Estimation of endoplasmic reticulum stress-inducing ability of nobiletin, a citrus polymethoxyflavonoid, in SK-N-SH human neuroblastoma cells

Abstract

Nobiletin, a citrus polymethoxyflavonoid compound, has been considered useful in the development of drugs and functional foods for various diseases, including dementia and diabetes. It is therefore important to understand its toxic effects. We previously reported that nobiletin treatment at a dose of 100 µM induced the expression of DDIT3 and TRIB3 genes and proteins, which are well known to contribute to apoptosis caused by endoplasmic reticulum (ER) stress, commonly in three cell lines, such as SK-N-SH human neuroblastoma cells. Therefore, their increased expression raises concerns that nobiletin might exert a toxic effect by inducing ER stress. In the present study, SK-N-SH cells were treated with 100 µM nobiletin or 1 µg/mL tunicamycin, a potent inducer of ER stress, for 3, 6, 12, and 24 hr. The maximum expression of those proteins appeared later and was much weaker in the nobiletin-treated cells than in the tunicamycin-treated cells. The expression level of BiP protein, one of the chaperons, which increases in response to ER stress, was not changed in the nobiletin-treated cells, whereas it was strongly induced 12 and 24 hr after the onset of tunicamycin treatment. In addition, cleavages of caspase-3 and poly (ADP-ribose) polymerase occurred 24 hr after the onset of tunicamycin treatment, whereas cleavage did not occur at any point during nobiletin treatment. Therefore, although nobiletin has the ability to induce the expression of DDIT3 and TRIB3, those increased levels, at doses up to at least 100 µM, cannot be enough to lead to ER stress resulting in apoptosis.