# Polymethoxyflavones from Gardenia oudiepe (Rubiaceae) induce cytoskeleton disruption-mediated apoptosis and sensitize BRAF-mutated melanoma cells to chemotherapy

Raimundo Gonçalves de Oliveira-Júnior, Nolwenn Marcoult-Fréville, Grégoire

Prunier, Laureen Beaugeard, Edilson Beserra de Alencar Filho, Eduard David

Simões Mourão, Sylvie Michel, Lucindo José Quintans-Júnior, Jackson

Roberto Guedes da Silva Almeida, Raphaël Grougnet, et al.

► To cite this version:

Raimundo Gonçalves de Oliveira-Júnior, Nolwenn Marcoult-Fréville, Grégoire Prunier, Laureen Beaugeard, Edilson Beserra de Alencar Filho, et al. Polymethoxyflavones from Gardenia oudiepe (Rubiaceae) induce cytoskeleton disruption-mediated apoptosis and sensitize BRAF-mutated melanoma cells to chemotherapy. Chemico-Biological Interactions, 2020, 325, pp.109109. 10.1016/j.cbi.2020.109109. hal-02946824

HAL Id: hal-02946824

https://hal.science/hal-02946824v1

Submitted on 5 Aug 2025

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<figure><img src="image_1.png" /></figure>
POLYMETHOXYFLAVONES FROM *Gardenia oudiepe* (RUBIACEAE) INDUCE CYTOSKELETON DISRUPTION-MEDIATED APOPTOSIS AND SENSITIZE BRAF-MUTATED MELANOMA CELLS TO CHEMOTHERAPY

Raimundo Gonçalves de Oliveira-Júnior<fnref n="1" />*, Nolwenn Marcoult-Fréville<fnref n="1" />, Grégoire Prunier<fnref n="1" />, Laureen Beaugeard<fnref n="1" />, Edilson Beserra de Alencar Filho<fnref n="2" />, Eduard David Simões Mourão<fnref n="2" />, Sylvie Michel<fnref n="3" />, Lucindo José Quintans-Júnior<fnref n="4" />, Jackson Roberto Guedes da Silva Almeida<fnref n="5" />, Raphaël Grougnet<fnref n="3" />#, Laurent Picot<fnref n="1" />#

<footnote n="1">¹ LIENSs UMRi CNRS 7266, La Rochelle Université, 17042 La Rochelle, France.</footnote>

<footnote n="2">² Department of Pharmacy, Universidade Federal do Vale do São Francisco, 56304-205, Petrolina, Pernambuco, Brazil.</footnote>

<footnote n="3">³ Equipe Produits Naturels, Analyse, Synthèse, UMR CNRS 8038, Université Paris Descartes, 75006 Paris, France.</footnote>

<footnote n="4">⁴ LANEF, Universidade Federal de Sergipe, 49100-000 São Cristóvão, Sergipe, Brazil</footnote>

<footnote n="5">⁵ NEPLAME, Universidade Federal do Vale do São Francisco, 56304-205, Petrolina, Pernambuco, Brazil.</footnote>

<footnote>*Corresponding authors at: 1) UMRi CNRS 7266 LIENSs, La Rochelle Université, Curie B101 Faculté des Sciences et Technologies, Avenue Michel Crépeau, 17042 La Rochelle, France. E-mail address: rgonca01@univ-lr.fr (Raimundo G. de Oliveira-Júnior).</footnote>

<footnote># Both authors have equally contributed to the supervision of this study.</footnote>
ABSTRACT

A series of 10 natural and semisynthetic flavonoids (1 to 10) were obtained from *Gardenia oudiepe* (Rubiaceae), an endemic plant from New Caledonia. Most of them were polymethoxylated flavones (PMFs) of rare occurrence. After a cell viability screening test, PMFs 2 and 3 showed significant cytotoxic activity against A2058 human melanoma cells (IC50 = 3.92 and 8.18 µM, respectively) and were selected for in-depth pharmacological assays. Both compounds inhibited cell migration and induced apoptosis and cell cycle arrest after 72h of treatment. Immunofluorescence assays indicated that these outcomes were possibly related to the induction of cytoskeleton disruption associated to actin and tubulin depolymerization. These data were confirmed by molecular docking studies, which showed a good interaction between PMFs 2 and 3 and tubulin, particularly at the colchicine binding site. As A2058 are considered as chemoresistant to conventional chemotherapy, compounds 2 and 3 (½IC50) were associated to clinically-used antimelanoma drugs (vemurafenib and dacarbazine) and combined therapies efficacy was assessed by the MTT assay. PMFs 2 restored the sensitivity of A2058 cells to dacarbazine treatment (IC50 = 49.38 µM vs. >100 µM). Taken together, these data suggest that PMFs from *G. oudiepe* could be potential leaders for the design of new antimelanoma drugs.

**Keywords:** apoptosis; cancer; cytoskeleton; flavonoids; melanoma; multidrug resistance.

Highlights

* PMFs from *G. oudiepe* inhibited proliferation and migration of A2058 cells;

* PMFs 2 and 3 induced apoptosis, cell cycle arrest and cytoskeleton disruption;

* Molecular docking showed a good interaction of PMFs on the colchicine binding site;

* PMF 2 sensitized melanoma cells to dacarbazine.
# 1. Introduction

Malignant melanoma is one of the most aggressive forms of skin cancer. Although it accounts for about 1% of all malignant skin tumors diagnosed in developed countries (e.g. USA), cutaneous melanoma is responsible for most of skin cancer deaths [1]. Overall, melanomas derive through malignant transformation of melanocytes, cells producing melanin pigment [2]. It is also well described that melanogenesis can affect the behavior of melanoma cells and consequently the antimelanoma therapy [3-6]. Cutaneous melanoma presents a high invasive and metastatic potential, which hinders its treatment by conventional therapies. When the diagnosis is delayed, melanoma metastasis spreads quickly into many organs (e.g. bones, liver, brain, lungs, etc.) and survival rate falls down dramatically [7,8]. Until recent years, the treatment of advanced melanoma was very limited, mostly focused on the use of dacarbazine, an alkylating agent used as monotherapy or combined with other conventional anticancer drugs [9]. Fortunately, new therapeutic options have emerged in the past decades. Promising results were obtained with small molecules targeting the RAF/MEK pathway (e.g. vemurafenib and dabrafenib, two BRAF inhibitors; and trametinib, a MEK inhibitor), mainly in BRAF-mutated metastatic melanoma [10-12]. However, multidrug resistance mechanisms are increasingly common in melanoma cells, requiring the use of first-line drugs at high doses or even combinations of several drugs (e.g. BRAF inhibitors could be combined with alkylating agents or MEK inhibitors) [13-15]. Depending on the clinical stage, these therapeutic approaches are no longer efficient and usually generate harmful side effects [16,17].

