sulindac and Cell-Transformation--Neoplastic

Fascinating progress has been made in the past 2 years in our understanding of the genetic alterations associated with colorectal cancer predisposition and development. First, the genotype-phenotype relationship of the cancer susceptibility syndrome associated with familial adenomatous polyposis has been shown to depend on mutation type. Second, hereditary nonpolyposis colorectal cancer syndromes have been recognized as being frequently associated with a defect in the DNA mismatch-repair pathway. A gene on chromosome 2 called hMSH2, which demonstrates homology with the bacterial repair gene MutS, has been shown to be altered in some families with hereditary nonpolyposis colorectal cancer. A defect on chromosome 3 may act by impairing the same pathway. Genotyping of particular loci, termed microsatellite, provides an easy identification of tumors deficient in mismatch repair. Third, the mechanisms by which the inactivation of tumor-suppressor genes such as p53 and APC may contribute to the tumorigenic process have begun to be elucidated. These different discoveries will have important impacts in the prevention and management of colorectal carcinoma, one of the most frequent human cancers.

Topics: Adenomatous Polyposis Coli; Animals; Cell Transformation, Neoplastic; Chromosome Mapping; Chromosomes, Human, Pair 2; Chromosomes, Human, Pair 3; Colorectal Neoplasms; Colorectal Neoplasms, Hereditary Nonpolyposis; DNA-Binding Proteins; DNA, Neoplasm; DNA, Satellite; Genes, APC; Genes, Tumor Suppressor; Genetic Predisposition to Disease; Humans; Mice; Mutation; MutS Homolog 2 Protein; Oncogenes; Proto-Oncogene Proteins; Sulindac; Syndrome

Topics: Adenomatous Polyposis Coli; Cell Transformation, Neoplastic; Follow-Up Studies; Humans; Sulindac

Sulindac represents a promising candidate agent for lung cancer chemoprevention, but clinical trial data have not been previously reported. We conducted a randomized, phase II chemoprevention trial involving current or former cigarette smokers (≥30 pack-years) utilizing the multi-center, inter-disciplinary infrastructure of the Cancer Prevention Network (CPN).. At least 1 bronchial dysplastic lesion identified by fluorescence bronchoscopy was required for randomization. Intervention assignments were sulindac 150mg bid or an identical placebo bid for 6 months. Trial endpoints included changes in histologic grade of dysplasia (per-participant as primary endpoint and per lesion as secondary endpoint), number of dysplastic lesions (per-participant), and Ki67 labeling index.. Slower than anticipated recruitment led to trial closure after randomizing participants (n=31 and n=30 in the sulindac and placebo arms, respectively). Pre- and post-intervention fluorescence bronchoscopy data were available for 53/61 (87%) randomized, eligible participants. The median (range) of dysplastic lesions at baseline was 2 (1-12) in the sulindac arm and 2 (1-7) in the placebo arm. Change in dysplasia was categorized as regression:stable:progression for 15:3:8 (58%:12%:31%) subjects in the sulindac arm and 15:2:10 (56%:7%:37%) subjects in the placebo arm; these distributions were not statistically different (p=0.85). Median Ki67 expression (% cells stained positive) was significantly reduced in both the placebo (30 versus 5; p=0.0005) and sulindac (30 versus 10; p=0.0003) arms, but the difference between arms was not statistically significant (p=0.92).. Data from this multi-center, phase II squamous cell lung cancer chemoprevention trial do not demonstrate sufficient benefits from sulindac 150mg bid for 6 months to warrant additional phase III testing. Investigation of pathway-focused agents is necessary for lung cancer chemoprevention.

Topics: Adult; Aged; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Transformation, Neoplastic; Chemoprevention; Female; Humans; Interdisciplinary Studies; Lung Neoplasms; Male; Middle Aged; Smoking; Sulindac; Treatment Outcome

Inheritance of a gene mutation leads to the initiation of 5 to 10% of most cancers, including colon cancer cases. We developed a chemoprevention strategy using a novel combination of the non-steroidal anti-inflammatory (NSAID) sulindac plus the anthelminthic benzimidazole, mebendazole. This oral drug combination was effective in the ApcMin/+ mouse model of Familial Adenomatous Polyposis (FAP). Treatment with 35 mg/kg daily mebendazole reduced the number of intestinal adenomas by 56% (P = 0.0002), 160 ppm sulindac by 74% (P < 0.0001), and the combination by 90% (P < 0.0001). The combination significantly reduced microadenomas, polyp number and size in both the small intestines and colon when compared to untreated controls or sulindac alone. Mebendazole as a single agent decreased COX2 expression, blood vessel formation, VEGFR2 phosphorylation, and worked synergistically with sulindac to reduce overexpression of MYC, BCL2, and various pro-inflammatory cytokines. Given the low toxicity of mebendazole, these preclinical findings support the consideration of clinical trials for high risk cancer patients using mebendazole either alone or in combination. The findings have implications for populations with moderate and above risk for developing cancer.

