colorimetric assay was performed as a preliminary evaluation of the cytotoxicity of the selected flavonoids according to the method described and validated by Strawa et al. [21].Briefly, melanoma cells and fibroblasts were seeded in 96-well microplates at a density of 1 × 10 4 cells per well.Fibroblasts were used as a control cell line in order to assess the selectivity of the compounds.Following 24 h of incubation (at 37 • C with 5% CO 2 ), the cells were exposed to different concentrations of compounds 1-37 and cisplatin (6.25, 12.5, 25, 50, 100, or 200 µM) for 24, 48, and 72 h.Subsequently, the cells were washed with PBS, incubated with MTT solution for 2 h, and lysed with dimethyl sulfoxide (DMSO) supplemented with 1% (v/v) Sorensen's glycine buffer.The absorbance was measured using a microplate reader (EPOCH 2, BioTek, Winooski, VT, USA) at a wavelength of 570 nm.The compounds were used at a final DMSO concentration of no more than 0.1% (v/v) in each well.The results were generated from at least three independent experiments.Cisplatin (cPt) was used as a positive control.The IC 50 values were calculated with GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA).

Neutral Red Uptake (NRU) Assay
The NRU (neutral red uptake) was conducted as previously described by Szoka and co-workers, with some modifications [22].Briefly, melanoma cells and fibroblasts were seeded in microplates at the density of 1 × 10 4 cells per well and allowed to adhere for 24 h.Then, cells were incubated with different concentrations of compounds 1-37 and cPt (6.25, 12.5, 25, 50, 100, or 200 µM) for 24, 48, and 72 h.After treatment, the culture medium was replaced with a neutral red solution (40 µg/mL).An additional 2 h of incubation was performed.The neutral red solution was then removed and the neutral red accumulated in the cells was dissolved in 50% EtOH with 1% of the glacial acetic acid.The absorbance was estimated at 540 nm and IC 50 values were calculated using GraphPad Prism software (GraphPad Software, San Diego, CA, USA).

Proliferation Assay
The inhibition of DNA biosynthesis was evaluated using [ 3 H]-thymidine incorporation into the DNA of melanoma cells (C32, A375) and fibroblasts treated with the studied compounds [21].The cells were briefly seeded in 24-well plates (1 × 10 5 cells per well) and allowed to adhere for 24 h.Subsequently, the studied cells were treated with 1 mL of DMEM with 0.5 µCi [ 3 H]-thymidine (6.7 Ci/mmol) and various concentrations (6.25-200 µM) of the most active compounds.Cisplatin (cPt) was used as a positive control.After incubation for 24, 48, and 72 h, the cells were rinsed three times with PBS.Then, cells were lysed in 1 mL of 0.1 M NaOH containing 1% SDS.The cell lysate was moved into scintillation vials containing scintillation liquid, and the radioactivity of the samples was measured using a liquid scintillation analyzer (Perkin-Elmer, Waltham, MA, USA).

Apoptosis Assay
The apoptosis assay was conducted as described before [22].Firstly, cells were placed in 6-well plates at a density of 1 × 10 5 cells per well and allowed to adhere for 24 h.Then, cells were treated with 25 µM, 50 µM, and 100 µM of the selected flavonoids (1, 6, 15, 16, 17, 37) and incubated for 48 h.Floating and adherent cells were collected and assayed using the manufacturer's protocol supported by the Dead Cell Apoptosis Kit for flow cytometry.Concisely, 100 µL of an annexin-binding buffer containing a 5 µL annexin V-FITC conjugate solution and 1 µg/mL PI (propidium iodide) was used to disperse the cells.After 15 min of incubation, an additional 400 µL of the mentioned buffer was added.The cells were analyzed with a DxFLEX flow cytometer (Beckman Coulter, Brea, CA, USA).

