| Transitional cell carcinoma (TCC) of the urinary bladder is the most common cancer in urinary system. Among these bladder tumors approximately 80% are diagnosed as superficial bladder carcinoma ( SBC) . With a strong propensity to recur, more than 70% of patients with SBC have a secondary primary lesion within 2 years and about one third of them show disease progression and e-ventually die from bladder cancer . Clinical data indicate that tumor grade ( differentiation) as well as intrinsic and/or acquired resistance to drugs is major cause for relapse of carcinoma.Protein kinase C ( PKC) is a gene family that functions in a signaling pathway associated with growth and differentiation. PKCa is closely related to tumor genesis and tumor progression, and hence may be a therapeutic target . Two reports have shown that therapy with antisense PKCa oligos inhibits tumor growth in nude mices bearing implanted human gioblastoma , bladder, lung and colon carcinomas . Furthermore, two recent Phase I clinical trials found that anti-sense PKCa oligo produced antitumor responses against ovarian cancer and non - Hodgkin' s lymphoma . But little is known about relationship between PKCa expression and SBC.Some studies support a correlation between PKC activation and multidrug resistance ( MDR). Researches have shown that PKC can directly phosphorylate P - gp and induce expression of the mdrl' s mRNA, while inhibition of PKC sensitizes the cancer cells to radiation - and drug - induced apoptosis. But the expression pattern of PKC isozymes differs in various MDR cell lines. In several in vitro experiments, PKCa overexpression accompanies MDR phenotype. Withno information about PKCa in MDR of SBC, we also want to ask whether PKCa overexpression results in intrinsic drug resistance in SBC.In the present study, we examined PKCa expression in 76 patients with SBC but without chemotherapy preoperatively, and investigated the association of above indexes with clinicopathological parameters and outcome. It has been known that activation of PKCa involves translocation . So the ratio of the amount of PKCa in membrane to that in cytosol ( M/C) was used as surrogate measure of PKCa activation . The changes in a bladder cell line drug sensitivity to adriamy-cin( ADM) were also shown to correlate with PKCa activation in vitro.Materials and Methods1. subjects: Surgical specimens were obtained between 2000 and 2003 from patients with newly diagnosed primary SBC. The clinical information was retrieved from the medical records. All patients routinely underwent cystoscope and computed tomography before surgery for preoperative staging. They included 54 male and 22 female patients, ranging in age from 46 to 74 years. Histological diagnosis revealed that all patients had transitional cell carcinoma. Their histo-logic classification and staging according to the UICC classification were as follows; Ta (n = 18) , Tis (n = 18), T1 (n=40); G1 (n=34), G2 (n=26) , G3 ( n = 16) , respectively. 76 samples of normal bladder tissue more than 5cm from the tumor were obtained from 76 patients in this study. Histologically, these bladder samples were determined to be free of malignant cells as normal control. No patient received any radiation or chemotherapy preoperatively. In this series, they were treated with transuretheral resection or segmental cystecto-my followed by standard ADM instillation. A 2 - year follow - up was undertaken for all the patients ( Median 14. 2 months, range 4-24 months). Recurrence was defined as cystoscopically visible tumors.2. Methods; Western blot analysisCells were washed three times in cold PBS, and harvested. Cells were lysed in ice-cold lysis buffer (50 mM Tris [pH 7.4] , 150 mM NaCl, 2 mMEDTA, 1 % NP - 40) supplemented with protease inhibitor cocktail (Sigma) for 10 min at 4 C. Whole cell extract was cleared by centrifugation for 15min at 12, 000 g. Tissue samples prepared as described above, as well as the cell lysates with 20 ug of proteins, were boiled in Laemmli sample buffer for 5 min, and resolved by SDS - PAGE. The proteins were transferred to Immunobilon transfer membrane ( Millipore). The membrane was probed with the indicated antibodies and subsequently horseradish peroxidase - conjugated secondary antibodies using standard procedure, and detected by enhanced chemiluminescence (ECL) system ( Amersham Biosciences). The protein expression level was quantified by measuring the immuno - density of the main band using image software ( UVP, UK). For quantization of PKCa protein, relative amount of PKCa was calculated with Takao' s method.Plasmid construct.The human PKCa full length cDNA clone was purchased from Invitrogen ( Clone ID: RG001129, GenBank accession no. X52479). The cDNA was sub-cloned into pDONR?201( Invitrogen) donor vector by PCR with Pyrobest DNA Polymerase ( Takara) using BP Clonase Reaction system (Invitrogen). Entry clone was identified by DNA sequencing analysis. pcDNA - PKCa - GFP re-combinant plasmid expressing PKCa with C - terminal fused Green Fluorescent Protein ( GFP) was constructed with entry clone and GFP expression vector pcD-NA - DEST47 (Invitrogen) using Gateway LR recombination reaction system (Invitrogen).Cell culture, transfection and selection of stable expression cloneRT - 4, the parental drug - sensitive SBC cell line ( ATCC) , was maintained in RPMI - 1640 medium (Sigma) supplemented with 10% fetal bovine serum (FBS) , 2mM glutamine, 25mM HEPES at 37 C in a humidified, 5% CO2 incubator.The cells were transfected with pcDNA - PKCa - GFP vector by Iipofecti-mine - Plus reagent (life Technologies, Gaithersburg, MD) , according to the manufacturer' s instructions. The stable expression clones ( RT - 4/PKCa -GFP) were established by the selection with Geneticin (Invitrogen) at a concentration of 400 μg/ml for two weeks. Positive clones were identified by GFPfluorescence microscopy (Olympus). Isolated clones were analyzed for PKCa -GFP expression by western blot analysis.MTT assayThe cytotoxity of Dox on RT -4 and RT -4 /PKCa - GFP cells was determined using a MTT assay. 100μl culture medium containing 1,000 cells was split onto a 96 -well plate. After 12 hours incubation, the varying amounts of Dox to the final concentration of 10nM, 30nM, 100nM, 300nM, lμM, 3μM,10μM,30μM ,100μM ,300μM and 1mM, were added into the medium, respectively, and the cells were incubated for additional 36h. There were 3 wells for each concentration, while the control wells were incubated with the normal medium instead at the same condition. Viability was determined by adding 20μl of 5 mg/ml MTT (Sigma - Aldrich) and incubating plates at 37°C for 4h. The medium was removed, 100μl of isopropanol acidified with 0.012M HC1 was then added and incubated at 37°C overnight to dissolve the formazan crystals formed by the cells. Finally, the absorbances were determined at a test wavelength of 540nM and a reference wavelength of 630nM, using a multiwell spectrophotome-ter ( BioTech). For each cell line, the IC50 value was defined as the dose of drug that causes 50% cells viability. The resistance index ( RI) was calculated according to reference.PKC activation and inhibition assayRT - 4/PKCα - GFP cell clone stably expressing PKCα - GFP was passaged for twice in the medium without Geneticin, and then incubated with lOnM PMA (Sigma) , a specific PKC activator, or 10 nM Calphostin C in serum - free medium for 2h. The cytotoxity of ADM was determined with an MTT assay. RT - 4/PKCa - GFP cells without the stimulation served as the experimental control. Translocation of PKCa was also studied by fluorescence microscopy.Expressions of mdrl ,LRP and MRP1 mRNAs with RT - PCRTotal RNA was extracted from frozen specimens using Trizol reagent according to the procedure provided by the supplier, and quantified by a spectropho-tometer . Reverse transcription was performed with an equal amount of total RNA from each sample using a BcaBEST RT - PCR kit , following the protocol supplied by the manufacturer. PCR was carried out using the primers for the in-dicated genes at the optimized conditions. Details of PCR conditions and gene specific primers are available upon request. The semi - quantitative analysis of the amplified PCR products was performed using a BAS2000 imaging plate . All samples were analyzed simultaneously. The relative amount of each MDR gene mRNA was normalized to β- actin signal from the same sample. Results were expressed as the ratio of MDR gene/β - actin, which was named the expression level for the respective MDR gene. Statistical analysisAll of the determinations were made in triplicate. For statistical analysis, the compared t test ,AVOVA and SNK test were used. Postoperative recurrence - specific survival was determined by the Kaplan - Meier method. The log -rank test was used to establish the statistical difference in recurrence between the patients with high and low levels of PKCa activation. Factors related to recurrence - specific survival in patients with SBC were also analyzed by univariate a-nalysis and multivariate analysis, respectively. A P value of 0. 05 or less was considered significant.ResultsPKCa expression in TCC of bladderThe total amounts of protein for PKCa were significantly greater in cancerous than in normal bladder tissues ( P < 0. 001) . The ratio of the absolute a-mount of PKCa in membrane to that in cytosol (M/C) significantly increased (tumor vs. normal; 0.82 ± 0.12 vs. 0. 25 ± 0.12, P <0.01).Relation of PKCa expression with clinicopathological parametersWith increase of tumor grade, the relative level of PKCa significantly increased in membrane( For foldchangesGlvs. G2vs. G3 ;0.79 ±0. 25vs. 1. 