The results for the primary fibroblasts AG1 mirrored that of the HFF

assay. As shown in Fig. 1A, both 20567609 FK-16 and 2583244 LL-37 significantly reduced the viability of LoVo and HCT116 cells in a dose-dependent manner. Noticeably, FK-16 displayed a better activity against colon cancer cells than LL-37. Moreover, FK-16 had a minimal effect on the viability of human normal colon mucosal epithelial NCM460 cells. A control peptide with scrambled sequence of FK-16 also had negligible effects on LoVo and HCT116, indicating FK-16 mediated selective killing of cancer cells. As HCT116 was more sensitive than LoVo to FK-16, HCT116 was selected for subsequent mechanistic study and treated with either 20 mM or 40 mM FK-16, at which,20% and,50% of cytotoxicity Tipifarnib chemical information occurred. FK-16-Induced Autophagy and Apoptosis targeting Atg5 and Atg7, both of which are required for the formation of autophagosomes at the early stage. Knockdown of Atg5 or Atg7 significantly reduced the cytotoxic effect of FK-16, suggesting that FK-16 induced autophagic cell death in colon cancer cells. Requirement of p53 for FK-16-induced Apoptosis and Autophagic Cell Death The tumor suppressor protein p53 is known to take part in both caspase-dependent and -independent cell death, including AIF/ Endo-dependent apoptosis and autophagy. Here, we demonstrated that FK-16 directly activated p53 to mediate both cell death pathways. As shown in Fig. 3A, FK-16 increased the nuclear level of p53 and upregulated the expression of PUMA and Bax. Consistently, FK-16 reduced the expression of Bcl-2. To elucidate the functional involvement of p53 in AIF/Endodependent apoptosis and autophagic cell death triggered by FK16, p53 was knocked down by siRNA. Knockdown of p53 not only restored the expression of Bax and Bcl-2, but also remarkably reduced FK-16-induced LC3-I/II, Atg5 and Atg7 expression as well as nuclear expression of AIF and EndoG, suggesting that p53 was a common activator of both cell death pathways. Accordingly, knockdown of p53 reduced phosphatidylserine externalization, the formation of LC3+ autophagic vacuole and the loss of cell viability induced by FK-16. Altered Expression of Bcl-2 and Bax during FK-16-induced Apoptosis and Autophagic Cell Death Since the Bcl-2 family has been reported to regulate both caspase-independent apoptosis and autophagy in other biological contexts, we next questioned if FK-16-induced colon cancer cell death was mediated through Bcl-2 and Bax, both of which were downstream of p53. Results showed that restoration of Bcl-2 expression by transfection with Bcl-2-encoding plasmid or genetic ablation of Bax using Bax2/2 HCT116 cells 3 FK-16-Induced Autophagy and Apoptosis abolished FK-16-induced expression of LC3-I/II, Atg5 and Atg7 as well as nuclear expression of AIF and EndoG, suggesting that downregulation of Bcl-2 and upregulation of Bax were required for FK-16-induced autophagic and apoptotic signaling. In line with these findings, restoration of Bcl-2 expression or genetic ablation of Bax reduced the formation of LC3+ autophagic vacuole and the loss of cell viability induced by FK-16. These findings indicate that FK-16 acted through the p53-Bcl-2/Bax pathway to simultaneously induce AIF/EndoG-dependent apoptosis and autophagy-mediated cell death. Reciprocal Regulation between Caspase-independent Apoptosis and Autophagic Cell Death To elucidate the potential crosstalk between autophagic cell death and AIF/EndoG-dependent apoptosis in the action of FK16, these two cellular processes were abolished by RNA interference. The knockdown effica