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Wei Jing,1 Na Song,2,3 Yun-Peng Liu,2,3 Xiu-Juan Qu,2,3 Ya-Fei Qi,4 Ce Li,2,3 Ke-Zuo Hou,2,3 Xiao-Fang Che,2,3 Xiang-Hong Yang41The First Department of Oncology, Shengjing Hospital of China Medical University, Shenyang, People’s Republic of China; 2Department of Medical Oncology, The First Hospital of China Medical University, Shenyang, People’s Republic of China; 3Key Laboratory of Anticancer Drugs and Biotherapy of Liaoning Province, The First Hospital of China Medical University, Shenyang, People’s Republic of China; 4Department of Pathology, Shengjing Hospital of China Medical University, Shenyang, People’s Republic of ChinaBackground: Although aberrant DNA methyltransferase 3a (DNMT3a) expression is important to the tumorigenesis of pancreatic ductal adenocarcinoma (PDAC), the role of DNMT3a in PDAC prognosis is not clarified yet due to the limited studies and lacking of underlying molecular mechanism.Methods: The expression of DNMT3a was examined by immunohistochemistry in PDAC tissues. Gene expression profiles assays were conducted to explore the impact of DNMT3a on biological processes and signal pathways. Cell cycle and apoptosis were measured by flow cytometry. Western blotting and real-time qPCR assays were used to explore the impact of DNMT3a on expression of protein and mRNA related to cell cycle, STAT3 signaling pathway and apoptosis.Results: DNMT3a was overexpressed and closely associated with poor outcomes of PDAC. DNMT3a knockdown restrained PDAC cell proliferation, induced cell cycle arrest and promoted apoptosis in vitro. Affymetrix GeneChip Human Transcriptome Array identified that the cell cycle-related process was most significantly associated with DNMT3a. DNMT3a knockdown induced G1-S phase transition arrest by decreasing the expression of cyclin D1, which was mediated by the reduction of IL8 and the subsequent inactivation of STAT3 signaling pathway. Furthermore, exogenous apoptosis was also promoted after DNMT3a knockdown, probably via up-regulation of DNA transcription and expression in CASP8.Conclusion: These findings indicate that DNMT3a plays an important role in PDAC progression. DNMT3a may serve as a prognostic biomarker and a therapeutic strategy candidate in PDAC.Keywords: DNA methyltransferase 3a, pancreatic ductal adenocarcinoma, prognosis, proliferation

Arsenic trioxide (ATO) is a strong inducer of apoptosis in malignant hematological cells. Inducible phosphatidyl inositol 3 kinase (PI3K)-Akt activation promotes resistance to ATO. In the present study, we evaluated whether E3 ubiquitin ligase Cbl-b, a negative regulator of PI3K activation, is involved in the action of ATO. The effect of ATO on cell viability was measured by the Trypan blue exclusion assay or by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was determined by flow cytometry and protein expression was assayed by Western blotting. ATO decreased the viability of HL60 cells and induced cellular apoptosis, which was accompanied by transient activation of Akt. The PI3K/Akt inhibitor, LY294002, significantly increased ATO-induced apoptosis (P < 0.05). In addition, ATO up-regulated the expression of Cbl-b proteins. Furthermore, ATO inhibited cell viability with an IC50 of 18.54 μM at 24 h in rat basophilic leukemia-2H3 cells. ATO induced cellular apoptosis with transient activation of Akt and Cbl-b was also up-regulated. Rat basophilic leukemia-2H3 cells transfected with a dominant negative (DN) Cbl-b mutation showed overexpression of Cbl-b (DN) and enhanced Akt activation. Compared with cells transfected with vector, ATO-induced apoptosis was decreased and G2/M phase cells were increased at the same concentration (P < 0.05). The PI3K/Akt inhibitor, LY294002, re-sensitized Cbl-b (DN) overexpressing cells to ATO and reversed G2/M arrest (P < 0.05). Taken together, these results suggest that Cbl-b potentiates the apoptotic action of ATO by inhibition of the PI3K/Akt pathway.