Cytotoxic Activity of Gallic Acid and Myricetin against Ovarian Cancer Cells by Production of Reactive Oxygen Species ^†

Abstract

Some studies demonstrate that gallic acid (GA) and myricetin (MYR) isolated from Rhus trilobata provide the therapeutic activity of this plant against cancer. However, few reports demonstrate that both compounds could also have therapeutic potential in ovarian cancer. Therefore, evaluating the cytotoxic activity of GA and MYR against ovarian cancer cells and determining the possible action mechanism present are important. For this purpose, SKOV-3 cells (ovarian adenocarcinoma; HTB-77™, ATCC^®) were cultivated according to the supplier’s instructions (37 °C and 5% CO₂) to determine the biological activity of GA and MYR by confocal/transmission electron microscopy, PI-flow cytometry, H₂DCF-DA, MTT, and Annexin-V assays. Possible molecular targets of the compounds were determined by the Similarity Ensemble approach. Results showed that GA and MYR treatments decreased the viability of SKOV-3 cells at 50 and 166 μg/mL, respectively (p ≤ 0.05, ANOVA vs. vehicle group). They also induced morphological changes (cytoplasmic reduction, nuclear chromatin condensation, cytoplasmic vesicles increment, polymerized actin, and stabilized tubulin), cell cycle arrest (GA: 8.3% G₂/M and MYR: 78% G₁), and apoptosis induction (GA: 18.9% and MYR: 8.1%), due to ROS generation (34 to 42%) for 24 h (p ≤ 0.05, ANOVA vs. vehicle group). In silico studies demonstrated that GA and MYR interact with carbonic anhydrase-IX and PI3K, respectively. In conclusion, GA and MYR show cytotoxic activity against SKOV-3 cells through ROS production, which modifies the cytoskeleton and induces apoptosis. Therefore, GA and MYR could be considered as base compounds for the development of new treatments in chemotherapy for ovarian cancer.

1. Introduction

Ovarian cancer is the sixth most frequent tumor in women and represents the fourth-highest cause of death in Mexico due to gynecological tumors [1]. The principal treatment for this disease is surgical resection, followed by a complementary treatment with chemotherapy, in which cytotoxic or cytostatic drugs are administered with antitumor activity [1,2]. However, chemotherapy for ovarian cancer has shown limited success and generation of resistance in neoplastic tissue, whereby the search for alternative treatments or new therapeutic agents for this disease is necessary. Plants used for alternative medicine represent an option in the search for active compounds for cancer treatment. Some examples of plant compounds with antineoplastic action and used in the treatment of ovarian cancer are paclitaxel (Taxus brevifolia) [3], vincristine (Catharanthus roseus) [4], and curcumin (Curcuma longa) [5], among others [1,2].

Polyphenols are another type of compound found in plants, which have attracted attention in recent decades for their beneficial effects on health, because polyphenols can fight diseases such as cancer through the induction of oxidative stress by the generation of reactive oxygen species (ROS) [6]. In the same sense, several studies have demonstrated that ROS can act as second messengers and regulate different biological processes in cells, such as restructuring the cytoskeleton, division, and death [7]. Some examples of polyphenols with anticancer properties are gallic acid (GA), quercetin, fisetin, kaempferol, and myricetin (MYR), among others [8]. However, several findings have demonstrated that GA and MYR could have interesting applications in ovarian cancer treatment [9,10].

Therefore, this study aimed to evaluate the cytotoxic activity of GA and MYR against ovarian cancer cells and to determine the possible action mechanism present.

2. Materials and Methods

2.1. Compounds Studied

The compounds evaluated were GA (G7384) and MYR (M6760) from Sigma-Aldrich© (≥96% purity, HPLC-grade) (St. Louis, MO, USA). The controls used were paclitaxel (T7402, Sigma^®, St. Louis, MO, USA) because this drug is utilized as treatment for ovarian cancer, 0.3% hydrogen peroxide (H₂O₂; H1009, Sigma^®, St. Louis, MO, USA) as an oxidative stress inducer, and 0.5% DMSO/1× PBS as a vehicle (D2650, Sigma^®, St. Louis, MO, USA) for the dilution of compounds. Reagents and equipment utilized complementary to the study are mentioned in the text.

2.2. Cell Culture Protocol

The cell line used was SKOV-3 (ovarian adenocarcinoma, HTB-77™) acquired from ATCC^® (Manassas, VA, USA). The cell culture was made according to the supplier’s instructions (37 °C and 5% CO₂). Cell were collected with 1× trypsin-EDTA (Sigma^®), and density was determined with 0.4% Trypan blue (Sigma^®).

