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

Figure 3

Pharmacological (bexarotene) and genetic inhibition (siRNA) of NRF2 causes transcriptional and translational downregulation of HER1. Luciferase assay showing transcriptional downregulation of HER1 following NRF2 inhibition by (a) bexarotene or (b) siRNA in PEO1, OVCAR3, and SKOV3 cell lines. Exponentially growing PEO1, SKOV3, and OVCAR3 excluding MCF7-AREc32 cell lines were transfected with either empty PGL3 basic vector or 1 μg PGL3 basic vector with cloned HER1 driving the expression of luciferase gene. Cotransfection with 0.2 μg pRL-CMV plasmid was performed as an internal transfection control as described in the Materials and Methods. At 24 h posttransfection, cells were (a) either left untreated or treated with 2.5 μM bexarotene. (b) Cells were either transfected with scrambled siRNA (Sc) or transfected with 20 pmol of NRF2 siRNA (Si) for 24 h. Following treatments, lysates were prepared and luciferase activity was measured using dual luciferase reporter assay (Promega) in multiplate reader (MODULUS, Promega). (c) Immunoblot analysis following treatment with bexarotene demonstrated protein downregulation of HER1 receptor and decrease of NRF2, HO-1, and HER1. Exponentially growing cells were either left untreated (UT) or treated with 2.5 μM bexarotene for 24 h before being harvested and processed for immunoblotting using relevant antibodies. Bar chart showing total NRF2, HO-1, and total HER1 levels in PEO1, OVCAR3, and SKOV3 cell lines by quantifying immunoblot signal intensities obtained in (c). (d) Immunoblot analysis following knockdown of NRF2 demonstrated protein downregulation of both HER1 receptor and decrease of NRF2 and HO-1 in PEO1, OVCAR3, and SKOV3 cell lines. Cells were either transfected with scrambled siRNA (Sc) or transfected with 75 pmol of NRF2 siRNA (Si). After 24 h and 48 h, cells were harvested and processed for immunoblotting using relevant antibodies. β-Actin of the same blot was used as loading control. Bar chart shows the levels of relevant proteins by quantifying immunoblot signal intensities obtained and expressed as fold change. Data shown in (a) and (b) are the means ± S.D. of triplicates, normalised to UT or scramble expressed in fold change with statistical significance determined by Student’s t-test (, , and ).

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