Expression Of C-FLIP MRNA In Badder Urothelial Carcinomas And Its Clinical Significance

PrefaceBladder cancer is a kind of malignant tumor frequently occurred in urinary system. In china,the occurrence of bladder cancer is 3-4 times more frequent in men than in women.Transitional cell carcinoma(TCC)is by far the most frequent histotype, although the distribution of histotypes varies in different populations.In Chinese people,90%of all bladder cancers were TCC.Most bladder carcinogens exert their action by direct contact with the bladder epithelium.Inhaled or ingested compounds, which are either directly carcinogenic or which can be transformed by the body in carcinogenic byproducts,are excreted via the urine,and in this way they reach the urinary bladder.There is also evidence that chronic inflammation,caused either by infections or stones,has a role in the promotion of bladder cancer.A caspase-like protein termed c-FLIP(cellular FADD-like interleukin-1β-converting enzyme inhibitory protein)impairs the Fas-mediated cell death signal. c-FLIP is expressed mainly in a long(c-FLIPL)form and a short spliced form (c-FLIPS).The short form consists of exclusively two tandem repeats of death effector domain(DED),which inhibits procaspase-8 activation.The longer isoform contains two DED in its NH2-terminal region and shares extensive homology with the caspase-8 catalytic domain in its COOH-terminal region.However,c-FLIPL sequence lost the conserved Cys residue in the catalytic site and a closely positioned His residue,which are essential for the caspase-8 catalytic activity.To explore the expression of c-FLIP in bladder transitional cell carcinoma and its'clinical significance,in this study,the expression levels of c-FLIP were evaluated by using RT-PCR in 30 bladder transitional cell carcinoma tissues,10 normal bladder tissues,Its correlations with the clinical stage,pathology type,prognosis were evaluated in positivecase. Method30 bladder cancer samples were Collected which are setected by operation in our university from 2002 to 2005,These samples were graded according to WHO/ISUP1998,which is 4gradeâ… ,12gradeâ… -â…¡,5gradeâ…¡,5stageâ…¡-â…¢,4gradeâ…¢.TNM staged were confirmed among these sample that is 6T_a,8T₁,9T₂, 7T₃.10Sampes from normal bladder tissues were collected after operation.All of samples are stored in -80℃after bathing by water.RT-RCR kit are obtained from Takara Shuzo Co.Ltd.c-FLIP-F,c-FLIP-R and B-actin primer are obtained from Takara;TRIZOL from GEBCOBRL.Expression levels of c-FLIP mRNA were determined by RT-PCR,Total RNA was refine according to kit directions,RNA concentration and purity were tested by Ultraviolet spectrum,the samples OD260/OD280 was larger than 1.575.2ul of RNA were used for each 20-ul RT-cDNA reaction.The reaction under the following condition:65℃for 1min,30℃for 5min,65℃for 30min,98℃for 5min,5℃for 5min.For c-FLIP mRNA detection,3ul of RT-cDNA product were amplified using the primers c-FLIP -F 5'-GTT CTT CGG GAC ACC TTC AGT-3' and c-FLIP -R 5'- TAT CCA CGC CAA ACA CGC TCT -3'.Each 25-ul reaction contained dd.H₂O17.1ul 10×buffer 2.5ul dNTPs 2ul Taq-E 0.2ul hTERT-F 0.1ul hTERT-R 0.1ul.each cycling conditions were as follows:94℃for 3min,32cycles of 94℃45s,57.6℃1min,and 72℃1min,followed by one cycle of 72℃for 7min.The primers of B-actine as inside contrast take part in the reaction.ResultThere were over expression of c-FLIP mRNA in bladder urothelial carcinomas tissues.The positive rate in the bladder urothelial carcinomas tissues was distinctively different from the normal bladder urothelial tissues(P＜0.05).There was no correlation between positive case and clinical stage,pathology grade.The survival rate in c-FLIP mRNA overexpression case was significantly lower than that of the low expression case(p＜0.05).DiscussionA caspase-like protein termed c-FLIP(cellular FADD-like interleukin-1β-converting enzyme inhibitory protein)impairs the Fas-mediated cell death signal. c-FLIP is expressed mainly in a long(c-FLIP_L)form and a short spliced form(c-FLIPs). The