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The consumption of glucosinolate (GL)-rich foods, including Brassica vegetables, such as mustard, broccoli, and maca, is associated with decreased risk of developing cancer. The GL content in maca, which is recognized as a "superfood", is approximately 100-times higher than that in other brassicas. Although maca is a potential dietary source of GLs, limited studies have examined the bioactivity of maca GLs using the combination of chemical characterization and bioassays. In this study, the fractions (Lm-II and Lm-III) rich in intact GLs (glucotropaeolin and glucolimnanthin) were isolated and characterized from maca ethanolic extracts using chromatography and mass spectrometry. Additionally, the growth-inhibitory effects of Lm-II and Lm-II fractions against hepatocellular carcinoma (HepG2/C3A) and colon adenocarcinoma (HT29) cell lines were examined in the absence or presence of myrosinase (MYR). Fractions lacking low molecular weight sugars dose-dependently exerted cytotoxic effects in the presence of MYR. The half-maximal inhibitory concentration values of Lm-II and Lm-III against HepG2/C3A were 118.8 and 69.9 µg/mL, respectively, while those against HT29 were 102.6 and 71.5 µg/mL, respectively. These results suggest that the anticancer properties of maca can be attributed to GLs and corroborate the categorization of maca as a "superfood."Supplemental data for this article is available online at https://doi.org/10.1080/01635581.2021.1952444.

Anticarsia gemmatalis is one of the main defoliators of soybean in Brazil. Bacillus thuringiensis (Bt) transgenic crops are used for their management. In this paper we used RNA-seq to explore the response of A. gemmatalis to Bt HD73, as well as to detect transcriptional differences after Bt infection between resistant and susceptible strains. A total of 3853 and 6224 differentially expressed genes (DGEs) were identified in susceptible and resistant larvae after Bt exposure, respectively. We identified 2143 DEGs between susceptible and resistant larvae and 1991 between susceptible and resistant larvae Bt exposed. Immunity-related genes, Bt toxins receptors, proteases, genes involved in metabolic processes, transporters, cuticle proteins and mobile elements have been identified. qRT-PCR data demonstrated upregulation of five genes in susceptible strain after Bt exposure. These results provide insights to understand the molecular and cellular mechanisms of response to Bt that could be used in strategies to control agricultural pests.

Chlorophyllin (Chl), a semisynthetic compound derived from chlorophyll, has been a focus in cancer prevention because it exerts important biological activities, such as antigenotoxic, antioxidative and anticarcinogenic activities. Given that most sporadic cancers are related to environmental chemicals exposure and based on evidence that Chl has the ability to protect cells against carcinogenic effects of these compounds, the aim of this study was to evaluate the protective effect of Chl against benzo[a]pyrene toxicity in HepG2/C3A cells. To investigate the ability of Chl to mitigate the cytotoxic effects of B[a]P, the cells were co-treated and the cell viability, cell growth kinetics, cell cycle, and apoptosis induction were evaluated. Besides, the mRNA levels of cell cycle components (cyclins and cyclin-dependent kinases - CDKs) and apoptotic genes were analyzed. Our results showed that Chl was able to reduce the cytotoxic and antiproliferative effects of B[a]P in a multi-specific manner, restoring the normal distribution of the cell cycle and inhibiting the cell death induced by the xenobiotic. The RT-qPCR analysis showed that Chl caused a downregulation in cyclin CCNA2 and cyclin-dependent kinases CDK1 and CDK2 mRNA level. B[a]P decreased the mRNA levels of genes involved in cell cycle control (CCNA2, CCNB1, CCND1 and CCNE1 cyclins; and CDK1 and CDK2 cyclin-dependent kinases) and apoptosis (BAX, CASP7, and TP53). Cells co-treated with 200 µM Chl and B[a]P also showed a downregulation of mRNA levels of the genes. Chl maintained the TP53 gene expression in B[a]P-treated cells near control levels. Thus, Chl is a good candidate as a chemoprotective agent that mitigates the cytotoxic effects B[a]P and, thus, might be a promising tool to prevent liver cancer.

Fluopsin C is an antibiotic compound derived from secondary metabolism of different microorganisms, which possesses antitumor, antibacterial, and antifungal activity. Related to fluopsin C antiproliferative activity, the aim of this study was to examine the following parameters: cytotoxicity, genotoxicity, cell cycle arrest, cell death induction (apoptosis), mitochondrial membrane potential (MMP), colony formation, and mRNA expression of genes involved in adaptive stress responses and cellular death utilizing a monolayer. In addition, a three-dimensional cell culture was used to evaluate the effects on growth of tumor spheroids. Fluopsin C was cytotoxic (1) producing cell division arrest in the G1 phase, (2) elevating expression of mRNA of the CDKN1A gene and (3) decrease in expression of mRNA H2AFX gene. Further, fluopsin C enhanced DNA damage as evidenced by increased expression of mRNA of GADD45A and GPX1 genes, indicating that reactive oxygen species (ROS) may be involved in the observed genotoxic response. Reticulum stress was also detected as noted from activation of the ribonuclease inositol-requiring protein 1 (IRE1) pathway, since a rise in mRNA expression of the ERN1 and TRAF2 genes was observed. During the cell death process, an increase in mRNA expression of the BBC3 gene was noted, indicating participation of this antibiotic in oncotic (ischemic) cell death. Data thus demonstrated for the first time that fluopsin C interferes with the volume of tumor spheroids, in order to attenuate their growth. Our findings show that fluopsin C modulates essential molecular processes in response to stress and cell death.

1α, 25, dihydroxyvitamin D

Made available in DSpace on 2020-12-12T02:45:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-07-17 Rubus rosifolius: Sm. (Rosaceae) is a plant traditionally used in Brazil and some other countries to treat diarrhea, stomach diseases, and as an analgesic, antimicrobial, antihypertensive, and as well as other pharmacological properties. The aim of this study was to examine cytotoxic and genotoxic effects of R. rosifolius leaves extract on HepG2/C3A cells and correlate these findings with the expression of mRNA to underlying mechanisms of action. At concentrations between 0.01 and 100 µg/ml, cytotoxic effects were not detected by the MTT assay. This was confirmed by mRNA induction of the CYP3A4 gene (by RT-qPCR assay). However, genotoxic effects occurred at treatments from 1 µg/ml extract (comet and micronucleus test). An increase in the number of cells in S phase was observed at 100 µg/ml, and an elevation in apoptotic cell number was found for all tested concentrations (10, 20, or 100 µg/ml) (cell cycle and apoptosis analysis by flow cytometry). The genotoxicity induced by the extract was the main cause of the rise in the number of cells undergoing apoptosis, as indicated by rise in mRNA of CASP7 gene, and elevation of cells in the S phase of the cell cycle at the higher tested concentrations, as an attempt to repair genetic damage that occurred. These observations suggest that, despite its pharmacological potential, the use of R. rosifolius leaves extract may pose a risk to the integrity of the genetic material of human cells. Post-Graduate Program on General and Applied Biology São Paulo State University–UNESP–Biosciences Institute Departamento de Biomedicina Centro Universitário De Rio Preto–UNIRP–São José Do Rio Preto Marilia Medical School Speech and Hearing Therapy Department São Paulo State University–UNESP–Faculty of Philosophy and Sciences Department of Biological Sciences Vale Do Itajaí University (UNIVALI) Faculty of Pharmaceutical Sciences University of Campinas Department of General Biology Londrina State University CECAV and Department of Genetics and Biotechnology University of Trás-os-Montes and Alto Douro Post-Graduate Program on General and Applied Biology São Paulo State University–UNESP–Biosciences Institute Speech and Hearing Therapy Department São Paulo State University–UNESP–Faculty of Philosophy and Sciences

This study aimed to evaluate the modes of action of harringtonine (HT) and homoharringtonine (HHT) alkaloids in cell with wild (HepG2/C3A) and mutant p53 (HuH-7.5). We performed assays for cytotoxicity, genotoxicity, induction of apoptosis, cell cycle phase, and membrane integrity. Obtained data were compared with the relative expression of mRNA of genes related to proliferation, apoptosis, cell cycle control, metabolism of xenobiotics, and reticulum endoplasmic stress. The relative expression of the genes showed an increase in apoptosis-inducing mRNAs, such as TNF and BBC3, as well as a reduction in BCL2 and BAK. The mRNAs of CYP2E1 and CYP2C19 xenobiotic metabolism genes increased in both lineages, while CYP3A4 increased only in the HuH-7.5 lineage. The mRNA expression of endoplasmic reticulum (ER) stress genes ( ERN1 and EIF2AK3) was shown to increase in HHT and HT treatments. A similar increase was recorded in the mRNA expression of the TRAF2 gene. The changes observed in this study support the hypothesis that ER stress was more strongly associated with TNF induction, causing cell death by apoptosis in p53 mutant cells. This result with wild and mutant p53 cells may have clinical implications in the use of these compounds.