Western Immunoblot
Floating cells were harvested by centrifugation of the conditioned media (300× g, 5 min).Cell pellet and adherent cells were lysed in a cell lysis buffer supplemented with inhibitors of proteases and phosphatases.The cell lysate was sonicated (2 × 10 s) on ice with an ultrasonic homogenizer (UP200S, Hielscher Ultrasonics GmbH, Teltow, Germany) and centrifuged for 10 min at 10,000× g.The supernatant was collected and stored in a freezer at −70 • C. Protein concentrations in the cell supernatants were determined using a Lowry assay.Obtained proteins were dissolved on SDS-PAGE gels (7.5%, 10%, or 12%) using the Mini-Protean Tetra system.The proteins were transferred to nitrocellulose membranes using the Mini Trans-Blot Cell wet blotting system.The 5% skim milk was used to block membranes.Then, membranes were probed for 24 h at 4 • C with the following antibodies: anti-phospho-RSK, anti-RSK, anti-PARP, anti-cleaved PARP, anti-caspase-8, anticaspase-9, anti-caspase-7, anti-caspase-3, and anti-actin.Membranes were incubated with an ECL-HRP (enhanced chemiluminescence-horseradish peroxidase) substrate, and the signal was spotted using the BioSpectrum Imaging System and VisionWorks LS software 7.0 (Ultra-Violet Products, Ltd., San Gabriel, CA, USA).The intensity of the bands was quantified by densitometric analysis using UVIBand software, version 15.08d (Uvitec Ltd., Cambridge, UK).

Immunofluorescence Microscopy
Firstly, to examine fluorescent signals, cells were placed in the bottom of 96-well black plates at 1 × 10 4 cells per well and permitted to adhere for 24 h.Subsequently, the cells were treated with compounds 1, 6, 15-17, and 37 at concentrations of 25 µM, 50 µM, or 25 µM cPt for 48 h.After incubating and removing the medium, cells were fixed in 4% paraformaldehyde for 15 min.Later, cells were rinsed three times with PBS and permeabilized with 0.1% Triton X-100 for 5 min at room temperature.The 10% heatinactivated goat serum in the PBS was used to block non-specific binding.Subsequently, cells were treated with the following antibodies overnight: anti-cleaved caspase 3 and anti-cleaved caspase-7.Next, cells were incubated with the Alexa Fluor 594 conjugated antibody for 1 h in a dark place.Cell nuclei were stained with Hoechst 33342 and Factin was stained with Phalloidin-Atto 488.Fluorescent signals were captured using a BD Pathway 855 confocal microscope (Becton Dickinson, Franklin Lakes, NJ, USA).The mean fluorescence intensity was calculated using ImageJ software (National Institutes of Health, Bethesda, Rockville, MD, USA).

Statistical Analysis
The statistical analysis for the IC 50 calculations was performed using nonlinear regression using GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA) and MS Excel 2019 software (Microsoft, Washington, DC, USA).All data were received as mean ± SD (from at least three independent replicates), and the significance of differences was determined using ANOVA.

Cell Viability Assays
In the first step, the potential cytotoxic effect of different concentrations of the selected flavonoids (Table 1, Figure 1) and cPt, a well-known anticancer agent, was studied using MTT and NRU tests on A375 and C32 melanoma cell lines and fibroblasts (Tables 2 and 3, Figures S1 and S2).

No. π C2-C3 B-Ring
Benzo-γ-Pyrane Ring     Values are expressed as µM and represented as the mean ± standard deviation (SD) from at least three independent measurements; A : cPt-cisplatin used as positive control.
An MTT assay was carried out separately for each sample for a preliminary evaluation of the cytotoxic effects of all samples on the viability of A375, C32, and fibroblast cells after 24, 48, and 72 h of incubation.The cytotoxic potential was expressed as a median inhibitory concentration (IC 50 ) value.At this point, six compounds showed the highest activity in both A375 and C32 cell lines: 1, 6, 15, 16, 17, and 37. Compound 17 (zapotin) displayed the strongest and a selective effect among all tested compounds after 48 h of incubation, where the IC 50 reached values of 31.1 ± 2.9, 26.5 ± 1.3, and 78.0 ± 5.2 µM for A375, C32, and normal cells, respectively.The cytotoxic activity of the most active compounds increased when the incubation time was increased from 24 h to 48 h.Nevertheless, this effect was not consistent when treatment was increased further from 48 h to 72 h.On the other hand, sixteen compounds (2, 3, 8-13, 21-23, 27, 29, 34-36) did not indicate any activity, even at the highest used concentration (200 µM).Values are expressed as µM and represented as the mean ± standard deviation (SD) from at least three independent measurements; A : cPt-cisplatin as a positive control.
Despite the fact that the MTT test is the most common method for the evaluation of cell viability, several studies have revealed inaccuracies that are inherent in this method.Some experiments demonstrate that flavonoids are redox-active against tetrazolium salt even in the absence of cells, which interferes with the MTT test [15].Thus, we performed an NRU assay to confirm the reliability of the obtained results with the MTT method.The data obtained from the NRU analysis indicate a strong activity of the six compounds mentioned in MTT test.After 48 h of exposure, in both A375 and C32 cells, the IC 50 of 17 (14.7 ± 1.2 and 14.7 ± 1.1 µM, respectively) showed comparable cytotoxicity to cPt (14.4 ± 1.1 and 15.0 ± 1.2 µM).Importantly, 5,6-dihydroxyflavone (15), 7,8-dihydroxyflavone (16), and zapotin (17) were more active in cancer cells than in fibroblasts.