Topics: Adenomatous Polyposis Coli; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Transformation, Neoplastic; Colon; Colonic Neoplasms; HCT116 Cells; HT29 Cells; Humans; Intestine, Small; Male; Mebendazole; Mice, Inbred C57BL; Mice, Nude; Sulindac; Xenograft Model Antitumor Assays

Cancer stem cells (CSCs) are shown to be responsible for initiation and progression of tumors in a variety of cancers. We previously showed that anthocyanin-containing baked purple-fleshed potato (PP) extracts (PA) suppressed early and advanced human colon cancer cell proliferation and induced apoptosis, but their effect on colon CSCs is not known. Considering the evidence of bioactive compounds, such as anthocyanins, against cancers, there is a critical need to study anticancer activity of PP, a global food crop, against colon CSCs. Thus, isolated colon CSCs (positive for CD44, CD133 and ALDH1b1 markers) with functioning p53 and shRNA-attenuated p53 were treated with PA at 5.0 μg/ml. Effects of baked PP (20% wt/wt) against colon CSCs were also tested in vivo in mice with azoxymethane-induced colon tumorigenesis. Effects of PA/PP were compared to positive control sulindac. In vitro, PA suppressed proliferation and elevated apoptosis in a p53-independent manner in colon CSCs. PA, but not sulindac, suppressed levels of Wnt pathway effector β-catenin (a critical regulator of CSC proliferation) and its downstream proteins (c-Myc and cyclin D1) and elevated Bax and cytochrome c, proteins-mediating mitochondrial apoptosis. In vivo, PP reduced the number of crypts containing cells with nuclear β-catenin (an indicator of colon CSCs) via induction of apoptosis and suppressed tumor incidence similar to that of sulindac. Combined, our data suggest that PP may contribute to reduced colon CSCs number and tumor incidence in vivo via suppression of Wnt/β-catenin signaling and elevation of mitochondria-mediated apoptosis.

Topics: Animals; Anthocyanins; Antineoplastic Agents; Apoptosis; Azoxymethane; bcl-2-Associated X Protein; beta Catenin; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Colonic Neoplasms; Cytochromes c; Food; Humans; In Situ Nick-End Labeling; Lentivirus; Male; Mice; Mitochondria; Neoplastic Stem Cells; RNA, Small Interfering; Solanum tuberosum; Sulindac; Tumor Suppressor Protein p53; Wnt Proteins

Phospho-sulindac is a sulindac derivative with promising anticancer activity in lung cancer, but its limited metabolic stability presents a major challenge for systemic therapy. We reasoned that inhalation delivery of phospho-sulindac might overcome first-pass metabolism and produce high levels of intact drug in lung tumors. Here, we developed a system for aerosolization of phospho-sulindac and evaluated the antitumor efficacy of inhaled phospho-sulindac in an orthotopic model of human non-small cell lung cancer (A549 cells). We found that administration by inhalation delivered high levels of phospho-sulindac to the lungs and minimized its hydrolysis to less active metabolites. Consequently, inhaled phospho-sulindac (6.5 mg/kg) was highly effective in inhibiting lung tumorigenesis (75%; P < 0.01) and significantly improved the survival of mice bearing orthotopic A549 xenografts. Mechanistically, phospho-sulindac suppressed lung tumorigenesis by (i) inhibiting EGF receptor (EGFR) activation, leading to profound inhibition of Raf/MEK/ERK and PI3K/AKT/mTOR survival cascades; (ii) inducing oxidative stress, which provokes the collapse of mitochondrial membrane potential and mitochondria-dependent cell death; and (iii) inducing autophagic cell death. Our data establish that inhalation delivery of phospho-sulindac is an efficacious approach to the control of lung cancer, which merits further evaluation.