There is evidence that metabolic profile changes after Roux-Y gastric bypass (RYGB), especially due to modifications in the gastrointestinal tract. In addition, previous studies have suggested that probiotics can modify the microbiome and produce metabolites important for metabolic health maintenance. In this sense, the aim of this study was to verify the influence of probiotic supplementation on the plasma metabolite profile after RYGB.This was a randomized, double-blind, placebo-controlled clinical trial conducted with 31 patients subjected to RYGB surgery, randomized in probiotic group that was supplemented with a probiotic supplement (FloraVantageReductions in trimethylamine-N-oxide (TMAO) and alanine were observed in both groups, however this reduction was greater in the probiotic group (TMAO 13.82%, p = 0.01 and alanine 14.03%, p = 0.03) at T2. Additionally, β-hydroxybutyrate (BHB) levels increased 10.77% in the probiotic group (p = 0.03) compared to the placebo group at T2.Supplementation with Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07 was able to associate with significant differences in relevant plasma metabolites associated with improved metabolic health.

Salinomycin (SAL) is a monocarboxylic polyether ionophore antibiotic isolated from Streptomyces albus. It exhibits an effective antitumor potential against numerous human cancer cells. This study aimed to assess the antiproliferative effects of SAL in human hepatocellular carcinoma HepG2/C3a cell line. We investigated the effects of SAL on cell growth, DNA damage induction, cell cycle changes and apoptosis; and relative changes in expression of cell cycle-related, apoptosis-related, and CYP450 genes. SAL induced cell cycle arrest in G2/M phase, upregulation of CDKN1A and GADD45A and downregulation of cyclin genes including CCNB1 and CCNA2. SAL effectively suppressed mRNA levels of CTNNB1 gene, an important oncogene that promotes tumorigenesis. The decrease of HepG2/C3A cells survival can also be due to downregulation of antiapoptotic BCL-2 expression, thus promoting the induction of apoptosis by SAL. This study also demonstrated the ability of SAL in modulating hepatic cytochrome P450 (CYP) mRNA expression, such that SAL caused the upregulation of CYP1A members and CYP3A5; and downregulation of CYP3A4. Taken together, these data contribute to the understanding of the mechanism of action of SAL, highlighting that metabolizing enzymes modulated by SAL can interfere with chemotherapy treatment and it must be considered in associated treatments.

Made available in DSpace on 2022-04-29T08:32:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-09-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Background: Obesity is considered a chronic inflammatory disease and transforming growth factor beta 1 (TGFβ1) might exert important roles in disease pathogenesis regulating adipocyte differentiation and immune-inflammatory environment. However, the role of this cytokine as a biomarker in obesity is poorly addressed. Therefore, the present study aimed to evaluate the impact of TGFB1 polymorphisms and TGFβ1 plasmatic levels in obesity Methods and results: TGFB1 promoter region polymorphisms (rs1800468, G-800A and rs1800469, C-509 T) were evaluated in 75 obese patients and 45 eutrophic patients through PCR–RFLP and plasmatic TGFβ1 was quantified through ELISA from 37 of the obese patients, and correlations with clinical and biochemical parameters were tested. Despite no association was found between TGFB1 polymorphisms and obesity susceptibility, several correlations with clinical data were noted. Among others, AC haplotype negatively correlated with plasmatic TGFβ1, while plasmatic TGFβ1 negatively correlated with C-reactive protein and positively correlated with liver abnormalities on ultrasound and, specifically, with steatosis presence and degree. Conversely, GT haplotype, which associates with higher TGFβ1 production, was also positively correlated with the same parameters of liver abnormalities. Further, plasmatic vitamin D negatively correlated with TGFβ1, while positively correlated with AC haplotype. Conclusion: Overall, the results indicate that TGFβ1 might exert important roles in obesity pathophysiology and correlate with biochemical and clinical parameters both at systemic protein as well as at genetic level. Importantly, the consistent positive correlation at both levels with steatosis might suggest this cytokine as a biomarker for this hepatic abnormality in obese patients. Department of General Biology Biological Sciences Center Londrina State University (UEL) Department of Pathological Sciences Biological Sciences Center Londrina State University (UEL) Clinical Surgery Department Federal University of Parana (UFPR) School of Medicine Department of Pathology São Paulo State University (UNESP) Laboratory of DNA Polymorphisms and Immunology Department of Pathological Sciences Biological Sciences Center State University of Londrina, PR445, Km 380 Celso Garcia Cid highway School of Medicine Department of Pathology São Paulo State University (UNESP) CNPq: 152170/2019-7 CNPq: 306386/2017-8

