Production and characterization of rhamnolipids by Pseudomonas aeruginosa isolated in the Amazon region, and potential antiviral, antitumor, and antimicrobial activity.

Biosurfactants encompass structurally and chemically diverse molecules with surface active properties, and a broad industrial deployment, including pharmaceuticals. The interest is growing mainly for the low toxicity, biodegradability, and production from renewable sources. In this work, the optimized biosurfactant production by Pseudomonas aeruginosa BM02, isolated from the soil of a mining area in the Brazilian Amazon region was assessed, in addition to its antiviral, antitumor, and antimicrobial activities. The optimal conditions for biosurfactant production were determined using a factorial design, which showed the best yield (2.28 mg/mL) at 25 °C, pH 5, and 1% glycerol. The biosurfactant obtained was characterized as a mixture of rhamnolipids with virucidal properties against Herpes Simplex Virus, Coronavirus, and Respiratory Syncytial Virus, in addition to antimicrobial properties against Gram-positive bacteria (Staphylococcus aureus and Enterococcus faecium), at 50 µg/mL. The antitumor activity of BS (12.5 µg/mL) was also demonstrated, with potential selectivity in reducing the proliferation of breast tumor cells, after 1 min of exposure. These results demonstrate the importance of studying the interconnection between cultivation conditions and properties of industrially important compounds, such as rhamnolipid-type biosurfactant from P. aeruginosa BM02, a promising and sustainable alternative in the development of new antiviral, antitumor, and antimicrobial prototypes.

Polymorphisms in drug-metabolizing genes and urinary bladder cancer susceptibility and prognosis: Possible impacts and future management.

Epidemiological studies have shown the association of genetic variants with risks of occupational and environmentally induced cancers, including bladder (BC). The current review summarizes the effects of variants in genes encoding phase I and II enzymes in well-designed studies to highlight their contribution to BC susceptibility and prognosis. Polymorphisms in genes codifying drug-metabolizing proteins are of particular interest because of their involvement in the metabolism of exogenous genotoxic compounds, such as tobacco and agrochemicals. The prognosis between muscle-invasive and non-muscle-invasive diseases is very different, and it is difficult to predict which will progress worse. Web of Science, PubMed, and Medline were searched to identify studies published between January 1, 2010, and February 2023. We included 73 eligible studies, more than 300 polymorphisms, and 46 genes/loci. The most studied candidate genes/loci of phase I metabolism were CYP1B1, CYP1A1, CYP1A2, CYP3A4, CYP2D6, CYP2A6, CYP3E1, and ALDH2, and those in phase II were GSTM1, GSTT1, NAT2, GSTP1, GSTA1, GSTO1, and UGT1A1. We used the 46 genes to construct a network of proteins and to evaluate their biological functions based on the Reactome and KEGG databases. Lastly, we assessed their expression in different tissues, including normal bladder and BC samples. The drug-metabolizing pathway plays a relevant role in BC, and our review discusses a list of genes that could provide clues for further exploration of susceptibility and prognostic biomarkers.

Effects of docetaxel on metastatic prostate (DU-145) carcinoma cells cultured as 2D monolayers and 3D multicellular tumor spheroids.

Docetaxel (DTX) is one of the chemotherapeutic drugs indicated as a first-line treatment against metastatic prostate cancer (mPCa). This study aimed to compare the impact of DTX on mPCa (DU-145) tumor cells cultured as 2D monolayers and 3D multicellular tumor spheroids (MCTS) in vitro. The cells were treated with DTX (1-96 µM) at 24, 48, or 72 hr in cell viability assays (resazurin, phosphatase acid, and lactate dehydrogenase). Cell death was assessed with fluorescent markers and proliferation by clonogenic assay (2D) and morphology, volume, and integrity assay (3D). The cell invasion was determined using transwell (2D) and extracellular matrix (ECM) (3D). Results showed that DTX decreased cell viability in both culture models. In 2D, the IC50 (72 hr) values were 11.06 µM and 14.23 µM for resazurin and phosphatase assays, respectively. In MCTS, the IC50 values for the same assays were 114.9 µM and 163.7 µM, approximately 10-fold higher than in the 2D model. The % of viable cells decreased, while the apoptotic cell number was elevated compared to the control in 2D. In 3D spheroids, only DTX 24 µM induced apoptosis. DTX (≥24 µM at 216 hr) lowered the volume, and DTX 96 µM completely disintegrated the MCTS. DTX reduced the invasion of mPCa cells to matrigel (2D) and migration from MCTS to the ECM. Data demonstrated significant differences in drug response between 2D and 3D cell culture models using mPCa DU-145 tumor cells. MCTS resembles the early stages of solid tumors in vivo and needs to be considered in conjunction with 2D cultures when searching for new therapeutic targets.

