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. 2021 Sep 29;73(6):761–774. doi: 10.1007/s10616-021-00495-y

Aglycone flavonoid brachydin A shows selective cytotoxicity and antitumoral activity in human metastatic prostate (DU145) cancer cells

Larissa Cristina Bastos de Oliveira 1, Higor Lopes Nunes 1, Diego Luis Ribeiro 2, Jessyane Rodrigues do Nascimento 3, Cláudia Quintino da Rocha 4, Ilce Mara de Syllos Cólus 1, Juliana Mara Serpeloni 1,5,✉
PMCID: PMC8554999  PMID: 34776627

Graphic abstract

In prostate cancer, flavonoids possess a wide variety of anticancer effects, focused on the antioxidant/pro-oxidant activity, inactivation of the androgen receptor, cell cycle arrest, apoptosis induction, metastasis inhibition, among others. This current research investigated the antitumoral in vitro activity of Brachydin A (BrA), a dimeric flavonoid isolated from Fridericia platyphylla, in human castration-resistant prostate cancer DU145. It was compared BrA selective effects in tumor prostate DU145 cells with non-tumor prostate epithelial PNT2 cells. Cell viability experiments (resazurin, neutral red, MTT, and LDH release assays) showed that BrA was sevenfold more cytotoxic to tumor cells than non-tumor prostate cells, with IC50 values of 77.7 µM and 10.7 µM for PNT2 and DU145 cells, respectively. Furthermore, BrA induced necrosis and apoptosis (triple fluorescence staining assay) without interfering with oxidative stress (CM-H2DCFDA) in DU145 cells. Also, BrA (15.36 µM) reduced cell proliferation on clonogenic assay (DU145 cells) but no change in cell number and protein content was observed when cell growth curve assay was used. Wound healing and transwell assays were used for checking the effects of BrA on cell migration and invasion, and BrA impaired these processes in PNT2 (wound healing) and DU145 cells (transwell). Our results inspire further studies to test BrA as a novel chemotherapeutic drug and to evaluate its effects on drug-resistant metastatic cancer cells.

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Keywords: Apoptosis, Fridericia platyphylla, Chemoprevention, Cytotoxicity, Phytochemical, PNT2 cells

Introduction

Prostate cancer (PCa) is the fourth most frequently diagnosed cancer (7.3%) in the world, preceded by female breast cancer (11.7%), lung (11.4%), and colorectal (10.0%) cancers. In men, PCa is the most frequently diagnosed cancer in 112 countries, followed by lung cancer in 36 countries (Sung et al. 2021). Currently, treatment for advanced PCa focus on Androgen Deprivation Therapy (ADT), which regulates the proliferation of prostate cells; however, the growth of prostate tumor cells is often androgen-independent (Kvízová et al. 2021) limiting the response to treatments which frequently results in drug resistance; therefore, the search for new therapies is inevitable.

Phytochemicals, bioactive chemical substances produced as secondary metabolites in plants, are highly preferred over synthetic drugs in inhibiting tumor growth and promoting cell death due to their low toxicity with minimal side effects (Bonta 2020). Indeed, flavonoid compounds were intensively tested for their capacity to enhance the effect of anticancer drugs and to combat multidrug resistance in different types of cancers, including PCa (Costea et al. 2020). Their molecular mechanisms in PCa are dependent on the antioxidant/pro-oxidant effects, androgen receptors, cell cycle arrest, apoptosis induction, angiogenesis blockage, metastasis inhibition, and genetic/epigenetic mechanisms (Costea et al. 2019).

The recent advancement and sensitivity of analytical techniques have overcome the difficulties in the separation and identification of phytochemicals, allowing easy identification of the specific bioactive compounds. Brachydins are unique dimeric flavonoids isolated from Fridericia platyphylla, also known by the synonym name Arrabidaea brachypoda, a Brazilian medicinal plant (da Rocha et al. 2014; Nascimento et al. 2021). They are composed of four independent rings (labeled A, B, C, and D) and two fused benzopyran rings, with different substituent groups on the C ring. Brachydin A (BrA) differs from brachydins B (BrB) and C (BrC) in the presence of hydroxyl radical on C ring (da Rocha et al. 2014) (Fig. 1).

Fig. 1.

Fig. 1

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Chemical structure of Brachydin A ((6S)-7-[(E)-2-(4-hydroxyphenyl)ethenyl]-1,8-dimethoxy-6-phenyl-6,7-dihydrochromeno[3,2-c]chromene-3,10-diol) 

Regarding its biological activities, Nunes et al. (2020) compared BrA with other molecules using a computational method based upon Tanimoto’s coefficient in PubChem, and this method revealed similarities with other molecules such as licoricidin (CID: 480865) and glyasperin D (CID: 480860), that were cytotoxic to hepatic (HepG2), breast (MCF-7), lung (A459), and colon (SW480) tumor cells. The authors also noticed the similarity with erycibenin (CID: 16093658) that inhibited proliferation of tumor lymphocytes.

