Supplementary MaterialsTable_1. SGC-7901 cells, as well as the Notch/PTEN/AKT axis was involved in the activating PAC-induced apoptosis. PAC treatment led to decreased levels of Notch (NTM), NICD, pPTEN, and pAKT compared to controls. PAC-induced inhibition of Notch-related protein expression levels and the resulting apoptosis were reversed by overexpression of Notch1 (NTM) or/and Notch2 (NTM). Moreover, PAC treatment clearly inhibited tumor growth in mice both bearing tumors derived from both MGC-803 and SGC-7901 cells. This work reveals that PAC induces the apoptosis by suppressing activation of Notch receptor proteolytic cleavage and subsequently blocking the PTEN/AKT signaling axis in gastric cancer cells. Thus, PAC is a potential alternative agent for the treatment of gastric cancer. M-22 (6). PAC is a novel azaphilone with a special pyranoquinone bicyclic core structure (6). Previous studies have shown that azaphilone molecules exert a variety of important biological activities (7C9). Indeed, PAC showed highly selective cytotoxicity in GC cells in our previous study (6). However, the molecular mechanism by which PAC inhibits GC cells remains unclear. Open in a separate window Figure 1 PAC inhibits the proliferation of gastric tumor cells. (A) Framework of penicilazaphilone C (PAC). (B) MGC-803 and SGC-7901 cells had been treated using the indicated concentrations of PAC for 24 h, and cell viability was assessed with an MTT assay. (C) Cells had been treated for 24 h as with (B), and disrupted cell membranes had been detected having a BrdU assay. (D) Cells had been treated as with (B) for 12, 24, and 36 h, and MTS-incorporating live cells had been recognized with an MTS proliferation assay. (E) Cells had been treated as with (B) for two weeks, and live cells had been detected having a colony development assay. (F) Cells had been treated as with (B) for 24 h; cell morphology was noticed under an inverted phase-contrast microscope and pictures were obtained. Data are expressed as the mean SD; * TLR9 0.05, ** 0.01, *** 0.001. The Notch signaling pathway is widely distributed among vertebrates and invertebrates and is highly evolutionarily conserved (10). Notch signaling affects multiple normal morphologenic processes, including the differentiation of pluripotent progenitor cells, apoptosis, cell proliferation, and cell boundary formation (11, 12). Humans and rodents have four Notch receptors (Notch l?4) and five ligands (Jagged 1, 2, Delta-like 1, 3, 4,) (13). The Notch receptor is composed of an extracellular region, a single transmembrane region, and an intracellular region containing an ankyrin domain and a RBP-JK-associated molecule domain (14). Notch signaling is activated by interaction of a ligand from adjacent cells with the Notch receptor (14). Two cleavage events release the intracellular domain of the Notch receptor (NICD) into the cytoplasm, after which it enters the nucleus and binds to the transcription factor CSL to activate the NICD/CSL transcription complex, thereby inducing expression of target genes, such as HES, HEY, and HERP, and exerting biological effects (13). In this study, we investigated the therapeutic effect of PAC on GC in detail to identify the underlying mechanism of PAC-mediated inhibition of GC cell proliferation. Materials and Methods Cell Lines and Culture The human GC cell lines MGC-803, HGC-27, AGS, MKN-45, and SGC-7901 and the normal gastric mucosa MLN8054 supplier cell line GES-1 were obtained from ATCC (Manassas, USA). The cells were cultured in DMEM or RPMI-1640 (HyClone, USA), MLN8054 supplier supplemented with 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin (HyClone, USA) at MLN8054 supplier 37C in a humidified incubator containing 5% CO2. Cells were passaged at 80C90% confluence and were used for experiments in the exponential growth phase..