Recent studies have investigated flavonoids as new candidates for the treatment of melanoma. They exhibited significant cytotoxicity against several tumor cell lines through down-regulation of growth factors (e.g. EGF and VEGF), alterations of survival signaling pathways (e.g. ERK, Akt-1, NF-κB, JNK, MAPK), cell cycle arrest and apoptosis induction by modulation of pro-apoptotic and anti-apoptotic proteins (e.g. caspases, Bcl-2, Bcl-xL, Bad,) and oxidative stress [18-22]. Among flavonoids with promising antitumor potential, polymethoxyflavones (PMFs) have been highlighted because of their pharmacological versatility, acting as multitarget drugs in numerous experimental models. In addition to modulating signaling pathways typically involved in apoptosis and cell proliferation [23,24], previous studies have shown that PMFs induce cytoskeleton disarrangement associated with depolymerization of actin filaments and microtubules [25,26]. PMFs are also capable of
sensitizing cancer cells to chemotherapy, enhancing the efficacy of clinically-used anticancer
drugs with no additional toxicity [27,28].

Although the anticancer effects of PMFs have been previously reported, their antimelanoma potential is still largely unexplored to date. In this context, the aim of this study was to investigate the antimelanoma effects of natural and semisynthetic flavones from *Gardenia oudiepe* (Rubiaceae), a non-endangered shrub endemic to New Caledonia. These molecules were obtained from bud exudates of the plant and exhibit rare substitution patterns on A and B rings. In this paper, we describe the influence of the methoxy/hydroxy substituents on the antimelanoma activity of a PMFs series. Besides, we provide unprecedented evidences about two flavones with pro-apoptotic activity mediated by caspase activation and cytoskeleton disruption in chemoresistant BRAF-mutated melanoma cells.

**2. Materials and Methods**

**2.1. Plant material**

Bud exudates of *G. oudiepe* were collected in October 2008 in Forêt Plate, North Province of New Caledonia. After plant identification, a voucher specimen (POU-0290) was deposited at the Herbarium of the Botanical and Tropical Ecology Department of the IRD Center, Noumea, New Caledonia.

**2.2. Isolation of flavones 1–7 from G. oudiepe**

Bud exudates of *G. oudiepe* (200.0 g) were dissolved in dichloromethane. After filtration and evaporation of the solvent, 52.0 g of exudates free from vegetal pieces were recovered. Chromatographic fractionations on normal phase silica gel using cyclohexane/dichloromethane and dichloromethane/methanol (increasing polarity) as eluent solvents provided flavones 1–7.

**2.3. Semi-synthesis of flavones 8–10 from kaempferol**

Flavones **8–10** were obtained as previously reported [29]. Compound **8** was obtained by methylation of kaempferol (100 mg) (Sigma-Aldrich®, France), using 4 equiv. of dimethyl sulfate (Me₂SO₄) and 2 equiv. of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). The reaction was performed at room temperature in dried acetone for 1 h. After precipitation and washing with iced water, obtained residues were solubilized with ethyl acetate (15 mL) and treated with a solution of 1 N HCl (3 mL). Finally, reaction product was extracted with ethyl acetate
(3 x 10 mL) and the organic phase was washed with a saturated solution of NaCl, then dried over Na₂SO₄. Paper filtration and solvent evaporation provided a mixture, which was purified by silica gel column chromatography using as eluent dichloromethane/methanol (95/5, v/v, yield: 70%).

For preparation of compounds **9** and **10**, kaempferol (80 mg, 0.28 mmol) was acetylated using acetic anhydride in dry pyridine. The reaction was performed by stirring for 15 min at room temperature. After precipitation and washing with iced water, recrystallization in CH₂Cl₂/CH₃OH (9/1, v/v) provided 108 mg (0.26 mmol) of triacetyl derivative **9** (yield: 95%). The same procedure for the semi-synthesis of **9** was applied for 4 h instead of 15 min. After precipitation and washing with iced water, recrystallization in CH₂Cl₂/CH₃OH (9/1, v/v) provided 120 mg (0.26 mmol) of tetra acetyl derivative **10** (yield: 95%). ¹H and ¹³C NMR data of **8 – 10** were in accordance with those previously reported [29,30].

## 2.4. *Cell line and culture conditions*

All pharmacological assays were performed on A2058 cells (ATCC® CRL-11147, LGC ATCC Standards, France). It is a melanoma cell line obtained from metastatic cells removed from lymph nodes of a 43-year-old Caucasian patient and considered as tumorigenic at 100% frequency in nude mice. This cell line expresses the oncogenic BRAF<sup>V600E</sup> mutation and alterations in *in PTEN*, *RB1* and *p53* genes, providing a clinically relevant model for evaluation not only of new anti-melanoma molecules, but also of new chemosensitizer agents [31,32]. Previously, we have demonstrated that this cell line is also resistant to conventional chemotherapy [33]. Cells were grown in DMEM culture medium and supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Dominique Dutscher, France), at 37 °C in humidified atmosphere with 5% CO₂.

## 2.5. *Cell viability assay*

Compounds **1 – 10** were solubilized in DMSO at 10⁻² M and diluted in DMEM to obtain 10 µM solutions. Final concentrations of DMSO were lower than 1%, tested as negative control and validated as non-cytotoxic concentration. Cell viability was determined by the MTT assay as previously reported [34]. Confluent cells were trypsinized and centrifuged at 1200 g for 5 min. The supernatant was discarded and cell pellet was suspended in culture cell medium to obtain a 4 x 10⁴ cel.mL⁻¹ suspension. Cells (2 x 10³/well) were added into 96-well plates with **1 – 10** (10 µM) and incubated for 72 h. After treatments, 20 µL of a MTT solution (5 g.L⁻¹) was added to each well and microplates were incubated for 4 h.
Formazan crystals were dissolved in DMSO (100 µL) and absorbances were measured using a FLUOstar Omega microplate reader (BMG Labtech, France). Cell viability was expressed as percentage of growth inhibition (GI%). Photomicrographs after treatments were obtained in a Nikon Eclipse TS100 inverted microscope (magnification X100) equipped with a numeric camera. The most bioactive flavones (2 and 3) were subsequently tested at increasing concentrations (1 – 20 µM) to determine their IC₅₀ by non-linear regression analysis.

