SUMMARY Esophageal adenocarcinoma continues to rise in incidence. Despite recognition of Barrett's metaplasia as the histological precursor, prognosis remains poor. The mitogen-activated protein kinases (MAPK) pathway is activated in Barrett's-associated dysplasia and adenocarcinoma and this activation is, in part, due to acid and bile acid reflux. We investigated the effects of sorafenib, an orally active Raf-inhibitor, on acid and bile acid-stimulated growth and signaling in SEG-1 cells, derived from a Barrett's esophageal cancer. SEG-1 cells were pretreated with sorafenib or vehicle and subsequently stimulated with acid or bile acid. MAPK signals, including phospho-ERK and phospho-p38, as well as cyclin D1 expression were assessed by Western blotting. Cell proliferation was measured by WST-1 colorimetric assay. Acid (pH 3.0-4.0) and bile acid (taurocholate 50-100 micromol/L) activated ERK and p38. Acid and bile acid exposure also increased levels of cyclin D1, a G1 to S cell cycle regulator. Furthermore, acid and taurocholate exposure increased cell proliferation. Sorafenib abrogated MAPK activation and cyclin D1 up-regulation and significantly inhibited cell growth. In summary, sorafenib inhibits acid or bile acid-stimulated Barrett's esophageal cancer cell proliferation by a mechanism involving the MAPK pathway. Our results suggest that sorafenib might be useful in the management of Barrett's-associated dysplasia and adenocarcinoma. These findings provide a foundation for in vivo studies to assess the efficacy of sorafenib in Barrett's-related neoplasia.

PKC-δ is a serine/threonine kinase that mediates diverse signal transduction pathways. We previously demonstrated that overexpression of PKC-δ slowed the G1 progression of Caco-2 colon cancer cells, accelerated apoptosis, and induced cellular differentiation. In this study, we further characterized the PKC-δ dependent signaling pathways involved in these tumor suppressor actions in Caco-2 cells overexpressing PKC-δ using a Zn2+ inducible expression vector. Consistent with a G1 arrest, increased expression of PKC-δ 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 p21Waf1, a cyclin-dependent kinase (cdk) inhibitor in PKC-δ transfectants compared with empty vector (EV) transfected cells, whereas the PKC-δ specific inhibitor rottlerin (3 μ M) or knockdown of this isoenzyme with specific siRNA oligonucleotides blocked p21Waf1 expression. Concomitantly, compared to EV control cells, PKC-δ upregulation decreased cyclin D1 and cyclin E proteins co-immunoprecipitating with cdk6 and cdk2, respectively. In addition, overexpression of PKC-δ 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-δ plays in cell growth and cell cycle regulation, we knocked down PKC-δ using specific siRNA oligonucleotides. PKC-δ specific siRNA oligonucleotides, but not irrelevant control oligonucleotides, inhibited PKC-δ protein by more than 80% in Caco-2 cells. Moreover, PKC-δ knockdown enhanced cell proliferation (∼1.4-2-fold, P<0.05) and concomitantly increased cyclin D1 and cyclin E expression (∼1.7-fold, P<0.05). This was a specific effect, as nontargeted PKC-ζ was not changed by PKC-δ siRNA oligonucleotides. Consistent with accelerated apoptosis in PKC-δ transfectants, compared to EV cells, PKC-δ 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-δ specific siRNA oligonucleotides inhibited Bax protein expression by more than 50%, indicating that PKC-δ regulates apoptosis through Bax. Taken together, these results elucidate two critical mechanisms regulated by PKC-δ 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-δ in colonic carcinogenesis.

