Abstract
Activation of protein kinase C (PKC) prevents apoptosis in certain cells; however, the mechanisms are largely unknown. Inhibitors of apoptosis (IAP) family members, including NAIP, cIAP-1, cIAP-2, XIAP/hILP, survivin, and BRUCE, block apoptosis by binding and potently inhibiting caspases. Activation of NF-kappa B contributes to cIAP-2 induction; however, the cellular mechanisms regulating cIAP-2 expression have not been entirely defined. In this study, we examined the role of the PKC and NF-kappa B pathways in the regulation of cIAP-2 in human colon cancers. We found that cIAP-2 mRNA levels were markedly increased in human colon cancer cells by treatment with the phorbol ester, phorbol-12-myristate-13-acetate (PMA), or bryostatin 1. Inhibitors of the Ca2+-independent, novel PKC isoforms, but not inhibitors of MAPK, PI3-kinase, or PKA, blocked PMA-stimulated cIAP-2 mRNA expression, suggesting a role of PKC in PMA-mediated cIAP-2 induction. Pretreatment with the PKC delta-selective inhibitor rottlerin or transfection with an antisense PKC delta oligonucleotide inhibited PMA-induced cIAP-2 expression, whereas cotransfection with a PKC delta plasmid induced cIAP-2 promoter activity, which, taken together, identifies a role for PKC delta in cIAP-2 induction. Treatment with the proteasome inhibitor, MG132 or inhibitors of NF-kappa B (e.g. PDTC and gliotoxin), decreased PMA-induced up-regulation of cIAP-2. PMA-induced NF-kappa B activation was blocked by either GF109203x, MG132, PDTC, or gliotoxin. Moreover, overexpression of PKC delta-induced cIAP-2 promoter activity and increased NF-kappa B transactivation, suggesting regulation of cIAP-2 expression by a PKC delta/NF-kappa B pathway. In conclusion, our findings demonstrate a role for a PKC/NF-kappa B-dependent pathway in the regulation of cIAP-2 expression in human colon cancer cells. These data suggest a novel mechanism for the anti-apoptotic function mediated by the PKC delta/NF-kappa B/cIAP-2 pathway in certain cancers.
Highlights
Colorectal cancer is the third leading cause of cancer-related deaths in the United States with ϳ130,000 new cases diagnosed per year [1]
To better delineate upstream signaling pathways responsible for cIAP-2 induction, the human colon cancer cell line Caco-2 was treated with the phorbol ester PMA, and RNA extracted for RNase Protection Assay (RPA) using a multiprobe containing cDNAs for a number of anti-apoptotic genes as well as the housekeeping genes L32 and GAPDH (Fig. 1)
We have shown that human colon cancer cells express members of the Inhibitors of apoptosis (IAP) family, including xIAP, cIAP-2, and cIAP-1
Summary
Colorectal cancer is the third leading cause of cancer-related deaths in the United States with ϳ130,000 new cases diagnosed per year [1]. Other cellular models of apoptosis have been used to demonstrate that, during the transduction of cell death signals, there is selective inhibition/activation of PKC isotypes, depending on cell type and apoptotic stimuli [19, 20]. We show that expression of one of the IAP family member, cIAP-2, is preferentially induced in human colon cancer cells following PMA treatment; inhibition of Ca2ϩindependent, novel PKC isoforms, but not inhibition of MAPK, PI3-kinase and PKA, blocked PMA-stimulated cIAP-2 mRNA expression, suggesting a role for PKC in PMA-mediated cIAP-2 induction. A role for NF-B activation, mediated through PKC, acts to induce cIAP-2 expression These results demonstrate that the PKC␦/NF-B pathway plays an important role in the regulation of the anti-apoptosis protein cIAP-2 in human colon cancer cells
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