sulindac-sulfide and Colorectal-Neoplasms

Distant metastatic colorectal cancer (CRC) is present in approximately 25% of patients at initial diagnosis, and eventually half of CRC patients will develop metastatic disease. The 5-year survival rate for patients with metastatic CRC is a mere 12.5%; thus, there is an urgent need to investigate the molecular mechanisms of cancer progression in CRC. High expression of human high-mobility group A2 (HMGA2) is related to tumor progression, a poor prognosis, and a poor response to therapy for CRC. Therefore, HMGA2 is an attractive target for cancer therapy. In this study, we identified aspirin and sulindac sulfide as novel potential inhibitors of HMGA2 using a genome-wide mRNA signature-based approach. In addition, aspirin and sulindac sulfide induced cytotoxicity of CRC cells stably expressing HMGA2 by inhibiting cell proliferation and migration. Moreover, a gene set enrichment analysis (GSEA) revealed that gene sets related to inflammation were positively correlated with HMGA2 and that the main molecular function of these genes was categorized as a G-protein-coupled receptor (GPCR) activity event. Collectively, this is the first study to report that aspirin and sulindac sulfide are novel potential inhibitors of HMGA2, which can induce cytotoxicity of CRC cells stably expressing HMGA2 by inhibiting cell proliferation and migration through influencing inflammatory-response genes, the majority of which are involved in GPCR signaling.

Topics: Aspirin; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cytotoxins; HMGA2 Protein; Humans; Neoplasm Proteins; Sulindac

Nonsteroidal anti-inflammatory drugs (NSAIDs) are well known for treating inflammatory disease and have been reported to have anti-tumorigenic effects. Their mechanisms are not fully understood, but both cyclooxygenase (COX) dependent and independent pathways are involved. Our goal was to shed further light on COX-independent activity.. Human colorectal cancer cells were observed under differential interference contrast microscopy (DICM), fluorescent microscopy, and micro-impedance measurement. Microarray analysis was performed using HCT-116 cells treated with sulindac sulfide (SS). PCR and Western blots were performed to confirm the microarray data and immunohistochemistry was performed to screen for Nesprin-2 expression. Micro-impedance was repeating including Nesprin-2 knock-down by siRNA.. HCT-116 cells treated with SS showed dramatic morphological changes under DICM and fluorescent microscopy, as well as weakened cellular adhesion as measured by micro-impedance. Nesprin-2 was selected from two independent microarrays, based on its novelty in relation to cancer and its role in cell organization. SS diminished Nesprin-2 mRNA expression as assessed by reverse transcriptase and real time PCR. Various other NSAIDs were also tested and demonstrated that inhibition of Nesprin-2 mRNA was not unique to SS. Additionally, immunohistochemistry showed higher levels of Nesprin-2 in many tumors in comparison with normal tissues. Further micro-impedance experiments on cells with reduced Nesprin-2 expression showed a proportional loss of cellular adhesion.. Nesprin-2 is down-regulated by NSAIDs and highly expressed in many cancers.. Our data suggest that Nesprin-2 may be a potential novel oncogene in human cancer cells and NSAIDs could decrease its expression.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Biomarkers, Tumor; Blotting, Western; Cell Adhesion; Cell Proliferation; Colorectal Neoplasms; Electric Impedance; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Male; Microfilament Proteins; Nerve Tissue Proteins; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA Stability; RNA, Messenger; RNA, Small Interfering; Sulindac; Tissue Array Analysis; Tumor Cells, Cultured

Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) expression is upregulated not only by NSAIDs such as sulindac sulfide, but also by several antitumorigenic dietary compounds, suggesting that NAG-1 is a specific target for the development of effective anticancer agents. Despite being a downstream target of p53, NAG-1 induction is both p53-dependent and p53-independent. It is not clear whether NAG-1 induction is the responsible factor in cancer cell apoptosis with mutated p53. In this study, we report that NAG-1 induction alone cannot determine apoptotic cell fate in colon cancer cells. Although docetaxel induced an increase in NAG-1 and apoptosis in both HCT-116 (wild-type p53) and HT-29 (mutant p53) colon cancer cells, NAG-1 knockdown with siRNA prevented docetaxel-induced cell death in only HCT-116 cells. Docetaxel decreased Bcl-2 in HCT-116 cells, which have functionally active p53, according to luciferase reporter gene analyses, and docetaxel-induced cell death and changes in Bcl-2 and NAG-1 expression were blocked by PFT-α, a p53 inhibitor. In HT-29 cells with functionally inactive p53, the docetaxel-induced Bcl-xL decrease, NAG-1 increase, and cell death were not blocked by PFT-α. On the other hand, sulindac sulfide at concentrations that significantly induced NAG-1 did not decrease cell viability comparable to docetaxel, and it did not affect the level of p53, Bax, Bcl-2, and Bcl-xL in either cell line. The present study demonstrates that p53-dependent NAG-1 induction is linked to cell death and that NAG-1 induction without accompanying alteration of antiapoptosis protein Bcl-2 family members may not lead to cancer cell death.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Cell Survival; Colorectal Neoplasms; Docetaxel; Genes, p53; Growth Differentiation Factor 15; HCT116 Cells; HT29 Cells; Humans; Molecular Targeted Therapy; Sulindac; Taxoids; Tumor Suppressor Protein p53; Up-Regulation

COX inhibitors appear to be promising agents in combination with cytostatics in the treatment of colorectal carcinoma (CRC). The aim of this study was to compare growth inhibitory effects of cytostatics (5-fluorouracil, 5-FU; oxaliplatin) and COX inhibitor sulindac sulfide (an active metabolite of sulindac), given alone or in combination, on several CRC cell lines.. A series of human CRC cell lines were incubated with various combinations of the test drugs used in concentrations from 3 to 200 microM. The cell survival was assessed by MTT assay. Isobolograms and median effect method of Chou and Talalay were used to assess the nature and quantitative aspects of interaction observed between studied drugs. Cell cycle progression and apoptosis were measured using flow cytometric methods. In addition, growth inhibitory effects of studied agents on CRC cell lines were compared with a normal (mouse fibroblast) cell line.. Sulindac sulfide synergistically potentiated the inhibitory effects of 5-FU and oxaliplatin on CRC survival, parallel to the induction of apoptosis. A dose reduction effect for synergistic activity of sulindac sulfide with studied cytostatics (in the range of 5- to 14-fold, when compared to single agent) suggested that the inhibitory effect of cytostatics on CRC survival may be obtained at low doses. In addition, sulindac sulfide appeared to be more specific against CRC cells than normal cells.. It was apparent that combination of 5-FU or oxaliplatin with sulindac sulfide results in a powerful inhibition of growth of colorectal carcinoma cells in vitro, which may be more specific for cancer than normal cells.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; BALB 3T3 Cells; Cell Cycle; Cell Growth Processes; Cell Survival; Colorectal Neoplasms; Drug Synergism; Fluorouracil; HT29 Cells; Humans; Mice; Organoplatinum Compounds; Oxaliplatin; Sulindac

To determine the role of 14-3-3 in colorectal cancer apoptosis induced by nonsteroidal anti-inflammatory drugs (NSAIDs), we evaluated the effects of sulindac on 14-3-3epsilon protein expression in colorectal cancer cells. Sulindac sulfide inhibited 14-3-3epsilon proteins in HT-29 and DLD-1 cells in a time- and concentration-dependent manner. Sulindac sulfone at 600 mumol/L inhibited 14-3-3epsilon protein expression in HT-29. Indomethacin and SC-236, a selective cyclooxygenase-2 (COX-2) inhibitor, exerted a similar effect as sulindac. Sulindac suppressed 14-3-3epsilon promoter activity. As 14-3-3epsilon promoter activation is mediated by peroxisome proliferator-activated receptor delta (PPARdelta), we determined the correlation between 14-3-3epsilon inhibition and PPARdelta suppression by NSAIDs. Sulindac sulfide inhibited PPARdelta protein expression and PPARdelta transcriptional activity. Overexpression of PPARdelta by adenoviral transfer rescued 14-3-3epsilon proteins from elimination by sulindac or indomethacin. NSAID-induced 14-3-3epsilon suppression was associated with reduced cytosolic Bad with elevation of mitochondrial Bad and increase in apoptosis which was rescued by Ad-PPARdelta transduction. Stable expression of 14-3-3epsilon in HT-29 significantly protected cells from apoptosis. Our findings shed light on a novel mechanism by which NSAIDs induce colorectal cancer apoptosis via the PPARdelta/14-3-3epsilon transcriptional pathway. These results suggest that 14-3-3epsilon is a target for the prevention and therapy of colorectal cancer.

Topics: 14-3-3 Proteins; Adenoviridae; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; bcl-Associated Death Protein; Colorectal Neoplasms; Cyclooxygenase 2 Inhibitors; HT29 Cells; Humans; Indomethacin; Mitochondria; PPAR delta; Promoter Regions, Genetic; Pyrazoles; Sulfonamides; Sulindac