## 2.6. Cell migration assay

Cell migration was determined by the wound healing assay as previously described [33]. Cells (2 x 10³/well) were grown in 24-well plates to 90% confluence. Cell monolayers were scratched with a sterile plastic tip, washed with PBS solution and incubated in a new cell culture medium containing low-toxic concentrations of 2 or 3 (½IC₅₀ = 2 and 4 µM, respectively) for 48 h. Cell migration was recorded using a ZEISS Axion Observer microscope in living cell mode (one photomicrograph every 10 min). Cell migration was calculated at 0, 24 and 48h of treatment by measuring the cell surface using ImageJ® software.

## 2.7. Apoptosis analysis

### 2.7.1. Annexin-V/6-CFDA staining

A2058 cells were incubated in 4-well slides (5 x 10³/well) for 24 h and then exposed to the PMFs 2 (4 µM, IC₅₀) and 3 (8 µM, IC₅₀) during 72 h. After treatments, cells were washed with PBS and double stained with annexin V-Cy3 (red fluorescence) and 6-carboxyfluorescein diacetate (6-CFDA, green fluorescence) solution, according to manufacturer's recommendations (Sigma-Aldrich®, France). Before visualization, DAPI was added to wells for DNA staining of necrotic cells. Staurosporine (1 µM, for 4 h) was used as standard drug. Cells were observed under fluorescence microscope (ZEISS Axion Observer, France).

### 2.7.2. Caspase-3 activity

Caspase-3 activity was determined using a commercial assay based on the hydrolysis of Ac-DEVD-pNA (CASP3C kit, Sigma-Aldrich®, France). Cells (2.5 mL of a cell suspension at at 5 x 10⁵ cel.mL⁻¹) were incubated in 75 cm² flasks and exposed to cell culture medium (12.5 mL, negative control) or compounds 2 (4 µM, IC₅₀) and 3 (8 µM, IC₅₀) for 72 h. After treatments, cells were washed with PBS, lysed with lysis buffer and the enzymatic
activity was measured according to manufacturer's recommendations. Results were expressed as µmol pNA/min/mL. Staurosporine (1 µM, for 4 h) was used as standard drug.

## 2.8. Cell cycle analysis

Cell cycle analysis was performed by flow cytometry, using propidium iodide (PI) staining. A2058 cells were grown in control culture medium or treated with the PMFs 2 (4 µM, IC₅₀) and 3 (8 µM, IC₅₀) for 72 h. Then, cells were stained in PBS containing PI 100 µg.mL⁻¹, RNAse A 100 µg.mL⁻¹ and 0.1% Triton X-100 (ThermoFisher Scientific, France), at 37 °C for 15 min. After staining, melanoma cells were analyzed using a FACS Cantoll flux cytometer (BD Biosciences, France) equipped with an air cooled blue LASER (λ = 488 nm, 20 mW). Light diffusion parameters (forward and lateral scatter lights) were adjusted to define the size threshold excluding cellular debris and cell clusters for single-cell fluorescence analysis. PI fluorescence was measured using a FL3 filter (λ = 670 nm) and analyzed using the BD FACS Diva Software (BD Biosciences, France). Distribution of A2058 cells in different cell cycle phases was determined according to their DNA content as measured by the fluorescence intensity of PI: diploid cells (2n): G0/G1 phase; replicative cells (2n&lt;DNA content&lt;4n): S phase; tetraploid cells (4n): G2/M phase; hypodiploid cells (DNA content &lt; 2n): apoptotic sub-G1 phase [33,35].

## 2.9. Cytoskeleton analysis

### 2.9.1. Actin staining with phalloidin

Cells were incubated in 4-well culture slides (1 x 10⁴/well) for 24 h. After incubation, cells were treated with compounds 2 (4 µM, IC₅₀) and 3 (8 µM, IC₅₀) for 24 h, washed with PBS and then fixed with 4% paraformaldehyde, for 15 min at 37 °C. Subsequently, cells were permeabilized with 0.1% Triton X100 for 10 min, washed with PBS and stained with a fluorescent conjugated phalloidin solution (Phalloidin-TRITC 10 µg.mL⁻¹, Sigma-Aldrich®, France) for 40 min at room temperature. After phalloidin staining, DAPI was added to all wells for nucleus staining (15 min, at room temperature). Finally, cells were washed with PBS to remove excess staining solution and immediately visualized under fluorescence microscope (ZEISS Axion Observer, France).

### 2.9.2. Immunofluorescence assay for α-tubulin staining
Melanoma cells were grown in 4-well culture slides (1 x 10⁴/well) for 24 h, washed with PBS and then exposed to PMFs **2** (4 µM, IC₅₀) and **3** (8 µM, IC₅₀) for 24 h. Cells were fixed with 4% paraformaldehyde in PBS, for 15 min at 37 °C. After fixation, cells were permeabilized and saturated with a 3% BSA solution containing 0.1% Triton X100 in PBS, and then incubated overnight at 4 °C with a mouse anti-α-tubulin-FITC monoclonal antibody (Sigma-Aldrich®, France) at 1:50 dilution in PBS. After incubation, cells were washed with PBS and nucleus staining was performed with a DAPI solution during 15 min at room temperature. Photomicrographs were taken on a Zeiss fluorescence microscope (ZEISS Axion Observer, France).

## 2.10. Docking study

Molecular docking procedures were performed to access the interaction modes for **2** and **3** with tubulin. Analyses were performed using both nocodazole and colchicine binding sites, which are slightly displaced in the region between the A and B domains of tubulin [36]. Crystallographic structures of tubulin complexed with colchicine and nocodazole were obtained from RCSB protein data bank [36,37] (https://www.rcsb.org/), under the codes PDB IDs: 4O2B and 5CA1, respectively. The use of colchicine and nocodazole complexes with tubulin aimed to compare the interaction modes of **2** and **3**, considering the specificities of these two crystallographic structures, both available on RCSB PDB data bank.

All the docking procedures were made using the Autodock Vina [38] module of Autodock and AutodockTools packages [39]. This module contains a more recent algorithm that provides better results in comparison with the original Lamarckian Genetic Algorithm of Autodock, with a very smallest computational cost [38].