2.3. Determination of Cell Viability by Formazan Salts

Cultures of 20,000 cells/well were cultivated with 200 μL of supplemented medium (Sigma^®, St. Louis, MO, USA), on 96-well plates (Corning^®, Corning, NY, USA), and incubated for 24 h. Treatments on adherent cells were with 10 to 200 μg/mL of samples or controls for 24 h. At the end of treatments, 5 mg/mL MTT (Sigma^®, St. Louis, MO, USA) was added for 4 h, and optical density was measured at λ: 590 nm in VariosKan^® Flash (Thermo-Scientific^®, Waltham, MA, USA). Half-maximal inhibitory concentration (IC₅₀) was calculated by regression analysis (percentage survival vs. log concentration). Verification of results was by phase-contrast microscopy (LSM 700, Zeiss^®, Pleasanton, CA, USA) [8].

2.4. Measurement of ROS and Apoptosis

Cultures of 20,000 cells/well were cultivated with 200 μL of supplemented medium (Sigma^®, St. Louis, MO, USA), under 96-well black plates (Corning^®, Corning, NY, USA) and incubated for 24 h. Adherent cells were treated with IC₅₀ of samples or controls for 24 h. Then, the culture medium was removed, and ROS/apoptosis was determined on independent assays as follows: (i) ROS: 25 μM H₂-DCF-DA (Sigma^®, St. Louis, MO, USA), for 15 min, at 37 °C, and λ: 488_ex/529_em nm; (ii) Apoptosis: 200 μL 1× binding buffer, 2 μL AnV-FITC (BioVision™, Milpitas, CA, USA), for 15 min, at 37 °C, λ: 485_ex/538_em nm. The optical density was measured with VariosKan^® Flash (Thermo-Scientific^®, Waltham, MA, USA). Verification of the results was by confocal microscopy (LSM 700, Zeiss^®, Pleasanton, CA, USA).

2.5. Evaluation of Cell Cycle by Flow Cytometry and Cell Morphology by Immunofluorescence/TEM

Cultures of 50,000 cells/well were cultivated with 2 mL of supplemented medium (Sigma^®, St. Louis, MO, USA), on 6-well plates (Corning^®, Corning, NY, USA), and incubated for 24 h. Adherent cells were treated with IC₅₀ of samples or controls for 24 h. Then, the culture medium was removed, and cell cycle, cell morphology, and cell ultrastructure were determined in independent assays by flow cytometry, immunofluorescence, and transmission electron microscopy (TEM), respectively, according to the method disclosed by Varela-Rodríguez et al. (2019) [8].

2.6. In Silico Analysis

Identification of target pharmacophores was carried out with the Similarity Ensemble approach (SEA) (http://sea.bkslab.org/, accessed on 1 February 2021) [11]; proteins with binding sites for the active compounds were found through an inverse protein–ligand approach. The parameters used were as follows: pKi, P-Value, or Max TC, for selecting the possible target protein.

2.7. Statistical Analysis

The presentation of the results was as mean ± S.D. of 3 independent assays (n = 3; triplicate). The statistical analysis was performed for parametric data with normal distribution using one-way ANOVA and comparisons were made among means with the negative control (vehicle group without treatment) through Tukey and Dunnett tests (Minitab^®, version 18; State College, PA, USA). Identified differences were considered significant when p ≤ 0.05.

3. Results and Discussion

Some studies demonstrate that polyphenols can induce oxidative stress in cancer cells and alter various biological processes [6]. In SKOV-3 cells, cell viability, apoptosis, and ROS were determined after treatment with AG and MYR for 24 h. GA and MYR presented an IC₅₀ of 50 and 166 μg/mL respectively, in comparison with the negative control (vehicle group) (p ≤ 0.05, Dunnett) (Figure 1A). However, these results were not like those exhibited for paclitaxel, which had a positive control (reference group of cancer drug) with IC₅₀ of 5 μg/mL. In the same conditions, cell death by apoptosis due to the presence of phosphatidylserine was detected in the external cell membrane (Figure 1D). GA and MYR induced 18.9 and 8.1% of apoptosis in comparison to the vehicle group (p ≤ 0.05, Dunnett) (Figure 1B). The induction of apoptosis was like that observed with paclitaxel, but there was less intensity for both compounds (Figure 1D). Additionally, GA and MYR increased intracellular ROS levels among 42 and 34% comparison, with 3.5% observed in the vehicle group (p ≤ 0.05, Dunnett) or 76.5% with the H₂O₂ group (Figure 1B,D).

