Flavonoid brachydin B decreases viability, proliferation, and migration in human metastatic prostate (DU145) cells grown in 2D and 3D culture models.

Brachydin B (BrB) is a unique dimeric flavonoid extracted from Fridericia platyphylla (Cham.) LG Lohmann with different biological activities. However, the antitumoral potential of this flavonoid is unclear. In our study, we evaluated the effects of the BrB flavonoid on cell viability (MTT, resazurin, and lactate dehydrogenase assays), proliferation (protein dosage and clonogenic assay), and migration/invasion (3D ECM gel, wound-healing, and transwell assays) of metastatic prostate (DU145) cells cultured both as traditional 2D monolayers and 3D tumor spheroids in vitro. The results showed that the BrB flavonoid promotes cytotoxic effects from ≥1.50 µM after 24 h of treatment in DU145 cells in monolayers. In 3D prostate tumor spheroids, BrB also induced cytotoxic effects at higher concentrations after longer treatment (48, 72, and 168 h). Furthermore, BrB treatment is associated with reduced DU145 clonogenicity in 2D cultures, as well as decreased area/volume of 3D tumor spheroids. Finally, BrB (6 µM) reduced cell migration/invasion in 2D monolayers and promoted antimigratory effects in DU145 tumor spheroids (≥30 µM). In conclusion, the antitumoral and antimigratory effects observed in DU145 cells cultured in 2D and 3D models are promising results for future studies with BrB using in vivo models and confirm this molecule as a candidate for metastatic prostate cancer therapy.

Chemical characterization of Brazilian savannah Byrsonima species (muricis) and their impact on genomic instability and chemopreventive effects.

The identification of new drugs with few or no adverse effects is of great interest worldwide. In cancer therapy, natural products have been used as chemopreventive and chemotherapeutic agents. Plants from the Brazilian savannah belonging to the Byrsonima genus are popularly known as muricis and have attracted much attention due to their various pharmacological activities. However, there are currently no data on these plants concerning their use as chemopreventive or chemotherapeutic agents in human cell lines. The present study assessed the potential of B. correifolia, B. verbascifolia, B. crassifolia, and B. intermedia extracts as natural alternatives in the prevention and/or treatment of cancer. The chemical constituents present in each extract were analyzed by electrospray ionization-mass spectrometry (ESI-MSN). The mutagenic/antimutagenic (micronucleus assay), genotoxic/antigenotoxic (comet assay), apoptotic/necrotic (acridine orange/ethidium bromide uptake), and oxidative/antioxidative (CM-H2DCFDA) effects of the extracts and their influence on gene expression (RTqPCR) were investigated in nonmetabolizing gastric (MNP01) and metabolizing hepatocarcinoma (HepG2) epithelial cells to evaluate the effects of metabolism on the biological activities of the extracts. The genotoxicity, mutagenicity, and apoptotic effects observed in HepG2 cells with B. correifolia and B. verbascifolia extracts are probably associated with the presence of proanthocyanidins and amentoflavone. In MNP01 cells, none of the four extracts showed mutagenic effects. B. crassifolia and B. intermedia extracts exhibited strong antimutagenicity and enhanced detoxification in HepG2 cells and antioxidant capacities in both types of cells, possibly due to the presence of gallic and quinic acids, which possess chemopreventive properties. This study identifies for the first time B. correifolia and B. verbascifolia extracts as potential agents against hepatocarcinoma and B. crassifolia and B. intermedia extracts as putative chemopreventive agents.

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

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.

Characterization of the invitro cytotoxic effects of brachydins isolated from Fridericia platyphylla in a prostate cancer cell line.

Brachydins (Br) A, B, and C are flavonoids extracted from Fridericia platyphylla (Cham.) L.G. Lohmann roots (synonym Arrabidaea brachypoda), whose extract previously exhibited cytotoxic and antitumor activity. In vitro cell culture of human prostate tumor cell line (PC-3) was used to determine cell viability as evidenced by MTT, neutral red, and LDH release using nine concentrations (0.24 to 30.72 µM) of each brachydin. A triple-fluorescent staining assay assessed the mechanism resulting in cell death. Genomic instability and protein expression were evaluated using comet assay and western blot analysis, respectively. The pro-oxidant status was analyzed using the5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) probe. The IC50 values for brachydins BrA, BrB, and BrC were 23.41, 4.28, and 4.44 µM, respectively, and all compounds induced apoptosis and necrosis. BrB and BrC increased p21 levels indicating a possible G1 cell cycle arrest. BrA (6 µM) and BrB (3.84 µM) decreased phospho-AKT (AKT serine/threonine kinase) expression, which also influenced cell cycle and proliferation. BrA, BrB, and BrC elevated cleaved PARP (poly (ADP-ribose) polymerase), a protein related to DNA repair and induction of apoptotic processes. Therefore, this study determined the IC50 values of brachydins in the PC-3 cell line as well as the influence on cell proliferation and cell death processes, such as apoptosis and necrosis, indicating the proteins involved in these processes. ABBREVIATIONS: ANOVA: Analysis of Variance; BrA: Brachydin A; BrB: Brachydin B; BrC: Brachydin C; CGEN: Genetic Heritage Management Council; CID: Compound identification number; CM-H2DCFDA, 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester; CO2: Carbon dioxide; DMSO: Dimethyl sulfoxide; DNA: Deoxyribonucleic acid; DTT: Dithiothreitol; DXR: Doxorubicin; ECL: Chemiluminescence; EDTA: Ethylenediaminetetraacetic acid; FBS: Fetal bovine serum; H2O2: Hydrogen peroxide; HRMS: High-Resolution Mass Spectrometry; IC50: Half maximal inhibitory concentration; LDH: Lactate dehydrogenase; MTT, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; Na3VO4: Sodium Orthovanadate; NaOH: Sodium hydroxide; NCBI: National Center for Biotechnology Information; NMR: Nuclear Magnetic Resonance; PBS: Phosphate buffer saline; PCR: Polymerase chain reaction; PSMF: Phenylmethylsulfonyl fluoride; RPMI: Roswell Park Memorial Institute Medium; SDS-PAGE: Sodium Dodecyl Sulfate-Polyacrylamide gel electrophoresis; STR: Short tandem repeat; TBS-T: Tris-buffered saline and Polysorbate 20; UPHLC: Ultra-Performance Liquid Chromatography.

Pouteria ramiflora (Mart.) Radlk. extract: Flavonoids quantification and chemopreventive effect on HepG2 cells.

Pouteria ramiflora (Mart.) Radlk., popularly known as curriola, is commonly used in Brazil as medicinal plant to treat worm infections, dysentery, pain, inflammation, hyperlipidemia, and obesity. At present the safety of this extract when used therapeutically in human remains to be determined. Thus, the aim of this study was to examine cytotoxicity, antiproliferative, and antimutagenic actions of this extract. The hydroalcoholic extract from P. ramiflora leaves consisted of flavonoids identified and quantified as myricetin-3-O-ß-D-galactopyranoside (13.55 mg/g) and myricetin-3-O-α-L-rhamnopyranoside (9.61 mg/g). The extract exhibited cytotoxicity at concentrations higher than 1.5 µg/ml in human hepatocarcinoma (HepG2)and 2.5 µg/ml in non-tumoral primary gastric (GAS) cells using the MTT assay, and at concentrations higher than 3 µg/ml in HepG2 and 3.5 µg/ml in GAS cells by the neutral red assay. The extract did not show antiproliferative effect as evidenced by the nuclear division index (NDI). However, in the presence of benzo[a]pyrene (BaP) (positive control), an enhanced cytostatic effect in the NDI and flow cytometry was noted. It is of interest that when the extract was co-incubated with BaP a significant decrease in DNA damage was observed indicating an antimutagenic action. This protective effect might be attributed to myricetin and gallic acid found in P. ramiflora extract. The low cytotoxicity action and protective effect observed in the present study encourage further studies regarding other biological effects of P. ramiflora, as well as its potential use as a chemopreventive agent.