Topics: Administration, Inhalation; Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Disease Models, Animal; Dose-Response Relationship, Drug; ErbB Receptors; Humans; Lung Neoplasms; Mitochondria; Mitogen-Activated Protein Kinases; Oxidative Stress; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; raf Kinases; Signal Transduction; Sulindac; Tumor Burden; Xenograft Model Antitumor Assays

Both selenium and non-steroidal anti-inflammatory drug (NSAID) sulindac are effective in cancer prevention, but their effects are affected by several factors including epigenetic alterations and gene expression. The current study was designed to determine the effects of the combination of selenium and sulindac on tumor inhibition and the underlying mechanisms.. We fed the intestinal tumor model Apc/p21 mice with selenium- and sulindac-supplemented diet for 24 weeks, and found that the combination of selenium and sulindac significantly inhibited intestinal tumorigenesis, in terms of reducing tumor incidence by 52% and tumor multiplicities by 80% (p<0.01). Mechanistic studies revealed that the combination of selenium and sulindac led to the significant induction of the expression of p27 and p53 and JNK1 phosphorylation, and led to the suppression of β-catenin and its downstream targets. Impressively, the data also showed that demythelation on p21 promoter was associated with tumor inhibition by the combination of selenium and sulindac.. The selenium is synergistic with sulindac to exert maximal effects on tumor inhibition. This finding provides an important chemopreventive strategy using combination of anti-cancer agents, which has a great impact on cancer prevention and has a promising translational potential.

Topics: Adenomatous Polyposis Coli Protein; Animals; Anti-Inflammatory Agents, Non-Steroidal; beta Catenin; Blotting, Western; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; DNA Methylation; Drug Synergism; Drug Therapy, Combination; Female; Intestinal Neoplasms; Male; Mice; Mice, Knockout; Mitogen-Activated Protein Kinase 8; Phosphorylation; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction; Selenium; Sulindac; Tumor Suppressor Protein p53

Pharmacological targeting of breast cancer stem cells (CSCs) is highly promising for the treatment of breast cancer, as the small population of CSCs appears responsible for tumor initiation and progression and also for resistance to conventional treatment. Here we report that the novel phosphosulindac (OXT-328, PS) selectively and effectively eliminates breast CSCs both in vitro and in vivo. PS reduced cell proliferation and induced apoptosis in various breast CSCs. Breast CSCs are resistant to conventional cancer drugs but are sensitive to PS. Long-term treatment of mixtures of cultured breast CSCs and breast cancer cells with PS preferentially eliminated the CSCs. PS impaired the ability of CSCs to form mammospheres and markedly suppressed the expression of CSC-related genes. More importantly, PS prevented by half (p = .06) the formation of tumors initiated by CSCs in immunodeficient mice, and inhibited by 83% (p < .05) the growth of already formed breast cancer xenografts, reducing the proportion of CSCs in them. PS suppressed the Wnt/β-catenin pathway by stimulating the degradation of β-catenin and its relocalization to the cell membrane and also blocked the epithelial-mesenchymal transition and the generation of breast CSCs. These results indicate that PS has a strong inhibitory effect against breast cancer, acting, at least in part, by targeting CSCs through a signaling mechanism involving Wnt signaling.

Topics: Animals; Antineoplastic Agents; Apoptosis; beta Catenin; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Coculture Techniques; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Immunocompromised Host; Mice; Molecular Targeted Therapy; Neoplastic Stem Cells; Organophosphorus Compounds; Sulindac; Wnt Proteins; Wnt Signaling Pathway; Xenograft Model Antitumor Assays

The non-steroidal anti-inflammatory drug sulindac is an effective chemopreventive agent in sporadic colorectal cancer but its potential benefit in mismatch repair deficient cancers remains to be defined. We wanted to determine whether genetic defects that are relevant for colorectal cancer, such as Msh2 or p53 deficiency, would influence the efficiency of sulindac chemoprevention or increase the side effects.. Msh2 or p53 deficient and wild-type mice received feed containing 160-320 ppm sulindac for up to 25 weeks with or without a concurrent treatment with the carcinogen azoxymethane. Colon tissue was analysed by histopathology and molecular biology methods.. We show that sulindac prevented azoxymethane-induced distal colon tumours in all mice. In the proximal colon, however, sulindac induced new inflammatory lesions on the mucosal folds, which further developed into adenocarcinoma in up to 18-25% of the p53 or Msh2 deficient mice but rarely in wild-type mice. This region in the proximal colon was characterised by a distinct profile of pro- and anti-inflammatory factors, which were modulated by the sulindac diet, including upregulation of hypoxia inducible factor 1α and macrophage inflammatory protein 2.. These data show that the sulindac diet promotes carcinogenesis in the mouse proximal colon possibly through chronic inflammation. Sulindac has both beneficial and harmful effects in vivo, which are associated with different microenvironments within the colon of experimental mice. Deficiency for the Msh2 or p53 tumour suppressor genes increases the harmful side effects of long-term sulindac treatment in the mouse colon.

