NRF2 Regulates HER1 Signaling Pathway to Modulate the Sensitivity of Ovarian Cancer Cells to Lapatinib and Erlotinib

Figure 7

Treatment with bexarotene causes inhibition of NRF2-dependent antioxidant response pathway and generates ROS. (A) Western analysis showing repression of NRF2 and HO-1 levels following bexarotene treatment in PEO1, OVCAR3, and SKOV3 cell lines. Exponentially growing cells were either left untreated, treated with 2.5 μM bexarotene, or a combination of 2.5 μM bexarotene together with 5 μM of lapatinib and erlotinib for 24 h before being harvested to prepare protein lysates and processed as described in Materials and Methods. β-Actin was used as loading control. The bars indicate NRF2 and HO-1 levels following quantification of immunoblot signal intensities obtained in (a) and normalised to the value of UT and expressed as fold change. The signal intensities of bands were quantified through integrated optical densitometry measurement. (b) Bexarotene treatment causes inhibition of NRF2-dependent transcription. Exponentially growing AREc32 cell line stably expressing 8× cis-antioxidant response elements driving the expression of luciferase gene in an NRF2-dependent manner were either left untreated (UT), treated with bexarotene alone, or with bexarotene and combination of lapatinib and erlotinib for different time points as indicated. Following this, cell lysates were prepared and assayed for luciferase activity (BrightGlo Luciferase System, Promega). (c) Bexarotene treatment causes increase in ROS levels. Exponentially growing AREc32 cell lines stably expressing 8× cis-antioxidant response elements driving the expression of luciferase gene in an NRF2-dependent manner were seeded in triplicates in opaque flat bottom black-walled 96-well plates for 24 h. Following this, cells were either left untreated (UT), treated with bexarotene alone, or with bexarotene and combination of lapatinib or erlotinib for different time points as indicated. Following incubations, cells were loaded with DCFDA fluorescent stain for 45 min and assayed for ROS as described in Materials and Methods. Data are the 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 according to the scale , , .

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