Search

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.

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.

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.

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.

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.

Soybean isoflavonoids have received significant attention due to their potential anticarcinogenic and antiproliferative effects and possible role in many signal transduction pathways. However, their mechanisms of action and their molecular targets remain to be further elucidated. In this paper, we demonstrated that two soybean isoflavones (genistein and daidzein) reduced the proliferation of the human colon adenocarcinoma grade II cell line (HT-29) at concentrations of 25 and 50-100 μM, respectively. We then investigated the effects of genistein and daidzein by RT-PCR on molecules that involved in tumor development and progression by their regulation of cell proliferation. At a concentration of 50 μM genistein, there was suppressed expression of β-catenin (CTNNBIP1). Neither genistein nor daidzein affected APC (adenomatous polyposis coli) or survivin (BIRC5) expression when cells were treated with concentrations of 10 or 50 μM. These data suggest that the down-regulation of β-catenin by genistein may constitute an important determinant of the suppression of HT-29 cell growth and may be exploited for the prevention and treatment of colon cancer.

The installation of oxidative process arises from an imbalance between oxidants and antioxidants compounds, in favor of excessive generation of free radicals. This process can affect cellular components, like endoplasmic reticulum (ER). The ER is extremely sensitive to changes in homeostasis, where, on different stimuli, may result in adaptation to survival or induction of apoptosis (unfolded protein response, “UPR” pathway). The oxidative damage caused by endogenous or exogenous agents or a dysregulation of the UPR pathway can lead to adverse conditions, whose chronicity has important implications for the etiology of chronic diseases, including cancer. Therefore, it becomes important to search for chemoprotective agents aiming their role in preventive medicine. Between these substances, selenium’s antioxidant activity seems to be effective in treating diseases which have the oxidative stress as its development. We evaluated the modulating action of l-(+)-selenomethionine (SeMet) in HepG2 cells against cellular stress induced by H2O2 through MTT assay, comet assay, and gene expression by qRT-PCR of genes related to oxidative stress, UPR pathway, and apoptosis. In MTT assay, the lower concentrations of SeMet showed a cytoprotective action against the damage caused by H2O2. Likewise, it was verified in the comet assay that the concentration of 50 ng/mL reduced the genotoxic damage caused by H2O2. SeMet at 50 ng/mL regulated the genes tested in the qRT-PCR, showing an antiapoptotic and antioxidant effect. These results suggest that SeMet positively modulates the genes of oxidative stress and ER, leading to a chemoprotective and antioxidant effect, becoming an alternative in preventive medicine.

The identification of antitumoral substances is the focus of intense biomedical research. Two structural analogues of thalidomide, LNO3 and L3, are two synthetic compounds that might possess such antitumor properties. We evaluated the toxicological effects of these substances, including cytotoxicity, genotoxicity and induction of apoptosis in HTC cells. Additionally, the production of free radicals (nitric oxide and superoxide) was investigated, and the expression of caspases genes 3, 8, and 9 were determined by RT-qPCR. The compounds exhibited cytotoxic effects that resulted in inhibited cell proliferation. LNO3 showed to be more effective and toxic than L3 in all assays. LNO3 stimulated the release of NO and superoxide, which was accompanied by the formation of peroxynitrite. Apoptosis was induced in a dose-dependent manner by both compounds; however, the expression of caspases 3, 8 and 9 was unchanged. These results suggested that L3 and LNO3 possess antiproliferative and pro-apoptotic effects in HTC cells. Additionally, although they exhibited cytotoxicity, L3 and LNO3 might be useful coadjuvants in tumor treatment studies.

Isoflavones are phenolic compounds widely distributed in plants and found in a high percentage in soybeans. They have important biological properties and are regarded as potential chemopreventive agents. The aim of this study was to verify the preventive effect of two soy isoflavones (genistein and daidzein) by a micronucleus assay, analysis of GST activity, and real-time RT-PCR analysis of GSTa2 gene expression. Mutagens of direct (doxorubicin) and indirect (2-aminoanthracene) DNA damage were used. Hepatoma cells (HTC) were treated with genistein or daidzein for 26 h at noncytotoxic concentrations; 10 μM when alone, and 0.1, 1.0 and 10 μM when combined with genotoxic agents. The micronucleus test demonstrated that both isoflavones alone had no genotoxic effect. Genistein showed antimutagenic effects at 10 μM with both direct and indirect DNA damage agents. On phase II enzyme regulation, the current study indicated an increase in total cytoplasmic GST activity in response to genistein and daidzein at 10 μM supplementation. However, the mRNA levels of GSTa2 isozymes were not differentially modulated by genistein or daidzein. The results point to an in vitro antimutagenic activity of genistein against direct and indirect DNA damage-induced mutagenicity.

