Piperlongumine inhibits antioxidant enzymes, increases ROS levels, induces DNA damage and G2/M cell cycle arrest in breast cell lines.

Piperlongumine (PLN) is a biologically active alkaloid/amide derived from Piper longum, with known promising anticancer activity. The aim of this study was to compare the antiproliferative activity of PLN in human breast MCF-7 adenocarcinoma cell line with effects in HB4a normal mammary epithelial non-tumor cell line. The parameters examined were cell growth, viability, reactive oxygen species (ROS) levels and DNA damage, as well as the effects on the modulating targets responsible through regulation of these pathways. PLN increased ROS levels and expression of the SOD1 antioxidant enzyme. PLN inhibited the expression of the antioxidant enzymes catalase, TRx1, and PRx2. The ability of PLN to inhibit antioxidant enzyme expression was associated with the oxidative stress response. PLN induced genotoxicity in both cell lines and upregulated the levels of GADD45A mRNA and p21 protein. The DNA damage response ATR protein was downregulated in both cell lines and contributed to an enhanced PLN genotoxicity. In HB4a cells, Chk1 protein, and mRNA levels were also decreased. In response to elevated ROS levels and DNA damage induction, the cells were arrested at the G2/M phase, probably in an attempt to promote cell survival. Although cell viability was reduced in both cell lines, only HB4a cells underwent apoptotic cell death, whereas other types of cellular death may be involved in MCF-7 cells. Taken together, these data provide insight into the anticancer mechanisms attributed to PLN effects, which acts as an inhibitor of DNA damage response (DDR) proteins and antioxidant enzymes.

In vitro evaluation of the cytotoxic and genotoxic effects of Al and Mn in ambient concentrations detected in groundwater intended for human consumption.

Environmental exposure to metals can induce cytotoxic and genotoxic effects in cells and affect the health of the exposed population. To investigate the effects of aluminum (Al) and manganese (Mn), we evaluated their cytogenotoxicity using peripheral blood mononuclear cells (PBMCs) exposed to these metals at previously quantified concentrations in groundwater intended for human consumption. The cell viability, membrane integrity, nuclear division index (NDI), oxidative stress, cell death, cell cycle, and DNA damage were analyzed in PBMCs exposed to Al (0.2, 0.6, and 0.8 mg/L) and Mn (0.1, 0.3, 1.0, and 1.5 for 48 h. We found that Al induced late apoptosis; decreased cell viability, NDI, membrane integrity; and increased DNA damage. However, no significant alterations in the early apoptosis, cell cycle, and reactive oxygen species levels were observed. In contrast, exposure to Mn altered all evaluated parameters related to cytogenotoxicity. Our data show that even concentrations allowed by the Brazilian legislation for Al and Mn in groundwater intended for human consumption cause cytotoxic and genotoxic effects in PBMCs. Therefore, in view of the results found, a comprehensive approach through in vivo investigations is needed to give robustness and validity to the results obtained, thus broadening the understanding of the impacts of metals on the health of environmentally exposed people.

Vitamin D3 and Salinomycin synergy in MCF-7 cells cause cell death via endoplasmic reticulum stress in monolayer and 3D cell culture.

1α, 25, dihydroxyvitamin D3 (1,25D), the active form of vitamin D3, has antitumor properties in several cancer cell lines in vitro. Salinomycin (Sal) has anticancer activity against cancer cell lines. This study aims to examine the cytotoxic and antiproliferative effect of Sal associated with 1,25D on MCF-7 breast carcinoma cell line cultured in monolayer (2D) and three-dimensional models (mammospheres). We also aim to evaluate the molecular mechanism of Sal and 1,25D-mediated effects. We report that Sal and 1,25D act synergistically in MCF-7 mammospheres and monolayer causing G1 cell cycle arrest, reduction of mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) overproduction with a long-lasting cytotoxic response represented by clonogenic and mammosphere assay. We observed the induction of cell death by apoptosis with upregulation in mRNA levels of apoptosis-related genes (CASP7, CASP9, and BBC3). Extensive cytoplasmic vacuolization, a morphological characteristic found in paraptosis, was also seen and could be triggered by endoplasmic reticulum stress (ER) as we found transcriptional upregulation of genes related to ER stress (ATF6, GADD153, GADD45G, EIF2AK3, and HSPA5). Overall, Sal and 1,25D act synergistically, inhibiting cell proliferation by activating simultaneously multiple death pathways and may be a novel and promising luminal A breast cancer therapy strategy.