Proliferation Assay
The observed decrease in cancer cell viability could have been the result of an impaired cell proliferation rate.Therefore, based on the cytotoxic tests, we selected the most active flavonoids (1, 6, 15-17, 37) and performed a proliferation assay (Table 4).Treatment with 17 showed a significant decrease in [ 3 H]-thymidine incorporation into newly synthesized DNA after 48 h of incubation in both A375 and C32 cell lines (IC 50 = 6.0 ± 0.2 and 9.1 ± 1.2 µM, respectively).It is worth mentioning that 17 affected DNA biosynthesis more in cancer cells than in normal cell lines.Values are expressed as µM and represented as the mean ± standard deviation (SD) from at least three independent measurements; cPt-cisplatin as positive control.
In the next step, the levels of the essential mediators of apoptosis were analyzed by the Western blot method (Figure 3, Figures S3 and S5-S18).The treatment of A375 cells with 1, 6, 37, and cPt for 48 h resulted in the cleavage of both initiator caspases, caspase-9 and caspase-8, and decreased levels of the caspases-3 and -7 zymogens.However, compound 6's action was inversely proportional to the used concentration.Treatment with compounds 15 and 16 increased the expression of cleaved caspase-8, but 16 induced a weak cleavage of caspase-9.On the other hand, after using 17, there were no visible signs of active initiator caspases.However, the expression of caspase-8 pro-form was significantly downregulated.Compounds 16 and 37, at higher concentrations, and cPt impaired the phosphorylation of the RSK protein (ribosomal kinase).significantly downregulated.Compounds 16 and 37, at higher concentrations, and cPt impaired the phosphorylation of the RSK protein (ribosomal kinase).
In the C32 cell line, flavonoids 1, 6, 15, and 37 and cPt generated cleaved PARP and caspase-9.The expression of caspase-8 zymogen was below the limit of detection in these cells.Furthermore, 17, 37, and cPt inhibited the phosphorylation of RSK.In Figures 4 and S4, the expression of cleaved caspase-3 and cleaved caspase-7 revealed by immunofluorescent staining in both melanoma cell lines is presented.In the A375 cell line, 48 h of treatment with 1 and 6 slightly increased the level of cleaved caspase-3.Furthermore, 1, 6, 15, and 37 notably extended caspase-3 expression in the C32 cells.Also, the level of the cleaved caspase-7 slightly increased in C32 cells after treatment with 6, 16, and 17.In the C32 cell line, flavonoids 1, 6, 15, and 37 and cPt generated cleaved PARP and caspase-9.The expression of caspase-8 zymogen was below the limit of detection in these cells.Furthermore, 17, 37, and cPt inhibited the phosphorylation of RSK.
In Figure 4 and Figure S4, the expression of cleaved caspase-3 and cleaved caspase-7 revealed by immunofluorescent staining in both melanoma cell lines is presented.In the A375 cell line, 48 h of treatment with 1 and 6 slightly increased the level of cleaved caspase-3.Furthermore, 1, 6, 15, and 37 notably extended caspase-3 expression in the C32 cells.Also, the level of the cleaved caspase-7 slightly increased in C32 cells after treatment with 6, 16, and 17.

Discussion
Despite the fact that surgery, radiation therapy, and chemotherapy are conventionally used to treat cancers, there is undoubtedly a great need to investigate natural products that may reduce the frequency of cancer incidences or the side effects of synthetic drugs [23].
The results available in the literature that describe the anticancer activity of flavonoids are diverse and sometimes contradictory.This dissimilarity may be connected with various experimental conditions (e.g., concentrations of substrates or conditions of incubation) or the limited number of compounds included in such studies.Thus, in this study, we tested a large set of flavonoid compounds for their cytotoxic, antiproliferative, and apoptotic effects under the same conditions to obtain values that are as close to those in the real world as possible.Compound 1 (apigenin), which may be considered the major flavone in the plant kingdom, is also one of the most studied phytoconstituents for its anticancer activity.Zhao and co-workers investigated the cytotoxic effect of apigenin using MTT assays.According to their results, apigenin at a concentration of 40 µM decreased A375 cell viability [24], which is in agreement with our results after 48 h of incubation (IC50 = 31.9± 1.0 µM).On the other hand, Cao et al. implied that apigenin at 10 and 20 µM significantly inhibited A375 cell motility and invasion in a dose-dependent manner [25].Even though MTT seems to be the most common test for detecting

