Response of Bladder Carcinoma Cells to TRAIL and Antisense Oligonucleotide, Bcl-2 or Clusterin Treatments

Purpose

Bladder transitional cell carcinoma is the second most common urological malignancy, of which 80% are superficial disease limited to the bladder. Superficial bladder transitional cell carcinoma has a high propensity for recurrence and progression after initial resection, necessitating adjuvant intravesical therapy. TRAIL (tumor necrosis factor-related apoptosis inducing ligand) can selectively induce apoptosis in most tumor cells while sparing normal cells. TRAIL drives not only the death receptor pathway, but also the mitochondrial pathway through Bid. Due to the anti-apoptotic functions of Bcl-2 and clusterin on the mitochondrial apoptotic pathway the effects of down-regulating these proteins were examined in partially TRAIL resistant bladder transitional cell carcinoma cell lines.

Materials and Methods

Antisense oligonucleotides targeting Bcl-2 and clusterin were used alone or combined with TRAIL and cytotoxicity was examined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolim bromide) proliferation assay. Apoptotic pathway signals were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blotting after the various combination treatments. All statistical tests were 2 sided.

Results

Although no direct correlation between TRAIL sensitivity and the relative expression levels of Bcl-2 and clusterin was found in the bladder transitional cell carcinoma cell lines examined, antisense oligonucleotide mediated the down-regulation of Bcl-2 and clusterin, increasing the sensitivity of the partially resistant cells to TRAIL. This was mediated through increased apoptotic signaling of the mitochondrial pathway, as evident by the increased activation of caspase-9 and 3, and cleaved DFF45. There was no benefit of combined antisense oligonucleotide therapy.

Conclusions

This study provides proof of principle that TRAIL combined with antisense oligonucleotide-Bcl-2 may have potential as a novel future treatment strategy for bladder transitional cell carcinoma.

Key Words: urinary bladder, carcinoma, transitional cell, apoptosis, tumor necrosis factors, genes, bcl-2

Abbreviations and Acronyms: ASO, antisense oligonucleotide, BCG, bacillus Calmette-Guerin, CD, cell death, Clus, clusterin, DFF, DNA fragmentation factor, DR, death receptor, MM, mismatch, MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolim bromide, TCCB, transitional cell carcinoma of the bladder, TNF, tumor necrosis factor, TRAIL, TNF-related apoptosis inducing ligand