Before docking procedures, an initial geometry optimization of the ligands was made through the PM3 semiempirical Hamiltonian, using the Gaussian 09W package [40], installed on CENAPAD-UFC cluster architecture (http://www.cenapad.ufc.br/). Redocking procedure was performed using the crystallographic structure of colchicine and nocodazole complexes, considering a grid box with dimensions of 14 x 14 x 14 Å and internal spacing of 1.0 Å (default of Vina), providing excellent results concerning energy and RMSD (Root Mean Square Deviation) values. The last parameter must be lower than 2.0 Å for an acceptable redocking analysis. Then, the same parameters were used for the docking procedures using **2** and **3**. 2D and 3D images were generated with Chimera 1.13.1rc [41] and Maestro free module of Schrodinger package [42]. Thus, conformers, orientations, interaction profiles and energies generated were analyzed to corroborate the experimental bioactivity data.
## 2.11. Chemosensitization of melanoma cells to chemotherapy

The chemosensitizing effect of compounds **2** and **3** was evaluated by measuring cell viability in 96-well plates. Cells (2 x 10³/well) were treated with low-toxic concentrations of **2** (2 µM, ½IC₅₀) and **3** (4 µM, ½IC₅₀) in combination with increasing concentrations of vemurafenib and dacarbazine (1 – 100 µM). Vemurafenib (Selleckchem®, France) and dacarbazine (Sigma-Aldrich®, France) were also tested alone to compare their antiproliferative activity with those of combined treatments. After 72 h of treatment, cell viability was determined using the MTT assay as described above and results were expressed as IC₅₀. The combination index (CI) was calculated using the software CompuSyn (version 1.0), according to Chou-Talalay method [43]. CI < 1.0 indicates synergism, CI > 1.0 indicates antagonism, and CI ≈ 1.0 indicates an additive effect.

## 2.12. Statistical analysis

All results are presented as mean ± standard error of the mean (SEM), from at least three independent measurements (n=3). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey's test or unpaired Student's *t* test, according to the case. Values of *p*<0.05 were considered statistically significant. All analyses were performed using GraphPad Prism 6.0 software (GraphPad Prism Software, Inc., San Diego, CA, USA).

# 3. Results and Discussion

## 3.1. Structure-activity relationship

Previous reports have shown that flavon-3-ol are potential candidates for new anticancer drugs and methylation of these compounds frequently improves their antiproliferative effect against tumor cells [26,44,45]. However, the influence of the hydroxy and methoxy substitution pattern of the flavone core remains unclear in terms of antimelanoma potential. This prompted us to screen a series of natural 3-methoxy-PMFs (**1-8**) together with two acetylated kaempferol derivatives (**9**, **10**) for antiproliferative activity toward A2058 melanoma cells at 10 µM. This aggressive cancer cell line expresses the oncogenic BRAF V600E mutation and is therefore considered as highly invasive and metastatic. The resistance of this cell line to conventional chemotherapy was previously demonstrated [33].
Considering the obtained results, some structure-activity relationships may be established (Table 1). PMFs **2**, with a structure including a 5,7-dihydroxy-6-methoxy A-ring, a 3-methoxy C-ring and one methoxy group on the B-ring on position C4', was the most active with 66.53% of growth inhibition. Compound **2**, with an additional methoxy group on C3' and a hydroxyl group on C5' still demonstrated 42.86% of growth inhibition. With the same A- and C-rings, less or more hindered B-ring led to dramatically less efficient derivatives **1** (bearing a single phenol on C4') and **4** (3',4', 5'-trimethoxy).

Flavones **8** and **6-7**, with the same B-and C-rings than **2** and **3**, respectively, but with modified pattern on A-ring (5,7-dihydroxy or 5-hydroxy-7-methoxy), were also much less active. Acetylated derivatives 9 and 10 did not shown any relevant antiproliferative activity.

In summary, 5,7-dihydroxy-6-methoxy A-ring and methoxy groups on C3 and C4' seem to be crucial for antimelanoma activity, in agreement with previous studies involving other types of tumor cell lines [46-49]. In order to decipher the mode of action of the most active PMFs, we decided to conduct a thorough pharmacological study of compounds **2** and **3**.
<table><caption>**Table 1.** Chemical structures and antiproliferative activity of compounds **1-10**.</caption><thead><tr><th>Substitution pattern</th><th>Compound<br>(PMF)</th><th>R<sub>1</sub></th><th>R<sub>2</sub></th><th>R<sub>3</sub></th><th>R<sub>4</sub></th><th>R<sub>5</sub></th><th>R<sub>6</sub></th><th>R<sub>7</sub></th><th>Antiproliferative<br>activity (%)</th></tr></thead><tbody><tr><td></td><td><b>1</b></td><td>OMe</td><td>OH</td><td>OMe</td><td>OH</td><td>H</td><td>OH</td><td>H</td><td>18.55 ± 5.16</td></tr><tr><td></td><td><b>2</b></td><td>OMe</td><td>OH</td><td>OMe</td><td>OH</td><td>H</td><td>OMe</td><td>H</td><td>66.53 ± 5.71</td></tr><tr><td></td><td><b>3</b></td><td>OMe</td><td>OH</td><td>OMe</td><td>OH</td><td>OMe</td><td>OMe</td><td>OH</td><td>42.86 ± 8.27</td></tr><tr><td></td><td><b>4</b></td><td>OMe</td><td>OH</td><td>OMe</td><td>OH</td><td>OMe</td><td>OMe</td><td>OMe</td><td>5.68 ± 5.00</td></tr><tr><td></td><td><b>5</b></td><td>OMe</td><td>OH</td><td>H</td><td>OH</td><td>H</td><td>OH</td><td>H</td><td>22.29 ± 6.69</td></tr><tr><td></td><td><b>6</b></td><td>OMe</td><td>OH</td><td>H</td><td>OH</td><td>OMe</td><td>OMe</td><td>OH</td><td>20.80 ± 6.50</td></tr><tr><td></td><td><b>7</b></td><td>OMe</td><td>OH</td><td>H</td><td>OMe</td><td>OMe</td><td>OMe</td><td>OH</td><td>29.48 ± 4.95</td></tr><tr><td></td><td><b>8</b></td><td>OMe</td><td>OH</td><td>H</td><td>OMe</td><td>H</td><td>OMe</td><td>H</td><td>29.79 ± 9.77</td></tr><tr><td></td><td><b>9</b></td><td>OAc</td><td>OH</td><td>H</td><td>OAc</td><td>H</td><td>OAc</td><td>H</td><td>7.04 ± 7.39</td></tr><tr><td></td><td><b>10</b></td><td>OAc</td><td>OAc</td><td>H</td><td>OAc</td><td>H</td><td>OAc</td><td>H</td><td>10.13 ± 5.27</td></tr></tbody></table>

Mean ± SEM of at least 3 determinations. PMF: polymethoxyflavone. All compounds were tested at 10 µM.

<figure><img src="image_2.png" /></figure>
## 3.2. PMFs inhibit cell growth

The antiproliferative effect of compounds **2** and **3** on A2058 melanoma cells was determined by the MTT assay. Cells were treated with increasing concentrations (1 – 20 µM) of **2** and **3** for 72h and then IC₅₀ values were calculated. PMFs **2** and **3** inhibited cell growth in a concentration-dependent manner, resulting in cell mortality increased by 90-92% upon treatment with 20 µM of both compounds (Figure 1). IC₅₀ values for **2** and **3** were 3.92 and 8.18 µM respectively, indicating a stronger antiproliferative activity when compared to several methoxyflavones previously evaluated against human melanoma cells [26].

