Activity Changes of the Peptic Lactoferrin Hydrolysate in Human Gastric Cancer AGS Cells in Response to Cu(II) or Mn(II) Addition.

Lactoferrin is an interesting bioactive protein in milk and can interact with various metal ions of trace elements such as copper, iron, manganese, and others. In this study, a lactoferrin hydrolysate (LFH) was generated from commercial bovine lactoferrin by protease pepsin, fortified with Cu2+ (or Mn2+) at two levels of 0.64 and 1.28 (or 0.28 and 0.56) mg/g protein, respectively, and then measured for the resultant bioactivity changes in the well-differentiated human gastric cancer AGS cells. The assaying results indicated that the LFH and Cu/Mn-fortified products had long-term anti-proliferation on the cells, while the treated cells showed DNA fragmentation and increased apoptotic cell proportions. Regarding the control cells, the cells treated with the LFH and especially Cu/Mn-fortified LFH had remarkably up-regulated mRNA expression of caspase-3 and Bax by respective 1.21-3.23 and 2.23-2.83 folds, together with down-regulated mRNA expression Bcl-2 by 0.88-0.96 folds. Moreover, Western-blot assaying results also indicated that the cells exposed to the LFH and Cu/Mn-fortified LFH (especially Mn at higher level) for 24 h had an enhanced caspase-3 expression and increased ratio of Bax/Bcl-2. It can thus be concluded that the used Cu/Mn-addition to the LFH may lead to increased bioactivity in the AGS cells; to be more specific, the two metal ions at the used addition levels could endow LFH with a higher ability to cause cell apoptosis by activating caspase-3 and increasing the Bax/Bcl-2 ratio.

Effect of Cu/Mn-Fortification on In Vitro Activities of the Peptic Hydrolysate of Bovine Lactoferrin against Human Gastric Cancer BGC-823 Cells.

Bovine lactoferrin hydrolysate (BLH) was prepared with pepsin, fortified with Cu2+ (Mn2+) 0.64 and 1.28 (0.28 and 0.56) mg/g protein, and then assessed for their activity against human gastric cancer BGC-823 cells. BLH and the four fortified BLH products dose- and time-dependently had growth inhibition on the cells in both short- and long-time experiments. These samples at dose level of 25 mg/mL could stop cell-cycle progression at the G0/G1-phase, damage mitochondrial membrane, and induce cell apoptosis. In total, the fortified BLH products had higher activities in the cells than BLH alone. Moreover, higher Cu/Mn fortification level brought higher effects, and Mn was more effective than Cu to increase these effects. In the treated cells, the apoptosis-related proteins such as Bad, Bax, p53, cytochrome c, caspase-3, and caspase-9 were up-regulated, while Bcl-2 was down-regulated. Caspase-3 activation was also evidenced using a caspase-3 inhibitor, z-VAD-fmk. Thus, Cu- and especially Mn-fortification of BLH brought health benefits such as increased anti-cancer activity in the BGC-823 cells via activating the apoptosis-related proteins to induce cell apoptosis.

Effect of the Plastein Reaction in Presence of Extrinsic Amino Acids on the Protective Activity of Casein Hydrolysate against Ethanol-Induced Damage in HHL-5 Cells.

Casein hydrolysates (CH) were prepared using papain and modified by the plastein reaction (CH-P) in the presence of extrinsic phenylalanine (CH-P-Phe) or tryptophan (CH-P-Trp). The in vitro protective activity of CH and its modified products against ethanol-induced damage in HHL-5 cells was investigated. The results showed that the modification by the plastein reaction reduced the amino group content of CH. However, the modification by the plastein reaction in the presence of extrinsic amino acids could enhance the antioxidant, proliferative, cell cycle arresting, and anti-apoptosis activity of CH. Biological activities of CH and its modified products in the HHL-5 cells varied depending on the hydrolysate concentration (1, 2, and 3 mg/mL) and treatment time (24, 48, and 72 h). Generally, higher biological activities were found after cell treatment with CH or its modified products at concentration of 2 mg/mL for 48 h compared to other treatments. In addition, CH modified in the presence of tryptophan (CH-P-Trp) showed higher biological activity than that modified in the presence of phenylalanine (CH-P-Phe). Based on the obtained results, it can be concluded that casein hydrolysates with enhanced biological activity and potential health benefits can be produced by papain and the plastein reaction with the incorporation of extrinsic amino acids.

Copper or Manganese Supplementation Endows the Peptic Hydrolysate from Bovine Lactoferrin with Enhanced Activity to Human Gastric Cancer AGS Cells.

A lactoferrin hydrolysate (LFH) was generated from bovine lactoferrin by pepsin, mixed with Cu2+ and Mn2+ at 0.64-1.28 and 0.28-0.56 mg/g protein, respectively; and then their in vitro effects on human gastric cancer AGS cells were assessed. With incubation times of 24 or 48 h, LFH and its Cu2+/Mn2+ mixtures at 10-30 mg/mL in dose-dependent manner inhibited cell growth; and more, these mixtures showed higher activities than LFH alone. Cell treatments of LFH and the mixtures (25 mg/mL) for 24 h could arrest cell cycle at G0/G1-phase, damage mitochondrial membrane integrity, and induce apoptosis, while the mixtures were also more powerful than LFH to exert these three effects. Higher Cu2+/Mn2+ supplementation level resulted in higher growth inhibition, cell cycle arrest, mitochondrial membrane potential disruption, and apoptosis induction; furthermore, Mn2+ was notable for its higher efficacy than Cu2+ to increase these four effects. Western-blot assay results revealed that four apoptosis-related proteins Bad, Bax, cytochrome c, and p53 were up-regulated, and both caspase-3 and caspase-9 also were cleaved and activated; moreover, two autophagy-related proteins LC3-II and cleaved Beclin-1 were down- and up-regulated, respectively. It is thus concluded that Cu2+ and especially Mn2+ could endow supplemented LFH with increased anti-cancer effects in AGS cells, with two proposed events as enhanced apoptosis induction (via activating apoptosis-related proteins) and autophagy inhibition (via activating autophagy-related proteins).