We found that knockdown of the subunits of AMPK reversed the inhibition of mTORC1 and ERK induced by low doses of berberine. vertical collection). B) A769662 (50 mM) does impact mitochondrial membrane potential (fluorescence percentage) measured with JC-1 or reduces ATP levels. C Dose-dependent inhibition Pectolinarigenin of [3H]-thymidine incorporation into DNA by increasing concentrations of A769662 in PANC-1 cells stimulated with neurotensin and insulin. Image Editing: Irrelevant lanes were removed (indicated by a thin, vertical black collection) from your acquired digital images and flanking lanes juxtaposed using Adobe Photoshop.(PDF) pone.0114573.s002.pdf (91K) GUID:?674208AF-4088-4007-989F-C103E410F16F Data Availability StatementThe authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information Pectolinarigenin documents. Abstract Natural products represent a rich reservoir of potential small chemical molecules exhibiting anti-proliferative and chemopreventive properties. Here, we display that treatment of pancreatic ductal adenocarcinoma (PDAC) cells (PANC-1, MiaPaCa-2) with the isoquinoline alkaloid berberine (0.3C6 M) inhibited DNA synthesis and proliferation of these cells and delay the progression of their cell cycle in G1. Berberine treatment also reduced (by 70%) the growth of MiaPaCa-2 cell growth when implanted into the flanks of nu/nu mice. Mechanistic studies exposed that berberine decreased mitochondrial membrane potential and intracellular ATP levels and induced potent AMPK activation, as demonstrated by phosphorylation of AMPK subunit at Thr-172 and acetyl-CoA carboxylase (ACC) at Ser79. Furthermore, berberine dose-dependently inhibited mTORC1 (phosphorylation of S6K at Thr389 and S6 at Ser240/244) and ERK activation in PDAC cells stimulated by insulin and neurotensin or fetal bovine serum. Knockdown of 1 1 and 2 catalytic subunit manifestation of AMPK reversed the inhibitory effect produced by treatment with low concentrations of berberine on mTORC1, ERK and DNA synthesis in PDAC cells. However, at higher concentrations, berberine inhibited mitogenic signaling (mTORC1 and ERK) and DNA synthesis through an AMPK-independent mechanism. Similar results were acquired with metformin used at doses that induced either moderate or pronounced reductions in intracellular ATP levels, which were virtually identical to the decreases in ATP levels acquired in response to berberine. We propose that berberine and metformin inhibit mitogenic signaling in PDAC cells through dose-dependent AMPK-dependent and self-employed pathways. Intro Pancreatic ductal adenocarcinoma (PDAC) is definitely a devastating disease, with overall 5-year survival rate of only 6% [1]. The incidence of this disease in the US is estimated to increase to Pectolinarigenin more than 44,000 fresh instances in 2014 and is now the fourth leading cause of tumor mortality in both men and women [2]. Total deaths due to PDAC are projected to increase dramatically to become the second leading cause of cancer-related Ctgf deaths Pectolinarigenin before 2030 [1] As the current therapies offer very limited survival benefits, novel strategies to treat and prevent this aggressive disease are urgently required [3]. G protein-coupled receptors (GPCRs) and their cognate agonists are progressively implicated as autocrine/paracrine growth factors for multiple solid tumors, including small cell lung malignancy, colon, prostate, breast and pancreas [4]C[8]. We showed that pancreatic malignancy cell lines communicate multiple GPCRs [9] and a variety of GPCR agonists, including neurotensin, angiotensin II and bradykinin, stimulated DNA synthesis in pancreatic malignancy cell lines, including PANC-1 and MiaPaca-2 [9]C[12]. Furthermore, a broad-spectrum GPCR antagonist [13], [14], inhibited the growth of pancreatic malignancy cells either or xenografted into nu/nu mice [15]. Additional studies demonstrated increased manifestation of GPCRs in pancreatic malignancy cells [16]C[19]. Subsequently, we recognized positive crosstalk between insulin/IGFI receptors and GPCR signaling systems in pancreatic malignancy cells, leading to mTORC1 signaling and ERK activation, and synergistic activation of DNA synthesis and cell proliferation [20]C[22]. These findings presume an added importance in view of the large number of Pectolinarigenin epidemiological studies linking long standing up type-2 diabetes.