Abstract
OBJECTIVE To explore the expression of the MDR1/P-glycoprotein, Fas and survivin and to examine their correlation with the biologic behavior of bladder transitional cell carcinoma (BTCC)
METHODS Immunohistochemistry was used to examine the expression of P-gp, survivin and Fas in BTCC (n=64) and normal bladder mucosa (n=12).
RESULTS The expression level of P-gp and survivin in BTCC was higher compared to normal bladder mucosa (P<0.01) and their expression was strongly correlated with clinical grading (P<0.01). In BTCC and normal bladder mucosa Fas expression was 50% and 100%, respectively (P< 0.01). Recurrent BTCC showed higher expression than primary BTCC (P< 0.01) and the expression of P-gp in BTCC had a reverse correlation with Fas expression but no correlation with survivin expression.
CONCLUSION The MDR of BTCC was strongly correlated with the expression of P-gp and Fas, but was not correlated with survivin expression. Thus, enhancing cancer sensitivity to chemotherapy by reversing multidrug resistance with reversal agents or up-regulating Fas expression by apoptotic enhancing agents, might be a potential therapy to prevent tumor recurrence and invasiveness.
keywords
Bladder transitional cell carcinomas (BTCC) are the most common Urologic tumors in China and the sixth most common malignancy in developed countries.[1] One characteristic feature of BTCC is the high recurrence incidence which is about 75~80% after operation. Combined surgical management, including radical cystectomy and transurethral resection and post-operation intravesical chemotherapy, are the main therapeutic approachs. Drug resistance is a persistent problem for cancer chemotherapy and also is a serious obstacle for successful mutimodality therapy. Induction of cancer cell apoptosis is an important mechanism for effective chemotherapy. [2] Up to now, there has been a lack of agreement about the association between cancer cell apoptosis and multidrug resistance so their precise underlying relationship remains an open question.[3,4] In the present study, we examined the expression of the MDRl/P-glycoprotein, Fas and survivin by immunohistochemistry in normal and cancerous bladder tissue. An association between the expression of MDRl/P-gp, survivin, Fas and the clinicopathologic parameters of the bladder cancers was also examined.
MATERIALS AND METHODS
Clinical specimens
Seventy-seven paraffin-embedded tissue specimens were obtained, after informed consent, from 76 patients treated at The No.2 Hospital of Tianjin Medical University between April 2004 and December 2004. All the specimans were pathologically vrified to be cancerous or normal bladder tissue. Among them, 64 cases were shown to be bladder transitional cell carcinoma. The cases were comprised of 40 males and 24 females and 48 primary and 16 recurrent tumors. Their ages ranged from 34 to 86 years with an average of 51. According to the World Health Organization/Intemational Society of Urologic Pathology (ISUP) (1998 classification), 12 were Grade 1, 28 Grade 2 and 24 Grade 3 BTCC. Based on the 1997 American Joint Committee on Cancer-International Union Against Cancer (AJCC-UICC) TNM system, 42 specimens were classified as Stage Ta-Tl and 22 as Stage T2-T4. In addition, 12 speimens were normal bladder tissues from transurethral resection of the prostate from patients with benign prostatic hyperplasia. Their ages ranged from 59 to 78 with an average of 68.
Method
Immunohistochemical staining
Consecutive 4 pm thick sections were cut from the paraffin blocks, after which En Vision immunostaining was used. The following antibodies were applied to the sections: MDRl/P-glycoprotein (1:50, mouse monoclonal antibody, ZM-0189, ZSBiO, Beijing, China), Fas (1:100, mouse monoclonal antibody, ZM-0295, ZSBiO, Beijing, China), survivin (1:80, rabbit polyclonal antibody, BA1420,BOSTER, Wuhan, China). The color reaction was developed with the addition of DAB. All procedures were implemented according to the supplier’s instructions. For negative controls, sections were processed as the above but with PBS instead of the primary antibodies.
Evaluation of MDRl/P-glycoprotein, Fas and survivin staining
The immunoreactivity to survivin was localized in the cytoplasm, while Fas and P-gp were localized in the cytoplasm and cell membrane. The staining was graded according to the proportion of positive cells and the degree of staining intensity achieved. The former was defined as follows : <10% as 0, 10~25% as 1, 25~50% as 2, >50% as 3 and the latter was : —(background) was defined as 0, +(weak yellow) as 1, ++ (yellow) as 2, +++(brown) as 3. The sum <2 was defined as negative expression, > 3 as positive expression.
Statistical analysis
All data were analyzed with SPSS 11.5 statistical software (including chi-square or adjusted chi-square) and P<0.05 was considered to have statistical significance.