<figure><img src="image_3.png" /></figure>

**Figure 1.** Effect of PMFs **2** and **3** on A2058 melanoma cells viability. Cells were cultured in the presence of increasing concentrations of molecules (1 – 20 µM) for 72h and then cell viability was determined by the MTT assay. Results are expressed as mean ± SEM of at least three independent measurements.

## 3.3. PMFs induce apoptosis

Apoptosis induction is one of the most common effects provoked by anticancer drugs. In this sense, the pro-apoptotic effect of **2** and **3** was determined by double fluorescence staining with Annexin-V and 6-CFDA. DAPI was also used for DNA staining of permeable cells. As shown in Figure 2A, PMFs **2** (4 µM, ~IC₅₀) and **3** (8 µM, ~IC₅₀) increased the number of annexin-V and 6-CFDA double-stained cells compared to control, suggesting a significant pro-apoptotic activity. Besides, characteristic apoptotic features were observed in A2058 cells treated with **2** and **3**, including rounding cell, chromatin condensation, nuclear
fragmentation and apoptotic bodies. In contrast, these morphological changes were not evidenced in untreated cells.

To confirm the pro-apoptotic effect of the flavones, the caspase-3 activity, a hallmark of apoptosis, was also determined. Caspase-3 is an executor caspase and displays important role in both intrinsic and extrinsic apoptosis pathways. It is indispensable for certain processes associated with cell dismantling, such as apoptotic chromatin condensation, DNA fragmentation and formation of apoptotic bodies [50]. After 72h of treatment, both PMFs increased the caspase-3 activity compared to untreated cells (*p*<0.05), confirming their capacity to induce apoptosis. Similar results were obtained for staurosporine (2 µM), a pro-apoptotic standard drug (Figure 2B).
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>**Figure 2.** Pro-apoptotic effect of PMFs **2** (4 µM, ~IC₅₀) and **3** (8 µM, ~IC₅₀) on A2058 melanoma cells. (A) Photomicrographs were obtained after 72h of treatment using Annexin-V/6-CFDA double staining + DAPI for DNA staining of permeable cells. (B) Caspase-3 activity was determined by colorimetric assay. Staurosporine (2 µM, 4h) was used as positive control. Data are expressed as mean ± SEM, *p*< 0.05 (vs. control group), according to ANOVA one-way followed by Tukey's post-test (n=3).</figcaption></figure>
### 3.4. PMFs induce cell cycle arrest

To assess whether PMFs-induced cell growth inhibition was mediated via alterations in cell cycle progression, we investigated the effect of flavones 2 and 3 on cell cycle phase distribution by flow cytometry. Cell cycle phases were determined according to the DNA content, stained with propidium iodide solution. Exposure to compound 2 (4 µM, ~IC₅₀) increased sub-G1 cell population, which consists of cells with typical DNA fragmentation patterns caused by internucleosomal hydrolysis of chromatin, corroborating the pro-apoptotic effect previously demonstrated. In addition, PMF 2 caused a significant G2/M arrest at the expense of G0/G1 phase cell population (Figure 3). No relevant effect on cell cycle was observed for compound 3-treated cells.

<figure><img src="image_6.png" /></figure>

**Figure 3.** Effects of PMFs 2 (4 µM, ~IC₅₀) and 3 (8 µM, ~IC₅₀) on cell cycle progression after 72h of treatment. After incubation, melanoma cells were stained with propidium iodide and measured by flow cytometry, as shown in the quantitative distribution of cells in different phases of cell cycle (A) and in the representative histograms (B). Data are expressed as mean ± SEM, *p*<0.05 (vs. control group) according to ANOVA one-way followed by Tukey's post-test, from at least three independent measurements (n=3).

### 3.5. PMFs inhibit cell migration
As A2058 cell line has high invasive and metastatic potential, we determined the effect of PMFs **2** and **3** on cell motility by wound healing assay. To eliminate any interference of cytotoxicity-associated effects, compounds were tested at low-cytotoxic concentrations, equivalent to half of the calculated IC₅₀ values (½IC₅₀ ~ 2 and 4 µM for PMFs **2** and **3**, respectively). At these concentrations, **2** and **3** exerted minimal cytotoxicity, showing growth inhibition (GI) = 11.54 ± 3.81 and 15.35 ± 4.89%, respectively, according to the MTT assay. As presented in Figure 4, the flavones **2** and **3** significantly reduced cell migration after 24 and 48h of exposure (*p*<0.05). Interestingly, PMF **3**-treated cells were completely immobile, suggesting some relevant effects on cell morphology that would be compromising cell motility.

Generally, cell migration involves drastic changes in cell shape, which are mostly driven by the cytoskeleton. It is a highly integrated multistep process that is initiated by the protrusion of the cell membrane, followed by cell remodeling and retraction movements. Firstly, chemoattractant agents stimulate intracellular signaling pathways responsible for regulating actin filaments reorganization. The actin cytoskeleton is essential for the formation of protrusive structures and transducing the mechanical forces necessary for cell translocation at the leading edge. Subsequently, contractile forces mediated by actin/myosin stress fibers promote cell retraction, allowing the translocation of the cytoplasm and nucleus toward the leading edge. In parallel, microtubules play an important role in cell remodeling contributing to retraction movements. Previous studies have revealed that movements are significantly inhibited in cells treated with drugs known to interfere with microtubules assembly, which makes them less dynamic and less able to remodel [51–54]. In this context, we hypothesized that PMFs **2** and **3** would be inhibiting cell migration through cytoskeleton disruption.
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>**Figure 4.** Effect of compounds 2 (2 µM, ~½IC₅₀) and 3 (4 µM, ~½IC₅₀) on cell mobility after 24h (A) and 48h (B) of treatment. Photomicrographs illustrate cell migration into the zone free of cells according to the treatment (C). Data are expressed as mean ± SEM, *p*<0.05 (ANOVA one-way followed by Tukey's post-test), from at least three independent measurements.</figcaption></figure>

### 3.6. PMFs promote cytoskeleton disruption

In order to assess the effect of flavones 2 and 3 on cytoskeleton integrity, A2058 cells were treated for 24h with both compounds at their respective IC₅₀ (~ 4 and 8 µM), and then actin filaments and microtubules structures were visualized by staining with phalloidin and
immunofluorescence using a FITC-conjugated anti-α-tubulin monoclonal antibody, respectively. As shown in Figure 5, untreated cells commonly appeared as fusiform cells with ramifications, presenting microfilaments that formed typical parallel stress fibers. In contrast, 2 and 3-treated cells showed rounding morphology with the presence of peripheral ring of actin near the nucleus, blebbing and accumulation of fragmented F-actin, suggesting that these molecules altered actin filaments organization. These changes may be related not only to the effect on cell motility, but also to the previously shown pro-apoptotic activity. When cells undergo apoptosis, they partially detach from the extracellular matrix, round up, contract, and disassemble peripheral adhesion complexes. Concomitantly, there is loss of stress fibers and actin is rearranged into a peripheral ring. Finally, dynamic membrane protrusions (blebs) are formed followed by actin depolymerization, resulting in the formation of apoptotic bodies. As actin depolymerization has been shown to induce an apoptotic response in various cell lines, altered actin filaments dynamic seems to act as sensor and mediator of apoptosis [55–57].