Genisteína e daidzeína, as principais isoflavonas de soja, são moléculas de baixo peso molecular com amplo espectro de atividades biológicas. Muitos estudos sugerem que estas isoflavonas tem ação protetora contra várias doenças crônicas incluindo câncer. Estas moléculas podem exercer seus efeitos através de múltiplos mecanismos que entre outros destacam-se: atividade antioxidante, inibição de crescimento de células neoplásicas, regulação do ciclo celular e apoptose, modulação de enzimas do metabolismo de xenobióticos (fase I e II) e modulação de várias vias de sinalização celular. No presente trabalho avaliamos os efeitos das isoflavonas genisteína e daidzeína sobre três linhagens de células tumorais: hepatoma de rato (HTC), hepatoma humano (HepG2) e carcinoma de cólon humano (HT29). Os parâmetros avaliados tiveram foco sobre atividade antiproliferativa (HTC, HepG2 e HT29), efeitos genotóxicos e antigenotóxicos (HTC), atividade sobre modulação de enzimas do metabolismo de xenobióticos (HTC, HepG2) e efeito sobre a via de sinalização celular Wnt/β-catenina (HT29). Os principais resultados obtidos no presente trabalho foram: (i) Em HTC, o ensaio do micronúcleo mostrou que nenhuma das isoflavonas utilizadas teve efeito genotóxico. Genisteína a 10 μM apresentou efeito antimutagênico quando associada aos agentes de danos ao DNA de ação direta (DXR) e indireta (2AA). Os níveis de GST mRNA não foram diferencialmente modulados por genisteína ou daidzeína. (ii) Em HepG2, genisteína e daidzeína (10 a 100 μM) inibiram crescimento celular de forma dose e tempo dependente. Resultados através de PCR em tempo real mostraram que a suplementação com 50 μM de genisteína causou uma redução na expressão de CYP1A1 enquanto a daidzeína promoveu aumento de expressão nas duas concentrações testadas (10 e 50 μM). Os níveis de GST mRNA não foram diferencialmente modulados por genisteína ou daidzeína. (iii) Em HT29, genisteína inibiu proliferação celular em concentrações de 25 a 100 μM. Daidzeína somente inibiu proliferação na concentração mais alta (100 μM). Genisteína (50 μM) reduziu significativamente a expressão do gene CTNNBIP1(β-catenin) e não interferiu na expressão de APC (Adenomatous polyposis coli) ou BIRC5 (survivina). Daidzeina não interferiu na expressão dos genes CTNNBIP1(β-catenin), APC (Adenomatous polyposis coli) ou BIRC5 (survivina) em nenhuma concentração testada. Estes estudos indicam que especialmente a genisteína apresenta grande potencial a ser explorado para tratamento e prevenção de câncer. Genistein and daidzein, the soy isoflavones, are low molecular weight molecules with a wide spectrum of biological activities. Studies suggest that these isoflavones have protective action against chronic diseases including cancer. These molecules may exert effects through multiple mechanisms as antioxidant activity, inhibition of growth of neoplastic cells, cell cycle regulation and apoptosis, the modulation of xenobiotic metabolizing enzymes (phase I and II) and modulation signaling pathways. In this study we evaluated the effects of isoflavones genistein and daidzein on three tumor cell lines: rat hepatoma (HTC), human hepatoma (HepG2) and human colon carcinoma (HT29). The parameters studied had focused on antiproliferative activity (HTC, HepG2 and HT29), genotoxic and antigenotoxic effects (HTC), modulation of xenobiotic metabolism enzymes (HTC, HepG2) and effect on the signaling pathway Wnt / β - catenin (HT29). The main results obtained in this study were: (i) in HTC, the micronucleus assay showed antimutagenic effect of genistein at 10 μM when associated with DNA damage direct (DXR) and indirect (2AA) agents. In the regulation of enzymes of phase II metabolism, the study indicated an increase in total GST activity in response to genistein and daidzein at 10 μM. GST mRNA levels were not differentially modulated by genistein or daidzein. (ii) in HepG2, genistein and daidzein (10 to 100 μM) inhibited cell growth in a dose and time dependent. Real-time PCR showed that 50 μM of genistein caused a reduction in the expression of CYP1A1 while daidzein increased expression at 10 and 50 μM. GST mRNA levels were not differentially modulated by genistein or daidzein. (iii) in HT29, genistein inhibited cell proliferation at concentrations 25 to 100 μM. Daidzein inhibited proliferation only at the highest concentration (100 μM). Genistein (50 μM) significantly reduced gene expression CTNNBIP1 (β-catenin) and did not affect the expression of APC (Adenomatous polyposis coli) or BIRC5 (survivin). Daidzein did not affect the expression of genes CTNNBIP1 (β-catenin), APC (Adenomatous polyposis coli) or BIRC5 (survivin) at any concentration tested. These studies indicate that genistein has especially great potential to be exploited for cancer treatment and prevention.

