Abstract
A, valproic acid, apicidin and MS275). NTR1 mRNA and protein expression was assessed by Northern and Western blots, respectively. NTR1 promoter activity was assessed by luciferase assay. (ii) The role of GSK-3β and ERK signaling pathways was determined using the selective inhibitors SB-216763 (for GSK-3β) and U0126 or PD98059 (for MEK/ERK) as well as siRNA to GSK-3β. (iii) To assess the functional effects of HDACI regulation of NTR, the effect of NaBT on NT-mediated induction of COX-2, c-myc and IL-8 expression was assessed by Northern blot and RNase protection assay. RESULTS. (i) Treatment with all HDACIs resulted in the potent down-regulation of endogenous NTR1 mRNA, protein and promoter activity. Northern blot analyses showed that HDACIs down-regulate NTR1 mRNA in a time-and dose-dependent fashion in all five NTR1-positive cell lines. Notably, NTR1mRNAwas dramatically decreased after 4 h of NaBT treatment in HT29 cells suggesting that NTR1 may be a direct target gene of HDACIs. (ii) Overexpression of GSK-3β decreased NTR1 promoter activity (> 30%); inhibition of GSK-3β increased NTR1 expression in HT29 and SW480 cells, indicating that GSK-3β is a negative regulator of ERK and NTR1. Consistent with our previous findings, HDACIs significantly decreased phosphorylated ERK while increasing GSK-3β. Selective MEK/ERK inhibitors suppressed NTR1 mRNA expression in a timeand dose-dependent fashion, and reduced NTR1 promoter activity by ~70%. (iii) Pretreatment with NaBT prevented NT-mediated COX-2 and c-myc expression and attenuated NT-induced IL-8 expression. CONCLUSIONS. HDACIs suppress endogenous NTR1 expression and function in CRC cell lines; this effect may involve both a direct and/or an indirect mechanism through the GSK-3β/ERK pathway. The down-regulation of NTR1 in CRCs may represent an important mechanism for the anti-cancer effects of HDACIs.
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