Topics: Adenocarcinoma; Animals; Anti-Inflammatory Agents, Non-Steroidal; Anticarcinogenic Agents; Apoptosis; Azoxymethane; Carcinogens; Cell Transformation, Neoplastic; Colon; Colonic Neoplasms; Drug Evaluation, Preclinical; Gene Expression Regulation; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation Mediators; Intestinal Mucosa; Mice; Mice, Inbred C57BL; Mice, Knockout; MutS Homolog 2 Protein; Precancerous Conditions; Sulindac; Tumor Suppressor Protein p53

Curcumin exhibits anti-inflammatory and antitumor activity and is being tested in clinical trials as a chemopreventive agent for colon cancer. Curcumin's chemopreventive activity was tested in a transgenic mouse model of lung cancer that expresses the human Ki-ras(G12C) allele in a doxycycline (DOX) inducible and lung-specific manner. The effects of curcumin were compared with the lung tumor promoter, butylated hydroxytoluene (BHT), and the lung cancer chemopreventive agent, sulindac. Treatment of DOX-induced mice with dietary curcumin increased tumor multiplicity (36.3 +/- 0.9 versus 24.3 +/- 0.2) and progression to later stage lesions, results which were similar to animals that were co-treated with DOX/BHT. Microscopic examination showed that the percentage of lung lesions that were adenomas and adenocarcinomas increased to 66% in DOX/BHT, 66% in DOX/curcumin and 49% in DOX/BHT/curcumin-treated groups relative to DOX only treated mice (19%). Immunohistochemical analysis also showed increased evidence of inflammation in DOX/BHT, DOX/curcumin and DOX/BHT/curcumin mice relative to DOX only treated mice. In contrast, co-treatment of DOX/BHT mice with 200 p.p.m. [DOSAGE ERROR CORRECTED] of sulindac inhibited the progression of lung lesions and reduced the inflammation. Lung tissue from DOX/curcumin-treated mice demonstrated a significant increase (33%; P = 0.01) in oxidative damage, as assessed by the levels of carbonyl protein formation, relative to DOX-treated control mice after 1 week on the curcumin diet. These results suggest that curcumin may exhibit organ-specific effects to enhance reactive oxygen species formation in the damaged lung epithelium of smokers and ex-smokers. Ongoing clinical trials thus may need to exclude smokers and ex-smokers in chemopreventive trials of curcumin.

Topics: Animals; Anticarcinogenic Agents; Butylated Hydroxytoluene; Cell Transformation, Neoplastic; Curcumin; Doxycycline; Genes, ras; Lung Neoplasms; Mice; Mice, Transgenic; Organ Specificity; Reactive Oxygen Species; Sulindac

We have previously reported that sulindac, a non-steroidal anti-inflammatory drug, inhibited tumor formation in the small intestine but increased tumors in the colon of Apc(Min/+) mice, a model of human familial adenomatous polyposis. To further explore intestinal regional responses, we studied effects of sulindac on additional gene-targeted mouse models of human intestinal tumorigenesis; these were (i) Apc(1638N/+) mouse (chain termination mutation in exon 15 of the Apc gene); (ii) Mlh1(+/-) mouse (DNA mismatch repair deficiency, a mouse model of human hereditary non-polyposis colorectal cancer) and (iii) double-heterozygous Mlh1(+/-)Apc(1638N/+) mutant mouse. Mice were fed AIN-76A control diet with or without 0.02% sulindac for 6 months. Intestinal regional tumor incidence, multiplicity, volume and degree of inflammation were used as end points. The results showed the following: (i) sulindac inhibited tumor development in the small intestine of Apc(1638N/+) mice; (ii) in contrast, sulindac increased tumors in the small intestine of Mlh1 mutant mice, a neoplastic effect which persisted in heterozygous compound Mlh1(+/-)Apc(1638N/+) mutant mice; (iii) sulindac increased tumors in the cecum of all mice regardless of genetic background; (iv) sulindac decreased inflammation in the small intestine of Apc(1638N/+) mice, but it increased inflammation in the small intestine of Mlh1(+/-) mice and Mlh1(+/-)Apc(1638N/+) mice and (v) sulindac enhanced inflammation in the cecum of all mutant mice. Findings indicate that the effects of sulindac in the intestine of these mutant mouse models are probably related to genetic background and appear to be associated with its inflammatory-inducing response.

