RESUMO
Osteosarcoma (OS) causes millions of death worldwide and, since there is no effective therapy, it is necessary to identify the molecular mechanism of OS, which can direct the development of new therapies. This study investigated the role of bone morphogenetic protein 9 (BMP9), a member of the transforming growth factor (TGF)-ß family, in OS development. This study first examined BMP9 expression in tissue from OS patients and normal subjects. The OS cell line (MG63) and tumor cells from OS patients were then transfected with BMP9 and cell proliferation and apoptosis were assessed. Western blot and reverse transcription-polymerase chain reaction were used to study the expression of cancer-related genes [B cell lymphoma (Bcl)-2, cleaved Caspase-3, Caspase-9, and poly ADP-ribose polymerase]. To confirm the in vivo impact of BMP9, mice were transplanted with OS tumor cells and then treated with BMP9 carried in attenuated Salmonella enterica serovar Typhimurium. Our study found that the OS tumor tissue had a lower expression of BMP9 compared to normal tissue. Transfection of BMP9 in OS and MG63 cells inhibited cell growth and promoted apoptosis. In vitro studies showed a decrease in Bcl-2 gene expression and an increase in Cyto-c, Caspase-3, and Caspase-9 expression. In vivo studies indicated that consistent treatment with BMP9 in OS mice results in inhibition of tumor growth. This study shows that BMP9 inhibition is associated with OS development and that enhanced expression of BMP9 may be a potential treatment method for OS.
Assuntos
Neoplasias Ósseas/genética , Fatores de Diferenciação de Crescimento/biossíntese , Osteossarcoma/genética , Animais , Apoptose/genética , Neoplasias Ósseas/patologia , Caspase 3/biossíntese , Caspase 3/genética , Caspase 9/biossíntese , Caspase 9/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Fator 2 de Diferenciação de Crescimento , Fatores de Diferenciação de Crescimento/genética , Humanos , Camundongos , Osteossarcoma/patologia , Poli(ADP-Ribose) Polimerases/genética , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Salmonella enterica/patogenicidade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The dibenzylbutyrolactone lignan (-)-cubebin, which is extracted from the seeds of the pepper Piper cubeba, has shown promise as an anti-inflammatory, analgesic, leishmanicidal, antiproliferative, and trypanocidal compound. Given the therapeutic potential of (-)-cubebin, this study aimed to investigate its safety profile by analyzing cytotoxicity, mutagenicity, cell proliferation kinetics, induction of apoptosis, and expression of pro-apoptotic genes in human colon adenocarcinoma cells (HT29) exposed to (-)-cubebin. MTT cytotoxicity assays demonstrated that (-)-cubebin was cytotoxic only at 280 µM, whereas it was not cytotoxic at 2.8, 14, or 28 µM. Data demonstrated that (-)-cubebin was not mutagenic as evidenced by a micronucleus (MN) assay, did not alter cell-growth kinetics over 4 d, and showed absence of induced apoptosis after 24 h. Further, CASP8 and CASP9 gene expression was not markedly changed in HT29 cells exposed to 28 µM or 70 µM (-)-cubebin for 12 h. Based on our observations, (-)-cubebin was cytotoxic at a concentration of 280 µM, suggesting that the use of this concentration should be avoided. However, lower concentrations exerted no apparent damaging effects, indicating that this lignan is safe to use for pharmacological purposes at certain concentrations.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos Fitogênicos/farmacologia , Neoplasias do Colo/tratamento farmacológico , Lignanas/farmacologia , Piper/química , Apoptose/efeitos dos fármacos , Caspase 8/biossíntese , Caspase 9/biossíntese , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA/efeitos dos fármacos , Células HT29 , Humanos , Testes para MicronúcleosRESUMO
Ultrasound, a non-invasive therapy method, is a potential tool for medical applications, but its biological effects on vascular smooth muscle cells (VSMCs) have not been characterized. The aim of this study was to explore the effect and possible apoptotic mechanism of VSMCs that were induced by low-frequency ultrasound (LFU). Cell viability and apoptosis of A7r5 cells were evaluated after treating A7r5 cells with a continuous 45-kHz 1.0-W/cm(2) ultrasound (exposure time of 0, 10, 20, 30, and 35 s) by MTT assay and flow cytometry. At the optimum ultrasound exposure condition (30 s), gene chip analysis was performed, and the apoptotic signaling pathway was confirmed by reverse transcription-polymerase chain reaction and Western blot. As measured by flow cytometry, LFU significantly induced A7r5 cell apoptosis. Comparing the ultrasound group with the control group, the protein expression of caspase-9 and caspase-3 was increased by 50 and 57%, respectively; the caspase-3 mRNA level was increased by 37.5%. These findings indicate that an intrinsic pathway plays a major role in apoptosis that is induced by LFU and that LFU can induce A7r5 cell apoptosis via caspase-9- and caspase-3-dependent pathways.
