tolfenamic-acid and Colonic-Neoplasms

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cyclooxygenase 2; DNA Topoisomerases, Type I; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Molecular Structure; Structure-Activity Relationship; Topoisomerase I Inhibitors; Topoisomerase Inhibitors

Curcumin (Cur) has been extensively studied in several types of malignancies including colorectal cancer (CRC); however its clinical application is greatly affected by low bioavailability. Several strategies to improve the therapeutic response of Cur are being pursued, including its combination with small molecules and drugs. We investigated the therapeutic efficacy of Cur in combination with the small molecule tolfenamic acid (TA) in CRC cell lines. TA has been shown to inhibit the growth of human cancer cells in vitro and in vivo, via targeting the transcription factor specificity protein1 (Sp1) and suppressing survivin expression. CRC cell lines HCT116 and HT29 were treated with TA and/or Cur and cell viability was measured 24-72 hours post-treatment. While both agents caused a steady reduction in cell viability, following a clear dose/ time-dependent response, the combination of TA+Cur showed higher growth inhibition when compared to either single agent. Effects on apoptosis were determined using flow cytometry (JC-1 staining to measure mitochondrial membrane potential), Western blot analysis (c-PARP expression) and caspase 3/7 activity. Reactive oxygen species (ROS) levels were measured by flow cytometry and the translocation of NF-kB into the nucleus was determined using immunofluorescence. Results showed that apoptotic markers and ROS activity were significantly upregulated following combination treatment, when compared to the individual agents. This was accompanied by decreased expression of Sp1, survivin and NF-kB translocation. The combination of TA+Cur was more effective in HCT116 cells than HT29 cells. These results demonstrate that TA may enhance the anti-proliferative efficacy of Cur in CRC cells.

Topics: Active Transport, Cell Nucleus; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Caspase 3; Caspase 7; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colonic Neoplasms; Curcumin; Flow Cytometry; HCT116 Cells; HT29 Cells; Humans; Inhibitor of Apoptosis Proteins; Membrane Potential, Mitochondrial; NF-kappa B; ortho-Aminobenzoates; Poly (ADP-Ribose) Polymerase-1; Reactive Oxygen Species; Sp1 Transcription Factor; Survivin

Tolfenamic acid is one of the fenamic acid-derived non-steroid anti-inflammatory drugs (NSAIDs) and has been shown to exhibit anti-cancer activities in several types of cancer. Both mutations and aberrant expression of β-catenin are highly associated with progression of cancer. Therefore, β-catenin is considered to be a promising molecular target for cancer prevention and treatment. The current study investigates the role of tolfenamic acid on β-catenin expression in colon cancer. Treatment with tolfenamic acid led to inhibition of cell growth and down-regulation of β-catenin expression in a dose- and time-dependent manner in human colon cancer cell lines. Reduction of β-catenin upon tolfenamic acid treatment was associated with ubiquitin-mediated proteasomal degradation, without affecting mRNA level and promoter activity of β-catenin. In addition, treatment with tolfenamic acid downregulated Smad2 and Smad3 expression, while overexpression of Smad2, but not Smad3, blocked tolfenamic acid-induced suppression of β-catenin expression. Tolfenamic acid also decreased expression of β-catenin target genes, including vascular endothelial growth factor (VEGF). Compared to adjacent normal tissue, intestinal tumor tissues of Apc(Min/+) mice exhibited increased expression of β-catenin, Smad2, Smad3, and VEGF, which were down-regulated with tolfenamic acid treatment at a dose of 50mg/kg body weight. In conclusion, our findings suggest that tolfenamic acid inhibits growth of colon cancer cells through downregulation of Smad2 and, subsequently, facilitating ubiquitin-proteasome-mediated β-catenin degradation in colon cancer.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; beta Catenin; Cell Line, Tumor; Colonic Neoplasms; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Mutant Strains; ortho-Aminobenzoates; Proteasome Endopeptidase Complex; Smad2 Protein; Smad3 Protein; Vascular Endothelial Growth Factor A

Tolfenamic acid (TA) is a non-steroidal anti-inflammatory drug (NSAID) that inhibits lung, esophageal, breast and pancreatic cancer cell and tumor growth, and this study investigated the anticancer activity of TA in colon cancer. TA inhibited growth and induced apoptosis in RKO, SW480, HT-29, and HCT-116 colon cancer cells, and TA (50 mg/kg/d) also inhibited tumor growth in athymic nude mice bearing RKO cells as xenografts. TA downregulated expression of Sp proteins (Sp1, Sp3, and Sp4) in colon cancer cells and this was accompanied by decreased expression of several Sp-regulated growth promoting (cyclin D1, hepatocyte growth factor receptor), angiogenic (vascular endothelial growth factor (VEGF) and its receptor 1), survival (survivin and bcl-2), and inflammatory (NFκBp65/p50) gene products. The mechanism of TA-mediated effects on Sp proteins was due to activation of caspases. These results now extend the number of NSAIDs that may have clinical potential for colon cancer chemotherapy and show that the anticancer activity of TA is due, in part, to targeting Sp transcription factors.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Blotting, Western; Cell Proliferation; Colonic Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; ortho-Aminobenzoates; Sp Transcription Factors; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Recent studies showed that the metal-coordinated non-steroidal anti-inflammatory drug (NSAID), copper indomethacin, reduced aberrant crypt formation in the rodent colon cancer model, while also exhibiting gastrointestinal sparing properties. In the present study, the stability and biological activity of three BiNSAIDs of the general formula [Bi(L)3]n, where L=diflunisal (difl), mefenamate (mef) or tolfenamate (tolf) were examined. NMR spectroscopy of high concentrations of BiNSAIDs (24h in cell medium, 37°C) indicated that their structural stability and interactions with cell medium components were NSAID specific. Assessment of cell viability using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium]bromide (MTT) assay showed that the toxicity ranking of the BiNSAIDs paralleled those of the respective free NSAIDs: diflH

Topics: Anti-Inflammatory Agents, Non-Steroidal; Bismuth; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Coordination Complexes; Diflunisal; Drug Evaluation, Preclinical; Drug Stability; Humans; Inhibitory Concentration 50; Mefenamic Acid; ortho-Aminobenzoates