Inhibition of inducible NF-κB activity reduces chemoresistance to 5-fluorouracil in human stomach cancer cell line

Abstract

5-fluorouracil (5-FU) is used for the treatment of stomach and colon cancer, but many tumors are resistant to this chemotherapeutic agent. 5-FU induces apoptosis of several cancer cell lines, while some chemotherapeutic agents are known to activate the transcriptional factor NF-κB, which strongly suppresses apoptosis in vitro. In the present study, we investigated the relationship between activation of NF-κB and chemoresistance to 5-FU in human stomach cancer cell lines, NUGC3 (5-FU sensitive) and NUGC3/5FU/L (5-FU resistant). Treatment with 5-FU for 9–12 h caused activation of inducible NF-κB in NUGC3/5FU/L cells but not in NUGC3 cells. 5-FU also resulted in an increase in the number of TUNEL-positive cells and enhanced caspase-3 activity 3- to 5-fold in NUGC3 cells but not NUGC3/5FU/L cells. Moreover we also demonstrated that the inhibition of inducible NF-κB activation by using a NF-κB decoy could induce apoptosis and reduce chemoresistance against 5-FU. Our results suggest that 5-FU chemoresistance can be overcome by inhibition of inducible NF-κB activation, and that the use of the NF-κB decoy combined with 5-FU treatment is a new molecular and gene therapeutic strategy aimed at treatment of human stomach cancers resistant to 5-FU.

Introduction

5-fluorouracil (5-FU)¹ is one of the most effective chemotherapeutic agents used for gastrointestinal tumors. It acts by blocking the enzyme thymidylate synthase and by inhibiting the synthesis of both RNA and DNA. Recent studies on the cytotoxic effects of 5-FU have concluded that 5-FU can induce apoptosis in sensitive cell lines, similar to other chemotherapeutic agents [1], [2]. Like many other anticancer therapies, chemoresistance to 5-FU treatment is still a major clinical problem in some cancer patients. Several investigators studied the metabolic and genetic mechanisms of such resistance [3], [4], [5], [6], but only a few studies have examined the molecular mechanisms of chemoresistance to 5-FU based on its apoptotic properties.

Discovered in 1986 as a DNA-binding activity that recognized the immunoglobulin light-chain intronic enhancer, nuclear factor-kappaB (NF-κB) has been studied intensively for its role in the control of expression of genes involved in immune and inflammatory functions [7]. Recently, the role of NF-κB in the regulation of apoptosis of normal and cancer cells has also been studied [8], [9]. NF-κB is activated in several types of cancer cells and constitutive activation of NF-κB protects these cells against apoptosis [10]. It has been also reported that NF-κB activation by tumor necrosis factor (TNF)-α [11], [12], chemotherapeutic drugs [12], [13], or ionizing radiations [12] can protect cells from apoptosis. Furthermore, transient inhibition of NF-κB using adenoviral delivery of a modified form of an inhibitor of NF-κB (IκB-α) or proteasome inhibitors can reduce the chemoresistance against CPT-11 in some cancer cell lines [14], [15]. These facts suggest that NF-κB could participate in resistance to cancer treatment.

Previous studies showed that NF-κB activation could be blocked by gene decoy strategy as part of a new class of anti-gene strategies, providing a potential therapeutic approach for the prevention of several diseases [16], [17], [18]. The decoy used for these purposes consists of a double-stranded oligodeoxynucleotide (ODN) containing a sequence corresponding to the consensus sequence of the κB-binding sites [19].

The aim of the present study was to abolish resistance of cancer cells to 5-FU by inhibiting NF-κB activity. For this purpose, we used two human stomach cancer cell lines, NUGC3; a 5-FU-sensitive cell line, and NUGC3/5FU/L; a 5-FU-resistant cell line that had acquired resistance as a consequence of repeated 5-day exposures to stepwise-increasing concentrations of 5-FU in vitro. NUGC3/5FU/L is 200-fold and over 16-fold resistant to 96- and 1-h exposures to 5-FU, respectively [3]. We evaluated the cytotoxicity of 5-FU in these cell lines and investigated the relationship between activation of NF-κB and chemoresistance to 5-FU. We also examined the effects of the inhibition of inducible NF-κB activation on chemoresistance against 5-FU by using a NF-κB decoy.