Obesity is considered a chronic inflammatory disease and transforming growth factor beta 1 (TGFβ1) might exert important roles in disease pathogenesis regulating adipocyte differentiation and immune-inflammatory environment. However, the role of this cytokine as a biomarker in obesity is poorly addressed. Therefore, the present study aimed to evaluate the impact of TGFB1 polymorphisms and TGFβ1 plasmatic levels in obesity METHODS AND RESULTS: TGFB1 promoter region polymorphisms (rs1800468, G-800A and rs1800469, C-509 T) were evaluated in 75 obese patients and 45 eutrophic patients through PCR-RFLP and plasmatic TGFβ1 was quantified through ELISA from 37 of the obese patients, and correlations with clinical and biochemical parameters were tested. Despite no association was found between TGFB1 polymorphisms and obesity susceptibility, several correlations with clinical data were noted. Among others, AC haplotype negatively correlated with plasmatic TGFβ1, while plasmatic TGFβ1 negatively correlated with C-reactive protein and positively correlated with liver abnormalities on ultrasound and, specifically, with steatosis presence and degree. Conversely, GT haplotype, which associates with higher TGFβ1 production, was also positively correlated with the same parameters of liver abnormalities. Further, plasmatic vitamin D negatively correlated with TGFβ1, while positively correlated with AC haplotype.Overall, the results indicate that TGFβ1 might exert important roles in obesity pathophysiology and correlate with biochemical and clinical parameters both at systemic protein as well as at genetic level. Importantly, the consistent positive correlation at both levels with steatosis might suggest this cytokine as a biomarker for this hepatic abnormality in obese patients.

The objective of this study was to evaluate the antibacterial effect of sophorolipid in combination with lactic acid against relevant bacteria isolated from the poultry industry. Staphylococcus aureus, Listeria monocytogenes, Salmonella enterica, and Escherichia coli were isolated from chicken meat and antibacterial tests with sophorolipid and lactic acid were performed. Checkerboard, time-kill, and scanning electron microscopy analyses were used to confirm the antibacterial action and the combined effects. Although no inhibitory effects were observed for E. coli and Salmonella, these compounds presented antibacterial activity against L. monocytogenes and S. aureus. Additionally, sophorolipid and lactic acid were not cytotoxic at the concentrations used in the tests. The combination of sophorolipid and lactic acid resulted in an additive interaction, reducing the concentration of the active compounds needed for effectiveness against S. aureus and L. monocytogenes, to 50% and 75%, respectively. These findings lead to the possibility of developing a new, sustainable, and natural antimicrobial solution that is considered noncytotoxic and has wide applicability in the poultry industry to reduce substantial losses in this sector.

Carnosic acid (CA) is a phenolic diterpene with many important biological activities including antimicrobial, antioxidant, anti-inflammatory properties, and anti-proliferative properties. The aim of the present study was to investigate cytotoxic activity, cell cycle, apoptotic, and molecular effects attributed to CA in non-tumoral IMR-90 (human fetal lung fibroblasts), as well as tumoral NCI-H460 (human non-small-cell lung cancer) cell lines. Cell proliferation was evaluated by Real-Time Cell Analysis system, while apoptosis and cell cycle were assessed using flow cytometry. RT-qPCR was used to estimate the relative expression of genes involved in cell cycle regulation, DNA damage and repair, and apoptosis induction. CA inhibited proliferation of IMR-90 and NCI-H460 cells via cell cycle arrest at G0/G1 and G2/M phases, according to the treatment concentration. The mRNA levels of genes encoding cyclins A2, B1, and B2 were downregulated in response to CA treatment of IMR-90 cells. Apoptosis was induced and proapoptotic gene PUMA was upregulated in both cell lines. mRNA levels of genes ATR, CCND1, CHK1, CHK2, MYC, GADD45A, H2AFX, MTOR, TP53, and BCL2, CASP3 were not markedly changed following CA treatments. Although CA exerted antiproliferative activity against NCI-H460 tumor cells, this phytochemical induced toxic effects in non-tumoral cells, and thus needs to be considered carefully prior to pharmacological use therapeutically.

Background By identifying the molecular mechanisms underlying sodium selenite (Na2SeO3) cytotoxicity during exposure in non-tumor cells (HaCaT cells), we will improve the current understanding of its antiproliferative effects and modulation of gene expression in the main pathways related to the cell cycle, cell death, oxidative stress, and DNA damage and repair. Methods Non-tumor HaCaT cells were treated with Na2SeO3 to induce cytotoxicity, and the effects were investigated using an MTT assay (cell viability), real-time cell analysis (profiling the cell index), flow cytometry (membrane integrity, cell cycle disruption, and apoptosis), a comet assay (genotoxicity, i.e., DNA damage), and RT-qPCR (mRNA expression of genes). Results Treatment with Na2SeO3 was cytotoxic at 10 μM, producing morphological changes in cells (cytoplasmic granulations); however, it did not have a genotoxic effect. Na2SeO3 induced cell membrane damage, cell death, and cell cycle arrest in HaCaT cells. It also altered the mRNA expression levels of PUMA, ATR, and mTOR genes. However, it had no effect on the mRNA expression of caspases or PARP1, BIRC5, BECN1, and c-MYC genes, suggesting that Na2SeO3 causes PUMA-dependent apoptosis in HaCaT cells. The mRNA expression of specific genes related to oxidative stress, DNA damage and repair, and cell cycle control were unchanged by Na2SeO3. Conclusions We demonstrated the cytotoxic effect of Na2SeO3 in HaCaT cells by analyzing mRNA expression patterns, changes in cell morphology, and proliferation kinetics.