Discussion
Despite the fact that surgery, radiation therapy, and chemotherapy are conventionally used to treat cancers, there is undoubtedly a great need to investigate natural products that may reduce the frequency of cancer incidences or the side effects of synthetic drugs [23].
The results available in the literature that describe the anticancer activity of flavonoids are diverse and sometimes contradictory.This dissimilarity may be connected with various experimental conditions (e.g., concentrations of substrates or conditions of incubation) or the limited number of compounds included in such studies.Thus, in this study, we tested a large set of flavonoid compounds for their cytotoxic, antiproliferative, and apoptotic effects under the same conditions to obtain values that are as close to those in the real world as possible.Compound 1 (apigenin), which may be considered the major flavone in the plant kingdom, is also one of the most studied phytoconstituents for its anticancer activity.Zhao and co-workers investigated the cytotoxic effect of apigenin using MTT assays.According to their results, apigenin at a concentration of 40 µM decreased A375 cell viability [24], which is in agreement with our results after 48 h of incubation (IC 50 = 31.9± 1.0 µM).On the other hand, Cao et al. implied that apigenin at 10 and 20 µM significantly inhibited A375 cell motility and invasion in a dose-dependent manner [25].Even though MTT seems to be the most common test for detecting cytotoxicity, no data are available for the C32 cell line using this method thus far.Nevertheless, the SRB test showed that 1 is a more potent cytotoxic compound against C32 cells than against A375 cells [26].These results follow our NRU assays.To determine structure-activity relationships as predictors of cytotoxicity, we compared the cytotoxic effects of closely related compounds.Another widely distributed flavone aglycone in plants is luteolin (6), which possesses one more OH group in the B-ring.According to our data, it affected A375 and C32 cell viability in both MTT (IC 50 = 38.23 and 42.10 µM, respectively, after 48 h of incubation) and NRU (IC 50 = 25.70 and 13.11 µM, respectively, after 48 h of incubation) tests; hence, 6 is a powerful cancer-inhibiting compound.In previous studies, luteolin possessed IC 50 values that reached 18.6 µg/mL (IC 50 = 65.0 µM, A375, 48 h of incubation) and 95.1 µg/mL (IC 50 = 332.5 µM, C32, 24 h of incubation) [1].In our study, luteolin (6) treatment for 48 h decreased A375 cell viability, with IC 50 values of 38.2 µM and 25.7 µM, as determined by MTT and NRU assays, respectively.The IC 50 values for 6 in C32 cells after 24 h treatment were very diverse depending on the assay used, reaching >200 µM in the MTT test and 41.3 µM in the NRU assay.Nevertheless, comparing compounds 15, 16, and 17 seems particularly promising for further stages of research.In our experiment, we demonstrated the anticancer activity of 15 (5,6-dihydroxyflavone) for the first time.This is extremely important because the hydroxyl groups in the A-ring of flavonoids may be responsible for their activity as well as for sharing the unsubstituted B ring, which was also confirmed by the high activity of 16 (7,8-dihydroxyflavone) (Tables 2 and 3).In contrast to 15, flavonoid 16 has already been reported as an anticancer agent in two human malignant melanoma cell lines (SK-MEL-2 and G-361).In the MTS test, the inhibition of the viability by 7,8dihydroxyflavone (16) was significant in both melanoma cell lines (IC 50 values were in the range of 200-250 µM) [27].Moreover, 17, selected in our survey as the most active in the cell viability test, is mentioned in only a few studies for its anticancer activity (e.g., HeLa, SCC, MCF-7, U251N, and PANC-1 cell lines) [21,[28][29][30].It is worth mentioning that increasing activity against cancer cells with 15-17 is not connected to higher toxic effects against normal cells.Compound 37 differs in its structural structure from other active flavonoids; it belongs to the group of isoflavones that have the B-ring attached to the C-ring in position 3 [31].Genistein (37) was a formerly mentioned inhibitor of the growth of another melanoma cell line (OCM-1) and in many other cancers, such as leukemia, lymphoma, ovarian, cervical, leiomyoma, melanoma, neuroblastoma, gastric, pancreatic, breast, and prostate cancer cells [32][33][34].
Reviewing the results obtained from the MTT and NRU tests, we concluded that flavonoid acetylation or glycosylation in carbons in the B-ring (e.g., 10-13) and benzo-γpyrane ring (e.g., 2-5, 27-29) reduce their activity in melanoma cells.Our conclusions are consistent with the available literature data regarding flavonoids and their derivative biological activities.In silico molecular docking analysis and ligand-active site interactions were studied by Nile et al. using inter alia quercetin, quercetin glycoside, quercetin 3,4 ′ -Odiglucoside and α-glucosidase, tyrosinase, and xanthine oxidase enzymes.Their results show that docked quercetin is active against glucosidase, tyrosinase, and xanthine oxidase, which is consistent with our in vitro study [35].
Furthermore, the methylation of hydroxyls is a typical modification found in flavonoid metabolites.Based on previous studies, higher methoxylated compounds are significantly more toxic than less methoxylated molecules.It was found that 7-methoxy-baicalein is more cytotoxic than baicalein.It is also assumed that the overall number of OH groups negatively correlates with the cytotoxic potential of the molecule (apigenin vs. quercetin) [36].Nevertheless, it seems that the overall number of OH groups in the C-and B-rings unfavorably influences the cytotoxic potential of the compounds.There is a lack of information about the A-ring.According to our results, the appearance of free hydroxyls in the A ring (15,16) or the methylation of OH groups in the B-ring (17) increased their function as potential anticancer agents.
