Hederagenin Induces Apoptosis in Cisplatin-Resistant Head and Neck Cancer Cells by Inhibiting the Nrf2-ARE Antioxidant Pathway

Figure 4

Hederagenin induces apoptosis by inhibiting the Nrf2-ARE pathway in HNC cells. (a, b) Western blot analysis of cleaved PARP (cPARP), p53, phospho-p53-Ser15 (pp53), Bcl-2, Bax, cleaved caspase-3 (cCasp3), Nrf2, HO-1, NQO-1, xCT, and Keap1 in HNC cells exposed to 80 μM hederagenin for 24 h (a) or the indicated period of time (b). β-Actin was used as a loading control. (c) Quantitative real-time PCR analysis of Nrf2 expression in cisplatin-resistant HNC cells exposed to 80 μM hederagenin for 24 h. (d) Western blot analysis of Nrf2, xCT, and p62 in HN3-cisR cells treated with 80 μM hederagenin and/or the proteasome inhibitor MG132 (5 μM). (e) Immunofluorescence staining of p62 (green) and Nrf2 (red) in nontreated HN3-cisR cells and HN3-cisR cells treated with 80 μM hederagenin for 24 h. DAPI was used as a nuclear counterstain. (f) Nrf2 levels in cytoplasmic and nuclear extracts of HN3-cisR cells exposed to 0, 50, or 80 μM hederagenin for 24 h. (g) Nrf2 transcriptional activity in cisplatin-resistant HNC cells treated with 0 (NT), 50, or 80 μM hederagenin for 24 h. (h) Changes in HO-1 and NQO1 mRNA levels in cisplatin-resistant HNC cells treated with 0 (NT), 50, or 80 μM hederagenin for 24 h. The error bars represent the standard error from three replicate experiments , relative to the NT control.

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