RESUMO
BACKGROUND AND PURPOSE: Gastrointestinal tumours overexpress voltage-gated calcium (CaV3) channels (CaV3.1, 3.2 and 3.3). CaV3 channels regulate cell growth and apoptosis colorectal cancer. Gossypol, a polyphenolic aldehyde found in the cotton plant, has anti-tumour properties and inhibits CaV3 currents. A systematic study was performed on gossypol blocking mechanism on CaV3 channels and its potential anticancer effects in colon cancer cells, which express CaV3 isoforms. EXPERIMENTAL APPROACH: Transcripts for CaV3 proteins were analysed in gastrointestinal cancers using public repositories and in human colorectal cancer cell lines HCT116, SW480 and SW620. The gossypol blocking mechanism on CaV3 channels was investigated by combining heterologous expression systems and patch-clamp experiments. The anti-tumoural properties of gossypol were estimated by cell proliferation, viability and cell cycle assays. Ca2+ dynamics were evaluated with cytosolic and endoplasmic reticulum (ER) Ca2+ indicators. KEY RESULTS: High levels of CaV3 transcripts correlate with poor prognosis in gastrointestinal cancers. Gossypol blockade of CaV3 isoforms is concentration- and use-dependent interacting with the closed, activated and inactivated conformations of CaV3 channels. Gossypol and CaV3 channels down-regulation inhibit colorectal cancer cell proliferation by arresting cell cycles at the G0/G1 and G2/M phases, respectively. CaV3 channels underlie the vectorial Ca2+ uptake by endoplasmic reticulum in colorectal cancer cells. CONCLUSION AND IMPLICATIONS: Gossypol differentially blocked CaV3 channel and its anticancer activity was correlated with high levels of CaV3.1 and CaV3.2 in colorectal cancer cells. The CaV3 regulates cell proliferation and Ca2+ dynamics in colorectal cancer cells. Understanding this blocking mechanism maybe improve cancer therapies.
Assuntos
Bloqueadores dos Canais de Cálcio , Canais de Cálcio Tipo T , Proliferação de Células , Neoplasias do Colo , Gossipol , Humanos , Gossipol/farmacologia , Gossipol/análogos & derivados , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Neoplasias do Colo/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Proliferação de Células/efeitos dos fármacos , Canais de Cálcio Tipo T/metabolismo , Canais de Cálcio Tipo T/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Cálcio/metabolismo , Linhagem Celular Tumoral , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Antineoplásicos/farmacologiaRESUMO
Low temperature and sodium butyrate (NaBu) are two of the most used productivity-enhancing strategies in CHO cell cultures during biopharmaceutical manufacturing. While these two approaches alter the balance in the reciprocal relationship between cell growth and productivity, we do not fully understand their mechanisms of action beyond a gross cell growth inhibition. Here, we used continuous culture to evaluate the differential effect of low temperature and NaBu supplementation on CHO cell performance and gene expression profile. We found that an increase in cell-productivity under growth-inhibiting conditions was associated with the arrest of cells in the G1/G0 phase. A transcriptome analysis revealed that the molecular mechanisms by which low temperature and NaBu arrested cell cycle in G1/G0 differed from each other through the deregulation of different cell cycle checkpoints and regulators. The individual transcriptome changes in pattern observed in response to low temperature and NaBu were retained when these two strategies were combined, leading to an additive effect in arresting the cell cycle in G1/G0 phase. The findings presented here offer novel molecular insights about the cell cycle regulation during the CHO cell bioprocessing and its implications for increased recombinant protein production. This data provides a background for engineering productivity-enhanced CHO cell lines for continuous manufacturing.
