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1.
PLoS One ; 15(10): e0240754, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33104743

RESUMO

Effects of aripiprazole on dopamine regulation are being tested as a treatment for patients with a dual diagnosis of schizophrenia and addictions, often cocaine dependence. Aripiprazole has one of the fewest side-effects among the second-generation antipsychotics. Nevertheless, severe aripiprazole hepatotoxicity was reported in persons with a history of cocaine and alcohol abuse. Here we report that therapeutically relevant aripiprazole concentrations, equal to laboratory alert levels in patients' serum, reduce the rate of hepatocytes' division. This could be an underlying mechanism of severe liver injury development in the patients with a history of alcohol and cocaine abuse, the two hepatotoxic agents that require increased ability of liver self-regeneration. Monitoring liver functions is, therefore, important in the cases when aripiprazole is co-prescribed or used with drugs with potential hepatotoxic effects.


Assuntos
Aripiprazol/farmacologia , Divisão Celular/efeitos dos fármacos , Fígado/citologia , Animais , Contagem de Células , Divisão Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Senescência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Ratos
2.
Exp Hematol ; 91: 39-45.e2, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32961298

RESUMO

Mutations in the gene encoding DNA methyltransferase 3A (DNMT3A) comprise the majority of mutations found in clonal hematopoiesis (CH), an age-related condition that was recently found to affect outcomes in patients undergoing hematopoietic stem cell transplant (HSCT). Recent studies have indicated that patients with CH have worse prognoses after HSCT, suggesting stress imposed by HSCT preconditioning agents may impact hematopoietic stem cell (HSC) dynamics in transplant recipients. In this study, we used a competitive transplantation mouse model to investigate how treatment with the common preconditioning agents 5-fluorouracil (5-FU) and busulfan (BU) affect the prevalence of Dnmt3a-/- HSCs and progenitor cells in competition with wild-type cells. We found that, though sufficient to deplete peripheral blood counts, 5-FU preconditioning did not significantly alter the frequency of Dnmt3a-null hematopoietic stem and progenitor cells (HSPCs) in mosaic mice. In contrast, mice treated with BU had a sevenfold decline in total bone marrow cells and an increase in Dnmt3a-null HSPCs that was detectable in peripheral blood. Indeed, even though all mosaic mice had a starting engraftment of ∼10%-40%, 85%-100% of HSPCs were Dnmt3a-null in four of seven mice after BU treatment, indicating these cells expand dramatically during recovery. Overall, these results suggest that individual preconditioning regimens have different effects on the expansion of Dnmt3a-mutant cells in patients with pre-existing CH. Thus, the presence of CH-associated mutants should be evaluated prior to selecting preconditioning regimens for HSCT.


Assuntos
Bussulfano/farmacologia , DNA (Citosina-5-)-Metiltransferases/deficiência , Hematopoese/genética , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Animais , Medula Óssea/patologia , Divisão Celular/efeitos dos fármacos , Linhagem da Célula , Células Clonais , Fluoruracila/farmacologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Camundongos , Quimera por Radiação
3.
Anticancer Res ; 40(10): 5517-5527, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988875

RESUMO

BACKGROUND/AIM: Drug resistance is a significant cause of high mortality in ovarian cancer (OC) patients. The reverse transcriptase inhibitor azidothymidine (AZT) has been utilized as a treatment for tumors, but its role in OC treatment has not been revealed. The aim of the present in vitro study was to examine the influence of AZT on the growth of human OC cells and the involved proteins. MATERIALS AND METHODS: The proliferation, cell cycle distribution, extent of apoptosis, mitotic index, and terminal restriction fragment length were examined in three OC cell lines, CaOV3, TOV112D, and TOV21G, treated with AZT. RESULTS: AZT inhibited growth of the TOV21G and CaOV3 cell lines by regulating cell cycle distribution. Specifically, AZT caused G2/M phase arrest on TOV21G cells and S phase arrest on CaOV3 cells. In addition, AZT treatment induced up-regulation of p21 and p16 in the TOV21G and CaOV3 cell line, respectively. CONCLUSION: AZT inhibited cell proliferation in serous and clear cell OC via the regulation of cell cycle distribution.


