cyclin-d1 and sulindac-sulfone

Phosphodiesterase type 5 (PDE5) plays a key role in regulating the intracellular cyclic GMP (cGMP) concentration, which influences anti-proliferative and pro-apoptotic mechanisms in multiple carcinomas. PDE5 inhibitors, such as exisulind and its analogs have anticancer activities. In this study, we found that suppressing PDE5 gene expression by PDE5 siRNA inhibited cell proliferation and induced apoptosis in OS-RC-2 human renal cell carcinoma cells. These effects were enhanced by 8-Br-cGMP, a cell membrane permeable cGMP derivative, and were inhibited by KT5823, a protein kinase G (PKG) inhibitor, indicating that PKG was activated by intracellular cyclic GMP. In addition, there was a reduction in both the mRNA and protein expression of cyclin D1, while p21 protein expression was increased; the reduction in cyclin D1 expression was blocked by the proteasome inhibitor, MG132, or c-Jun N-terminal kinase (JNK) inhibitor; both β-catenin and JNK were phosphorylated by activated PKG. Furthermore, p21 protein expression was decreased in Sp1 siRNA transfected-cells treated with 8-Br-cGMP, indicating that p21 may be partly controlled by the PKG activation through Sp1. Furthermore, we found that PKG Iβ was responsible for the anticancer activities. Our findings indicate that the downregulation of PKG-activated genes, such as cyclin D1 partly accounts for the pro-apoptotic effects in PDE5 siRNA-transfected OS-RC-2 cells.

Topics: Antineoplastic Agents; Apoptosis; beta Catenin; Carbazoles; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Gene Expression Regulation; Humans; JNK Mitogen-Activated Protein Kinases; Phosphodiesterase 5 Inhibitors; Phosphorylation; Signal Transduction; Sp1 Transcription Factor; Sulindac

Nonsteroidal anti-inflammatory drugs mediate anticancer effects by modulating cyclooxygenase-2 (COX-2)-dependent and/or COX-2-independent mechanism(s); however, the toxicity issue is a concern with single agents at higher doses. In this study, we determined the combined effect of celecoxib, a COX-2 inhibitor, along with exisulind (sulindac sulfone/Aptosyn) at low doses in prostate cancer.. We used a sequential regimen of N-methyl-N-nitrosourea + testosterone to induce prostate cancer in Wistar-Unilever rats. Following carcinogen treatment, celecoxib and exisulind individually and their combination at low doses were given in NIH-07 diet for 52 weeks. We determined the incidence of prostatic intraepithelial neoplasia, adenocarcinomas, rate of tumor cell proliferation, and apoptosis. Immunohistochemical and Western blot analysis were done to determine COX-2, epidermal growth factor receptor (EGFR), Akt, androgen receptor, and cyclin D1 expression. Serum prostaglandin E2 and tumor necrosis factor-alpha levels were determined using enzyme immunoassay/ELISA assays.. The rats that received celecoxib in combination with exisulind at low doses showed a significant decrease in prostatic intraepithelial neoplasia and adenocarcinomas as well as an enhanced rate of apoptosis. An overall decrease in COX-2, EGFR, Akt, androgen receptor, and cyclin D1 expression was found associated with tumor growth inhibition. Reduced serum levels of COX-2 protein, prostaglandin E2, and tumor necrosis factor-alpha indicated anti-inflammatory effects. A strong inhibition of total and phosphorylated form of EGFR (Tyr(992) and Tyr(845)) and Akt (Ser(473)) was significant in rats given with these agents in combination.. In this study, we show for the first time that the combination of celecoxib with exisulind at low doses could prevent prostate carcinogenesis by altering key molecular events.

