TPA-enhanced motility and invasion in a highly metastatic variant (L-10) of human rectal adenocarcinoma cell line RCM-1: selective role of PKC-alpha and its inhibition by a combination of PDBu-induced PKC downregulation and antisense oligonucleotides treatment

We previously found that 12-O-tetradecanoylphorbol-13-acetate (TPA)-enhance d invasiveness was associated with augmentation of cell motility but not th at of metalloproteinase activity in a highly metastatic variant (L-10) of t he human colon adenocarcinoma cell line RCM-1 and that this enhancement was possibly mediated by protein kinase C (PKC). In this study, we first inten ded to determine the specific isoforms of PKC involved in this TPA-enhanced L-10 cell motility that leads to invasion, and then investigated the way t o inhibit the enhanced motility and invasion by using antisense oligodeoxyn ucleotides (ODN) targeting the isoform. An activator of conventional PKC is oforms (cPKC), thymeleatoxin, enhanced L-10 cell motility and invasion like TPA, and an inhibitor of cPKC, Go-6976, efficiently inhibited TPA-enhanced motility and invasion. TPA treatment induced a shift of PKC-alpha, but not other isoforms, from the cytosol to the membrane fraction, indicating the activation of the isoform. During the assay period, only activation but not downregulation of PKC-alpha occurred with the low concentration of TPA use d in our assays. Antisense ODNs specific for PKC-alpha efficiently reduced its expression at the protein levels and inhibited L-10 cell motility in th e absence of TPA. With TPA treatment, however, the remaining PKC-alpha was sufficient for activation leading to enhanced invasion. Only a combination of depletion of PKC by prolonged stimulation with a high concentration of p horbol 12,13 dibutyrate (PDBu) and treatment with antisense ODNs effectivel y inhibited L-10 cell invasion even in the presence of TPA. These results s uggested that downregulation of PKC isoforms by treatment with antisense OD Ns alone is insufficient to suppress the isoform-mediated cellular events i n the presence of PKC activators, and thus that some additional treatments are necessary for the successful downregulation of them.