Despite the recent identification, brachydins have been poorly investigated regarding their biological activities. For example, BrB showed antimicrobial activity against Candida albicans and enhanced the bioactivity of both norfloxacin and ethidium bromide against S. aureus SA1199-B (Sousa Andrade et al. 2020). Furthermore, BrB and BrC inhibited Tripanosoma cruzi invasion and its intracellular development in host cells with similar potencies to the antiprotozoal drug benznidazole (Rocha et al. 2014). BrA, BrB, and BrC presented in vitro anti-inflammatory activities in arthritic synoviocytes (Salgado et al. 2020), a result that corroborates the traditional use of roots for osteoarthritis treatment. Finally, our group previously showed that all three brachydins induced cytotoxicity in metastatic prostate PC3 cells in vitro (Nunes et al. 2020). Considering the promising results, here we investigated the biological effects of BrA in human non-tumor prostate cells (PNT2) and tumor metastatic prostate cells (DU145), integrating cell viability/proliferation, ROS overproduction, and cell migration/motility analysis.

Materials and methods

Brachydin A

The full details of collection, extraction, isolation, structure elucidation and identification of BrA (MW: 524 g/mol) were described by Rocha et al. (2017) and Nascimento et al. (2021). Briefly, the structure elucidation was determined using Nuclear Magnetic Resonance and High-Resolution Mass Spectrometry. The purity of BrA was determined to employ Ultra-Performance Liquid Chromatography (UHPLC-HMRS) analysis and was greater than 98%. The BrA was sent to the Mutagenesis and Oncogenetics Laboratory from Londrina State University (UEL; Londrina, PR, Brazil) and was promptly dissolved in Dimethyl Sulfoxide (DMSO; Labsynth, Diadema, SP, Brazil) and phosphate-buffered saline (PBS, pH 7.4) to obtain a final stock-solution of DMSO 0.25% in cell culture.

Cell lines and culture conditions

The human androgen-independent prostate carcinoma cell line (DU145) isolated from a brain metastatic patient was purchased from ATCC® (American Type Culture Collection - Cat. Nº HTB-81TM; Manassas, VA, USA). The non-tumor prostatic epithelial cells PNT2 cells were obtained from the BCRJ (Rio de Janeiro Bank Cell Collection - Cat. Nº 0366; Rio de Janeiro, RJ, Brazil). Both cell lines were cultivated using Roswell Park Memorial Institute 1640 (RPMI 1640 - Gibco, Grand Island, NY, USA) medium supplemented with 10% Fetal Bovine Serum (FBS – Gibco, Grand Island, NY, USA), 0.024% sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA) and 1% antibiotic-antimycotic solution (10,000 units/mL penicillin, 10,000 µg/mL streptomycin and 25 µg/mL amphotericin B; Gibco) and kept in an incubator Thermo Scientific 3110 Series II CO2 Water Jacketed (Thermo Fisher Scientific; Carlsbad, CA, USA) with an atmosphere of 5% CO2 at 37 °C and 96% relative humidity. When the cultures reached confluency (70–90%), the cells were washed twice with PBS, detached with TrypLE™ Express Enzyme 1× (Gibco, Grand Island, NY, USA), and sub-cultured. All the treatments, except for cell proliferation curves and wound healing assays, were performed in FBS-free RPMI 1640 Medium.

Experimental design

Treatments were performed with Negative Control (NC; PBS), Solvent Vehicle (SV; 0.25% DMSO) and different BrA concentrations for DU145 cells (0.24; 0.75; 0.96; 1.50; 3.84; 6.00; 15.36; 24.00 and 30.72 µM) and PNT2 cells (1.0, 2.5, 5.0, 10, 20, 30, 40, 50 and 60 µM), such as established by our previous work (Nunes et al. 2020). DU145 and PNT2 cells (1.0 × 104/well) were seeded in a 96-well plate (Greiner Bio-One - Monroe, NC, USA), incubated for 24 h for stabilization and treated with SV and BrA for 1 to 72 h depending on the assay. As the cell viability from the SV group showed no statistical differences compared to NC group, all experiments were compared against to the SV group. All experiments were conducted between the 3rd–8th cell passage, and three independent cultures (n = 3) were performed.