Assuntos
Aorta/metabolismo , Apoptose/efeitos da radiação , Miócitos de Músculo Liso/metabolismo , Animais , Aorta/citologia , Aorta/efeitos da radiação , Caspase 3/biossíntese , Caspase 9/biossíntese , Linhagem Celular , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Miócitos de Músculo Liso/efeitos da radiação , RNA Mensageiro/biossíntese , Ratos , SomRESUMO
We investigated in vitro the effect of low-frequency and low-energy ultrasound (LFLEU) on apoptosis of an overexpressed HSP27 human aortic smooth muscle cell (HASMC) line. A frequency of 42.6 kHz was used in all experiments. HASMC were exposed to ultrasound and cell viability was evaluated by MTT reduction. Overexpressed HSP27-HASMC was constructed on a pcDNA3.1 vector. Apoptosis was determined 24 h after treatment by flow cytometry; gene display was evaluated with Affimax chips, and HSP27 mRNA and protein expression levels were measured by RT-PCR and Western blotting. The apoptosis rate (at 30 s) was significantly lower in HASMC transfected with HSP27 (7.14 ± 1.73%), compared with cells transfected with a mock plasmid (17.31 ± 2.72%) or a control group (14.23 ± 2.77%), indicating a protective function for apoptosis induced by LFLEU. Gene display analysis showed that caspase-9 expression in HSP27 cell lines was downregulated and caspase-3 upregulated. However, RT-PCR and Western blotting analysis indicated that both caspase-9 and caspase-3 were inhibited at both the mRNA and protein levels. We suggest that overexpressed HSP27 is capable of protecting the LFLEU from apoptosis and that the pathway for this protection is via downregulated caspase-9 and caspase-3 expression.
Assuntos
Apoptose/efeitos da radiação , Caspase 3/biossíntese , Caspase 9/biossíntese , Proteínas de Choque Térmico HSP27/genética , Músculo Liso Vascular/diagnóstico por imagem , Aorta/diagnóstico por imagem , Aorta/metabolismo , Caspase 3/genética , Caspase 9/genética , Linhagem Celular , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Regulação para Baixo , Expressão Gênica/efeitos da radiação , Proteínas de Choque Térmico HSP27/biossíntese , Humanos , Músculo Liso Vascular/citologia , RNA Mensageiro/biossíntese , Som , Estresse Fisiológico , UltrassonografiaRESUMO
BACKGROUND: Chronic hepatitis C (CHC) has emerged as a leading cause of cirrhosis in the U.S. and across the world. To understand the role of apoptotic pathways in hepatitis C virus (HCV) infection, we studied the mRNA and protein expression patterns of apoptosis-related genes in peripheral blood mononuclear cells (PBMC) obtained from patients with HCV infection. METHODS: The present study included 50 subjects which plasma samples were positive for HCV, but negative for human immunodeficiency virus (HIV) or hepatitis B virus (HBV). These cases were divided into four groups according to METAVIR, a score-based analysis which helps to interpret a liver biopsy according to the degree of inflammation and fibrosis. mRNA expression of the studied genes were analyzed by reverse transcription of quantitative polymerase chain reaction (RT-qPCR) and protein levels, analyzed by ELISA, was also conducted. HCV genotyping was also determined. RESULTS: HCV infection increased mRNA expression and protein synthesis of caspase 8 in group 1 by 3 fold and 4 fold, respectively (p < 0.05). In group 4 HCV infection increased mRNA expression and protein synthesis of caspase 9 by 2 fold and 1,5 fold, respectively (p < 0.05). Also, caspase 3 mRNA expression and protein synthesis had level augumented by HCV infection in group 1 by 4 fold and 5 fold, respectively, and in group 4 by 6 fold and 7 fold, respectively (p < 0.05). CONCLUSIONS: HCV induces alteration at both genomic and protein levels of apoptosis markers involved with extrinsic and intrinsic pathways.
