Bovine lactoferricin B induces apoptosis of human gastric cancer cell line AGS by inhibition of autophagy at a late stage W.-R. Pan, P.-W. Chen, Y.-L.

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Bovine lactoferricin B induces apoptosis of human gastric cancer cell line AGS by inhibition of autophagy at a late stage W.-R. Pan, P.-W. Chen, Y.-L. S. Chen, H.-C. Hsu, C.-C. Lin, W.-J. Chen Journal of Dairy Science Volume 96, Issue 12, Pages 7511-7520 (December 2013) DOI: 10.3168/jds.2013-7285 Copyright © 2013 American Dairy Science Association Terms and Conditions

Figure 1 The purity of bovine lactoferrin (bLF), bLF acid hydrolysate (bLFh), and 3 lactoferricin B (LFcinB) peptides were analyzed by 15% Tricine SDS-PAGE and stained with Coomassie Brilliant Blue G-250. M=molecular weight marker. Journal of Dairy Science 2013 96, 7511-7520DOI: (10.3168/jds.2013-7285) Copyright © 2013 American Dairy Science Association Terms and Conditions

Figure 2 Effects of bovine lactoferrin acid hydrolysate (bLFh) and lactoferricin B (LFcinB) peptides on the cell viability of gastric cancer cell line AGS (A) and a nontransformed murine fibroblast cell line 3T3 (B). Cells were treated with different concentrations (0 to 200μM) of bLFh and LFcinB peptides each for 24h, followed by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Each concentration was repeated in 8 wells for 3 independent experiments. Journal of Dairy Science 2013 96, 7511-7520DOI: (10.3168/jds.2013-7285) Copyright © 2013 American Dairy Science Association Terms and Conditions

Figure 3 Effects of lactoferricin B25 (LFcinB25) on AGS cell (gastric cancer cell line) morphology. The AGS cells were treated with 64μM LFcinB25, and cell morphology changes were assessed from 0 to 24h using an inverted microscope with 200× magnification. Journal of Dairy Science 2013 96, 7511-7520DOI: (10.3168/jds.2013-7285) Copyright © 2013 American Dairy Science Association Terms and Conditions

Figure 4 Lactoferricin B25 (LFcinB25) treatment of AGS cells (gastric cancer cell line) led to the accumulation of cells in the sub-G1 phase (A and B) of the cell cycle. Flow cytometry was applied to analyze the AGS cell cycle in the absence or presence of LFcinB25 (64μM) for 0 to 24h, with visualization by propidium iodide (PI) staining. Color version available in the online PDF. Journal of Dairy Science 2013 96, 7511-7520DOI: (10.3168/jds.2013-7285) Copyright © 2013 American Dairy Science Association Terms and Conditions

Figure 5 Western blot analysis for the activation of apoptosis-related proteins. The AGS cells (gastric cancer cell line) were treated with lactoferricin B25 (64μM) for 0 to 48h, and the cellular extracts were detected by Western blotting for cytochrome C, the pro- and cleaved forms of caspase-3, caspase-7, caspase-8, caspase-9, and poly(ADP-ribose) polymerase (PARP) to see if LFcinB25 induced apoptosis of AGS cells through the caspase-dependent intrinsic or extrinsic pathways. Lane Con=untreated control. β-Actin was used as internal control. Journal of Dairy Science 2013 96, 7511-7520DOI: (10.3168/jds.2013-7285) Copyright © 2013 American Dairy Science Association Terms and Conditions

Figure 6 Western blot analysis for the activation of autophagy-related proteins. The AGS cells were treated with lactoferricin B25 (LFcinB25; 64μM) for 0 to 48h, and their cellular extracts were detected by Western blotting for JNK-1, Bcl-2, phosphorylated Bcl-2, Beclin 1, cleaved Beclin 1, LC3 I, and LC3 II to see if LFcinB25 induced autophagy of AGS cells. Lane Con=untreated control. β-Actin was used as internal control. Journal of Dairy Science 2013 96, 7511-7520DOI: (10.3168/jds.2013-7285) Copyright © 2013 American Dairy Science Association Terms and Conditions