Background Cell culture (spheroid and 2D monolayer cultures) is an essential tool in drug discovery. Piperlongumine (PLN), a naturally occurring alkaloid present in the long pepper (Piper longum), has been implicated in the regulation of GSTP1 activity. In vitro treatment of cancer cells with PLN increases ROS (reactive oxygen species) levels and induces cell death, but its molecular mode of action has not been entirely elucidated. Methods In this study, we correlated the antiproliferative effects (2D and 3D cultures) of PLN (CAS 20069–09-4, Sigma-Aldrich) with morphological and molecular analyses in HepG2/C3A cell line. We performed assays for cytotoxicity (MTT), comet assays for genotoxicity, induction of apoptosis, analysis of the cell cycle phase, and analysis of the membrane integrity by flow cytometry. Relative expression of mRNA of genes related to proliferation, apoptosis, cell cycle control, metabolism of xenobiotics, and reticulum endoplasmic stress. Results PLN reduced the cell proliferation by the cell cycle arrest in G2/M. Changes in the mRNA expression for CDKN1A (4.9x) and CCNA2 (0.5x) of cell cycle control genes were observed. Cell death occurred due to apoptosis, which may have been induced by increased expression of proapoptotic mRNAs (BAK1, 3.1x; BBC3, 2.4x), and by an increase in 9 and 3/7 active caspases. PLN induced cellular injury by ROS generation and DNA damage. DNA damage induced MDM2 signaling (3.0x) associated with the appearance of the monastral spindle in mitosis. Genes associated with ROS degradation also showed increased mRNA expression (GSR, 2.0x; SOD1, 2.1x). PLN induce endoplasmic reticulum stress with the increase in the mRNA expression of ERN1 (4.5x) and HSPA14 (2.2x). The xenobiotic metabolism showed increased mRNA expression for CYP1A2 (2.2x) and CYP3A4 (3.4x). In addition to 2D culture, PLN treatment also inhibited the growth of 3D culture (spheroids). Conclusion Thus, the findings of our study show that several gene expression biomarkers (mRNAs) and monastral spindle formation indicated the many pathways of damage induced by PLN treatment that contributes to its antiproliferative effects. Graphical abstract

Oxidative stress (OS) is involved in the onset of various pathological processes, and sodium selenite (Na2SeO3) is known to have antioxidant activity. This study evaluated the cellular response of human HepG2/C3A cells supplemented with Na2SeO3 when exposed to hydrogen peroxide (H2O2)-induced OS. We analyzed cytotoxicity, cell proliferation, and genotoxicity in comparison with molecular data of mRNA and protein expression. The MTT and comet assays revealed that Na2SeO3 conferred cytoprotective and anti-genotoxic effects. In contrast, RTCA (Real-Time Cell Analysis) and flow cytometry analysis revealed that Na2SeO3 did not inhibit H2O2-induced anti-proliferative effects or cell cycle arrest (G2/M). Cells exposed simultaneously to Na2SeO3 and H2O2 showed overexpression of GPX1 mRNA, indicating that Na2SeO3 influenced the cellular antioxidant system. Furthermore, downregulation of CAT mRNA and SOD1 and PRX2 proteins induced by H2O2, was minimal after the Na2SeO3+H2O2 treatment. Although normalization of CCN2B mRNA expression by Na2SeO3 was observed after the Na2SeO3+H2O2 treatment, this was not observed for other genes such as CDKN1A, CDKN1C, and CDKN2B, which are related to cell cycle control, nor for GADD45A, which is involved in the cellular response to DNA damage. Furthermore, both CDKN1B and CDKN1C expression were downregulated in HepG2/C3A cells treated with Na2SeO3 only. Our results indicate that cellular response to Na2SeO3 involved the modulation of the antioxidant system. Na2SeO3 was unable completely recover HepG2/C3A cells from H2O2-induced oxidative damage, as evidenced by analysis of cell proliferation kinetics, cell cycle assay, and expression of key genes involved in cell cycle progression and response to DNA damage.