Non-steroidal anti-inflammatory drugs (NSAIDs) have shown chemopreventive properties in colorectal cancer, involving both cyclooxygenase (COX)-dependent and -independent mechanisms. Apart from their selectivity for COX isoenzymes, NSAIDs differ in their acidic character which supports ability to uncouple oxidative phosphorylation. To assess the possible contribution of uncoupling to their antineoplastic properties, we compared the effect of sulindac sulfide (SS), an acidic NSAID and NS-398, a non-acidic tricyclic, on mitochondrial function and apoptosis in colorectal cancer cell lines (HT29, Caco-2, HCT15 and HCT116). Although cell lines displayed a different COX status, SS and NS-398 caused growth arrest in a dose-related manner. High dose (10(-4)M) of SS but not of NS-398, increased the percentage of subG1 cell population while reducing mitochondrial transmembrane potential (DeltaPsim). Cyclosporin A (CsA, 1 microM) prevented collapse of DeltaPsim induced by 10(-4)M SS but not by 7.5 microM FCCP used as a protonophoric control. SS and FCCP increased the cytosolic release of Smac/DIABLO which was differently affected by CsA pretreatment depending on the uncoupler. Finally, 7.5 microM FCCP failed to induce apoptosis whereas CsA prevented apoptosis induced by SS from 16% in HCT15 to 41% in HCT116. The present study shows that despite the ability of sulindac sulfide to behave as a protonophoric uncoupler, CsA-sensitive opening of mitochondrial permeability transition pore contributes little to its pro-apoptotic effect in colorectal cancer cells.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Apoptosis Regulatory Proteins; Carrier Proteins; Chemoprevention; Colorectal Neoplasms; Humans; Intracellular Signaling Peptides and Proteins; Mitochondria; Mitochondrial Proteins; Nitrobenzenes; Oxidation-Reduction; Phosphorylation; Sulfonamides; Sulindac; Tumor Cells, Cultured; Uncoupling Agents

We previously showed that nonsteroidal anti-inflammatory drugs (NSAID) such as sulindac sulfide, which has chemopreventive activity, modulate the expression of several genes detected by microarray analysis. Activating transcription factor 3 (ATF3) was selected for further study because it is a transcription factor involved in cell proliferation, apoptosis, and invasion, and its expression is repressed in human colorectal tumors as compared with normal adjacent tissue. In this report, we show that ATF3 mRNA and protein expression are up-regulated in HCT-116 human colorectal cancer cells following treatment with NSAIDs, troglitazone, diallyl disulfide, and resveratrol. To ascertain the biological significance of ATF3, we overexpressed full-length ATF3 protein in the sense and antisense orientations. Overexpression of ATF3 in the sense orientation decreased focus formation in vitro and reduced the size of mouse tumor xenografts by 54% in vivo. Conversely, overexpression of antisense ATF3 was protumorigenic in vitro, however, not in vivo. ATF3 in the sense orientation did not modulate apoptosis, indicating another mechanism is involved. With microarray analysis, several genes relating to invasion and metastasis were identified by ATF3 overexpression and were confirmed by real-time reverse transcription-PCR, and several of these genes were modulated by sulindac sulfide, which inhibited invasion in these cells. Furthermore, overexpression of ATF3 inhibited invasion to a similar degree as sulindac sulfide treatment, whereas antisense ATF3 increased invasion. In conclusion, ATF3 represents a novel mechanism in which NSAIDs exert their anti-invasive activity, thereby linking ATF3 and its gene regulatory activity to the biological activity of these compounds.

Topics: Activating Transcription Factor 3; Allyl Compounds; Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Chromans; Colorectal Neoplasms; Cyclooxygenase Inhibitors; Disulfides; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Male; Mice; Mice, Nude; Microarray Analysis; Neoplasm Invasiveness; Resveratrol; RNA, Messenger; Stilbenes; Sulindac; Thiazolidinediones; Transcription Factors; Transplantation, Heterologous; Troglitazone; Up-Regulation

Cyclooxygenase (COX) inhibitors have antitumorigenic activity and increase the expression of the early growth response gene Egr-1, a tumor suppressor gene and transcription factor. In this study, we have investigated the gene regulatory and anti-invasive activity of two traditional nonsteroidal anti-inflammatory drugs (NSAID), sulindac sulfide and indomethacin. These compounds inhibited tumor cell invasion and induced Egr-1 expression in lung adenocarcinoma A549 cells. Overexpression of Egr-1 reduced cellular invasion in the Matrigel system, whereas suppression of Egr-1 by small interference RNA (siRNA) attenuated the inhibition of Matrigel invasion by these compounds, indicating that Egr-1 is responsible for the decrease in invasion reported following treatment with NSAIDs. Egr-1-overexpressing cells were analyzed for genes involved in invasion and metastasis. Thrombospondin-1 (TSP-1) an antiangiogenic and anti-invasion protein was up-regulated by Egr-1 overexpression, which was confirmed following treatment with sulindac sulfide. Furthermore, the induction of TSP-1 by sulindac sulfide was blocked by Egr-1 siRNA. When TSP-1 was sequestered by the addition of anti-TSP-1 antibody, the inhibition of invasion by sulindac sulfide was attenuated, indicating that TSP-1 is involved in the inhibition of invasion by NSAIDs. We used the Min mouse model to determine if sulindac sulfide would increase Egr-1 and TSP-1 in vivo, because this model is widely used to study the effects of NSAIDs on tumor formation. Treatment of Min mice with concentrations of sulindac sulfide that inhibit tumor formation increased the expression of Egr-1 and TSP-1 in colonic tissues and in the polyps of these mice. This is the first report suggesting that COX inhibitors suppress tumor cell invasion via TSP-1, which occurs downstream of Egr-1.