Complexos de cobre (II) são promissores candidatos para o desenvolvimento de novos tratamentos para diversos tipos de câncer, incluindo o câncer de mama. O presente trabalho avaliou a atividade antitumoral in vitro e os efeitos toxicogenéticos in vivo de um novo complexo de cobre (II). O composto Bis(((Z)-4-((4-chlorophenil)amino)-4- oxobut-2-enoil)oxi)cobre (RC1) foi sintetizado e posteriormente utilizado nos ensaios biológicos. Um total de 2,5x104 células 4T1 foram plaqueadas e submetidas à tratamento com as concentrações de 3,125; 6,25; 12,5; 25; 50; 100; 250; 500 e 1000 μg/mL do composto RC1, durante 24, 48 e 72 horas. A partir dos resultados de citotoxicidade a IC50 do composto RC1 foi calculada e utilizada nos demais ensaios in vitro. Para análise da genotoxicidade utilizou-se o ensaio do cometa e para análise do tipo de morte celular a análise morfológica com coloração diferencial de brometo de etídio e acridina, e a marcação em citometro de fluxo com kit de detecção de apoptose PE Anexina V. Ainda por citometria foi analisada a integridade da membrana das células 4T1, bem como o ciclo celular, após 24 horas de tratamento com o composto RC1. O ensaio de expressão gênica foi realizado por qPCR e genes relacionados à danos, reparo do DNA e apoptose foram estudados. In vivo o potencial toxicogenético foi avaliado em camundongos Swiss com as concentrações de 3, 6 e 12mg/kg do composto RC1 por meio dos ensaios de cometa, micronúcleo e fagocitose. Os resultados demonstraram que o composto RC1 induziu citotoxicidade nas células 4T1, possivelmente, via danos no DNA que levara ao aumento da expressão de ATM e p21, induzindo parada de ciclo celular em G1. O mecanismo de morte celular do RC1 em células 4T1 foi a apoptose desencadeado pelo aumento na expressão de BAX e CASP- 7, sem alterar a integridade de membrana das células 4T1. In vivo, o composto foi genotóxico em camundongos e causou aumento da frequência de danos genômicos (cometa), mas não cromossômicos (micronúcleo), no DNA e quando comparado ao quimioterápico de referência, cisplatina, foi menos genotóxico. O RC1 também foi capaz de aumentar a frequência de fagocitose esplênica. Esses resultados indicam que o novo complexo de cobre (II), descrito pioneiramente neste trabalho, apresenta potencial terapêutico no tratamento do câncer de mama. Copper (II) complexes are promising candidates for the development of new treatments for various types of cancer, including breast cancer. The present paper reported on the evaluation of the in vitro antitumor activity and toxicogenic effects in vivo of a new copper (II) complex. The Bis(((Z)-4-((4-chlorophenyl)amino)-4-oxobut-2- enoyl)oxy)copper (RC1) compound was synthesized and subsequently used in the biological assays. A total of 2.5 x 104 of 4T1 cells were plated and treated with the RC1 compost at concentrations of 3.125; 6.25; 12.5; 25; 50; 100; 250; 500 and 1000 μg/mL for 24, 48 and 72 hours. The IC50 of RC1 compound was calculated from the cytotoxicity results and used in the others in vitro assays. The comet assay was the method employed for analysis of the genotoxicity, as for the identification of the type of cell death, the method was the morphological analysis with differential staining of bromide and acridine, and the marking in flow cytometry with apoptosis detection kit PE Annexin V. 4T1 cells membrane integrity and the cell cycle, was analyzed after 24 hours of treatment with compound RC1 by flow cytometry. The gene expression assay was performed by qPCR and genes related to DNA-damage, DNA repair and apoptosis were studied. The in vivo toxicogenic potential was evaluated in Swiss mice with the concentrations of 3, 6 and 12mg/kg of RC1 compound RC1 by the comet, micronucleus and phagocytosis assays. The results demonstrated that RC1 compound induced cytotoxicity in 4T1 cells, which appears to occur because of DNA damage, which increased ATM and p21 expression, inducing cell cycle arrest in G1. The mechanism of cellular death of RC1 in 4T1 cells was apoptosis, that occurred by the increase in expression of BAX and CASP-7, without altering the membrane integrity of 4T1 cells. In in vivo, the compound was genotoxic in mice and caused an increase in the frequency of genomic damage (comet), but not chromosome (micronucleus), and when compared to the reference chemotherapy, cisplatin, it was less genotoxic. RC1 was also able to increase the frequency of splenic phagocytosis. These results indicate that the new copper (II) complex, firstly described in this work, presents therapeutic potential in the treatment of breast cancer.