Topics: Animals; Anticarcinogenic Agents; Antineoplastic Combined Chemotherapy Protocols; Celecoxib; Cyclin D1; Cyclooxygenase 2; Cyclooxygenase Inhibitors; ErbB Receptors; Humans; Male; Methylnitrosourea; Prostatic Neoplasms; Pyrazoles; Rats; Rats, Wistar; Receptors, Androgen; Sulfonamides; Sulindac; Testosterone

Sulindac sulfone (FGN-1, Aptosyn), a metabolite of the nonsteroidal anti-inflammatory drug sulindac, lacks cyclooxygenase inhibitory activity. Although its ability to inhibit tumorigenesis in both carcinogen-treated animals and patients with familial adenomatous polyposis has been attributed to the induction of apoptosis, its complete mechanism of action remains unclear. The purpose of the present study was to determine the ability of sulindac metabolites to regulate cellular levels of beta-catenin and downstream targets of the adenomatous polyposis coli (APC)/beta-catenin pathway in vitro. Sulindac sulfone was consistently more potent than the sulfide metabolite in all analyses, significantly decreasing the expression of total cellular beta-catenin (50% of control), pro-caspase 3 (49%), cyclin D1 (51%), and PPARdelta (65%) in SW480 cells. No significant alteration in pro-caspase 3 or beta-catenin expression was found in HCA7, LS174, or Caco-2 cells treated with sulindac sulfone. A dose-dependent reduction in TCF-mediated transcriptional activity was also observed in SW480 cells. These data demonstrate that sulindac sulfone can modulate the APC/beta-catenin pathway in vitro and that its efficacy is dependent upon the mutational status of APC and beta-catenin.

Topics: Adenomatous Polyposis Coli Protein; Antineoplastic Agents; beta Catenin; Cell Line, Tumor; Colon; Colonic Neoplasms; Cyclin D1; Cyclooxygenase Inhibitors; Humans; Mutation; PPAR gamma; Sulindac; Transcription, Genetic

The activation of protein kinase G (PKG) by cGMP has become of considerable interest as a novel molecular mechanism for the induction of apoptosis in cancer cells, because sulindac sulfone (exisulind, Aptosyn) and certain derivatives that inhibit cGMP-phosphodiesterases and thereby increase cellular levels of cGMP appear to induce apoptosis via this mechanism. However, other effects of these compounds have not been excluded, and the precise mechanism by which PKG activation induces apoptosis has not been elucidated in detail. To directly examine the effects of PKG on cell growth and apoptosis, we generated a series of mutants of PKG Ialpha: PKG IalphaS65D, a constitutively activated point mutant; PKG IalphaDelta, a constitutively activated N-terminal truncated mutant; and PKG IalphaK390R, a dominant-negative point mutant. A similar series of mutants of PKG Ibeta were also constructed (Deguchi et al., Mol. Cancer Ther., 1: 803-809, 2002). The present study demonstrates that when transiently expressed in SW480 colon cancer, the constitutively activated mutants of PKG Ibeta, and to a lesser extent PKG Ialpha, inhibit colony formation and induce apoptosis. We were not able to obtain derivatives of SW480 cells that stably expressed these constitutively activated mutants, presumably because of toxicity. However, derivatives that stably overexpressed wild-type PKG Ibeta displayed growth inhibition, whereas derivatives that stably expressed the dominant-negative mutant (KR) of PKG Ibeta grew more rapidly and were more resistant to Aptosyn-induced growth inhibition than vector control cells. Stable overexpression of PKG Ibeta was associated with decreased cellular levels of beta-catenin and cyclin D1 and increased levels of p21(CIP1). Reporter assays indicated that activation of PKG Ibeta inhibits the transcriptional activity of the cyclin D1 promoter. We also found that transient expression of the constitutively activated mutants of PKG Ibeta inhibited cell migration. Taken together, these results indicate that activation of PKG Ibeta is sufficient to inhibit growth and cell migration and induce apoptosis in human colon cancer cells and that these effects are associated with inhibition of the transcription of cyclin D1 and an increase in the expression of p21(CIP1).