Topics: Adaptor Proteins, Signal Transducing; Adenomatous Polyposis Coli; Adenomatous Polyposis Coli Protein; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Cecum; Cell Transformation, Neoplastic; Colorectal Neoplasms, Hereditary Nonpolyposis; Disease Models, Animal; Humans; Inflammation; Intestine, Small; Mice; Mutation; MutL Protein Homolog 1; Nuclear Proteins; Sulindac

Lung cancer is the leading cause of cancer death in the world, and greater than 90% of lung cancers are cigarette smoke-related. Current treatment options are inadequate, because the molecular basis of cigarette-induced lung cancer is poorly understood.. Here, we show that human primary or immortalized bronchial epithelial cells exposed to cigarette smoke for eight days in culture rapidly proliferate, show anchorage-independent growth, and form tumors in nude mice. Using this model of the early stages of smoke-induced tumorigenesis, we examined the molecular changes leading to lung cancer. We observed that the embryonic signaling pathways mediated by Hedgehog and Wnt are activated by smoke. Pharmacological inhibition of these pathways blocked the transformed phenotype.. These experiments provide a model in which the early stages of smoke-induced tumorigenesis can be elicited, and should permit us to identify molecular changes driving this process. Results obtained so far indicate that smoke-induced lung tumors are driven by activation of two embryonic regulatory pathways, Hedgehog (Hh) and Wnt. Based on the current and emerging availability of drugs to inhibit Hh and Wnt signaling, it is possible that an understanding of the role of Hh and Wnt in lung cancer pathogenesis will lead to the development of new therapies.

Topics: Animals; Bronchi; Cell Adhesion; Cell Line; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Epithelial Cells; Female; Hedgehog Proteins; Humans; Lung Neoplasms; Mice; Mice, Nude; Models, Biological; Phenotype; Signal Transduction; Smoke; Smoking; Sulindac; Transplantation, Heterologous; Veratrum Alkaloids; Wnt Proteins

Nonsteroidal anti-inflammatory drugs (NSAIDs) have chemopreventive potential against colorectal carcinomas (CRCs). Inhibition of cyclooxygenase (COX)-2 underlies part of this effect, although COX-2-independent mechanisms may also exist. Nonsteroidal anti-inflammatory drugs appear to inhibit the initial stages of the adenoma-carcinoma sequence, suggesting a link to the APC/beta-catenin/TCF pathway (Wnt-signalling pathway). Therefore, the effect of the NSAID sulindac on nuclear (nonphosphorylated) beta-catenin and beta-catenin/TCF-mediated transcription was investigated. Nuclear beta-catenin expression was assessed in pretreatment colorectal adenomas and in adenomas after treatment with sulindac from five patients with familial adenomatous polyposis (FAP). Also, the effect of sulindac sulphide on beta-catenin/TCF-mediated transcription was studied. Adenomas of FAP patients collected after treatment with sulindac for up to 6 months showed less nuclear beta-catenin expression compared to pretreatment adenomas of the same patients. Sulindac sulphide abrogated beta-catenin/TCF-mediated transcription in the CRC cell lines DLD1 and SW480, and decreased the levels of nonphosphorylated beta-catenin. As a result, the protein levels of the positively regulated TCF targets Met and cyclin D1 were downregulated after sulindac treatment. This study provides in vivo and in vitro evidence that nuclear beta-catenin localisation and beta-catenin/TCF-regulated transcription of target genes can be inhibited by sulindac. The inhibition of Wnt-signalling provides an explanation for the COX-2-independent mechanism of chemoprevention by NSAIDs.