Assuntos
Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Zidovudina/farmacologia , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia
4.
J Biosci Bioeng ; 130(5): 525-532, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32800439

RESUMO

Chinese hamster ovary (CHO) cells are used as host cells for biopharmaceutical production, including monoclonal antibodies (mAbs). Arresting the cell cycle with chemical compounds is an effective approach to improve biopharmaceutical productivity. In a previous study, potential new cell cycle-arresting compounds were screened from marine-derived microorganism culture extracts, and it was suggested that staurosporine might improve mAb productivity in CHO cells via cell cycle arrest. The purpose of this study was to demonstrate the effectiveness of staurosporine as a cell-cycle arresting compound to improve mAb productivity. The optimal staurosporine concentration range was initially investigated using batch cultures. Thereafter, the effects on the culture profile and mAb productivity were evaluated using fed-batch cultures. Staurosporine at concentrations ≥10 nM induced cell death, but at concentrations ≤5 nM did not. In the range of 2-4 nM, cell growth was inhibited, whereas the specific production rate (Qp) and cell longevity were improved in a dose-dependent manner. The Qp and maximum mAb concentration with 4 nM staurosporine improved by 36.3 and 5.2%, respectively, compared to those with control conditions. Cell viability post-culture without staurosporine was 40.0 ± 0.3%, whereas with 4 nM staurosporine, it was 90.1 ± 1.0%. Flow cytometric analysis indicated cell-cycle arrest at the G1/G0 phase with 4 nM staurosporine addition. The present study highlighted the efficacy of staurosporine in improving mAb production by causing cell-cycle arrest. Further research into staurosporine analogs and how to use them will lead to development of more effective industrial production technologies of biopharmaceuticals.


Assuntos
Anticorpos Monoclonais/biossíntese , Proteínas Recombinantes/biossíntese , Estaurosporina/farmacologia , Animais , Técnicas de Cultura Celular por Lotes , Células CHO , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Proteínas Recombinantes/genética
5.
Sci Rep ; 10(1): 14076, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826930

RESUMO

Marine bacterial strains are of great interest for their ability to produce secondary metabolites with anticancer potentials. Isolation, identification, characterization and anticancer activities of isolated bacteria from El-Hamra Lake, Wadi El-Natrun (Egypt) were the objectives of this study. The isolated bacteria were identified as a moderately halophilic alkaliphilic strain. Ethyl acetate extraction was performed and identified by liquid chromatography-mass spectrophotometry (LC-MS-MS) and nuclear magnetic resonance analysis (NMR). Cytotoxicity of the extract was assessed on the HepG2 cell line and normal human peripheral lymphocytes (HPBL) in vitro. Halomonas sp. HA1 extract analyses revealed anticancer potential. Many compounds have been identified including cyclo-(Leu-Leu), cyclo-(Pro-Phe), C17-sphinganine, hexanedioic acid, bis (2-ethylhexyl) ester, surfactin C14 and C15. The extract exhibited an IC50 of 68 ± 1.8 µg/mL and caused marked morphological changes in treated HepG2 cells. For mechanistic anticancer evaluation, 20 and 40 µg/mL of bacterial extract were examined. The up-regulation of apoptosis-related genes' expression, P53, CASP-3, and BAX/BCL-2 at mRNA and protein levels proved the involvement of P53-dependant mitochondrial apoptotic pathway. The anti-proliferative properties were confirmed by significant G2/M cell cycle arrest and PCNA down-regulation in the treated cells. Low cytotoxicity was observed in HPBL compared to HepG2 cells. In conclusion, results suggest that the apoptotic and anti-proliferative effects of Halomonas sp. HA1 extract on HepG2 cells can provide it as a candidate for future pharmaceutical industries.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Extratos Celulares/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Halomonas/química , Neoplasias Hepáticas/patologia , Antineoplásicos/isolamento & purificação , Divisão Celular/efeitos dos fármacos , Extratos Celulares/isolamento & purificação , Quebras de DNA de Cadeia Simples , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Linfócitos/efeitos dos fármacos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Ribotipagem , Regulação para Cima/efeitos dos fármacos
6.
Proc Natl Acad Sci U S A ; 117(34): 20943-20949, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32817465

RESUMO

The reiterative process of lateral root (LR) formation is widespread and underlies root system formation. However, early LR primordium (LRP) morphogenesis is not fully understood. In this study, we conducted both a clonal analysis and time-lapse experiments to decipher the pattern and sequence of pericycle founder cell (FC) participation in LR formation. Most commonly, LRP initiation starts with the specification of just one FC longitudinally. Clonal and anatomical analyses suggested that a single FC gradually recruits neighboring pericycle cells to become FCs. This conclusion was validated by long-term time-lapse live-imaging experiments. Once the first FC starts to divide, its immediate neighbors, both lengthwise and laterally, are recruited within the hour, after which they recruit their neighboring cells within a few hours. Therefore, LRP initiation is a gradual, multistep process. FC recruitment is auxin-dependent and is abolished by treatment with a polar auxin transport inhibitor. Furthermore, FC recruitment establishes a morphogenetic field where laterally peripheral cells have a lower auxin response, which is associated with a lower proliferation potential, compared to centrally located FCs. The lateral boundaries of the morphogenetic field are determined by phloem-adjacent pericycle cells, which are the last cells to be recruited as FCs. The proliferation potential of these cells is limited, but their recruitment is essential for root system formation, resulting in the formation of a new vascular connection between the nascent and parent root, which is crucial for establishing a continuous and efficient vascular system.


Assuntos
Arabidopsis/genética , Raízes de Plantas/crescimento & desenvolvimento , Arabidopsis/metabolismo , Transporte Biológico/fisiologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Ácidos Indolacéticos/farmacologia , Meristema/metabolismo , Morfogênese/genética , Organogênese Vegetal/fisiologia , Floema/metabolismo , Raízes de Plantas/metabolismo , Transdução de Sinais/efeitos dos fármacos
7.
Anticancer Res ; 40(8): 4695-4700, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727794

RESUMO

BACKGROUND/AIM: We investigated the anti-proliferative effect of quercetin on liver cancer cell lines. MATERIALS AND METHODS: Thirteen liver cancer cell lines were cultured followed by treatment with varying concentrations of quercetin (0-100 µM) or quercetin and 5-FU, and the cell viability was analysed by the MTT assay. Flow cytometry was also used to examine cell cycle progression after treatment with quercetin. RESULTS: The addition of quercetin resulted in a dose- and time-dependent suppression of cell proliferation. In some cell lines, treatment with quercetin and 5-FU caused an additional or synergistic effect. Most cell lines displayed cell cycle arrest at different phases of the cell cycle. CONCLUSION: Quercetin inhibits the proliferation of liver cancer cells via induction of apoptosis and cell cycle arrest.


Assuntos
Neoplasias Hepáticas/tratamento farmacológico , Quercetina/farmacologia , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/patologia
8.
PLoS One ; 15(7): e0236373, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32702063

RESUMO

The diagnosis of patients with malignancies relies on the results of a clinical cytological examination. To enhance the diagnostic qualities of cytological examinations, it is important to have a detailed analysis of the cell's characteristics. There is, therefore, a need for developing a new auxiliary method for cytological diagnosis. In this study, we focused on studying the charge of the cell membrane surface of fixed cells, which is one of important cell's characteristics. Although fixed cells lose membrane potential which is observed in living cells owing to ion dynamics, we hypothesized that fixed cells still have a cell membrane surface charge due to cell membrane components and structure. We used 5 cell lines in this study (ARO, C32TG, RT4, TK, UM-UC-14). After fixation with CytoRich Red, we measured the cell membrane surface charge of fixed cells in solution using zeta potential measurements and fixed cells on glass slides, visualizing it using antibody-labeled beads and positively-charged beads. Furthermore, we measured the cell membrane surface charge of fixed cells under different conditions, such as different solution of fixative, ion concentration, pH, and pepsin treatments. The zeta potential measurements and visualization using the beads indicated that the cell membrane surface of fixed cells was negatively charged, and also that the charge varied among fixed cells. The charge state was affected by the different treatments. Moreover, the number of cell-bound beads was small in interphase, anaphase, and apoptotic cells. We concluded that the negative cell membrane surface charge was influenced by the three-dimensional structure of proteins as well as the different types of amino acids and lipids on the cell membrane. Thus, cell surface charge visualization can be applied as a new auxiliary method for clinical cytological diagnosis. This is the first systematic report of the cell membrane surface charge of fixed cells.


