Gemcitabine has limited clinical benefits for pancreatic ductal adenocarcinoma (PDAC). The phosphatidylinositol-3-kinase (PI3K)/AKT and mammalian target of rapamycin (mTOR) signaling pathways are frequently dysregulated in PDAC. We investigated the effects of NVP-BEZ235, a novel dual PI3K/mTOR inhibitor, in combination with gemcitabine and endothelial monocyte activating polypeptide II (EMAP) in experimental PDAC. Cell proliferation and protein expression were analyzed by WST-1 assay and Western blotting. Animal survival experiments were performed in murine xenografts. BEZ235 caused a decrease in phospho-AKT and phospho-mTOR expression in PDAC (AsPC-1), endothelial (HUVECs), and fibroblast (WI-38) cells. BEZ235 inhibited in vitro proliferation of all four PDAC cell lines tested. Additive effects on proliferation inhibition were observed in the BEZ235-gemcitabine combination in PDAC cells and in combination of BEZ235 or EMAP with gemcitabine in HUVECs and WI-38 cells. BEZ235, alone or in combination with gemcitabine and EMAP, induced apoptosis in AsPC-1, HUVECs, and WI-38 cells as observed by increased expression of cleaved poly (ADP-ribose) polymerase-1 (PARP-1) and caspase-3 proteins. Compared to controls (median survival: 16 days), animal survival increased after BEZ235 and EMAP therapy alone (both 21 days) and gemcitabine monotherapy (28 days). Further increases in survival occurred in combination therapy groups BEZ235 + gemcitabine (30 days, P = 0.007), BEZ235 + EMAP (27 days, P = 0.02), gemcitabine + EMAP (31 days, P = 0.001), and BEZ235 + gemcitabine + EMAP (33 days, P = 0.004). BEZ235 has experimental PDAC antitumor activity in vitro and in vivo that is further enhanced by combination of gemcitabine and EMAP. These findings demonstrate advantages of combination therapy strategies targeting multiple pathways in pancreatic cancer treatment.
243 Background: The phosphatidylinositol-3-kinase (PI3K)/AKT and mammalian target of rapamycin (mTOR) signaling pathway dysregulation is a prominent feature of pancreatic ductal adenocarcinoma (PDAC). Gemcitabine (GEM), a standard systemic treatment for PDAC, has limited clinical benefits. The present study investigated the effects of NVP-BEZ235 (BEZ235), a novel dual PI3K/mTOR inhibitor, in combination with gemcitabine and endothelial monocyte activating polypeptide II (EMAP) in experimental PDAC. Methods: Protein expression and cell proliferation were analyzed by Western blotting and WST-1 assay. Animal experiments were performed in murine xenografts. Results: BEZ235 inhibited phospho-AKT (Ser473) and phospho-mTOR (Ser2448) expression in PDAC (AsPC-1), endothelial (HUVECs) and fibroblast (WI-38) cells. NVP-BEZ235 also caused a significant dephosphorylation of downstream mTORC1 target proteins phospho-p70 S6K (Thr389) and phospho-4E-BP1 (Thr37/46). In vitro 72-hour proliferation of four PDAC cell lines was significantly inhibited by BEZ235. Additive effects on proliferation inhibition were observed in the BEZ235 and GEM combination in PDAC cells and in combination of BEZ235 or EMAP with gemcitabine in HUVECs and WI-38 cells. BEZ235, alone or in combination with GEM and EMAP, induced apoptosis in AsPC-1, HUVECs and WI-38 cells as observed by increased expression of cleaved poly (ADP-ribose) polymerase-1 (PARP-1) and caspase-3 proteins. PDAC in vivo therapy demonstrated that compared to controls (median survival: 16 days), animal survival increased after BEZ235 and EMAP therapy alone (both 21 days) and GEM monotherapy (28 days). Further increases in survival occurred in combination therapy groups BEZ235+GEM (30 days, p=0.007), BEZ235+EMAP (27 days, p=0.02), GEM+EMAP (31 days, p=0.001) and BEZ235+GEM +EMAP (33 days, p=0.004). Conclusions: BEZ235 has experimental PDAC antitumor activity in vitro and in vivo that can be enhanced in combination with cytotoxic (GEM) and antiendothelial (EMAP) agents. These findings demonstrate advantages of combination therapy strategies targeting multiple pathways in pancreatic cancer treatment.