Metalloproteinase 9 immunostaining profile is positively correlated with tumor grade, extraprostatic extension and biochemical recurrence in prostate cancer.

Metastasis is the main problem in the treatment of prostate cancer (PCa), and for it to occur, proteolytic enzymes must remodel the extracellular matrix (ECM) surrounding the tumor. The most important group of enzymes with this action include the matrix metalloproteinases (MMPs), which act on various substrates cleaving ECM components. The present study aimed to evaluate the protein immunostaining profiles of matrix metalloproteinase 2 (MMP-2) and 9 (MMP-9) in PCa Brazilian patients using the indirect immunohistochemical methodology. The tissue samples (n = 178), 60 from malignant tumor, 58 from adjacent non-tumor, and 60 from ECM, were evaluated according to the immunostaining intensity. The malignant tumor cytoplasmic MMP-2 immunostaining was more intense than in ECM (p = 0.001), but it did not correlate with any clinical-pathological parameter. The MMP-9 staining was similar in tumor cytoplasm, adjacent non-tumor cytoplasm and ECM, but showed significant positive correlations with ISUP grade (p = 0.044; Tau=0.249), extraprostatic extension (p = 0.025; Tau=0.309), and biochemical recurrence (p = 0.048; Tau=0.306). A significant positive correlation was also observed between MMP-2 and MMP-9 in all cell compartments analyzed. Although further research is warranted to elucidate the precise mechanisms underlying these observations, our findings suggest MMP-9 as a promising candidate marker for tissue invasion that could be used in predicting the progression and prognosis of PCa.

Allelic variants and immunostaining profile in CXCL12/CXCR4 axis: An investigation of association with prognosis in prostate cancer.

Prostate cancer (PCa) is the malignant neoplasm that most commonly affects men and is an important cause of death. It can be detected by changes in serum levels of Prostate Specific Antigen (PSA) and digital rectal examination, but often symptoms do not appear until advanced stages and metastases. The C-X-C Motif Chemokine Ligand 12/C-X-C Motif Chemokine Receptor 4 (CXCL12/CXCR4) axis acts in cell migration and may be involved in the metastatic process. In this context, the aim of this study was to evaluate the allelic variants rs1801157 (CXCL12) and rs2228014 (CXCR4) and the immunostaining of CXCR4 protein as candidates for prognostic markers in PCa. Samples (n = 60) were divided according to prognostic parameters (with and without metastasis at diagnosis) in tree groups: better prognosis, worse prognosis with metastasis at diagnosis and worse prognosis without metastasis at diagnosis, and immunostaining was evaluated by indirect immunohistochemistry, considering tumoral and adjacent tissues from the same patient (n = 120). A significant association was found between the C allele of rs2228014 (CXCR4) and the extraprostatic extension. For CXCR4 immunostaining a weak labeling and a cytoplasmic localization predominated, as well as a significant difference between malignant versus adjacent tissue, with higher protein expression in the malignant tissue. A significant association was found between CXCR4 tumor immunostaining with TNM staging (T2b-T2c) and PSA level (> 20 ng/mL). None of the allelic variants affected CXCR4 immunostaining. Prognostic groups did not differ in allelic variant frequency or immunostaining profile. Findings suggest that CXCR4 receptor may be one of the ways to worsen the prognosis of prostatic cancer.

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.

Association between Polymorphisms of Hemochromatosis (HFE), Blood Lead (Pb) Levels, and DNA Oxidative Damage in Battery Workers.