Assuntos
Linhagem Celular/ultraestrutura , Membrana Celular/ultraestrutura , Células Cultivadas/ultraestrutura , Citodiagnóstico , Anáfase/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Fixadores/farmacologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Pepsina A/farmacologia , Propriedades de Superfície
9.
Sci Rep ; 10(1): 12532, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32719397

RESUMO

Rho GTPases are important regulators of many cellular functions like cell migration, adhesion and polarity. The molecular switches are often dysregulated in cancer. We detected Rho-dependent upregulation of the orphan seven-transmembrane receptor G-protein-coupled receptor family C group 5 member A (GPRC5A). GPRC5A is highly expressed in breast cancer whereas in lung cancer, it is often downregulated. Here, we analyzed the function of GPRC5A in breast epithelial and breast cancer cells. Activation or expression of RhoA/C led to GPRC5A-dependent inhibition of proliferation and reduction of the colony forming capacity of benign breast epithelial cells. This effect is based on an inhibition of EGFR signalling. Knockout of retinoic acid induced 3 (RAI3, the gene for GPRC5A) in breast cancer cells increased cell division, whereas Rho activation had no effect on proliferation. Knockout of RAI3 in benign breast epithelial cells led to decrease of EGFR expression and diminished proliferation.


Assuntos
Receptores Acoplados a Proteínas-G/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Proteína de Ligação a GTP rhoC/metabolismo , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Receptores ErbB/metabolismo , Humanos , Ligantes , Modelos Biológicos , Mutação/genética , Fosforilação/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos
10.
PLoS Comput Biol ; 16(6): e1007812, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32497127

RESUMO

Apoptotic cell death can be initiated through the extrinsic and intrinsic signaling pathways. While cell cycle progression promotes the responsiveness to intrinsic apoptosis induced by genotoxic stress or spindle poisons, this has not yet been studied conclusively for extrinsic apoptosis. Here, we combined fluorescence-based time-lapse monitoring of cell cycle progression and cell death execution by long-term time-lapse microscopy with sampling-based mathematical modeling to study cell cycle dependency of TRAIL-induced extrinsic apoptosis in NCI-H460/geminin cells. In particular, we investigated the interaction of cell death timing and progression of cell cycle states. We not only found that TRAIL prolongs cycle progression, but in reverse also that cell cycle progression affects the kinetics of TRAIL-induced apoptosis: Cells exposed to TRAIL in G1 died significantly faster than cells stimulated in S/G2/M. The connection between cell cycle state and apoptosis progression was captured by developing a mathematical model, for which parameter estimation revealed that apoptosis progression decelerates in the second half of the cell cycle. Similar results were also obtained when studying HCT-116 cells. Our results therefore reject the null hypothesis of independence between cell cycle progression and extrinsic apoptosis and, supported by simulations and experiments of synchronized cell populations, suggest that unwanted escape from TRAIL-induced apoptosis can be reduced by enriching the fraction of cells in G1 phase. Besides novel insight into the interrelation of cell cycle progression and extrinsic apoptosis signaling kinetics, our findings are therefore also relevant for optimizing future TRAIL-based treatment strategies.


Assuntos
Apoptose , Ciclo Celular , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Transdução de Sinais , Algoritmos , Teorema de Bayes , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Geminina/química , Células HCT116 , Humanos , Cinética , Modelos Estatísticos
11.
BMC Cancer ; 20(1): 552, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32539694

