tolfenamic-acid and Colorectal-Neoplasms

Intervention strategies in familial adenomatous polyposis (FAP) patients and other high-risk colorectal cancer (CRC) populations have highlighted a critical need for endoscopy combined with safe and effective preventive agents. We performed transcriptome profiling of colorectal adenomas from FAP patients and the polyposis in rat colon (Pirc) preclinical model, and prioritized molecular targets for prevention studies in vivo. At clinically relevant doses in the Pirc model, the drug Clotam (tolfenamic acid, TA) was highly effective at suppressing tumorigenesis both in the colon and in the small intestine, when administered alone or in combination with Sulindac. Cell proliferation in the colonic crypts was reduced significantly by TA, coincident with increased cleaved caspase-3 and decreased Survivin, β-catenin, cyclin D1 and matrix metalloproteinase 7. From the list of differentially expressed genes prioritized by transcriptome profiling, Mmp7, S100a9, Nppb and Aldh1a3 were defined as key oncogene candidates downregulated in colon tumors after TA treatment. Monthly colonoscopies revealed the rapid onset of tumor suppression by TA in the Pirc model, and the temporal changes in Mmp7, S100a9, Nppb and Aldh1a3, highlighting their value as potential early biomarkers for prevention in the clinical setting. We conclude that TA, an "old drug" repurposed from migraine, offers an exciting new therapeutic avenue in FAP and other high-risk CRC patient populations.

Topics: Adenoma; Adenomatous Polyposis Coli; Aldehyde Oxidoreductases; Animals; beta Catenin; Biomarkers, Tumor; Calgranulin B; Carcinogenesis; Caspase 3; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Down-Regulation; Gene Expression Profiling; Humans; Male; Matrix Metalloproteinase 7; Oncogenes; ortho-Aminobenzoates; Rats

Recent studies demonstrate that tolfenamic acid (TA) induces apoptosis and suppresses the development and progression of several types of cancers. However, the underlying mechanisms are complex and remain to be fully elucidated. Nuclear factor-kappaB (NF-κB) plays a critical role in inflammation, cancer development and progression. Although non-steroidal anti-inflammatory drugs modulate NF-κB signaling pathway in different ways, the link between NF-κB and TA-induced apoptosis of colorectal cancer cells has yet to be thoroughly investigated. In this study, we examined the effects of TA on the NF-κB pathway and apoptosis. TA activated NF-κB transcriptional activity and binding affinity of NF-κB to DNA. TA-induced NF-κB activation was mediated by an increased phosphorylation and proteosomal degradation of IκB-α and subsequent p65 nuclear translocation. We also observed that TA stabilized p65 and increased nuclear accumulation via an increase of p65 phosphorylation at Ser276 residue, which was mediated by p38 mitogen-activated protein kinase and extracellular signal-regulated kinase. The knockout of p53 blocked TA-induced transcriptional activation of NF-κB, but not the p65 nuclear accumulation. TA increased transcriptional activity of p53 and the binding affinity of p53 with p65, which are mediated by p38 mitogen-activated protein kinase and extracellular signal-regulated kinase-stimulated Ser276 phosphorylation. TA-induced apoptosis was ameliorated by the knockout of p65 and p53 and the point mutation of p65 at Ser276 residue. We demonstrate a novel molecular mechanism by which TA induced the NF-κB and apoptosis in human colorectal cancer cells.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Colorectal Neoplasms; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Humans; I-kappa B Proteins; NF-kappa B; NF-KappaB Inhibitor alpha; ortho-Aminobenzoates; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Phytochemicals; Protein Binding; Protein Transport; Transcription Factor RelA; Transcriptional Activation; Tumor Suppressor Protein p53

Several studies have shown substantial evidences that non-steroidal anti-inflammatory drugs (NSAIDs) exert anticancer effects by generating reactive oxygen species (ROS). Tolfenamic acid (TA) is one of the traditional NSAIDs widely used for treatment of migraine. TA has anti-cancer activities in several human cancer models. In this study, we report that generation of ROS by TA leads to apoptosis through modulation of several pathways in human colorectal cancer cells. TA induced rapid generation of intracellular ROS and led to an increase of phosphorylation of H2AX, a tail moment of comet and distribution of fragmented genomic DNA traces. Treatment of N-acetyl-l-cysteine (NAC) abolished TA-induced phosphorylation of H2AX and apoptosis. Treatment of TA resulted in an increase of nuclear factor-kappaB (NF-κB) transcriptional activity through inhibitor of kappa B (IκB-α) degradation and subsequent p65 nuclear translocation. In addition, TA increased apoptosis-inducing activating transcription factor 3 (ATF3) expression. However, the treatment of NAC abolished TA-mediated NF-κB activation and ATF3 expression and chemical inhibition of NF-κB or knockdown of p65 significantly attenuated TA-induced ATF3 expression. Our finding indicates that ROS-mediated DNA damage and subsequent activation of NF-κB and ATF3 expression plays a significant role in TA-induced apoptosis in human colorectal cancer cells.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Colorectal Neoplasms; DNA Damage; Humans; ortho-Aminobenzoates; Reactive Oxygen Species; Signal Transduction