<figure><img src="image_10.png" /><figcaption>**Figure 5.** Effect of PMFs 2 (4 µM, IC₅₀) and 3 (8 µM, IC₅₀) on actin filaments of A2058 cells. After 24h of treatment, cells were fixed, permeabilized and actin filaments were stained with TRITC-conjugated phalloidin. DAPI was used for DNA staining. Photomicrographs were obtained under fluorescence microscope. Note the stress fiber formation in control cells</figcaption></figure>
and the decreasing of actin filaments and perinuclear accumulation of F-actin in PMF 2 and 3-
treated cells.

On the other hand, actin depolymerization can be also understood as a consequence of cytoskeleton disruption frequently associated to microtubules depolymerization. For this reason, we investigated the effect of PMFs 2 and 3 on tubulin dynamic. Melanoma cells were treated for 24h with 2 (4 µM, ~IC₅₀) and 3 (8 µM, ~IC₅₀), fixed with 4.0% paraformaldehyde, permeabilized and saturated with a PBS solution containing 0.1% triton X-100 and 3.0% BSA, and then stained with an anti-α-tubulin antibody, directly conjugated with FITC. DAPI was used to stain DNA content and cells were visualized under fluorescence microscope. In this assay, cells were also treated with nocodazole 50 nM (a tubulin destabilizing binder) and paclitaxel 50 nM (a tubulin stabilizing binder), allowing to compare different effects on microtubules with the ones observed for the flavonoids. Cells treated with 2 and 3 presented tubulin depolymerization, similar to nocodazole, as well as rounding morphology with accumulation of tubulin near the nucleus and blebbing. Paclitaxel-treated cells showed DNA fragmentation, a typical phenomenon in apoptosis, but microtubules were visibly stabilized (Figure 6).

Microtubules display important cellular functions including chromosomal segregation during cell division, intracellular transport and remodeling, and cell motility. Consequently, drugs that interfere with microtubule dynamics are able to block mitosis and cell migration, and induce cytoskeleton disruption-mediated apoptosis. In this context, tubulins are considered as valuable targets for anticancer drugs. Most of anti-tubulin drugs act on three well characterized sites, inhibiting tubulin polymerization (vinca alkaloids and colchicine sites) or preventing the microtubule disassembly (taxanes site) [58,59]. Recently, methoxylated flavones have been pointed as lead compounds in the design of new tubulin inhibitors. Although substitutions in the A-ring are apparently not relevant for this activity, previous studies have shown that methoxy groups on C3 and C4' are essential to promote tubulin depolymerization [26,45,60,61]. Considering that PMFs 2 and 3 have structural requirements for anti-tubulin activity and that their effects on microtubules were similar to nocodazole (a colchicine site binder), molecular docking studies were performed to better understand the interaction between tubulin with the flavones.
<figure><img src="image_11.png" /><figcaption>**Figure 6.** Effect of PMF 2 (4 µM, IC₅₀) and 3 (8 µM, IC₅₀), nocodazole (50 nM) and paclitaxel (50 nM) on microtubules organization of A2058 cells. After 24h of treatment, melanoma cells were fixed, permeabilized and α-tubulin was stained with a FITC-conjugated anti-α-tubulin monoclonal antibody. Photomicrographs were obtained under fluorescence microscope. Note intact microtubule network in control cells, while PMFs 2 and 3-treated cells showed tubulin depolymerization (rounding morphology and perinuclear accumulation of tubulin), similar to nocodazole-treated cells.</figcaption></figure>

### 3.7. *Molecular docking studies*

Molecular docking procedures were performed to access the interaction modes for 2 and 3 with tubulin. The analyses used both nocodazole and colchicine binding sites, which are
considered to be located in the same region, although slightly displaced between the A and B domains of tubulin [36] (Figure 7). It can be responsible for different modes of interaction with specific amino acids.

<figure><img src="image_12.png" /><figcaption>**Figure 7.** Binding sites of nocodazole (left) and colchicine (right) in tubulin. Both ligands are in magenta.</figcaption></figure>

Initially, the original crystallographic complexes involving nocodazole and colchicine, with the tubulin, were submitted to a redocking procedure to check the efficiency of the Vina algorithm in these systems. Figure 8 (A and B) shows the best molecular geometries of colchicine and nocodazole after redockings (blue), comparing with the respective native geometries (orange). Binding energies (BE) and RMSD values for the best conformations of colchicine and nocodazole are given in Table 2. The excellent redocking results validate the Vina algorithm to verify the possible interaction modes for the tested flavonoids (RMSD = 0.188 Å and BE = -10 Kcal/mol for colchicine; RMSD = 1.085 Å and BE = -9.0 Kcal/mol for nocodazole).
<figure><img src="image_13.png" /><figcaption>**Figure 8.** A1 and A2: Native (orange) and post-redocking (blue) conformations of colchicine and nocodazole in 3D view; B1 and B2: post-redocking complexes in 2D visualization, showing the interactions with specific amino acids (in green, hydrophobic *aa*; in cyan, polar *aa*; in blue, positively charged *aa*; in red, acid *aa*).</figcaption></figure>

