Figure 8.

Treatment with lapatinib, erlotinib, and bexarotene causes inhibition of NRF2-dependent transcription and depletion of total glutathione levels. (a) Single and combination of lapatinib and erlotinib cause inhibition of NRF2-dependent transcription. Exponentially growing MCF7-AREc32 cell lines stably expressing cis-regulatory antioxidant response elements driving the expression of luciferase gene in an NRF2-dependent manner were treated with 1 nM HRG alone or with cotreatment of 5 μM lapatinib and erlotinib either individually or in combination for different time points as indicated. Following this, cell lysates were prepared and assayed for luciferase activity as described in Materials and Methods. Data shown are mean values ± S.D of quadruplicates, normalised to untreated (UT) and expressed as fold change with statistical significance determined by one-way ANOVA followed by Tukey's post hoc test. Asterisks indicate significant differences between individual groups as indicated and according to the scale ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. (b) Single and combination of lapatinib and erlotinib cause decrease in glutathione level. Exponentially growing cells were seeded in 60 mm tissue culture plates for 24 h and either left untreated (UT) or treated with media containing 1 nM heregulin alone (HRG) or with cotreatment of 5 μM lapatinib and erlotinib or their combination with 2.5 μM bexarotene (COMB) for 72 h before being harvested to prepare protein lysates and processed for glutathione assay. Data are mean values ± S.D of triplicates and expressed as fold change to the UT. Statistical significance was determined by one-way ANOVA followed by Tukey's post hoc test according to the scale ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001.