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Angiotensin Type2Receptor-mediated Apoptosis of EJ Bladder Cancer Cells

Author: WanPei
Tutor: TanWanLong
School: Southern Medical University,
Course: Surgery
Keywords: bladder cancer angiotensin type Ⅱ receptor adenovirus gene therapy
CLC: R737.14
Type: Master's thesis
Year: 2013
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Abstract

IntroductionBladder cancer is one of the common malignancies of the urinary tract. It has high morbidity in China. Traditional therapies including operation, radiotherapy and chemotherapy have limited value because of the damage to normal cells and the high recurrence rate. With the significant advances of molecular biology have been made in recent years, gene therapy has become a new strategy for the treatment of bladder cancer.Angiotensin Ⅱ (AngⅡ), a kind of bioactive peptide of the renin-angiotensin system (RAS), regulates cardiovascular homeostasis and tissue growth by binding to AT1receptor (AT1R) and AT2receptor (AT2R) on specific organs. AT2R is highly expressed in fetal tissues, while its expression is dramatically decreased after birth. When injury occurs in cardiovascular system or nervous system, AT2R is re-expressed in these tissues during the wound healing. Studies that have shown antiproliferative effect of AT2R suggest that AT2R might have potential in antigrowth in cancer. Increase the level of AT2R in numerous cancer cell lines, such as prostate cancer, lung cancer, pheochromocytoma and colorectal cancer cells, induces apoptosis and growth of cancer cells can be suppressed.In our experiments, recombinant adenoviral vectors expressing AT2R were transduced into bladder cancer cells to elevate the expression of AT2R. We have examined the effects of overexpression of AT2R on proliferation and apoptosis of bladder cancer cell lines and have discussed the probable intracellular mechanisms.Materials and MethodsCell CultureOne human bladder cancer cell line(EJ cell line) was cultured by biological technology college of Southern Medical University. DMEM was purchased from HYCLONE, Fetal bovine serum(FBS) and LipofectaminrM2000were purchased from Invitrogen. Polybrene was purchased from Santa Cruz Biotechnology. RIPA lysis buffer was purchased from Beijing Dingguochangsheng biological technology company.Recombinant Adenoviral ConstructsWe prepared two recombinant adenoviral vectors following the methods introduced by Li:an adenoviral vector containing the enhanced green fluorescent protein gene controlled by a cytomegalovirus promoter (Ad-CMV-EGFP) and an adenoviral vector containing genomic AT2R (G-AT2R) DNA and enhanced green fluorescent protein gene controlled by cytomegalovirus promoter (Ad-G-AT2R-EGFP).These were provided by Li Hongwei from the laboratory of biological technology college of Southern Medical University.293T cells were seeded into150mm2tissue culture plates.10multiplicity of infection (MOI, virus/cell) recombinant adenoviral vectors were transfected into293T cells.36to48h later, cytopathic effect (CPE) was observed from the cells. After freeze thawing and centrifugation, cells were purified through two cesium chloride gradients ultracentrifugation. We calculated the viral particles and purity according to the OD260nm and OD280nm of adenoviral DNA and tested the virus titer by Plaque assay (pfu/ml).Cell TreatmentsBladder cancer cells (EJ cell line,106/ml per hole) were seeded into150mm2tissue culture plates. On the next day, control vector Ad-CMV-EGFP or Ad-G-AT2R-EGFP was transduced into the bladder cancer cells. The cells grown in Dulbecco’s modified Eagle’s medium (DMEM)supplemented with10%fetal bovine serum (FBS).Then, observed the cell morphology24h,48h and72h later. Transduced cells were used48to72h later for apoptosis assessment.RNA isolation and concentration measurementTotal RNA was isolated from the cells using TRIzol kit. The concentration, OD260nm and OD280nm of the isolate RNA was tested by ultraviolet spectrophotometer.Expression of AT2R analysisThe expression of AT2R was detected by two methods:(1) observation of the expression of GFP in the bladder cancer cells transduced with adenoviral vectors;(2) expression of AT2R was analyzed using real-time PCR after the bladder cancer cells were transduced48hours later.Cell Cycle AnalysesAfter washing by PBS and centrifugation, the cells were fixed by addition of ice-cold70%ethanol for at least18h. Then, the cells were resuspended in300uL of0.1%PI (50mg/L propridium iodide,0.1%sodium-citrate,0.1%tritonX-100) and kept30min in the dark at room temperature. Cell cycle analyses were measured by flow cytometry and ModFit LT software (Verity).Apoptosis AssessmentTo detect apoptotic cells, we used a DeadEnd Colorimetric terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) System (Promega). Procedures were conducted according to the instructions of the kit. Cells which had brown color particles in the nucleus were TUNEL-positive cells.RNA Isolation and Reverse Transcription-PCREJ cells were cultured in their respective media and harvested for reverse transcription-PCR (RT-PCR). Total RNA was isolated from the cells using TRIZOL reagent (Invitrogen). Isolated RNA underwent DNase I treatment to remove genomic DNA and was then converted into cDNA with iScript cDNA synthesis kit (Bio-Rad). PCR of AT2R and18sRNA was done under the following conditions:dena-turing for15s at95℃, annealing for1min at60℃, and ex-tension for30s at72℃, with a total of40cycles. The oligonucleotide sequences of forward and reverse primers used for AT2R were as follows:forward,5’-CCACCCTTGCCACTACTAGCA-3’; reverse5’-CATTGTTGCCAGAGAT GTTCACA-3’. Oligonucleotideprimers for18sRNAwhich were also used for below real-time