Background: Genistein (5,7-Dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) is the most abundant isoflavone in soybean, which has been associated with a lower risk of development of cancer and cardiovascular diseases. Of particular interest regarding cancer preventive properties of flavo-noids is their interaction with cytochrome P450 enzymes (CYPs). However, contradictory data report the effect of genistein on expression of СYPs enzymes. Objective: The aim of this study was to investigate the effects of genistein on cytochrome P450 (CYP) gene expression levels in human hepatocellular carcinoma (HepG2/C3A) and colon adenocarcinoma (HT29) cells. Methods: Real-time RT–PCR was used to examine the expression of genes families involved in xenobi-otic metabolism, such as CYP1 (CYP1A1, CYP1B1), CYP2 (CYP2E1, CYP2D6), CYP3 (CYP3A4); and of a family involved in the catabolism of the all-trans-retinoic acid (ATRA), CYP26 (CYP26A1, CYP26B1). Results: RT-qPCR data analysis showed that after 12 h of exposure of HepG2/C3A cells to genistein (5 and 50 µM) there was an upregulation of CYP1A1 and CYP1B1 and downregulation of CYP2D6, CYP26A1 and CYP26B1 mRNA levels. There was no change in the mRNA levels of CYP P450 genes in HT29 cells. Conclusion: Our results suggest that treatment with genistein in non-toxic concentrations may impact the expression level of CYPs involved in the biotransformation of xenobiotics and drug metabolizing en-zymes. Moreover, the downregulation of ATRA metabolism-related genes opens a new research path for the study of genistein as retinoic acid metabolism blocking agent for treating cancer and other patholo-gies.

Leydig cells are the major testosterone-producing cells of the male reproductive system, and damage to these cells can impair fertility of men. Bisphenol A (BPA) is one of the chemicals with the highest volume of production worldwide. The aim of this study was to evaluate the effects of BPA on the growth, viability, and testosterone production of TM3 murine Leydig cells after exposure to BPA for 24 or 48 h. BPA reduced testosterone production, cell viability and cell growth in a concentration-dependent manner. The highest tested concentration of BPA (100 μM) increased cellular death, as indicated by an increased sub-G1 phase population and a larger number of cells labeled with Hoechst 3342. This concentration of BPA also decreased the number of metabolically active mitochondria as revealed by rhodamine staining. Therefore, our data show that BPA is toxic to Leydig TM3 cells and impairs their steroidogenic function.

Made available in DSpace on 2018-12-11T17:22:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-08-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Financiadora de Estudos e Projetos Background/Aims: Compared with non-obese individuals, obese individuals commonly store more vitamin D in adipose tissue. VDR expression in adipose tissue can influence adipogenesis and is therefore a target pathway deserving further study. This study aims to assess the role of 1,25(OH)2D3 in human preadipocyte proliferation and differentiation. Methods: RTCA, MTT, and trypan blue assays were used to assess the effects of 1,25(OH)2D3 on the viability, proliferation, and adipogenic differentiation of SGBS cells. Cell cycle and apoptosis analyses were performed with flow cytometry, triglycerides were quantified, and RT-qPCR was used to assess gene expression. Results: We confirmed that the SGBS cell model is suitable for studying adipogenesis and demonstrated that the differentiation protocol induces cell maturation, thereby increasing the lipid content of cells independently of treatment. 1,25(OH)2D3 treatment had different effects according to the cell stage, indicating different modes of action driving proliferation and differentiation. In preadipocytes, 1,25(OH)2D3 induced G1 growth arrest at both tested concentrations without altering CDKN1A gene expression. Treatment with 100 nM 1,25(OH)2D3 also decreased MTT absorbance and the lipid concentration. Moreover, increased normalized cell index values and decreased metabolic activity were not induced by proliferation or apoptosis. Exposure to 100 nM 1,25(OH)2D3 induced VDR, CEBPA, and CEBPB expression, even in the preadipocyte stage. During adipogenesis, 1,25(OH)2D3 had limited effects on processes such as VDR and PPARG gene expression, but it upregulated CEBPA expression. Conclusions: We demonstrated for the first time that 1,25(OH)2D3 induces changes in preadipocytes, including VDR expression and growth arrest, and increases the lipid content in adipocytes treated for 16 days. Preadipocytes are important cells in adipose tissue homeostasis, and understanding the role of 1,25(OH)2D3 in adipogenesis is a crucial step in ensuring adequate vitamin D supplementation, especially for obese individuals. São Paulo State University (UNESP) School of Medicine Department of Pathology Maastricht University Department of Bioinformatics - BiGCaT NUTRIM School for Nutrition and Translational Research in Metabolism State University of Londrina (UEL) Department of Biochemistry and Biotechnology State University of Londrina (UEL) Department of General Biology Maastricht University Maastricht Centre for Systems Biology (MaCSBio) Centro de Ciências Bio. Dept. de Bio. Geral Lab. de Genética Toxicológica Univ. Estadual de Londrina Rodovia Celso Garcia Cid, PR 445 Km 380, Campus Universitário São Paulo State University (UNESP) School of Medicine Department of Pathology CNPq: 150067/2017-8 CAPES: 9933/2014-00

