Enhancement of 5-Fluorouracil Efficacy on High COX-2 Expressing HCA-7 Cells by Low Dose Indomethacin and NS-398 but not on Low COX-2 Expressing HT-29 Cells

The antiproliferative effect of 5-fluorouracil (5-FU) in the presence of low dose non-steroidal anti-inflammatory drugs (NSAIDs) on high cyclooxygenase-2 (COX-2)-expressing HCA-7 and low COX-2-expressing HT-29 colon carcinoma cell lines was investigated. Pharmacogenetic parameters were studied to characterize the 5-FU sensitivity of the two cell lines. Thymidylate synthase (TS) and methylenetetrahydrofolate reductase (MTHFR) polymorphisms were determined by PCR analysis. Cell proliferation was measured by SRB assay, cell cycle distribution and apoptosis by FACS analysis. Cyclooxygenase expression was detected by Western blot and also by fluorescence microscopy. Prostaglandin E2 (PGE2) levels were investigated with ELISA kit. The HT-29 cell line was found to be homozygous for TS 2R and 1494ins6 and T homozygous for MTHFR 677 polymorphisms predicting high 5-FU sensitivity (IC50: 10 μM). TS 3R homozygosity, TS 1496del6 and MTHFR 677CT heterozygosity may explain the modest 5-FU sensitivity (IC50: 1.1 mM) of the HCA-7 cell line. Indomethacin and NS-398 (10 µM and 1.77 µM, respectively) reduced the PGE2 level in HCA-7 cells (>90%). Low concentrations of NSAIDs without antiproliferative potency increased the S-phase arrest and enhanced the cytotoxic action of 5-FU only in HCA-7 cells after 48-hours treatment. The presented data suggested that the enhancement of 5-FU cytotoxicity by indomethacin or NS-398 applied in low dose is related to the potency of NSAIDs to modulate the cell-cycle distribution and the apoptosis; however, it seems that this effect might be dependent on cell phenotype, namely on the COX-2 expression.

[1]  M. Marshall,et al.  Cell cycle effects of nonsteroidal anti-inflammatory drugs and enhanced growth inhibition in combination with gemcitabine in pancreatic carcinoma cells. , 2001, The Journal of pharmacology and experimental therapeutics.

[2]  R. Ueda,et al.  MRP8/ABCC11 directly confers resistance to 5-fluorouracil , 2007, Molecular Cancer Therapeutics.

[3]  J. Morrow,et al.  Regulation of Constitutive Cyclooxygenase-2 Expression in Colon Carcinoma Cells* , 2000, The Journal of Biological Chemistry.

[4]  Ji Hyun Han,et al.  Selective COX-2 inhibitor, NS-398, inhibits the replicative senescence of cultured dermal fibroblasts , 2004, Mechanisms of Ageing and Development.

[5]  R. Matthews,et al.  A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase , 1995, Nature Genetics.

[6]  Y. Ou,et al.  Indomethacin induces apoptosis in 786-O renal cell carcinoma cells by activating mitogen-activated protein kinases and AKT. , 2007, European journal of pharmacology.

[7]  F. Shanahan,et al.  Prostaglandin E2 stimulates Fas ligand expression via the EP1 receptor in colon cancer cells , 2008, British Journal of Cancer.

[8]  G. Peters,et al.  5-Ethyl-2′-deoxyuridine, a modulator of both antitumour action and pharmacokinetics of 5-fluorouracil , 1999, Journal of Cancer Research and Clinical Oncology.

[9]  A. Bennett,et al.  Studies on the mechanism by which indomethacin increases the anticancer effect of methotrexate , 1987, British journal of pharmacology.

[10]  Ying Liu,et al.  Analysis of P53 mutations and their expression in 56 colorectal cancer cell lines. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[11]  I. B. Borel Rinkes,et al.  NS-398, a selective cyclooxygenase-2 inhibitor, reduces experimental bladder carcinoma outgrowth by inhibiting tumor cell proliferation. , 2005, Urology.

[12]  J. Houghton,et al.  p 53 Dependence of Fas Induction and Acute Apoptosis in Response to 5-Fluorouracil-Leucovorin in Human Colon Carcinoma Cell Lines 1 , 2000 .

[13]  S. Eksborg,et al.  Celecoxib Prevents Neuroblastoma Tumor Development and Potentiates the Effect of Chemotherapeutic Drugs In vitro and In vivo , 2007, Clinical Cancer Research.

[14]  S. Minoura,et al.  Indomethacin increases the cytotoxicity of cis-platinum and 5-fluorouracil in the human uterine cervical cancer cell lines SKG-2 and HKUS by increasing the intracellular uptake of the agents , 2001, International Journal of Clinical Oncology.

[15]  D. Elder,et al.  The cyclooxygenase 2-selective inhibitor NS398 inhibits proliferation of oral carcinoma cell lines by mechanisms dependent and independent of reduced prostaglandin E2 synthesis. , 2003, Clinical cancer research : an official journal of the American Association for Cancer Research.

[16]  H. Chan,et al.  Purification, characterization and selective inhibition of human prostaglandin G/H synthase 1 and 2 expressed in the baculovirus system. , 1994, Biochimica et biophysica acta.

[17]  N. Kawai,et al.  Cyclooxygenases and colon cancer. , 2002, Prostaglandins & other lipid mediators.