RESULTS
Expression of MDR1/P-glycoprotein, Fas and survivin in BTCC
The results of the immunohistochemical staining were as follows: the MDRl/P-glycoprotein, Fas and survivin staining was positive in 53.1, 50.1 and 93.8% of the cases of bladder transitional cell carcinomas compared to 3, 12 and 0% respectively of the tissues from normal bladders. Survivin was more highly expressed in bladder transitional cell carcinoma than in normal bladder tissue (χ2=53.437, P=0.001),but expression of Fas was higher in normal bladder compared to cancerous bladder (χ2=3.2, P=0.074). However expression of MDR1/ P-glycoprotein did not differ significantly between normal and cancerous tissues (Figs.1~4) (χ2=3.2, P= 0.074), but MDRl/P-glycoprotein expression did significantly correlate with pathological grading and tumor recurrence (P<0.01). Survivin correlated only with pathological grading (P<0.01). Fas expression had no correlation with the clinical pathological characteristics (P>0.05, Tablel).
Survivin positive expression in grade 2 BTCC (x 400).
Fas positive expression in normal bladder (x 200).
Fas positive expression in grade 2 BTCC (x 200)
P-GP positive expression in grade 2 BTCC (x 200).
Expression of P-gp, survivin and Fas in BTCC and correlation with clinical pathological characteristics
Expression of the MDRl/P-glycoprotein in BTCC and its correlation with Fas and survivin
The expression of P-gp in BTCC showed a reverse correlation-with Fas expression, but no correlation with survivin expression ( Table 2). The survivin expression level were increased gradually with an ascending pathological grade.
MDRl/P-glycoprotein expression correlation with survivin and Fas
DISCUSSION
The appearance of tumor cells resistant to multiple anticancer agents is a serious obstacle in cancer treatment. The overexpression of MDRl/P-glycoprotein is one of most critical molecular proteins for limiting drug resistance. Overexpression of P-gp has been found in human bladder cancer cells selected by drug resistance against P-gp-targeting drugs.[5] hi patients with bladder cancers, expression of P-gp is often increased after chemoradiotherapeutic treatment.[6] Furthermore, recent studies have found that over-expression of the MDR1 gene occurred more frequently in recurrent bladder cancers.[7,8] In our study, we found that the expression of the MDRl/P-glycoprotein was markedly elevated in recurrent bladder cancers after intravesical chemotherapy compared to untreated primary tumors, indicating that this increased expression of the MDRl/P-glycoprotein might be partly involved in drug resistance and intravesical recurrence in these patients. Moreover, over-expression of the MDRl/P-glycoprotein was significantly correlated with different pathological grading in these bladder cancers but not with their clinical stage, These findings are consistent with previous studies.[9] Results from this study provide further support for enhancing chemotherapy sensitivity through the use of agents such as Verapamil, CyclosporinA, etc. that reverse resistance.
The formation of multidrug resistance in cancer cells is a multiple gene regulation process, that might involve overexpression of an anti-apoptotic gene and/or abnormal expression of the apoptosis-regulating gene. Recent studies have shown that changes in apoptosis-regulating genes correlated with a multidrug-resistance mechanism, such as Bcl-2 and bax.[10] Survivin is a recently discovered protein belonging to a gene family of inhibitors of apoptosis (IAP). It is selectively expressed in cancer cells but undetectable in nonproliferating normal tissues, suggesting an important role in tumorigenesis. In our study, increased expression of survivin was positively correlated with an ascending pathological grade. Furthermore, there was no significant correlation between MDRl/P-glycoprotein and survivin expression, findings which are consistent with a previous study.[11]
Fas (CD95/APO-1) is a cell-surface "death receptor" that mediates apoptosis upon engagement by its ligand. Tumor cells frequently exhibit decreased expression of Fas. In our study, MDRl/P-glycoprotein expression was reversely correlated with Fas, indicating that chemotherapy resistance was possiblly associated with Fas-mediated apoptotic inhibition. One study found that Fas gene transduction could reverse the MDR of the human drug-resistant gastric cancer cell, SGC7901/VCR.[12]
In conclusion, this study further demonstrates that MDRl/P-glycoprotein mediated multidrug resistance might be an important reason of chemotherapy failure and tumor recurrence in bladder transitional cell carcinomas. The formation of multidrug resistance was possiblly associated with Fas-mediated apoptotic inhibition, whereas no definite association was shown with the anti-apoptotic protein survivin. Thus, enhancing cancer sensitivity to chemotherapy, reversal of multidrug resistance by agents that reverse multidrug resistance or up-regulating Fas expression by apoptotic agents, might be a potential goal to prevent tumor recurrence and invasiveness.
- Received March 1, 2006.
- Accepted April 24, 2006.
- Copyright © 2006 by Tianjin Medical University Cancer Institute & Hospital and Springer
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