RESUMO

BACKGROUND: The oncogene LSF (encoded by TFCP2) has been proposed as a novel therapeutic target for multiple cancers. LSF overexpression in patient tumors correlates with poor prognosis in particular for both hepatocellular carcinoma and colorectal cancer. The limited treatment outcomes for these diseases and disappointing clinical results, in particular, for hepatocellular carcinoma in molecularly targeted therapies targeting cellular receptors and kinases, underscore the need for molecularly targeting novel mechanisms. LSF small molecule inhibitors, Factor Quinolinone Inhibitors (FQIs), have exhibited robust anti-tumor activity in multiple pre-clinical models, with no observable toxicity. METHODS: To understand how the LSF inhibitors impact cancer cell proliferation, we characterized the cellular phenotypes that result from loss of LSF activity. Cell proliferation and cell cycle progression were analyzed, using HeLa cells as a model cancer cell line responsive to FQI1. Cell cycle progression was studied either by time lapse microscopy or by bulk synchronization of cell populations to ensure accuracy in interpretation of the outcomes. In order to test for biological specificity of targeting LSF by FQI1, results were compared after treatment with either FQI1 or siRNA targeting LSF. RESULTS: Highly similar cellular phenotypes are observed upon treatments with FQI1 and siRNA targeting LSF. Along with similar effects on two cellular biomarkers, inhibition of LSF activity by either mechanism induced a strong delay or arrest prior to metaphase as cells progressed through mitosis, with condensed, but unaligned, chromosomes. This mitotic disruption in both cases resulted in improper cellular division leading to multiple outcomes: multi-nucleation, apoptosis, and cellular senescence. CONCLUSIONS: These data strongly support that cellular phenotypes observed upon FQI1 treatment are due specifically to the loss of LSF activity. Specific inhibition of LSF by either small molecules or siRNA results in severe mitotic defects, leading to cell death or senescence - consequences that are desirable in combating cancer. Taken together, these findings confirm that LSF is a promising target for cancer treatment. Furthermore, this study provides further support for developing FQIs or other LSF inhibitory strategies as treatment for LSF-related cancers with high unmet medical needs.


Assuntos
Benzodioxóis/farmacologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Quinolonas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Apoptose/genética , Benzodioxóis/uso terapêutico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Cromossomos Humanos/efeitos dos fármacos , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Microscopia Intravital , Terapia de Alvo Molecular/métodos , Neoplasias/genética , Neoplasias/patologia , Quinolonas/uso terapêutico , RNA Interferente Pequeno/metabolismo , Imagem com Lapso de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
12.
PLoS One ; 15(5): e0232482, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32357366

RESUMO

The study was designed to assess whether plant extracts / phytochemical (D-Pinitol) synergistically combine with antituberculosis drugs and act on Mycobacterium smegmatis (M. smegmatis) as well as assess their mode of action on Mycobacterium tuberculosis (M.tb) Filamenting temperature sensitive mutant Z (FtsZ) protein. Resazurin microtitre plate assay (Checker board) was performed to analyze the activity of plant extracts against M. smegmatis. Synergistic behaviour of plant extracts / D-Pinitol with Isoniazid (INH) and Rifampicin (RIF) were determined by time-kill and checker board assays. Elongation of M. smegmatis cells due to this treatment was determined by light microscopy. The effect of Hexane methanol extract (HXM) plant extracts on cell viability was determined using PI/SYTO9 dual dye reporter Live/Dead assay. Action of HXM plant extracts / D-Pinitol on inhibition of FtsZ protein was done using Guanosine triphosphatase (GTPase) light scattering assay and quantitative Polymerase Chain Reaction (qPCR). The Hexane-methanolic plant extract of Acacia nilotica, Aegle marmelos and Glycyrrhiza glabra showed antimycobacterial activity at 1.56 ± 0.03, 1.32 ± 0.02 and 1.25 ± 0.03 mg/mL respectively and that of INH and RIF were 4.00 ± 0.06 µg/mL and 2.00 ± 0.04 µg/mL respectively. These plant extracts and major phytochemical exudate D-Pinitol was found to act synergistically with antimycobacterial drugs INH and RIF with an FIC index ~ 0.20. Time-Kill kinetics studies indicate that, these plant extracts were bacteriostatic in nature. D-Pinitol in conjunction with INH and RIF exhibited a 2 Log reduction in the growth of viable cells compared to untreated. Attempt to elucidate their mode of action through phenotypic analysis indicated that these plant extracts and D-Pinitol was found to interfere in cell division there by leading to an abnormal elongated cellular morphology. HXM extracts and D-Pinitol synergistically combined with the first line tuberculosis drugs, INH and RIF, to act on M. smegmatis. The increase in the length of M. smegmatis cells on treatment with D-Pinitol and HXM extract of the plants indicated that they hinder the cell division mechanism thereby leading to a filamentous phenotype, and finally leading to cell death. In addition, the integrity of the bacterial cell membrane is also altered causing cell death. Further gene expression analysis showed that these plant extracts and D-Pinitol hampers with function of FtsZ protein which was confirmed through in vitro inhibition of FtsZ-GTPase enzymatic activity.