Topics: Adenoma; Adenomatous Polyposis Coli; Adult; Anti-Inflammatory Agents, Non-Steroidal; beta Catenin; Cell Nucleus; Cell Transformation, Neoplastic; Chemoprevention; Colorectal Neoplasms; Cytoskeletal Proteins; Female; Humans; Mitogens; Proto-Oncogene Proteins; Signal Transduction; Sulindac; Trans-Activators; Transcription, Genetic; Tumor Cells, Cultured; Wnt Proteins; Zebrafish Proteins

Topics: Animals; Annexin A5; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Cell Division; Cell Line; Cell Line, Transformed; Cell Survival; Cell Transformation, Neoplastic; Combinatorial Chemistry Techniques; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Epithelial Cells; Genes, ras; Inhibitory Concentration 50; Microscopy, Fluorescence; Phenotype; Proto-Oncogene Proteins; Signal Transduction; Structure-Activity Relationship; Sulindac; Wnt Proteins; Zebrafish Proteins

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Blotting, Western; Cell Line, Transformed; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Genes, ras; Guanosine Triphosphate; Inhibitory Concentration 50; Kinetics; Magnetic Resonance Spectroscopy; Mitogen-Activated Protein Kinases; Models, Molecular; Phosphorylation; Protein Binding; Proto-Oncogene Proteins c-raf; ras Proteins; Signal Transduction; Spectrometry, Fluorescence; Sulindac

Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to reduce the risk of colorectal cancer (CRC). They are also known to induce the regression of colorectal adenomas, which are precursors to CRC. Despite these evidences, the exact mechanism by which NSAIDs exerts its anti-oncogenic effect is not completely understood. Using a focus formation assay, here we show that sulindac sulfide, a NSAID, specifically inhibits cell transformation mediated by oncogenic Ha-Ras, but not by other established oncogene products such as v-Src, Galpha12, and Galpha13. Our results suggest that the ability of sulindac sulfide to suppress transformation is confined to specific oncogenic pathways. Further studies of the sulindac-resistant oncogenic pathways may lead to identification of novel therapeutic agents that are effective in the prevention or treatment of CRC.

Topics: 3T3 Cells; Animals; Antineoplastic Agents; Cell Division; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Genes, ras; Genes, src; Kinetics; Mice; Sulindac; Transfection

The nonsteroidal anti-inflammatory drug Sulindac has chemopreventive and antitumorigenic properties. Its metabolites induce apoptosis and inhibit signaling pathways critical for malignant transformation, including the Ras pathway. Here we show that the new Sulindac derivative IND 12 reverses the phenotype of Ras-transformed MDCK-f3 cells and restores an untransformed epithelioid morphology characterized by growth in monolayers with regular cell-cell adhesions. Moreover, IND 12 treatment induces the expression at membranes of the cell adhesion protein E-cadherin and increases the level of the E-cadherin-bound beta-catenin. As a consequence, IND 12-treated MDCK-f3 cells lose their invasion capacity and regain the ability to aggregate. In the presence of IND 12, MDCK-f3 cells show regenerated expression and activity ratios of the small GTPases Rac and Rho normally found in untransformed MDCK cells. Strikingly, IND 12 treatment decreases the levels of phosphorylated mitogen-activated protein kinases, which are downstream substrates of the Ras-regulated Raf/mitogen-activated protein kinase pathway, and the level of Ras-induced activation of gene expression. Our findings identify a novel drug with high potential in cancer therapy by targeting Ras-induced cell transformation.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; beta Catenin; Cadherins; Cell Aggregation; Cell Line, Transformed; Cell Transformation, Neoplastic; Cytoskeletal Proteins; Dogs; Enzyme Inhibitors; Epithelial Cells; Genes, ras; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Sulindac; Trans-Activators

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that have been implicated in a variety of biologic processes. The PPARdelta isotype was recently proposed as a downstream target of the adenomatous polyposis coli (APC)/beta-catenin pathway in colorectal carcinogenesis. To evaluate its role in tumorigenesis, a PPARdelta null cell line was created by targeted homologous recombination. When inoculated as xenografts in nude mice, PPARdelta -/- cells exhibited a decreased ability to form tumors compared with PPARdelta +/- and wild-type controls. These data suggest that suppression of PPARdelta expression contributes to the growth-inhibitory effects of the APC tumor suppressor.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Base Sequence; Cell Transformation, Neoplastic; Colorectal Neoplasms; DNA Primers; Humans; Receptors, Cytoplasmic and Nuclear; Sulindac; Transcription Factors; Tumor Cells, Cultured

