High glucose modulates antiproliferative effect and cytotoxicity of 5-fluorouracil in human colon cancer cells

Abstract

5-Fluorouracil (5-FU)-based chemotherapy is widely used for the treatment of colorectal cancer (CRC). While optimal doses of 5-FU are generally established based on a patient's estimated body surface area, the plasma concentrations of 5-FU vary among patients. In addition, hyperglycemia in patients with CRC has been reported as a risk factor in poor prognosis. The aim of the present study was to investigate whether hyperglycemia affects antiproliferative effect of 5-FU on the human colon cancer cells (SW480, SW620, LoVo, and HCT116). Growth inhibition of 5-FU was accessed by WST-8 assay. The effect of high glucose (HG, 15 mM) and 5-FU on the cellular proliferation was evaluated by flow cytometry analysis using 5-ethynyl-2'-deoxy-uridine (EdU) incorporation plus 7-AAD. Cell death was determined by flow cytometry using Annexin V-FITC and PI. The results showed that HG, compared to physiological normal glucose (NG) concentration (5 mM), leads to increased cell proliferation and increased GI50 of 5-FU in the four colon cancer cell lines. When the cells were pretreated with a low-dose 5-FU in NG condition, subsequent HG treatment eliminated inhibitory effect of 5-FU in cancer cell growth. In the presence of 5-FU (0.5 μg/mL for LoVo and HCT116; 1 μg/mL for SW480 and SW620), culture with HG for 72 h does not significantly altered cell cycle profile in the four cell lines but significantly increased DNA replication in SW620 (21%) and LoVo (17%). Flow cytometric analysis showed that HG protects cells against 5-FU-induced cell death in SW480. Finally, HG did not alter intracellular level of reactive oxygen species (ROS), although 5-FU indeed induced higher intracellular level of ROS. In conclusion, HG attenuates growth inhibition of 5-FU and our results indicate that decreased cell death and increased DNA replication may account for the attenuating effect of a HG environment on 5-FU-induced tumor growth inhibition.

FIG. 1.

Proliferation of four colon cancer cells under the treatment of high glucose and 5-FU. Cells were plated in DMEM with normal glucose concentration (NG, 5 mM, —■—) or high glucose concentration (HG, 15 mM, ) for 24 h. Then, the cells were treated with various concentration of 5-fluorouracil (5-FU) for 3 days. The cellular proliferation was assessed by WST-8 assay as described. Cellular proliferation of each colon cancer cell was significantly enhanced by the treatment of high glucose. The proliferation diminished with the increase of 5-FU dose. High glucose eliminated the growth inhibition of 5-FU when the dose of 5-FU was at least higher than GI50 for each cells. These analyses were mean±standard deviation (SD) of three independent experiments from at least three replications. **p<0.01, *p<0.05.

FIG. 2.

High glucose quenched the growth inhibition of short pretreated 5-FU. Cells were plated with the NG medium after 24 h, a dose of 5-FU lower than GI50 for each cell (0.5 μg/mL for SW480, SW620, and LoVo; 0.2 μg/mL for HCT116) was added and cultured for another 24 h. Then, the medium were replaced with either the NG (5 mM, gray column) or the HG (15 mM, black column) medium with 5-FU of three different concentration and incubated for another 3 days as indicated in horizontal axis. The survival fraction was assessed by WST-8 assay. The data were normalized with the OD450 read of the well in which the cells were pretreated with 5-FU for 24 h in the NG medium but without 5-FU in the NG medium during subsequent 3-day incubation. The data indicated that high glucose may abolish the cytotoxicity of 5-FU when the cells were pretreated with a lower concentration of 5-FU. The data were represented as the mean±SD from three independent experiments in at least four replications. **p<0.01.

FIG. 3.

High glucose increased DNA replication. (A) Detection of DNA replication by EdU click chemistry in SW620. Unsynchronized cells either untreated (left) or treated (right) with 1 μg/mL of 5-FU in the NG medium for 3 days. The incorporated EdU was detected with Alexa Fluor 488 tagged azide using Click-it kit (Molecular Probes). Bivariate distributions demonstrated DNA content (7-AAD fluorescence) versus incorporation of EdU. The mean fluorescence intensities of EdU-labeled mid-S phase cells (gating was shown in the right plot) as well as unlabeled cells were also shown. A comparable plot was also obtained from SW480, LoVo, and HCT116. (B) High glucose significantly increased the fluorescence intensity of EdU in LoVo and HCT116. The mean fluorescence intensity of EdU-labeled mid-S phase cells was normalized with that of the EdU-unlabeled cells. (C) In the presence of 5-FU, high glucose significantly increased the fluorescence intensity of EdU in SW620 and LoVo. The data represent the mean±SD of three independent experiments. *p<0.05. **p<0.01. EdU, 5-ethynyl-2′-deoxy-uridine.

FIG. 4.

Effect of high glucose on 5-FU-induced cell death in the colon cancer cells. Cells were treated with 5-FU (1 μg/mL for SW480 and SW620; 0.5 μg/mL for LoVo and HCT116) for 3 days. Cells were then stained with Annexin V-FITC in combination with PI and analyzed by flow cytometry. (A) Represented bivariate plot of Annexin V-FITC and PI for SW480 were shown. Percentages show Annexin-positive/PI-positive cells (late apoptotic/necrotic, top right quadrant) and Annexin-positive/PI-negative cells (early apoptotic, bottom right quadrant). (B) Annexin V-positive cells were counted for the representation of apoptotic cells. Each bar represents the mean±SD of three independent experiments. *p<0.05; **p<0.01.