The membranes were then incubated overnight at 4 C with one of the following the specific primary antibodies: anti-p-AMPK1-thr172, anti-AMPK, anti-p-ACC-ser79, anti-ACC, anti-p-LKB1-ser428, anti-LKB1, anti-CaMKK, anti-TRPV1 (Cell Signaling Technology, Danvers, MA, USA). GUID:?602A2814-57E4-4F95-8560-0E418E0BBB30 S6 Fig: Western blot of pAMPK, AMPK, KU 0060648 pACC and ACC in HepG2 cells treated with capsaicin and capsaicin + BAPTA. (TIF) pone.0211420.s006.tif (2.2M) GUID:?5FF21816-AC1B-4316-B23D-F48C2A74F23E S7 Fig: Western blot of pAMPK and AMPK in HepG2 cells with AMPK knocked-down and treated with capsaicin. (TIF) pone.0211420.s007.tif (1.9M) GUID:?48E843CD-955B-4C30-A95F-DAAB79034A53 S8 Fig: Western blot of pAkt, Akt, pmTOR and mTOR in HepG2 cells treated with capsaicin. (TIF) pone.0211420.s008.tif (2.3M) GUID:?E5A8F049-0AD3-4E99-80FC-AAEC7B2D4440 S9 Fig: Western blot of LC3, p62, procaspase 9 and procaspase 3 in HepG2 cells treated with capsaicin. (TIF) pone.0211420.s009.tif (2.4M) GUID:?E8A3BF9B-B9D7-4F9E-BAC2-651AA2A222D1 Data Availability StatementAll relevant data are within the manuscript and its Supporting Information files. Abstract Capsaicin is usually a natural compound present in chili and red peppers and the responsible of their spicy flavor. It has recently provoked interest because of its antitumoral effects in many cell types although its action mechanism is not clearly comprehended. As metabolic dysregulation is one of the hallmarks of cancer cells and the key metabolic sensor in the ADAMTS9 AMP-activated kinase (AMPK), in this study we explored the ability of capsaicin to modulate AMPK activity. We found that capsaicin activated AMPK in HepG2 cells by increasing AMPK phosphorylation and its downstream target ACC. Mechanistically, we decided that capsaicin activated AMPK through the calcium/calmodulin-dependent protein kinase kinase , CaMKK as either the CaMKK inhibitor STO-609 or CaMKK knock down with siRNA abrogated the activation of AMPK. Moreover, capsaicin decreased cell viability, inhibited Akt/mTOR pathway and increased reactive oxygen species (ROS) in HepG2 cells. AMPK activation was involved in the underpinning mechanism of capsaicin-induced cell death. Introduction Natural compounds and dietary products provide an interesting area of research because of their low toxicity and potent efficacy. Capsaicin (CAP) is a natural alkaloid and the main active ingredient of spicy peppers belonging to genus. It is used as additive in food in many cultural cuisines and it is responsible for the warm or burning sensation experienced on contact with chili peppers. Although traditionally associated with analgesic effects, it has been recently proposed that capsaicin also displays antitumor activity in various cell types and enhances the sensitivity of cancer cells to cytotoxic drugs [1C3]. In addition, laboratory data support the notion that capsaicin could act as an anti-obesity drug by increasing energy expenditure [4C6]. It has recently been shown that the intake of capsaicin reduces the insulin resistance caused by obesity in rats [7, 8]. Moreover, epidemiological KU 0060648 data reveal that consumption of foods made up of capsaicin is associated with a lower prevalence of obesity [9, 10]. Cancer cells undergo a metabolic reprogramming in order to satisfy energy demands of a continuous growth. Even in the presence of oxygen, tumors maintain anaerobic glycolysis to ensure enough levels of carbohydrate intermediates for anabolic reactions, as described by Otto Warburg nine decades ago . Furthermore, recent research indicates that metabolites themselves can be oncogenic by altering cell signaling and blocking cellular differentiation . Therefore, to impact metabolic reactions in cancer cells may be a new therapeutic strategy for this disease. Hepatocellular carcinoma (HCC) remains one of the most common and lethal malignancies worldwide despite the development of various therapeutic strategies. The prognosis for patients with advanced HCC remains extremely poor due to the high rates KU 0060648 of recurrence and metastasis. The liver is the major metabolic organ and dysregulation of metabolic balance has been reported to cause liver diseases including cancer . The key metabolic sensor for the cell energy status is the enzyme AMP-activated kinase (AMPK). Its activation leads to the implementation of catabolic pathways in order to restore ATP levels. Activation of AMPK is usually regulated by phosphorylation and allosteric modulation. Phosphorylation at the conserved residue of Thr172 in the catalytic domain name increases about 500-fold AMPK activity. The main upstream kinases that phosphorylate AMPK are liver kinase B1 (LKB1) and the kinase that phosphorylates Ca2+/calmodulin dependent kinase type , (CaMKK, also known as CaMKK2) . In addition, AMP exerts an allosteric activation by increasing the AMPK activity by 5-fold . The importance of AMPK as a therapeutic target in cancer is beginning to be unveiled. Clinical data suggest a greater benefit of anticancer therapy in patients with type 2 diabetes mellitus treated with metformin, an activator of AMPK. . It has also been recently observed that AMPK may be involved in the appearance of resistant phenotypes. For example, the loss of LKB1 in breast cancer cells increases the aggressiveness, migration ability and appearance of stem-like phenotype whereas the activation of LKB1, and consequently of AMPK, reduces the formation of mamospheres and the expression of pluripotent factors . Therefore, AMPK is emerging as a new therapeutic target which, through.