Figure 1.
L-carnitine treatment fails to increase ATP concentration in cancer cells.
(a) Cancer cells are resistant to oligomycin in the presence of D-glucose (2 g/L) but not L-glucose (2 g/L). Human HepG2 cancer cells were cultured in the presence or absence of either D-glucose or L-glucose in the culture medium and treated with various doses of oligomycin (0.1, 0.25, 0.5. 1.0 µg/ml) for 6 h and ATP content was assessed. Mean+SD (n = 3). *P<0.01, versus control. DM: DMSO. (b) LC does not increase intracellular ATP content in cancer cells. Human hepatic HepG2 and SMMC-7721 cells were cultured in the normal culture medium respectively and treated with different doses of LC for 6 h, ATP content was detected. LC: L-carnitine. (c) Thymotytes are sensitive to oligomycin in the presence of D-glucose (2 g/L). Mouse thymocytes were treated with oligomycin (1 mg/ml) for different time points (1, 3, 6, 9 h), total ATP content was detected. (d) LC efficiently increases cellular ATP content. Mouse thymocytes were treated with LC (1 mM) for various times, cellular ATP content was assassed. Veh: vehicle.
Figure 2.
LC treatment inhibits cancer cell proliferation in vivo and in vitro.
(a) LC selectively inhibits HepG2 tumor growth compared with normal tissues. BALB/c nude mice were s.c. inoculated in the left armpit of each mouse with HepG2 cells (1×106 cells/mouse). When the tumor size reaches 50–75 mm3, nude mice were i.p. injected with 400 mg/kg for consecutive 15 days. Normal Balb/c mice were treated as the nude mice. Body and organ weight were detected. B. W: Body weight. Mean+SD (n = 8). *P<0.01, **P<0.05, versus each control respectively. % inhibition = body weight or organ weight in the LC-treated group/ average body weight or organ weight in the control group×100. (b) LC inhibits HepG2 cell proliferation in a dose-dependent manner. HepG2 cells were treated with various doses of LC (1.25, 2.5, 5, 10 mM) for 24 h or 48 h, cell proliferation was detected by MTS assay. Mean+SD (n = 3). *P<0.01, **P<0.05, compared with the control. (c) LC induces cell cycle arrest at Go/G1 phase. HepG2 cells were treated with different doses of LC for 24 h, cell cycle were detected by flow cytometry. Representative results were shown. (d, e) LC slightly induces cell death. HepG2 cells were exposed to various doses of LC for 24 h and cell apoptosis was detected by flow cytometry. Summary of the data were shown in (d) and representive flow images were shown in (e). **P<0.05, versus control. (f) LC does not dramatically affect thymocyte cell viability. Mouse thymocytes were treated with various doses of LC (2.5, 5, 10 mM) for 24 h, cell viability was detected by MTS assay and cell number was count.
Figure 3.
LC treatment selectively induces expression of p21cip1 gene, mRNA and protein in cancer cells.
(a) LC induces p21cip1 gene expression but not p27 and GAPDH. HepG2 cells were treated with LC (2.5, 5.0, 10 mM) for 24 h; cells were collected for gene expression profile analysis. In the gene chip, there are 2 probes for p21cip1 and 1 probe for p27kip1. All the fold increases of p21cip1 and p27kip1 gene expression versus control were shown. (b) LC dose-dependently induces p21cip1 mRNA expression but not p27kip1 in HepG2 cells. HepG2 cells were incubated with different concentrations of LC (2.5, 5, 10 mM) for either 12 h or 24 h; the cells were collected for mRNA assay of p21cip1 and p27kip1 by real-time PCR. Fold increase of the LC-treated versus control was shown. Mean+SD (n = 3). *P<0.01, **P<0.05, compared with control. (c) LC dose-dependently and time-dependently induces p21cip1 protein accumulation in HepG2 cancer cells. HepG2 and SMMC7721 cells were treated with various doses of LC for 48 h or HepG2 cells were exposed to 5 mM of LC for 12, 24, 36, 48 h; p21 and p27 proteins were detected by Western blot. (d) LC dose-dependently decreases Rb phosphorylation. HepG2 cells were treated with LC for 48 h; Rb and phosphorylated Rb were dectected by Western blot. Typical Western images were shown (left) and band intensity was quantified (right).
Figure 4.
LC induces Histone acetylation in cultured cells.
(a) LC dose-dependently induces accumulation of acetylated histones. HepG2 cells were exposed to LC (1.25, 2.5, 5.0 mM) for 48 h, acetylated histones H3 and H4 were detected by Western blot. GAPDH was used as a loading control. (b) LC time-dependently induces accumulation of acetylated histones. HepG2 cells were treated with LC (5 mM) for different time points (12 h, 24 h, 36 h, 48 h) and acetylated histones were detected. (c) LC treatment increases lysine-acetylated protein accumulation in human HepG2 and SMMC-7721 cancer cells. Human HepG2 and SMMC-7721 cells were treated with LC (10 mM) for 12 h, and then cells were collected for Western blot to detect acetylated proteins with lysine acetylated antibody. (d) LC dose-depentently increases accumulation of acetylated histones in mouse thymocytes. Mouse thymocytes were treated with LC for 24 h, histone acetylation was detected by Western Blot. Buty (1 mM) was used as a positive control.
Figure 5.
Computional molecular docking of LC with HDAC.
(a) The chemical structure of LC was shown. (b) The docking model of L-carnitine in active site of HDAC.
Figure 6.
LC directly inhibits HDAC activity in vitro and in cultured cells.
(a) LC directly inhibits HDAC activity in vitro. HepG2 cell lysates (1, 5, 30 µg protein) were treated with various doses of LC for 1 h, HDACI/II activity was detected. HDAC inhibitors Buty and TSA were used as positive controls. *P<0.05, versus vehicle control. (b, c) LC inhibits HDAC activites in cultured cells. HepG2 and SMMC-7721 cancer cells were treated with various doses of LC for either 6 h or 12 h, HDACI/II activities were detected. Buty and TSA were used as positive controls. *P<0.05, versus vehcile control. (d) Acetyl-LC inhibits HDAC1/2 activities in vitro and in cultured cells. HepG2 cells and cell lysates were exposed to various doses of Acetyl-LC for 6 h and 1 h respectively, HDACI/II activities were detected. Cell-: in cultured cells. (e, f) LC directly increases histone acetylation and lysine-acetylated protein accumulation in vitro. HepG2 cell lysates were treated with LC for 1.5 h, Acetyl-H3, –H4 and lysine-acetylated proteins were detected by Western Blot. TSA (1 µM) was used as o pisitive control. (g, h) As in (e, f), mouse thymocyte lysates were used instead of HepG2 cell lysates. Buty (1 mM) was used as a control.
Figure 7.
LC treatment induces accumulation of acetylated histones in chromatin associated with p21cip1 gene but not p27kip1 gene.
(a, b) HepG2 cells were treated with LC (10 mM) and Buty (1 mM) for 12 h; cells were collected for CHIP assay as described in the Materials and Methods part. The PCR data and fold enrichment of p21cip1 and p27kip1 promoter gene in LC- or Buty-treated versus vehicle control were shown in (a) and (b) respectively. IP: immunoprecipitation.; Neg: negative.