Figure 1.
Purification and analysis of components.
A) SDS-PAGE analysis of purified HMW-bLf indicating the presence of pure ≥250 kDa protein in elutes (lanes 1 and 2). B) The purified protein was confirmed to be bLf through Western blotting using anti-bLf specific antibody. C) Dissociation of HMW-bLf in 1 M NaCl into the dimeric (∼160 kDa) and monomeric (∼78 kDa) forms (lane S1). These were confirmed to be bLf bands by Western blot for the same dissociated sample (S1). The absence of any other lower bands and the detection of all the constituent bands by anti-bLf specific antibody indicate that HMW-bLf is an oligomer formed by the interactions of monomeric bLf molecules.
Figure 2.
Physico-chemical characterization of HMW-bLf.
A) Fourier transform infra-red (FTIR) spectra of HMW-bLf indicating characteristic peaks and compared with other forms of bLf. B) Differential scanning calorimetry thermograms of the different bLf forms C) SDS-PAGE showing the comparative resistance of HMW-bLf to Omnizyme (human digestive enzyme cocktail) treatment. It also gives an indication that HMW-bLf is an oligomer made of bLf monomers; because upon digestion HMW-bLf besides dimers and trimers does not release any other fragments lower than ∼78 kDa apart from the ones produced from the digestion of commercially pure NM-bLf.
Figure 3.
A B and C represent the cellular cytotoxicity measured by LDH release assay induced by HMW-bLf in a concentration dependent manner, in MDA-MB-231 (human breast carcinoma) SW480 (human colorectal adenocarcinoma) and FHs 74 Int (normal intestinal cells) cells. D and E show the cell death (mortality count) as measured by Flow cytometry using propidium iodide staining (* p<0.05 and ** p<0.01). Other forms of bLf were used for comparison.
Figure 4.
HMW-bLf decreases the cellular proliferation of MDA-MB-231 and SW480 (A and B respectively) cells in a concentration dependent manner. The cell fate was also monitored by analyzing the cell morphology (C – MDA-MB-231 and D – SW480) which clearly indicates cell death. (* p<0.05 and ** p<0.01)
Figure 5.
A – Representative confocal microscopic images show HMW-bLf internalization in MDA-MB-231 and SW480 in a time dependent manner. The degradation of actin an indicator of apoptosis was observed after 6 h of treatment with HMW-bLf. The reduction in the intensity of the Alexa 568 signal indicates the degradation of the actin cytoskeleton. B and C are high magnification images HMW-bLf internalization in SW480 (B) and MDA-MB231(C) with separate panels showing nucleus, actin and HMW-bLf alone. Arrows in 4B points out to the cells that have taken up HMW-bLf showing perturbed actin structure, and arrowhead points out to the cell with intact actin structure and is without HMW-bLf uptake in 4 h (SW480). Arrows in 4C point out to the beginning of nuclear degradation at 6 h (MDA-MB-231).
Figure 6.
A and B represent the increased caspase-3 activity measurements upon treatment with HMW-bLf in MDA-MB-231 and SW480 cells, respectively. (* P<0.05 and ** P<0.01). Panels C and D are the respective Western blots showing an increase in cleaved caspase-3 expression upon treatments in MDA-MB-231 and SW480 cells.