On the Mechanisms of 12-Otetradecanoylphorbol-13-acetate-induced Growth Arrest in Pancreatic Cancer Cells

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Abstract

Objectives:

Protein kinase C (PKC) is involved in cell growth, differentiation, and apoptosis. We investigated the effects of the PKC activator, the tetradecanylphorbol acetate (TPA), in human pancreatic cancer cells.

Methods:

Cell proliferation was measured by thymidine incorporation. Expression of cell cycle proteins was investigated by Western blot. Real-time reverse transcriptase-polymerase chain reaction was used to measure p21WAF1 messenger RNA expression, whereas knockdown of its expression was accomplished with a specific small interferring RNA. Cell cycle phases were determined by flow cytometry.

Results:

TPA time and concentration dependently inhibited thymidine incorporation in Panc-1 and CD18 cells and induced G2/M cell cycle arrest. The TPA decreased cyclin A and B expression, increased cyclin E, and markedly increased the expression of p21WAF1 at both the messenger RNA and protein levels. TPA-induced p21WAF1 expression and growth inhibition were blocked by the PKC inhibitor, bisindoylmaleimide. TPA induced extracellular signal-regulated kinase1/2 phosphorylation, whereas the MEK inhibitor, PD98059, blocked the TPA-induced p21WAF1 expression. Small interferring RNA targeted to p21WAF1 blocked TPA-induced p21WAF1 protein expression but not TPA-induced cell growth arrest.

Conclusions:

TPA-induced p21WAF1 expression is mediated by the MEK/ERK pathway but is not involved in TPA-induced growth inhibition. In contrast, cyclin A and cyclin B are likely involved in TPA-induced G2/M arrest because both proteins are involved in S phase and G2/M transition during cell proliferation.

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