Supplementary Material for: Peperomin E Induces Promoter Hypomethylation of Metastatic-Suppressor Genes and Attenuates Metastasis in Poorly Differentiated Gastric Cancer

<b><i>Background/Aims:</i></b> Peperomin E (PepE), a natural secolignan isolated from the whole plant of <i>Peperomia dindygulensis</i>, has been reported by ourselves and others to display potent anti-cancer effects in many types cancer cells, especially gastric cancer. However, the effects of PepE on the metastasis of poorly-differentiated gastric cancer cells and the underlying molecular mechanisms have not been well elucidated. <b><i>Methods:</i></b> We evaluated PepE effects on gastric cancer cell invasion and migration <i>in vitro</i> via wound healing and transwell assays and those on growth and metastasis <i>in vivo</i> using an orthotopic xenograft NOD-SCID mouse model. DNA methyltransferase (DNMT) activity was determined using a colorimetric DNMT activity/inhibition assay kit. PepE binding kinetics to DNMTs were determined using the bio-layer interferometry binding assay. Gene and protein levels of DNMTs, AMPKα-Sp1 signaling molecules, and metastatic-suppressor genes in PepE-treated gastric cancer cells were determined using quantitative reverse transcription-PCR arrays and western blotting. The effect of PepE on Sp1 binding to the <i>DNMT</i> promoter was determined by electrophoretic mobility-shift assay. Global DNA methylation levels were determined using liquid chromatography coupled with electrospray ionization tandem mass spectrometry. The methylation status of silenced metastatic-suppressor genes (MSGs) in gastric cancer cells was investigated by methylation-specific PCR. <b><i>Results:</i></b> PepE can dose-dependently suppress invasion and migration of poorly-differentiated gastric cancer cells <i>in vitro</i> and <i>in vivo</i> with low toxicity against normal cells. Mechanistically, PepE not only covalently binds to the catalytic domain of DNMT1 and inhibits its activity (IC₅₀ value 3.61 μM) but also down-regulates DNMT1, 3a, and 3b mRNA and protein expression in in gastric cancer cells, by disruption of the physical interaction of Sp1 with the <i>DNMT1, 3a</i>, and <i>3b</i> promoter and mediation of the AMPKα-Sp1 signaling pathway. The dual inhibition activity of PepE toward DNMTs renders a relative global DNA hypomethylation, which induces MSG promoter hypomethylation (e.g., E-cadherin and TIMP3) and enhances their expression in gastric cancer cells. <b><i>Conclusion:</i></b> Collectively, our data indicated that PepE may represent a promising therapeutic lead compound for intervention in gastric cancer metastasis and may also exhibit potential as a DNA methylation inhibitor for use in epigenetic cancer therapy.