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

ABSTRACT

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.