Made available in DSpace on 2018-12-11T17:11:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-07-01 Studies have shown that metabolic disorders, serum inflammatory markers and weight gain (obesity) are correlated with vitamin D deficiency. Therefore, the present study correlated the serum calcidiol (s25(OH)D3) levels in a sample of individuals from southern Brazil with variables related to metabolic disorders, obesity and lifestyle habits and assessed the cytotoxic effect of calcitriol on adipose tissue-derived mesenchymal stem cells (ADSCs). The results showed a 79.23% prevalence of hypovitaminosis D in the study population and a correlation (p

Studies that evaluated the mechanisms of action of Plumbagin (PLB) and its toxicity may contribute to future therapeutic applications of this compound. We investigate biomarker important in the mechanisms of action correlate the expression of mRNA with the cytotoxic and genotoxic effects of PLB on HepG2/C3A. In the analysis of cytotoxicity, PLB decreased cell viability and membrane integrity at concentrations ≥ 15μM. Xenobiotic-metabolizing system showed strong mRNA induction of CYP1A1, CYP1A2, and CYP3A4, suggesting extensive metabolization. PLB induced apoptosis and an increase in the mRNA expression of genes BBC3, CASP3, and CASP8. At a concentration of 15μM, there was a reduction in the expression of PARP1 mRNA and an increase in the expression of BECN1 mRNA, suggesting that PLB may also induce cell death by autophagy. PLB induced an arrest at the G2/M phase due to DNA damage, as observed in the comet assay. This damage is associated with the increased mRNA expression of genes p21, GADD45A, and H2AFX and with changes in the expression of proteins H2AX, p21, p53, Chk1, and Chk2. These results allow a better understanding of the cellular action of PLB and of its toxicity, thereby contributing to the development of PLB-based drugs, with markers of mRNA expression possibly playing a role as indicators for monitoring toxicity in human cells.

Aims Fructooligosaccharides (FOSs) known for their health properties and β-(2→6)-levan-type FOSs have shown prebiotic and immunomodulatory activities that overcome those of commercial β-(2→1)-FOSs, but costs do not favour their use. Moreover, FOSs can reach the bloodstream through the diet, and little is known about their direct effect on cells. The aim of this work was to produce high-content FOSs by Bacillus subtilis natto CCT7712 in a bioreactor using commercial sucrose and to evaluate their antiproliferative effects in OVCAR-3 cells. Methods and results FOS production reached 173·60 g l-1 , 0·2 vvm aeration and uncontrolled pH. Levan-type FOSs, composed of β-(2 → 6) links and mainly GF3 (6-nystose), were identified using RMN spectroscopy, FT-IR and ESI-MS. FOSs decreased the viability and proliferation of OVCAR-3 cells, and the effects were associated with an increased pro-inflammatory response by the induction of IL-8 and TNF-α, and the repression of ER-β genes. The metabolic profiles showed disruption of cellular homeostasis that can be associated with a decrease in proliferation. Conclusions The high production of levan-type FOSs from B. subtilis natto CCT7712 in a bioreactor was achieved, and they showed antiproliferative potential in OVCAR-3 cells. Significance and impact of the study FOS could be a good target for future therapeutic studies and commercial use.

Plumbagin (PLB) is a phytochemical being used for centuries in traditional medicines. Recently, its capacity to inhibit the development of human tumors has been observed, through the induction of apoptosis, cell cycle arrest, and inhibition of angiogenesis and metastasis. Here we evaluated the mechanism of action of PLB in the kidney adenocarcinoma 786-O cell line, which are metabolizing cells important for toxicology studies. After the treatment with PLB, we observed increased apoptosis and cell cycle arrest in S and G2/M phases, starting at 5 µM. In addition, PLB was cytotoxic, genotoxic and induced loss of cell membrane integrity. Regarding gene expression, treatment with 7.5 µM PLB reduced the amount of MTOR, BCL2 and ATM transcripts, and increased CDKN1A (p21) transcripts. Phosphorylation levels of yH2AX was increased and MDM2 protein level was reduced following the treatment with PLB, demonstrating its genotoxic effect. Our results suggest that PLB acts in molecular pathways related to the control of proliferation and cell death in 786-O cells.

