Aicar Inhibits the Growth of Uveal Melanoma Cells via an Amp- Kinase Independent Pathway

Abstract

Purpose: : AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase, which senses cellular energy change, and helps regulate cellular metabolism. 5-Aminoimidazole-4-carboxamide riboside (AICAR) is taken up by cells, converted to ZMP (an analog of AMP) and is used as a pharmacologic activator of AMPK. It has recently been shown that AICAR can cause growth arrest in various cancer cell lines. We chose to investigate the effect and mechanism of action of AICAR on the growth of primary and metastatic uveal melanoma cell lines.

Methods: : Growth Inhibition Assay: Primary and metastatic uveal melanoma cell lines were cultured in the presence of 1, 2 and 4mM AICAR for 3-5 days. In separate experiments, cells were pretreated with 0.1 uM Iodotubericin (inhibits conversion of AICAR to ZMP) or 2 uM dipyridamole (inhibits the entry of AICAR into the cells). At the end of the incubation, cell growth was assessed with the MTT assay. Flow cytometry for cell cyle analysis: Uveal melanoma cells were treated with 2 mM AICAR for 48 hrs, then fixed with 70% ethanol and stained with propidium iodide for subsequent flow cytometry. Western blotting: Uveal melanoma cell lines were treated with 1,2 and 4 mM AICAR for 12, 24 and 48 hrs. Protein extracts were analyzed by Western blotting with antibodies against total AMPK and Phospho-AMPK, Proliferating Cell Nuclear Antigen (PCNA) and cyclin depedent kinase inhibitors p21, p27 and p53.

Results: : All six cell lines tested were inhibited by treatment with AICAR in a dose-dependent manner (65-80 % for the highest dose, p<0.05). Pre-treatment with Iodotubericidin did not abolish the effect seen with AICAR alone, while Dipyridamole (inhibitor of AICAR cellular entry) abolished AICAR-induced cell growth inhibition. Cell cycle analysis revealed arrest of melanoma cells in G1 phase after incubation with AICAR. Finally, treatment with AICAR resulted in a decrease in expression of PCNA in a dose dependent manner. We did not observe any change in the expression of p21, p27 and p53.

Keywords: melanoma • metabolism