Occupational exposure to lead (Pb) continues to be a serious public health concern and may pose an elevated risk of genetic oxidative damage. In Brazil, car battery manufacturing and recycling factories represent a great source of Pb contamination, and there are no guidelines on how to properly protect workers from exposure or to dispose the process wastes. Previous studies have shown that Pb body burden is associated with genetic polymorphisms, which consequently may influence the toxicity of the metal. The aim of this study was to assess the impact of Pb exposure on DNA oxidative damage, as well as the modulation of hemochromatosis (HFE) polymorphisms on Pb body burden, and the toxicity of Pb, through the analysis of 8-hydroxy-2'-deoxyguanosine (8-OHdG), in subjects occupationally exposed to the metal. Male Pb-exposed workers (n = 236) from car battery manufacturing and recycling factories in Brazil participated in the study. Blood and plasma lead levels (BLL and PLL, respectively) were determined by ICP-MS and urinary 8-OHdG levels were measured by LC-MS/MS, and genotyping of HFE SNPs (rs1799945, C → G; and 1800562, G → A) was performed by TaqMan assays. Our data showed that carriers of at least one variant allele for HFE rs1799945 (CG + GG) tended to have higher PLL than those with the non-variant genotype (ß = 0.34; p = 0.043); further, PLL was significantly correlated with the levels of urinary 8-OHdG (ß = 0.19; p = 0.0060), while workers that carry the variant genotype for HFE rs1800562 (A-allele) showed a prominent increase in 8-OHdG, as a function of PLL (ß = 0.78; p = 0.046). Taken together, our data suggest that HFE polymorphisms may modulate the Pb body burden and, consequently, the oxidative DNA damage induced by the metal.

Three-dimensional cell cultures as preclinical models to assess the biological activity of phytochemicals in breast cancer.

The demand for the development of three-dimensional (3D) cell culture models in both/either drug screening and/or toxicology is gradually magnified. Natural Products derived from plants are known as phytochemicals and serve as resources for novel drugs and cancer therapy. Typical examples include taxol analogs (i.e., paclitaxel and docetaxel), vinca alkaloids (i.e., vincristine, vinblastine), and camptothecin analogs (topotecan, irinotecan). Breast cancer is the most frequent malignancy in women, with a 70% chance of patients being cured; however, metastatic disease is not considered curable using currently available chemotherapeutic options. In addition, phytochemicals present promising options for overcoming chemotherapy-related problems, such as drug resistance and toxic effects on non-target tissues. In the toxicological evaluation of these natural compounds, 3D cell culture models are a powerful tool for studying their effects on different tissues and organs in similar environments and behave as if they are in vivo conditions. Considering that 3D cell cultures represent a valuable platform for identifying the biological features of tumor cells as well as for screening natural products with antitumoral activity, the present review aims to summarize the most common 3D cell culture methods, focusing on multicellular tumor spheroids (MCTS) of breast cancer cell lines used in the discovery of phytochemicals with anticancer properties in the last ten years.

An Overview Regarding Pharmacogenomics and Biomarkers Discovery: Focus on Breast Cancer.

Breast cancer represents a health concern worldwide for being the leading cause of cancer- related women's death. The main challenge for breast cancer treatment involves its heterogeneous nature with distinct clinical outcomes. It is clinically categorized into five subtypes: luminal A; luminal B, HER2-positive, luminal-HER, and triple-negative. Despite the significant advances in the past decades, critical issues involving the development of efficient target-specific therapies and overcoming treatment resistance still need to be better addressed. OMICs-based strategies have marked a revolution in cancer biology comprehension in the past two decades. It is a consensus that Next-Generation Sequencing (NGS) is the primary source of this revolution and the development of relevant consortia translating pharmacogenomics into clinical practice. Still, new approaches, such as CRISPR editing and epigenomic sequencing are essential for target and biomarker discoveries. Here, we discuss genomics and epigenomics techniques, how they have been applied in clinical management and to improve therapeutic strategies in breast cancer, as well as the pharmacogenomics translation into the current and upcoming clinical routine.

Anticancer activities of Brachydin C in human prostate tumor cells (DU145) grown in 2D and 3D models: Stimulation of cell death and downregulation of metalloproteinases in spheroids.