Cell viability

Resazurin (AlamarBlue®) reduction assay

The resazurin (alamarBlue®; Sigma-Aldrich, St. Louis, MO, USA) reduction assay was performed according to Riss et al. (2004). After treatment, 40 µL of 0.5 mM resazurin solution (diluted in PBS) was added to each well of the plate (20% of final volume). After 6 h of incubation with resazurin solution (Eilenberger et al. 2018), the plates were analyzed in a microplate spectrophotometer (Biotek Eon, Winooski, VT, USA) at λ = 570 nm (resorufin) and λ = 600 nm (resazurin). The absorbances obtained were multiplied by the oxidation factor at each wavelength. Cell viability (%) was calculated from the difference between the resulting values of 570 nm and 600 nm, and data normalization was performed with SV as 100% cell viability.

Neutral red (NR) assay

The NR (CAS: 553-24-2, Sigma-Aldrich, St. Louis, MO, USA) assay was conducted following Repetto et al. (2008). After the treatments, the cells were washed with PBS and incubated in NR solution (40 µg/mL) for 3 h at 37 °C. Next, the NR solution was removed, cells were washed again in PBS and the destaining solution (0.1 mL, 50% ethanol, 49% deionized water, and 1% glacial acetic acid; v/v/v/; Sigma-Aldrich, St. Louis, MO, USA) was added to each well. Absorbance was read using a spectrophotometer (Biotek Eon, Winooski, VT, USA) at λ = 540 nm. Data normalization was performed with SV as 100% cell viability.

MTT assay

MTT assay was performed according to the protocol reported by Mosmann (1983). After treatments, the MTT working solution (0.50 mg/mL in PBS; m/v) was added to each well, and the plate was incubated for 4 h at 37 °C. After the incubation, the purple formazan crystals were dissolved in DMSO (Sigma-Aldrich) for 5 min. The absorbance was evaluated at λ = 570 nm using an automated microplate spectrophotometer (Biotek Eon, Winooski, VT, USA) using SV as 100% of cell viability.

LDH assay

After treatments, samples were analyzed using a commercial LDH kit following the manufacturer’s recommendations and methods previously standardized in our laboratory (Specian et al. 2016). The absorbance was measured at λ = 490 and 680 nm, using a microplate spectrophotometer (Biotek Eon, Winooski, VT, USA). For each sample, LDH activity was calculated from the difference between the resulting values of 680 nm and 490 nm, and data normalization was performed with Positive control (PC) as 100% LDH release.

Selective-index

To determine the cytotoxic selectivity of the BrA flavonoid, the selectivity index (SI) was calculated based on the resazurin assay results using following equation:

SI=IC50non-tumorcellPNT2IC50tumorcellDU145

where SI ≥ 10 was considered as higher selective compound, while 2–10 are potentially selective to tumor cells.

Redox imbalance

The intracellular ROS levels were measured using the fluorescent probe CM-H2DCFDA (5-[and-6]-chloromethyl-2,7-dichlorodihydrofluorescein diacetate, acetyl ester) (Molecular Probes; Invitrogen, Carlsbad, CA, USA). DU145 cells (1 × 104/well) were seeded in 96-well culture black bottom plates (Uniscience do Brasil, Osasco, SP, Brazil) for 24 h and treated with SV (DMSO 0.25%), PC (1 mM H2O2) and BrA (0.24; 0.75; 0.96; 1.50; 3.84; 6.00; 15.36; 24.00 and 30.72 µM) for 1, 3, 6, 12, and 24 h. After the treatments, cells were washed with PBS buffer and incubated with CM-H2DCFDA working solution (5 µM) for 30 min, at 37 °C. The probe solution was removed by aspiration, the plates were washed with PBS, and fluorescence intensity (FI) was recorded using a VictorTMX3 Multilabel Plate Reader (PerkinElmer, São Paulo, SP, Brazil) at excitation λ = 485 nm and emission λ = 528 nm. The results were expressed in arbitrary units (A.U.).

Cell death (triple staining apoptosis/necrosis assay)

For cell death analysis, the triple staining apoptosis/necrosis assay was performed using the Propidium Iodide (IP; Sigma-Aldrich), Hoescht 33,342 (Ho; Sigma-Aldrich), and Fluorescein Diacetate (DAF; Sigma-Aldrich) dyes. PNT2 and DU145 cells (2.5 × 104/well) were seeded in 24-well plates (Greiner Bio-One - Monroe, NC, USA), stabilized for 24 h and treated with SV (0.5% DMSO), PC (0.345 µM DXR) or BrA for 24 h before labelling with fluorochromes (final culture concentration = 4.0 µg/mL Ho, 7.5 µg/mL DAF, and 1.0 µg/mL PI). In total, 600 cells per treatment were analyzed using the Olympus BX 43 (Olympus Microscopy, Europa) at ×400 magnification, considering viable, apoptotic, or necrotic cells in percentage (%) (Proietti De Santis et al. 2003).

Cell proliferation/survival

Clonogenic assay

The clonogenic assay was carried out according to Franken et al. (2006). Briefly, DU145 and PNT2 cells (250 cells) were seeded in a 6-well plate (Greiner Bio-One). After 24 h, cells were treated with SV (DMSO 0.25%) and BrA (0.24; 1.50; 6.00 e 15.36 µM), and the plates were kept immobile in an incubator for 7 days. Subsequently, the plates were washed with PBS, and a fixative solution (20 mL of ethanol 99% and 40 mL of acetone; v/v; Sigma-Aldrich, St. Louis, MO, USA) was added for 10 min, followed by staining with Giemsa 5% (Sigma-Aldrich, St. Louis, MO, USA) for 5 min. Next, the plates were washed with distilled water by submersion and left at room temperature (20 °C) for drying and colony counting. The colonies were analyzed and counted in binocular stereomicroscope and the results normalized using the SV group as 100% of surviving fraction.

Cell proliferation curves

Cell growth curves based on cell counting and total protein content were prepared according to the method described by Costa Lopes et al. (2000) and modified by our research team (Serpeloni et al. 2015, 2020). Briefly, DU145 cells (2.5 × 104/well) were seeded in 24-well plates (Greiner Bio-One), stabilized for 24 h, and treated with SV (DMSO 0.25%), DXR (0.345 µM) and BrA (0.24; 1.50; 6.00 e 15.36 µM) for 24 to 96 h. The cells were harvested after 24, 48, 72 and 96 h of treatment and 20 µL of the cell suspension was used for analysis on Countess™ II Automated Cell Counter (Thermo Fisher Scientific Inc., Waltham, MA, USA). After 2 h, culture media of the same plate was removed, and the proteins precipitated for 15 min in 5% trichloroacetic acid (TCA; Sigma-Aldrich, St. Louis, MO, USA). After washing, 1% bromophenol blue (Sigma-Aldrich, St. Louis, MO, USA) dissolved in 1% acetic acid (Sigma-Aldrich) was added to the wells. After 30 min incubation, the plates were washed with distilled water, and 400 µL of Tris-Base (Sigma-Aldrich, St. Louis, MO, USA) were added to the wells for color extraction. The absorbance of extracted bromophenol blue was recorded at λ = 550 nm in a spectrophotometer (Biotek Eon, Winooski, VT, USA) and used to calculate the total protein content.

Cell migration

Horizontal migration (wound-healing) assay

The wound-healing assay was performed according to the protocol of Martinotti and Ranzato (2020). In summary, DU145 and PNT2 cells (6.0 × 105/well) were seeded in 12-well plates (Greiner Bio-One) and stabilized for 24 h. Next, the culture medium was removed, and we created a gap by scratching a confluent monolayer using a 200 µL tip. Then, the plate was gently washed with PBS to remove the dettached cells, before treatment with SV (DMSO 0.25%) and BrA (1.5; 3.84 and 6.0 µM for DU145 cells and 1.0; 2.5, and 5.0 µM for PNT2 cells). The photomicrographs were obtained at 0, 24, and 48 h after the treatments, and the images were analyzed using TScratch software version 1.0 (The Mathworks, Natick, MA, USA, 2010). The results were presented as wound size (%), considering the SV group at 0 h as 100%.

Vertical cell migration/invasion assay (transwell)

DU145 cells (5.0 × 104/well) were seeded on the insert membrane with incomplete RPMI medium containing the treatments with SV group (0.25% DMSO) and BrA (3.84 and 6.0 µM), and the underside of the plate was filled only with a complete RPMI medium. Next, the cells were incubated for 24 h, after which the cells located at the membrane top were removed with a cotton swab and washed with PBS. The cells that migrate through the membrane were fixed with methanol, stained with 0.1% violet crystal (Merck; Darmstadt, Germany), and analyzed using an inverted Olympus CKX41 microscope (Tokyo, Japan) at ×400 magnification. The colour of  migrated cells in the lower surfaces of the filters were extracted, and absorbance at λ = 590 nm was measured. The results were presented considering the SV control as 100% migration.

The cell invasion assay was performed using the Corning® BioCoat™ insert Matrigel Invasion kit (Nova York, NY, USA) in 24-well plates where DU145 cells were seeded and processed as previously described by the migration assay, and the results were presented considering the SV control as 100% invasion.