Familial adenomatous polyposis is an inherited colorectal cancer syndrome characterized by the presence of multiple adenomatous colorectal polyps. Molecular studies have revealed that germline mutations in the APC gene are the underlying cause of the disease. The nonsteroidal anti-inflammatory agent sulindac has been shown to reduce the number of colorectal adenomas. Most sulindac trials in the large bowel have focused on the distal colon and relatively little is known about its effect on the proximal colon. Moreover, it is unknown whether the site of the APC mutation affects the efficacy of sulindac.. This study investigated whether there were regional differences in the effect of sulindac on the colon and whether response to sulindac was dependent on the site of mutation in the APC gene. In an open prospective study 17 patients with familial adenomatous polyposis were treated with 300 mg oral sulindac daily for four months followed by a washout phase of six months. Ten of the patients had an intact colon and seven had rectal stumps only. The number, size, and the degree of dysplasia of the adenomas were evaluated by colonoscopy at entry, end of treatment and end of the study.. Overall, a statistically significant decrease in the number of adenomas was observed (120 +/- 112 to 28 +/- 64, P = 0.007). After cessation of sulindac treatment the number of adenomas increased to 48 +/- 44.5, but remained significantly lower than the values observed at baseline. In the ten patients with intact colons, adenomas decreased by sevenfold in the proximal colon (103 +/- 73 to 15.1 +/- 47.4, P = 0.011) and twofold in the distal colon (80 +/- 52 to 29.6 +/- 37.2, P = 0.005). The size of adenomas and the grade of dysplasia also decreased. No correlation could be seen between the APC mutation site and the response to treatment.. These data indicate that sulindac reduces the number of adenomas in the entire colon and that the effect seems to be more pronounced in the proximal colon.

Topics: Adenomatous Polyposis Coli; Administration, Oral; Adolescent; Adult; Aged; Cell Transformation, Neoplastic; Colonoscopy; Female; Genes, APC; Genotype; Germ-Line Mutation; Humans; Male; Middle Aged; Sulindac; Treatment Outcome

The nonsteroidal anti-inflammatory drug sulindac and the ornithine decarboxylase inhibitor difluoromethylornithine (DFMO) are both potent inhibitors of colon carcinogenesis in experimental models of this disease. The combination of these two agents is undergoing evaluation as a strategy for colon cancer chemoprevention in humans with resected colon polyps. We evaluated the effects of the major sulfide and sulfone metabolites of sulindac and DFMO alone, or in combinations, on the growth and survival of Caco-2 colon cancer-derived cells and in clones of these cells transfected with an activated K-ras oncogene. Both the sulfide and sulfone metabolites of sulindac reduced cell viability, measured by colony-forming assays, primarily by inducing apoptosis. Expression of an activated K-ras oncogene caused cells treated with either sulindac sulfide or sulfone to undergo apoptosis earlier than nontransfected controls. However, clonogenic survival, measured 2 weeks after drug treatment, was the same in both Caco-2 and ras-transfected Caco-2 cells treated with sulindac metabolites. A 24-h treatment with DFMO caused a dose-dependent decrease in the colony-forming ability of cells expressing an activated K-ras but had no effect on the viability of the parental Caco-2 cells. The DFMO-dependent decrease in colony formation in K-ras-activated cells occurred in the absence of apoptosis. Assessment of cell survival by colony-forming assays indicated that these two agents acted in an additive manner when combined. These data indicate that K-ras can influence the kinetics of apoptosis induction by sulindac metabolites and cell survival in response to DFMO. However, cytotoxicity induced by these agents occurs via unique mechanisms. These studies suggest that the combination of DFMO and sulindac may be useful in human cancer prevention strategies.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Caco-2 Cells; Cell Survival; Cell Transformation, Neoplastic; Chemoprevention; Eflornithine; Enzyme Inhibitors; Genes, ras; Humans; Sulindac

Epidemiological and model studies with laboratory animals have provided evidence that nonsteroidal anti-inflammatory drugs reduce the risk of colon cancer. Sulindac, a nonsteroidal anti-inflammatory drug, has been shown to inhibit azoxymethane (AOM)-induced colon carcinogenesis in rats when administered continuously before, during, and after carcinogen treatment (initiation and postinitiation periods) or when given continuously beginning 14 weeks after carcinogen administration (promotion/ progression stage). The present study was designed to investigate the chemopreventive efficacy of sulindac sulfone (exisulind), the sulfone metabolite of sulindac, when administered during the promotion/progression stage of colon carcinogenesis in comparison to the effect during the initiation and postinitiation periods. We have also studied the modulating effect of exisulind on colonic tumor apoptosis. At 5 weeks of age, groups of male F344 rats were fed diets containing 0%, 0.06%, and 0.12% exisulind. At 7 weeks of age, groups of animals were injected s.c. with AOM (15 mg/kg body weight, once weekly for 2 weeks). Animals intended for the promotion/progression study and receiving 0% exisulind were switched to an experimental diet containing 0.12% exisulind at 14 weeks after the second AOM treatment. All rats remained on their respective dietary regimens until the termination of the study, 50 weeks after the second AOM injection. Colon tumors were evaluated histopathologically for tumor type. Administration of 0.06% and 0.12% exisulind during the initiation and postinitiation periods significantly inhibited the incidence and multiplicity of invasive and/or noninvasive adenocarcinomas of the colon. The inhibition of colon tumorigenesis by exisulind was associated with a significant retardation of body weight gain shortly after sulfone administration and increased apoptosis in the colon tumors. In contrast, administration of the higher dose (0.12%) of exisulind during the promotion/progression stage had only minimal effects on colon tumorigenesis and apoptosis in the colon tumors, suggesting that early administration, but not late administration, may be required for chemopreventive efficacy of this drug.

