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.
Highlights
Hepatocellular carcinoma (HCC) remains sixth most prevalent and third most common cause of cancer-related deaths in the globe[1,2]
Consistent with in vitro results, MYC mRNA and protein expression were remarkably reduced in Huh[7] xenograft tumors in PGG treated mice (Fig. 1g,h). These results demonstrated that PGG suppresses MYC expression in HCC cells
Based on in vitro results, we further examined whether MYC mRNA expression is associated with Glycine-N-methyl transferase (GNMT) in tumors from HCC patients (n = 60). Quantitative real-time PCR (qRT-PCR) result analysis showed that the expression levels of MYC mRNA in HCC tissues were significantly negatively correlated with GNMT mRNA levels as determined by Pearson’s correlation (Fig. 2g)
Summary
Hepatocellular carcinoma (HCC) remains sixth most prevalent and third most common cause of cancer-related deaths in the globe[1,2]. The reduced expression of GNMT in human HCC cell lines and tumor tissues of HCC patients was first reported by Chen et al in 199812. 3 patients with a missense mutation of GNMT gene in the coding region has been reported and shown to affect catalytic activity[15,16]. Hypermethylation studies showed that 3′ region of the TSS (transcription start site) of GNMT was hypermethylated to some extent in 3 HCC cell lines and seven out of the 35 primary tumors. Demethylating drug treatment did not show significant induction of GNMT mRNA in HCC cells. The precise mechanisms underlying PGG mediated GNMT induction remains elusive. We investigated the GNMT induction mechanism of PGG to improve the understanding of GNMT downregulation in HCC. Our data revealed MYC participation in downregulation of GNMT expression in HCC
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