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)-enhanced invasiveness was associated with augmentation of cell motility but not that of metalloproteinase activity in a highly metastatic variant (L-10) of the human colon adenocarcinoma cell line RCM-1 and that this enhancement was possibly mediated by protein kinase C (PKC). In this study, we first intended 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 to inhibit the enhanced motility and invasion by using antisense oligodeoxynucleotides (ODN) targeting the isoform. An activator of conventional PKC isoforms (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 used in our assays. Antisense ODNs specific for PKC-alpha efficiently reduced its expression at the protein levels and inhibited L-10 cell motility in the 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 phorbol 12,13 dibutyrate (PDBu) and treatment with antisense ODNs effectively inhibited L-10 cell invasion even in the presence of TPA. These results suggested that downregulation of PKC isoforms by treatment with antisense ODNs alone is insufficient to suppress the isoform-mediated cellular events in the presence of PKC activators, and thus that some additional treatments are necessary for the successful downregulation of them.
Shimao
Y
Y
Department of Pathology, Miyazaki Medical College, Kiyotake, Japan.
Nabeshima
K
K
Inoue
T
T
Koono
M
M
D000230:Adenocarcinoma / Q000201:enzymology / Q000473:pathology*; D001483:Base Sequence; D002227:Carbazoles / Q000494:pharmacology; D015536:Down-Regulation; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009281:Naphthalenes / Q000494:pharmacology; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D010703:Phorbol Esters / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D012004:Rectal Neoplasms / Q000201:enzymology / Q000473:pathology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D002227:Carbazoles; D007211:Indoles; D007527:Isoenzymes; D009281:Naphthalenes; D016376:Oligonucleotides, Antisense; D010703:Phorbol Esters; C081021:Go 6976; D015240:Phorbol 12,13-Dibutyrate; C080287:thymeleatoxin; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate