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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

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Pathology & Oncology Research

Abstract

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.

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Abbreviations

5-FU:

5-fluorouracil

CRC:

colorectal cancer

COX-2:

cyclooxygenase-2

dUMP:

2’-deoxyuridine 5’-monophosphate

ELISA:

enzyme-linked immunosorbent assay

FACS:

fluorescence activated cell sorter

FBS:

fetal bovine serum

IC50 :

50% inhibitory concentration

MTHFR:

5,10-methylenetetrahydrofolate

NSAIDs:

non-steroidal anti-inflammatory drugs

PBS:

phosphate-buffered saline

PGE2 :

prostaglandin E2

SRB:

sulphorhodamine B

TBS:

Tris-buffered saline

TS:

thymidylate synthase

UTP:

uridine 5’-triphosphate

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Acknowledgements

Authors thank Dr. E. Hitre for critical reading of this manuscript and Cs. Polényi Makácsné, A. Nagy, J. Kútvölgyi, A. Éber Mousáné for their technical assistance. This study was supported by the Jedlik Ányos Grant (NKFP1-00024/2005).

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Authors and Affiliations

  1. National Institute of Oncology, Budapest, Hungary

    Andrea Réti, Éva Pap, Vilmos Adleff, Viktor L. Komlósi, Judit Kralovánszky & Barna Budai

  2. 1st Institute of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary

    Gábor Barna & András Jeney

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  1. Andrea Réti
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Correspondence to Judit Kralovánszky.

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Réti, A., Barna, G., Pap, É. et al. 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. Pathol. Oncol. Res. 15, 335–344 (2009). https://doi.org/10.1007/s12253-008-9126-9

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