<table><caption>**Table 2.** Binding energies and RMSD values for the docked compounds (PMFs **2** and **3**, colchicine and nocodazole).</caption><thead><tr><th>Entry</th><th>Compound</th><th>Binding Energy (Kcal/mol)</th><th>RMSD native (Å)</th><th>RMSD u.b. (Å)</th><th>RMSD l.b. (Å)</th></tr></thead><tbody><tr><td>1</td><td>Colchicine</td><td>-10.0</td><td>0.188</td><td>-</td><td>-</td></tr><tr><td>2</td><td>Nocodazole</td><td>-9.0</td><td>1.085</td><td>-</td><td>-</td></tr><tr><td>3</td><td><b>2</b><sub>col</sub> (conf. 1)</td><td>-8.4</td><td>-</td><td>0.000</td><td>0.000</td></tr><tr><td>4</td><td><b>2</b><sub>col</sub> (conf. 2)</td><td>-8.0</td><td>-</td><td>7.429</td><td>1.320</td></tr></tbody></table>
<table><tbody><tr><td>5</td><td><b>2</b><sub>col</sub> (conf. 3)</td><td>-7.7</td><td>-</td><td>2.008</td><td>1.256</td></tr><tr><td>6</td><td><b>3</b><sub>col</sub> (conf. 1)</td><td>-8.2</td><td>-</td><td>0.000</td><td>0.000</td></tr><tr><td>7</td><td><b>3</b><sub>col</sub> (conf. 2)</td><td>-8.1</td><td>-</td><td>1.938</td><td>0.576</td></tr><tr><td>8</td><td><b>3</b><sub>col</sub> (conf. 3)</td><td>-7.8</td><td>-</td><td>7.653</td><td>1.418</td></tr><tr><td>9</td><td><b>3</b><sub>col</sub> (conf. 4)</td><td>-7.8</td><td>-</td><td>2.241</td><td>1.265</td></tr><tr><td>10</td><td><b>3</b><sub>col</sub> (conf. 5)</td><td>-7.3</td><td>-</td><td>7.866</td><td>1.561</td></tr><tr><td>11</td><td><b>2</b><sub>noc</sub> (conf. 1)</td><td>-5.0</td><td>-</td><td>0.000</td><td>0.000</td></tr><tr><td>12</td><td><b>2</b><sub>noc</sub> (conf. 2)</td><td>-4.1</td><td>-</td><td>3.062</td><td>1.427</td></tr><tr><td>13</td><td><b>3</b><sub>noc</sub> (conf. 1)</td><td>-2.6</td><td>-</td><td>0.000</td><td>0.000</td></tr><tr><td>14</td><td><b>3</b><sub>noc</sub> (conf. 2)</td><td>-1.9</td><td>-</td><td>2.010</td><td>0.473</td></tr></tbody></table>

*"col" indicates the docking using the original protein of colchicine complex as standard. "noc" indicates the use of nocodazole complex. "RMSD native" is related to the crystallographic conformation of colchicine; "RMSD u.b." matches each atom in one conformation with itself in the other conformation, ignoring any symmetry; "RMSD 1.b." matches each atom in one conformation with the closest atom of the same element type in the other conformation, being calculated as the maximum value between atoms c1,c2 and c2,c1.

On the other hand, considering the energy values after docking procedures for PMFs **2** and **3**, it can be observed that the obtained geometries considering the exact binding site of colchicine (entries 3-10 in Table 2) are much more stable than the dockings at nocodazole exact binding site (entries 11-14 in Table 2). This may be explained by the fact that the molecular structure of colchicine has higher similarity with the flavones, considering the type of atoms (carbons and oxygens, only one nitrogen in colchicine), the relative size of rings, and the presence of six-member rings substituted with oxygens (methoxyl, hydroxyl and carbonyl in colchicine and PMFs) (Figure 8 and 9). Thus, the interaction profile with amino acids is more favorable in exact region of colchicine interactions.
<figure><img src="image_14.png" /><figcaption>**Figure 9.** A1: Most stable pose of **2**; A2: Best poses of **2** (in cyan the most stable); A3: Best pose of **2** in 2D visualization; B1: Most stable pose of **3**; B2: Best poses of **3** (in cyan the most stable); B3: Best pose of **3** in 2D visualization.</figcaption></figure>

Considering the exposed above, Figures 9 and 10 show the most stable geometries for **2** and **3** after docking in exact binding site of colchicine. It is important to note that the “symmetrical” structural profile of **2** and **3** (two opposite aromatic A and B-rings with methoxy/hydroxy groups) generated two conformational orientations as possible results. Both are in a same place but inverted by 180°, with near binding energy values (conf1/conf2 for **2**<sub>col</sub> and conf1/conf3 for **3**<sub>col</sub>, Table 2). Interestingly, all the *RMSD* *l.b.* values are less than 2.0 Å (Table 2), indicating that the same atoms types occupy similar positions, even with inverted structure. In fact, a compound can assume more than a unique stable conformation in the binding site, and the similar atoms types and locations in the possible conformers for **2** and **3** (carbons and oxygens), show that the best occupation region for these flavonoids does not suffer significant variation, possessing a good sample profile (Figure 9).
<figure><img src="image_15.png" /><figcaption>**Figure 10.** Superimposed structures of native colchicine (orange) and best poses of **2** (cyan) and **3** (green). In the protein 3D images, the A domain is in red and B domain in blue.</figcaption></figure>

Additionally, considering the first best geometries of the compounds, the interacting amino acids can be observed through the 2D models (Figure 9). The colchicine binding site is predominantly hydrophobic, which favors the interaction with methoxylated flavonoids. On the other hand, the presence of some hydroxyl groups on flavonoid structures leads at least to one hydrogen bond with a donor character, as visualized in 2D maps. The common amino acids interacting with colchicine, **2** and **3** are: LEU242, CYS241, VAL238, interacting with the trimethoxy group of colchicine and dihydroxy-methoxy group of **2** and **3**, in the best pose; ALA180, THR179, interacting with the other extremity of the ligands; ILE318, ALA316, ALA354, LYS352, LEU255, LYS254, ASP251, ALA250, LEU248, interacting with central points of the compounds.

Analyzing and comparing the binding energies between the ligands, colchicine presents the most stable pose at their specific binding site (-10 Kcal/mol), followed by **2**, which is slightly more stable than **3** for the best pose. Then, considering the overall of this first computational analysis by docking algorithms, the molecular binding profiles are in agreement with experimental results and corroborate the mechanism of action for **2** and **3** in tubulin depolymerization.
### 3.8. Chemosensitizing effect

Advanced melanoma is constantly associated with intrinsic or adaptively acquired mechanisms of drug resistance. Targeted therapies involving the use of BRAF and MEK inhibitors showed promising results in the past years, but their therapeutic efficacy is still limited. In most cases, the pharmacological response is temporary and patients develop tumor collapse within one year of treatment [62]. Recently, combined therapies have been employed in the treatment of advanced melanoma using drugs that act on different molecular targets in an attempt to maximize their effectiveness. Although a promising strategy, these drug associations display high toxicity and can lead to rapid liver, kidney and gastrointestinal failure [63]. However, the same approach has been performed using natural products as chemosensitizing agents. Plant-derived molecules are usually well tolerated and have a better safety profile, which encourages their use in adjuvant antimelanoma therapy [64]. In this context, we evaluated the chemosensitizing potential of PMFs 2 and 3 on A2058 melanoma cells.