Brachydin C (BrC) has demonstrated in vitro cytotoxic and antiproliferative effects in prostate cancer cells. In the present study, we compare the anticancer effects of BrC in DU145 cells grown in common bidimensional cultures (2D) and multicellular tumor spheroids (MCTS), often denominated 3D in vitro models, that can better mimic the microenvironment of tissues. BrC IC50 values obtained in the resazurin assay after 24 h of treatment were 47.31 µM (2D) and 229.8 µM (3D) and these cytotoxic effects were time-dependent only in 3D. BrC (5.0-60 µM) interfered with the growth of MCTS and reduced cell viability after 11 days of treatment, a result that is not attributable to oxidative stress evaluated using the CM-H2 DCFDA probe. BrC (6.0 µM) impaired horizontal (wound-healing) and vertical cell migration and invasion (transwell assay) in 2D and BrC (5.0-60 µM) in 3D (ECM Gel®). BrC modulated the expression of genes BIRC5, TNF-α, CASP3, NKX3.1, MMP9, MMP11, CDH1, and ITGAM and downregulated proteins CASP7, BAX, and TNF-α in Western blotting analysis. In conclusion, BrC stimulated cell death and decreased epithelial-mesenchymal transition. Furthermore, DU145 MCTS displayed higher resistance to BrC-induced cell death than 2D cultures, a difference that should be considered in future approaches in prostatic cancer studies.

Flavone cirsimarin impairs cell proliferation, migration, and invasion in MCF-7 cells grown in 2D and 3D models.

The present study investigates the mechanisms underlying the in vitro antitumoral activity of cirsimarin (CIR 10 to 320 µM), a flavone extracted from the aerial parts of Scoparia dulcis L., on MCF-7 cells cultured in 2D and multicellular tumor spheroids (3D). CIR (from 40 µM) decreased cell viability in the resazurin assay and colony formation in the 2D model. In the same way, in the 3D model, CIR (from 40 µM) induced cell death (triple staining assay) and decreased spheroid integrity after 16 days with no induction of intracellular reactive species (CM-H2DCFDA). In 2D, CIR decreased the invasion (transwell) and horizontal migration (wound healing), while in 3D, CIR diminished cell migration (ECM® gel) and induced DNA damage (comet assay) possibly related to cell death. CIR mediated antitumoral effects in 3D spheroids by negative modulation of genes associated with cell proliferation (CCND1, CCNA2, CDK2, CDK4, and TNF) and death (BCL-XL, BAX, CASP9, and BIRC5). BIRC5 and CDKs inhibitors have been proposed as versatile anticancer drugs, which makes our results quite interesting. TNF negative modulation may also be related to the downregulation of MMP9 and MMP11 and anti-migration/invasion of MCF-7 cells cultured in 2D and 3D models. These are relevant properties for long-term strategies to avoid metastasis and improve the prognosis of breast cancer.

The Antitumoral/Antimetastatic Action of the Flavonoid Brachydin A in Metastatic Prostate Tumor Spheroids In Vitro Is Mediated by (Parthanatos) PARP-Related Cell Death.

Metastatic prostate cancer (mPCa) is resistant to several chemotherapeutic agents. Brachydin A (BrA), a glycosylated flavonoid extracted from Fridericia platyphylla, displays a remarkable antitumoral effect against in vitro mPCa cells cultured as bidimensional (2D) monolayers. Considering that three-dimensional (3D) cell cultures provide a more accurate response to chemotherapeutic agents, this study investigated the antiproliferative/antimetastatic effects of BrA and the molecular mechanisms underlying its action in mPCa spheroids (DU145) in vitro. BrA at 60-100 µM was cytotoxic, altered spheroid morphology/volume, and suppressed cell migration and tumor invasiveness. High-content analysis revealed that BrA (60-100 µM) reduced mitochondrial membrane potential and increased apoptosis and necrosis markers, indicating that it triggered cell death mechanisms. Molecular analysis showed that (i) 24-h treatment with BrA (80-100 µM) increased the protein levels of DNA disruption markers (cleaved-PARP and p-γ-H2AX) as well as decreased the protein levels of anti/pro-apoptotic (BCL-2, BAD, and RIP3K) and cell survival markers (p-AKT1 and p-44/42 MAPK); (ii) 72-h treatment with BrA increased the protein levels of effector caspases (CASP3, CASP7, and CASP8) and inflammation markers (NF-kB and TNF-α). Altogether, our results suggest that PARP-mediated cell death (parthanatos) is a potential mechanism of action. In conclusion, BrA confirms its potential as a candidate drug for preclinical studies against mPCa.