A busca por compostos a partir de fontes naturais, como os fitoquímicos, tem crescido, já que podem ser uma nova abordagem no tratamento e prevenção do câncer. Assim, avaliamos o modo de ação dos alcaloides harringtonina (HT) e homoharringtonina (HHT) em linhagens celulares de hepatócitos humano p53 selvagem (HepG2/C3A) e mutante (HuH-7.5), realizando testes de viabilidade celular , citometria de fluxo para apoptose, ciclo celular e integridade de membrana, genotoxicidade e expressão relativa de mRNA para genes de apoptose, ciclo celular, metabolização de xenobióticos, estresse de retículo e de controle de proliferação.HT e HHT mostraram aumento significativo na indução de apoptose inicial nas duas linhagens, além alterarem o ciclo celular com aumento significativode células em G2/M nas duas linhagens quando tratadas com HHT e aumento significativo de células em S na linhagem C3A quando tratadas com HT. A expressão relativa dos genes mostrou um aumento de genes indutores de apoptose como TNF, BBC3 e redução em BCL2 e BAK, mostrando que os compostos induzem a morte celular pela apoptose. Genes metabolizadores mostraram-se alterados quando tratados com os compostos, onde houve aumento de CYP2E1, CYP2C19 e CYP3A4, sendo este ultimo apenas na linhagem HuH-7.. Expressões relativas de genes de estresse de retículo, ERN1 e EIF2AK3 mostraram aumenta significativamente nos tratamentos com HT e HHT, que podem estar ligadas com a ação principal desses compostos relacionada a inibição da expressão proteica nas células . O estresse de retículo também pode causar aumento de outros genes, como o TRAF2, que pôde ser visto aumentado neste estudo em ambos os tratamentos, como também induzem fatores de necrose tumoral correlacionados com o aumento observado para o gene TNF. Desse modo, sugerimos que os compostos induzem o estresse de retículo pela alteração proteica causando morte celular pelas vias intrínseca e extrínseca atribuído ao aumento de BBC3 e TNF respectivamente. The search for compounds from natural sources, such as phytochemicals, has grown, as they may be a new approach in the treatment and prevention of cancer. Thus, we evaluated the mode of action of the harringtonine (HT) and homoharringtonine (HHT) alkaloids in wild-type (HepG2 / C3A) and mutant (HuH-7.5) human hepatocyte cell lines, performing cell viability tests, flow cytometry for apoptosis , cell cycle and membrane integrity, genotoxicity and relative mRNA expression for apoptosis, cell cycle, xenobiotic metabolism, reticulum stress and proliferation control. HT and HHT showed a significant increase in the induction of initial apoptosis in both strains, in addition to altering the cell cycle with significant increase of G2 / M cells in the two strains when treated with HHT and significant increase of S cells in the C3A line when treated with HT. The relative expression of the genes showed an increase in apoptosis inducing genes such as TNF, BBC3 and reduction in BCL2 and BAK, showing that the compounds induce cell death by apoptosis. Metabolizing genes were altered when treated with the compounds, where CYP2E1, CYP2C19 and CYP3A4 increased, the latter being only in the HuH-7 lineage. Relative expressions of reticulum, ERN1 and EIF2AK3 stress genes showed a significant increase in treatments with HT and HHT, which may be linked to the principal action of such compounds related to inhibition of protein expression in cells. Reticulum stress may also cause increase in other genes, such as TRAF2, which could be seen increased in this study in both treatments, as well as induce tumor necrosis factors correlated with the observed increase for the TNF gene. Thus, we suggest that the compounds induce the reticulum stress by the protein alteration causing cell death by the intrinsic and extrinsic pathways attributed to the increase of BBC3 and TNF respectively.

