| ObjectiveThe proto-oncogene Bcl-2 was initially isolated from the t(14; 18)(q32;q21) chromosomal translocations in non-Hodgkin's B-cell lymphomas founded by Tsujimoto in 1985. The Bcl-2 gene encodes a 26-kDa protein that is known to be localized mainly at the outer mitochondrial membrane, as well as to the endoplasmic reticulum and nuclear membranes. Among some genes controlling the apoptotic process, the Bcl-2 proto-oncogene is known to be a key regulator of apoptosis, functioning as an anti-apoptotic protein with the ability to protect against a variety of physiologic or pathologic insults and environmental stimuli including chemotherapeutic agents, heat shock, radioactive ray, hydrogen dioxide, deprivation of growth factor and neurotoxin. Biochemical and genetic evidence indicates that Bcl-2 blocks most forms of apoptosis by preventing mitochondrial changes, such as the release of cytochrome c and an apoptosis-inducing factor from the intermembrane space into the cytoplasm. Bcl-2 is considered a new type of drug resistance gene for its ability to elevate the resistance of tumour cell to chemotherapy.Reactive oxygen species(ROS), such as O20-,·OH, and H2O2, are the principal species of intracellular oxidants. They are generated as by-products of electron transport through the mitochondrial respiratory chain. ROS can react with almost every critical cellular macromolecule, including DNA, lipid, protein, and carbohydrates, and cause functional as well as structural alterations in these biomolecules, which ultimately lead to cell death and tissue damage. ROS can lead to cell death by either necrosis or apoptosis, depending on the intensity of the oxidative stimuli. The localization of Bcl-2 at the site of oxygen free radical generation, and evidence that ROS are able to cause apoptosis in various cell lines have raised the possibility that Bcl-2 might prevent apoptosis by either acting as an antioxidant or by suppressing production of free radicals. Experimental data from in vitro and in vivo studies suggest that Bcl-2 may block apoptosis through regulation of cellular antioxidant defense mechanisms and, in this context, has been considered to act as a free radical scavenger.Although several lines of evidence suggest that Bcl-2 overexpressing cells have been shown to express relatively high levels of antioxidant enzymes and GSH, which lower steady-state concentrations of ROS and/or repair oxidative cellular damage, but the exact molecular mechanisms by which Bcl-2 regulates cellular ROS remain unresolved. Recently, increasing evidence supports the role of NF-κB in regulation of antiapoptotic gene expression and promotion of cell survival. The transcriptional regulation of antioxidant enzymes, such as CAT, SOD, and GPx, is mediated partially by NF-κB. Interestingly, the DNA binding activity of NF-κB and its transcriptional activity are constitutively elevated in Bcl-2-overexpressing cells, compared with those in vectortransfected counterparts. It is reasonable to speculate that potentiation of cellular antioxidant capacity by Bcl-2 maybe involving activation of NF-κB.As the most frequent urinary system tumour in our country, bladder carcinoma is inclined to recurrence and progression post operation. Prevention of bladder carcinoma recurrence and progression has long puzzled clinician and become an important direction in urinary study. In this study, we reconstructed the first time a stable human Bcl-2 transfected cell line, BIU87/Bcl-2, that was derived from the transfection of human bladder carcinoma cell line BIU87 with a plasmid vector containing recombinant Bcl-2 [pcDNA3.1(+)-Bcl-2]. We examined the apoptosis and intracellular ROS in BIU87, BIU87/neo, and BIU87/Bcl-2 cells induced by adriamycin using flow cytometry in order to discuss the effect of Bcl-2 on apoptosis and intracellular ROS. We examined intracellular activity of SOD and CAT in BIU87/neo and BIU87/Bcl-2 cells induced by adriamycin using spectrophotometer for the purpose to discuss the effect of Bcl-2 on intracellular antioxidase. We also extracted protein of cytoplasm and nucleus in BIU87/neo and BIU87/Bcl-2 cells treated with adriamycin and examined the expression of NF-κB p50, p65, and IκBαto discuss the effect of Bcl-2 on NF-κB activation, thus reveal the pathway of Bcl-2 regulates intracellular ROS in bladder carcinoma.MethodA Bcl-2-expressing plasmid [pcDNA3.1(+)-Bcl-2] was prepared using the standard recombinant DNA methods. The transfection of the expression vector pcDNA3.1(+)-Bcl-2 to the BIU87 cell line was performed by a lipofection method using Lipofectamine 2000. G418-resistant transformant was obtained from the cell line, and stable monoclonal transformant expressing the human Bcl-2 were selected by RT-PCR and Western blot analysis. After BIU87, BIU87/neo and BIU87/Bcl-2 cells were treated with adriamycin at different concentrations for 24 hours, the cell viabilities were quantified by MTT method, the apoptosis rates and the level of intracellular ROS were determined using flow cytometric analysis, and the morphological changes were detected by using acridine orange fluorescent staining method. We extracted protein of endochylema and nucleus in BIU87/neo and BIU87/Bcl-2 cells treated with adriamycin respectively and examined the expression of NF-κB p50, p65 in nucleus and IκBαin cytoplasm using Western blot analysis. We also detected the intracellular activity of superoxide dismutase and catalase using xanthine oxidase method and visible light method.Results1. Establishment of Bcl-2 over-expression bladder carcinoma cell line and it's sensitivity to ADR(1)Identification of eukaryotic expression vectorBeing inserted by incision enzyme EcoR I and Xho I, the plasmid pcDNA3.1(+)-Bcl-2 expressed an 847bp cDNA fragment. Nucleic acid sequence analysis confirmed further that Bcl-2 located between the site of EcoR I and Xho I in pcDNA3.1(+).