Statistical analysis

All data are expressed as mean ± standard deviation and were analyzed by One-Way Analysis of Variance (ANOVA) followed by the Tukey’s test (p ≤ 0.05), using GraphPad Prism® 7.00 software (La Jolla, CA, USA). Sample homogeneity was tested using Bartlett’s test.

Results

Cell viability

In the resazurin assay, results showed a dose-dependent inhibition on cell viability after 24 h treatment with BrA in PNT2 (Fig. 2a), and DU145 (Fig. 2b) cells with the dosage ranged from 1.0 to 60 µM (PNT2) and 0.24 to 30.62 µM (DU145). The viability of PNT2 cells significantly decreased after 24 h of treatment with 40 µM BrA. In tumor DU145 cells, a cytotoxic effect was observed at lower concentrations (6.0 µM) than in the non-tumor cell line. The IC50 for prostate tumor DU145 cells was 10.7 µM and, for non-tumor PNT2 cells, a 50% viability reduction was not achieved, with an estimated IC50 value of 77.7 µM. The Selectivity Index (SI) for BrA was estimated in 7.3 against tumor DU145 cells, which is considered a good candidate as antitumor drug for PCa. To further investigate BrA effects in metastatic prostate DU145 cells, NR (Fig. 2c), MTT (Fig. 2d), and LDH release (Fig. 2e) assays were performed to evaluate cell viability. BrA decreased the percentage of viable cells in the 15.36 µM concentration in NR (IC50 9.89 µM), 6.00 µM MTT assay (IC50 6.08) and 15.36 µM in the LDH test (IC50 12.78 µM).

Fig. 2.

Fig. 2

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The resazurin assay has assessed cell viability (%) in a PNT2 and b DU145 cells treated with different concentrations of brachydin A (BrA) for 24 h. Additional cell viability assays in DU145 cells treated with BrA for 24 h were assessed by c NR, d MTT, and e LDH release. The results are expressed as mean ± standard deviation of three independent experiments (n = 3). *p ≤ 0.05 vs. SV group (DMSO 0.25%) after ANOVA followed by Tukey’s test

Cell death

Treatments with BrA (1–60 µM) did not induce cell death either by apoptosis or necrosis in non-tumor prostate PNT2 cells after 24 h of treatment. For the metastatic prostate DU145 cells, it was observed increased cell death by necrosis after BrA (1.5, 6.00, and 24.00 µM) treatments. Additionally, the percentage of apoptotic cells increased after BrA 24.00 µM treatment (Table 1).

Table 1.

Percentage (%) of viable, apoptotic, and necrotic PNT2 and DU145 cells after 24 h treatment with different concentrations of brachydin A (BrA) and their respective controls

Treatments Cells (%)
Viable Necrosis Apoptosis
PNT2 cells
PC (DXR 0.345 µM) 90.83 ± 0.50a 6.67 ± 0.76a 2.50 ± 0.00a
SV (DMSO 0.25%) 94.66 ± 2.24 3.67 ± 1.89 1.67 ± 0.71
BrA (µM)
10 97.09 ± 0.24 1.83 ± 0.00 1.08 ± 0.35
20 95.00 ± 0.82 2.83 ± 0.24 2.17 ± 1.06
30 94.67 ± 1.53 3.00 ± 1.18 2.33 ± 1.18
40 94.42 ± 2.24 3.58 ± 2.00 2.00 ± 0.47
50 93.20 ± 1.77 4.83 ± 1.65 2.42 ± 0.12
60 92.17 ± 5.54 5.75 ± 2.71 2.08 ± 0.82
DU145 cells
PC (DXR 0.345 µM) 96.50 ± 1.00 2.33 ± 1.61 1.17 ± 0.76
SV (DMSO 0.25%) 94.67 ± 3.51 1.17 ± 1.04 4.16 ± 3.32
BrA (µM)
1.50 94.33 ± 2.57 4.17 ± 2.46a 1.50 ± 0.50
6.00 91.00 ± 0.50a 6.83 ± 1.04a 2.17 ± 1.44
24.00 71.00 ± 2.12a 23.75 ± 1.06a 5.25 ± 1.06a
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Data are presented as the percentage (%) of cell morphology type found with triple staining

SV Solvent Vehicle, PC positive control, DXR doxorubicin, BrA brachydin A

ap ≤ 0.05 vs. SV group after ANOVA followed by Tukey’s test

Redox imbalance

Reactive intracellular (RS) species were quantified in tumoral prostate DU145 cells to verify if pro-oxidant effects could be involved in BrA cytotoxicity. Results showed no redox imbalance comparing BrA and SV (0.25% DMSO) groups at any treatment time (1, 3, 6, 12, or 24 h). Only 1mM H2O2 (positive control) showed a significant increase in the RS levels for all treatment times (Fig. 3).

