Abstract
Adaphostin (NSC680410), a small molecule congener of tyrphostin AG957, has been demonstrated previously to have significant anti-proliferative effects in several leukemia models. However, this effect of adaphostin in adherent cells/solid tumor models has not been examined. In this study, we investigated the anti-proliferative effects of adaphostin in the human prostate cancer cell line PC-3. Specifically, we explored the potential molecular mechanism(s) by which adaphostin elicits its anti-proliferative effect(s). We demonstrate that adaphostin inhibits the proliferation of PC-3 cells by inducing a G(1) phase cell cycle arrest. This adaphostin-induced G(1) arrest was associated with an increase in the expression of p21 and p27 and a decrease in the expression of G(1)-specific cyclins (cyclin A, D1, and D3) and cyclin-dependent kinases 4 and 6. Consequently, a dramatic decrease in the phosphorylation of retinoblastoma protein was also observed. Additionally, we found that adaphostin treatment induced a decrease in the phosphorylation of nucleophosmin, a major nuclear phosphoprotein, and that this decreased phosphorylation was a result of the p21- and p27-mediated inactivation of cyclin E-cyclin-dependent kinase 2 complex kinase activity. Furthermore, we have determined that the adaphostin-mediated cell cycle arrest of PC-3 cells is dependent upon activation of the p38 MAPK. We also demonstrate that the hepatocyte growth factor receptor-c-Met is involved in the adaphostin-mediated signaling events that regulate p38 MAPK. Taken together, these results identify for the first time a signaling cascade of adaphostin-mediated G(1) phase-specific cell cycle arrest in PC-3 cells. These findings suggest that the tyrphostin member has a broader spectrum of activity than originally predicted.
Highlights
Adaphostin (NSC680410), a small molecule congener of tyrphostin AG957, has been demonstrated previously to have significant anti-proliferative effects in several leukemia models
These results suggest that the adaphostin-mediated up-regulation of p21 and p27, which leads to an inhibition of CDK2-cyclin E complex kinase activity, results in dislation of p21 and p27 was required for the adaphostin-mediated down-regulation of NPM phosphorylation, we transfected p21and p27-specific siRNA along with control scrambled siRNA into PC-3 cells in the presence or absence of adaphostin
To determine whether MAPK signaling plays a role in the above-described adaphostin-induced cell cycle arrest, we examined the MAPK signaling events induced by adaphostin treatment on PC-3 cells
Summary
Drug—Adaphostin (NSC680410) was derived and obtained through the Developmental Therapeutics Program, NCI, National Institutes of Health. Plated cells were harvested in 25 M BrdUrd for 3 h; subsequently, cells were washed, trypsinized, and resuspended in Cytofix/Cytoperm buffer followed by incubation with DNase, fluorescein isothiocyanate-BrdUrd antibody, and 7-amino-actinimycin D as recommended by the manufacturer. PC-3 cells were transfected in 60-mm tissue culture plates using Lipofectamine reagent (Invitrogen) with 200 nM concentrations each of double-stranded oligonucleotide as per the manufacturer’s instructions. PC-3 cells were transfected in 60-mm tissue culture plates using Lipofectamine reagent (Invitrogen) with 5 g of plasmid as per the manufacturer’s instructions. After 24 h of transfection, media were replaced with fresh warm media followed by adaphostin treatment for the desired concentration and time. PC-3 cells were transfected with 0.5 g of p21 or p27 promoter-luciferase plasmid along with -galactosidase (for normalizing luciferase activity) using Lipofectamine reagent (Invitrogen) following the manufacturer’s instructions. The cells were collected, extracted, and analyzed for luciferase activity using dual-luciferase reporter assay system (Promega)
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