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.

Selective anticancer effects of Serjania marginata Casar. extract in gastric cells are mediated by antioxidant response.

Gastric cancer is the fifth most common malignancy worldwide. Serjania marginata Casar. (SM) displays anti-inflammatory properties and has been used to treat gastrointestinal disorders. In the current study, we examined whether the hydroethanolic extract of SM leaves exerted cytotoxic, mutagenic, and protective effects in non-tumor gastric epithelium cells (MNP01) and gastric adenocarcinoma cells (ACP02) in vitro and analyzed whether its action was selective. Initially, cell viability (MTT assay), cell cycle kinetics (flow cytometry), and cell proliferation (total protein content) were analyzed. In addition, genomic instability (cytokinesis-block micronucleus cytome assay), anti/pro-oxidant status (CM-H2 DCFDA probe), and transcriptional expression (RT-qPCR) of genes related to cell cycle, cell death, and antioxidant defense were also evaluated. The SM extract was cytotoxic toward MNP01 and ACP02 cells at concentrations greater than 300 and 100 µg·ml-1 , respectively, and decreased protein content only toward ACP02 cells at 200 µg ml-1 . In ACP02 cells, the SM extract at 100 µg·ml-1 associated with doxorubicin (DXR; 0.2 µg ml-1 ) clearly promoted cell cycle arrest at the G2/M phase. The extract alone was not mutagenic to either cell type and reversed DXR-induced DNA damage and H2 O2 -induced oxidative stress in MNP01 cells. The gene expression experiments showed that SM hydroethanolic extract exerts an antioxidant response via NFE2L2 activation in non-tumor gastric cells, and cell cycle arrest (G2/M) in ACP02 gastric cancer cells via the TP53 pathway. The selective action of SM indicates that it is a promising therapeutic agent to treat gastric diseases and merits further studies.

COVID-19: The question of genetic diversity and therapeutic intervention approaches.

Coronavirus disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2), is the largest pandemic in modern history with very high infection rates and considerable mortality. The disease, which emerged in China's Wuhan province, had its first reported case on December 29, 2019, and spread rapidly worldwide. On March 11, 2020, the World Health Organization (WHO) declared the COVID-19 outbreak a pandemic and global health emergency. Since the outbreak, efforts to develop COVID-19 vaccines, engineer new drugs, and evaluate existing ones for drug repurposing have been intensively undertaken to find ways to control this pandemic. COVID-19 therapeutic strategies aim to impair molecular pathways involved in the virus entrance and replication or interfere in the patients' overreaction and immunopathology. Moreover, nanotechnology could be an approach to boost the activity of new drugs. Several COVID-19 vaccine candidates have received emergency-use or full authorization in one or more countries, and others are being developed and tested. This review assesses the different strategies currently proposed to control COVID-19 and the issues or limitations imposed on some approaches by the human and viral genetic variability.

Phytochemical Profile, and Antiproliferative and Proapoptotic Effects of Pouteria ramiflora (Mart.) Radlk. Leaf Extract, and Its Synergism with Cisplatin in HepG2 Cells.

