Cell death by the quinoxaline dioxide DCQ in human colon cancer cells is enhanced under hypoxia and is independent of p53 and p21

Abstract

Introduction: We have shown that the radio sensitizer DCQ enhances sensitivity of HCT116 human colon cancer cells to hypoxia. However, it is not known whether the p53 or p21 genes influence cellular response to DCQ. In this study, we used HCT116 that are either wildtype for p53 and p21, null for p53 or null for p21 to understand the role of these genes in DCQ toxicity.

Methods: HCT116 cells were exposed to DCQ and incubated under normoxia or hypoxia and the viability, colony forming ability, DNA damage and apoptotic responses of these cells was determined, in addition to the modulation of HIF-1α and of p53, p21, caspase-2, and of the ataxia telangiectasia mutated (ATM) target PIDD-C.

Results: DCQ decreased colony forming ability and viability of all HCT116 cells to a greater extent under hypoxia than normoxia and the p21-/-cell line was most sensitive. Cells had different HIF-1α responses to hypoxia and/or drug treatment. In p53+/+, DCQ significantly inhibited the hypoxia-induced increases in HIF-1α protein, in contrast to the absence of a significant HIF-1α increase or modulation by DCQ in p21-/- cells. In p53-/- cells, 10 μM DCQ significantly reduced HIF-1α expression, especially under hypoxia, despite the constitutive expression of this protein in control cells. Higher DCQ doses induced PreG1-phase increase and apoptosis, however, lower doses caused mitotic catastrophe. In p53+/+ cells, apoptosis correlated with the increased expression of the pro-apoptotic caspase-2 and inhibition of the pro-survival protein PIDD-C. Exposure of p53+/+ cells to DCQ induced single strand breaks and triggered the activation of the nuclear kinase ATM by phosphorylation at Ser-1981 in all cell cycle phases. On the other hand, no drug toxicity to normal FHs74 Int human intestinal cell line was observed.

Conclusions: Collectively, our findings indicate that DCQ reduces the colony survival of HCT116 and induces apoptosis even in cells that are null for p53 or p21, which makes it a molecule of clinical significance, since many resistant colon tumors harbor mutations in p53.

Figure 1

DCQ reduces the viability of HCT116 cells more so under hypoxia than normoxia. (A) The effect of hypoxia on HCT116 (p53^+/+, p53^-/-, p21^-/-) cell viability after 6, 12, or 24 hrs of exposure to 1% O₂. Cells were plated in 96 well plates at 1.2 × 10⁵ cells/ml and treated at 50% confluency. Viability was determined using Cell Titer 96 non-radioactive proliferation assay. (B) Dose-dependent decrease in the viability of cells exposed to DCQ for 6 hrs and cultured under normoxia or hypoxia. Values are averages ± SD of two independent experiments each done in triplicates; (*) indicates p < 0.05 (one way ANOVA). ■ Normoxia □ Hypoxia. The experiment was repeated three times each in quadruplicates.

Figure 2

DCQ reduces the clonogenic survival of HCT116 cells more so under hypoxia than normoxia. (A) Clonogenic survival of DCQ-treated cells exposed to normoxic or hypoxic conditions. At 50% confluency, cells were treated for 12 hrs with different DCQ concentrations in normoxia or hypoxia, after which they were replated at low densities and colonies (more than 50 cells) were stained and counted after 10-14 days in culture. Surviving fractions were calculated as mentioned in "Methods". (*) indicates p < 0.05 (one way ANOVA). (B) Effect of DCQ on HIF-1α protein expression. Cells were plated in 100 mm dishes and treated for 6 hrs with DCQ while in normoxia or hypoxia. Whole cell lysates were immunoblotted for HIF-1α. GAPDH was used to ensure equal loading. Relative densitometry values are presented at the bottom of the blots. All ratios were normalized to GAPDH and calculated relative to the control cells cultured under oxia. The experiment was repeated three times each in triplicates.

Figure 3

DCQ induces mitotic catastrophe and apoptosis in HCT116 cells. (A) Low concentrations of DCQ triggered mitotic catastrophe in all HCT116 cell lines. Cells were cultured on coverslips and treated at 50% confluency with 2.5 μM DCQ for 48 hrs after which they were fixed and stained with Hoechst and viewed under a fluorescent microscope using UV. (**) indicates p < 0.001 (one way ANOVA) with respect to the Ctrl. (B) Higher concentrations of DCQ (5 and 10 μM) induced increases in the PreG₁ phase population more so under hypoxia. Treatment with DCQ in normoxia or hypoxia was for 6 hrs, after which cells were harvested immediately and DNA was stained with PI for analysis with FACScan flow cytometry. The percentage of Pre G₁ cells was calculated using Cell Quest. (C) Annexin V assay showing the apoptotic/necrotic response of p53^+/+ cells exposed to 5 or 10 μM DCQ for 6 hr in normoxia or hypoxia. Apoptosis was assayed 24 hr after drug treatment, and appeared to be enhanced in hypoxia at higher drug concentrations. Quadrant A = apoptotic cells, B = apoptotic+necrotic, C = normal, D = necrotic. The experiment was repeated twice each in duplicates.

Figure 4

DCQ is not cytotoxic to normal intestinal cells. DCQ at concentrations of up to 10 μM did not reduce FHs74 Int human normal intestinal cell viability. At 50% confluency, cells were exposed to DCQ for 6 hr or were left untreated. Viability was assessed by the Cytotox 96 non-radioactive assay (A) and by the MTT-based Promega assay (B). Values are averages ± SE of two independent experiments each done in triplicates. The experiment was repeated three times each in triplicates.

Figure 5

DCQ induces DNA damage and increases ATM expression in p53^+/+ HCT116 cells. SSB and DSB induced by DCQ in p53^+/+ cell line. (A) Examples of comets induced by DCQ in cells subjected to the alkaline comet assay. Cells treated with DCQ for 6 hrs in normoxia or hypoxia were collected directly after treatment, subjected to the alkaline comet assay and images were taken using a fluorescent microscope at 40× (oil immersion) magnification. The comets observed by each treatment are directly proportional to the amount of SSBs induced. (B) The mean of the parameters (% DNA in comet' s tail and tail moment) are shown in the graphs above. More than 50 cells per treatment were photographed and quantified using TriTek CometScore software. (*) indicates p < 0.05 (one way ANOVA) with respect to control. (C) DCQ-induced phosphorylation of ATM in p53^+/+ cells at 6 hrs as an indication of DSB. After treatment, cells were fixed and subjected to immunocytochemical detection of ATM phosphorylated on Ser-1981, and stained with PI to detect at the same time p-ATM in each phase of the cell cycle. The mean of the FL-1 intensity ± SD (reflecting the level of p-ATM expression) at the G₁, S and G₂M phases of the cell cycle are shown in the table. The experiment was repeated twice each in duplicates.

Figure 6

DCQ modulates the protein expression levels of key mediators of apoptosis and mitotic catastrophe. At 50% confluency, cells were treated with 5 or 10 μM DCQ for 6 hrs. Whole cell lysates were then immunoblotted with the different primary antibodies and probed with GAPDH to ensure equal loading. (A) p53 and p21 protein expression and (B) caspase-2 and PIDD-C protein expression in HCT116 cell lines in response to DCQ treatment under normoxic or hypoxic conditions. (C) Relative densitometry values of analyzed proteins are plotted. All values were normalized to GAPDH and calculated relative to the control cells cultured under normoxia. The experiment was repeated twice each in duplicates.