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.

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 a dietary supplement composed of hydrolyzed milk proteins and vanillin on the reduction of infection and oxidative stress induced by chemotherapy.

This study evaluates the antioxidant and antibacterial activity of a mixture of lactoferrin hydrolysate (LfH), whey protein hydrolysate (WPH) and vanillin in vitro and in vivo to design a chemoprotective supplement for reducing the infection and oxidative stress induced by chemotherapy. The designed supplement showed significant antibacterial activity against E. coli. The supplement with the highest concentration exhibited considerable antioxidant activity in (2,2-diphenyl-1-picrylhydrazyl) DPPH free radicals, (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) ABTS, and reducing power assays. In the biochemical analysis of liver homogenate, the supplement 3 increased the level of enzymes Catalase (CAT), Glutathione peroxidase (GPx), Superoxide dismutase (SOD), and also the Ferric Reducing Ability of Plasma (FRAP) while decreased thiobarbituric acid reactive substances (TBARS) in comparison to paclitaxel group, indicative of activity against oxidative stress. Antibacterial and antioxidant activity of the designed supplement makes it a good candidate for use as a functional food to reduce the side effects of chemotherapy. PRACTICAL APPLICATIONS: A dietary supplement composed of lactoferrin hydrolysate (LfH), whey protein hydrolysate (WPH) and vanillin showed antibacterial activity against E. coli and S. aureus in vitro. The studied supplement also exhibited significant antioxidant properties in the model system and anti-oxidative stress activity in mice exposed to paclitaxel. This supplement has a potential for use in the food matrix to reduce the chemotherapy side effects and to act as a chemoprotective agent.

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).

In vitro effectiveness of recombinant human lactoferrin and its hydrolysate in alleviating LPS-induced inflammatory response.

This study aimed to evaluate the potential anti-inflammatory role of the most produced form of lactoferrin expressed in various expression systems (Fe-saturated recombinant human Lf, rhLf) and its hydrolysate in concentrations resembles that found in mature human milk. Co-culture model consisted of CaCo-2 and RAW 246.7 cell lines was used to evaluate the potential anti-inflammatory activity of rhLf and its hydrolysate. During this experiment, CaCo-2 monolayer permeability and integrity was assayed through the measurement of transepithelial electrical resistance (TEER values). Also, the production of reactive oxygen species (ROS), nitric oxide (NO) and different cytokines (IL-8, IL-1ß, IL-6, IL-10, IL-12p70, and TNF-α) were measured. The treatment with rhLf and its hydrolysate protected the monolayer integrity against LPS effect and reduced IL-8 and ROS production. This effect was dependent on the dose and 2mgmL-1 of rhLf hydrolysate was more effective. The addition of rhLf and its hydrolysate to infant formula is a prominent step towards improving both infant formula functionality and newborn health. Thus, these functional ingredients could be incorporated in infant foods. In this context, ongoing researches are conducted to clarify this effect whether by using synthetic peptides or by using LPS-sepsis animal.

Antihypertensive mechanism of lactoferrin-derived peptides: angiotensin receptor blocking effect.

Looking for antihypertensive mechanisms beyond ACE inhibition, we assessed whether lactoferrin (LF)-derived peptides can act as receptor blockers to inhibit vasoconstriction induced by angiotensin II or endothelin-1. The lactoferricin B (LfcinB)-derived peptide LfcinB20-25 (RRWQWR), the low molecular weight LF hydrolysate (LFH < 3 kDa), and two peptides identified in LFH < 3 kDa (LIWKL and RPYL) were tested in ex vivo assays of vasoactive responses. The peptide RPYL was tested in radioligand receptor binding assays. Both LFH < 3 kDa and individual peptides inhibited angiotensin II-induced vasoconstriction. RPYL showed the highest ex vivo inhibitory effect and also inhibited binding of [(125)I]-(Sar(1),Ile(8))-angiotensin II to AT1 receptors. By contrast, neither LFH < 3 kDa nor RPYL inhibited endothelin-1 and depolarization-induced vasoconstrictions. In conclusion, LF-derived peptides selectively inhibit angiotensin II-induced vasoconstriction by blocking angiotensin AT1 receptors. Therefore, inhibition of angiotensin II-induced vasocontriction is suggested as a mechanism contributing along with ACE inhibition to the antihypertensive effect of some LF-derived peptides.