Identification of Retinoid-Modulated Proteins in Squamous Carcinoma Cells Using High-Throughput Immunoblotting

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Abstract
Retinoids have shown clinical efficacy in cancer chemoprevention and therapy presumably by modulating the growth, differentiation, and apoptosis of normal, premalignant, and malignant cells. To better understand the mechanisms by which retinoids exert their effects, we used a high-throughput Western blotting method (Becton-Dickinson PowerBlot) to evaluate changes in the levels of cellular signaling proteins in head and neck squamous cell carcinoma cells treated with the cytostatic all-trans-retinoic acid or with the proapoptotic retinoids 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid or N-(4-hydroxyphenyl)retinamide. Treatments of the head and neck squamous cell carcinoma cells with these retinoids for 24 h resulted in increased levels of 14, 22, and 22 proteins and decreased levels of 5, 10, and 7 proteins, respectively. The changes in the levels of the following proteins were confirmed by conventional western immunoblotting: all-trans-retinoic acid increased ELF3, topoisomerase II α, RB2/p130, RIG-G, and EMAPII and decreased MEF2D and cathepsin L. N-(4-Hydroxyphenyl)retinamide up-regulated ELF3, c-Jun, Rb2/p130, JAK1, p67phox, Grb2, O6-methylguanine-DNA methyltransferase, and Ercc-1. 6-[3-(1-Adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid increased Rb2/p130, c-Jun, Sp1, Sin, and tomosyn and decreased cathepsin L, Mre11, and topoisomerase II α. Some of these proteins were also modulated by these retinoids in other human cancer cell lines. A subset of the proteins were modulated similarly by the different retinoids, whereas changes in other proteins were unique for each retinoid. These results suggest that the mechanisms by which these retinoids modulate proteins are distinct but may overlap. Some of the retinoid-modulated proteins identified in this study may be novel candidates for mediating different responses to retinoids.