Induction of endoplasmic reticulum stress by the pro-apoptotic retinoid N-(4-hydroxyphenyl) retinamide via a reactive oxygen species-dependent mechanism in human head and neck cancer cells

N-(4-hydroxyphenyl) retinamide (4HPR), which has shown efficacy in cancer chemo-prevention and therapy, induces the mitochondrial apoptosis pathway via increased generation of reactive oxygen species (ROS). ROS is also known to be able to induce an endoplasmic reticulum (ER) stress response, which can contribute to apoptosis but may also antagonize it. Therefore, we used human head and neck squamous cell carcinoma (HNSCC) cells to determine whether 4HPR affects ER stress. Different experimental approaches have indicated that 4HPR induces ER stress response: electron microscopy, which showed extensive ER dilation; splicing of the X-box binding protein 1 (XBP-1), a marker of unfolded protein response (UPR) activation; and quantitative real-time PCR and immunoblotting, which revealed the up-regulation of several ER-stress associated mRNAs and proteins, including the chaperone heat shock protein HSPA1A. Most of these effects of 4HPR were abrogated by cotreatment of cells with the antioxidant 3-tert-butyl-4-hydroxyanisole (BHA) indicating that they were downstream of the increase in ROS. Furthermore, siRNA-mediated silencing and chemical inhibition of HSPA1A, which exerts either pro- or anti-apoptotic effects, decreased 4HPR-induced apoptosis. These results demonstrate that 4HPR induces ER stress and uncovered a pro-apoptotic role for HSPA1A in 4HPR-induced apoptosis.