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

Abstract

PKC-d is a serine/threonine kinase that mediates diverse signal transduction pathways. We previously demonstrated that overexpression of PKC-d slowed the G1 progression of Caco-2 colon cancer cells, accelerated apoptosis, and induced cellular differentiation. In this study, we further characterized the PKC-d dependent signaling pathways involved in these tumor suppressor actions in Caco-2 cells overexpressing PKC-d using a Zn 2 þ inducible expression vector. Consistent with a G1 arrest, increased expression of PKC-d caused rapid and significant downregulation of cyclin D1 and cyclin E proteins (50% decreases, Po0.05), 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-d transfectants compared with empty vector (EV) transfected cells, whereas the PKC-d specific inhibitor rottlerin (3 lM) or knockdown of this isoenzyme with specific siRNA oligonucleotides blocked p21Waf1 expression. Concomitantly, compared to EV control cells, PKC-d upregulation decreased cyclin D1 and cyclin E proteins co-immunoprecipitating with cdk6 and cdk2, respectively. In addition, overexpression of PKC-d increased binding of cdk inhibitor p27Kip1 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-d plays in cell growth and cell cycle regulation, we knocked down PKC-d using specific siRNA oligonucleotides. PKC-d specific siRNA oligonucleotides, but not irrelevant control oligonucleotides, inhibited PKCd protein by more than 80% in Caco-2 cells. Moreover, PKC-d knockdown enhanced cell proliferation (B1.4-2-fold, Po0.05) and concomitantly increased cyclin D1 and cyclin E expression (B1.7-fold, Po0.05). This was a specific effect, as nontargeted PKC-f was not changed by PKC-d siRNA oligonucleotides. Consistent with accelerated apoptosis in PKC-d transfectants, compared to EV cells, PKC-d 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-d specific siRNA oligonucleotides inhibited Bax protein expression by more than 50%, indicating that PKC-d regulates apoptosis through Bax. Taken together, these results elucidate two critical mechanisms regulated by PKC-d 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-d in colonic carcinogenesis.

Evaluation of apoptotic cell death mechanisms induced by hypericin-mediated photodynamic therapy in colon cancer cells

“…Bcl-2 upregulation is related to the worst prognosis in colorectal tumorigenesis, while Bax expression tumors have a better prognosis (Cerda et al, 2006). Suppressed Bcl-2 and induced Bax expression are associated with altered apoptotic profiles in Caco-2 cells (Cerda et al, 2006).…”

Protein kinase C-θ regulates KIT expression and proliferation in gastrointestinal stromal tumors

“…The protein kinase C family has crucial regulatory roles in cell growth and cell-cycle progression (Frey et al, 1997;Ashton et al, 1999;Jiang et al, 2002;Deeds et al, 2003;Cerda et al, 2006). Various studies have shown that PKC suppression generally inhibits cell-cycle progression, whereas PKC activation stimulates cellcycle progression (Frey et al, 1997;Deeds et al, 2003;Cerda et al, 2006).…”

“…Various studies have shown that PKC suppression generally inhibits cell-cycle progression, whereas PKC activation stimulates cellcycle progression (Frey et al, 1997;Deeds et al, 2003;Cerda et al, 2006). Therefore, we evaluated cell-cycle checkpoints after PKCy silencing in our GIST models (Figure 2c).…”

Protein Kinase Cγ Mediates Regulation of Proliferation by the Serotonin 5-Hydroxytryptamine Receptor 2B

“…PKC␣ has been reported to increase cellular proliferation in hepatocellular carcinoma (40) and goblet cells (41), whereas PKC regulates Kit expression and induces proliferation in gastrointestinal stromal tumors (42). Conversely, PKC␤ and PKC␦ down-regulate proliferation of vascular smooth muscle cells (43,44), PKC␦ also inhibits Caco-2 cell proliferation (45).…”