Table 1.
The IC-50 valuesa for chemotherapeutic agents in ESFT cell lines incubated with indicated agents for 48 hours in normoxia and hypoxia.
Figure 1.
The effect of hypoxia on proliferation of ESFT cells.
Proliferation was measured as the decrease in fluorescence of the plasma membrane-labelling dye CellVue™ Claret (see text for details). The graphs are semi-log plots of log10 (CVC fluorescence) vs. time, and differences between slopes were compared by linear regression analysis. Values represent the mean ± SEM of n = 6 determinations. Open circles = normoxia, closed circles = hypoxia.
Table 2.
Effects of fenretinide on proliferation and apoptosis levels of ESFT cells in normoxia and hypoxia over 48 hours.
Figure 2.
Fenretinide induces ROS production in both normoxia and hypoxia.
(A) Representative histograms of the effects of hypoxia and treatment with fenretinide on ROS production in the RDES cell line. (B) Hypoxia caused a significant increase in basal levels of ROS in both RDES and SKES-1 cell lines in the absence of fenretinide. (C–D) The magnitudes of ROS increase following treatment of ESFT cells with fenretinide (3 µM) were similar in normoxia and hypoxia over a period of 30 min–2 hrs. Values are mean ± SEM of n = 3 independent experiments. *Value significantly different from zero time control at p≤0.01.
Figure 3.
GSH levels and the activity of GSH-regulatory enzymes in RDES and SKES-1 cells.
Cells were harvested at the time points indicated for determination of GSH levels and the activities of GRD, GCS, and GGT1 as described in the text. Each bar represents the mean ± SEM of n = 6 determinations. Grey bars = normoxia; Black bars = hypoxia. *Hypoxia values significantly different from those in normoxia at p≤0.01.
Figure 4.
Pearson correlation modelling of the relationships between GSH and its regulatory enzyme activities in ESFT.
The parameters were measured in ESFT cells cultured under normoxic conditions in the absence of fenretinide. (A–C) Relationships between the enzymatic activities of GSH regulatory enzymes. (D–F) Correlation between activities of GSH-regulatory enzymes and GSH levels in ESFT cells.
Figure 5.
Effects of siRNA-targeted knockdown of GRD and GCS on sensitivity of RDES (A–E) and SKES-1 (F–J) cells to fenretinide.
Western blots (Panels A & F) show decreased GRD and GCS protein levels in RDES and SKES-1 cells electroporated with siRNA. Enzymatic activities of GRD and GCS were decreased in both RDES (Panel B) and SKES-1 (Panel G) cells following electroporation with siRNA, *p<0.0001. GSH levels were also significantly decreased in RDES (Panel C) and SKES-1 (Panel H) cells following electroporation with siRNA, *p<0.0001. Sensitivity of ESFT cells to fenretinide was improved by decreasing GSH levels in both RDES (Panels D & E) and SKES-1 (Panels I & J) cells in normoxia and hypoxia. For panels D,E,I, and J, white bars = electroporated control; light grey bars = control non-specific siRNA; grey bars = siGRD; dark grey bars = siGCS; Black bars = siGRD+siGCS (400 nM each), *p≤0.01; **p≤0.0001.
Figure 6.
The effect of increasing GSH levels by NAC supplementation on sensitivity of RDES (Panels A–C) and SKES-1 (Panels D–F) cells to fenretinide.
NAC supplementation significantly increased GSH levels in RDES (Panel A) and SKES-1 (Panel D) by between 10 and 50% compared to un-supplemented controls. The effects of increasing GSH levels in RDES (Panels B–C) and SKES-1 (Panels E–F) cells were cell line-specific. *Number of viable cells was significantly different between normoxia and hypoxia, specific IC-50 values are reported in the text.