59 ±0. 84vs. 2. 45 ± 0. 76, P < 0. 01) , decreased in cytosol ( For fold changes Glvs. G2vs. G3:1.24 ±0.38vs. 0. 80 ±0.26vs. 0.58 ±0.22 ,P <0.01) , and the value of M/C significantly increased ( G1 vs. G2 vs. G3: 0. 15 ± 0. 03 vs. 0.42 ± 0.06 vs. 0.93 ± 0.17, P <0.01). But PKCa expression was not significantly associated with tumor stage, age, gender, or tumor multigenesis ( P >0.05).Correlation between PKCa expression and recurrence - free survival in patients with SBCSBC patients receiving standard ADM treatment after surgery were evaluated for the postoperative clinical course. The postoperative tumor free period was estimated by the Kaplan - Meier analysis. Patients with SBC were divided into two groups; patients with high M/C of PKCa ( greater than the median value 0. 56) and patients with low M/C of PKCa (less than the median value). Those with low M/C had a longer recurrence - free interval compared with those with high M/C during the 2 - year follow - up ( P = 0. 035). By univariate analysis, high M/C for PKCa , high tumor grade , tumor multiplicity and high absolute level of PKCa predicted shortened recurrence - free survival (P =0.035,0.013 , 0.024 , and 0. 038respectively) , while multivariate analysis showed that high M/C for PKCa was an independent factor in shortened recurrence - free survival for patients with SBC( RR =3.98,95% CI 1. 22 -5.68 ,P =0. 030).Effect of PKCa on the drug resistance of RT — 4 cell lineTo investigate the effect of PKCa on the drug resistance of bladder cells, we established stable cell clones expressing PKCa — GFP using RT - 4 drug -sensitive bladder carcinoma cell line. Two highest expression clones were identified by western blot analysis. It has been known that activation of PKCa involves translocation of PKCa from the cytoplasm to the plasma membrane. In order to further identify the character of expressed PKCa - GFP, fluorescence microscopy was conducted to examine the distribution of PKCa - GFP under the activation/inhibition stimulation as described in ' Materials and Methods'. As the result , translocations of PKCa - GFP between the cytosol and the membrane were observed in both conditions . These data demonstrated that stably expressed PKCa - GFP has the same character as endogenous PKCa.We next testified the effect of PKCa activity on the drug resistance of RT -4 by measurement of the resistance of RT - 4/PKCa - GFP cells to the cytotoxity of ADM. MTT assay was conducted on RT -4 and RT -4/PKCa - GFP cells with and without PMA or Calphostin C treatment, following the incubation with ADM at various concentrations . In the normal growth condition, RT - 4/PKCa - GFP cells showed a stronger growth ability than parental RT - 4 cell line un-der the addition of ADM( P <0. 01) .suggesting that RT -4 cell overexpressing PKCa has the drug resistance potential against ADM. After PMA induction for 2h, resistance potential of RT -4/PKCa - GFP cells with PMA was 21. 25 -fold higher than those without treatment(P <0.001) , demonstrating that the activation of PKCa significantly raises the drug resistance potential of RT - 4/ PKCa - GFP cells. Conversely, Calphostin C treated RT -4/PKCa - GFP cells revealed a remarkably decreased growth ability compared with RT - 4/PKCa -GFP cells without treatment( P <0. 01) .demonstrating that inhibition of PKCa activity significantly reduces the drug resistance of RT -4/PKCa - GFP cells. Taken together, our data indicate that PKCa may be involved in the regulation of the drug resistance potential of RT -4 cells via PKC pathway.Expression of MDR - related genesBy RT — PCR, we found that the resistance of RT4/ PKCa — GFP was mainly associated with overexpression. of mdr - 1. Level of mdr - 1 expression in RT -4/PKCa - GFP cells was 4. 6 - fold higher than parental RT -4 cell line( P < 0. 001). After PKCa activation, level of mdr - 1 expression was 12. 8 - fold higher than parental cell line ( P < 0. 0001 ) , while there was no significant difference between the two cell lines after PKCa inhibition (P >0. 05). But we did not find the significant changes of MRP and LRP mRNA levels during above course.Conclusions1. The ratio of membrane to cytoplasm ( M/C) for PKCa level even in patients with SBC showed a well consistence with histologic grade. The result supports our speculation that abnormally activated PKCa in SBC may play a role in tumor differentiation, on the other hand, indicates that the ratio of membrane to cytoplasm (M/C) for PKCa level may be a reasonable and sensitive index for predicting response to ADM.2. Abnormal activation of PKCa may be used as surrogate biomarker for prognosis and serve as a therapeutic target for a patient population with a tendency to early recur after chemotherapy. M/C for PKCa may be a reasonable and...
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