In the next step of our experiment, we evaluated the inhibition of DNA biosynthesis by measuring [ 3 H]-thymidine incorporation into the DNA of C32, A375, and normal cells.Thymidylate deficiency and fluctuations in cellular nucleotide pools slow tumor growth by impairing DNA replication and repair [37,38].Again, using 17 caused the most notable decrease in the assay after 48 h of incubation for both A375 and C32 cell lines (IC 50 = 6.0 ± 0.2 and 5.3 ± 0.4 µM, respectively), with very low toxic effects against normal cells.Recently, Strawa and co-workers noticed that 17 evokes DNA biosynthesis inhibition in an oral squamous carcinoma (SCC-25) cell line [21].
In cancers, like many other diseases, an imbalance in apoptotic mechanisms occurs.Flavonoids are known as substances that can be aspirants for the ancillary therapy of cancer via modulating apoptotic pathways [39,40].A summarized scheme of action by 1, 6, 16-17, and 37 in A375 and C32 cells is given in Figure 5.A graphical representation of the substitutions and chemical groups relevant to the cytotoxic activity of these flavonoids is shown in Figure 6.It was previously reported that treatment with apigenin (1) induces the intrinsic and extrinsic pathways of apoptosis.As Bo et al. presented, increased levels of active caspase-3 and caspase-9 and the cleavage product of poly-(ADP-ribose) polymerase (cPARP) are present in human colon carcinoma HCT-116 cells treated with apigenin [41].However, 1 activates kinase RSK, which is responsible for the inactivation of cytosolic pro-apoptotic proteins and the activation of transcription factors that mediate their synthesis [42,43].This phenomenon may become one of the mechanisms by which melanoma cells defend themselves to evade apoptosis.In our study, 1 provoked the activation of caspases-8, -7, -9, and -3 in the A375 cells and caspase-3 in the C32 cells.Compound 6, which differs from apigenin by the presence of a hydroxyl group at C4 ′ , caused a dosedependent activation of caspases in the A375 cell line.The lower concentration (25 µM) induced extrinsic (cleavage of caspase-8) and intrinsic (cleavage of caspase-3, -7, and -9) pathways of apoptosis.However, the presence of cleaved initiator caspases was not found in cells treated with a higher concentration of 6.Also, in C32 cells, treatment with 6 did not cause an increase in the cleavage of initiator caspases.In contrast, it induced the processing of caspase-3 and caspase-7 and promoted apoptosis but inhibited RSK proteins.These results may suggest that 6 shows two dose-dependent mechanisms of action subsequent to cell death at the appointed time of incubation (48 h).Another study demonstrated the activation of caspases-3 and -9 in non-small-cell lung cancer cells A549 and H460 or oral squamous cancer cells SCC-4 [44][45][46].Flavonoid 15, a non-studied till now compound, induced caspase-8 activation in A375 melanoma cells.Furthermore, it possessed a bidirectional and dose-dependent action on RSK proteins, suppressing their phosphorylation at higher concentrations.On the other hand, 15 activated the intrinsic apoptosis pathway by the induction of caspase-9 in C32 cells.A previous investigation indicated that 16 is an activator of caspase-3 and c-PARP in SK-MEL-2 and G-361 melanoma cells [27].Consistent with these findings, 16 induced apoptosis in both A375 and C32 cell lines.This has been formerly shown by Western blot analysis, in which 17 degraded PARP-1, a substrate of caspase 3, in HeLaPKCεA/E cells [47].According to our immunofluorescence and Western blot results, treatment with 17 increased cleaved caspases-3 and -7 levels and led to the processing of PARP.The flow cytometric analysis of C32 melanoma cells showed that 17 (at 25 µM) caused the most significant increase in the percentage of early apoptotic cells (19.7 ± 1.6%) and the percentage of late apoptotic cells (32.8 ± 2.4%).However, 17 induced a massive RSK phosphorylation in A375 cells while inhibiting it in C32 cells.To summarize, the anticancer potential of 17 is most probably based on DNA biosynthesis inhibition (in both A375 and C32) and the inhibition of RSK activity (C32).As previous studies reported, genistein (37) acts on cancer progression mainly by targeting the NF-κB, Akt, and caspase pathways [48][49][50].Based on our investigation, 37 led to apoptosis by the activation of caspases-3, -7, -8, and -9.Other studies of this compound reported that it could induce apoptosis in various human cancer cells (e.g., HT-29) via caspase-3 activation [34,51,52].This may prove the importance of the B-ring attached to the C-ring in position 3 for cytotoxic activity.Indeed, in our study, the anticancer effects and their mechanism for compounds 37 and 1, which differ in the positioning of the 4-hydroxyphenyl group, were similar.However, 37 and 1 inversely affected the phosphorylation of RSK.
In sum, the hydroxylation of the B-ring in the 3 ′ position does not seem to affect the strength and mechanism of the anticancer action of flavonoids (1; 5,7,3 ′ -hydroxyflavone vs. 6; 5,7,3 ′ ,4 ′ -tetrahydroxyflavone).Our conclusions agree with those of previous studies [53].Notably, many reports have provided evidence of the influence of flavonoid glycosylation on their activity [46].Glycosylated flavonoids (6 vs. 10-13 or 1 vs. 2, 3) possess weaker cytotoxic activity than aglycones.The weak antiproliferative effects of flavonoid glycosides might be derived from steric blocking involved in cell entry and receptor binding [47].Flavonoid A-ring poly-O-methylation contributed to enhancing their biological activity [46], which is also consistent with our scores (e.g., 25 vs. 24).It is worth noting that 5-OH seems to play a crucial role in membrane-dependent processes associated with the bioactivity of flavonoids [48].In the present experiment, the most potent compounds have a hydroxyl group in the A rings at the C5 position (1, 6, 15, and 37).However, further explanations should be provided.