The nonsteroidal anti-inflammatory drug tolfenamic acid has been shown to suppress cancer cell growth and tumorigenesis in different cancer models. However, the underlying mechanism by which tolfenamic acid exerts its antitumorigenic effect remains unclear. Previous data from our group and others indicate that tolfenamic acid alters expression of apoptosis- and cell-cycle arrest-related genes in colorectal cancer cells. Here, we show that tolfenamic acid markedly reduced the number of polyps and tumor load in APC(min)(/+) mice, accompanied with cyclin D1 downregulation in vitro and in vivo. Mechanistically, tolfenamic acid promotes endoplasmic reticulum (ER) stress, resulting in activation of the unfolded protein response (UPR) signaling pathway, of which PERK-mediated phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) induces the repression of cyclin D1 translation. Moreover, the PERK-eIF2α-ATF4 branch of the UPR pathway plays a role in tolfenamic acid-induced apoptosis in colorectal cancer cells, as silencing ATF4 attenuates tolfenamic acid-induced apoptosis. Taken together, these results suggest ER stress is involved in tolfenamic acid-induced inhibition of colorectal cancer cell growth, which could contribute to antitumorigenesis in a mouse model.

Topics: Activating Transcription Factor 4; Adenomatous Polyposis Coli Protein; Animals; Anti-Inflammatory Agents, Non-Steroidal; Blotting, Western; Cell Transformation, Neoplastic; Colonic Polyps; Colorectal Neoplasms; Cyclin D1; eIF-2 Kinase; Endoplasmic Reticulum Stress; Humans; Immunoprecipitation; Mice; Mice, Inbred C57BL; Mice, Knockout; ortho-Aminobenzoates; Phosphorylation; Protein Serine-Threonine Kinases; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured; Unfolded Protein Response

Tolfenamic acid (TA) is a non-steroidal anti-inflammatory drug associated with anti-tumorigenic and pro-apoptotic properties in animal and in vitro models of cancer. However, the underlying cellular mechanisms by which TA exerts its effects are only partially understood. Activating transcription factor 3 (ATF3) is a member of the ATF/CREB subfamily of the basic region-leucine zipper family and has been known as a tumor suppressor in human colorectal cancer cells. The present study was performed to observe whether ATF3 mediates TA-induced apoptosis and to elucidate the molecular mechanism of ATF3 transcription induced by TA. TA treatment and ectopic expression of ATF3 increased apoptosis, whereas knockdown of ATF3 resulted in significant repression of TA-activated apoptosis. The TA treatment also induced ATF3 promoter activity. Internal deletion and point mutation of the predicted ATF/C/EBP binding site in ATF3 promoter abolished luciferase activation by TA. Overexpression of ATF2 resulted in significant increase in ATF3 promoter activity, and electrophoretic mobility shift assay identified this region as a core sequence to which ATF2 binds. TA treatment resulted in an increase in ATF2 phosphorylation, which was followed by a subsequent increase in ATF3 transcription. Knock down of ATF2 abolished TA-induced ATF3 expression. We further provide evidence that TA leads to increases in phospho-p38 MAPK, JNK and ERK levels. Inhibition of these pathways using selective inhibitors and dominant negative constructs ameliorated TA-induced ATF3 expression and promoter activities. The current study shows that TA stimulates ATF3 expression and subsequently induces apoptosis. These pathways are mediated through phosphorylation of ATF2, which is mediated by p38 MAPK-, JNK- and ERK-dependent pathways.

Topics: Activating Transcription Factor 2; Activating Transcription Factor 3; Base Sequence; Cell Line, Tumor; Colorectal Neoplasms; DNA; Gene Expression Regulation; Humans; MAP Kinase Signaling System; ortho-Aminobenzoates; Phosphorylation; Promoter Regions, Genetic

Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to prevent colorectal tumorigenesis. Although antitumor effects of NSAIDs are mainly due to inhibition of cyclooxygenase activity, there is increasing evidence that cyclooxygenase-independent mechanisms may also play an important role. The early growth response-1 (EGR-1) gene is a member of the immediate-early gene family and has been identified as a tumor suppressor gene. Tolfenamic acid is a NSAID that exhibits anticancer activity in a pancreatic cancer model. In the present study, we investigated the anticancer activity of tolfenamic acid in human colorectal cancer cells. Tolfenamic acid treatment inhibited cell growth and induced apoptosis as measured by caspase activity and bioelectric impedance. Tolfenamic acid induced EGR-1 expression at the transcription level, and analysis of the EGR-1 promoter showed that a putative ETS-binding site, located at -400 and -394 bp, was required for activation by tolfenamic acid. The electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed that this sequence specifically bound to the ETS family protein epithelial-specific ETS-1 (ESE-1) transcription factor. Tolfenamic acid also facilitated translocation of endogenous and exogenous ESE-1 to the nucleus in colorectal cancer cells, and gene silencing using ESE-1 small interfering RNA attenuated tolfenamic acid-induced EGR-1 expression and apoptosis. Overexpression of EGR-1 increased apoptosis and decreased bioelectrical impedance, and silencing of endogenous EGR-1 prevented tolfenamic acid-induced apoptosis. These results show that activation of ESE-1 via enhanced nuclear translocation mediates tolfenamic acid-induced EGR-1 expression, which plays a critical role in the activation of apoptosis.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Apoptosis; Binding Sites; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; DNA-Binding Proteins; Early Growth Response Protein 1; Gene Expression Regulation, Neoplastic; Humans; Models, Biological; ortho-Aminobenzoates; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ets; RNA, Small Interfering; Transcription Factors