Efficiency of novel nanocombinations of bovine milk proteins (lactoperoxidase and lactoferrin) for combating different human cancer cell lines

Abstract

Bovine lactoperoxidase (LPO) and lactoferrin (LF) are suitable proteins to be loaded or adsorbed to chitosan nanoparticles (NPs) for preparing stable nanoformulations with potent anticancer activity. In the present study, nanocombinations of LPO and LF revealed improvement in their stability and activity compared to single (free or nanoformulated) bovine proteins. The coating or loading of LPO-loaded NPs with LF resulted in the highest synergistic cytotoxicity effect against Caco-2, HepG-2, MCF-7 and PC-3 cells in comparison with other NPs and free proteins without causing toxicity toward normal cells. This synergistic improvement in the anticancer activity was apoptosis-dependent that was confirmed by severe alterations in cellular morphology, high percentage of annexin-stained cells and sub-G1 populations as well as nuclear staining with orange fluorescence of treated cancer cells. Additionally, significant alterations in the expression of well characterized cellular proliferation and apoptosis guards (NF-κB, Bcl-2 and p53) in these NPs-treated cancer cells compared to 5-fluorouracil (5-FU) treated cells. Our findings provide for the first time that these new synergistic nanoformulated forms of LPO and LF were superior in their selective apoptosis-mediating anticancer effect than free form of these proteins and 5-FU. LF coating or loading of LPO-loaded NPs present as promising therapy for cancer.

Figure 1

Purification of LPO and LF and scanning electron micrograph of the most active LPO and LF NPs. (a) Elution profile of LPO and LF on a Mono S column. (b) 12% SDS-PAGE for bovine LPO and LF; Lane I is protein marker, lane II is purified LF and lane III is is purified LPO. (c) Morphology of the most active NPs (I) LPO + LF-loaded NPs and (II) LF coated LPO-loaded NPs.

Figure 2

Stability of bovine milk protein activities as free and nanoformulated forms throughout 9 weeks. (a) LPO. (b) LF.

Figure 3

Normal and cancer cell morphology shown by phase contrast microscope. Human cancer cell lines (I) including fibroblast cells, Caco-2 cells, HepG-2 cells, MCF-7 cells and PC-3 cells following 72 h treatment with (II) LPO-loaded NPs, (III) LF-coated NPs, (IV) LPO + LF-loaded NPs, (V) LF coated LPO-loaded NPs and (VI) 5-FU.

Figure 4

Investigation of the apoptotic effect of the most effective NPs using double nuclear staining. (a) Flow charts of annexin-PI analysis of cancer cells lines (I) including fibroblast cells, Caco-2 cells, HepG-2 cells, MCF-7 cells and PC-3 cells following 72 h treatment with (II) LPO-loaded NPs, (III) LF-coated NPs, (IV) LPO + LF-loaded NPs, (V) LF coated LPO-loaded NPs and (VI) 5-FU. (b) The percentage of total apoptosis in NPs-treated normal and cancer cells in comparison with untreated cells. (c) Fluorescence images of ethidium bromide-acridine orange staining of cancer cells lines (I) including Caco-2 cells, HepG-2 cells, MCF-7 cells and PC-3 cells following 72 h treatment with (II) LPO + LF-loaded NPs, (III) LF coated LPO-loaded NPs and (IV) 5-FU.

Figure 5

Cell cycle analysis of the two most effective NPs-treated human cancer cell lines. (a) Flow charts of cell cycle analysis of human cancer cell lines (I) including Caco-2 cells, HepG-2 cells, MCF-7 cells and PC-3 cells following 72 h treatment with (II) LPO + LF-loaded NPs and (III) LF coated LPO-loaded NPs. (b) Quantitative distribution of LPO + LF-loaded NPs- and LF coated LPO-loaded NPs-treated cancer cell populations in different phases of cell cycle in comparison with untreated cancer cells.

Figure 6

Relative changes in the expression levels of three key genes in human cancer cell lines after treatment with LPO + LF-loaded NPs, LF-coated LPO-loaded NPs and 5-FU for 72 h. (a) NF-κB. (b) Bcl-2. (c) p53.