Assuntos
Técnicas de Cultura de Células , Cricetinae , Animais , Células CHO , Fase de Repouso do Ciclo Celular , Cricetulus , Proteínas Recombinantes/metabolismo , Ciclo CelularRESUMO
This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief. Given the comments of Dr Elisabeth Bik regarding this article "... the Western blot bands in all 400+ papers are all very regularly spaced and have a smooth appearance in the shape of a dumbbell or tadpole, without any of the usual smudges or stains. All bands are placed on similar looking backgrounds, suggesting they were copy/pasted from other sources, or computer generated", the journal requested the authors to provide the raw data. However, the authors were not able to fulfil this request and therefore the Editor-in-Chief decided to retract the article.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzopiranos/farmacologia , Indenos/farmacologia , MicroRNAs/biossíntese , Neuroma Acústico/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , MicroRNAs/efeitos dos fármacos , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Regulação para CimaRESUMO
The acquisition of a castration-resistant prostate cancer phenotype by prostate cancer cells is the alteration that has the worst prognosis for patients. The aim of this study was to evaluate the role of the microRNAs-23b/-27b as well as the possible CCNG1 target gene in tissue samples from patients with localized prostate cancer that progressed to castration-resistant prostate cancer and in a castration-resistant prostate cancer cell line (PC-3). The microRNAs and target gene expression levels of the surgical specimens were analyzed by quantitative real-time polymerase chain reaction. The prostate cancer cell line, PC-3, was transfected with pre-miR-23b, pre-miR-27b, and their respective controls using Lipofectamine RNAiMAX and exposed or not to flutamide. After transfections, expression levels of both the microRNAs and the gene, CCNG1, were analyzed by quantitative real-time polymerase chain reaction. The apoptosis and cell cycle assays were performed on the mini MUSE cytometer. MicroRNAs-23b/-27b were underexpressed in surgical specimens of prostate cancer; however, their target gene, CCNG1, was overexpressed in 69% of the cases. After transfection with the microRNAs-23b/-27b and flutamide, we observed a reduction in gene expression compared with cells that were treated only with microRNAs or only with flutamide. In the apoptosis assay, we demonstrated cell sensitization following transfection with microRNAs-23b/-27b and potentiation when co-administered with flutamide. The number of cells in apoptosis was almost three times higher with the simultaneous treatments (miR + flutamide) compared with the control (p < 0.05). In the cell cycle assay, only flutamide treatment showed better results; a higher number of cells were found in the G0-G1 phase, and a lower percentage of cells completed the final phase of the cycle (p < 0.05). We conclude that microRNAs-23b/-27b are downexpressed in prostate cancer, and their target gene, CCNG1, is overexpressed. We postulated that microRNAs-23b/-27b sensitize the PC-3 cell line and that after the addition of flutamide in the apoptosis assay, we would observe synergism in the treatments between miR and flutamide. In the cell cycle assay, the use of flutamide was sufficient to decrease the number of cells in mitosis. Therefore, we postulate that microRNAs, along with other drugs, may become very useful therapeutic tools in the treatment of castration-resistant prostate cancer.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclina G1/genética , Flutamida/metabolismo , MicroRNAs/genética , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Linhagem Celular Tumoral , Fase G1/efeitos dos fármacos , Fase G1/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Mitose/efeitos dos fármacos , Mitose/genética , Próstata/efeitos dos fármacos , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Fase de Repouso do Ciclo Celular/genética , Transfecção/métodosRESUMO
Continuous increases in the rates of tumor diseases have highlighted the need for identification of novel and inexpensive antitumor agents from natural sources. In this study, we investigated the effects of enriched fraction from hydroalcoholic Brazilian red propolis extract against Hep-2 cancer cell line. Initially 201 fractions were arranged in 12 groups according to their chromatographic characteristics (A-L). After an in vitro cell viability screening, J and L were further selected as promising enriched fractions for this study. The chemical characterization was performed and Biochanin A, Formononetin, and Liquiritigenin compounds were quantified. Through MTT viability assay and morphological changes observed by Giemsa and DAPI staining, the results showed that red propolis inhibited cancer cells growth. Flow cytometry results indicated effects that were partly mediated through programmed cell death as confirmed by externalization of phosphatidylserine, DNA cleaved assay, increase at SUB G1-G0 phase in cell cycle analysis and loss of mitochondrial membrane potential. In conclusion, our results demonstrated that red propolis enriched fractions promoted apoptotic effects in human cancer cells through the mechanisms involving mitochondrial perturbation. Therefore, red propolis fractions contain candidate agents for adjuvant cancer treatment, which further studies should elucidate the comprehensive mechanistic pathways.
Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Própole/farmacologia , Apoptose/efeitos dos fármacos , Brasil , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Flavanonas/farmacologia , Fase G1/efeitos dos fármacos , Genisteína/farmacologia , Humanos , Isoflavonas/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Fase de Repouso do Ciclo Celular/efeitos dos fármacosRESUMO
The kodkod population is in constant decrease and the somatic cell nuclear transfer (SCNT) might help to preserve the genetic pool of this species. The cell cycle synchronization of donor cells plays a crucial role in SCNT. The objective of this research was to evaluate two different methods for quiescence induction, serum starvation (SS) and contact inhibition (CI), both for 1, 3 and 5 days, on skin fibroblast from domestic cat and kodkod. Flow cytometry analysis revealed that in domestic cat, SS and CI, both at 3 and 5 days, increased the percentage of fibroblasts in G0/G1 compared to growing cells (GC) (p < .05). In kodkod, only SS for 3 and 5 days and CI for 1 and 3 days increased the percentage of fibroblasts in G0/G1 compared to GC (p < .05). Viability analysis by differential staining revealed that SS for 5 days decreased the proportion of live fibroblasts in domestic cat and kodkod (p < .05). Regarding gene expression analysis, in domestic cat fibroblasts, no differences were found in the BAX/BCL2 ratio in SS and CI (both at 1, 3 and 5 days) compared to GC. In kodkod fibroblasts, BAX/BCL2 ratio was increased in CI at 3 and 5 days compared to SS at 3 and 5 days (p < .05). In conclusion, in kodkod fibroblasts SS for 5 days and CI after 3 days might have a negative impact on cellular viability. According to these results, we suggest SS for 3 days for cell cycle synchronization in kodkod fibroblasts.
Assuntos
Ciclo Celular/fisiologia , Felidae/fisiologia , Fibroblastos/citologia , Técnicas de Transferência Nuclear/veterinária , Animais , Apoptose/genética , Sobrevivência Celular , Clonagem de Organismos/veterinária , Inibição de Contato , Meios de Cultura Livres de Soro , Perfilação da Expressão Gênica , Fase de Repouso do Ciclo CelularRESUMO
Extracts of Serjania lethalis A. St.-Hil leaves and stems were tested in order to identify potential agents against Leishmania amazonensis. The hexane fraction (HF) and dichloromethane subfractions (DDF and MDF) showed leishmanicidal effect. The anti-promastigote IC50 values were 10.29 (HF), 11.41 (DDF) and 28.33µg/mL (MDF); whereas those against amastigote were 7.2 (HF), 8.1 (DDF) and 6.5µg/mL (MDF). Among the fractions and subfractions assayed, only HF altered the cell cycle of the parasite, increasing 3-fold the number of cells in the sub-G0/G1 phase. HF also changed the parasite mitochondrial membrane potential (ΔΨm) and the percentage of annexin-V-propidium iodide positive promastigotes. Our evaluations of the IC50 values showed that HF, DDF and MDF decreased NO production in infected macrophages stimulated with IFN-γ and LPS. Moreover, HF increased the production of TNF-α in Leishmania infected macrophages. This paper reports for the first time the leishmanicidal activity of extracts and fractions of Serjania lethalis leaves and also characterizes its leishmanicidal and immunomodulatory properties.
Assuntos
Antiprotozoários/farmacologia , Leishmania mexicana/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Magnoliopsida/química , Extratos Vegetais/farmacologia , Animais , Anexina A5/análise , Fase G1/efeitos dos fármacos , Hexanos/química , Imunomodulação , Concentração Inibidora 50 , Interferon gama/imunologia , Leishmania mexicana/crescimento & desenvolvimento , Leishmania mexicana/fisiologia , Lipopolissacarídeos/imunologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/parasitologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Cloreto de Metileno/química , Camundongos , Óxido Nítrico/biossíntese , Extratos Vegetais/química , Folhas de Planta/química , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Chronic Myeloid Leukemia (CML) is sustained by a small population of cells with stem cell characteristics known as Leukemic Stem Cells that are positive to BCR-ABL fusion protein, involved with several abnormalities in cell proliferation, expansion, apoptosis and cell cycle regulation. Current treatment options for CML involve the use of Tirosine Kinase Inhibitor (Imatinib, Nilotinib and Dasatinib), that efficiently reduce proliferation proliferative cells but do not kill non proliferating CML primitive cells that remain and contributes to the persistence of the disease. In order to understand the role of Cyclin Dependent Kinase Inhibitors in CML LSC permanence after TKI treatment, in this study we analyzed cell cycle status, the levels of several CDKIs and the subcellular localization of such molecules in different CML cell lines, as well as primary CD34(+)CD38(-)lin(-) LSC and HSC. Our results demonstrate that cellular location of p18(INK4c) and p57(Kip2) seems to be implicated in the antiproliferative activity of Imatinib and Dasatinib in CML cells and also suggest that the permanence of quiescent stem cells after TKI treatment could be associated with a decrease in p18(INK4c) and p57(Kip2) nuclear location. The differences in p18(INK4c)and p57(Kip2)activities in CML and normal stem cells suggest a different cell cycle regulation and provide a platform that could be considered in the development of new therapeutic options to eliminate LSC.
Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p18/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Células-Tronco Neoplásicas/patologia , Inibidores de Proteínas Quinases/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dasatinibe/farmacologia , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Mesilato de Imatinib/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Transporte Proteico/efeitos dos fármacos , Fase de Repouso do Ciclo Celular/efeitos dos fármacosRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Euphorbia umbellata latex (sap) has normally been used in folk medicine in southern Brazil to treat different types of cancers. AIM OF STUDY: To carry out a biomonitored investigation of partitioned latex using in vitro assay, to identify the main mechanisms related with the action of the most active fraction as well as to develop a phytochemical study with this material. MATERIALS AND METHODS: Biological screening was performed with hexane, chloroform, ethyl acetate and methanol fractions from the latex of E. umbellata using MTT, trypan blue, and neutral red assays to determine the cytotoxicity against HRT-18, HeLa and Jurkat cells and flow cytometry, DNA quantification, acridine orange and Hoechst 33342 staining to investigate mechanisms of action for the hexane extract. The phytochemical study of the hexane fraction was performed by chromatographic procedures and the substances were identified by NMR analysis. The isolated terpenes were evaluated using MTT to determine the cytotoxicity against Jurkat cells. RESULTS: All the fractions presented concentration and time dependent cytotoxicity. The hexane fraction showed the highest cytotoxicity; whereas the Jurkat cell was the lineage with the highest sensitivity (IC50 1.87µg/mL). Fragmentation of DNA and apoptosis are two mechanisms related with the toxicity of hexane fraction. The hexane fraction arrested the cell cycle in the G0/G1 phase, and the selectivity index was 4.30. Phytochemical study of the hexane fraction led to isolation of euphol (main compound) and germanicol acetate. Both substances demonstrated some slight cytotoxic activity against Jurkat cells after 72h; however the activity was minimal compared to vincristine (anticancer standard drug). CONCLUSION: The current research proves that the fractions of the latex from E. umbellata have a cytotoxic effect against three different cancer cells lines. The hexane fraction showed high in vitro cytotoxic effects against Jurkat cells demonstrating that the effect may be due to non-polar constituents. The two isolated terpenes (euphol and germanicol acetate) showed poor cytotoxic activity indicating that the anticancer properties of the extract may be caused by other substances present in the hexane fraction.
Assuntos
Citotoxinas/farmacologia , Euphorbia/química , Extratos Vegetais/farmacologia , Apoptose/efeitos dos fármacos , Brasil , Linhagem Celular Tumoral , Citotoxinas/química , Fragmentação do DNA/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Células HeLa , Humanos , Células Jurkat , Medicina Tradicional/métodos , Compostos Fitoquímicos/química , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/química , Fase de Repouso do Ciclo Celular/efeitos dos fármacosRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: The stem barks of Lafoensia pacari have been traditionally used not only by South Amerindians but also by Brazilian and Paraguayan populations for treating a variety of unhealthy conditions to which their biological potential has been scientifically documented in several reports over the last decade. Although its anticancer usage is also popular, no scientific support for such activity has been found. AIM: To provide scientific evidence for the anticancer popularity of Lafoensia pacari. MATERIALS AND METHODS: Extracts prepared according to the popular use along with a methanol extract and its four fractions were produced from Lafoensia pacari stem barks. The chromatogram profile of each one was obtained by HPLC. Several tumor cell lines were exposed to these solutions in in vitro assays and the effects evaluated by morphological, growth, and cell cycle status changes. RESULTS: High toxicity determined by the lactate dehydrogenase levels with a significant drop in the cell proliferation index were found for all cell lines included in this study after exposition to Lafoensia pacari extract and fractions. The morphological features along with the expression of annexin V have strongly suggested apoptosis induction, which has been confirmed by G0/G1 cell cycle arrest. CONCLUSIONS: The data have clearly shown that exposition of human tumor cell lines to Lafoensia pacari stem barks extract leads to apoptosis induction due to cell cycle arrest in G0/G1 phases, supporting its anticancer use.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Lythraceae/química , Extratos Vegetais/farmacologia , Antineoplásicos Fitogênicos/isolamento & purificação , Brasil , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Medicina Tradicional , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Paraguai , Casca de Planta , Caules de Planta , Fase de Repouso do Ciclo Celular/efeitos dos fármacosRESUMO
New d-ribofuranoside derivatives containing two five membered heterocycles, isoxazole and triazole or two triazole rings, were synthesized. The final products as well as the synthetic precursors were physically and spectroscopically characterized. These new diheterocyclic derivatives together with other d-riboside compounds were assessed for their impact on PC3 cell line viability. We found that exposure of prostate cancer cells to some of these compounds caused a significant inhibition of cell growth and a G0/G1 cell cycle arrest, which was concomitant with alterations in the expression of proteins involved in cell cycle progression. Furthermore, the inhibitory activity was improved in di-heterocycles when the carbohydrate moiety was protected with a cyclopentylidene group compared to the isopropylidene analogues.
