RESUMO
BACKGROUND/AIM: To identify the best of three isatin-based scaffolds in terms of anticancer activity. MATERIALS AND METHODS: Synthesis of isatin-based scaffolds was performed through a reaction to form Schiff bases. In silico analyses consisted of a target prediction with the Swiss Target Prediction tool and a molecular docking by AutoDock Vina. Anticancer activity and cytotoxicity were determined using the WST1 viability assay. RESULTS: Three scaffolds (IA, IB, and IC) were synthesized and confirmed with good reaction yields. The Swiss Target Prediction tool showed a trend towards kinases. Molecular docking assays demonstrated higher affinity of IC towards CDK2. Anticancer activity assays identified IC as the most active against the cancer cell lines. Cytotoxicity results in non-cancer cells suggested a lack of selectivity. CONCLUSION: The scaffold IC was identified as the best in terms of anticancer activity and these effects may be due to inhibition of CDK2, as evidenced by molecular docking.
Assuntos
Antineoplásicos/farmacologia , Quinase 2 Dependente de Ciclina/metabolismo , Isatina/farmacologia , Simulação de Acoplamento Molecular/métodos , Neoplasias/tratamento farmacológico , Bases de Schiff/química , Antineoplásicos/química , Apoptose , Proliferação de Células , Humanos , Isatina/química , Neoplasias/metabolismo , Neoplasias/patologia , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Breast cancer is the most common neoplasm among women but thanks to innovative therapies, patients' prognosis has considerably improved. In this aspect, nanotechnology has been applied for cancer therapy aiming to reduce its usual side effects. In this study we aimed to evaluate the effects of nanoemulsions containing photosensitizer and chemotherapeutic agents associated with photodynamic therapy in a breast cancer in vivo model. Our results showed that synergistic treatments in which chloroaluminum phthalocyanine (NE-Pc) administered together with Doxorubicin (Dox) in the presence of laser irradiation (NE-PcDoxo + PDT) led to a reduction of 4 T1 induced breast cancer in mice, decline of tumor VEGF expression, increase in Caspase-3 expression, tissue necrosis and massive decrease in proliferative cells, as shown by Ki67 immunostaining. Furthermore, this associated treatment induced overexpression of apoptotic genes ABL1, CD70, CRADD, FASL, and NME5 and a reduction in expression of anticancer drug target genes CDK2, ERBB2, FIGF, IGF2, PARP4 and PGR. These results validate this treatment as a promising alternative to improve the currently applied anticancer strategies.
Assuntos
Antineoplásicos/química , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/radioterapia , Doxorrubicina/química , Indóis/química , Nanocápsulas/química , Compostos Organometálicos/química , Fármacos Fotossensibilizantes/química , Animais , Antineoplásicos/farmacologia , Caspase 3/genética , Caspase 3/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Doxorrubicina/farmacologia , Descoberta de Drogas , Quimioterapia Combinada , Emulsões/química , Feminino , Humanos , Indóis/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Experimentais , Compostos Organometálicos/farmacologia , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
INTRODUCTION AND OBJECTIVES: Liver regeneration plays a valuable significance for hepatectomies, and is mainly attributed to hepatocyte proliferation. MicroRNA-125a-3p was reported to be highly associated with liver regeneration process. We studied the underlying mechanism of the functional role of miR-125a-3p in liver regeneration. MATERIALS AND METHODS: The miR-125a-3p mimics and inhibitor vector were constructed and transfected into primary human liver HL-7702 cells, the transfected cell viability was detected using cell counting kit-8 (CCK-8). Cell cycle distribution was analyzed by flow cytometry. With Targetscan and OUGene prediction, the potential targets of miR-125 were verified by real-time quantitative PCR (qPCR) and luciferase reporter assays in turn. The overexpression vector of proline-rich acidic protein 1 (PRAP1) was constructed and co-transfected with miR-125a-3p mimics into HL-7702 cells, detecting the changes of proliferative capacity and cell cycle distribution. Western blot and qPCR performed to analyze gene expressions. RESULTS: Overexpressed miR-125a-3p notably increased the hepatocyte viability at 48h, and decreased the number of G1 phase cells (p<0.05). However, miR-125a-3p inhibition suppressed the development of hepatocytes. PRAP1 was the target of miR-125a-3p. After co-transfection with PRAP1 vector, hepatocyte viability was decrease and the G1 phase cell number was increased (p<0.05). More importantly, overexpressed PRAP1 notably decreased the mRNA and protein levels of cyclin D1, cyclin-dependent kinase 2 (CDK2) and cell division cycle 25A (CDC25A). CONCLUSION: The elevated miR-125a-3p positively correlated with hepatocyte viability and cell cycle progression due to the modulation of PRAP1, and miR-125a-3p may contribute to improving liver regeneration.