The intra-articular injection of adipose-derived stem cells decreases pain and reduces inflammation in knee osteoarthritis, with or without the addition of platelet-rich plasma also improves functionality.

The increase of individuals with Osteoarthritis (OA) has generated an increase in public spending in the treatments, which are still not that effective. So, the purpose of this study was to analyze and compare four types of interventions: platelet-rich plasma (PRP), adipose-derived stem cells (ADSCs), ADSCs + PRP and the standard surgical video arthroscopy (All groups passed through standard arthroscopy before intervention). The evaluation was performed by applying the questionnaires Western Ontario McMaster Universities, Short Form Health Survey 36 and Visual Analog Pain Scale, also by analyzing the synovial fluid (inflammatory cytokines, enzymatic, colorimetric and viscosity analysis), this evaluation happened in two moments: before the surgical procedures and after 6 months of the interventions and also was made a comparison to standard arthroscopy. The questionnaires results showed a greater improvement in the scores of the domains analyzed in the ADSCs + PRP group, followed by the ADSCs and PRP group. In the evaluation of inflammatory cytokines, there was a significant reduction in the cytokine IL-1b only in the ADSCs + PRP group (46%) and ADSCs (31%), of IL-6 in the ADSCs + PRP group (72%), of IL-8 in the ADSCs + PRP group (50%) and ADSCs (31%), and TNF in the ADSCs + PRP group (46%). There was also a significant increase in the amount of total proteins (79%) in the control group and polymorphonuclear cells (47%) in the ADSCs + PRP group. Taking all the results into account, we infer that therapies with ADSCs + PRP and only ADSCs are safe and effective over 6 months for the improvement of pain, functional capacity and joint inflammation in volunteers with OA. It is also considered that the use of ADSCs + PRP, particularly, is a promising alternative to help manage this disease, due to the better results presented among the four propose interventions.

The enzymatic disaggregation by trypsin does not alter cell quality and genomic stability of adipose-derived stem cells cultivated for human cell therapy.

There is no consensus between the protocols used for the isolation, maintenance and cultivation of Adipose-derived stem cells (ADSCs) for therapeutic purposes. Thus, was evaluated the maintenance method of ADSCs submitted to enzymatic disaggregation by trypsin. Was made (1st until 10th passage) immunophenotyping, cell differentiation assays, comet assay, differential cell death, apoptosis, cell viability and membrane integrity by flow cytometry.The results showded that trypsinization,did not induce genomic instability, also did not alter the tail moment. The cell death assay, showed that only on the 10th passage there was a significant reduction and was cofirmed by flow cytometry that is apoptosis. The viability showded significant reduction only in 10th passage, this was related to the loss of integrity of membrane, proven by flow cytometry. The quantities varied along the passages (11 × 105 to 2 × 105). Qualitatively, it can be observed that as the number of cells decreases, there is also a reduction in the juxtaposition of ADSCs and increased of the cell size, it is started in 6th passage. In view of the results, it is suggested for more safety, that ADSCs cultured until the 5th passage being used in human transplantation procedures.

Synthesis, Characterization, Antiproliferative Activity of Galloyl Derivatives and Investigation of Cytotoxic Properties in HepG2/C3A Cells.

