These studies clearly underscore the importance of P2X7 receptor/inflammasome in PDAC progression and suggested that P27XR would be a potential target in pancreatic cancer prevention. Different P2X7R antagonists have been used extensively in animal models of inflammation and pain, and some of them are under extensive clinical trials. fed modified AIN-76A diets containing 0, 50 or 100 ppm A438079 Rabbit Polyclonal to p14 ARF and AZ10606120 for 38 weeks. Pancreata were collected, weighed, and evaluated for PanINs and PDAC. Control diet-fed male mice showed 50% PDAC incidence. Dietary A438079 and AZ10606120 showed 60% PDAC Caftaric acid incidence. A marginal increase of PanIN 3 (carcinoma and studies support the pro tumorigenic role of P2X7 purinoceptor gene in various cancers, including human pancreatic cancer [11, 15]. To further understand the role of P2X7R and the inflammasome in pancreatic tumor progression, we carried out transcriptomic analysis of LSL-Kras pancreatic tumors by next generation sequencing. Our results show that P2X7R (~20-fold) (Figure ?(Figure1A),1A), its key inflammasome components: caspase-1 (15-fold) (Figure ?(Figure1B),1B), IL-1 (~45-fold) (Figure ?(Figure1C),1C), and in addition to (data not shown) IL-18 (~35-fold), IL-33 (~93 folds), TNF- (~13-fold) and COX-2 (~41-fold) are increased in pancreatic tumors compared to normal pancreas. Further analyses of mouse PC tissues by immunohistochemistry and/or immunofluorescence (Figure ?(Figure1D)1D) suggest that P2X7R is a critical contributor to the progression of pancreatic tumor growth through inflammatory signaling (Figure ?(Figure1E1E). Open in a separate window Figure 1 Expression of P2X7R and inflammasome markers in pancreatic tumors(ACC) NGS analysis showing mRNA overexpression of P2X7R (A), caspase-1 (B) and IL-1 in the pancreatic tumors from genetically engineered mice compared to normal pancreas from wild type mice. (D) IHC analysis of P2X7R expression in normal pancreas (upper left panel) and pancreatic tumor (upper right panel), IHF analysis of P2X7R expression Caftaric acid in normal pancreas (upper left panel) and pancreatic tumor (upper right panel). (E) Schematic representation of P2X7R-NLRP-caspase-IL1 inflammasome cascade. Significant overexpression of P2X7R, caspase-1, IL-1 mRNA and P2X7R protein expressions were seen in the pancreatic tumors compared to normal pancreas. Synthesis of A438079 and Caftaric acid AZ10606120 We synthesized P2X7R inhibitors A438079 and AZ10606120 for the MTD and chemoprevention efficacy studies from the procedures described in previous publications and the patent application filed by Jones, was marginally increased in both drug-treated groups (Tables ?(Tables11 and ?and2).2). Pancreas of male GEM fed AIN76 A diet showed a 24.3 3.4 % (Table ?(Table1)1) incidence of PDAC within the pancreas, while in female mice it was a 25.6 3.4 % (Table ?(Table2).2). The carcinoma percentage within the pancreas was significantly increased (up to 2-fold in males; Table ?Table1)1) by both drugs in GEM. Female GEM treated with higher dose of A438079 and lower dose of AZ10606120 showed reduced carcinoma (Table ?(Table2).2). Although higher dose of AZ10606120 showed reduced carcinoma, due to early termination this group is not used for comparison (~45% of mice). Modulation of predictive specific signature marker(s) by A438079 and AZ10606120 in pancreatic cancer The pancreatic tumor tissues obtained from efficacy studies were used to determine the predictive signature markers and dose response effects of A438079 and AZ10606120. Signature markers associated with tumor growth using the pancreas from wild type mice and 45-week-old p48Cre/+-LSL-KrasG12D/+ mice were analyzed by transcriptome analysis (Figure ?(Figure1).1). Furthermore, we completed relevant biomarker analyses of the pancreatic tumor tissues from lower dose untreated and treated male mice to compare the effects of P2X7R inhibitors on tumor growth and their responses on signature markers in comparison to untreated mouse tumors by real-time PCR analysis and immunohistochemistry (Figures ?(Figures4,4, ?,5,5, ?,6,6, ?,7).7). Dietary A438079 significantly reduced mRNA expressions of P2X7R, IL-33, NLRP3 and p21 while non-significant reduction was seen for caspase-1, caspase-3, NLRP-1, PCNA and p53 in the pancreatic tumor tissues (Figure ?(Figure4).4). Dietary AZ10606120 significantly increased mRNA expressions of NLRP-2 (Figure ?(Figure5).5). A non-significant decrease was seen for caspase-1, caspase-3, and p21 with increase in p53 in the pancreatic tumor Caftaric acid tissues (Figure ?(Figure5).5). A438079 had no effects on mRNA expression of NLRP-6 whereas AZ10606120 did not show significant change in the mRNA expressions of IL-33, NLRP-1, NLRP-6 and p21 (Figures ?(Figures4,4, ?,5).5). Immunohistochemistry results revealed that A438079 Caftaric acid significantly reduced protein expression of P2X7R, CDc25c and caspase-3 while a non-significant decrease was seen for p53, PCNA and COX-2 (Figures ?(Figures6,6, ?,7).7). Immunohistochemistry results revealed that AZ10606120 significantly reduced the protein expression of CDc25c and caspase-3 while a non-significant decrease was seen for P2X7R and COX-2 (Figures ?(Figures6,6, ?,7).7). AZ10606120 had no effects on PCNA but significantly increased p53 (Figures ?(Figures6,6, ?,77). Open in a separate window Figure 4 Biomarker modulation by A438079 in pancreatic tumors(ACJ) Effect of A438079 (50 ppm) on mRNA expression of P2X7R (A), Caspase-1 (B), Caspase-3 (C), IL-33 (D), NLRP1 (E), NLRP2 (F),.