Assuntos
Proteínas de Bactérias/genética , Proteínas do Citoesqueleto/genética , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/genética , Plantas Medicinais , Antituberculosos/administração & dosagem , Proteínas de Bactérias/antagonistas & inibidores , Divisão Celular/efeitos dos fármacos , Proteínas do Citoesqueleto/antagonistas & inibidores , Sinergismo Farmacológico , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos/efeitos dos fármacos , Humanos , Técnicas In Vitro , Inositol/administração & dosagem , Inositol/análogos & derivados , Isoniazida/administração & dosagem , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium smegmatis/citologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Extratos Vegetais/administração & dosagem , Rifampina/administração & dosagem , Temperatura
13.
J Biosci Bioeng ; 130(1): 106-113, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32253091

RESUMO

Monoclonal antibodies (mAbs) are active pharmaceutical ingredients in antibody drugs, produced mainly using recombinant Chinese hamster ovary (CHO) cells. The regulation of recombinant CHO cell proliferation can improve the productivity of heterologous proteins. Chemical compound approaches for cell cycle regulation have the advantages of simplicity and ease of use in industrial processes. However, CHO cells have genetic and phenotypic diversity, and the effects of such compounds might depend on cell line and culture conditions. Increasing the variety of cell cycle inhibitors is a promising strategy to overcome the dependency. Marine microorganisms are a vast and largely undeveloped source of secondary metabolites with physiological activity. In this study, we focused on secondary metabolites of marine microorganisms and evaluated their effectiveness as cell cycle inhibitory compounds. Of 720 extracts from microorganisms (400 actinomycetes and 320 filamentous fungi) collected from the Okinawan Sea, we identified nine extracts that decreased the specific growth rate and increased the specific production rate without reducing cell viability. After fractionating the extracts, the components of active fractions were estimated using time-of-flight mass spectrometry analysis. Then, four compounds, including staurosporine and undecylprodigiosin were deduced to be active compounds. These compounds have been reported to exert a cell cycle inhibitory effect on mammalian cells. These compounds might serve as additives to improve mAb production in CHO cells. This study indicates that secondary metabolites of marine microorganisms are a useful source for new cell cycle inhibitory compounds that can increase mAb production in CHO cells.


Assuntos
Actinobacteria/química , Ciclo Celular/efeitos dos fármacos , Fungos/química , Inibidores do Crescimento/farmacologia , Água do Mar/microbiologia , Actinobacteria/genética , Actinobacteria/isolamento & purificação , Actinobacteria/metabolismo , Animais , Células CHO , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Fungos/genética , Fungos/isolamento & purificação , Fungos/metabolismo , Inibidores do Crescimento/metabolismo , Prodigiosina/análogos & derivados , Prodigiosina/metabolismo , Prodigiosina/farmacologia , Estaurosporina/metabolismo , Estaurosporina/farmacologia
15.
Molecules ; 25(7)2020 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-32272719