Melanoma cells were treated with increasing concentrations of vemurafenib (a BRAF inhibitor) and dacarbazine (an alkylating agent), alone or combined to low-toxic concentrations of 2 (2 µM, ~ ½IC₅₀) and 3 (4 µM, ~ ½IC₅₀). After 72h of treatment, cell viability was measured by the MTT assay. As shown in Figure 11 and Table 3, A2058 cells were sensitive to vemurafenib, which exhibited IC₅₀ = 5.93 µM, but resistant to dacarbazine (IC₅₀ > 100 µM). When combined to PMFs 2 or 3, vemurafenib showed no improved antiproliferative effect. In contrast, PMF 2 restored the sensitivity of melanoma cells to dacarbazine treatment, which showed IC₅₀ = 49.38 µM.

<table><caption>**Table 3.** Effect of monotreatments (vemurafenib or dacarbazine) and combined treatments (vemurafenib or dacarbazine + PMFs 2 or 3) on A2058 melanoma cells. Data are presented as IC₅₀ values and 95% confidence intervals.</caption><thead><tr><th>Monotreatment</th><th>IC<sub>50</sub> (µM)</th><th>Combined treatment</th><th>IC<sub>50</sub> (µM)</th></tr></thead><tbody><tr><td rowspan="2">Vemurafenib</td><td rowspan="2">5.93 (4.71–7.47)</td><td>Vemurafenib + PMF 2</td><td>7.98 (6.72–9.47)</td></tr><tr><td>Vemurafenib + PMF 3</td><td>6.75 (5.61–8.12)</td></tr><tr><td rowspan="2">Dacarbazine</td><td rowspan="2">&gt;100</td><td>Dacarbazine + PMF 2</td><td>49.38 (35.43–68.81)</td></tr><tr><td>Dacarbazine + PMF 3</td><td>&gt;100</td></tr></tbody></table>

PMFs 2 and 3 were tested at low-toxic concentrations (½IC₅₀ ~ 2 and 4 µM, respectively). Vemurafenib and dacarbazine were tested in increasing concentrations (1 – 100 µM).
Combination index (CI) was calculated using the Chou-Talalay theorem in order to better characterize the sensitizing effect of flavone 2. According to CI values, combined therapies can provide additive (CI ~ 1), synergistic (CI < 1) or antagonistic (CI > 1) effects. Compound 2 showed CI values of approximately 1.0 when associated with almost all tested dacarbazine concentrations, indicating an additive effect (Figure 11C). A synergistic effect was observed at dacarbazine 20 µM. Taken together, these results suggest that G. oudiepe is a potential source of antimelanoma PMFs, acting as cytotoxic or/and chemosensitizing agents on metastatic and chemoresistant tumor cells.

<figure><img src="image_16.png" /></figure>

**Figure 11.** Chemosensitizing effect of PMFs 2 and 3 on A2058 cells. (A): antiproliferative effect of vemurafenib (Vemu, 1–100 µM) alone or in the presence of compounds 2 and 3 (~ ½IC₅₀). (B): antiproliferative effect of dacarbazine (Daca, 1–100 µM) alone or in the presence of compounds 2 and 3 (~ ½IC₅₀). (C): combination index for the association of dacarbazine with PMF 2. (D) photomicrographs show reduction of cell density promoted by combined therapy, compared to monotherapies and control group. Results are expressed as mean ± SEM, *p*<0.05 according to unpaired Student's *t* test, from at least three independent measurements.
Melanoma cells are a useful experimental model for assessing the chemosensitizing potential of new anticancer agents. This is because their tumor progression is closely related to multiple resistance mechanisms that hinder the efficacy of both new and older therapies (e.g. BRAF<sup>V600E</sup>, NRAS, p53, PTEN and MEK mutations) [65]. Although the antiproliferative effect of PMFs has been previously demonstrated in several tumor cell lines, their anticancer potential is not yet sufficiently explored in melanoma cells. The results presented in this study corroborate previous pharmacological reports, providing evidences about the increase in antimelanoma activity according to the methylation degree of the studied flavones [23-26]. PMFs 2 and 3 present structural similarities with other anti-tubulin agents, which contributed to their effect in disarranging cell cytoskeleton and, consequently, inducing cell death. In another perspective, the chemosensitizing effect induced by low toxic concentration of PMF 2 (1/2IC<sub>50</sub>) provides a new alternative for the treatment of metastatic melanoma through its combination with conventional anticancer drugs, reducing their therapeutic doses, limiting their toxic effects on non-tumoral tissues and possibly delaying multidrug resistance mechanisms.

**4. Conclusions**

The antimelanoma potential of ten natural and semisynthetic flavones from *G. oudiepe* was evaluated. PMFs 2 and 3 were the most active derivatives. They inhibited cell proliferation and migration, and induced cell arrest in G2/M phase. Both molecules also induced apoptosis after 72h of treatment. These outcomes are possibly related to the induction of cytoskeleton disruption associated with actin and tubulin depolymerization. These data were confirmed by molecular docking studies, which showed a good interaction between PMFs 2 and 3 and tubulin, particularly at the colchicine binding site. In addition, the flavones also restored the sensitivity of A2058 cells to dacarbazine treatment. These results suggest that the most active PMFs from *G. oudiepe* could be candidates for the treatment of metastatic melanoma or used as adjuvant in antimelanoma therapy.

**Acknowledgments**

This work was accomplished through technical-scientific collaboration by Franco-Brazilian Network on Natural Products (FB2NP). The authors thank the North Province of New Caledonia for facilitation of field investigations, Dr. Vincent Dumontet for plant collection and Chouaha Bouzidi for the management of the chemical library.
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IC₅₀ on melanoma cells

PMF 2 = 4 µM

PMF 3 = 8 µM

<figure><img src="image_17.png" /><figcaption>Polymethoxyflavones (PMFs)</figcaption></figure>

Control cell

<figure><img src="image_18.png" /></figure>

Treated cell

<figure><img src="image_19.png" /></figure>

α-tubulin staining

<figure><img src="image_20.png" /></figure>

Microtubule disassembly

<figure><img src="image_21.png" /></figure>

Apoptosis induction

<figure><img src="image_22.png" /></figure>

Cell migration inhibition

<figure><img src="image_23.png" /></figure>

Cell cycle arrest

<figure><img src="image_24.png" /></figure>

Chemosensitization

<figure><img src="image_25.png" /></figure>

Colchicine binding site