BACKGROUND: Appropriate substituents in the galloyl group could lead to significant biological properties. OBJECTIVES: Novel galloyl-substituted compounds bearing 2-substituted-1, 3, 4-oxadiazol-5-yl, 5- substituted-1,2,4-triazol-3-yl, and carboxamide groups were synthesized and evaluated for their antiproliferative activity. Additionally, galloyl hydrazide (2) was evaluated by performing cytotoxicity, membrane integrity, cell cycle, and apoptosis assays in HepG2/C3A cells. METHODS: General procedure was used for the synthesis of galloyl-substituted (3-9, 11) and characterized by their spectroscopic data (1H and 13C NMR). The antiproliferative activity of all novel galloyl derivatives was evaluated against nine human tumors and one nontumoral cell line. Three response parameters (GI50, TGI, and LC50) were calculated. The cytotoxicity test was performed for the resazurin assay. The membrane integrity, cell cycle, and apoptosis assays were performed by flow cytometry. RESULTS: The substitution of the methoxy group of the galloyl ring system for a carboxamide group (3, 4, 5, and 6) produced compounds with moderate antitumoral activity, particularly 6, against six human cancer cell lines, K-562, PC-3, NCI-ADR/RES, OVCAR, 786-0 and NCI-H460, with GI50 values ≤ 9.45 µg/mL. Triazole derivatives 7 and 8 exhibited higher antitumoral activity toward OVCAR, MCF-7 and leukemia K-562 cell lines, exhibiting GI50 values less than 10 µg/mL. Compound 11 displayed significant activity against PC-3 (GI50 = 4.31 µg/mL), OVCAR (GI50 = 8.84 µg/mL) and K-562 (GI50 = 8.80 µg/mL) cell lines. Galloyl hydrazide (2) had cytotoxic activity in HepG2/C3A cells (IC50 = 153.7 µg/mL). In membrane permeability, cell count, cell cycle, and apoptosis assays, as determined using the IC50 of compound (2) in HepG2/C3A cells, increased membrane permeability, decreased cell count, altered cell cycle, and initial apoptosis was observed compared to the control group. CONCLUSION: Thus, our results showed for the first time the synthesis, antiproliferative activity, and cytotoxicity of galloyl-substituted compounds. Galloyl-substitution does not have a very strong synergistic effect in the inhibition of cancer cell proliferation compared with galloyl hydrazide (2). Compound 2 demonstrated promising activity in HepG2/C3A hepatocarcinoma cells.

The Use of Natural Fiber-Rich Food Product Is Safe and Reduces Aberrant Crypt Foci in a Pre-Clinical Model.

BACKGROUND: Colorectal cancer is a highly prevalent disease, requiring effective strategies for prevention and treatment. The present research aimed to formulate a natural fiber-rich food product (NFRFP) and to evaluate its safety, toxicogenetics, and effects on aberrant crypt foci induced by 1,2-dimethyl-hydrazine in a preclinical model. METHODS: A total of 78 male Wistar rats were distributed in six experimental groups: negative control, positive control (1,2-Dimethylhydrazine-40 mg/Kg), and four groups fed with 10% NFRFP: NFRFP, pre-treatment protocol, simultaneous treatment, and post-treatment protocol. RESULTS: The NFRFP was shown to be a good source of fibers and did not change biometric, biochemical, hematological, and inflammatory parameters, and did not induce signs of toxicity and genotoxicity/carcinogenicity. NFRFP exhibited a chemopreventive effect, in all protocols, with damage reduction (% DR) of 75% in the comet test. NFRFP reduced the incidence of aberrant crypt outbreaks by 49.36% in the post-treatment protocol. CONCLUSIONS: The results suggest the applicability of NFRFP in the human diet due to potential production at an industrial scale and easy technological application in different products, since it could be incorporated in food without altering or causing small changes in final product sensory characteristics.

Transcriptional profiling analysis of susceptible and resistant strains of Anticarsia gemmatalis and their response to Bacillus thuringiensis.

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.

Dimethoxycurcumin reduces proliferation and induces apoptosis in renal tumor cells more efficiently than demethoxycurcumin and curcumin.

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.

mRNAs biomarker related to the control of proliferation and cell death in HepG2/C3A spheroid and monolayer cultures treated with piperlongumine

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 20069­09-4, 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

Cytotoxicity of sodium selenite in HaCaT cells induces cell death and alters the mRNA expression of PUMA, ATR, and mTOR genes.

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.

Carnosic acid exhibits antiproliferative and proapoptotic effects in tumoral NCI-H460 and nontumoral IMR-90 lung cells.

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.

Up and down-regulation of mRNA in the cytotoxicity and genotoxicity of Plumbagin in HepG2/C3A.

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.

The α -Tomatine Exhibits Antiproliferative Activity, Rupture of Cell Membranes and Induces the Expression of APC Gene in the Human Colorectal Adenocarcinoma Cell Line (Ht-29)

Abstract The α-tomatine is a steroidal glycoalkaloid found in immature tomatoes (Lycopersicon esculentum) that has important biological functions including the inhibition of cancer cell growth and preventing metastasis. This study aimed to evaluate the effects of α-tomatine on cytotoxicity, cellular proliferation, apoptosis, and mRNA expression of APC, CCNA2, β-catenin, CASP9, BAK, BAX and BCL-XL in colorectal adenocarcinoma cell line HT-29. HT29 cells were treated with three concentrations of α-tomatine (0.1, 1 and 10 µg/mL), although only the 1 µg/mL concentration of α-tomatine was used to evaluate genetic expression patterns by real time-PCR. Results showed that α-tomatine was cytotoxic only at the 10 µg/mL concentration. Cell proliferation was significantly inhibited after the first 24 hours of treatment only with concentrations of 10 µg/mL. In contrast, there were no significant differences in apoptosis for any treatment. In the gene expression studies, only APC expression was significantly altered by α-tomatine treatment. In conclusion, α-tomatine has antiproliferative activity in the first 24h of treatment, does not induce apoptosis in this cell line and causes disruption of cell membranes, thereby increasing the expression of APC gene related to cell cycle.