Assuntos
Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Alcenos/farmacologia , Carboidratos , Linhagem Celular Tumoral , Humanos , Isoxazóis/farmacologia , Masculino , Triazóis/farmacologiaRESUMO
Hexachlorobenzene (HCB) is an organochlorine pesticide widely distributed in the environment. In this study we have demonstrated that HCB induced loss of cell viability and alterations in cell cycle regulation in FRTL-5 rat thyroid cells. Analysis of cell cycle distribution by flow cytometric analysis demonstrated that HCB induced cell cycle arrest at G2/M and at G0/G1 phase, inhibiting cell cycle progression at the G1/S phase, after 24 h and 72 h of treatment. HCB-treatment resulted in an increase in transforming growth factor-beta (TGF-ß1) mRNA levels, a negative regulator of cell growth in thyroid epithelial cells. Time-dependent studies showed that both cytosolic and nuclear p27 protein levels were increased by 5 µM HCB. After 24 h of treatment, total p27 in whole cells lysate was increased. Dose-dependent studies, demonstrated that HCB (0.005, 0.05, 0.5 and 5 µM) increased p27, both in the cytosol and nucleus. HCB (5 µM) induced a concomitant decrease in nuclear cyclin D1 protein levels, in a time-dependent manner. We have also demonstrated that TGF-ß1 Smad signaling is involved in HCB-induced alterations of p27 and cyclin D1 protein levels. On the other hand, ERK1/2 activation is not involved in the alteration of cell cycle regulatory proteins.
Assuntos
Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Hexaclorobenzeno/toxicidade , Praguicidas/toxicidade , Glândula Tireoide/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , RNA Mensageiro/metabolismo , Ratos , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia , Fatores de Tempo , Fator de Crescimento Transformador beta1/genética , Regulação para CimaRESUMO
The aim of this study was to investigate the renal protective effect of icariin in 5/6 nephrectomized rats and the molecular mechanisms involved. Forty male Sprague-Dawley rats were randomly divided into 5 groups: sham-operated group, 5/6 nephrectomy model group, icariin groups (20 and 40 mg/kg), and benazepril group. After 12-weeks treatment, 24-h urine and serum were collected, and urine protein, serum creatinine, and blood urea nitrogen were determined. The rats were then sacrificed and fresh kidney tissues were prepared to obtain single cell suspensions. Cell cycle distribution and cell apoptosis were determined by annexin V-FITC/propidium iodide (PI) double staining using a flow cytometer. mRNA expression of Bcl-2 and Bax was examined using quantitative real-time PCR. After 12-weeks treatment, urinary protein, serum creatinine, and blood urea nitrogen in the icariin-treated group were much lower than in the untreated group compared with 5/6 nephrectomy model. Icariin reduced the percentage of S phase cells, increased the percentage of G0/M phase cells, and inhibited apoptosis in the renal cells. mRNA expression of Bcl-2 and Bax was decreased. In conclusion, icariin has a renal protective effect in 5/6 nephrectomized rats, which may be related mainly to alterations in cell cycle distribution and expression of apoptotic genes.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Flavonoides/farmacologia , Expressão Gênica/efeitos dos fármacos , Rim/efeitos dos fármacos , Nefrectomia , Substâncias Protetoras/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Benzazepinas/farmacologia , Nitrogênio da Ureia Sanguínea , Creatinina/sangue , Creatinina/urina , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Rim/citologia , Rim/metabolismo , Rim/cirurgia , Masculino , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Fase S/efeitos dos fármacos , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Parathyroid hormone-related peptide (PTHrP) is distributed in most fetal and adult tissues, and its expression correlates with the severity of colon carcinoma. Recently we obtained evidence that in Caco-2 cells, a cell line from human colorectal adenocarcinoma, exogenous PTHrP increases the number of live cells, via ERK1/2, p38 MAPK, and PI3-kinase and induces the expression of cyclin D1, a cell cycle regulatory protein. In this study, we further investigated the role of PTHrP in the regulation of the cell cycle progression in these intestinal cells. Flow cytometry analysis revealed that PTHrP treatment diminishes the number of cells in the G0/G1 phase and increases the number in both S and G2/M phases. The hormone increases the expression of CDK6 and diminishes the amount of negative cell cycle regulators p27Kip1, p15INK4B, and p53. However, PTHrP does not modify the expression of cyclin D3, CDK4, and p16INK4A. In addition, inhibitors of ERK1/2 (PD98059), p38 MAPK (SB203580), and PI3Kinase (LY294002) reversed PTHrP response in Caco-2 cells. Taken together, our results suggest that PTHrP positively modulates cell cycle progression and changes the expression of proteins involved in cell cycle regulation via ERK1/2, p38 MAPK, and PI3K signaling pathways in Caco-2 cells.
