Nonsteroidal anti-inflammatory drug sulindac sulfide suppresses structural protein Nesprin-2 expression in colorectal cancer cells.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Nesprin-2 is down-regulated by NSAIDs and highly expressed in many cancers. GENERAL SIGNIFICANCE: Our data suggest that Nesprin-2 may be a potential novel oncogene in human cancer cells and NSAIDs could decrease its expression.

A novel COX-independent mechanism of sulindac sulfide involves cleavage of epithelial cell adhesion molecule protein.

Non-steroidal anti-inflammatory drugs (NSAIDs) are extensively used over the counter to treat headaches and inflammation as well as clinically to prevent cancer among high-risk groups. The inhibition of cyclooxygenase (COX) activity by NSAIDs plays a role in their anti-tumorigenic properties. NSAIDs also have COX-independent activity which is not fully understood. In this study, we report a novel COX-independent mechanism of sulindac sulfide (SS), which facilitates a previously uncharacterized cleavage of epithelial cell adhesion molecule (EpCAM) protein. EpCAM is a type I transmembrane glycoprotein that has been implemented as an over-expressed oncogene in many cancers including colon, breast, pancreas, and prostate. We found EpCAM to be down-regulated by SS in a manner that is independent of COX activity, transcription regulation, de novo protein synthesis, and proteasomal degradation pathway. Our findings clearly demonstrate that SS drives cleavage of the extracellular portion of EpCAM near the N-terminus. This SS driven cleavage is blocked by a deleting amino acids 55-81 as well as simply mutating arginine residues at positions 80 and 81 to alanine of EpCAM. Proteolysis of EpCAM by SS may provide a novel mechanism by which NSAIDs affect anti-tumorigenesis at the post-translational level.

Changes in hepatic gene expression in dogs with experimentally induced nutritional iron deficiency.

BACKGROUND AND OBJECTIVE: We investigated hepatic gene expression in dogs with experimentally induced nutritional iron deficiency (ID). Our hypothesis was that ID would result in decreased hepcidin gene expression, and possibly in altered expression of other genes associated with iron metabolism. METHODS: Liver biopsies were collected from each of 3 dogs before induction of ID, at the point of maximal ID, and after resolution of ID. Using Affymetrix microarray technology and analytical tools specifically designed for microarray data, we identified genes that had at least a 2-fold change in expression in response to ID. Four genes were selected for further analysis by reverse transcriptase PCR (RT-PCR). RESULTS: Dogs with ID had markedly decreased expression of the hepcidin gene (mean decrease of 40-fold for one probe and >100-fold for another probe) and increased expression of the transferrin receptor gene (mean increase of >7-fold). There was also mildly decreased expression of the "similar to calreticulin" gene and a gene of unknown function. Results of RT-PCR analysis were consistent with microarray findings. CONCLUSION: Changes in hepcidin and transferrin receptor gene expression were consistent with the known biology of iron metabolism. The decrease in expression of a gene identified as "similar to calreticulin," while not statistically significant, was consistent with the findings of other investigators that suggest iron plays a role in calreticulin expression.

Molecular characterisation of canine nonsteroidal anti-inflammatory drug-activated gene (NAG-1).

Nonsteroidal anti-inflammatory drug (NSAID)-activated gene (NAG-1), a divergent member of the transforming growth factor beta superfamily, was previously identified as a gene induced by several anti-tumorigenic compounds, including NSAIDs and peroxisome proliferator-activated receptor gamma (PPARgamma) ligands in humans. In this study, canine NAG-1 was characterised from a canine genomic database. Gene induction by some NSAIDs and PPARgamma ligands was demonstrated in canine osteosarcoma cell lines. Phylogenetic analysis indicates that canine NAG-1 is more homologous with the corresponding mouse and rat genes than with human NAG-1. Expression of canine NAG-1 was increased by treatment with piroxicam and SC-560 (NSAIDs) and the PPARgamma ligand rosiglitazone. This study demonstrates that canine NAG-1 is up-regulated by some anti-tumorigenic compounds in osteosarcoma cell lines and may provide an important target of chemotherapy in canine cancer.

Anti-proliferative effect of horehound leaf and wild cherry bark extracts on human colorectal cancer cells.

Marubium vulgare (horehound) and Prunus serotina (wild cherry) have been traditionally used for the treatment of inflammatory-related symptoms such as cold, fever, and sore throat. In this report, we show that extracts of anti-inflammatory horehound leaves and wild cherry bark exhibit anti-proliferative activity in human colorectal cancer cells. Both horehound and wild cherry extracts cause suppression of cell growth as well as induction of apoptosis. We found that horehound extract up-regulates pro-apoptotic non-steroidal anti-inflammatory drug-activated gene (NAG-1) through transactivation of the NAG-1 promoter. In contrast, wild cherry extract decreased cyclin D1 expression and increased NAG-1 expression in HCT-116 and SW480 cell lines. Treatment with wild cherry extract resulted in the suppression of beta-catenin/T cell factor transcription, as assessed by TOP/FOP reporter constructs, suggesting that suppressed beta-catenin signaling by wild cherry extract leads to the reduction of cyclin D1 expression. Our data suggest the mechanisms by which these extracts suppress cell growth and induce apoptosis involve enhanced NAG-1 expression and/or down-regulation of beta-catenin signaling, followed by reduced cyclin D1 expression in human colorectal cancer cells. These findings may provide mechanisms for traditional anti-inflammatory products as cancer chemopreventive agents.

Molecular cloning and expression of canine hepcidin.

BACKGROUND: Hepcidin is a recently identified acute phase protein with antimicrobial and iron regulatory functions. It has been suggested that hepcidin may be the key mediator of anemia of chronic disease. Our research group is interested in developing a diagnostic assay to measure hepcidin in dogs. OBJECTIVES: The objectives of this study were to clone and sequence the canine hepcidin gene and to gather preliminary data about tissue expression of hepcidin in dogs. METHODS: RNA was extracted from fresh canine liver tissue and cDNA was generated and amplified. Standard reverse transcription polymerase chain reaction techniques were used with degenerate primers based on sequence homology between several other species. The amino acid (AA) sequence was compared with known sequences in other species. Tissue expression of canine hepcidin was determined by Western blot. RESULTS: The canine hepcidin cDNA sequence encoded a highly conserved protein of 85 AAs with 8 cysteine residues at the C-terminal end. This protein was likely the precursor form (pro-hepcidin) of a smaller secreted peptide. Phylogenetic analysis showed that human hepcidin was more homologous with canine than with rodent hepcidin. In dogs, as in people, hepcidin was expressed most strongly in the liver. Western blotting showed a clear band of approximately 9 kDa, consistent with pro-hepcidin. Weak expression was also detected in canine kidney and lung tissues. CONCLUSION: The results of this study establish the basis for future investigation involving canine hepcidin and suggest that the dog may be a suitable model for studying the role of hepcidin in human health and disease.

Anti-tumor activity of non-steroidal anti-inflammatory drugs: cyclooxygenase-independent targets.

Non-steroidal anti-inflammatory drugs (NSAIDs) are used extensively for analgesic and antipyretic treatments. In addition, NSAIDs reduce the risk and mortality to several cancers. Their mechanisms in anti-tumorigenesis are not fully understood, but both cyclooxygenase (COX)-dependent and -independent pathways play a role. We and others have been interested in elucidating molecular targets of NSAID-induced apoptosis. In this review, we summarize updated literature regarding cellular and molecular targets modulated by NSAIDs. Among those NSAIDs, sulindac sulfide and tolfenamic acid are emphasized in this review because these two drugs have been well investigated for their anti-tumorigenic activity in many different types of cancer.