[18]  W. Isaacs,et al.  Cyclooxygenases in cancer: progress and perspective. , 2004, Cancer letters.

[19]  D. Beer,et al.  Indomethacin-induced apoptosis in esophageal adenocarcinoma cells involves upregulation of Bax and translocation of mitochondrial cytochrome C independent of COX-2 expression. , 2000, Neoplasia.

[20]  T. Williams,et al.  Indomethacin Causes Prostaglandin D2-like and Eotaxin-like Selective Responses in Eosinophils and Basophils* , 2002, The Journal of Biological Chemistry.

[21]  S. Fujimura,et al.  Indomethacin enhances the cytotoxicity of VCR and ADR in human pulmonary adenocarcinoma cells. , 1997, The Tohoku journal of experimental medicine.

[22]  Zhe-Sheng Chen,et al.  Transport of Bile Acids, Sulfated Steroids, Estradiol 17-β-d-Glucuronide, and Leukotriene C4 by Human Multidrug Resistance Protein 8 (ABCC11) , 2005, Molecular Pharmacology.

[23]  Shan Wang,et al.  The COX-2 Selective Inhibitor-Independent COX-2 Effect on Colon Carcinoma Cells is Associated with the Delta1/Notch1 Pathway , 2008, Digestive Diseases and Sciences.

[24]  Y. Mizutani,et al.  Synergistic cytotoxicity and apoptosis of JTE-522, a selective cyclooxygenase-2 inhibitor, and 5-fluorouracil against bladder cancer. , 2002, The Journal of urology.

[25]  E. Hitre,et al.  Influence of thymidylate synthase gene polymorphisms on the survival of colorectal cancer patients receiving adjuvant 5-fluorouracil , 2005, Pharmacogenetics and genomics.

[26]  F. Fitzpatrick,et al.  Cyclooxygenase-2 and carcinogenesis. , 2000, Biochimica et biophysica acta.

[27]  R. Wickremasinghe,et al.  Antioxidants enhance the susceptibility of colon carcinoma cells to 5-fluorouracil by augmenting the induction of the bax protein. , 2001, Cancer letters.

[28]  J. Waxman,et al.  COX inhibitors and breast cancer , 2006, British Journal of Cancer.

[29]  A. Knox,et al.  Cyclooxygenase (COX) inhibitors induce apoptosis in non-small cell lung cancer through cyclooxygenase independent pathways. , 2003, Lung cancer.

[30]  Min Zhang,et al.  Selective cyclooxygenase 2 inhibitor NS-398 induces apoptosis in myeloma cells via a Bcl-2 independent pathway , 2005, Leukemia & lymphoma.

[31]  D. Dewitt,et al.  Differential inhibition of prostaglandin endoperoxide synthase (cyclooxygenase) isozymes by aspirin and other non-steroidal anti-inflammatory drugs. , 1993, The Journal of biological chemistry.

[32]  K. Schulze-Osthoff,et al.  Cyclooxygenase-2 (COX-2) inhibitors sensitize tumor cells specifically to death receptor-induced apoptosis independently of COX-2 inhibition , 2003, Oncogene.

[33]  G. Weckbecker.  Biochemical pharmacology and analysis of fluoropyrimidines alone and in combination with modulators. , 1991, Pharmacology & therapeutics.

[34]  M. Clynes,et al.  Enhancement of chemotherapeutic drug toxicity to human tumour cells in vitro by a subset of non-steroidal anti-inflammatory drugs (NSAIDs). , 1998, European journal of cancer.

[35]  H. Han,et al.  Short‐period hypoxia increases mouse embryonic stem cell proliferation through cooperation of arachidonic acid and PI3K/Akt signalling pathways , 2008, Cell proliferation.

[36]  M. Hanazono,et al.  Indomethacin preferentially augments 5-fluorouracil cytotoxicity in Colon 26 tumors by increasing the intracellular inflow of 5-fluorouracil , 1999, International Journal of Clinical Oncology.

[37]  P. Laurent-Puig,et al.  Thymidylate synthase and methylenetetrahydrofolate reductase gene polymorphisms: relationships with 5-fluorouracil sensitivity , 2004, British Journal of Cancer.

[38]  G. Milano,et al.  A role for dihydropyrimidine dehydrogenase and thymidylate synthase in tumour sensitivity to fluorouracil. , 1994, European journal of cancer.

[39]  W. Bodmer,et al.  p53 mutations in colorectal cancer. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[40]  M. Clynes,et al.  Structure-activity relationship of indomethacin analogues for MRP-1, COX-1 and COX-2 inhibition. identification of novel chemotherapeutic drug resistance modulators. , 2002, European journal of cancer.

[41]  R. Ramer,et al.  Decisive role of cyclooxygenase-2 and lipocalin-type prostaglandin D synthase in chemotherapeutics-induced apoptosis of human cervical carcinoma cells , 2008, Oncogene.

[42]  N. Hayashi,et al.  Inhibition of angiotensin II activity enhanced the antitumor effect of cyclooxygenase-2 inhibitors via insulin-like growth factor I receptor pathway. , 2003, Cancer research.

[43]  S. Mercadante.  The use of anti-inflammatory drugs in cancer pain. , 2001, Cancer treatment reviews.