Conclusions
The data presented above highlight the structure-activity relations determination of the chemical substitutions of flavonoid compounds responsible anticancer effects.The in vitro studies highlight the importance of the flavonoid cor hydroxyl groups.In conclusion, our results indicate that apigenin, luteolin, dihydroxyflavone, and genistein reduce the viability of amelanotic melanoma A375 C32 cell lines via the intrinsic and extrinsic pathways of apoptosis.On the other hand, dihydroxyflavone and zapotin likely act after the inhibition of DNA biosynthesis.studies also indicate that the RSK pathway is involved in the response of melanoma c to cytotoxic concentrations of certain flavonoids, which may provide a basis for stu on the combined use of flavonoids with inhibitors of RSK kinases.Neverthel additional investigations using in vivo assays to test these anticancer effects are requ to fully understand their mechanism of action.

Conclusions
The data presented above highlight the structure-activity relationship determination of the chemical substitutions of flavonoid compounds responsible for anticancer effects.The in vitro studies highlight the importance of the flavonoid core in hydroxyl groups.In conclusion, our results indicate that apigenin, luteolin, 5,6-dihydroxyflavone, and genistein reduce the viability of amelanotic melanoma A375 and C32 cell lines via the intrinsic and extrinsic pathways of apoptosis.On the other hand, 7,8-dihydroxyflavone and zapotin likely act after the inhibition of DNA biosynthesis.Our studies also indicate that the RSK pathway is involved in the response of melanoma cells to cytotoxic concentrations of certain flavonoids, which may provide a basis for studies on the combined use of flavonoids with inhibitors of RSK kinases.Nevertheless, additional investigations using in vivo assays to test these anticancer effects are required to fully understand their mechanism of action.

Table 2 .
Cytotoxic activity of compounds 1-37 measured using MTT test.IC 50 values are expressed as µM.