294 Background: Gemcitabine (Gem), the most active drug for locally advanced, non-operable and metastatic PDAC, has limited benefits as single agent or in combination. Endothelial monocyte activating polypeptide II (EMAP, E) enhances Gem effects in PDAC. We evaluated the antitumor activities of EMAP in combination with doxorubicin (Dox) or docetaxel (DT) in PDAC. Methods: In vitro cell proliferation, protein expression and apoptosis were analyzed by WST-1 assay, Western blotting and FACS analysis. In vivo local tumor growth and animal survival experiments were performed in murine xenografts. Results: In vitro PDAC cell proliferation was not affected by EMAP, compared to a small inhibition by Dox, DT and Gem. EMAP combination to these agents did not increase the antiproliferative effects. In endothelial cells (ECs), EMAP, Dox, DT and Gem all inhibited proliferation (59, 79, 96 and 85% at 10 μM, respectively); addition of EMAP caused additive antiproliferative effects. In PDAC cells, no agent caused measurable apoptosis, but in ECs all agents either alone or in combination increased the apoptosis. In vivo, Dox, DT, Gem and EMAP all decreased local tumor growth, and addition of EMAP enhanced inhibitory effects of DT and Gem, but not of Dox (92, 63, 60, 42, 73, 85 and 68 % inhibition after Dox, DT, Gem, E, Dox+E, DT+E and Gem+E, respectively); DT followed by Gem led to 72% inhibition without EMAP, and to 99% with EMAP (p=0.001). Inhibition of intra-tumoral proliferative activity and increase of apoptotic index were enhanced in all EMAP combination groups. Compared to controls (median survival: 21 days), EMAP (20 d) had no, but Dox (31 d) and DT (35 d) had extended survival benefit. EMAP enhanced the DT effect (44 d, p=0.009) but not that of Dox (31 d, p=0.04). In a sequential therapy experiment, median survival after controls, Gem, DT, Gem followed by DT, DT followed by Gem, Gem+E, DT+E, Gem/DT+E and DT/Gem+E was 17, 25, 29, 39, 39, 28, 35, 34 and 41 days, respectively. Conclusions: The antiendothelial agent EMAP enhances antitumor effects of not just gemcitabine. Therefore, combination approaches with EMAP-like agents could render other drugs such as taxanes or their doublets sufficiently effective for clinical applications in PDAC therapy. No significant financial relationships to disclose.
255 Background: Gemcitabine (Gem, G) has limited benefits as single agent or in combination for pancreatic ductal adenocarcinomas (PDACs). The phosphatidylinositol-3-kinase (PI3K)/AKT and mammalian target of rapamycin (mTOR) signaling pathways are frequently dysregulated in diverse human cancers including PDAC. NVP-BEZ235 (BEZ, B) is a novel dual PI3K/mTOR inhibitor that has been shown to have antitumor activity in multiple tumor types. Endothelial monocyte activating polypeptide II (EMAP, E) is an antiendothelial and antiangiogenic agent that enhances Gem and docetaxel activity in PDAC. We tested the combination benefits of BEZ and Gem in addition to EMAP in experimental PDAC. Methods: In vitro cell proliferation and protein expression were analyzed by WST-1 assay and Western blotting. In vivo animal survival experiments were performed in NOD-SCID PDAC xenografts. Results: Cultured cells of PDAC (AsPC-1), endothelial (HUVECs), and fibroblast origin (WI-38) all expressed AKT and mTOR protein. BEZ inhibited in vitro cell proliferation of AsPC-1 and HUVECs cells, with some additive effects in combination with Gem or EMAP, after 72 hours of incubation. In AsPC-1, treatment of BEZ (100 nM), Gem (100 nM) and EMAP (1 μM) caused 34, -7, -16, 62, 51, 3, and 59 percent inhibition in proliferation in the B, G, E, B+G, B+E, G+E and B+G+E groups. In HUVECs, percent inhibition in proliferation was 35, 33, 15, 55, 35, 31 and 53 in the B, G, E, B+G, B+E, G+E and B+G+E groups, respectively. Compared to controls (median survival: 16 days), an animal survival increase after BEZ and EMAP therapy alone (both 21 days) and Gem therapy alone (28 d) was observed. Further increases in survival occurred in combination therapy groups B+G (30 d, p=0.007), B+E (27 d, p=0.02), G+E (31 d, p=0.001) and B+G+E (33 d, p=0.004). Conclusions: Bez has experimental PDAC antitumor activity in vitro and in vivo that is further enhanced by combination of Gem and EMAP. These findings demonstrate advantages of combination therapy strategies targeting multiple pathways in pancreatic cancer treatment. No significant financial relationships to disclose.
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