Fig. 3.

Fig. 3

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Intracellular reactive oxygen species (ROS) production in metastatic prostate DU145 cells treated with brachydin A (0.24, 1.50, 6.00, and 15.34 µM) for 1, 3, 6, 12, and 24 h. The results are expressed as arbitrary units (A.U.) of fluorescence intensity. H2O2: hydrogen peroxide 1 mM (positive control). SV solvent control (DMSO 0.25%). Data represent the mean ± standard deviation of three independent experiments (n = 3). *p ≤ 0.05 vs. SV group (DMSO 0.25%) after ANOVA followed by Tukey’s test

Cell proliferation/survival

The antiproliferative activity of BrA was initially evaluated using the clonogenic assay in non-tumor PNT2 (Fig. 4a) and tumor prostate DU145 cells (Fig. 4b). Results showed decreasing in the colony formation at 15.36 µM of BrA only in DU145. Next, to evaluate the BrA potential to inhibit cell proliferation in tumor prostate cells, we performed cell growth curves by cell counting (Fig. 4c) and total protein content quantification (Fig. 4d) in DU145 cells. In this context, BrA revealed no antiproliferative effects after a 96-h treatment.

Fig. 4.

Fig. 4

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Colony formation in PNT2 and DU145 prostate cells after brachydin A (BrA) treatments in a clonogenic assay. Surviving fraction (%) of a PNT2 cells and b DU145 cells after treatment with different concentrations of BrA (0.24, 1.50, 6.00, and 15.34 µM) and SV group. Additional cell proliferation assays in DU145 cells treated with different concentrations of BrA for 96 h as assessed by d the number of cells and e quantification of total proteins. SV: solvent control (0.25% DMSO). Data represent the mean ± standard deviation obtained in three independent experiments (n = 3). *p ≤ 0.05 vs. SV group (DMSO 0.5%) after ANOVA followed by Tukey’s test

Cell migration and invasion

The results in Fig. 5a, b showed that BrA impaired horizontal migration of PNT2 but not DU145 cells, in the wound healing assay after 48 h of treatment compared with SV (0.25% DMSO) group. After 48 h, the % of wound closure was 67% for BrA 6.00 µM in PNT2 cells, whereas it was 87% and 93% in DU145 cells treated with BrA 3.84 and 6.00 µM respectively. Although the wound was not completely closed in the DU145 cells, there was no statistical difference in relation to the SV. The representative images demonstrated that the number of tumor prostate DU145 cells that migrated to fill the scratch is lower than in BrA treatments (1.50, 3.84, and 6.00 µM) compared to SV group. Representative images of the wound closure after migration in PNT2 and DU145 cells are depicted in Fig. 5c and d, respectively.

Fig. 5.

Fig. 5

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Brachydin A (BrA) impaired the migration of PNT2 and DU145 prostate cancer cells cultured as 2D monolayer. Percentage of wound area after 0, 24, and 48 h of a PNT2 and b DU145 cells treated with BrA. Representative figures of c PNT2 and d DU145 cells migration in ×4 objective. Bar scale = 200 μm

Additionally, BrA (6.00 µM) decreased vertical migration (Fig. 6a) but not invasion (Fig. 6b) of tumor prostate DU145 cells by the transwell system, showing that the presence of the extracellular matrix influences the vertical movement of metastatic cells. Figure 6c and d showed representative images of cell migration/invasion assays, respectively.

Fig. 6.

Fig. 6

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Cell migration and invasion assays in tumor prostate DU145 cells. Percentage (%) of migrated (a) and invaded (b) DU145 cells in transwell chambers after 24 h treatment with BrA (3.84 and 6.00 µM) and SV (0.25% DMSO) group. The bars represent the mean ± standard deviation (absorbance of extracted coloration) from measurements were carried out in triplicate (n = 3). *Significantly different from SV control at the respective treatment time (*p < 0.05; ANOVA followed by Tukey’s post-test). Representative images of transwell migration (c) and invasion (d) of DU145 cells after treatments with BrA

Discussion

Preclinical screening models can result in potential compounds for anticancer drug development, which help to decide whether a molecule like phytochemicals should be taken further in clinical trials and be used in cancer therapy (Choudhari et al. 2020). Around 5000 flavonoids have been identified distributed in a wide range of plants commonly present in the human diet (Imran et al. 2019), and assessed for their antitumoral properties (Kopustinskiene et al. 2020). Flavonoids have been shown to decrease the cell viability of cancer cells through induction of cell cycle arrest and activation of apoptosis (Hazafa et al. 2020; Izzo et al. 2020). For example, polyphenolic compounds from Lespedeza Bicolor inhibit progression of PC3 and 22Rv1 prostate tumor cells via induction of apoptosis and cell cycle arrest (Dyshlovoy et al. 2020).

