The Effects of NT-1044, a Novel AMPK Activator, on Endometrial Cancer Cell Proliferation, Apoptosis, Cell Stress and In Vivo Tumor Growth.

OBJECTIVES: Anti-diabetic biguanide drugs such as metformin may have anti-tumorigenic effects by behaving as AMPK activators and mTOR inhibitors. Metformin requires organic cation transporters (OCTs) for entry into cells, and NT-1044 is an AMPK activator designed to have greater affinity for two of these transporters, OCT1 and OCT3. We sought to compare the effects of NT-1044 on cell proliferation in human endometrial cancer (EC) cell lines and on tumor growth in an endometrioid EC mouse model. METHODS: Cell proliferation was assessed in two EC cell lines, ECC-1 and Ishikawa, by MTT assay after exposure to NT-1044 for 72 hours of treatment. Apoptosis was analyzed by Annexin V-FITC and cleaved caspase 3 assays. Cell cycle progression was evaluated by Cellometer. Reactive oxygen species (ROS) were measured using DCFH-DA and JC-1 assays. For the in vivo studies, we utilized the LKB1fl/flp53fl/fl mouse model of endometrioid endometrial cancer. The mice were treated with placebo or NT-1044 or metformin following tumor onset for 4 weeks. RESULTS: NT-1044 and metformin significantly inhibited cell proliferation in a dose-dependent manner in both EC cell lines after 72 hours of exposure (IC50 218 µM for Ishikawa; 87 µM for ECC-1 cells). Treatment with NT-1044 resulted in G1 cell cycle arrest, induced apoptosis and increased ROS production in both cell lines. NT-1044 increased phosphorylation of AMPK and decreased phosphorylation of S6, a key downstream target of the mTOR pathway. Expression of the cell cycle proteins CDK4, CDK6 and cyclin D1 decreased in a dose-dependent fashion while cellular stress protein expression was induced in both cell lines. As compared to placebo, NT-1044 and metformin inhibited endometrial tumor growth in obese and lean LKB1fl/flp53fl/fl mice. CONCLUSIONS: NT-1044 suppressed EC cell growth through G1 cell cycle arrest, induction of apoptosis and cellular stress, activation of AMPK and inhibition of the mTOR pathway. In addition, NT-1044 inhibited EC tumor growth in vivo under obese and lean conditions. More work is needed to determine if this novel biguanide will be beneficial in the treatment of women with EC, a disease strongly impacted by obesity and diabetes.

Dual-acting peptide with prolonged glucagon-like peptide-1 receptor agonist and glucagon receptor antagonist activity for the treatment of type 2 diabetes.

Type 2 diabetes is characterized by reduced insulin secretion from the pancreas and overproduction of glucose by the liver. Glucagon-like peptide-1 (GLP-1) promotes glucose-dependent insulin secretion from the pancreas, while glucagon promotes glucose output from the liver. Taking advantage of the homology between GLP-1 and glucagon, a GLP-1/glucagon hybrid peptide, dual-acting peptide for diabetes (DAPD), was identified with combined GLP-1 receptor agonist and glucagon receptor antagonist activity. To overcome its short plasma half-life DAPD was PEGylated, resulting in dramatically prolonged activity in vivo. PEGylated DAPD (PEG-DAPD) increases insulin and decreases glucose in a glucose tolerance test, evidence of GLP-1 receptor agonism. It also reduces blood glucose following a glucagon challenge and elevates fasting glucagon levels in mice, evidence of glucagon receptor antagonism. The PEG-DAPD effects on glucose tolerance are also observed in the presence of the GLP-1 antagonist peptide, exendin(9-39). An antidiabetic effect of PEG-DAPD is observed in db/db mice. Furthermore, PEGylation of DAPD eliminates the inhibition of gastrointestinal motility observed with GLP-1 and its analogues. Thus, PEG-DAPD has the potential to be developed as a novel dual-acting peptide to treat type 2 diabetes, with prolonged in vivo activity, and without the GI side-effects.

A potent and highly selective VPAC2 agonist enhances glucose-induced insulin release and glucose disposal: a potential therapy for type 2 diabetes.

Pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) activate two shared receptors, VPAC1 and VPAC2. Activation of VPAC1 has been implicated in elevating glucose output, whereas activation of VPAC2 may be involved in insulin secretion. A hypothesis that a VPAC2-selective agonist would enhance glucose disposal by stimulating insulin secretion without causing increased hepatic glucose production was tested using a novel selective agonist of VPAC2. This agonist, BAY 55-9837, was generated through site-directed mutagenesis based on sequence alignments of PACAP, VIP, and related analogs. The peptide bound to VPAC2 with a dissociation constant (K(d)) of 0.65 nmol/l and displayed >100-fold selectivity over VPAC1. BAY 55-9837 stimulated glucose-dependent insulin secretion in isolated rat and human pancreatic islets, increased insulin synthesis in purified rat islets, and caused a dose-dependent increase in plasma insulin levels in fasted rats, with a half-maximal stimulatory concentration of 3 pmol/kg. Continuous intravenous or subcutaneous infusion of the peptide reduced the glucose area under the curve following an intraperitoneal glucose tolerance test. The peptide had effects on intestinal water retention and mean arterial blood pressure in rats, but only at much higher doses. BAY 55-9837 may be a useful therapy for the treatment of type 2 diabetes.