RESUMO

Two novel series of compounds based on the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine and 4,5,6,7-tetrahydrobenzo[b]thiophene molecular skeleton, characterized by the presence of a 3',4',5'-trimethoxyanilino moiety and a cyano or an alkoxycarbonyl group at its 2- or 3-position, respectively, were designed, synthesized, and evaluated for antiproliferative activity on a panel of cancer cell lines and for selected highly active compounds, inhibition of tubulin polymerization, and cell cycle effects. We have identified the 2-(3',4',5'-trimethoxyanilino)-3-cyano-6-methoxycarbonyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine derivative 3a and its 6-ethoxycarbonyl homologue 3b as new antiproliferative agents that inhibit cancer cell growth with IC50 values ranging from 1.1 to 4.7 µM against a panel of three cancer cell lines. Their interaction with tubulin at micromolar levels leads to the accumulation of cells in the G2/M phase of the cell cycle and to an apoptotic cell death. The cell apoptosis study found that compounds 3a and 3b were very effective in the induction of apoptosis in a dose-dependent manner. These two derivatives did not induce cell death in normal human peripheral blood mononuclear cells, suggesting that they may be selective against cancer cells. Molecular docking studies confirmed that the inhibitory activity of these molecules on tubulin polymerization derived from binding to the colchicine site.


Assuntos
Antineoplásicos/química , Produtos Biológicos/química , Piridinas/química , Moduladores de Tubulina/química , Tubulina (Proteína)/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Produtos Biológicos/farmacologia , Morte Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colchicina/química , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Fase G2/efeitos dos fármacos , Células HeLa , Humanos , Células K562 , Leucócitos Mononucleares/efeitos dos fármacos , Camundongos , Simulação de Acoplamento Molecular/métodos , Piridinas/farmacologia , Relação Estrutura-Atividade
16.
Int J Mol Sci ; 21(7)2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-32218176

RESUMO

MiPEPs are short natural peptides encoded by microRNAs in plants. Exogenous application of miPEPs increases the expression of their corresponding miRNA and, consequently, induces consistent phenotypical changes. Therefore, miPEPs carry huge potential in agronomy as gene regulators that do not require genome manipulation. However, to this end, it is necessary to know their mode of action, including where they act and how they enter the plants. Here, after analyzing the effect of Arabidopsis thaliana miPEP165a on root and aerial part development, we followed the internalization of fluorescent-labelled miPEP165a into roots and compared its uptake into endocytosis-altered mutants to that observed in wild-type plants treated or not with endocytosis inhibitors. The results show that entry of miPEP165a involves both a passive diffusion at the root apex and endocytosis-associated internalization in the differentiation and mature zones. Moreover, miPEP165a is unable to enter the central cylinder and does not migrate from the roots to the aerial part of the plant, suggesting that miPEPs have no systemic effect.


Assuntos
Arabidopsis/efeitos dos fármacos , Endocitose , Arabidopsis/citologia , Arabidopsis/metabolismo , Transporte Biológico , Divisão Celular/efeitos dos fármacos , Difusão , Endocitose/efeitos dos fármacos , Fenótipo , Raízes de Plantas/citologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas
17.
Proc Natl Acad Sci U S A ; 117(12): 6330-6338, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32161132

RESUMO

Cytokinesis-the division of a cell into two daughter cells-is a key step in cell growth and proliferation. It typically occurs in synchrony with the cell cycle to ensure that a complete copy of the genetic information is passed on to the next generation of daughter cells. In animal cells, cytokinesis commonly relies on an actomyosin contractile ring that drives equatorial furrowing and separation into the two daughter cells. However, also contractile ring-independent forms of cell division are known that depend on substrate-mediated traction forces. Here, we report evidence of an as yet unknown type of contractile ring-independent cytokinesis that we termed wave-mediated cytofission. It is driven by self-organized cortical actin waves that travel across the ventral membrane of oversized, multinucleated Dictyostelium discoideum cells. Upon collision with the cell border, waves may initiate the formation of protrusions that elongate and eventually pinch off to form separate daughter cells. They are composed of a stable elongated wave segment that is enclosed by a cell membrane and moves in a highly persistent fashion. We rationalize our observations based on a noisy excitable reaction-diffusion model in combination with a dynamic phase field to account for the cell shape and demonstrate that daughter cells emerging from wave-mediated cytofission exhibit a well-controlled size.


