Radiosensitization by 2-benzoyl-3-phenyl-6,7-dichloroquinoxaline 1,4-dioxide under oxia and hypoxia in human colon cancer cells

Abstract

Background: The sensitizing effects of 2-benzoyl-3-phenyl-6,7-dichloroquinoxaline 1,4-dioxide (DCQ) and ionizing radiation (IR) were determined in four colon cancer cells and in FHs74Int normal intestinal cells.

Methods: Cell cycle modulation, TUNEL assay, clonogenic survival and DNA damage were examined under oxia or hypoxia. Effects on apoptotic molecules and on p-Akt and Cox-2 protein expression were investigated.

Results: The four cell lines responded differently to DCQ+IR; HT-29 cells were most resistant. Combination treatment caused significant increases in preG1 (apoptosis) in HCT-116, while G2/M arrest occurred in DLD-1. DCQ potentiated IR effects more so under hypoxia than oxia. Pre-exposure of DLD-1 to hypoxia induced 30% apoptosis, and G2/M arrest in oxia. The survival rate was 50% lower in DCQ+IR than DCQ alone and this rate further decreased under hypoxia. FHs74Int normal intestinal cells were more resistant to DCQ+IR than cancer cells.Greater ssDNA damage occurred in DLD-1 exposed to DCQ+IR under hypoxia than oxia. In oxia, p-Akt protein expression increased upon IR exposure and drug pre-treatment inhibited this increase. In contrast, in hypoxia, exposure to IR reduced p-Akt protein and DCQ restored its expression to the untreated control. Apoptosis induced in hypoxic DLD-1 cells was independent of p53-p21 modulation but was associated with an increase in Bax/Bcl-2 ratio and the inhibition of the Cox-2 protein.

Conclusion: DCQ is a hypoxic cell radiosensitizer in DLD-1 human colon cancer cells.

Figure 1

Effect of DCQ, IR and their combinations on cell cycle regulation in four different human colon cancer cell lines (SW-480, HT-29, HCT116 and DLD-1). Cells were treated with DCQ (0, 5, 10 μM), IR (2 Gy) or combinations. Immediately after radiation or drug treatment, cells were replenished with fresh medium containing no drug and incubated for another 24 hours. Control cells were treated with DMSO (0.1%). Cell cycle changes were assessed using Propidium Iodide stain with flow cytometry as described in "Materials and Methods". The percentage of cells in preG₁ and G₂/M phases were plotted as a function of DCQ dose. Results are representative of at least two independent experiments each performed in duplicates.

Figure 2

Effect of DCQ, IR and their combinations on cell cycle regulation in HCT116 cells exposed to oxic or hypoxic conditions. Cells were treated with 5 μM DCQ or DMSO (0.1%) and exposed to hypoxia or incubated in oxia for 1 hour, then irradiated (2 Gy). Immediately after radiation or drug treatment, cells were replenished with fresh medium containing no drug and incubated for another 24 hours. Cell cycle changes were assessed using Propidium iodide stain with flow cytometry as described in "Materials and Methods". Bar graphs are a summary of at least three independent experiments each performed in duplicates.

Figure 3

Effect of DCQ, IR and their combinations on cell cycle regulation in SW-480 cells exposed to oxic or hypoxic conditions. Cells were treated with 5 μM DCQ or DMSO (0.1%) and exposed to hypoxia or incubated in oxia for 4 hours, then irradiated (2 Gy). Immediately after radiation or drug treatment, cells were replenished with fresh medium containing no drug and incubated for another 24 hours. Cell cycle changes were assessed using Propidium iodide stain with flow cytometry as described in "Materials and Methods". Bar graphs are a summary of at least three independent experiments each performed in duplicates.

Figure 4

Combination effects of DCQ and IR in DLD-1 cells under oxic and hypoxic conditions. Cells were treated with 5 μM DCQ or DMSO (0.1%) and exposed to hypoxia or incubated in oxia for 1 hour, then irradiated (2 Gy). Immediately after radiation or drug treatment, cells were replenished with fresh medium containing no drug and incubated for another 24 hours. Cell cycle changes were assessed using Propidium iodide stain with flow cytometry as described in "Materials and Methods". Bar graphs are a summary of at least three independent experiments each performed in duplicates.

Figure 5

TUNEL assay showing that the combination of DCQ and IR induces apoptosis in DLD-1 cells under oxic and hypoxic conditions. Cells were treated with 5 μM DCQ or DMSO (0.1%) and exposed to hypoxia or incubated in oxia for 1 hour, then irradiated (2 Gy). Immediately after radiation or drug treatment, cells were replenished with fresh media containing no drugs and left in the incubator for 24 hours. The extent of DNA fragmentation was determined by TUNEL assay and measured by flow cytometry. The percentage of apoptotic cells was determined using CellQuest. Results are representative of at least two independent experiments.

Figure 6

Survival curves of DLD-1 cancer cells and FHs74Int normal cells exposed to DCQ alone or DCQ and irradiation. A. DLD-1 cells were exposed to 1 hour oxia or hypoxia in the presence of DCQ and the surviving fraction was determined as a percentage with respect to the untreated cells. B. DLD-1 cells were exposed to 1 hour oxia or hypoxia in the presence of DCQ and then irradiated (2 Gy) and the surviving fraction was determined as a percentage with respect to the irradiated cells. C. Absolute survival rates of DLD-1 cells exposed to DCQ, IR or their combinations under oxic and hypoxic conditions. D. FHs74Int cells were exposed to 1 hour oxia in the presence of DCQ and then irradiated. After irradiation, cells were re-plated and the colonies were stained with crystal violet and counted 10 days later. Each data point was calculated as percent of untreated cells of two independent experiments each performed in duplicates.

Figure 7

Induction of DNA damage in DLD-1 cells after treatment with DCQ, IR or combinations under oxic and hypoxic conditions. Cells were treated with 5 μM DCQ for 1 hour, 2 Gy IR or combinations. Immediately after treatment, DNA damage was assessed using alkaline single cell microgel electrophoresis (Comet) assay as mentioned in the "Materials and methods" section. A. The figure shows different grades of DNA fragmentation in DLD-1 cells. Magnification: 100×. B. An average of 100 cells per slide were counted and analyzed, and the mean of damaged cells is represented as the percentage of control untreated cells. C. Quantitative measurements of various comet assay end-points as analyzed using Comet Score software.

Figure 8

Effects of DCQ and IR on the expression levels of p53, p-p53, p21 (A), Bax/Bcl2 (B), p-Akt and Cox-2 (C) proteins. DLD-1 cells were treated under oxic and hypoxic conditions with 5 μM DCQ, 2 Gy IR or combinations. After 24 hours, 40 μg cell lysates were subjected to SDS-PAGE. Fold induction of protein levels was based on densitometry measurments. Protein levels in treated cells were defined as percentage of control. All plots were re-probed with GAPDH to ensure equal protein loading.