PCR, were obtained from Applied Biosystems, Inc.Western Blot AnalysisWestern blot were conducted with cell samples, primary antibodies and secondary antibodies. Cells were splitting by RIPA lysis buffer and centrifuged to obtain the supernate containing protein. Primary antibodies including anti-total p38MAPK, anti-total p53, anti-phosphorylated P38(PP38), anti-phosphorylated p53(pp53), and anti-activated caspase3were from Cell Signaling Technology. Anti-β-actin and the secondary antibodies horseradish peroxidase-conjugated anti-rabbit IgG were from Sigma-Aldrich.Data AnalysisFor all experiments, viral transduction was done in triplicate wells and repeated at least thrice. Data are presented as mean±SE of three or more independent determinations. Statistical significance was assessed by using a one-or two-way ANOVA as appropriate followed by a post hoc test (Bonferroni) to compare individual means. Differences were considered significant at P<0.05.ResultsRecombinant adenoviral vectors constructs and transductionTwo kinds of recombinant adenoviral vectors (Ad-CMV-EGFP, Ad-G-AT2R-EGFP) were purified through two cesium chloride gradients ultracentrifugation. Concentrations (Ad-CMV-EGFP:1x109; Ad-G-AT2R-EGFP:1.2x109) were calculated by OD260.Purity of recombinant adenovirus was preferable (OD260/OD280>1.3).EJ cell line was sensitive to adenovirus. The transduction efficiency changed with the dose of adenovirus. The fluorescence micrographs presented in Fig.l showed that EJ cells transduced with Ad-G-AT2R-EGFP (10ifu/cell)24h later produced a high level of expression of AT2R immunoreactivity and GFP. EJ cells with Ad-G-AT2R-EGFP (100ifu/cell) produced inhibition. This indicated that10ifu/cell was the better infected dose.(Not only can achieve higher infection rates, but also will not cause significant effect on cell growth)RNA isolation and concentration measurementRNA was isolated using TRIzol and its concentration was tested by ultraviolet spectrophotometer. RNA from cells transduced with Ad-CMV-EGFP was0.75ug/ul(OD260/0D280=1.95) while RNA from cells transduced with Ad-G-AT2R-EGFP was0.8ug/ul(OD260/0D280=1.92). The outcome indicated that the purity of RNA was preferable for real-time PCR test.Expression of AT2R in EJ cells after transductionThe results of real-time PCR showed that the expression of AT2R in EJ cells with Ad-G-AT2R-EGFP was remarkably higher compared with EJ cells with Ad-CMV-EGFP, which indicated that the recombinant adenoviral vectors with AT2R gene lead to the overexpression of AT2R.Effect of Increased AT2R Expression on Cell Cycle Progression and Cell Proliferation in EJ CellsAnalysis of the cell cycle of EJ cells transduction with Ad-G-AT2R-EGFP (100ifu/cell,24hours) indicated a significant reduction in the number of G2-phase cells and a significant increase in the number of G1-phase cells when compared with EJ cells transduced with Ad-CMV-EGFP (10ifu/cell,24hours). There were no significant changes in the number of S-phase cells.AT2R over expression Induces Apoptosis in Bladder Cancer CellsTransduced with Ad-G-AT2R-EGFP for2days, a large number of EJ cells presented apoptotic-like morphologic characteristics when compared with cells with Ad-CMV-EGFP. Apoptotic cells (brown color particles in the nucleus) were detected among EJ cells with Ad-G-AT2R-EGFP when compared with EJ cells without adenovirus and cells treated with control vector Ad-CMV-EGFP using the DeadEnd Colorimetric TUNEL System kit.AT2R-Induced Apoptosis Is Not Mediated by p38MAPKand Caspase-3via an Extrinsic Cell Death Signaling Pathway The results of western blot indicated that43-kDa protein that cross-reacts with anti-total p38MAPK (pp38) is present in EJ cells. The blot also revealed that in the Ad-G-AT2R-EGFP-treated cells, basal levels of pp38MAPK were not changed in a dose-independent manner when compared with Ad-CMV-EGFP-treated cells or mock-transduced cells. Basal levels of total p38MAPK were not changed. Ad-CMV-EGFP-treated cells showed low levels of apoptosis.Ad-G-AT2R-EGFP (10ifu/cell)-transduced EJ cells displayed remarkable higher caspase-3-like and pp53activity compared with Ad-CMV-EGFP (10ifu/cell)-transduced cells or mock-transduced cells, while there was no significant change of p53activity either in cells with Ad-G-AT2R-EGFP or in cells with Ad-CMV-EGFP.Conclusion:Bladder cancer is one of the common malignancies of the urinary tract. Compared with traditional therapies, gene therapy has become a new treatment modality. Renin-angiotensin system shows its important role in regulating the growth and proliferation of cancer cells. Functional Ang II type2receptors (AT2R) have antigrowth and antiproliferation effects in these cells. To investigate the effects of AT2R in bladder cancer cells, we constructed recombinant adenoviral vectors expressing AT2R. The vectors were transduced into bladder cancer cell line (EJ cell line) and led to overexpression of AT2R in the cells. The results of cell cycle and apoptosis analyses indicated that increased level of AT2R induced the arrest of cells proliferation, a significant reduction of S-phase cells and an enrichment of Gi-phase cells. In addition, overexpression of AT2R induced apoptosis in the EJ cells.According to the results of western blot and reverse transcription-PCR; we consider that the potential of AT2R to induce apoptosis exclusive activated p38mitogen-activated protein kinase, caspase-3and p53. The mechanism of overexpression AT2R induces EJ cell apoptosis need to be further research. These outcomes suggest that AT2R has the ability to induce apoptosis in bladder cancer cells and that AT2R may become the novel target in gene therapy for bladder cancer.

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