RESUMO
MP [4-(3',3'-dimethylallyloxy)-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniae isolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27(KIP1) protein and p21(CIP1) mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21(CIP1), p16(INK4a) and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation of p73, JunB, FKHR, and Bim. The results indicate that MP may be a potential treatment for cervical cancer.
Assuntos
Apoptose/efeitos dos fármacos , Benzofuranos/administração & dosagem , Endófitos/química , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Xylariales/química , Proteínas Reguladoras de Apoptose/genética , Benzofuranos/isolamento & purificação , Proteínas de Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cycadopsida , Inibidor de Quinase Dependente de Ciclina p21/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27/efeitos dos fármacos , Proteínas de Ligação a DNA/efeitos dos fármacos , Citometria de Fluxo , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/efeitos dos fármacos , Genes p16/efeitos dos fármacos , Células HeLa , Humanos , Proteínas Nucleares/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/efeitos dos fármacos , Transcrição Gênica , Proteína Tumoral p73 , Proteínas Supressoras de Tumor/efeitos dos fármacosRESUMO
Arsenic is carcinogenic, possibly partly through epigenetic mechanisms. We evaluated the effects of arsenic exposure and metabolism on DNA methylation. Arsenic exposure and methylation efficiency in 202 women in the Argentinean Andes were assessed from concentrations of arsenic metabolites in urine (inorganic arsenic, methylarsonic acid [MMA], and dimethylarsinic acid [DMA]), measured by HPLC-ICPMS. Methylation of CpGs of the tumor suppressor gene p16, the DNA repair gene MLH1, and the repetitive elements LINE1 was measured by PCR pyrosequencing of blood DNA. Genotyping (N = 172) for AS3MT was performed using Sequenom™, and gene expression (N = 90) using Illumina DirectHyb HumanHT-12 v3.0. Median arsenic concentration in urine was 230 µg L(-1) (range 10.1-1251). In linear regression analysis, log(2)-transformed urinary arsenic concentrations were positively associated with methylation of p16 (ß = 0.14, P = 0.0028) and MLH1 (ß = 0.28, P = 0.0011), but not with LINE1. Arsenic concentrations were of borderline significance negatively correlated with expression of p16 (r(s) = -0.20; P = 0.066)), but not with MLH1. The fraction of inorganic arsenic was positively (ß = 0.026; P = 0.010) and DMA was negatively (ß = -0.017, P = 0.043) associated with p16 methylation with no effect of MMA. Carriers of the slow-metabolizing AS3MT haplotype were associated with more p16 methylation (P = 0.022). Arsenic exposure was correlated with increased methylation, in blood, of genes encoding enzymes that suppress carcinogenesis, and the arsenic metabolism efficiency modified the degree of epigenetic alterations.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Intoxicação por Arsênico/genética , Arsênio/análise , Metilação de DNA/efeitos dos fármacos , Exposição Ambiental/análise , Genes p16/efeitos dos fármacos , Proteínas Nucleares/genética , Adolescente , Adulto , Argentina , Intoxicação por Arsênico/sangue , Intoxicação por Arsênico/metabolismo , Intoxicação por Arsênico/urina , Feminino , Marcadores Genéticos/genética , Haplótipos , Humanos , Metiltransferases/genética , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Estatísticas não Paramétricas , Abastecimento de ÁguaRESUMO
The testicular feminized (Tfm) mouse carries a nonfunctional androgen receptor (AR) and reduced circulating testosterone levels. We used Tfm and castrated mice to determine whether testosterone modulates markers of aging in cardiomyocytes via its classic AR-dependent pathway or conversion to estradiol. Male littermates and Tfm mice were divided into 6 experimental groups. Castrated littermates (group 1) and sham-operated Tfm mice (group 2, N = 8 each) received testosterone. Sham-operated Tfm mice received testosterone in combination with the aromatase inhibitor anastrazole (group 3, N = 7). Castrated littermates (group 4) and sham-operated untreated Tfm mice (group 5) were used as controls (N = 8 and 7, respectively). An additional control group (group 6) consisted of age-matched non-castrated littermates (N = 8). Cardiomyocytes were isolated from the left ventricle, telomere length was measured by quantitative PCR and expression of p16INK4α, retinoblastoma (Rb) and p53 proteins was detected by Western blot 3 months after treatment. Compared with group 6, telomere length was short (P < 0.01) and expression of p16INK4α, Rb and p53 proteins was significantly (P < 0.05) up-regulated in groups 4 and 5. These changes were improved to nearly normal levels in groups 1 and 2 (telomere length = 0.78 ± 0.05 and 0.80 ± 0.08; p16INK4α = 0.13 ± 0.03 and 0.15 ± 0.04; Rb = 0.45 ± 0.05 and 0.39 ± 0.06; p53 = 0.16 ± 0.04 and 0.13 ± 0.03), but did not differ between these two groups. These improvements were partly inhibited in group 3 compared with group 2 (telomere length = 0.65 ± 0.08 vs 0.80 ± 0.08, P = 0.021; p16INK4α = 0.28 ± 0.05 vs 0.15 ± 0.04, P = 0.047; Rb = 0.60 ± 0.06 vs 0.39 ± 0.06, P < 0.01; p53 = 0.34 ± 0.06 vs 0.13 ± 0.03, P = 0.004). In conclusion, testosterone deficiency contributes to cardiomyocyte aging. Physiological testosterone can delay cardiomyocyte aging via an AR-independent pathway and in part by conversion to estradiol.
Assuntos
Envelhecimento/metabolismo , Senescência Celular/fisiologia , Estradiol/metabolismo , Miócitos Cardíacos/fisiologia , Receptores Androgênicos/metabolismo , Testosterona/farmacologia , Envelhecimento/patologia , Animais , Biomarcadores/análise , Genes p16/efeitos dos fármacos , Masculino , Camundongos , Modelos Animais , Orquiectomia , Distribuição Aleatória , Proteína do Retinoblastoma/metabolismo , Encurtamento do Telômero/efeitos dos fármacos , Testosterona/deficiência , Proteína Supressora de Tumor p53/metabolismoRESUMO
This study was undertaken to investigate, by immunohistochemistry, the expression of some tumor suppressor genes such as p16, p21 and Retinoblastoma (Rb) during 4-Nitroquinoline 1-oxide induced rat tongue carcinogenesis. Male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution through their drinking water for 4, 12 or 20 weeks. Ten animals were used as negative control. Neither histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure, nor statistically significant differences (p>0.05) in expression of all the tumor suppressor genes were found when compared to the negative control. However, the levels of Rb were increased (p<0.05) in pre-neoplastic lesions at 12 weeks following carcinogen exposure. In well-differentiated squamous cell carcinoma induced after 20 weeks of treatment with 4NQO, p16 and Rb were expressed in some tumor cells. Taken together, the results support the belief that the expression of Rb is closely event-related to malignant transformation and conversion of the oral mucosa, being a reliable biomarker linked to oral cancer pathogenesis.