Alzheimer’s disease (AD) is the most common form of dementia and has no cure. Therapeutic strategies focusing on the reduction of oxidative stress, modulation of amyloid-beta (Aβ) toxicity and inhibition of tau protein hyperphosphorylation are warranted to avoid the development and progression of AD. The aim of this study was to screen the crude extracts (CEs) and ethyl-acetate fractions (EAFs) of Guazuma ulmifolia, Limonium brasiliense, Paullinia cupana, Poincianella pluviosa, Stryphnodendron adstringens and Trichilia catigua using preliminary in vitro bioassays (acetylcholinesterase inhibition, antioxidant activity and total polyphenol content) to select extracts/fractions and assess their protective effects against Aβ25–35 toxicity in SH-SY5Y cells. The effect of the EAF of S. adstringens on mitochondrial membrane potential, lipid peroxidation, superoxide production and mRNA expression of 10 genes related to AD was also evaluated and the electropherogram fingerprints of EAFs were established by capillary electrophoresis. Chemometric tools were used to correlate the in vitro activities of the samples with their potential to be evaluated against AD and to divide extracts/fractions into four clusters. Pretreatment with the EAFs grouped in cluster 1 (S. adstringens, P. pluviosa and L. brasiliense) protected SH-SY5Y cells from Aβ25-35-induced toxicity. The EAF of S. adstringens at 15.62 μg/mL was able completely to inhibit the mitochondrial depolarization (69%), superoxide production (49%) and Aβ25-35-induced lipid peroxidation (35%). With respect to mRNA expression, the EAF of S. adstringens also prevented the MAPT mRNA overexpression (expression ratio of 2.387x) induced by Aβ25–35, which may be related to tau protein hyperphosphorylation. This is the first time that the neuroprotective effects of these fractions have been demonstrated and that the electropherogram fingerprints for the EAFs of G. ulmifolia, L. brasiliense, P. cupana, P. pluviosa and S. adstringens have been established. The study expands knowledge of the in vitro protective effects and quality control of the evaluated fractions.

Curcumin (Cur), is a pigment with antiproliferative activity but has some pharmacokinetic limitations, which led researchers to look for more effective structure analogs. This work investigated the effects of Cur and compared them with the two analogs, demethoxycurcumin (DeMC) and dimethoxycurcumin (DiMC), to elucidate their mechanisms of action. The cytotoxic, antiproliferative, and genotoxic effects these compounds were correlated based on gene expression analysis in the human renal adenocarcinoma cells (786-O). Cur decreased CYP2D6 expression and exhibited cytotoxic effects, such as inducing monopolar spindle formation and mitotic arrest mediated by the increase in CDKN1A (p21) mRNA. This dysregulation induced cell death through a caspase-independent pathway but was mediated by decrease in MTOR and BCL2 mRNA expression, suggesting that apoptosis occurred by autophagy. DeMC and DiMC had similar effects in that they induced monopolar spindle and mitotic arrest, were genotoxic, and activated GADD45A, an important molecule in repair mechanisms, and CDKN1A. However, the induction of apoptosis by DeMC was delayed and regulated by the decrease of antiapoptotic mRNA BCL.XL and subsequent activation of caspase 9 and caspase 3/7. DiMC treatment increased the expression of CYP1A2, CYP2C19, and CYP3A4 and exhibited higher cytotoxicity compared with other compounds. It induced apoptosis by increasing mRNA expression of BBC3, MYC, and CASP7 and activation of caspase 9 and caspase 3/7. These data revealed that different gene regulation processes are involved in cell death induced by Cur, DeMC, and DiMC. All three can be considered as promising chemotherapy candidates, with DiMC showing the greatest potency.

Background Hepatocellular Carcinoma (HCC) is extremely aggressive and presents low rates of response to the available chemotherapeutic agents. Many studies have focused on the search for alternative low-cost natural compounds with antiproliferative potential that selectively respond to liver cancer cells. Purpose This study assessed the in vitro direct action of trans-chalcone (TC) on cells of the human HCC HuH7.5 cell line. Methods We subjected the HuH7.5 tumor cells to TC treatment at increasing concentrations (12.5–100 µM) for 24 and 48 h. Cell viability was verified through MTT and 50% inhibitory concentration of cells (IC50 23.66 µM) was determined within 48 h. We quantified trypan blue proliferation and light microscopy, ROS production, mitochondrial depolarization and autophagy, cell cycle analysis, and apoptosis using Muse® cell analyzer and immunocytochemical markings of p53 and β-catenin. Results Data showed an effective dose- and time-dependent TC-cytotoxic action at low micromolar concentrations without causing toxicity to non-cancerous cells, such as erythrocytes. TC-treatment caused mitochondrial membrane damage and cell cycle G0/G1 phase arrest, increasing the presence of the p53 protein and decreasing β-catenin, in addition, to inducing cell death by autophagy. Additionally, TC decreased the metastatic capacity of HuH7.5, which affected the migration/invasion of this type of cell. Conclusion In vitro TC activity in the human HCC HuH7.5 tumor cell line is shown to be a potential molecule to develop new therapies to repair the p53 pathway and prevent the overexpression of Wnt/β-catenin tumor development inducing autophagy cell death and decreasing metastatic capacity of HuH7.5 cell line.