In total, tumor tissues of 33 AsUCs and 25 non-AsUCs as well as four normal-appearing bladder mucosal samples from non-cancer patients were studied. Tissues were harvested in a way that neoplastic parts were separated from adjacent normal parts. All 33 patients with AsUC had lived or been living in the arseniasis endemic area in southwest Taiwan where people drank the arsenic-contaminated artesian well water for at least 10 years (2).
Glutathione contents. GSH is the major antioxidant in cells and functions as a
free radical scavenger, which may detoxify toxins or chemotherapeutic agents.Thus, the intracellular GSH content may be altered in urothelial carcinogenesis.
Tissue extracts were prepared from frozen tissues by a standard extraction protocol. Briefly, about 0.5 cm3 tumor chip was used. Total protein was extracted by solubilizing with the Laemmli buffer (PIERCE Life Science Co., Rockford, IL). Protein contents of tissue extracts were determined using a commercial BCA kit (PIERCE Life Science Co., Rockford, IL). Cells (3×106) were resuspended in 0.5 ml 5% metaphosphoric acid (Sigma Chemical Co., St.
Louis, MO) on ice for 20 min. Cells were then lysed by three cycles of freeze-and-thaw. The supernatant was collected by centrifugation. Cellular GSH content was examined using the GSH-400 kit (Oxis International, Portland, OR). Briefly, 50 ml supernatant were incubated with 0.4 ml reaction buffer (200 mM potassium phosphate, 0.2 mM diethylene-triamine pentaacetic acid and 0.025% lubrol, pH 7.8). Then 25 ml chromogenic reagent in 0.2 N HCl and 25 ml 30% NaOH were sequentially added and mixed thoroughly. The mixture was reacted in the dark at room temperature for at least 10 min. The colorimetric density of the target chromogen was determined by a spectrophotometer (DU640i; Beckman, Fullerton, CA) at 400 nm.
Western blotting. Protein extracts (50 mg) were separated on 10%
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SDS-PAGEs and transferred to microporous polyvinylidene difluoride membranes (Millipore, Bedford, MA). After blocking with the TBST buffer (10 mM Tris-base; pH=7.5, 100 mM NaCl, 0.1 % Tween 20) plus 1% bovine serum albumin (BSA), the membranes were incubated with human specific anti-P53 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Bcl-2 (Santa Cruz Biotechnology), anti-c-Fos (Santa Cruz Biotechnology) and anti-GST-π (Signet Laboratories Inc., Dedham, MA) antibodies at 4°C for 12 hrs. The membranes were then washed three times with the TBST buffer (20 min each) and incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit immunoglobulin (Santa Cruz Biotechnology) for 1 hr at room temperature, respectively. After 3 washes, these proteins were detected by Western Blotting Luminol Reagent (Santa Cruz Biotechnology). The protein band intensities were digitalized and quantified using the Imagemaster VDS version 3.0 software (ImagemasterVDS; AmershamPharmacia). The band intensities of proteins were normalized to that of β-actin, which was run in parallel blots.
Statistical methods. Glutathione levels between the two groups were
compared with the Student’s t-test. Three separate experiments with triplicate data were performed and data was presented as mean ± standard error of the means (SEM). Fisher’s exact test or Chi-square test was used to compare protein expressions determined by Western blotting analysis. All tests were two-sided with p<0.05 as being statistically significant.69
Results
The mean glutathione (GSH) concentrations in both non-AsUC (22.8±1.8 µM/µg protein) and AsUC tissues (16.4±1.6 µM/µg protein) were significantly lower than that of normal mucosal tissues (33.4±7.2 µM/µg protein; p=0.04 and 0.002, respectively) by using the Student's t-test (Fig. 1). Further, GSH levels were significantly higher in non-AsUC than in AsUC (p=0.012). These results demonstrated cellular GSH contents may be down-regulated in urothelial carcinogenesis.
Protein levels of GST-π, Bcl-2, P53 and c-Fos were determined by Western blotting. All AsUC expressed GST-π and P53 proteins. A few cases of non-AsUC did not express the two proteins. The expressions of Bcl-2 and c-Fos were significantly higher in AsUC than in non-AsUC by using the Fisher's exact test (p=0.004 and p=0.02, respectively)(Table I). However, there were no significant differences in GST-π (p=0.18) or P53 (p=0.29) levels between the two groups.
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Discussion
It has been shown that AsUC and non-AsUC may be different from each other in clinicopathological parameters (11), which suggests that carcinogenic processes of the two may also be different. In this study, we showed that reduction of intracellular GSH levels may be an important feature in urothelial carcinogenesis. The reasons why the GSH levels of non-AsUC were significantly higher than in AsUC, however, are unclear. The glutathione/glutathion-S-transferase (GSH/GST) system in normal and tumor tissues plays a role in the susceptibility to endogenous and/or exogenous toxic compounds. GSH contents and GST-π activity have been analyzed in several tissues including human bladder tumors (12). These results revealed the reduced glutathione levels of bladder carcinoma were significantly lower in patients as compared with the control group (12;13). Interestingly, similar results have also been found in this study. The low GSH concentration of the urothelia may be responsible for the vulnerability of UC to chemical carcinogenesis. Previous studies have reported overexpression of GST-π and elevations of GSH in some arsenic-resistant cancer cell lines (14). The same features were also detected in arsenic-resistant UC cell lines in our previous report (15). There were no significant differences in the GST-π protein levels between AsUC and non-AsUC in this study. However, some studies have suggested it is the GST-π activity or other isoforms of GST (such as α, µ and θ isoforms), not GST-π total protein levels, that is involved in arsenic-mediated carcinogenesis (12;16;17). Further study is necessary to clarify the issue.
The expressions of oncoproteins Bcl-2 and c-Fos were significantly up-regulated in AsUC compared to non-AsUC. Hu, et al., (2002) have suggested that protein level of Bcl-2 was one of practical biomarkers to screen arseniasis-mediated skin carcinoma (18). In our series, all 33 cases of AsUC expressed Bcl-2 detected by Western blotting. In contrast, 6 of the 25 non-AsUC (24%) did not express Bcl-2 protein, which appears compatible to the findings of Hu, et al. Recent studies showed that Bcl-2 protein was positive expression in UC by immunochemical staining (19;20). However, since up to 75% of non-AsUC expresses Bcl-2 in this study. Therefore, Bcl-2 may be a sensitive marker but not a marker with good specificity for arseniasis. AP-1, a heterodimeric transcription factor of c-Fos and c-Jun, can mediate many biological effects of tumor promoters and was an important regulator of cell growth. Arsenic has been shown to modulate the mitogen-activated protein kinase cascade in several cell systems, resulting in the activation of
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transcription factors, including AP-1 (21). Recently, many studies have demonstrated the same results that arsenic induction of AP-1 DNA binding activity was accompanied by up-regulation of c-Fos and c-Jun nuclear proteins in bladder epithelial cells (22). We have also observed that more AsUC than non-AsUC expressed c-Fos. Of particular relevance to the present studies was a report that c-Fos expression was as a concomitant factor associated with arsenic-mediated carcinogenesis (23). However, a larger sample size is needed to validate the hypothesis that c-Fos up-regulation is closed associated with AsUC.
We did not find a statistic difference in the expression of wild-type P53 protein levels between the two groups of tumors. Tchounwou et al., (2003) have also shown that the level of P53 protein did not significantly differ between arsenic trioxide-treated and control liver carcinoma cells (24).
Contrarily, many studies have shown that high protein levels of mutant-type P53 were detected in AsUC (7;18). It is thus possible that arsenic-mediated urothelial carcinogenesis involves P53 mutation but not transcriptional or translational modification of P53 protein levels. Further studies were needed to determine if mutant-type P53 involved in arsenic-mediated urothelial carcinogenesis.
In summary, our results suggest that cellular GSH contents are down-regulated in urothelial carcinogenesis, especially in arsenic-related tumors. The Bcl-2 and c-Fos oncoproteins may play important roles in arsenic-mediated urothelial carcinogenesis. Carcinogenic pathway of AsUC is at least partly different from that of non-AsUC.
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Legends
Figure 1. Cellular GSH contents were down-regulated in urothelial
carcinogenesis. The mean GSH concentrations in both non-AsUC (22.8±1.8 µ M/ µ g. protein; p=0.04) and AsUC tumors (16.4±1.6 µ M/ µ g protein; p=0.002) were significantly lower than normal mucosal tissues (33.4±7.2 µ M/ µ g. protein) by using the Student's t-test. Of note, GSH levels of AsUC were significant lower than that of non-AsUC (p=0.012).
0 5 10 15 20 25 30 35 40 45 50
Glutathione Contents (µM/ µg tissue protein) Normal Urothelium Non-As UC As UC
* *** **
Fig. 1
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Table I. Comparison of protein expressions of GST-π, P53, Bcl-2 and c-Fos in two groups of tumors by Western blotting
Molecular markers GST-π P53 Bcl-2 c-Fos Expression +d –e + – + – + –
AsUCa
(n=33) 33 0 19 14 33 0 30 3 Non-AsUCb
(n=25) 23 2 10 15 19 6 16 9 Total 56 2 29 29 52 6 46 12
p value
c 0.18 0.29 0.004** 0.02*aAsUC = arsenic-related urothelial carcinoma
bNon-AsUC = non-arsenic related urothelial carcinoma
cFisher’s exact test (two-sided) was used to compare protein expressions in the two groups of tumors
dThe detectable intensity of protein by Western blotting was scored as "+"
eThe undetectable intensity of protein by Western blotting was scored as "-"
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