Assuntos
Apoptose , Hepatite C Crônica/imunologia , Hepatite C Crônica/patologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Cirrose Hepática/patologia , Adulto , Biomarcadores/sangue , Western Blotting , Caspase 3/biossíntese , Caspase 8/biossíntese , Caspase 9/biossíntese , Feminino , Perfilação da Expressão Gênica , Genótipo , Hepacivirus/classificação , Hepacivirus/genética , Hepatite C Crônica/complicações , Hepatite C Crônica/virologia , Humanos , Fígado/patologia , Cirrose Hepática/etiologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Índice de Gravidade de DoençaRESUMO
To better understand the T-cell hyporesponsiveness of patients with paracoccidioidomycosis, we tested the hypothesis that the T cells were committed to apoptosis. We show here that T cells of patients with paracoccidioidomycosis overexpress caspase 9 and caspase 8 but express low Bcl-2 levels and that interleukin-2 was unable to revert the hyporesponsiveness. These data suggest that the T cells would in vivo be driven to a tolerant state and apoptosis.
Assuntos
Caspase 8/biossíntese , Caspase 9/biossíntese , Paracoccidioidomicose/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Linfócitos T/imunologia , Adulto , Idoso , Humanos , Tolerância Imunológica , Interleucina-2/imunologia , Pessoa de Meia-Idade , Adulto JovemRESUMO
Hexachlorobenzene (HCB) is a widespread environmental pollutant. Chronic exposure of humans to HCB produces a number of effects, such as triggering of porphyria, increased synthesis of liver microsomal enzymes, neurological symptoms, immunological disorders and thyroid dysfunctions. In rats, HCB induced hepatic porphyria, neurotoxic effects, and toxic effects on the reproductive system, thyroid function, and immune system. HCB is also known to cause tumors of the liver, thyroid and mammary gland in laboratory animals. The aim of this study was to investigate parameters of thyroid growth regulation, mainly cell proliferation and apoptosis in thyroid tissue from HCB (0.1, 1, 10, 100, and 500 mg/kg body weight)-treated female Wistar rats. The current study demonstrates that only the exposure to the highest HCB dose for 30 days, has adverse effects on thyroid endpoints examined related to thyroid gland morphology, and 3,3'5,5'-tetraiodothyronine (T(4), thyroxine) serum levels, without changes in thyroid-stimulating hormone concentrations or in thyroid gland weight. Morphological changes, included flattened epithelium and increased colloid size compared with control tissue. Transforming growth factor (TGF-beta1) mRNA levels, evaluated by RT-PCR, revealed a significant upregulation after exposure to HCB (1, 10, 100 mg/kg body weight). Cell proliferation evaluated by 5'-Br deoxiuridine incorporation into DNA, was not altered at any dose. HCB (1, 10, 100 mg/kg body weight) induces apoptosis, evaluated by in situ end labeling of fragmented DNA, terminal deoxynucleotidyl transferase-mediated deoxy uridine triphosphate nick-end labeling, in rat thyroid glands. This process is associated with dose-dependent increases in cytochrome c release from the mitochondria and procaspase-9 processing to its active product. Caspase-8 was not activated. These studies indicate that doses of HCB that do not disrupt thyroid economy induce TGF-beta1 expression and apoptosis in the thyroid gland, involving the mitochondrial pathway.