Topics: Antineoplastic Agents; Apoptosis; Cell Division; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Cyclic GMP-Dependent Protein Kinases; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Activation; Humans; Mutation; Sulindac

Exisulind and its analogues are inhibitors of cyclic GMP phosphodiesterases (PDEs) that have been shown to activate and induce protein kinase G, resulting in the induction of apoptosis in colon cancer cells. These drugs also reduce beta-catenin protein levels and decrease cyclin D1 mRNA levels in SW480 cells. Herein we report on studies pertaining to exisulind regulation of beta-catenin levels and activity in colon tumor cells. Exisulind and its higher-affinity PDE analogues, (Z)-5-fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)-indenylacetamide hydrochloride (CP461) and (Z)-1H-indene-3-acetamide, 5-fluoro-2-methyl-N-(phenylmethyl)-1-[(3,4,5-trimethoxyphenyl)methylene] (CP248), reduced beta-catenin, including the nuclear beta-catenin in SW480 cells (EC(50) approximately 200 microM, 1 microM, and <1 microM, respectively). The 50% reduction of beta-catenin was seen in 8-14 hr. There was no change in beta-catenin mRNA. Exisulind-induced beta-catenin reduction was blocked by the proteasomal inhibitor MG132 (Z-leu-Leu-Leu-CHO), indicating that the effect of exisulind involved ubiquitin-proteasomal degradation. A consequence of reduced beta-catenin in SW480 cells was that exisulind, CP461, and CP248 caused a concentration- and time-dependent decrease in cyclin D1 levels (EC(50) approximately 300 microM, 1 microM, and <1 microM, respectively) in 4 hr. The effect was via decreased cyclin D1 mRNA levels. Exisulind-induced degradation of beta-catenin was not blocked by the inhibition of caspase-3 activity and/or apoptosis, and some SW480 cells showed a reduction in beta-catenin levels before the appearance of early apoptosis indicators. Expression of the N-terminal 170 amino acid fragment of beta-catenin reduced the effects of beta-catenin degradation, cyclin D1 reduction, and the apoptosis response to exisulind. These results indicate that exisulind-induced beta-catenin degradation precedes the induction of apoptosis and that the down-regulation of inappropriate beta-catenin-activated genes accounts in part for the pro-apoptotic effects of exisulind and CP461 in colon tumor cells.

Topics: Adenomatous Polyposis Coli; Antineoplastic Agents; Apoptosis; beta Catenin; Caspase 3; Caspases; Colonic Neoplasms; Cyclin D1; Cysteine Endopeptidases; Cytoskeletal Proteins; Down-Regulation; Humans; Multienzyme Complexes; Proteasome Endopeptidase Complex; Protein Biosynthesis; Signal Transduction; Sulindac; Trans-Activators; Tumor Cells, Cultured; Ubiquitin

Nonsteriodal anti-inflammatory drugs (NSAIDs) are among the most commonly used medications in the United States and elsewhere, mainly for the treatment of arthritis. The NSAID sulindac causes regression and prevents the recurrence of premalignant colonic polyps in patients with familial adenomatous polyposis and inhibits colon carcinogenesis in rodents. Sulindac and sulindac sulfone, a metabolite of sulindac that lacks cyclooxygenase (cox) inhibitory activity, also inhibit mammary carcinogenesis in rats. To obtain insights into the relevance of these findings to human breast cancer, we examined the mechanism of action of sulindac and its sulfide and sulfone metabolites on the normal human mammary epithelial cell line MCF-10F and the human breast cancer cell line MCF-7. Of the three compounds, the sulfide was the most potent inhibitor of cell growth, although the sulfone and sulfoxide were also active at higher concentrations. Treatment of MCF-10F and MCF-7 cells with 100 microM sulindac sulfide resulted in accumulation of cells in the G1 phase of the cell cycle and induction of apoptosis. Apoptosis occurred within 24 h as determined by the TUNEL assay and DNA laddering was observed at 72 h. The accumulation of cells in G1 was associated with decreased levels of expression of cyclin D1 but no effect was seen on the expression of CDK4 or the immediate early response gene c-jun. Treatment with sulindac sulfide caused a striking induction of the CDK inhibitor p21WAF1 in MCF-10F cells. The MCF-7 cell line expressed a high basal level of p21WAF1 which did not change significantly after drug treatment. The pro-apoptotic gene BAX was not induced in either MCF-10F or MCF-7 cells by sulindac sulfide. Stable overexpression of cyclin D1, which frequently occurs in breast cancers, did not protect mammary epithelial cells from inhibition by the sulfide. These studies suggest that this class of compounds warrants further study with respect to breast cancer prevention and treatment.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Apoptosis; Breast; Breast Neoplasms; Cell Division; Cell Line; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Epithelial Cells; Female; Humans; Sulindac