Objectives To further elucidate anti-cancer mechanisms of metformin again pancreatic cancer we evaluated inhibitory effects of metformin on pancreatic tumorigenesis in a genetically-engineered mouse model and investigated its possible anti-inflammatory and anti-angiogenesis effects. of genetic events driving pancreatic carcinogenesis we have developed a genetically engineered Slit2 LSL-and transgene constructs. The following PCR conditions were used for and = 0.52 (length × width × depth) and the tumor Alvimopan (ADL 8-2698) burden was calculated as tumor weight (mg) / body weight (mg) × 100. The mRNA and protein expression for the target genes were first quantitated relative to the expression Alvimopan (ADL 8-2698) of the Alvimopan (ADL 8-2698) housekeeping gene β-actin and then normalized to the background expression in saline-treated mice (Control). The differences among the three groups were tested using Kruskal-Wallis test. When the Kruskal-Wallis test was significant pairwise comparisons were assessed with the Wilcoxon rank-sum test for each pair of groups; all comparisons are reported for the reader to interpret. All statistical analyses were done with SAS 9.2 software and two sided values ≤ 0.05 were considered significant. Results Activation of KRASG12D and knocking out Trp53F2-10 at mouse pancreas We have developed a unique method of enabling an investigator-generated invasive and undifferentiated form of pancreatic cancer in a mouse model as described originally by Hingorani mutations in human pancreatic cancer 30 in progenitor cells of the mouse pancreas. We found that physiological expression of and alleles in progenitor cells of the developing mouse pancreas. These and mutations. The mice develop one highly aggressive undifferentiated pancreatic cancer at the place where the adenoviral Cre was injected in approximately three weeks and liver metastases are observed within four weeks (data not shown). The median survival of these mice is two months. A total of 30 mice were randomly divided into three groups (Figure 1and 2and Supplementary Table 1). Liver metastases were observed in all groups (Figures 3by suppressing NFκB activation via AMPK activation32. Non-phosphorylated STAT3 has been shown to play important roles in cellular function including binding to NFκB to mediate its nuclear import33. We examined the effect of metformin Alvimopan (ADL 8-2698) on NFκB and STAT3 activation by looking for changes in the level of total protein as well as changes in their phosphorylation levels. We observed that one-week pretreatment of metformin significantly reduced phospho-NFκB at the serine phosphorylation site and phospho-STAT3 at the tyrosine phosphorylation site but total protein levels were unchanged (Figure 4and STAT3 in pancreatic tumors Figure 5 Immunohistochemistry staining for phospho-NFκB phospho-STAT3 Sp1 and VEGFα in Control Met_1wk and Met_3wk groups Effects of metformin on anti-inflammation NFκB a master transcriptional gene has been known to activate downstream inflammatory mediators such as TGF-β1 TNF-α and IL-1β.38-40 In addition activated NFκB shows an important role in the up-regulation of MCP-1 which is a potent chemokine involved in the accumulation and function of macrophages.40-42 We investigated the effects of metformin on the mRNA expression of these downstream regulatory genes of NFκB signaling pathway in mouse pancreatic tissue. Metformin treatment significantly reduced mRNA Alvimopan (ADL 8-2698) expression of TNF-α (up to 65% < 0.01) TGF-β1 (up to 70% < 0.05) MCP-1 (up to 77% < 0.01) and IL-1β (up to 80% < 0.01) compared to the untreated Alvimopan (ADL 8-2698) control samples (Figure 6). Figure 6 Metformin decreased mRNA expression of the downstream inflammatory mediators in pancreatic tumors Effects of metformin on anti-angiogenesis It has previously been demonstrated that AMPK activation can contribute to increased VEGF expression43 44 and angiogenesis.45 46 VEGF is a well-established stimulator of vascular permeability and angiogenesis whereas TSP-1 originally isolated from platelets and megakaryocytes is a potential angiogenic inhibitor.47 PAI-1 expression is positively correlated with TSP-1 and can either enhance or inhibit angiogenesis depending upon its concentration.48 The IHC staining showed that the protein level of VEGFα was not significantly changed among the three groups (Figure 5). We also determined the mRNA expression of these three important.