In the present study we evaluated the antitumoral properties of the flavonoid BrA using a metastatic tumoral prostate (DU145) cell and evaluated its potential selectivity for non-tumor prostate cells using PNT2 as an experimental model. The metastatic prostate DU145 cell line show low prostatic acid phosphatase (PAP) expression and do not show androgen receptor (AR) or prostate-specific antigen (PSA) mRNA and protein expression (Namekawa et al. 2019). Androgen-independent cells (i.e., PC3 and DU145 cells) are less sensitive to the chemotherapeutic drug docetaxel in comparison with AR-dependent prostate cancer cells (Yang et al. 2019), which makes the search for compounds capable of inducing cytotoxicity in these cells a trending topic. On the other hand, the PNT2 cell line represents a model of non-tumor prostate epithelial cells (Johnson et al. 2014).

Most antitumor drugs initially go through cell viability/proliferation assays, and the decision to pursue further research with these agents is often based on these initial results (Shenoy et al. 2017). The resazurin reduction assay is based on a cell-permeable dye converted by active cellular metabolism in resofurin, which is an inexpensive test with higher sensitivity than tetrazolium assays (Riss et al. 2004). In this assay, BrA showed cytotoxic effects that are cell-type context-dependent and more selective for DU145 cells than for PNT2 non-tumor cells, with IC50 values of 11.5 µM and 70.63 µM, for tumoral and non-tumor cells, respectively. The SI value 7.26 showed BrA flavonoid as a promising molecule for chemotherapy, especially for metastatic PCa, once it showed seven-time more potency to tumoral cells compared to non-tumor prostate cells. Therefore, as the use of assays that rely on reducing viable cells to test natural compounds has been questioned (Shenoy et al. 2017), three other assays were performed to investigate the effects of BrA on DU145 cells viability (NR, MTT, and LDH release).

The flavonoid BrA showed the IC50 value of 9.9 µM (NR), 6.1 µM (MTT assay), and 12.8 µM (LDH release). Comparing these values using MTT assay in our previous research, BrA demonstrated IC50 values equal to 23.41 µM in tumor prostate PC3 cells in vitro (Nunes et al. 2020). The values obtained with DU145 are lower than with PC3 cell line and may indicate possible selectivity of BrA for the metastatic tumor type. BrA IC50 was greater than 20 µM against intracellular amastigotes of L. amazonensis, without displaying host cell toxicity, a similar concentration observed in our results for tumor prostate DU145 cells but not in normal prostate PNT2 cell. The evaluation of aspects of the general metabolism of viable cells, such as lysosomal integrity (NR), membrane integrity (LDH), and metabolic capacity (resazurin), may explain the differences observed. The results obtained suggest that BrA, in general, may have a more relevant effect on the cell reducing capacity (mitochondrial and subcellular reductases), since the effect on viability was detected at lower concentrations in resazurin and MTT assays (Rampersad 2012; Van Tonder et al. 2015; Präbst et al. 2017).

Fluorescent staining assay is normally used to determine cell viability and distinguish cell death mechanisms, apoptosis, or necrosis, through differences in plasma membrane integrity and permeability. The results obtained in the cell death assay using triple staining demonstrated increasing in the % of apoptotic and necrotic cells after treatments with BrA at 6 and 24 µM. The most exciting thing is that at the same BrA concentrations, no cell death induction was observed in non-tumor prostate PNT2 cells. These results confirm those obtained in the cell viability tests, where the cytotoxic effect from BrA after 24 h treatment was observed only in tumor prostate DU145 cells, but not in the PNT2 cells.

Cellular proliferation represents the ability of healthy cells to divide, and different assays are used to quantify the rate of growth of a population of cells (Präbst et al. 2017). BrA effects on proliferation of non-tumor and tumor prostate cells were tested by the colony formation assay, considered as a gold standard test for assessment of cellular radiosensitivity and chemosensitivity (Mirzayans et al. 2018). Our results showed that BrA flavonoid (15.36 µM) strongly decreased the proliferation of tumor DU145 cells while no differences observed in non-tumor prostate PNT2 cells. This difference in sensitivity to chemical compounds among tumors and normal cells is essential for chemotherapy candidates once that the capacity for continued proliferation is a prerequisite for the function of normal tissues. In contrast, for tumor cells, the capacity for unlimited proliferation is required to prevent recurrences (Franken et al. 2006).