short form consists of exclusively two tandem repeats of death effector domain (DED),which inhibits procaspase-8 activation.The longer isoform contains two DED in its NH₂-terminal region and shares extensive homology with the caspase-8 catalytic domain in its COOH-terminal region.However,c-FLIP_L sequence lost the conserved Cys residue in the catalytic site and a closely positioned His residue,which are essential for the caspase-8 catalytic activity.Fas is a transmembrane receptor that belongs to the tumor necrosis factor(TNF) receptor(TNFR)superfamily.Its cognate ligand,FasL,is a transmembrane protein found in a soluble trimeric form after cleavage by metalloproteinases.This soluble homotrimeric FasL complex is not able to induce cell death unless it is produced as a homohexameric complex.Fas carries an intracellular conserved stretch of 80 amino acids called the death domain,which serves as a docking platform to trigger an intracellular signal.Indeed,on binding of FasL or multivalent agonistic antibodies,the Fas death domain recruits the adaptor molecule Fas-associated death domain protein(FADD),which in turn aggregates the initiator caspase-8 and caspase-10.This intracellular complex is called death-inducing signaling complex(DISC).The fact that caspases are brought together in close vicinity leads to their autocleavage and activation of the downstream apoptotic signal.Fas-mediated apoptosis can be determined by a critical threshold of Fas stimulation or by the activation of specific caspases.The caspases are synthesized as inactive proenzymes and are activated by proteolytic cleavage of aspartic residues. Cellular FLICE inhibitory protein(FLIP)inhibits caspase 8 activity,and,therefore, inhibits Fas-mediated apoptosis.Alternative splicing leads to long(55 kD)and short form(28 kD)variants of FLIPs,both of which can interact with FADD and caspase 8 via DED motifs,and can be recruited to the Fas DISC.Therefore,FLIP has been implicated in the regulation of the Fas pathway during the initial phase of apoptosis.To explore the expression of c-FLIP in bladder transitional cell carcinoma and its' clinical significance,in this study,the expression levels of c-FLIP were evaluated by using RT-RCR in 30 bladder transitional cell.carcinoma tissues,10 normal bladder tissues.We analyzed the gene expression of c-FLIP in 30 bladder transitional cell carcinoma differing in stage and grade,and in 10 normal bladder tissues.There were over expression of c-FLIP mRNA in bladder transitional cell carcinoma whereas normal bladder tissues were all negative.The positive rate in the bladder transitional cell carcinoma was ditinctively different from the normal bladder tissues(p＜0.05).Our data indicate that c-FLIP mRNA levels do not correlate with clinical stage,pathology grade in bladder transitional cell carcinoma.The reason is that a number of factors,including both cell mutation and gene abnormal expression,inactivation of suppressor gene and elimination of printing gene etc,can be invoke to explain the lack of relationship between them.All the patients were followed up range 3.6 to 59.5 months.The survival rate in c-FLIP overexpression case was significantly lower than that of the lowexpression case (p＜0.05).The overexpression of c-FLIP mRNA was significantly higher in the bladder transitional cell carcinoma.It might serve as a prognostic marker for estimating the biologic characteristics of the bladder transitional cell carcinoma.In conclusion,our findings indicate that bladder tumor c-FLIP mRNA expression level correlates with outcome in patients with bladder transitional cell carcinoma.A larger study will be necessary to determine whether c-FLIP mRNA expression is predictive of outcome independent of clinical stage,pathology grade in bladder transitional cell carcinoma.ConclusionThe overexpression of c-FLIP mRNA was significantly higher in bladder urothelial carcinomas.It might serve as a prognostic marker for estimating the biologic characteristics of the bladder urothelial carcinomas.