RESUMO
This present study explored the association of Pyruvate Kinase isozyme M2 (PKM2), Phosphofructokinase 1 (PFK-1) and Adenine nucleotide translocator 1 (ANT1) with cervical carcinoma. A case-control method was designed by the collected 95 cervical biopsy samples, which were divided into 30 controls and 60 cases. Cases were subdivided into mild cervical carcinoma (MCC-25), intermediate cervical carcinoma (ICC-20), and severe cervical carcinoma (SCC-20) by method of cervical pathology. The expression of PKM2, PFK-1, and ANT1 was examined by methods of immunohistochemistry and western blotting (WB). The results showed that the positive proportions of PKM2 and PFK-1 in case group were higher than that of control, and the increased positive proportions of PKM2 and PFK-1 were also revealed with the order of Control, MCC, ICC, SCC (P<0.05). Further, the results of WB confirmed the enhanced expressions of PKM2 and PFK-1 in case group and the increasing trend of PKM2 and PFK-1 expressions in Control, MCC, ICC, and SCC groups. In addition, the WB result of ANT1 showed a lower level of expression in SCC group, while the positive proportion of ANT1 was not significant between cases group and control. In conclusion, PKM2 and PFK-1 genes are associated closely with cervical carcinoma. The enhanced expressions of PKM2 and PFK-1 indicate one developing signal of cervical carcinoma.
Assuntos
Translocador 1 do Nucleotídeo Adenina/análise , Proteínas de Transporte/análise , Colo do Útero/química , Proteínas de Membrana/análise , Fosfofrutoquinase-1/análise , Hormônios Tireóideos/análise , Neoplasias do Colo do Útero/química , Western Blotting , Estudos de Casos e Controles , Colo do Útero/patologia , Feminino , Glicólise , Humanos , Imuno-Histoquímica , Proteínas de Ligação a Hormônio da TireoideRESUMO
This study explored the reduction of adenosine triphosphate (ATP) levels in L-02 hepatocytes by hexavalent chromium (Cr(VI)) using chi-square analysis. Cells were treated with 2, 4, 8, 16, or 32 µM Cr(VI) for 12, 24, or 36 h. Methyl thiazolyl tetrazolium (MTT) experiments and measurements of intracellular ATP levels were performed by spectrophotometry or bioluminescence assays following Cr(VI) treatment. The chi-square test was used to determine the difference between cell survival rate and ATP levels. For the chi-square analysis, the results of the MTT or ATP experiments were transformed into a relative ratio with respect to the control (%). The relative ATP levels increased at 12 h, decreased at 24 h, and increased slightly again at 36 h following 4, 8, 16, 32 µM Cr(VI) treatment, corresponding to a "V-shaped" curve. Furthermore, the results of the chi-square analysis demonstrated a significant difference of the ATP level in the 32-µM Cr(VI) group (P < 0.05). The results suggest that the chi-square test can be applied to analyze the interference effects of Cr(VI) on ATP levels in L-02 hepatocytes. The decreased ATP levels at 24 h indicated disruption of mitochondrial energy metabolism and the slight increase of ATP levels at 36 h indicated partial recovery of mitochondrial function or activated glycolysis in L-02 hepatocytes.
Assuntos
Trifosfato de Adenosina/metabolismo , Carcinógenos Ambientais/toxicidade , Cromo/toxicidade , Hepatócitos/efeitos dos fármacos , Trifosfato de Adenosina/química , Análise de Variância , Animais , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Distribuição de Qui-Quadrado , China , Corantes , Hepatócitos/metabolismo , Humanos , Mitocôndrias Hepáticas/metabolismo , Sais de Tetrazólio , TiazóisRESUMO
The present study explored the ability of the voltage-dependent anion channel 1 (VDAC1) mRNA and ROS levels to modulate the effects of hexavalent chromium Cr(VI) on the adenosine triphosphate (ATP) level and the rate of apoptotic cell death. Cultured L-02 cells were pretreated with 20mM N-acetyl-cysteine (NAC) for 24h or transiently transfected with small interfering RNAs targeting VDAC1 (siVDAC1) for 48h; cells that were not pretreated were used as the control. The cells were subsequently treated with 0, 2, 8, or 32µM Cr(VI) for 24h. Then, levels of VDAC1 mRNA, ROS, and ATP and the apoptosis rate were measured by reverse-transcription quantitative PCR, fluorometry, a bioluminescence assay, and flow cytometry, respectively. The results showed that Cr(VI) at 32µM led to increase in the ROS level, VDAC1 mRNA expression, and the apoptosis rate and a decrease in the ATP level; pretreatment with NAC led to the down-regulation in the levels of ROS, VDAC1 mRNA and apoptosis and the significant up-regulation in the ATP levels. Interestingly, after the pretreatment with siVDAC1 to inhibit VDAC1 mRNA expression, the increased apoptosis rates and decreased ATP levels were reversed as well. These results suggested that changes in the ROS or VDAC1 mRNA levels were associated with changes in the ATP level and apoptosis rate. Furthermore, correlation analysis confirmed the association between both the ROS and VDAC1 levels and both the ATP level and the apoptosis rate. In conclusion, Cr(VI) induced ROS- and VDAC1-mediated decreases in ATP levels and increases in the apoptosis rate.