cancer than that of MSI negative colorectal cancer.But iNOS expression according to MSI is not reported.COX-2 and iNOS have been shown to contribute to the process of carcinogenesis in various cancers including colorectal cancer.But conflicting reports exist the interaction between COX-2 and iNOS.The aims of this study was to evaluate COX-2 and iNOS expression according to MSI and evaluate interaction between COX-2 and iNOS.The MMRproficient colorectal cancer cell line, SW480 and MMR-deficient colorectal cancer lines, HCTll6 (MLH1-), HCT15 (MSH6-), and LoVo (MSH2-) cell lines are cultured.The COX-2 and iNOS mRNA and protein expression is evaluated by RT-PCR and Western blot.COX-2 inbibiotor (NS398)and iYOS inhibitor (1400W) treated to cell lines.And than COX-2 and iNOS mRNA and protein expression was evaluated 2 days and 5 days after treated with inhi~itors.The COX-2 and iNOS mRNA expression is not different related to MSI.The COX-2 protein expression is not decreased in MMR-deficient cancer cell lines compared to MMR-proficient, cancer cell line.But iNOS protein expression is relatively increased in MMRdeficient cancer cell lines compared to MMR-proficient cancer cell line.The COX-2 protein expression is decreased in all cancer cell lines treated with NS398.The iNOS protein expression is increased in SW480, decreased in LoVo and not changed in HCTll6, HCT15 after treated with NS398.The COX-2 and iNOS protein expression is not changed in SW480 but, decreased in HCTll6, HCT15 and LoVo after treated with 1400W.The COX-2 suppressed iNOS expression in SW480.But, the iNOS suppressed COX-2 expression in HCT15 but stimulated in HCTll6.The both COX-2 and iNOS stimulated iNOS and COX-2 expression in LoVo.The iYOS protein expression is relatively increased in MMR-deficient colorectal cancer cell lines compared to MMR-deficient colorectal cancer cell line.The relationship between COX-2 and iNOS is variable results according to cancer cell lines, microsatellite instability and mutated MMR.Ahhought further sudidies are needed, this result suggest that iNOS inhibitor is more effective than the COX-2 inhibitor in MMR mutated cancers for chemoprevention.

Ik-l-induced NF-KB transcriptional activation.Conclusion: IL-1-induced COX-2 expression in IMF occurs, in part, vua a PKCg-dependent pathway.Contrary to other cell types, neither inhibition of PKC nor treatment with antioxidants affected NF-/cB transcription in IMF.These data suggest that PKC~ and ROS function via a pathway that works in conjunction with NF-KB and that both of these pathways are necessary for IL-l-induced COX-2 expression.

BACKGROUND & AIMS:Previous studies showed decreased protein kinase C (PKC)-delta expression in azoxymethane-induced rat and sporadic human colonic tumors. To elucidate the role of PKC-delta on the neoplastic phenotype of human colon cancer cells, we established stable transfectants of this isoenzyme in CaCo-2 cells.METHODS:Human PKC-delta complementary DNA was subcloned into 2 distinct metallothionein-regulated expression vectors. Polyclonal populations of PKC-delta transfectants were characterized by Western blotting. PKC-delta activity was measured in situ using a PKC-delta-specific substrate. Proliferation was determined by Coulter counter, and cell cycle distribution was analyzed by flow cytometry. In vitro transformation was assessed by growth in soft agar and differentiation by changes in alkaline phosphatase and sucrase isomaltase. Apoptosis was evaluated by 4',6-diamidino-2-phenylindole dihydrochloride and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining.RESULTS:In the presence of Zn(2+), PKC-delta transfectants expressed a 4-fold increase in the protein and a 2-fold increase in activity of PKC-delta. PKC-delta transfectants exhibited a 30% decrease (P < 0.05) in cell growth and an enhanced differentiation phenotype. Increased PKC-delta expression induced a significant G0/G1 arrest, inhibited anchorage-independent growth (50%, P < 0.05), and caused a 2-fold increase in apoptosis (P < 0.05).CONCLUSIONS:Our studies show that increased expression of PKC-delta inhibits anchorage-dependent and -independent growth, while inducing cellular differentiation and limiting survival of this human colon cancer cell line.