Abstract
L-carnitine (LC) is generally believed to transport long-chain acyl groups from fatty acids into the mitochondrial matrix for ATP generation via the citric acid cycle. Based on Warburg’s theory that most cancer cells mainly depend on glycolysis for ATP generation, we hypothesize that, LC treatment would lead to disturbance of cellular metabolism and cytotoxicity in cancer cells. In this study, Human hepatoma HepG2, SMMC-7721 cell lines, primary cultured thymocytes and mice bearing HepG2 tumor were used. ATP content was detected by HPLC assay. Cell cycle, cell death and cell viability were assayed by flow cytometry and MTS respectively. Gene, mRNA expression and protein level were detected by gene microarray, Realtime PCR and Western blot respectively. HDAC activities and histone acetylation were detected both in test tube and in cultured cells. A molecular docking study was carried out with CDOCKER protocol of Discovery Studio 2.0 to predict the molecular interaction between L-carnitine and HDAC. Here we found that (1) LC treatment selectively inhibited cancer cell growth in vivo and in vitro; (2) LC treatment selectively induces the expression of p21cip1 gene, mRNA and protein in cancer cells but not p27kip1; (4) LC increases histone acetylation and induces accumulation of acetylated histones both in normal thymocytes and cancer cells; (5) LC directly inhibits HDAC I/II activities via binding to the active sites of HDAC and induces histone acetylation and lysine-acetylation accumulation in vitro; (6) LC treatment induces accumulation of acetylated histones in chromatin associated with the p21cip1 gene but not p27kip1 detected by ChIP assay. These data support that LC, besides transporting acyl group, works as an endogenous HDAC inhibitor in the cell, which would be of physiological and pathological importance.
Citation: Huang H, Liu N, Guo H, Liao S, Li X, et al. (2012) L-Carnitine Is an Endogenous HDAC Inhibitor Selectively Inhibiting Cancer Cell Growth In Vivo and In Vitro. PLoS ONE 7(11): e49062. doi:10.1371/journal.pone.0049062 Editor: Wei-Guo Zhu, Peking University Health Science Center, China Received December 21, 2011; Accepted October 9, 2012; Published November 5, 2012 Copyright: ?2012 Huang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.

Introduction
Carnitine is biosynthesized from the amino acids lysine and methionine and its biologically active form is L-carnitine (LC). It is generally believed that carnitine transports long-chain acyl groups from fatty acids into the mitochondrial matrix, where they can be broken down through b-oxidation to acetyl-CoA to obtain usable energy via the citric acid cycle [1?]. Therefore LC is required for the generation of metabolic energy in living cells. It has been well known that most cancer cells predominantly generate energy by a high rate of glycolysis followed by lactic acid fermentation in the cytosol, rather than by a comparatively low rate of glycolysis followed by oxidation of pyruvate in mitochondria like most normal cells. This is known as Warburg’s effect in cancer cells [4,5]. Rapidly growing malignant cells typically haveglycolytic rates that are up to 200 times higher than those of their normal tissues of origin. Even though Warburg effect has been challenged and further developed, this theory remains the most frequently cited evidence that tumors display dysfunctional metabolism [6]. Based on this theory that the citric cycle is detrimental in most cancer cells [7,8], we hypothesize that LC would lead to disturbance of cellular metabolism in cancer cells but not in normal cells. In this study, we investigated the effects of LC on cytotoxicity both in cancer and normal cells. We found that LC selectively inhibited cancer cell growth both in vitro and in vivo. We further investigated the mechanism of LC-mediated cytotoxicity and found that physiological concentrations of LC could directly inhibit HDAC activities.

Materials and Methods Materials and agents
LC, R-carnitine, oligomycin (No. 04876), butyrate (Buty) and trichostatin A (TSA) were purchased from Sigma-Aldrich (St. Louis, MO). HDACTM I/II assay and screening system was purchased from Promega corporation (Madison, WI). L-glucose and D-glucose were obtained from Alfa Aesar (Karlsruhe, Germany). Fetal bovine serum (FBS) was purchased from Invitrogen Co. (Carlsbad, CA). Rabbit monoclonal antibodies against Acetyl-H3 (Lys9) (C5B11), rabbit polyclonal antibodies against PARP, acetylated-Lysine, acetyl-H4 (Lys8), and mouse monoclonal antibodies against p21 Waf1/Cip1 (DCS60) were all purchased from Cell Signaling (Beverly, MA). Antibodies against Rb (G401), Phospho-Rb (S780) were purchased from Bioworld Technology, Inc. Mouse monoclonal antibody p27 (F-8), rabbit polyclonal antibodies against GAPDH (FL-335) and horseradish peroxidase (HRP)-labeled secondary antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Enhanced chemiluminescence reagents were purchased from Amersham Biosciences (Piscataway, NJ).

Cell death assay
This was performed using Annexin V-FITC and propidium iodide (PI) double staining, followed by flow cytometry as previously described [9]. In brief, cultured HepG2 cells were harvested and washed with cold PBS and resuspended with the binding buffer, followed by Annexin V-FITC incubation for 15 min and PI staining for another 15 min at 4uC in dark. The stained cells were analyzed with flow cytometry within 30 min.

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