Assuntos
Proliferação de Células/genética , Hepatócitos/metabolismo , Regeneração Hepática/genética , Fígado/fisiologia , MicroRNAs/genética , Proteínas da Gravidez/genética , Western Blotting , Ciclo Celular/genética , Linhagem Celular , Sobrevivência Celular/genética , Ciclina D1/genética , Ciclina D1/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Fase G1 , Humanos , Reação em Cadeia da Polimerase , Proteínas da Gravidez/metabolismo , RNA Mensageiro/metabolismo , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismoRESUMO
Cyclin-dependent kinase 2 (CDK2) is an important member of the CDK family exerting its most important function in the regulation of the cell cycle. It catalyzes the transfer of the gamma phosphate group from an ATP (adenosine triphosphate) molecule to a Serine/Threonine residue of a peptide substrate. Due to the importance of this enzyme, and protein kinases in general, a detailed understanding of the reaction mechanism is desired. Thus, in this work the phosphoryl transfer reaction catalyzed by CDK2 was revisited and studied by means of hybrid quantum mechanics/molecular mechanics (QM/MM) calculations. Our results suggest that the base-assisted mechanism is preferred over the substrate-assisted pathway when one Mg2+ is present in the active site, in agreement with a previous theoretical study. The base-assisted mechanism resulted to be dissociative, with a potential energy barrier of 14.3 kcal/mol, very close to the experimental derived value. An interesting feature of the mechanism is the proton transfer from Lys129 to the phosphoryl group at the second transition state, event that could be helping in neutralizing the charge on the phosphoryl group upon the absence of a second Mg2+ ion. Furthermore, important insights into the mechanisms in terms of bond order and charge analysis were provided. These descriptors helped to characterize the synchronicity of bond forming and breaking events, and to characterize charge transfer effects. Local interactions at the active site are key to modulate the charge distribution on the phosphoryl group and therefore alter its reactivity.
Assuntos
Quinase 2 Dependente de Ciclina/química , Quinase 2 Dependente de Ciclina/metabolismo , Modelos Químicos , Modelos Moleculares , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Fosforilação , Conformação Proteica , Teoria Quântica , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The cell cycle comprises a series of steps necessary for cell growth until cell division. The participation of proteins responsible for cell cycle regulation, known as cyclin dependent kinases or Cdks, is necessary for cycle progression. Cyclin dependent kinase 2 (Cdk-2) is one of the most studied Cdks. This kinase regulates the passage through the G1/S phase and is involved in DNA replication in the S phase. Cdks have been extensively studied in mammals, but there is little information about these proteins in crustaceans. In the present work, the nucleotide and amino acid sequence of Cdk-2 from the white shrimp (Cdk-2) and its expression during hypoxia and reoxygenation are reported. Cdk-2 is a highly conserved protein and contains the serine/threonine catalytic domain, an ATP binding site and the PSTAIRE sequence. The predicted Cdk-2 structure showed the two-lobed structure characteristic of kinases. Expression of Cdk-2 was detected in hepatopancreas, gills and muscle, with hepatopancreas having the highest expression during normoxic conditions. Cdk-2 expression was significantly induced after hypoxia for 24â¯h in muscle cells, but in hypoxia exposure for 24 followed by 1â¯h of reoxygenation, the expression levels returned to the levels found in normoxic conditions, suggesting induction of cell cycle progression in muscular cells during hypoxia. No significant changes in expression of Cdk-2 were detected in these conditions in hepatopancreas and gills.