Assuntos
Actinas/metabolismo , Divisão Celular/fisiologia , Animais , Divisão Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Movimento Celular , Forma Celular , Tamanho Celular , Dictyostelium/citologia , Dictyostelium/genética , Modelos Biológicos , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia
18.
Biochem Biophys Res Commun ; 525(4): 948-953, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32173527

RESUMO

The SOS response is considered to be an extremely important feature of bacterial cells. It helps them to survive bad times, including helping to develop resistance to antibiotics. The SOS response blocks the cell division. For Escherichia coli it is well known that the SulA protein directly interacts with FtsZ - a key division protein. Now it is believed that fission blocking is based on FtsZ sequestration by the SulA protein, which leads to decrease in effective concentration of FtsZ in the cell below a critical value, which in vitro leads to dismantling of FtsZ polymers. In this work, we demonstrate that in order to block the division of E. coli, it is sufficient to have a relatively small amount of SulA in the cell. Moreover, the analysis of structures formed by FtsZ in E. coli cells under the conditions of SulA protein expression or the SOS response showed that there is no complete disassembly of FtsZ polymers, although Z-rings indeed are not formed. The results of the work indicate that the well-known sequestration mechanism is not comprehensive to explain blocking of the division process by SulA in vivo.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/citologia , Divisão Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Resposta SOS em Genética/fisiologia , Espectrometria de Fluorescência
19.
Neuropeptides ; 80: 102029, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32127176

RESUMO

Differentiation, self-renewal and quiescence of Hematopoietic stem cells (HSCs) is tightly regulated in order to protect the HSCs from the strain of constant cell division and depletion of the stem cell pool. The neurotransmitter Neuropeptide Y (NPY) is released from sympathetic nerves in the bone marrow and has been shown to indirectly affect HSC function through effects on bone marrow (BM) multipotent Mesenchymal Stromal Cells (MSCs), osteoblasts (OBs) and macrophages. Although the absence of NPY has been shown to be accompanied by severe BM impairment and delayed engraftment of HSCs, the direct effects of NPY on HSCs have never been assessed. Here, we aimed to explore the effect of NPY on the regulation of HSCs. All NPY receptors Y1, Y2, Y4 and Y5 were found to be highly expressed on most HSCs and mature hematopoietic cell subsets. In culture, in particularly expression of the Y1 receptor was shown to decrease in time. Doses of 300 nM NPY suppressed HSC proliferation in cell cultures, as confirmed by an increase of HSCs in G0 phase and an increase in the gene expression levels of FOXO3, DICER1, SMARCA2 and PDK1, which all have been shown to play an important role in the regulation of cell quiescence. These data support the idea that NPY may have a direct effect on the regulation of HSC fate by modulating cell quiescence.


Assuntos
Proliferação de Células/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Neuropeptídeo Y/farmacologia , Receptores de Neuropeptídeo Y/metabolismo , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Neuropeptídeo Y/metabolismo , Receptores de Neuropeptídeo Y/efeitos dos fármacos
20.
Integr Cancer Ther ; 19: 1534735419900555, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32009489

RESUMO

A colon cancer growth inhibitor partially purified from the isolated cell wall membrane fraction of Chlorella sorokiniana, here referred to as Chlorella membrane factor (CMF), was evaluated for its antitumor and immunomodulatory effects in cell culture and in a colon carcinoma mouse model. The CMF treatment dose- and time-dependently inhibited colon carcinoma cell growth in 2-dimensional cultures. Treatment with CMF also significantly inhibited the growth of colon carcinoma spheroids in 3-dimensional cell culture in coculture with T lymphocytes. In a mouse CT26 colon carcinoma peritoneal dissemination model, intraperitoneal injection of CMF (10 or 30 mg dry weight/kg body weight, every other day) dose-dependently and significantly attenuated the growth of tumor nodules via induction of tumor cell apoptosis. Evaluation of immune cell populations in ascites showed that CMF treatment tended to increase T lymphocytes but lower granulocyte populations. The present study suggests that the cell wall membrane fraction of Chlorella sorokiniana contains a bioactive material that inhibits colon carcinoma growth via direct cell growth inhibition and stimulation of host antitumor immunity. Hence, it is suggested that the Chlorella cell wall membrane extract or a bioactive substance in the extract is an attractive complementary medicine for cancer therapy.


Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Chlorella/química , Neoplasias do Colo/tratamento farmacológico , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Parede Celular , Colo/patologia , Neoplasias do Colo/patologia , Imunidade , Injeções Intraperitoneais , Camundongos , Extratos Vegetais/administração & dosagem
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