Assuntos
Trifosfato de Adenosina/metabolismo , Carcinógenos/toxicidade , Cromo/toxicidade , Poluentes Ambientais/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Canal de Ânion 1 Dependente de Voltagem/genética , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , RNA Mensageiro/metabolismoRESUMO
<p><b>OBJECTIVE</b>To study in vitro sperm damage caused by trichloroethylene in male rats.</p><p><b>METHODS</b>Sperms of Sprague-Dawley (SD) rats were collected 4 hours after being contaminated by trichloroethylene of 0, 2, 4, 6, 8, and 10 mmol/L in vitro. Giemsa staining was performed to observe the morphological changes of sperms, and flow cytometer was used to detect the changes in mitochondrial membrane potential.</p><p><b>RESULTS</b>The sperm motilities in 6, 8, and 10 mmol/L trichloroethylene groups decreased significantly compared with that in control group (P <0.01); the sperm aberration rates in 8 and 10 mmol/L trichloroethylene groups were significantly higher than that in control group (P<0.01). With the increase in exposure dose, the proportion of sperms with reduced mitochondrial membrane potential increased, and there were significant differences in sperm apoptosis rate between the 4, 6, 8, and 10 mmol/L trichloroethylene groups and control group (P<0.01).</p><p><b>CONCLUSION</b>In vitro exposure to trichloroethylene can reduce sperm motility and increase the aberration rate and apoptosis rate of sperms in male SD rats.</p>
Assuntos
Animais , Masculino , Ratos , Apoptose , Potencial da Membrana Mitocondrial , Ratos Sprague-Dawley , Motilidade dos Espermatozoides , Espermatozoides , Biologia Celular , Tricloroetileno , ToxicidadeRESUMO
This study explored the reduction of adenosine triphosphate (ATP) levels in L-02 hepatocytes by hexavalent chromium (Cr(VI)) using chi-square analysis. Cells were treated with 2, 4, 8, 16, or 32 μM Cr(VI) for 12, 24, or 36 h. Methyl thiazolyl tetrazolium (MTT) experiments and measurements of intracellular ATP levels were performed by spectrophotometry or bioluminescence assays following Cr(VI) treatment. The chi-square test was used to determine the difference between cell survival rate and ATP levels. For the chi-square analysis, the results of the MTT or ATP experiments were transformed into a relative ratio with respect to the control (%). The relative ATP levels increased at 12 h, decreased at 24 h, and increased slightly again at 36 h following 4, 8, 16, 32 μM Cr(VI) treatment, corresponding to a "V-shaped" curve. Furthermore, the results of the chi-square analysis demonstrated a significant difference of the ATP level in the 32-μM Cr(VI) group (P < 0.05). The results suggest that the chi-square test can be applied to analyze the interference effects of Cr(VI) on ATP levels in L-02 hepatocytes. The decreased ATP levels at 24 h indicated disruption of mitochondrial energy metabolism and the slight increase of ATP levels at 36 h indicated partial recovery of mitochondrial function or activated glycolysis in L-02 hepatocytes.
