Suppressive effect of nobiletin, a citrus polymethoxyflavonoid that downregulates thioredoxin-interacting protein expression, on tunicamycin-induced apoptosis in SK-N-SH human neuroblastoma cells.

Increased expression of thioredoxin-interacting protein (TXNIP) has recently been proved to be a crucial event for irremediable endoplasmic reticulum (ER) stress resulting in the programmed cell death (apoptosis) of pancreatic ß-cells. The present study demonstrated that treatment with 1-10 µg/ml tunicamycin, a potent revulsant of ER stress, drastically induced TXNIP expression accompanied by the generation of cleaved caspase-3 as an indicator of apoptosis in SK-N-SH human neuroblastoma cells. This result substantiated that TXNIP is also involved in neurodegeneration triggered by ER stress. Moreover, we evaluated the effects of nobiletin, a citrus polymethoxyflavonoid, on tunicamycin-induced apoptosis and TXNIP expression in SK-N-SH cells, because we reported previously that this flavonoid might be able to reduce TXNIP expression. Co-treatment of SK-N-SH cells with 100 µM nobiletin and 1 µg/ml tunicamycin for 24h strongly suppressed apoptosis and increased TXNIP expression induced by 1 µg/ml tunicamycin treatment alone. In addition, we proved that the ability of 100 µM nobiletin treatment to reduce TXNIP expression is exerted from 3h after the onset of treatment. Therefore, the protective and ameliorative effects of nobiletin on neuronal degeneration and impaired memory, which several studies using animal models have demonstrated, might arise in part from nobiletin's ability to repress TXNIP expression.

Characteristics of nobiletin-mediated alteration of gene expression in cultured cell lines.

Nobiletin, a polymethoxylated flavonoid that is highly contained in the peels of citrus fruits, exerts a wide variety of beneficial effects, including anti-proliferative effects in cancer cells, repressive effects in hyperlipidemia and hyperglycemia, and ameliorative effects in dementia at in vitro and in vivo levels. In the present study, to further understand the mechanisms of these actions of nobiletin, the nobiletin-mediated alterations of gene expression in three organ-derived cell lines - 3Y1 rat fibroblasts, HuH-7 human hepatocarcinoma cells, and SK-N-SH human neuroblastoma cells - were first examined with DNA microarrays. In all three cell lines, treatments with nobiletin (100 µM) for 24 h resulted in more than 200% increases in the expression levels of five genes, including the endoplasmic reticulum stress-responsive genes Ddit3, Trib3, and Asns, and in less than 50% decreases in the expression levels of seven genes, including the cell cycle-regulating genes Ccna2, Ccne2, and E2f8 and the oxidative stress-promoting gene Txnip. It was also confirmed that in each nobiletin-treated cell line, the levels of the DDIT3 (DNA-damage-inducible transcript 3, also known as CHOP and GADD153) and ASNS (asparagine synthetase) proteins were increased, while the level of the TXNIP (thioredoxin-interacting protein, also known as VDUP1 and TBP-2) protein was decreased. All these findings suggest that nobiletin exerts a wide variety of biological effects, at least partly, through induction of endoplasmic reticulum stress and suppressions of oxidative stress and cell proliferation.