Assuntos
Sistema de Sinalização das MAP Quinases , Proteína Relacionada ao Hormônio Paratireóideo/fisiologia , Células CACO-2 , Ciclina D3/genética , Ciclina D3/metabolismo , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor de Quinase Dependente de Ciclina p15/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fase de Repouso do Ciclo Celular , Proteína Supressora de Tumor p53/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Recent studies have demonstrated that the anti-diabetic drug, metformin, can exhibit direct antitumoral effects, or can indirectly decrease tumor proliferation by improving insulin sensitivity. Despite these recent advances, the underlying molecular mechanisms involved in decreasing tumor formation are not well understood. In this study, we examined the antiproliferative role and mechanism of action of metformin in MCF-7 cancer cells treated with 10 mM of metformin for 24, 48, and 72 hours. Using BrdU and the MTT assay, it was found that metformin demonstrated an antiproliferative effect in MCF-7 cells that occurred in a time- and concentration-dependent manner. Flow cytometry was used to analyze markers of cell cycle, apoptosis, necrosis and oxidative stress. Exposure to metformin induced cell cycle arrest in G0-G1 phase and increased cell apoptosis and necrosis, which were associated with increased oxidative stress. Gene and protein expression were determined in MCF-7 cells by real time RT-PCR and western blotting, respectively. In MCF-7 cells metformin decreased the activation of IRß, Akt and ERK1/2, increased p-AMPK, FOXO3a, p27, Bax and cleaved caspase-3, and decreased phosphorylation of p70S6K and Bcl-2 protein expression. Co-treatment with metformin and H2O2 increased oxidative stress which was associated with reduced cell number. In the presence of metformin, treating with SOD and catalase improved cell viability. Treatment with metformin resulted in an increase in p-p38 MAPK, catalase, MnSOD and Cu/Zn SOD protein expression. These results show that metformin has an antiproliferative effect associated with cell cycle arrest and apoptosis, which is mediated by oxidative stress, as well as AMPK and FOXO3a activation. Our study further reinforces the potential benefit of metformin in cancer treatment and provides novel mechanistic insight into its antiproliferative role.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Proteínas de Neoplasias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Feminino , Proteína Forkhead Box O3 , Humanos , Hidrogênio/farmacologia , Oxidantes/farmacologiaRESUMO
BACKGROUND: Human cutaneous leishmaniasis is caused by distinct species, including Leishmania amazonensis. Treatment of cutaneous leishmaniasis is far from satisfactory due to increases in drug resistance and relapses, and toxicity of compounds to the host. As a consequence for this situation, the development of new leishmanicidal drugs and the search of new targets in the parasite biology are important goals. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the mechanism of death pathway induced by the calpain inhibitor MDL28170 on Leishmania amazonensis promastigote forms. The combined use of different techniques was applied to contemplate this goal. MDL28170 treatment with IC50 (15 µM) and two times the IC50 doses induced loss of parasite viability, as verified by resazurin assay, as well as depolarization of the mitochondrial membrane, which was quantified by JC-1 staining. Scanning and transmission electron microscopic images revealed drastic alterations on the parasite morphology, some of them resembling apoptotic-like death, including cell shrinking, surface membrane blebs and altered chromatin condensation pattern. The lipid rearrangement of the plasma membrane was detected by Annexin-V labeling. The inhibitor also induced a significant increase in the proportion of cells in the sub-G0/G1 phase, as quantified by propidium iodide staining, as well as genomic DNA fragmentation, detected by TUNEL assay. In cells treated with MDL28170 at two times the IC50 dose, it was also possible to observe an oligonucleossomal DNA fragmentation by agarose gel electrophoresis. CONCLUSIONS/SIGNIFICANCE: The data presented in the current study suggest that MDL28170 induces apoptotic marker expression in promastigotes of L. amazonensis. Altogether, the results described in the present work not only provide a rationale for further exploration of the mechanism of action of calpain inhibitors against trypanosomatids, but may also widen the investigation of the potential clinical utility of calpain inhibitors in the chemotherapy of leishmaniases.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Fragmentação do DNA/efeitos dos fármacos , DNA de Protozoário/metabolismo , Dipeptídeos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Protozoários/biossíntese , DNA de Protozoário/genética , Fase G1/efeitos dos fármacos , Humanos , Leishmania , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/enzimologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Protozoários/genética , Fase de Repouso do Ciclo Celular/efeitos dos fármacosRESUMO