Assuntos
Animais , Humanos , Trifosfato de Adenosina/metabolismo , Carcinógenos Ambientais/toxicidade , Cromo/toxicidade , Hepatócitos/efeitos dos fármacos , Análise de Variância , Trifosfato de Adenosina/química , Técnicas de Cultura de Células , Distribuição de Qui-Quadrado , China , Corantes , Sobrevivência Celular/efeitos dos fármacos , Hepatócitos/metabolismo , Mitocôndrias Hepáticas/metabolismo , Sais de Tetrazólio , TiazóisRESUMO
<p><b>OBJECTIVE</b>To construct DNA methyltransferase 1 (DNMT1) low expression 16HBE cell line and observe the variation of cell cycle and global genomic DNA methylation.</p><p><b>METHODS</b>The method of Lenti-virus induced RNA interference was applied to introduce four different shRNA fragment into 16HBE cells. Flow cytometry and 5-mC immunofluorescence methods were used to observe the cell cycle and global DNA methylation status of DNMT1 low expression 16HBE cells.</p><p><b>RESULTS</b>The DNMT1 protein relative expression level of 16HBE-shDNMT1-4 cell line was down regulated about 44% (P < 0.05) compared with the control. No obvious differences of cell cycle and global genome DNA methylation status were observed between the 16HBE and 16HBE-shDNMT1.</p><p><b>CONCLUSION</b>The DNMT1 gene low expression cell is successfully constructed, and there are no obvious changes happened on the cell cycle and global genomic DNA methylation.</p>
Assuntos
Humanos , Ciclo Celular , Linhagem Celular , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases , Genética , Metabolismo , Metilação de DNA , Regulação para Baixo , Células Epiteliais , Metabolismo , Interferência de RNA , RNA Interferente Pequeno , GenéticaRESUMO
<p><b>OBJECTIVE</b>To investigate the effects of hydroquinone (HQ) on expression of Polymerase eta (Pol eta) and DNA damage in human hepatic cells (L-02), and to explore the role and possible mechanism of Pol eta involved in the process of DNA damage-tolerance.</p><p><b>METHODS</b>After L-02 hepatic cells were exposed to HQ with various concentrations (0, 5, 10, 20, 40, 80 and 160 micromol/L) for 24 h, cell survival rate was detected by MTT assay; DNA impairment was detected by single cell gel electrophoresis (SCGE); Real-time fluorescent quantitative PCR and Western blotting methods were used to measure the expression of Pol eta at the mRNA and protein level in L-02 hepatic cells exposed to HQ with various concentrations (0, 5, 10, 20, 40, 80 and 160 micromol/L).</p><p><b>RESULTS</b>MTT assay showed that HQ with concentrations from 0 to 80 micromol/L had little effect on the survival rate of L-02 (P>0.05); whereas the survival rate of the group of 160 micromol/Lwas significantly higher than that of the control (P<0.01) after being treated with HQ for 24 h; the higher dose of HQ presented, the more degrees of DNA damage were produced. It was found that HQ in a low concentration (1-80 micromol/L) could induce the expression of Pol eta which was in proportion to the increasements of HQ concentration; the expression levels of mRNA and protein were reached to the maximum when treated with 80 micromol/L; the expression of Pol eta decreased (the relative quantity values were 2.32 +/- 0.16 and 1.20 respectively) once the concentration of HQ exceeded 160 micromol/L as compared with the group of 80 micromol/L, but it was higher than that of the control.</p><p><b>CONCLUSION</b>This study suggested that Pol eta might involve in the process of DNA damage-tolerance induced by HQ in the hepatic cells.</p>
Assuntos
Humanos , Sobrevivência Celular , Células Cultivadas , Dano ao DNA , Reparo do DNA , DNA Polimerase Dirigida por DNA , Metabolismo , Hepatócitos , Metabolismo , Hidroquinonas , MutagênicosRESUMO
<p><b>OBJECTIVE</b>To investigate the effects of hydroquinone (HQ) on expression of ubiquitin-ligating enzyme Rad18 in human hepatic cells (L-02), and to explore the role and possible mechanism of Rad18 involved in toxicity of HQ to hepatic cells.</p><p><b>METHODS</b>After L-02 hepatic cells were exposed to HQ with various concentrations (0, 5, 10, 20, 40, 80 and 160 micromol/L) for 24 h, cell survival rate was measured by MTT assay; DNA impairment was evaluated by single cell gel electrophoresis (SCGE); The expression levels of Rad18 mRNA and protein were detected by Real-time fluorescent quantitative polymerase chain reaction (QPCR) technique and Western blot method respectively.</p><p><b>RESULTS</b>HQ with concentration from 0 to 80 micromol/L had little effect on survival rate of L-02 (P > 0.05); Whereas the survival rate in the group of 160 micromol/L was significantly lower than in the control with the significant difference (P < 0.01) after treated with HQ for 24 h; The higher dose of HQ presented, the more degrees of olive tail moment (OTM) were produced and a dose-dependent relationship was shown. HQ in a low concentration (0 to approximately 40 micromol/L) could induce increase in the expression of Rad18 mRNA and protein which was in proportion to the increment of HQ concentration; the expression of Rad18 mRNA was enhanced increasingly, while the expression of Rad18 protein unchanged basically once the concentration of HQ exceeded 40 micromol/L; Besides, there was a positive correlation between OTM and the expression level of Rad18 mRNA (r = 0.919, P < 0.01).</p><p><b>CONCLUSION</b>HQ could regulate up the expression of Rad18 in L-02 hepatic cells.</p>