Silenced PITX1 promotes chemotherapeutic resistance to 5-fluorocytosine and cisplatin in gastric cancer cells

Abstract

Resistance to chemotherapeutic drugs leads to a poor prognosis in gastric cancer (GC). The present study aimed to assess the association between pituitary homeobox paired homeodomain transcription 1 (PITX1) expression and the sensitivity of GC cells to the chemotherapeutic drugs 5-fluorouracil (5-FU) and cisplatin (CDDP). In the present study, the gastric cancer cell lines GES-1, AGS, BGC-823, MCG-803 and SGC-7901 were used. The expression of PITX1 was determined via reverse transcription-quantitative polymerase chain reaction in GC cell lines. AGS and BGC-823 cells, which exhibit a decreased PITX1 expression, were transfected with a PITX1 cDNA construct and its control vector. MCG-803 and SGC-7901 cells, which exhibit an increased PITX1 expression, were transfected with siRNA against PITX1 and its control scramble sequence. A Cell Counting kit-8 assay was performed to determine the impact of PITX1 expression on the sensitivity of GC cells to 5-FU and CDDP. The Cancer Genome Atlas database was used to analyze the expression of PITX1 with GC prognosis in the Asian population and to assess the potential mechanism of PITX1 in 5-FU and CDDP resistance. The results revealed that the overexpression of PIXT1 increased the sensitivity of GC cells to 5-FU/CDDP. The combination of 5-FU/CDDP and PITX1 overexpression also reduced the proliferation of GC cells. Additionally, PIXT1 knockdown decreased the sensitivity of GC cells to 5-FU/CDDP. TCGA data revealed that a lower expression of PITX1 is exhibited in Asian GC patients than in normal individuals. GC patients with a lower expression of PITX1 had a poor prognosis. The expression of PITX1 affected the sensitivity of GC cells to 5-FU/CDDP, indicating that PITX1 may increase the efficacy of treatment in GC patients.

Keywords: 5-fluorouracil; cisplatin; gastric cancer; pituitary homeobox paired homeodomain transcription 1; prognosis.

Figure 1.

The expression of PITX1 in gastric cancer cell lines determined via reverse transcription-quantitative polymerase chain reaction. *P<0.05 vs. GES-1. PITX1, pituitary homeobox paired homeodomain transcription 1.

Figure 2.

The viability of four gastric cancer cell lines was determined following treatment with different concentrations of 5-FU and CDDP. The viability of four gastric cancer cells treated with different concentration of (A) 5-FU and (B) CDDP was measured using a Cell Counting Kit-8 assay. 5-FU, 5-fluorouracil; CDDP, cisplatin; IC30, 30% inhibitory concentration.

Figure 3.

PITX1 overexpression affects the sensitivity of AGS and BGC-823 cell lines to 5-FU. (A) The expression of PITX1 was detected in AGS-pPITX1 cells via western blotting and RT-qPCR, and in BGC-823-pPITX1 cells via RT-qPCR. PITX1 expression was normalized to that of endogenous β-actin. (B) A Cell Counting kit-8 assay was utilized to assess the proliferation of AGS and BGC-823 cells treated with 5-FU, the pPITX1 construct, the 5-FU+pcDNA3.1 vector, the 5-FU+pPITX1 construct and the pcDNA3.1 vector. *P<0.05. All experiments were performed in triplicate and repeated at least three times. PITX1, pituitary homeobox paired homeodomain transcription 1; 5-FU, 5-fluorouracil; pPITX1, overexpression of PITX1; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; OD, optical density.

Figure 4.

Silenced PITX1 reduces the sensitivity of MCG-803 and SGC-7901 cells to 5-FU. (A) The expression of PITX1 was detected via western blotting and RT-qPCR in MCG-803 cells. SGC-7901 PITX1 expression was also determined via RT-qPCR The PITX1 mRNA level was normalized to that of endogenous β-actin. (B) A Cell Counting kit-8 assay was performed to analyze the proliferation of MCG-803 and SGC-7901 cells treated with 5-FU, siPITX1, siCtrl+5-FU, siPITX1+5-FU and siCtrl. *P<0.05. All experiments were performed in triplicate and repeated at least three times. PITX1, pituitary homeobox paired homeodomain transcription 1; 5-FU, 5-fluorouracil; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; si, small interfering RNA; Ctrl, control; OD, optical density.

Figure 5.

Differentially expressed PITX1 affects the sensitivity of gastric cancer cell lines to CDDP. (A) A Cell Counting kit-8 assay was performed to analyze the proliferation of (A) AGS and BGC-823, and (B) MCG-803 and SGC-7901 cells treated with CDDP, a pPITX1 construct, a CDDP+pcDNA3.1 vector, a CDDP+pPITX1 construct and a pcDNA3.1 vector. *P<0.05. All experiments were performed in triplicate and repeated at least three times. PITX1, pituitary homeobox paired homeodomain transcription 1; CDDP, cisplatin; pPITX1, overexpression of PITX1; OD, optical density.

Figure 6.

Evaluation of PITX1 expression and the survival of patients with GC in Asian populations based on TCGA data. (A) PITX1 mRNA expression was assessed in Asian patients with GC from the TCGA database. (B) Kaplan-Meier survival curve analysis for the overall survival of Asian patients with GC separated by PITX1 expression. PITX1 mRNA levels were measured as FPKM. PITX1, pituitary homeobox paired homeodomain transcription 1; GC, gastric cancer; TCGA, The Cancer Genome Atlas; FPKM, kilobase per million mapped reads. **P<0.01.

Figure 7.

Kyoto Encyclopedia of Genes and Genomes analysis of PITX1 co-expressed genes based on the TCGA database. (A) Functional analysis of the genes related to PITX1 from the TCGA database according to differential gene screening. (B) The represented genes that participated in necroptosis were positively correlated with PITX1. PITX1, pituitary homeobox paired homeodomain transcription 1; TCGA, The Cancer Genome Atlas; CASP8, caspase-8; FADD, Fas-associated protein with death domain; MLKL, mixed lineage kinase domain like pseudokinase; RIP3, receptor-interacting serine/threonine-protein kinase 3; Cor, correlation.