Induction of GNMT by 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranoside through proteasome-independent MYC downregulation in hepatocellular carcinoma

Abstract

Glycine-N-methyl transferase (GNMT) a tumor suppressor for hepatocellular carcinoma (HCC) plays a crucial role in liver homeostasis. Its expression is downregulated in almost all the tumor tissues of HCC while the mechanism of this downregulation is not yet fully understood. Recently, we identified 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranoside (PGG) as a GNMT promoter enhancer compound in HCC. In this study, we aimed to delineate the mechanism by which PGG enhances GNMT expression and to investigate its effect on GNMT suppression in HCC. Microarray and pathway enrichment analysis revealed that MYC was a major target of PGG. PGG suppressed MYC mRNA and protein expression in Huh7 and Hep G2 cells in a dose- and time-dependent fashion. Furthermore, MYC expression was also reduced in xenograft tumors in PGG treated mice. Moreover, shRNA-mediated knocked-down or pharmacological inhibition of MYC resulted in a significant induction of GNMT promoter activity and endogenous GNMT mRNA expression in Huh7 cells. In contrast, overexpression of MYC significantly inhibited GNMT promoter activity and endogenous GNMT protein expression. In addition, antibodies against MYC effectively precipitated the human GNMT promoter in a chromatin immunoprecipitation assay. Lastly, GNMT expression was negatively correlated with MYC expression in human HCC samples. Interestingly, PGG not only inhibited MYC gene expression but also promoted MYC protein degradation through proteasome-independent pathways. This work reveals a novel anticancer mechanism of PGG via downregulation of MYC expression and establishes a therapeutic rationale for treatment of MYC overexpressing cancers using PGG. Our data also provide a novel mechanistic understanding of GNMT regulation through MYC in the pathogenesis of HCC.

Figure 1

mRNA expression profiling reveals that MYC is target of PGG. (a) Genes that were affected by PGG for 1.5-fold (both upregulated and downregulated) were considered as the differentially expressed genes (DEGs). Venn diagram showed that 168 DEGs were persistently affected by PGG from 6 to 48 hours of treatment. (b) Pathways and genes identified by DAVID Functional Clustering analysis of the 168 DEGs were shown. Bold red letter indicated MYC is involved in all pathways. (c) MYC mRNA expression in PGG (0.1 mg/mL) treated Huh7 and HepG2 cells were determined by qRT-PCR after 24 hours. Data presented as fold to solvent control. The graph shows the means ± SD (n = 3). (d,e) Immunoblot assay of MYC protein in indicated cells treated with PGG for 24 hours at indicated concentrations (d) and treated with PGG (0.1 mg/mL) for indicated time points (e). β-actin expression was used as loading control. (f) Alterations in the mRNA levels of MYC target genes in Huh7 cells 24 hours after PGG (0.1 mg/mL) treatment were detected by qRT-PCR. Data presented as fold to solvent control. The graph shows the means ± SD (n = 3). (g,h) MYC mRNA (g) and protein (h) expression in Huh7 xenograft tumor tissues (samples described in previous study) were determined by qRT-PCR and immunoblot assay (n ≥ 4 mice from each group). β-actin expression was used as loading control. Each lane of immunoblot represented the protein sample extracted from Huh7 xenograft tumor of mice in the vehicle-treated group and PGG treated group. Right panel shows quantification of Myc signal intensities in left panel. The graph shows the means ± SEM. ***P < 0.001, *P < 0.05 (Student’s t-test).

Figure 2

MYC inhibits GNMT expression. (a) Huh7 cells were co-transfected with the GNMT promoter reporter, TK renilla reporter and pcDNA-MYC or control vector plasmid for 72 hours, then harvested for luciferase assay. Luciferase activity were measured and normalized to renilla reporter activity. Data were represented as fold to vector control. The graph shows the means ± SD (n = 3). (b) Huh7 cells were transfected with the pcDNA-MYC or control vector plasmid. Cells were harvested after 72 hours of transfection and relative level of MYC and GNMT mRNA were determined by qRT-PCR. Data normalized to internal control and presented as fold to vector control. The graph shows the means ± SD (n = 3). (c) Huh7 cells were co-transfected with the GNMT promoter reporter, TK renilla reporter and shMYC or shLacZ plasmids for 72 hours, then harvested for luciferase assay. Data were represented as in (a). The graph shows the means ± SD (n = 3). (d) Relative GNMT and MYC mRNA level in Huh7-shMYC and Huh7-shLacZ stable cells were determined by qRT-PCR. Data were represented as fold to Huh7-shLacZ control group. The graph shows the means ± SD (n = 3). (e) Effect of indicated concentrations of JQ1 on GNMT promoter expression in H7GPL cells after 48 hours of treatment. Relative luciferase activity Relative luciferase activity was calculated by normalizing luciferase activity to cell viability and presented as fold to control. Results are means ± SD (n = 3). (f) Effect of indicated concentrations of JQ1 on GNMT mRNA (upper panel) expression in Huh7 cells after 48 hours of treatment. Lower panel shows the MYC protein expression after JQ1 treatment. Results are means ± SD (n = 3). (g) The mRNA levels of MYC and GNMT in the human HCC tumor samples were determined by qRT-PCR (n = 60). (h) TCGA data set (n = 373) were assessed for MYC and GNMT correlation using the Pearson’s correlation analysis. (i,j) The mRNA expression of GNMT (i) and Myc (j) in the liver tissues from mice challenged with AFB1 were determined by qRT-PCR. Results are means ± SD. ***P < 0.001, **P < 0.01; *P < 0.05 (Student’s t-test).

Figure 3

MYC interacts with the promoter of GNMT. (a) Schematic representation of the GNMT promoter-driven luciferase reporter constructs (1.8Kb-Luc; −1812/+14) and two 5′-deletion mutant promoter constructs (1.4Kb-Luc; −1367/+14, deleted of E-box and 147b-Luc; −133/+14, the core promoter). The indicated constructs are described previously. (b) Huh7 cells transiently co-transfected with the reporter plasmids containing the 5′-flanking region of human GNMT promoter described in (a) with TK renilla reporter and pcDNA-MYC or control vector plasmids. Luciferase activity was measured 72 hours post-transfection and normalized to renilla reporter activity. Data were represented as fold to vector control. The graph shows the means ± SD (n = 3). (c) Huh7 cells transiently co-transfected with the reporter plasmids containing the 5′-flanking region of human GNMT promoter described in (a) with TK renilla reporter for 72 hours and treated with PGG or PBS solvent control. Luciferase activity was measured 24 hours after treatment and normalized to renilla reporter activity. Data were represented as fold to solvent control. The graph shows the means ± SD (n = 3). (d) Huh7 cells were co-transfected with the GNMT promoter reporter, SP1 mutated GNMT promoter reporter (described previously), TK renilla reporter and pcDNA-MYC or control vector plasmid for 72 hours, then harvested for luciferase assay. Data were measured and represented as described above. The graph shows the means ± SD (n = 3). (e) ChIP-qPCR analysis was performed in Huh7 cells. Enrichment of the GNMT promoter region (containing the core promoter −133/+14) and CCND1 promoter region (containing MYC response element) were calculated by qPCR quantification normalized to input. Results are means ± SD (n = 3). **P < 0.01; *P < 0.05 (Student’s t-test).

Figure 4

PGG affects GNMT promoter activity through inhibition of MYC expression. (a) Huh7 cells were co-transfected with the GNMT promoter reporter, TK renilla reporter and shMYC or shLacZ plasmids for 72 hours. Cells were treated with PGG (0.1 mg/mL) or solvent for 24 hours and harvested for luciferase assay and immunoblot analysis. Luciferase activity were measured and normalized to renilla reporter activity. Data were represented as fold to solvent control. The graph shows the means ± SD (n = 3). (b) Protein level of MYC in above mentioned cells were measured by Immunoblot assay. β-actin expression was used as loading control. (c) Huh7 cells were co-transfected with the GNMT promoter reporter, TK renilla reporter and pcDNA-MYC or control vector plasmid for 72 hours. Cells were treated with PGG (0.1 mg/mL) or solvent for 24 hours and harvested for luciferase assay. Luciferase activity were measured and normalized to renilla reporter activity. Data were represented as fold to solvent control. The graph shows the means ± SD (n = 3) (d) MYC mRNA expression in above mentioned cells were determined by qRT-PCR. Data normalized to internal control and presented as fold to vector control. The graph shows the means ± SD (n = 3). (e) Protein level of MYC in above mentioned cells were measured by Immunoblot assay. β-actin expression was used as loading control. ***P < 0.001, **P < 0.05 (Student’s t-test).

Figure 5

PGG induced decreases of MYC through proteasome independent degradation in Huh7 cells. (a) Huh7 cells were treated with PGG (0.1 mg/mL) for 3 hours and then co-exposed to cycloheximide (CHX 50 ug/ml) for indicated time intervals and harvested for immunoblot analysis for MYC expression. β-actin expression was used as loading control. (b) Graph shows quantification of MYC signal intensities in (a). (c) MYC overexpressed Huh7 cells were pre-incubated with indicated compounds for 1 hour and then co-treated with PGG for 24 hours. MYC protein level was determined by immunoblotting. β-actin expression was used as loading control. (d) Effect of indicated compounds (PGG 0.1 mg/mL, chloroquine (CQ) 100 μM) on GNMT promoter expression in H7GPL cells after 24 hours of treatment. Relative luciferase activity was calculated by normalizing luciferase activity to cell viability and presented as fold to control. Results are means ± SD (n = 3). (e) MYC overexpressed and control-Huh7 cells were treated with PGG (0.1 mg/mL) or solvent for 72 hours. Cell viability was determined by alamarBlue® viability assay. Results are means ± SD (n = 3). (f) Huh7-shLacZ and Huh7-shMYC stable cells were treated with various concentrations of PGG for 72 hours. Cell viability were determined by using MultiTox-Glo Multiplex Cytotoxicity Assay kit. The percentages of viable cells compared with the cells without PGG treatment are plotted. Results are means ± SD (n = 3). ***P < 0.001 (Student’s t-test).

Figure 6

The proposed molecular mechanisms of GNMT induction of PGG. The model shows that PGG inhibiting MYC mRNA and inducing MYC proteolysis and therefore inducing GNMT expression through MYC inhibition. The red arrows denote suppression whereas the blue arrow denotes activation.