The hedgehog pathway inhibitor cyclopamine increases levels of p27, and decreases both expression of IGF-II and activation of Akt in PC-3 prostate cancer cells.
The hedgehog signalling inhibitor cyclopamine has been shown to induce growth inhibition and cell cycle arrest in prostate cancer cell lines, but the mechanism of action has not been clearly defined, and observations between laboratories have not always been consistent. We first observed that albumin can protect PC-3 prostate cancer cells from cyclopamine-induced growth inhibition, suggesting that cyclopamine binds to albumin, and that only free cyclopamine is active. We then conducted a phospho-site protein kinase screen to elucidate the mechanism of cyclopamine-induced growth inhibition. Treatment of PC-3 cells with 5 or 10 microM cyclopamine for 72h resulted in a decrease in cell viability of approximately 50% and approximately 75%, respectively. A phospho-site protein kinase screen showed that cyclopamine decreased levels of phospho-Thr(187)-p27 by 71%. This phospho-site on p27 positively regulates its ubiquitin degradation; therefore a decrease in phospho-Thr(187)-p27 should correlate with increased levels of p27. Consistent with this hypothesis, treatment of PC-3 cells with cyclopamine resulted in a approximately 3-fold increase in p27 protein levels. Cdk-2 phosphorylates Thr(187)-p27, and immunoblotting demonstrated that cyclopamine treatment of PC-3 cells reduces the expression of cdk-2. Furthermore, cyclopamine decreased the levels of phosphorylated (activated) Akt, which is known to increase p27 degradation via Skp-2-induced ubiquitination. The mechanism by which cyclopamine decreases phosphorylated Akt is currently under investigation, but it may involve our observed cyclopamine-induced reduction in IRS-1 and IGF-II expression. These results demonstrate novel molecular correlates of cyclopamine-induced growth inhibition of prostate cancer cells.
Levitt
Randy J
RJ
Lady Davis Institute for Medical Research, Jewish General Hospital, Division of Experimental Medicine and Department of Oncology, McGill University, 3999 Cote Ste. Catherine, Montreal, Que., Canada H3T 1E2.
Zhao
Yunhua
Y
Blouin
Marie-José
MJ
Pollak
Michael
M
D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism*; D053823:Hedgehog Proteins / Q000037:antagonists & inhibitors*; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D008297:Male; D010766:Phosphorylation; D011471:Prostatic Neoplasms / Q000201:enzymology / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors*; D015398:Signal Transduction / Q000187:drug effects; D013912:Threonine / Q000378:metabolism; D015854:Up-Regulation; D014704:Veratrum Alkaloids / Q000494:pharmacology*
D053823:Hedgehog Proteins; D014704:Veratrum Alkaloids; D050760:Cyclin-Dependent Kinase Inhibitor p27; D013912:Threonine; D007335:Insulin-Like Growth Factor II; D051057:Proto-Oncogene Proteins c-akt; D051357:Cyclin-Dependent Kinase 2; C000541:cyclopamine