Several studies have demonstrated that ROS can act as second messengers and modify the cytoskeleton’s restructuring and cell division [7]. Therefore, analyses of cytoskeleton protein such as actin and tubulin were carried out, as well as different phases of the cell cycle during treatments with GA and MYR in SKOV-3 for 24 h. Some morphological changes observed in the treatments with both compounds were rounding and cellular individualization (PCM at 40X; Figure 1D), as well as cytoplasmatic reduction, chromatin condensation in the nucleus, and numerous cytoplasmic vesicles (TEM at 1000X; Figure 1D). These features were like the paclitaxel group. However, the characteristics found differed with the vehicle group, which presented mitotic division and chromosomal segregation (TEM at 1000X; Figure 1D).

Additionally, these changes suggest the presence of apoptotic events and corroborate the results mentioned above. Subsequently, microfilaments and microtubule networks were analyzed in the cell cytoskeleton through immunofluorescence to observe changes in the structuration of actin and tubulin of SKOV-3 cells (Figure 2). Both compounds in actin caused polymerization and a decrease in membrane prolongations or filaments, while in tubulin, they caused stabilization and an increase in microtubules (Figure 2). These changes were found in the paclitaxel group but were absent in the vehicle group (Figure 2). These findings can have important implications for cell functions such as transport, traffic, support, and division [7]; however, further studies to verify this claim are needed.

On the other hand, changes in phases of the cell cycle were observed by flow cytometry (Figure 1C). GA increased the G₂/M phase (8.3%), while MYR increased the G₀/G₁ phase (78%), in comparison with the vehicle group, or the paclitaxel group that increased the G₂/M phase (15.3%) (p ≤ 0.05, Tukey) (Figure 1C). Possibly, these findings can be associated with the fall in cell division detected by TEM, morphological changes seen by PCM, and the increase in intracellular ROS observed in SKOV-3 cells (Figure 1B,D). Finally, in silico analyses were performed with the chemical structures of GA and MYR in SEA to find the protein targets of these compounds. Results indicate that GA could inhibit carbonic anhydrase-IX through ROS induction [10], while MYR appears to be a protein kinases inhibitor, such as PI3K [9] (

Table 1. Therapeutic targets of GA and MYR.

Compound Name	Target Key	Target Protein	Organism	Description	pKi (L.E.)	p-Value	Max Tc * (Affinity)
GA (ZINC1504)	CAH9_HUMAN + 5 (SP: Q16790)	Carbonic anhydrase IX (CA9)	Eukaryote (Human)	Enzyme (E-lyase)	5.13 (0.60 kcal/mol/atom)	9.984 × 10−5	1 (6990 nM)
MYR (ZINC3874317)	PIK3CG_HUMAN + 5 (SP: P48736)	PI3K	Eukaryote (Human)	Enzyme (E-other)	5.33 (0.32 kcal/mol/atom)	0.5057	1

* Affinity binding of compound vs. protein ≤ 10 μM. GA, gallic acid; MYR, myricetin; SP, Swiss-Prot protein sequence database (UniProt); PI3K, Phosphatidylinositol 4,5-bisphosphate 3-kinase.

). The carbonic anhydrase-IX is an enzyme responsible for regulating the internal pH of cells through catalysis [12]; PI3K is an enzyme capable of phosphorylating inositol (phosphatidylinositol), activating AKT, and regulating diverse cell functions such as growth, proliferation, motility, survival, and intracellular trafficking [13] (Table 1). However, none of these molecular interactions have been studied in-depth. Therefore, the protein targets proposed of GA–carbonic anhydrase-IX and MYR–PI3K require of more in-depth studies to demonstrate and confirm these possible interactions.

4. Conclusions

GA and MYR were demonstrated to be capable of acting against SKOV-3 ovarian adenocarcinoma cells through ROS production, which modifies the actin/tubulin cytoskeleton, induces cell cycle arrest, and activates cell death by apoptosis. In silico studies with the SEA model allowed us to propose that carbonic anhydrase-IX and PI3K enzymes could be the targets for GA and MYR, respectively. However, docking, and experimental studies are necessary to confirm this proposal. Therefore, GA and MYR could be considered as base compounds for the development of new treatments in chemotherapy for ovarian cancer.