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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.

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.

1α, 25, dihydroxyvitamin D

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.

ResumoA osteoartrite (OA) pode deixar o indivíduo incapacitado para realizar suas atividades da vida diária devido ao quadro álgico. Essa é uma importante questão de saúde pública que se agrava no mundo inteiro e no Brasil, uma vez que a população passa pelo processo de envelhecimento, e isso causa um aumento nos gastos públicos com o acompanhamento e manutenção dos tratamentos que podem perdurar por anos e mesmo assim não serem resolutivos. Assim, torna-se necessária a busca por terapias inovadoras e eficazes que possam reduzir esses custos. Nesse contexto, o presente estudo relata a primeira aplicação de terapia celular com células-tronco mesenquimais do tecido adiposo no tratamento de OA refratária ao tratamento conservador realizada no Sistema Único de Saúde (SUS). Na avaliação, foram usados os instrumentos Escala Visual Analógica (EVA), os questionários de qualidade de vida Short Form Health Survey (SF-36) e Western Ontario and McMaster Universities (WOMAC), específicos para avaliação da OA, e fez-se uma análise do líquido sinovial (citocinas inflamatórias). A terapia celular melhorou as pontuações no WOMAC, SF-36, e EVA, e reduziu o processo inflamatório. Observou-se redução de 0,73 × do TNF, de 0,71 × da IL-1b, de 0,68 × da IL-8, e de 0,70 × da IL-10. Já para a IL-6, observou-se aumento de 1,48 ×. Portanto, considera-se este tipo de terapia celular promissora no auxílio do manejo desta doença, pois melhorou o quadro álgico do paciente, reduziu os marcadores inflamatórios, e possibilitou o retorno às atividades da vida diária, o que resultou em uma melhora de sua qualidade de vida.

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.

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 × 10

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

Additional file 1: Table S1. Primers used in Real Time PCR reactions.

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.

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.

