Induction of cyclin-kinase inhibitors p21Cip1 and p27Kip1 by chemopreventive isoprenoids perillyl alcohol, farnesol, and geraniol in human pancreatic adenocarcinoma cells.

Abstract

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The isoprenoid drugs perillyl alcohol (POH), geraniol (GOH), and farnesol (FOH) have been previously demonstrated to have anticancer properties both in vitro and in vivo models of human pancreatic ductal adenocarcinoma. Furthermore, the anticancer effect in vitro occurs with an increase in levels of the cyclin-kinase inhibitor protein p27Kip1 and a simultaneous decrease in levels of Cyclin A protein. It was hypothesized that removing p27Kip1 protein from cells would cause resistance to the isoprenoid antiproliferative effect. RNA interference (RNAi) was used to reduce protein levels of p27Kip1 in MIA PaCa-2 human pancreatic ductal adenocarcinoma cells, thus mitigating its potential influence in cell response. Proliferation rates of transfected cells were compared to nontransfected controls following 24-hour exposure to isoprenoids (300, 500 μM POH; 200, 400 μM GOH; 60, 90 μM FOH). Following transfection of anti-p27Kip1 siRNA, and immediately prior to isoprenoid exposure, endogenous levels of p27Kip1 protein were <6% of nontransfected levels, with the downregulation lasting at least 72 hours following removal of transfection media. However, the proliferation rate of transfected cells demonstrated only modest resistance relative to nontransfected cells. RNAi caused a 17.5%, 20.32%, and 15.56% reduction in isoprenoid-induced growth inhibition in POH, GOH, and FOH, respectively. With this result, protein levels of p21Cip1 were examined in MIA PaCa-2 and BxPC-3 human ductal adenocarcinoma cells, following exposure for 24 hours of POH (300,500 μM), GOH (200,400 μM), and FOH (60, 90 μM). Protein levels were compared using Western analysis and normalized using β-actin. Significant increases (p < 0.05, n≥3) were observed in p21Cip1 protein levels for all three isoprenoids. Increases were 3.5 and 4.5 fold for POH (300, 500 μM), 3.86 and 5-fold for GOH (200,400 μM), and 3 and 4.21-fold for FOH (60,90 μM) These results suggest that these three drugs operate through a similar mechanism of action, mediated in part by a combination of the increased expression of both p27Kip1 and p21Cip1, resulting in cells undergoing G0/G1 arrest.