Additionally, the growth curves based on counting proliferating cells is one of the most classical approach to evaluate the influence of a compound at different times of treatments, and automated cell counters were developed to facilitate and accelerate the process (Pereira et al. 2020). Besides this method, we also evaluated cell growth by quantifying total protein content, with the goal to further evaluate the antiproliferative effects of BrA in DU145 cells. However, our results suggest that the flavonoid did not interfere in cell growth compared to SV control and we believe that the longer treatment time employed in the clonogenic assay (7 days, versus 4 days in the proliferation curves) was more appropriate to observe the selective antiproliferative effect of BrA on tumor cells.

Distant metastasis is recurrent in prostate cancer patients; therefore, searches for compounds that reduce cell migration is a remarkable research field. Cell migration, especially, is a process of tumor progression in which tumor cells move from the primary site to colonize secondary sites, which may be via cell-cell adhesion, proteolytic degradation of the ECM, cytoskeleton remodeling, or growth factors (Vinci et al. 2013). Wound healing assays and transwell cell migration and invasion assays provides detailed information of migratory cell behaviors and thus may reveal molecular mechanisms of cell migration (Justus et al. 2014; Liang et al. 2007) reported that the average time to close a wound is usually between 8 and 18 h. Our results obtained in tumor DU145 cells showed that after 24 h the wound had already been closed in the SV group but remained with spaces in the BrA treatments. After 48 h, all wounds were closed, but the number of cells that migrated is noticeably smaller than in BrA treatments. This migration inhibition in tumor prostate DU145 can once again characterize the antitumoral effects of BrA.

The SNRNP200, SRRM1, and SRSF3 genes, whose proteins are components of the splicing machinery, are associated with relevant clinical parameters (e.g., Gleason score, T-stage, metastasis, and biochemical recurrence) and were overexpressed in prostate-derived cell lines including DU145 compared to the non-tumor prostate cell line PNT2 and, when silenced, these genes slow the migration of tumor cells (Jiménez-Vacas et al. 2020). Paired Box 3 (PAX3) protein is also overexpressed in tumors (DU145, LNCAP, and PC3 cells) but not in PNT2 cells, and the transwell tumor cells migration was reduced after PAX3 silencing (Zeng et al. 2020). Our results corroborate these important studies, showing that PNT2 non-tumor cells have less migration capacity than DU145 cells, which are further reduced in the presence of BrA. To further investigate this influence of BrA on cell migration, transwell migration and invasion assays were performed on DU145 cells, and the results demonstrated that, even when evaluated through different tests, BrA showed antimigratory activity, influenced by cell-cell anchoring (wound healing assay) or by chemotaxis (transwell).

Taken together, BrA showed cytotoxicity and significant antiproliferative effects in DU145 metastatic prostate cells, and the IC50 values reveal a much-reduced effect on non-tumor prostate PNT2 cells. This cytotoxic effect was seven-time more selective to tumor prostate cells and did not seem related to the ROS induction. Besides, the anticancer effects of BrA can also be related to an impaired cell vertical migration but not cell invasion. Thus, BrA may be considered as a novel chemotherapeutic agent for prostate cancer treatment and should also be evaluated in combination with other chemotherapeutic drugs to evaluate their effects on further sensitizing drug-resistant metastatic cancer cells.

Author contributions

Conceptualization and design: JMS; IMSC; LCBO; Provision of study materials and funding acquisition: JMS; CQR; DLR; Collection and assembly of data: LCBO; JRN. Data analysis and interpretation: LCBO; JMS; IMSC. Writing—original draft and editing: LCBŌ; DLR; IMSC; JMS. Supervision and project administration: DLR; IMSC; JMS.

Funding

This work was supported by Brazilian National Council for Scientific and Technological Development (CNPq: Grants 426246/2018-7 and 401516/2016-4) and Coordination for the Improvement of Higher Education Personnel - [Finnance Code 01].

Declarations

Conflict of interest

The authors have no conflict of interest to report.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Larissa Cristina Bastos de Oliveira, Email: larissa.oliveira@uel.br.

Higor Lopes Nunes, Email: higorlopesn@gmail.com.

Diego Luis Ribeiro, Email: diegoluisribeiro@usp.br.

Jessyane Rodrigues do Nascimento, Email: jessyane.nascimento@unesp.br.

Cláudia Quintino da Rocha, Email: rocha.claudia@ufma.br.

Ilce Mara de Syllos Cólus , Email: colus@uel.br.

Juliana Mara Serpeloni, Email: julianaserpeloni@uel.br.

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