(2)Expression of Bcl-2 in BIU87, BIU87/neo and BIU87/Bcl-2After transfection of BIU87 cells with pcDNA3.1(+)-Bcl-2, we selected a stable mono-clonal Bcl-2 transfectant from the G418-resistant cells by Western blotting using a highly specific anti-human Bcl-2 antibody and established the monoclonal cell line BIU87/Bcl-2. RT-PCR and Western blot analysis of Bcl-2 expression in the BIU87(parent), control vector(pcDNA3.1(+)-neo), and Bcl-2 vector(pcDNA3.1(+)-Bcl-2) transfected cells confirmed very high level expression of the protein in the latter.(3)Effect of adriamycin treatment on the inhibition of the transfected cell linesTo examine whether Bcl-2 over-expression decreases sensibility of adriamycin on BIU87, we examined the % inhibition of BIU87, BIU87/neo and BIU87/Bcl-2 cell line after adriamycin treatment for 24 hours. The % inhibition of BIU87/Bcl-2 cell line after adriamycin treatment was lower than BIU87 and BIU87/neo cell line, and there is no significant difference between the latter two cell line.2. The effect of Bcl-2 on adriamicin induced BIU87 cells apoptosis and elevation of intracellular ROS level(1)Bcl-2 inhibits adriamycin-mediated apoptosisTo investigate the effect of Bcl-2 on adriamycin-mediated apoptosis, BIU87, BIU87/neo and BIU87/Bcl-2 cell lines were treated with adriamycin at concentrations of 6.25, 12.5 or 25μ/ml for 24 hours. Flow cytometric determination of the DNA content of control and adriamycin treated propidium iodide(PI)-stained BIU87, BIU87/neo and BIU87/Bcl-2 cells indicated that a sub-G1 cell population appeared at 24 hours following adriamycin treatment. The results show that with the raising of the concentration of adriamycin the apoptosis rates were added in the three cell lines. When be treated by adriamycin, the apoptosis rate of BIU87/Bcl-2 cell line was lower than the other two ones. The results indicated that Bcl-2 over-expression can inhibit adriamycin-induced BIU87 apoptosis. Morphological changes were detected by using acridine orange fluorescent staining method, which is another hallmark of apoptosis. In our study, flavovirens hyperchromatic plaque could be seen in nucleus in BIU87, BIU87/neo and BIU87/Bcl-2 cell lines when they were treated by 12.5μg/ml of adriamycin respectively, which demonstrated apoptosis occurred in the three cell lines under the induce of adriamycin.(2)Bcl-2 inhibits adriamycin-mediated elevation of intracellular ROS levelAfter BIU87, BIU87/neo and BIU87/Bcl-2 cell lines were treated with adriamycin at concentrations of 6.25, 12.5 or 25μg/ml for 24 hours, Flow cytometry was used to determinate intracellular ROS level. The results show that with the raising of the concentration of adriamycin the intracellular ROS levels were added in the three cell lines. When be treated by adriamycin, the intracellular ROS level raising of BIU87/Bcl-2 cell line was lower than the other two ones.(3)Correlation analysis of intracellular ROS level and apoptosis rateSpearman analysis suggest that there is a positive correlation between intracellular ROS level and apoptosis rate in BIU87, BIU87/neo and BIU87/Bcl-2 cell lines after they were treated by different concentration of adriamicin for 24 hours.3. The effect of Bcl-2 on NF-κB activation and antioxidase activity changing induced by adriamicin in BIU87 cells(1)Detection of NF-κB activationWestern blot analysis results show that after be treated with adriamycin, cytoplasmic IκBαlevels and nuclear p50 and p65 protein were all increased in BIU87/neo and BIU87/Bcl-2 cells. Compared with BIU87/neo cells, a significant decrease in IκBαin the cytoplasm with a concomitant increase in p50 and p65 in the nucleus can be seen in BIU87/Bcl-2 cells(2)Changes of SOD and CAT activityCompared with control cells, BIU87/neo and BIU87/Bcl-2 cells were treated with adriamycin for 24 hours showed that the activity of SOD as well as CAT was decreased. Compared with BIU87/neo cells, BIU87/Bcl-2 cells showed increases on SOD and CAT activity.Conclusions1. Stable over-expression Bcl-2 gene bladder carcinoma cell line BIU87/Bcl-2 can be established successfully by a lipofection method.2. Over-expression of Bcl-2 decreases the sensitivity to adriamicin in bladder carcinoma cell line BIU87.3. Over-expression of Bcl-2 blocks adriamicin induced apoptosis in bladder carcinoma cell line BIU87 involving its ability to decrease intracellular ROS level.4. Bcl-2 promotes the activation of NF-κB through the degradation of IκBαin bladder carcinoma cell line BIU87.5. Bcl-2 inhibits the decrease of SOD and CAT induced by ADR in bladder carcinoma cell line BIU87.6. The way of Bcl-2 decreases intracellular ROS level may be as follows: Bcl-2 promotes activition of NF-κB, leading to increase of intracellular antioxygen enzyme and then lower intracellular ROS level. |
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