Regulatory Effects and Mechanism of GLUT1 on 5-Fu Resistance in Human Colon Cancer Cells
SHAO Huaming*, YANG Yan
This study was to investigate the regulatory effect and mechanism of GLUT1 (glucose transporter 1) on 5-Fu (5-fluorouracil) resistance in human colon cancer cells. In this study, the gene and protein expression of GLUT1 in colon cancer tissues and adjacent tissues in 17 patients with 5-Fu chemo-sensitive colon cancer and 13 patients with 5-Fu chemo-resistant colon cancer was detected by RT-qPCR (real-time fluorescence quantitative polymerase chain reaction) and Western blot. 5-Fu-resistant HT-29 cells (HT-29/5-Fu) were induced by increasing 5-Fu concentration intermittently. HT-29/5-Fu cells and their parents HT-29 cells were cultured in vitro. The inhibitory effect of 5-Fu on the proliferation of HT-29/5-Fu cells and HT-29 cells was detected by MTT assay. The gene and protein expression of GLUT1 in HT-29/5-Fu cells and HT-29 cells was detected by RT-qPCR and Western blot. The HT-29/5-Fu cells were divided into normal control group (NC), nonsense control group (pRS-scrambled) and GLUT1 interfering group (pRS-GLUT1). The gene and protein expression of GLUT1 was detected by RT-qPCR and Western blot. The cell cycle and apoptosis were detected by flow cytometry. The gene and protein expression of MRP1, ABCB1, GST-π, Bcl-2 and Bax in HT-29/5-Fu cells and HT-29 cells was detected by RT-qPCR and Western blot. This study showed as below: (1) The gene and protein expression of GLUT1 in 5-Fu-resistant colon cancer tissues was significantly increased than that in 5-Fu-sensitive colon cancer tissues (P<0.05); (2) The inhibitory rate of 5-Fu (5-100 pg/mL) on HT-29 cells was significantly increased than that of HT-29/5-Fu (P<0.05); (3) The gene and protein expression of GLUT1 in HT-29/5-Fu cells was significantly increased than that in HT-29 cells (P<0.05); (4) The mRNA and protein expression of GLUT1 in pRS-GLUT1 group was significantly decreased than that in NC group and pRS-scrambled group (P<0.05); (5) The absorbance in pRS-GLUT1 group was significantly decreased than that in NC group and pRS-scrambled group (P<0.05); (6) The proportion of cells in G0/G1 phase in pRSGLUT1 group was significantly increased, and the proportion of cells in S phase in pRS-GLUT1 group was significantly decreased than that in NC group and pRS-scrambled group (P<0.05); (7) The apoptosis rate in pRS-GLUT1 group was significantly increased than that in NC group and pRS-scrambled group (P<0.05); (8) The mRNA and protein expression of MRP 1, GST-π, ABCB 1 and Bcl-2 in pRS-GLUT1 group was significantly decreased than that in NC group and pRS-scrambled Group (P<0.05), and the mRNA and protein level of Bax in pRS-GLUT1 group was significantly higher than that in NC group and pRS-scrambled group (P<0.05). This study proved that GLUT1 gene silencing can increase the sensitivity of resistant colon cancer cells to 5-Fu, which may be related to regulating cell cycle, promoting apoptosis, inhibiting the expression of drug resistance related proteins, and regulating Bcl2/Bax apoptosis pathway in colon cancer cells.
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