Growth Arrest and Apoptosis induced by the Histone Deacetylase Inhibitor, Belinostat (PXD101), in T-24 Bladder Cancer Cells

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Background: We have shown that up-regulation of the inhibitor of apoptosis, survivin, in the urine correlates with the presence of transitional cell carcinoma (TCC) of the bladder and that survivin is highly expressed in bladder tumors where its expression correlates with decreased time to recurrence and poorer prognosis. Histone deacetylase (HDAC) enzyme inhibitors mediate gene expression and chromatin assembly, and induce growth arrest and apoptosis of tumor cells, thus representing a new strategy for the treatment of cancers. Because treatment with HDAC inhibitors reduces survivin levels in several cancers including prostate cancer, we will determine if the HDAC inhibitor, belinostat (PXD101) reduces survivin levels in T-24 cells. Reduction of survivin levels in T-24 cells treated with survivin siRNA is accompanied by decreased cell growth, a specific G2/M arrest, activation of apoptosis and altered levels of proteins including caspases and Bcl-2 related proteins, therefore we will test whether similar changes are induced in T-24 cells treated with belinostat.
Methods: T-24 cells were incubated with belinostat. Viable cell count and proliferation curves were constructed. Cell cycle arrest analysis was conducted with FACS. Changes in apoptosis signaling pathways were assessed by Western blots.
Results: Treatment of T-24 cells with belinostat (5 microM) for 48 and 72 hours induced 56.7 ± 4.2% and 64.8 ± 3.2% decreases in proliferation, respectively. Viable cell counts at 24 and 48 hours (belinostat, 5 microM) were decreased by 30.6 ± 4.8% and 89.0 ± 3.0% respectively. The IC50 of belinostat for proliferation and viable cell counts was approximately 1 microM at 48 and 72 hrs. Belinostat (2 and 5 microM, 48 hrs) increased the number of apoptotic cells (sub-G1 cells) 1.3-2.3 fold. Belinostat (1, 2 and 5 microM, 48 hrs) decreased G1 cells by 41-54% and increased S and G2/M phase cells 2.5-3.5 fold. Treatment with belinostat for 24 hrs decreased protein levels of survivin (38.2 ± 17% and 58.2 ± 6.0% at 1 and 5 microM), pro-caspase 2 (40.4 ± 11%; 5 microM), and pro-caspase 8 (55.2 ± 12.4%; 5 microM), while the Bcl2 related protein, Bak, and caspase 3 were unchanged. At 48 hrs, belinostat treatment (1 and 5 microM) decreased survivin and pro caspase 2 and 3 levels, whereas Bak and pro-caspases 8 levels were only decreased with 5 microM belinostat. The presence of cleaved (activated) caspase 2, 3, and 8 was visualized at 48 hrs with 1 and 5 microM belinostat.

Conclusions:
We have shown that treatment of T-24 bladder cancer cells with the HDAC inhibitor, belinostat, in addition to causing a profound decrease of cell growth and viability, a specific G2/M phase arrest, and an increase in apoptotic cells, causes a decrease in survivin levels and induces the appearance of cleaved (activated) caspase products with a concomitant decrease in pro-caspases, indicating that its action is dependent in part on an activation of survivin sensitive death receptor signaling pathways. It would appear that the action of this HDAC inhibitor is dependent in part on an activation of survivin sensitive death receptor signaling pathways.

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