As plantas são fontes de diversas moléculas que apresentam propriedades terapêuticas e destacam-se na busca por drogas quimioterápicas alvo-especificas e/ou seletivas. A Piperlongumina (PLN), um alcaloide derivado da Piper longum Linn, apresenta importantes propriedades biológicas, entre elas, a seletividade antiproliferativa para células tumorais. Neste trabalho, avaliou-se diferentes parâmetros como a citotoxicidade, danos a membrana celular, interferência na cinética de proliferação e ciclo celular, indução de apoptose, danos ao DNA, capacidade de gerar estresse oxidativo e o padrão de expressão de mRNA e proteínas relacionados a esses processos em células não tumorais e tumorais de mama (HB4a e MCF-7) e pulmão humano (IMR-90 e NCI-H460), também foram avaliados a capacidade de PLN e ela combinada com Olaparib (inibidor de PARP) em gerar quebras de fita dupla no DNA, além de seus mecanismos de ação em linhagens tumorais de pulmão P53 positiva e negativa. A PLN apresentou citotoxicidade para todas as linhagens com concentrações e tempos distintos. Induziu danos a membrana celular e reduziu a proliferação celular em todas as linhagens estudadas, com efeitos mais pronunciados em células não tumorais e P53 negativas. Ocasionou retenção de ciclo celular na fase G2/M em todas as linhagens excetuando-se a linhagem não tumoral de pulmão, na qual observamos retenção em G1. Além disso, induziu estresse oxidativo nas linhagens de mama, IMR-90 e NCI-H460, danos ao DNA e apoptose, sendo o estresse oxidativo a possível causa dos danos encontrados nas linhagens de pulmão A549 (p53 +/+) e H1299 (p53 -/-). Em linhagens de mama, a PLN induziu aumento de expressão de mRNA relacionados a apoptose, danos ao DNA e estresse oxidativo e diminuiu a expressão de mRNA de genes relacionados ao ciclo celular e do gene TP53, não havendo mudança na quantidade desta proteína após 24 h na linhagem HB4a e ativação na linhagem MCF-7. Em nível proteico, a PLN induziu redução das proteínas Catalase, TRx1, PRx1 e ATR e aumento de p21 nestas linhagens, em MCF-7, as proteínas Chk1 e p53 apresentaram redução e aumento de regulação respectivamente. A ação antiproliferativa apresentada em ambas linhagens, principalmente HB4a, se deve pela capacidade de PLN em causar inibição de proteínas relacionadas a proteção contra estresse oxidativo, levando a geração de danos ao DNA, inibindo o ciclo celular. Nas linhagens de pulmão IMR-90, vários genes relacionados ao ciclo celular tiveram expressão reduzidas assim como TP53, nesta linha, a retenção de ciclo foi mediada pelo gene CDKN1B, um precursor da proteína p27 que controla a progressão do ciclo celular na fase G1. Em IMR-90 também encontramos a proteína SOD2 aumentada além de redução em TRx1. Desta maneira, as mudanças nos padrões de índice celular observados foram atribuíveis à prisão do ciclo celular e apoptose devido ao aumento do estresse oxidativo e danos no DNA. Foi evidenciado que a PLN causa quebras de fita dupla, principalmente em células P53 negativas suplementadas com 10% de soro bovino fetal (SBF), ao mesmo tempo, observamos que a PLN potencializou sua ação com o Olaparib, provavelmente devido a capacidade de suprimir mecanismos da recombinação homologa. Estes resultados nos chama a atenção para os efeitos colaterais que a PLN pode causar nas células não tumorais, principalmente in vivo, visto que é descrita em muitos artigos como seletiva para células tumorais. Plants are sources of several molecules that present therapeutic properties and are highlighted in the search for target-specific and/or selective chemotherapy drugs. Piperlongumine (PLN), an alkaloid derived from Piper longum Linn, presents important biological properties, among them, antiproliferative selectivity for tumor cells. In this work, different parameters such as cytoxicity, cell membrane damage, interference in the kinetics of proliferation and cell cycle, apoptosis induction, DNA damage, ability to generate oxidative stress and the pattern of expression of mRNA and proteins related to these processes in non-tumor and tumoral cells (HB4a and MCF-7) and human lung (IMR-90 and NCI-H460), also evaluated the capacity of PLN and it combined with Olaparib (PARP inhibitor) in generating dual tape breaks in the DNA, in addition to their action mechanisms in tumoral lung lineages positive and negative P53. PLN has presented cytotoxity for all strains with distinct concentrations and times. It induced damage to the cell membrane and reduced cell proliferation in all the lineages studied, with more pronounced effects on non-tumor cells and negative P53. Occasioned cell cycle retention phase G2/M in all as lineages excepting no tumor cell line lung, in which we observe retention in G1. In addition, it induced oxidative stress in the breast, IMR-90 and NCI-H460 lineages, DNA and apoptosis damage, oxidative stress is the possible cause of the damage found in the pulmonary lineage A549 (p53 +/+) and H1299 (p53 -/-). In breast lineage, PLN induced increased expression of mRNA related to apoptosis, DNA damage and oxidative stress and decreased the mRNA expression of genes related to the cell cycle and the TP53 gene, there is no change in the amount of this protein after 24 h in the HB4a lineage and activation in the MCF-7 lineage. On a protein level, PLN induced a reduction in the proteins Catalase, TRx1, PRx1 and ATR and increase in p21 in these lineages, in MCF-7, the Chk1 and p53 proteins have reduced and increased regulation respectively. The antiproliferative action presented in both lineages, mainly HB4a, is due to the capacity of PLN in causing inhibition of proteins related to oxidative stress protection, leading to the generation of damage to DNA, inhibiting the cell cycle. In the IMR-90 lung cell line, several genes related to the cellular cycle had reduced expression as well as TP53, in this line, the cycle retention was mediated by the gene CDKN1B, a precursor of the p27 protein that controls the progression of the cell cycle in the G1 phase. In IMR-90 we also found the SOD2 protein increased in addition to a reduction in TRx1. In this way, changes in the observed cell index patterns were attributable to the arrest of the cell and apoptosis cycle due to increased oxidative stress and DNA damage. It was evidenced that PLN causes double-strand breaks, mainly in negative P53 supplemented cells with 10% fetal bovine serum (SBF), at the same time, we observe that PLN has enhanced its action with Olaparib, probably due to the ability to suppress mechanisms of homologous recombination. These results draw attention to the side effects that PLN can cause in non-tumor cells, especially in vivo, since it is described in many articles as selective for tumor cells.

The β-glucans (β-G) are polysaccharides produced by various organisms, and sulfation of β-G renders them more soluble. With the objective to assess the effects of sulfated and non-sulfated β-G extracted from Agaricus brasiliensis in MCF-7 cells, assays were used to evaluate cytotoxicity, genotoxicity, cell proliferation and mRNA expression. The sulfated and non-sulfated β-G showed dose-dependent cytotoxicity at concentrations of 5 and 10 μg/mL, by the MTT assay. However, only cytotoxicity was observed after 24 h by the Red Neutral test for sulfated β-G, with no genotoxicity for either β-G in comet assay. Proliferation was decreased only at 72 h at a concentration of 100 μg/mL of sulfated β-G. Treatment with 5 μg/mL of sulfated β-G for 6 h reduced the expression of pro-apoptotic genes and stress signaling genes, cell cycle arrest, damage and cell migration. The 5 μg/mL of non-sulfated β-G for 6 h reduced the expression of the stress response gene and signaling damage. These results indicate that the cytotoxicity in the MTT is not cell death, and that, in general, sulfated β-G have greater cytotoxicity compared to non-sulfated β-G.