Different species of the genus Pouteria have been used in folk medicine for the treatment of inflammation, fever, ulcers, diabetes, and diarrhea. We analyzed the phytochemical profile of the hydroethanolic extract from Pouteria ramiflora leaves by electrospray ionization ion trap tandem mass spectrometry and high-performance liquid chromatography-diode array detection, and examined whether it alone and in combination with cisplatin interfered with cell proliferation and death processes in HepG2 (human hepatocellular carcinoma) and FGH (human gingival fibroblasts) cells. Five compounds were identified in the extract: gallic acid, myricetin-3-O-α-l-arabinopyranoside, quercetin-3-O-ß-d-galactopyranoside, myricetin-3-O-α-l-rhamnopyranoside, and myricetin-3-O-ß-d-galactopyranoside. The extract was cytotoxic to both cell lines by inducing apoptotic cell death and acted in synergy with cisplatin; such effect was stronger in HepG2 cells than in FGH cells, demonstrating some selectivity to tumor cells. In HepG2 cells, the extract exerted antiproliferative effect mediated by induction of cell cycle arrest at the S and G2/M phases. Association of the extract with cisplatin enhanced the latter's antiproliferative effect, arrested the cell cycle at the S phase by CDK2 modulation, and reduced the number of anti-cyclin D1-stained HepG2 cells. Simultaneous treatment with the extract and cisplatin increased the latter's cytotoxicity, apoptotic cell death, and BAX expression in HepG2 cells. Altogether, the results reported herein indicate that P. ramiflora extract is a possible adjuvant to cancer therapy, which can circumvent the cisplatin-mediated resistance mechanisms in 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.

Association of polymorphisms of PTEN, AKT1, PI3K, AR, and AMACR genes in patients with prostate cancer.

Polymorphic variants in the PTEN (rs2735343), PI3K (rs2699887), AKT1 (rs2494750), AR (rs17302090), and AMACR (rs3195676) genes were evaluated as possible molecular markers of susceptibility, prognosis, and progression of prostate cancer (PCa), in a case-control study. Samples consisted of 277 patients with PCa and 277 controls from Londrina, PR, Brazil. SNPs were analyzed by real-time PCR. A family history of cancer, including PCa, as well as level of schooling were risk factors for PCa. The data were obtained via logistic regression, using odds ratios with a CI 95%. The genotypes of AKT1 and AKT1+AR demonstrated an association with protection for the disease. The combination of SNPs with the histopathological tumor data between allele variants of AMACR, AKT1+AR, and AKT1+AMACR indicated an association with protection against seminal vesicle invasion. The polymorphisms AKT1+AR and PI3K+AR were associated with protection against tumor bilaterality. The genotype combinations PTEN+AMACR and PTEN+AR were associated with the risk of extracapsular extension. Of the five genes studied, two were associated with protection for PCa, four were associated with protection for some prognostic variables, and only one was associated with risk. Thus, these SNPs are candidates for markers to discriminate men with better or worse prognosis for PCa.

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.

Phytochemical study and evaluation of cytotoxicity, mutagenicity, cell cycle kinetics and gene expression of Bauhinia holophylla (Bong.) Steud. in HepG2 cells in vitro.

Bauhinia holophylla (Bong.) Steud. (Fabaceae) is a plant used in Brazilian folk medicine to treat diabetes and inflammation. This study evaluated the phytochemical properties, cytotoxic, apoptotic, mutagenic/antimutagenic effects and alterations in gene expression (RNAm) in HepG2 cells treated with the B. holophylla extract. The phytochemical profile highlight the presence of flavonoids isorhamentin and quercetin derivates. The MTT assay was used to evaluate the cytotoxicity of different concentrations for different treatment times. Three concentrations (7.5, 15, 30 µg/mL) were chosen for assessment of apoptosis (AO/EB), mutagenicity (micronucleus), and cell cycle kinetics (flow cytometry). Thereafter, the concentration of 7.5 µg/mL was chosen to evaluate the protective effects against DNA damage induced by benzo[a]pyrene (B[a]P). At concentrations higher than 7.5 µg/mL (between 10 and 50 µg/mL), the extract was cytotoxic, induced apoptosis, and caused antiproliferative effects. However, it did not induce micronucleus and a reduction of apoptotic and micronucleated cells was observed in treatments that included the extract and B[a]P. The protective effect is attributable to the presence of flavonoids, described as antioxidants, inhibitors of DNA adduct and activators of detoxifying enzymes. The results of the present study such as absence of cytotoxic and mutagenic effects and protective effects against known carcinogens suggest that B. holophylla has potential for use soon as herbal medicine.