Assuntos
Proteínas de Artrópodes/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Hipóxia/enzimologia , Oxigênio/metabolismo , Penaeidae/enzimologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Sequência de Bases , Quinase 2 Dependente de Ciclina/química , Quinase 2 Dependente de Ciclina/genética , Brânquias/enzimologia , Hepatopâncreas/enzimologia , Músculos/enzimologia , Penaeidae/metabolismo , FilogeniaRESUMO
BACKGROUND: Cyclin-dependent kinase 2 (CDK2) has been studied due to its role in the cell-cycle progression. The elucidation of the CDK2 structure paved the way to investigate the molecular basis for inhibition of this enzyme, with the coordinated efforts combining crystallography with functional studies. OBJECTIVE: Our goal here is to review recent functional and structural studies directed to understanding the role of CDK2 in cancer and senescence. METHODS: There are over four hundreds of crystallographic structures available for CDK2, many of them with binding affinity information. We use this abundance of data to analyze the essential features responsible for the inhibition of CDK2 and its function in cancer and senescence. RESULTS: The structural and affinity data available CDK2 makes it possible to have a clear view of the vital CDK2 residues involved in molecular recognition. A detailed description of the structural basis for ligand binding is of pivotal importance in the design of CDK2 inhibitors. Our analysis shows the relevance of the residues Leu 83 and Asp 86 for binding affinity. The recent findings revealing the participation of CDK2 inhibition in senescence open the possibility to explore the richness of structural and affinity data for a new era in the development of CDK2 inhibitors, targeting cellular senescence. CONCLUSION: Here, we analyzed structural information for CDK2 in combination with inhibitors and mapped the molecular aspects behind the strongest CDK2 inhibitors for which structures and ligandbinding affinity data were available. From this analysis, we identified the significant intermolecular interactions responsible for binding affinity. This knowledge may guide the future development of CDK2 inhibitors targeting cancer and cellular senescence.
Assuntos
Quinase 2 Dependente de Ciclina/química , Quinase 2 Dependente de Ciclina/metabolismo , Neoplasias/enzimologia , Asparagina/metabolismo , Sítios de Ligação , Senescência Celular , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Leucina/metabolismo , Modelos Moleculares , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Hexachlorobenzene (HCB) is an organochlorine pollutant widely distributed in the environment around the entire world. Previous reports from our group and others have demonstrated that this compound is as an endocrine disruptor. We have also reported that HCB presents a co-carcinogenic effect in N-Nitroso-N-methyl-urea-induced mammary tumours in rats. In this work, we studied the effects of HCB on cell cycle progression and cell cycle regulating protein expression in the estrogen-sensitive breast cancer cell line, MCF-7. Here, we show that HCB alters cell cycle in a concentration-dependent way. The lowest assessed concentration (0.005µM) promotes the cell cycle progression, enhances cyclin D1 expression, and reduces the nuclear localization of p27 accompanied by an increased interaction between p27 and c-Src kinase. On the other hand, 5µM HCB delays the cell cycle progression and promotes the formation of the cyclin E-CDK2-p27 protein complex. Our results show that HCB stimulates cell proliferation through cell cycle modulation and c-Src involvement in MCF-7 cells. Here, we report for the first time that differential mechanisms of action of HCB on mammary cell cycle progression are triggered at different concentrations of this pollutant.
Assuntos
Ciclo Celular/efeitos dos fármacos , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Hexaclorobenzeno/toxicidade , Proteínas Oncogênicas/metabolismo , Quinases da Família src/metabolismo , Proteína Tirosina Quinase CSK , Divisão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclina D1/genética , Ciclina D1/metabolismo , Ciclina E/genética , Quinase 2 Dependente de Ciclina/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Humanos , Células MCF-7 , Proteínas Oncogênicas/genética , Fosforilação , Quinases da Família src/genéticaRESUMO
The toxicologic effects of copper (Cu) on tumor cells have been studied during the past decades, and it is suggested that Cu ion may trigger antiproliferative effects in vitro. However, in normal cells the toxicologic effects of high exposures of free Cu are not well understood. In this work, Cu uptake, the expression of genes associated with cell cycle regulation, and the levels of ROS production and related oxidative processes were evaluated in Cu-treated mammary epithelial MCF10A nontumoral cells. We have shown that the Cu additive is associated with the activation of cyclin D1 and cyclin B1, as well as cyclin-dependent kinase 2 (CDK2). These nontumor cells respond to Cu-induced changes in the oxidative balance by increase of the levels of reduced intracellular glutathione (GSH), decrease of reactive oxygen species (ROS) generation, and accumulation during progression of the cell cycle, thus preventing the cell abnormal proliferation or death. Taken together, our findings revealed an effect that contributes to prevent a possible damage of normal cells exposed to chemotherapeutic effects of drugs containing the Cu ion.
