Valproic acid inhibits cell size and cell proliferation by AMPK-mediated mTOR signaling pathway in JEG-3 cells

Abstract

Valproic acid is commonly used to treat seizure disorders, bipolar disorder, migraine prophylaxis, and neuropathic pain. Despite its effectiveness and widespread use, valproic acid has been proven to exert considerable teratogenic potential, such as neural tube defects and malformations of the heart in both the humans and animals. However, the molecular mechanism of the teratogenic effects of valproic acid has not been fully elucidated. Because adverse effects in fetus by teratogens are obviously detectable only after birth and there are the limits of teratogenicity testing using rodents, such as thalidomide tragedy, new strategies for pre-determining teratogenic effects are required. Here, we try to elucidate the indirect teratogenicity of valproic acid in a human placenta-derived cell line (JEG-3) using a transcriptomic approach. In this study, using human whole genome oligonucleotide microarray, we identified 2,076 up- and 1,730 down-regulated genes which were changed more than 1.5-fold in JEG-3 cells exposed to valproic acid. Many of these genes have associations with lysosome, transport, tight junction, splicesome, cell cycle and mammalian target of rapamycin (mTOR)-signaling pathway. Among these, we focused on the adenosine monophosphate (AMP)-dependent kinase or AMP-activated kinase (AMPK)-mediated mTOR signaling pathway, and hypothesized that the negative control of mTOR signaling by AMPK might induce inhibition of the growth of JEG-3 cell exposed to valproic acid. First, flow cytometry analysis showed that valproic acid induced the inhibition of cell growth caused by G1 phase arrest. Second, the expression of genes related to mTOR signaling was changed. Using quantitative real-time RT-PCR data, it was confirmed that PTEN, PIK3CB, PIK3CD, PIK3R3, IRS2, and PRKAA2 (AMPKα2) were overexpressed, and that GβL and AKT1 were under-expressed in valproic acid treated JEG-3 cells compared to a control. We also confirmed the protein expression and the activation of AMPK and raptor after valproic acid exposure. Thus, this study suggests that valproic acid affects AMPK, and then AMPK may influence cell growth through mTOR signaling, and particularly, mTOR activity is suppressed by raptor activation. From this point of view, these genes may provide potential biomarkers that may contribute to decrease the number of candidate drugs showing teratogenicity in large-scale tests using placenta cells.