Several studies have demonstrated that a balanced diet can contribute to better human health. For this reason, soy-based food and pure isoflavones (pills) are one of the most consumed. The association of this consumption and lower risks of chronic diseases and cancer is well established for the Asian population and has been attracting the attention of people worldwide, especially women at menopause who seek to alleviate the symptoms associated with the lack of estrogen. Despite positive epidemiological data, concerns still exist because of conflicting results found in scientific literature with relation to the role of isoflavones in breast and hormone-related cancers. The aim of our study was to investigate the cytotoxicity, induction of apoptosis, and changes in apoptosis-related genes of maximal physiological serum levels of the isoflavone genistein (Gen) in MCF-7 tumoral cells and in HB4a non-tumoral cells. In addition, induction of cell cycle arrest was also investigated. Only supraphysiological levels of Gen (50 and 100 µM) were cytotoxic to these cell lines. Concentrations of 10 and 25 µM did not induce apoptosis and significant changes in expression of the studied genes. Positive results were found only in cell cycle analysis: G0/G1 delay of MCF-7 cells in both concentrations of Gen and at 25 µM in HB4a cells. It is the first study investigating effects of Gen in the HB4a cell line. Thus, despite the lack of apoptosis induction (generally found with high concentrations), Gen at physiologically relevant serum levels still exerts chemopreventive effects through the modulation of cell cycle.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/fisiopatologia , Genisteína/farmacologia , Glycine max/química , Extratos Vegetais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Genisteína/sangue , Humanos , Células MCF-7 , Extratos Vegetais/sangue , Fase de Repouso do Ciclo Celular/efeitos dos fármacosRESUMO
BACKGROUND: Glibenclamide (Gli) binds to the sulphonylurea receptor (SUR) that is a regulatory subunit of ATP-sensitive potassium channels (KATP channels). Binding of Gli to SUR produces the closure of KATP channels and the inhibition of their activity. This drug is widely used for treatment of type 2-diabetes and it has been signaled as antiproliferative in several tumor cell lines. In previous experiments we demonstrated the antitumoral effect of Gli in mammary tumors induced in rats. The aim of the present work was to investigate the effect of Gli on MDA-MB-231 breast cancer cell proliferation and to examine the possible pathways involved in this action. RESULTS: The mRNA expression of the different subunits that compose the KATP channels was evaluated in MDA-MB-231 cells by reverse transcriptase-polymerase chain reaction. Results showed the expression of mRNA for both pore-forming isoforms Kir6.1 and Kir6.2 and for the regulatory isoform SUR2B in this cell line. Gli inhibited cell proliferation assessed by a clonogenic method in a dose dependent manner, with an increment in the population doubling time. The KATP channel opener minoxidil increased clonogenic proliferation, effect that was counteracted by Gli. When cell cycle analysis was performed by flow cytometry, Gli induced a significant cell-cycle arrest in G0/G1 phase, together with an up-regulation of p27 levels and a diminution in cyclin E expression, both evaluated by immunoblot. However, neither differentiation evaluated by neutral lipid accumulation nor apoptosis assessed by different methodologies were detected. The cytostatic, non toxic effect on cell proliferation was confirmed by removal of the drug.Combination treatment of Gli with tamoxifen or doxorubicin showed an increment in the antiproliferative effect only for doxorubicin. CONCLUSIONS: Our data clearly demonstrated a cytostatic effect of Gli in MDA-MB-231 cells that may be mediated through KATP channels, associated to the inhibition of the G1-S phase progression. In addition, an interesting observation about the effect of the combination of Gli with doxorubicin leads to future research for a potential novel role for Gli as an adjuvant in breast cancer treatment.
