Decreased c-Myc expression and activity by a growth inhibitory vitamin K analog.

3831 We previously showed that a synthetic K vitamin analog, Cpd 5, which is a potent growth inhibitor, inhibited c-myc expression and induced c-Myc phosphorylation (Proc. AACR 2003, 44: 617). To further explore Cpd 5 inhibitory mechanism(s), we examined Cpd 5 effects on c-Myc at the post-translational level. Cpd 5 induced c-Myc phosphorylation through prolonged ERK phosphorylation since it was antagonized by MEK inhibitor, U0126. c-Myc is known to be phosphorylated by GSK-3, which promotes c-Myc degradation. Western blot showed that Cpd 5 increased both GSK-3α and GSK-3β expression in Cpd 5 treated Hep 3B human hepatoma cells. Using GSK-3 inhibitor, SB26153, we found that phosphorylation of c-Myc was dramatically decreased due to inhibition of GSK-3 activity. Further, c-Myc protein levels reduced in Cpd 5 treated cells were restored by SB26153 action. The results show that Cpd 5 can increase GSK-3 activity to phosphorylate c-Myc, resulting in reduction of c-Myc protein levels, probably due to enhanced degradation. To investigate the consequences of reduced c-Myc levels, we measured expression of two c-Myc target genes, ornithine decarboxylase (ODC) and growth arrest gene Gadd45. Cpd 5 inhibited ODC expression in a time-dependent fashion, paralleling the Cpd 5 inhibition of c-Myc levels. Cpd 5 was also found to inhibit c-Myc DNA binding activity by 30–40%. c-Myc is known to repress Gadd45. Contrary to Cpd 5 inhibition of ODC, Cpd 5 strongly induced Gadd45 expression but had no effect on Gadd45 in c-myc null rat fibroblast cells, indicating Cpd 5 de-repressed the Gadd45 gene expression by inhibiting c-myc. These data show that Cpd 5 inhibited c-Myc levels and altered expression of its target genes, ODC and Gadd 45. Both of these may contribute to Cpd 5 mediated growth inhibition.