Of late, many studies are attempting to find new molecules with anticancer properties, especially those with the capability to inhibit cell growth. The aim of this work was to evaluate nerolidol, a plant-based compound, as its cytotoxicity, genotoxicity, antiproliferative and apoptotic induction, cell cycle, mitochondrial membrane potential and RT-qPCR of transcripts related to those pathways in the human hepatocellular carcinoma cell line (HepG2/C3A). Only cis-nerolidol (C-NER) demonstrated cytotoxicity (100-250 μM) activity and was selected to conduct the following experiments. C-NER did not show genotoxic activity, but altered the mitochondrial membrane potential, reduced cell proliferation by arresting cell cycle in G1 phase and induced cell death. RT-qPCR showed that C-NER down-regulated genes related to apoptosis (BAK1, BAX, CAPN1, CASP8, CASP9, PARP1 and TP53), cell cycle (CCND1, CCNE1, CDK1 and CDK2), xenobiotic metabolism (CYP2D6 and CYP3A4) and paraptosis (IGF1R receptor). Up-regulation was seen in case of genes related to cell survival (BBC3 and MYC) and reticulum stress protein response (EIF2AK3 and ERN1) and xenobiotic metabolism (CYP1A2 and CYP2C19). We deduced that the antiproliferative activity of C-NER is attributable to its modulation of the cyclins and cyclin-dependent kinases as these proteins are necessary for G1/S phase transition. EIF2AK3, ERN1, CYP2C19 and CYP1A2 up-regulation suggests that endoplasmic reticulum stress was induced owing to the increased activity of cytochrome P450 enzymes. Caspase-independent cell death was also observed, indicating that another type of cell death, paraptosis, was triggered. Our results indicate that C-NER has considerable potential in anticancer therapy because it modulates important molecular targets of cell survival and proliferation.

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.

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. Keywords: Folate, Breast cancer, Cytotoxicity, RTCA, Impedance, qPCR

As β-glucanas (β-G), polissacarídeos produzidos por vários organismos diferentes, apresentam propriedades benéficas, como potencial antioxidante, estimulo do sistema imune e proteção do material genético. No presente estudo, β-G sulfatadas e não-sulfatadas extraídas de Agaricus brasiliensis apresentaram no Ensaio do MTT, que avalia um parâmetro de metabolismo, citotoxicidade dose-dependente sendo a sulfatada mais citotóxica, e quando associado à doxorrubicina (DXR), β-G sulfatadas potencializaram a ação do quimioterápico. No Ensaio do Vermelho Neutro, que avalia o potencial de incorporar e armazenar esse corante nos lisossomos por células viáveis, apenas em 24 horas, os tratamentos de 50 e 100 µg/mL de β-G sulfatada apresentaram redução significativa de incorporação, nos demais tempos e concentração não houve diferença, assim como não houve diferença para β-G não-sulfatadas com relação ao controle. Também não apresentaram ação hemolítica ou mutagênica e nem proteção contra danos causados pela DXR nos ensaios de hemólise e cometa, respectivamente. Não houve alteração significativa da proliferação celular em 96 horas para o tratamento com β-G não-sulfatada, e apenas a concentrações de 100 µg/mL de β-G sulfatada após 72 horas apresentou redução significativa de proliferação no ensaio do RTCA. O estudo da expressão demonstrou que ambas β-G, principalmente a sulfatada, não causam danos no DNA como observado no ensaio do genotoxicidade. As β-G não causam estresse às células e na concentração e tempo estudados, não interferem na expressão dos genes de ciclo celular avaliados. Os resultados sugerem que ambas formas de β-G interferem no metabolismo e não causam morte celular, e que β-G sulfatadas possuem maior toxicidade. The β-glucan (β-G), polysaccharides produced by many different organisms, have beneficial properties, such as antioxidant activity, stimulation of the immune system, and protection of the genetic material. In the present study, β-G sulfated and non-sulfated extracted from Agaricus brasiliensis presented in the MTT assay, that evaluates a parameter of metabolism, dose-dependent cytotoxicity being the most cytotoxic sulfated, and when combined with doxorubicin (DXR), sulfated β-G potentiated the action of chemotherapy. In Test of Neutral Red, that assesses the potential to incorporate and store this dye in the lysosomes by viable cells, only at 24 hours, the treatments of 50 and 100 µg/mL of sulfated β-G showed a significant reduction of incorporation, so there was no difference in β-G non-sulfated compared to control. Also showed no hemolytic or mutagenic action nor protection against damage caused by DXR in hemolysis and comet assays, respectively. There was no significant change in cell proliferation at 96 hours for treatment with β-G not-sulfated, and only at concentrations of 100 µg/mL sulfated β-G after 72 hours showed a significant reduction in proliferation assay RTCA. The study showed that expression of both β-G, mainly sulfated, not cause DNA damage as observed in genotoxicity testing. The β-G not cause stress to the cells and studied the concentration and time, do not interfere with the expression of cell cycle genes evaluated. The results suggest that both forms of β-G interfere with the metabolism and do not cause cell death, and sulfated β-G have greater toxicity.