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KRAS mutation is associated with the progression and growth of pancreatic cancer and contributes to chemo-resistance, which poses a significant clinical challenge in pancreatic cancer. Here, we developed a RT22-ep59 antibody (Ab) that directly targets the intracellularly activated GTP-bound form of oncogenic KRAS mutants after it is internalized into cytosol by endocytosis through tumor-associated receptor of extracellular epithelial cell adhesion molecule (EpCAM) and investigated its synergistic anticancer effects in the presence of gemcitabine in pancreatic cancer. We first observed that RT22-ep59 specifically recognized tumor-associated EpCAM and reached the cytosol by endosomal escape. In addition, the anticancer effect of RT22-ep59 was observed in the high-EpCAM-expressing pancreatic cancer cells and gemcitabine-resistant pancreatic cancer cells, but it had little effect on the low-EpCAM-expressing pancreatic cancer cells. Additionally, co-treatment with RT22-ep59 and gemcitabine synergistically inhibited cell viability, migration, and invasion in 3D-cultures and exhibited synergistic anticancer activity by inhibiting the RAF/ERK or PI3K/AKT pathways in cells with high-EpCAM expression. In an orthotopic mouse model, combined administration of RT22-ep59 and gemcitabine significantly inhibited tumor growth. Furthermore, the co-treatment suppressed cancer metastasis by blocking EMT signaling in vitro and in vivo. Our results demonstrated that RT22-ep59 synergistically increased the antitumor activity of gemcitabine by inhibiting RAS signaling by specifically targeting KRAS. This indicates that co-treatment with RT22-ep59 and gemcitabine might be considered a potential therapeutic strategy for pancreatic cancer patients harboring KRAS mutation. [ABSTRACT FROM AUTHOR]

Deregulation of the phosphatidylinositol 3-kinase (PI-3K)/PDK-l/Akt signaling cascade is associated with pancreatic cancer tumor invasion, angiogenesis, and tumor progression. As such, it has been postulated that PDK-1/Akt signaling inhibitors may hold promise as novel therapeutic agents for pancreatic cancer. Disadvantages of currently available Akt inhibitors include tumor resistance, poor specificity, potential toxicity, and poor bioavailability. Previous studies have demonstrated that OSU-03012, a celecoxib derivative, specifically inhibits PDK-1 mediated phosphorylation of Akt with IC50 values in the low μM range. Human pancreatic cancer cell lines AsPC-1, BxPC-3, Mia-PaCa 2, and PANC-1 were cultured in media containing varying concentrations of OSU-03012, 5-fluorouracil (5-FU), and gemcitabine, and changes in Akt phosphorylation and cell viability were evaluated using western blotting and a 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay, respectively. Treatment with OSU-03012 resulted in decreased PDK-1-mediated Akt phosphorylation and cell growth inhibition for all cell lines with IC50 values ranging between 1.0 and 2.5 μM. Resistance to 5-FU and gemcitabine was observed in cell lines AsPC-1 and BxPC-3. Further analyses indicate that OSU-03012 induces both proapoptotic and antiproliferative effects in these cells. Taken together, these data suggest that OSU-03012 has potential value as a novel therapy for pancreatic cancer. [Copyright &y& Elsevier]

Pancreatic cancer remains a highly chemoresistant malignancy. Gemcitabine, the most effective first-line agent available, acts by disrupting cellular replication. Caspases belong to a family of proteases that function as key components of the apoptotic death machinery. We investigated the mechanisms by which gemcitabine blocks proliferation and whether it can induce apoptosis in pancreatic cancer cells. Quiescent pancreatic cancer cells (BxPC-3) were stimulated to proliferate (10% fetal calf serum) with or without gemcitabine, PS-341 (26S proteasome inhibitor), or both. Proliferation was measured by MTT assay and apoptosis by propidium iodine staining. To determine activation of the apoptotic regulatory cell proteins, caspase-3 and cleavage of poly(ADP-ribose)polymerase (PARP) into its 85-kDa fragment were assessed by Western blotting. Gemcitabine at even low doses (10 μmol/L) significantly inhibited cellular proliferation, whereas PS-341 (10 nmol/L) had no effect. With combined treatment, PS-341 potentiated the antiproliferative effects of gemcitabine (P=0.001). At 48 hours, the apoptotic fraction was greatly enhanced by the presence of PS-341 compared with gemcitabine alone. Caspase-3 accumulated as early as 30 minutes and was associated with cleavage of PARP to its apoptotic fragment. Gemcitabine, a nucleoside analogue, may in part exert its antiproliferative effects by directing pancreatic cancer cells to a default pathway of apoptosis. 26S proteasome inhibition potentiates this effect, suggesting its potential clinical value against chemoresistance in pancreatic cancer. [Copyright &y& Elsevier]