Assuntos
Antineoplásicos/farmacologia , Glibureto/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Fase G1/efeitos dos fármacos , Humanos , Hipoglicemiantes/farmacologia , Canais KATP/genética , Potencial da Membrana Mitocondrial/efeitos dos fármacos , RNA Mensageiro/metabolismo , Fase de Repouso do Ciclo Celular/efeitos dos fármacosRESUMO
STGC3 is a potential tumor suppressor in nasopharyngeal carcinoma. We previously found that CNE2 cells that re-expressed STGC3 formed smaller tumors in female mice than in male mice. Here, we investigated the sexual dimorphism of STGC3 as a tumor-suppressor in female and male nude mice injected subcutaneously with pcDNA3.1(+)-STGC3/CNE2 cells. ER-α was positively expressed in vitro in the CNE2 cells. The pcDNA3.1(+)-STGC3/CNE2 cell growth rate decreased after treatment with ß-estradiol in vitro. There were significant differences in tumor size or mass between pcDNA3.1(+)-STGC3/CNE2 and control cases (P < 0.05), but there were significant differences in tumor size between female and male nude mice in the STGC3 transfection groups, and the pcDNA3.1(+)-STGC3/CNE2 tumor growth rate in the female nude mice was the lowest in all cases (P < 0.05). There were no significant differences between female and male nude mice in control groups. Furthermore, a greater number of cells were blocked in the G(0)/G(1) phase in pcDNA3.1(+)-STGC3/ CNE2 tumor xenografts in the female mice. Protemic analysis found 9 differentially expressed proteins in the pcDNA3.1-STGC3/CNE2 xenograft tissues in females and males. A heat shock 70 protein 8 isoform 2 variant was identified as a down-regulated protein associated with cell cycle control and its downstream factor cyclin D1 was also decreased in STGC3-repressed xenografts in female mice. The data above suggest that STGC3 and its associated proteins play an important role in nasopharyngeal carcinoma gender differences.
Assuntos
Carcinoma/patologia , Genes Supressores de Tumor , Neoplasias Nasofaríngeas/patologia , Proteínas/genética , Animais , Carcinoma/metabolismo , Linhagem Celular Tumoral , Ciclina D1/metabolismo , Estradiol/fisiologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Proteínas de Choque Térmico HSC70/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/metabolismo , Transplante de Neoplasias , Proteínas/metabolismo , Proteínas/fisiologia , Fase de Repouso do Ciclo Celular , Caracteres Sexuais , Fatores Sexuais , Transcriptoma , Carga TumoralRESUMO
Amblyomin-X is a Kunitz-type serine protease inhibitor (Kunitz-type SPI) designed from the cDNA library of the Amblyomma cajennense tick, which displays in vivo anti-tumor activities. Here, the mechanisms of actions of Amblyomin-X in vascular endothelial growth factor A (VEGF-A)-induced angiogenesis were characterized. Topical application of Amblyomin-X (10 or 100 ng/10 µl; each 48 h) inhibited VEGF-A-induced (10 ng/10 µl; each 48 h) angiogenesis in the dorsal subcutaneous tissue in male Swiss mice. Moreover, similar effect was observed in the VEGF-A-induced angiogenesis in the chicken chorioallantoic membrane (CAM). Additional in vitro assays in t-End cells showed that Amblyomin-X treatment delayed the cell cycle, by maintaining them in G0/G1 phase, and inhibited cell proliferation and adhesion, tube formation and membrane expression of the adhesion molecule platelet-endothelial cell adhesion molecule-1 (PECAM-1), regardless of mRNA synthesis. Together, results herein reveal the role of Kunitz-type SPI on in vivo VEGF-A-induced angiogenesis, by exerting modulatory actions on endothelial cell proliferation and adhesion, especially on membrane expression of PECAM-1. These data provide further mechanisms of actions of Kunitz-type SPI, corroborating their relevance as scientific tools in the design of therapeutic molecules.