Pancreatic cancer may be the fourth leading cause of cancer related deaths in the United States. these selectively inhibited the proliferation of CD133+ but not CD24+CD44+ESA+ cells. We also examined the effect of low concentrations of metformin on cell invasion and tumor formation demonstrating and anticancer action. Metformin was associated with a reduction of phospho-Erk and phospho-mTOR impartial of Akt SB-715992 and AMPK phosphorylation. CD133+ pancreatic cancer cells are considered to be cancer stem cells that contribute to recurrence SB-715992 metastasis and resistance to adjuvant therapies in pancreatic cancer. Our results provide a basis for combination of metformin with current therapies to improve the prognosis of this disease. Introduction Pancreatic cancer is among the most aggressive of solid malignancies. Each year 43 920 patients are newly diagnosed with the disease resulting in 37 390 deaths per annum in the United States and making pancreatic cancer the fourth leading cause of cancer related death in both males and females . There has been little advance in treatment and SB-715992 the prognosis remains dismal     with a 5 year survival rate of only about 3% and a median survival of less than 6 months. Among patients who undergo potentially curative resection 5 year survival is less than 24% because of local recurrence and metastasis   . Novel therapeutic strategies are therefore urgently needed for this highly malignant disease. Metformin is usually a drug widely used for the treatment of type II diabetes. Recently epidemiologic data revealed that metformin but not other antidiabetic drugs decreases the incidence of pancreatic cancer in patients with diabetes mellitus  . Interestingly there was no correlation between the protective effect and patients’ blood sugar levels . A defensive effect was also observed in a excess fat hamster tumorigenesis model of pancreatic cancer using N-nitrosobis-(2-oxopropyl) amine . Several studies have established a direct action of metformin on many types of cancer cells including those of pancreatic cancer  . Metformin may therefore be a potential therapeutic agent in the treatment of pancreatic cancer though its mechanism of anticancer action is ambiguous. experiments have revealed a dose SB-715992 dependent effect of metformin on cancer cell proliferation. The typically used concentrations in such studies are 5-30 mM which are much higher than the plasma and tissue concentrations measured in individuals who have received recommended therapeutic doses and less than 1 mM of metformin has little effect on cancer cell proliferation  . Here we show that low concentrations of metformin have effects on different subpopulations of pancreatic cancer cells according to their differential expression of surface markers. CD133+ and CD24+CD44+ESA+ cells are considered pancreatic cancer stem cells and the proliferation of CD133+ but not CD24+CD44+ESA+ cells was selectively inhibited by low concentrations of metformin. Metformin was associated with reductions of phospho-Erk and phospho-mTOR impartial of Akt and AMPK phosphorylation. Although low concentration metformin had no effect on the proliferative capacity of pancreatic SB-715992 cancer cells in general their invasive capacities and pancreatic cancer xenograft growth were significantly inhibited. Materials and Methods Cell culture We obtained AsPC-1 and SW1990 cells from the American Type Culture Collection. AsPC-1 pancreatic adenocarcinoma cells were derived from the ascites of a 62-year-old Caucasian female patient with pancreatic adenocarcinoma; SW1990 pancreatic adenocarcinoma cells were derived from metastasis in the spleen of a 56-year-old Caucasian male patient with pancreatic adenocarcinoma. Both cell types were produced in Dulbecco’s altered Eagle medium (DMEM) EC-PTP (Invitrogen Carlsbad CA) supplemented 10% fetal bovine serum (FBS) (Gibco Billings MT) and penicillin/streptomycin (Invitrogen) at 37°C with 5% CO2. Flow cytometry For surface marker detection cells were resuspended in 100 SB-715992 μL Hank’s balanced salt answer with 1% FBS (Gibco). For isolation of CD133+ cells for western blot analysis cells were resuspended in 100 μL Hank’s balanced salt answer with 1% FBS. Fc.