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.

Espécies Reativas de Oxigênio (EROs) estão envolvidas em vários processos patológicos. Esses efeitos são minimizados no organismo por compostos com atividade antioxidante como o Selênio. O Selenito de Sódio (SS) é uma forma inorgânica de selênio e sua capacidade protetora precisa ser melhor esclarecida. Por isso, o estudo investigou o efeito do SS contra o peroxido de hidrogênio (H2O2) em células HepG2/C3A analisando citotoxicidade e proliferação celular comparando com dados molecular (mRNAs e proteínas). O SS apresentou atividade citoprotetora e antigenotóxica, entretanto, não foi observado alteração na curva de proliferação celular e na alteração das fases de ciclo celular (retenção em G2/M) nos tratamentos associados (SS+ H2O2) quando comparado com H2O2. O SS modulou o sistema de defesa antioxidante nas células induzidas ao estresse oxidativo pelo H2O2, onde observou-se um aumento da expressão relativa do mRNA GPX1 no tratamento associado mostrando resposta antioxidante. Além disso, alterações do mRNA CAT e proteína SOD1 e PRX2 pelo H2O2, foram minimizadas pelo tratamento associado também. Observou-se normalização dos níveis do mRNA CCN2B nos tratamentos associados, entretanto, esse efeito não foi evidenciado na expressão dos outros genes controladores de ciclo celular, onde observou-se aumento na expressão dos mRNAs CDKN1A, CDKN1C, CDKN2B e GADD45A nos tratamentos com H2O2 independente de SS. Desta maneira, nossos resultados demonstram que a proteção do SS se deve pela modulação do sistema de defesa antioxidante, entretanto este efeito não foi eficiente ao ponto de modular a expressão de genes que controlam o ciclo celular impedindo a continuidade do crescimento celular. Reactive Oxygen Species (ROS) are involved in various pathological processes. These effects are minimized in the body by compounds with antioxidant activity such as Selenium. Sodium selenite (SS) is an inorganic form of selenium and its cytoprotective and antioxidant capacity need to be clarified. Therefore, the study investigated the effect of SS against hydrogen peroxide (H2O2) in HepG2/C3A cells analyzing cytotoxicity and cell proliferation compared with molecular data (mRNA and protein). The SS showed antigenotoxic and cytoprotective activity, however, was not observed change in cell proliferation curve and change of cell cycle phases (G2/M arrest) in the associated treatments (SS+H2O2) when compared with H2O2. The SS modulate the antioxidant defense system in cells to oxidative stress induced by H2O2, where there was an increase in the relative expression GPX1 mRNA in the associated treatment showing antioxidant response. In addition, changes in the CAT mRNA and SOD1 and PRX2 protein H2O2 were also minimized by the associated treatment too. There was normalization of the levels of CCN2B mRNA in the associated treatments, however, this effect was not observed in the expression of other cell cycle controlling genes, which increase was observed in the expression of CDKN1A, CDKN1C, CDKN2B and GADD45A mRNAs in treatments with H2O2 independent of SS. Thus, our results show that SS protection is provided by modulation of antioxidant defense system, however this effect was not efficient to the point of modulating the expression of genes that control cell cycle blocking the continuity of cell growth.

O câncer de mama é a neoplasia que mais acomete as mulheres de todo mundo e a que representa as maiores taxas de mortalidade nesse grupo. A maioria das drogas antineoplásicas atuais não apresentam seletividade e, portanto, afetam tanto as células tumorais quanto as saudáveis. Dessa forma, a busca por novas drogas anticâncer é de grande interesse. O monastrol é uma molécula sintética que atua como inibidor alostérico da cinesina mitótica Eg5. Estudos mostram que esta cinesina está superexpressa em células tumorais quando comparadas com células normais. Dessa forma, o objetivo deste trabalho foi investigar o efeito antiproliferativo do monastrol em célula humana tumoral de adenocarcinoma mamário (MCF-7) e não tumoral de epitélio de mama (HB4a). As células MCF-7 e HB4a foram expostas ao monastrol nas concentrações de 5, 25, 50, 75 e 100 µM para a análise de citotoxicidade (ensaio MTT) e proliferação celular (Real-time Cell Analyzer); e nas concentrações de 25 e 50 µM para os demais testes. Foram avaliadas por citometria de fluxo a dinâmica do ciclo celular (Iodeto de propídeo) e indução de apoptose (Anexina V), além da obtenção de índice mitótico e apoptótico por análise morfológica com coloração de Hoechst 33342. Os níveis de mRNA de genes do ciclo celular (TP53, EG5, CCNA2, CCNB1, CCND1, CCNE1, CDC25A, CDC25C, CDKN1A, CDKN1B e CDKN1C), sinalizador de danos (GADD45A) e apoptose (BAX, BAK, BCL2 e BCLXL) foram verificados por RT-qPCR. No ensaio de citotoxicidade, o monastrol reduziu a viabilidade em relação ao controle apenas na linhagem tumoral MCF-7 a partir da concentração de 50 µM. Na avaliação da proliferação celular em tempo real também houve uma queda na proliferação das células MCF-7, enquanto as células HB4a não apresentaram alterações na cinética de crescimento. A análise da apoptose mostrou que o monastrol não induz morte celular por apoptose em nenhuma das linhagens celulares. Já na análise do ciclo celular, a exposição das células MCF-7 ao monastrol (50 µM) levou ao aumento da população G1 e G2/M com concomitante diminuição de S, enquanto na linhagem celular HB4a houve aumento de G2/M e diminuição de G1. Ambas células apresentaram aumento dos níveis de índice mitótico. A análise da expressão gênica demonstrou que o monastrol aumentou em 5 vezes os níveis de RNAm do gene CDKN1A nas células MCF-7 e reduziu em 2 vezes os níveis de RNAm de CDKN1C na linhagem HB4a. Estes resultados sugerem que o monastrol possui maior citotoxicidade sobre as células tumorais MCF-7, quando comparados com as células não tumorais HB4a., tornando-o um possível quimioterápico anticâncer. Breast cancer is the cancer that affects more women around the world and that is the highest mortality rates in this group. Most current anticancer drugs do not exhibit selectivity, and therefore affect both the tumor cells and healthy ones. Thus, the search for new anticancer drugs is of great interest. The monastrol is a synthetic molecule that acts as an allosteric inhibitor of the mitotic kinesin Eg5. Studies show that this kinesin is overexpressed in tumor cells when compared to normal cells. Thus, the objective of this study was to investigate the antiproliferative effect of monastrol in human tumor cell breast adenocarcinoma (MCF-7) and non-tumor breast epithelium (HB4a). MCF-7 and HB4a cells were exposed to monastrol in concentrations of 5, 25, 50, 75 and 100 µM for the cytotoxicity assay (MTT assay) and cell proliferation (Real-Time Cell Analyzer); and at concentrations of 25 and 50 µM for the other tests. In flow cytometry are assessed dynamics of the cell cycle (propidium iodide) and induction of apoptosis (Annexin V), as well as for morphological analysis obtaining mitotic and apoptotic index by staining with Hoechst 33342. The mRNA levels of cell cycle gene (P53, EG5, CCNA2, CCNB1, CCND1, CCNE1, CDC25A, CDC25C, CDKN1A, CDKN1B and CDKN1C), the damage indicator (GADD45A) and apoptosis (BAX, BAK, BCL2 and BCLXL) were verified by RT-qPCR. In the cytotoxicity assay, monastrol reduced viability only in MCF-7 tumor, compared to the control, from the concentration of 50 uM. In real-time cell analyzer was also a decrease in the proliferation of MCF-7 cells, while the HB4a curve showed no change in growth kinetics. Monastrol does not induce cell death by apoptosis in any of the cell lines. In the analysis of the cell cycle, monastrol (50 µM) increased the population of MCF-7 cells in G1 and G2 / M with a concomitant decrease in S, whereas the cell line HB4a an increase of the G2 / M population and decrease of the G1. Both cells showed increases in mitotic index levels. The gene expression analysis showed that monastrol increased 5 fold the levels of mRNA CDKN1A gene in MCF-7 cells and reduced the mRNA levels CDKN1C in lineage HB4a by 2-fold. These results suggest that monastrol has higher cytotoxicity on MCF-7 tumor cells compared to non-tumor cells HB4a., making it possible anticancer chemotherapy.