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).