Mitotic spindle defects and DNA damage induced by dimethoxycurcumin lead to an intrinsic apoptosis pathway in HepG2/C3A cells.

Dimethoxycurcumin (DiMC), a synthetic analog of curcumin, was shown to have antiproliferative activity in human tumor cell lines. Therefore, we investigated its cytotoxic, antiproliferative, genotoxic, and apoptotic effect and correlated these evaluations with the expression of transcripts and proteins in the human hepatocellular carcinoma cell line (HepG2/C3A). Treatment with DiMC resulted in increased CYP2E1, CYP2C19 and CYP1A2 transcripts levels and was cytotoxic (≥10 µM). DiMC caused mitotic arrest by inducing monopolar spindle formation and was genotoxic increasing expression of the CDKN1A, GADD45A and PARP1 gene, key effectors in the cell cycle arrest and DNA repair pathways, respectively. This genotoxicity was caused by generation of reactive oxygen species and reduction of antioxidant proteins levels. Furthermore, we observed a decrease in important proteins involved in DNA repair. In addition to the observed apoptotic morphology and the presence of annexin labeling, we observed increased expression of BAK1 and CASP7 genes and caspase 3/7 protein activity, showing that these effects caused apoptosis through the intrinsic pathway in HepG2/C3A cells. Our results indicate that DiMC modulates important molecular targets leading to cell death even in metabolic competent cells models has considerable potential in anticancer therapy.

Molecular pathways related to the control of proliferation and cell death in 786-O cells treated with plumbagin.

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.

In vitro evaluation of the protective effects of plant extracts against amyloid-beta peptide-induced toxicity in human neuroblastoma SH-SY5Y 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 (Aß) 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 Aß25-35 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 Aß25-35-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 Aß25-35-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 Aß25-35, 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.

Effects of folic acid on the antiproliferative efficiency of doxorubicin, camptothecin and methyl methanesulfonate in MCF-7 cells by mRNA endpoints.

There is a lack of consensus on whether the role of folate in cancer cells is protective or harmful. The use of folates in combination with cancer-targeting therapeutic regimens requires detailed information to ensure their safe and proper use. Therefore, we evaluated the effects of folic acid (FA) in combination with the chemotherapeutic compounds doxorubicin (DXR), camptothecin (CPT) and methyl methanesulfonate (MMS) on the growth of MCF-7 cells. The data generated from the RTCA assays demonstrated that FA did not affect proliferation in MCF-7 cells treated with DXR and CPT; however, FA reduced the efficacy of MMS treatment. RTCA data also confirmed that DXR and CPT exert their cytotoxic effects in a time-dependent manner and that CPT induced a significantly greater decrease in MCF-7 cell proliferation compared with DXR. The MTT assay failed to detect a reduction in cell proliferation in cells treated with MMS. We quantified the mRNA expression levels of genes associated with cellular stress response, cell cycle and apoptosis pathways using RT-qPCR. The addition of FA to DXR or CPT promoted a similar shift in the gene expression profile of MCF-7 cells compared with cells treated with DXR or CPT without FA; however, this shift did not alter the bioactivity of these drugs. Rather, it indicated that these drugs promoted cell death by alternative mechanisms. In contrast, the addition of FA to MMS reduced the efficacy of the drug without changing the gene expression profile. None of the genes encoding folate receptors that were analyzed were differentially expressed in cells treated with or without FA. In conclusion, supplementation with 450 µM FA was not cytotoxic to MCF-7 cells. However, the addition of FA to anti-cancer drugs must be performed cautiously as the properties of FA might lead to a reduction in drug efficacy.

Response of HepG2/C3A cells supplemented with sodium selenite to hydrogen peroxide-induced oxidative stress.

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.

Genistein Affects Expression of Cytochrome P450 (CYP450) Genes in Hepatocellular Carcinoma (HEPG2/C3A) Cell Line.

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 flavonoids 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 xenobiotic 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 enzymes. 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 pathologies.