AIL for 48 hours with or without the 1 ng/mL DR5/Fc chimera, or the 20 M zVAD-fmk pan-caspase inhibitor. Sub-G1 populations were analyzed by flow cytometry. PANC-1 cells were MedChemExpress Aglafoline transfected with 5 nM miR-221 mimic or 5 nM negative control. After 24 hours, the cells were incubated with or without 40 mM metformin and/or 10 ng/mL TRAIL for 48 hours. Sub-G1 populations were analyzed by flow cytometry. Data are the 
means SD of 3 determinations.Metformin Suppresses MiR-221 and Sensitizes TRAIL It has been reported that metformin induced G1-phase arrest with induction of p27 expression as one of the mechanisms in human cancer cells. Similarly, in our results, metformin induced G1-phase arrest, and up-regulated p27 protein expression in human pancreatic cancer PANC-1 cells. It was reported that down-regulation of miR-221 inhibited the growth of pancreatic cancer cells through up-regulation of PTEN, p27, p57, and PUMA. Among these target molecules of miR-221, p27 is a CDK inhibitor which induces G1-phase arrest in cancer cells. Therefore, we hypothesized that metformin could downregulate miR-221 expression, resulting in the induction of p27 with G1-phase arrest in pancreatic cancer cells. In this study, we found that metformin down-regulated the expression of miR-221 in pancreatic cancer cells. Furthermore, both the G1-phase arrest and the induction of p27 by metformin were suppressed by a miR-221 mimic in pancreatic cancer cells. From the results, we show for the first time that metformin-induced G1-phase arrest is at least partially caused by p27 induction through down-regulation of miR221. Meanwhile metformin decreased expressions of cyclin D1 and CDK4 proteins at 10 mM or more, consistent with previous reports. Therefore, the down-regulation of cyclin D1 and CDK4 protein expressions might contribute to G1-phase arrest by metformin at lower doses. It was reported that miR-221, one of the most well-known OncomiRs, was up-regulated in multiple malignancies including pancreatic cancer. Therefore, miR-221 is considered to be an attractive target for selective treatment against cancer. Interestingly, the previous study showed that miR-221 was elevated in the internal mammary arteries of subjects with type 2 diabetes and there was a significant inverse correlation between the oral dose of metformin and the level PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19783827 of miR-221, raising the possibility that our results may be physiological. On the other hand, previous studies reported that miR-221 also contributed to TRAIL resistance in human cancer cells. We therefore hypothesized that metformin may be able to improve the sensitivity of TRAIL via down-regulation of miR-221 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19785045 in pancreatic cancer cells. To confirm this hypothesis, we first examined the effect of metformin on TRAIL sensitivity in human pancreatic cancer cells. In human pancreatic cancer PANC-1 cells, AsPC-1 cells and MIA PaCa-2 cells, metformin enhanced the sensitivity of TRAIL. We next examined whether miR-221 was involved in the enhancement of TRAIL sensitivity by metformin. In the present study, the miR-221 mimic did not suppress the apoptosis induced by the combination of metformin and TRAIL human pancreatic cancer PANC-1 cells. As the possible reason of the difference from previous studies, we speculate that the anti-apoptotic pathway from miR-221 might not exist in human pancreatic cancer cells tested. Therefore, it is suggested that down-regulation of miR-221 by metformin is involved in G1-phase arrest, but not the apoptosis mentioned