Topics: Adenocarcinoma; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Line, Tumor; Colorectal Neoplasms; Cyclooxygenase Inhibitors; Early Growth Response Protein 1; Genes, Tumor Suppressor; Humans; Indomethacin; Lung Neoplasms; Mice; Neoplasm Invasiveness; RNA, Messenger; RNA, Small Interfering; Sulindac; Thrombospondin 1

The mechanisms underlying the anti-tumorigenic properties of cyclooxygenase inhibitors are not well understood. One novel hypothesis is alterations in gene expression. To test this hypothesis sulindac sulfide, which is used to treat familial adenomatous polyposis, was selected to detect gene modulation in human colorectal cells at physiological concentrations with microarray analysis. At micromolar concentrations, sulindac sulfide stimulated apoptosis and inhibited the growth of colorectal cancer cells on soft agar. Sulindac sulfide (10 microm) altered the expression of 65 genes in SW-480 colorectal cancer cells, which express cyclooxygenase-1 but little cyclooxygenase-2. A more detailed study of 11 genes revealed that their expression was altered in a time- and dose-dependent manner as measured by real-time RT-PCR. Northern analysis confirmed the expression of 9 of these genes, and Western analysis supported the conclusion that sulindac sulfide altered the expression of these proteins. Cyclooxygenase-deficient HCT-116 cells were more responsive to sulindac sulfide-induced gene expression than SW-480 cells. However, this response was diminished in HCT-116 cells overexpressing cyclooxygenase-1 compared with normal HCT-116 cells suggesting the presence of cyclooxygenase attenuates this response. However, prostaglandin E2, the main product of cyclooxygenase, only suppressed the sulindac sulfide-induced expression of two genes, with little known biological function while it modulated the expression of two more. The most likely explanation for this finding is the metabolism of sulindac sulfide to inactive metabolites by the peroxidase activity of cyclooxygenase. In conclusion, this is the first report showing sulindac sulfide, independent of cyclooxygenase, altered the expression of several genes possibly linked to its anti-tumorigenic and pro-apoptotic activity.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Blotting, Northern; Blotting, Western; Cell Division; Cell Separation; Cloning, Molecular; Colorectal Neoplasms; Cyclooxygenase 1; Cyclooxygenase Inhibitors; Dinoprostone; DNA; Dose-Response Relationship, Drug; Flow Cytometry; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Indomethacin; Isoenzymes; Membrane Proteins; Oligonucleotide Array Sequence Analysis; Prostaglandin-Endoperoxide Synthases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sulindac; Time Factors; Transcription, Genetic; Tumor Cells, Cultured

Sulindac sulfide, a metabolite of the nonsteroidal antiinflammatory drug (NSAID) sulindac sulfoxide, is effective at reducing tumor burden in both familial adenomatous polyposis patients and in animals with colorectal cancer. Another sulindac sulfoxide metabolite, sulindac sulfone, has been reported to have antitumor properties without inhibiting cyclooxygenase activity. Here we report the effect of sulindac sulfone treatment on the growth of colorectal carcinoma cells. We observed that sulindac sulfide or sulfone treatment of HCA-7 cells led to inhibition of prostaglandin E2 production. Both sulindac sulfide and sulfone inhibited HCA-7 and HCT-116 cell growth in vitro. Sulindac sulfone had no effect on the growth of either HCA-7 or HCT-116 xenografts, whereas the sulfide derivative inhibited HCA-7 growth in vivo. Both sulindac sulfide and sulfone inhibited colon carcinoma cell growth and prostaglandin production in vitro, but sulindac sulfone had no effect on the growth of colon cancer cell xenografts in nude mice.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Blotting, Western; Cell Division; Chromatography, Gas; Collagen; Colorectal Neoplasms; Cyclooxygenase 1; Cyclooxygenase 2; Dinoprostone; Dose-Response Relationship, Drug; Drug Combinations; Electrophoresis, Polyacrylamide Gel; Isoenzymes; Laminin; Membrane Proteins; Mice; Mice, Nude; Neoplasm Transplantation; Prostaglandin-Endoperoxide Synthases; Proteoglycans; Sulindac; Time Factors; Tumor Cells, Cultured