Inhibition of DNA methyltransferase by oligonucleotide antisense modifies cell characteristics in gastric cancer cell lines

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DNA (cytosine-5-)-methyltransferase 1 (DNMT1) plays an important role in the maintenance of DNA methylation patterns. Antisense strategies silencing DNMT1 activity was reported to inhibit the tumorigenicity, or induce p16 and p21 expression in cancer cells. In the present study, we show the inhibition of DNMT1 by an antisense oligonucleotide we designed, and we evaluated characteristic alteration of gastric cancer cells by the inhibition of DNMT1, in vitro and in vivo. Antisense oligonucleotide of DNMT1 [AS/MT; 5′-CGGTACGCGCCGGCATCT-3′ complemental sequences with -2 - +16 sequences of DNMT1 mRNA; X63692] and nonsense of DNMT1 (NS/MT; random sequences from AS/MT) were synthesized. Two gastric cancer cell lines MKN-45 and TMK-1 were evaluated. Suppressed protein expression of DNMT1 by AS/MT was confirmed by immunocytochemistry using rabbit polyclonal antibodies for DNMT-1 (Santa Cruz Biotechnology, CA). DNA synthesis was estimated with BrDU uptake incorporation. Western blotting was performed to quantify protein expression of Rb, E-cadherin, c-fos. In vivo experiment was designed using peritoneal dissemination model in nude mouse with TMK-1 cells. In MKN-45 and TMK-1, successful suppression of DNMT1 protein by incubation with 10 uM of AS/MT for 48h was confirmed by immunocytochemistry staining and NS/MT did not alter protein expression of DNMT1. Inhibition of cell growth was induced by AS/MT, dose-dependently, and 10uM of AS/MT for 48h inhibited TMK-1 proliferation up to 82.7% in survival rate. On the other hand, morphological alterations were observed by AS/MT in both cell lines, changing their cell shape from the original ones to dispersed, fibroblast-like cells with neurite-like processus, accompanying with increased adhesive ability. NS/MT did not demonstrate either growth inhibition or morphological change. Western blotting showed that AS/MT increased E-cadherin, and decreased c-fos. In vivo experiment showed increased cancer ability of peritoneal dissemination by AS/MT in nude mouse. Total wet tumor weight of AS/MT group (350 + 47.4 g) showed significant increase compared with the control group (248 + 41.5 g) (p=0.0065). Inhibition of DNMT1 by AS/MT was confirmed, and AS/MT showed growth inhibition and morphological alteration in vitro. In addition, AS/MT treatment increased cancer ability to attach on and invade into peritoneum in mouse in vivo. These findings suggested that general inhibition of DNA methylation may result in altered characteristics of malignant cells.