Evaluation of Drug Resistance in the Tamoxifen-treated MKN-45 Gastric Cancer Cell Line via the Epithelial-mesenchymal Transition Signaling Pathway

Abstract

One of the major challenges in gastric cancer (GC) chemotherapy is the phenomenon of multi-drug resistance (MDR). The epithelial-mesenchymal transition (EMT) and its key molecules, transforming growth factor-β (TGFβ) and SMAD2, play a central role in MDR occurrence. Tamoxifen (TAM), a triphenylethylene derivative, can overcome MDR in human gastric cancers. The aim of this study was to investigate the effect of TAM on 5-FU resistance of GC by suppressing the TGFβ1/SMAD2 signaling pathway and EMT. The MKN-45 cell line was subjected to treatment with 5-FU, TAM and a combination of both. The MTT assay was used to investigate the cytotoxic effects of 5-FU and TAM, and the DNA laddering technique was used to assess DNA fragmentation and apoptosis. Real-time RT-PCR examined the change in gene expression in EMT-related genes (SNAI2, VIM, TGFβ1 and SMAD2). The results of the present study indicated that not only TAM treatment significantly decreased the IC50 of 5-FU (P≤0.05), but also the addition of TAM to 5-FU induced apoptosis in the MKN-45 cell line. Treatment with TAM and 5-FU significantly inhibited TGFβ1 and TGFβ1-induced expression of EMT markers (VIM and SNAI2) in MKN-45 cells (P≤0.05). The reduction of TGFβ1 targets downstream of the SMAD2 signaling pathway reversed the process of EMT and significantly increased the sensitivity of MKN-45 cells to 5-FU. The results of the present study suggested that reversal of EMT-mediated MDR via the TGFβ1/SMAD signaling pathway using TAM may be a potential new therapeutic strategy to overcome chemoresistance to 5-FU during GC chemotherapy.

Keywords: Epithelial-mesenchymal transition; TGFβ1/ SMAD2 signaling pathway; drug resistance; Tamoxifen; gastric cancer.

Fig. 1

Dose-dependent growth inhibition and modulation IC50 of 5-FU by TAM in MKN-45 gastric cancer cells. MKN-45 gastric cancer cell line was treated with different concentrations of TAM and 5-FU drugs for 48 hours. The viability of MKN-45 cells treated with 5-FU (1-A) and TAM (1-B) drugs alone and simultaneously (1-C) for 48 hours of incubation was analyzed using the MTT method. The result showed that with TAM and 5-FU treatment, cell viability decreased in a dose-dependent manner and TAM treatment remarkably lowers the IC50 of MKN-45 cells to 5-FU. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to untreated control.

Fig. 2

Apoptosis induction by TAM in MKN-45 gastric cancer cells; Apoptosis of MKN-45 treated with TAM(C) and 5-FU (B) drugs alone and 5-FU+TAM (D) for 48 hours of incubation was assayed using DNA laddering. The results showed DNA fragmentation in GC cells treated with TAM or 5-FU alone or combined, but not in untreated cells (A). In addition, TAM treatment remarkably intensifies the DNA fragmentation of 5-FU-treated MKN-45 cells.

Fig. 3

The levels expression of TGFβ1 (A), SMAD2 (C), SNAI2 (B), and VIM (D) genes in MKN-45 treated with TAM and 5-FU drugs alone and in combination. The results showed down-regulating of TGFB1, SNAI2, and VIM genes in GC cells treated with TAM or 5-FU alone or in combination, but not in the SMAD2 gene. In addition, the combination treatment of TAM and 5-FU significantly reduced transcription of these genes in MKN-45 cells compared to the treatment of each drug alone. *P < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to untreated control; Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene.