The Pim-1 kinase regulates cell survival proliferation and differentiation and is

The Pim-1 kinase regulates cell survival proliferation and differentiation and is overexpressed frequently in many malignancies including leukemia and skin cancer. or skin malignancy. Overall our results offered preclinical proof of concept for 2′-HCA as a potent anticancer principle arising from direct targeting of CVT 6883 the Pim-1 kinase. and has been used in traditional Chinese medicine for treating dyspepsia gastritis blood circulation disturbances inflammatory diseases and malignancy in Korea China and India (5). A number of recent studies have CVT Rabbit polyclonal to CDKN2A. 6883 shown several potential health benefits of cinnamon for diabetes neuropathy cardiovascular disease and malignancy (6). Cinnamon extracts have been shown to inhibit proliferation of lymphoma melanoma hepatocellular CVT 6883 malignancy cervix malignancy and colon cancer cells and melanoma (4 7 These extracts also reportedly suppress angiogenesis by targeting VEGFR2 (10 11 2 (2′-HCA Fig. 1Aa) is usually a major component of the essential oil of cinnamon and is present at levels of approximately 0.01-0.8 mg/g in commercial cinnamon powder (4 12 It is regarded as a major bioactive component of cinnamon (13) and reportedly inhibits proliferation of several human cancer cell lines including breast colon leukemia ovarian and lung tumor cells (14-18). 2′-HCA also attenuates the xenograft growth of colon HCT116 malignancy cells and allograft growth of oral RK3E-ras-Fluc malignancy cells (16 18 However its direct molecular target(s) and mechanism(s) of action have not been clearly elucidated. Physique CVT 6883 1 2 (2′-HCA) inhibits Pim-1 activity. Aa chemical structure of 2′-HCA. Ab 2 inhibits Pim-1 activity. Active Pim-1 (50 ng) was mixed with 2′-HCA (0 2.5 5 or 10 μM) and then … Proviral insertion in murine lymphomas-1 (Pim-1) is a proto-oncogene involved in pivotal cellular processes (19). Pim-1 is usually overexpressed in various tumors and has been linked to a poor prognosis (20). Its deregulation also interferes with apoptosis and cell cycle-related pathways to promote neoplastic transformation in many forms of malignancy. Over-expression of Pim-1 in mice leads to a greater susceptibility to spontaneous tumor formation while increasing susceptibility to radiation- and chemically-induced tumorigenesis (21). Accordingly desire for developing small molecule inhibitors of Pim-1 has been growing (21). We sought to further investigate the underlying mechanism of the anti-tumorigenic effects of 2′-HCA and its relationship with Pim-1. In this study we observed that Pim-1 is usually a direct molecular target of 2′-HCA and revealed direct molecular binding using X-ray co-crystallography. The significance of these findings was further explored using a number of and methods. Materials and Methods Reagents 2 was purchased from Tokyo Chemical Industry (Tokyo Japan). Active Pim-1 and the CVT 6883 Pim-1 substrate peptide (KRRRLASLR) were purchased from SignalChem (Richmond Canada). Antibodies specific for detecting total Bad phosphorylated Bad (Ser112) total ERKs phosphorylated ERKs (Thr202/Tyr204) total RSK phosphorylated RSK (Thr356/Ser360) PARP caspase-3 and caspase-9 were purchased from Cell Signaling Technology (Beverly MA). Antibodies against total Pim-1 PARP Bad p27KIP1 p21CIP1 and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz CA). Cell culture All cell CVT 6883 lines were purchased from your American Type Culture Collection and were cytogenetically tested and authenticated before being frozen. Each vial of frozen cells was thawed and managed in culture for a maximum of 8 weeks. Enough frozen vials were available for each cell collection to ensure that all cell-based experiments were conducted on cells that had been tested and in culture for 8 weeks or less. The human erythroleukemia (HEL) cell collection was cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS Atlanta Biologicals Flowery Branch GA) and 1% antibiotic-antimycotic (Thermo Fisher Scientific Inc. Waltham MA). HaCaT skin keratinocytes and A431 human epidermoid carcinoma cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM Thermo Fisher Scientific Inc.) supplemented with 10% FBS and 1% antibiotic-antimycotic. JB6 P+ mouse skin epidermal cells were cultured in.