Protein kinase C delta inhibits Caco-2 cell proliferation by selective changes in cell cycle and cell death regulators

• Published in: Oncogene Vol. 25; no. 22; pp. 3123 - 3138
• Main Authors: CERDA, S. R, MUSTAFI, R, LITTLE, H, COHEN, G, KHARE, S, MOORE, C, MAJUMDER, P, BISSONNETTE, M
• Format: Journal Article
• Language: English
• Published: Basingstoke Nature Publishing 25-05-2006; Nature Publishing Group
• Subjects: Acetic acid; Ageing, cell death; Apoptosis; BAX protein; Bcl-2 protein; bcl-2-Associated X Protein - metabolism; Biological and medical sciences; Caco-2 Cells; Carcinogenesis; Cell cycle; Cell cycle, cell proliferation; Cell death; Cell differentiation; Cell growth; Cell physiology; Cell Proliferation; Cell transformation and carcinogenesis. Action of oncogenes and antioncogenes; Colon cancer; Colorectal cancer; Cyclin D1; Cyclin D1 - metabolism; Cyclin E; Cyclin E - metabolism; Cyclin-dependent kinase; Cyclin-Dependent Kinase 2 - metabolism; Cyclin-Dependent Kinase 6 - metabolism; Cyclin-dependent kinase inhibitor p21; Cyclin-Dependent Kinase Inhibitor p21 - metabolism; Cyclin-dependent kinase inhibitor p27; Cyclin-dependent kinases; Down-Regulation; Enzyme inhibitors; Enzyme Inhibitors - pharmacology; Fundamental and applied biological sciences. Psychology; G1 Phase - physiology; Gene expression; GTP-binding protein; Humans; Immunoprecipitation; Kinases; Molecular and cellular biology; Oligonucleotides; Phorbol 12-myristate 13-acetate; Post-transcription; Protein folding; Protein kinase C; Protein Kinase C-delta - physiology; Protein-serine/threonine kinase; Proteins; Proto-Oncogene Proteins c-bcl-2 - metabolism; RNA, Small Interfering - pharmacology; Signal Transduction; Tetradecanoylphorbol Acetate - pharmacology; Tumor suppressor genes; Tumors; Cell proliferation; Bcl-2; Protein kinase C; p21Waf1; Enzyme; Transferases; cyclins; CDKN1A Gene; Cyclin; PKC; Malignant tumor; Carcinogenesis; Colonic disease; Colon cancer; colon cancer cells; Cell death; C-Onc gene; Cell cycle; Bax; Digestive diseases; Intestinal disease; Protooncogene
• ISSN: 0950-9232; 1476-5594
• DOI: 10.1038/sj.onc.1209360

PKC-delta is a serine/threonine kinase that mediates diverse signal transduction pathways. We previously demonstrated that overexpression of PKC-delta slowed the G1 progression of Caco-2 colon cancer cells, accelerated apoptosis, and induced cellular differentiation. In this study, we further characterized the PKC-delta dependent signaling pathways involved in these tumor suppressor actions in Caco-2 cells overexpressing PKC-delta using a Zn2+ inducible expression vector. Consistent with a G1 arrest, increased expression of PKC-delta caused rapid and significant downregulation of cyclin D1 and cyclin E proteins (50% decreases, P<0.05), while mRNA levels remained unchanged. The PKC agonist, phorbol 12-myristate 13-acetate (TPA, 100 nM, 4 h), induced two-fold higher protein and mRNA levels of p21(Waf1), a cyclin-dependent kinase (cdk) inhibitor in PKC-delta transfectants compared with empty vector (EV) transfected cells, whereas the PKC-delta specific inhibitor rottlerin (3 microM) or knockdown of this isoenzyme with specific siRNA oligonucleotides blocked p21(Waf1) expression. Concomitantly, compared to EV control cells, PKC-delta upregulation decreased cyclin D1 and cyclin E proteins co-immunoprecipitating with cdk6 and cdk2, respectively. In addition, overexpression of PKC-delta increased binding of cdk inhibitor p27(Kip1) to cdk4. These alterations in cyclin-cdks and their inhibitors are predicted to decrease G1 cyclin kinase activity. As an independent confirmation of the direct role PKC-delta plays in cell growth and cell cycle regulation, we knocked down PKC-delta using specific siRNA oligonucleotides. PKC-delta specific siRNA oligonucleotides, but not irrelevant control oligonucleotides, inhibited PKC-delta protein by more than 80% in Caco-2 cells. Moreover, PKC-delta knockdown enhanced cell proliferation ( approximately 1.4-2-fold, P<0.05) and concomitantly increased cyclin D1 and cyclin E expression ( approximately 1.7-fold, P<0.05). This was a specific effect, as nontargeted PKC-zeta was not changed by PKC-delta siRNA oligonucleotides. Consistent with accelerated apoptosis in PKC-delta transfectants, compared to EV cells, PKC-delta upregulation increased proapoptotic regulator Bax two-fold at mRNA and protein levels, while antiapoptotic Bcl-2 protein was decreased by 50% at a post-transcriptional level. PKC-delta specific siRNA oligonucleotides inhibited Bax protein expression by more than 50%, indicating that PKC-delta regulates apoptosis through Bax. Taken together, these results elucidate two critical mechanisms regulated by PKC-delta that inhibit cell cycle progression and enhance apoptosis in colon cancer cells. We postulate these antiproliferative pathways mediate an important tumor suppressor function for PKC-delta in colonic carcinogenesis.