Assuntos
Antioxidantes/metabolismo , Cobre/metabolismo , Cobre/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Ciclina B1/metabolismo , Ciclina D1/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Humanos , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Regulação para Cima/efeitos dos fármacosRESUMO
Binding recognition is in the core of how nature controls processes in living cells, how enzyme-substrate binding leads to catalysis and how drugs modulate enzymes and receptors to convey a desirable physiological response. Thus, understanding binding recognition in a systematic manner is paramount, not only to understand biological processes but also to be able to design and discover new bioactive compounds. One such way to analyze binding interactions is through the development of binding interaction fingerprints. Here, we present the methodology to develop interaction fingerprints with three different software platforms along with two representative examples.
Assuntos
Mapeamento de Interação de Proteínas/métodos , Quinase 2 Dependente de Ciclina/metabolismo , Mapas de Interação de Proteínas , Receptores Opioides/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Casearia sylvestris is a tree found in tropical America. In Brazil it is known mainly as Guaçatonga. Literature reports suggest that the leaves and other plant parts have been used by indigenous populations from South America in preparations, mainly aqueous or hydroethanolic macerations or decoctions, most times taken orally for the primary treatment of several diseases, including cancer. AIM OF THE STUDY: This article reports the results of an investigation about the antiproliferative effects of Casearia sylvestris on tumor cells in vitro and in vivo. MATERIAL AND METHODS: Aqueous ethanolic maceration and column chromatography were done to obtain a crude aqueous ethanolic extract (CAE) and a chloroform fraction (f-CHCl3). The human breast cancer cell line MCF-7 was used in culture. In vitro, non-cytotoxic concentrations were determined by MTT assay and the antiproliferative effect was assessed by the colony forming unit assay using non-cytotoxic concentrations. Effects on the cell cycle were observed through flow cytometry using a propidium iodide kit. Casearin C was identified in f-CHCl3 by chromatography and H(1) nuclear magnetic resonance. The effect on some key proteins of DNA damage (phosphorylation on the histone H2AX) and cell cycle control (p53, p16, cdk2) was evaluated through immunoblot. Antiproliferative effects in vivo were measured in tumor tissue from Ehrlich ascites-bearing mice through the (3)H-thymidine uptake assay and the trypan blue exclusion method. RESULTS: In vitro, EC50 values found at 24 h on MCF-7 cells were 141 µg/mL for CAE and 66 µg/mL for f-CHCl3. Inhibition on proliferation was recorded at concentrations as low as 4 µg/mL in the case of the f-CHCl3 (up to 40%) and up to 50% when CAE was added at 9 µg/mL. The cell cycle arrest was demonstrated by the reduction in terms of number of cells in phases G2/M and S, up to 38.9% and 51.9% when cells were treated with CAE, and 53.9% and 66.2%, respectively, when cells were treated with f-CHCl3. The number of cells in G1 was increased when the cells were treated with CAE (21.4%) or f-CHCl3 (27.8%). Key proteins of cell cycle control were affected. The treatments caused activation of p53, p16 and DNA damage found by the appearance of bands corresponding to γ-H2AX. The treatments caused inhibition of cdk2. CAE and particularly f-CHCl3 caused significant inhibition on tumor growth in mice (40% and 60%, respectively). Uptake of (3)H-thymidine, thus proliferation was reduced in tumor cells from mice treated with CAE (>30%) or f-CHCl3 (up to 50%) compared to cells from control animals. Data from the trypan blue assay indicating a lower number of tumor cells in treated animals. From the overall, data from this study are in line with the traditional claims for the antitumor effect of Casearia sylvestris. CONCLUSIONS: This investigation suggests that whether the extracts from Casearia sylvestris are cytotoxic at high concentrations, lower concentrations have antiproliferative effect and could be useful to complement conventional cytotoxic chemotherapy, and should be evaluated further.