Proteasome mediated degradation of Id-1 is associated with TNFα-induced apoptosis in prostate cancer cells

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Abstract
Overexpression of the helix–loop–helix protein Id-1 has been reported in over 20 types of cancer. While a number of factors have been demonstrated to regulate Id-1 gene transcription, little is known about the mechanisms responsible for its degradation. In this study, we have demonstrated that Id-1 protein stability was regulated by TNFα in prostate cancer cells. We found that exposure of prostate cancer cell lines, DU145 and PC-3, to TNFα resulted in a rapid and significant downregulation of the Id-1 protein level. The fact that neither the Id-1 promoter activity nor the Id-1 mRNA level was affected by the TNFα treatment suggested that the decrease in Id-1 protein was not due to the suppression of gene transcription. In addition, the half-life of the Id-1 protein was decreased in both cell lines in the presence of TNFα, and the addition of an ubiquitin/proteasome inhibitor (MG-132) prior to the TNFα treatment completely blocked the effect of the TNFα-induced Id-1 protein degradation. Furthermore, introduction of a Flag-tag sequence into the N-terminus region of the Id-1 protein, which has been shown to stabilize the protein, was able to protect the Id-1 protein from TNFα-induced degradation. These results suggest that TNFα downregulated Id-1 through activation of the ubiquitin/proteasome degradation pathway in prostate cancer cells. Interestingly, in both DU145 and PC-3 cells, the decrease of Id-1 protein was associated with the activation of apoptotic pathway, as evidenced by the increased expression of cleaved PARP and caspase 3. In addition, TNFα failed to downregulate Id-1 in a sub-line of LNCaP cells that was resistant to TNFα-induced apoptosis. These results further suggest that the downregulation of Id-1 may facilitate TNFα-induced apoptosis in prostate cancer cells. In conclusion, our findings indicate that Id-1 protein may be regulated by TNFα through the ubiquitin/proteasome degradation pathway and the stability of the Id-1 protein appears to correlate with the sensitivity of TNFα-induced apoptosis.