Topics: Adenocarcinoma; Administration, Oral; Animals; Anti-Inflammatory Agents, Non-Steroidal; Anticarcinogenic Agents; Apoptosis; Azoxymethane; Carcinogens; Cell Transformation, Neoplastic; Colonic Neoplasms; Cyclooxygenase Inhibitors; Disease Progression; Dose-Response Relationship, Drug; Drug Administration Schedule; Male; Neoplasm Invasiveness; Rats; Rats, Inbred F344; Sulindac; Weight Gain

In this study, we use primary embryonic fibroblasts derived from cyclooxygenase-deficient transgenic embryos to further investigate the role of the two cyclooxygenases, cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2), in the process of neoplastic transformation. Cells with either, neither, or both of the cyclooxygenases were transformed by Ha-ras and/or SV40. Our results show that when a cyclooxygenase enzyme is present, the transformed cells have marked increases in COX-2 and/or COX-1 expression. Nevertheless, each type of cell, deficient in either or both cyclooxygenases, can be readily transformed at almost equal efficiency. Different nonsteroidal antiinflammatory drugs (NSAIDs) were used to examine their possible antineoplastic effects on the transformed cells, which have various levels of expression of COX-1 or COX-2. Our results show that NSAIDs suppress the colony formation in soft agar in a dosage-dependent manner in the absence of the cyclooxygenase(s). Thymidine incorporation and apoptosis analyses further demonstrate that the NSAIDs are effective in the cyclooxygenase-null cells. Our findings with cyclooxygenase knockout cells confirm recent reports that some of the antiproliferative and antineoplastic effects of NSAIDs are independent of the inhibition of either COX-1 or COX-2. They also show that transformation is independent of the status of cyclooxygenase expression, suggesting that the involvement of the cyclooxygenases in tumorigenesis may occur at later steps.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Arachidonic Acid; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cyclooxygenase 1; Cyclooxygenase 2; Dinoprostone; Embryo, Mammalian; Fibroblasts; Ibuprofen; Indomethacin; Isoenzymes; Membrane Proteins; Mice; Mice, Knockout; Nitrobenzenes; Oncogene Proteins, Viral; Piroxicam; Prostaglandin-Endoperoxide Synthases; Proto-Oncogene Proteins p21(ras); RNA, Messenger; Simian virus 40; Sulfonamides; Sulindac

The non-steroidal anti-inflammatory drug sulindac is used in cancer prevention and therapy, but the molecular aspects of its anti-tumor effect remain unresolved. In vivo the prodrug sulindac, is converted into the metabolite sulindac sulfide. We found that sulindac sulfide strongly inhibits Ras induced malignant transformation and Ras/Raf dependent transactivation. Sulindac sulfide decreases the Ras induced activation of its main effector, the c-Raf-1 kinase. In vitro sulindac sulfide directly binds to the Ras gene product p21ras in a non-covalent manner. Moreover, we can show that sulindac sulfide inhibits the interaction of p21ras with the p21ras binding domain of the Raf protein. In addition, sulindac sulfide can impair the nucleotide exchange on p21ras by CDC25 as well as the acceleration of the p21ras GTPase reaction by p120GAP. Due to its action at the most critical site in Ras signaling we propose sulindac sulfide as a lead compound in the search for novel anti-cancer drugs which directly inhibit Ras mediated cell proliferation and malignant transformation.

Topics: 3T3 Cells; Animals; Antineoplastic Agents; Cell Transformation, Neoplastic; Cells, Cultured; Humans; Mice; Oncogene Protein p21(ras); Proto-Oncogene Proteins c-raf; Rats; Signal Transduction; Sulindac; Transcriptional Activation