healthmedicinet

BLf induces cytotoxicity and reduces the proliferative ability of MDA-MB-231 and MCF-7
cells

The effect of both Apo-bLf and Fe-bLf on cytotoxicity and proliferation were evaluated
in the breast cancer cells MDA-MB-231 and MCF-7. Cells were treated with bLf for 24
and 48 h at concentrations of 0, 5, 10, 20, 30 and 40 nM. In order to quantify cell
death, Lactate dehydrogenase (LDH) cytotoxicity assays were employed to determine
cytotoxicity.

Apo-bLf was the most effective at inducing cell cytotoxicity with significant increases
in cytotoxicity at concentrations of 20, 30 and 40 nM in MDA-MB-231 (133.05%, p?=?0.003, 112.53%, p?=?0.003 and 110.17%, p?=?0.007, respectively) and MCF-7 cells (68.58%, p?=?0.001, 90.68%, p?=?0.00001 and 99.31%, p?=?0.000006, respectively) after 48 h (Fig. 1a). Furthermore, Apo-bLf demonstrated significant toxicity after 24 h in MCF-7 cells
at concentrations of 20 nM (25.93%, p?=?0.02), 30 nM (40.09%, p?=?0.001) and 40 nM (61.97%, p?=?0.001) (Fig. 1b). Fe-bLf demonstrated significant increases in cytotoxicity in MDA-MB-231 cells at
concentrations of 10, 20 and 30 nM (51.81%, p?=?0.04, 61.76%, p?=?0.01 and 42.01%, p?=?0.048) after 48 h (Fig. 1a) yet no significant cytotoxicity in MCF-7 cells (Fig. 1b).

Fig. 1. Cytotoxicity and proliferation in MDA-MB-231, MCF-7 and MCF-10-2A cells following
Apo-bLf and Fe-bLf treatments. Lactate dehydrogenase assay (LDH) results demonstrating
cytotoxicity in cells after 24 and 48 h Apo and Fe-bLf treatments in MDA-MB-231 (a), MCF-7 (b) and MCF-10-2A cells (e). CyQUANT® assay results represent cell proliferation levels after 24 and 48 h Apo
and Fe-bLf treatments in MDA-MB-231 (c) and MCF-7 cells (d) including high (20% FBS) and low (untreated) controls. Data represented as mean
with?+?SEM (n?=?6). *?=?p??0.05 compared with untreated (0 nM) group, **?=?p??0.01 compared with untreated (0 nM). Statistical analysis performed using Student’s
t-test

CyQUANT® assay results indicated a reduction in proliferation following treatments
with both Apo-bLf and Fe-bLf after 48 h in MDA-MB-231 cells (Fig. 1c). Apo-bLf significantly reduced proliferation at concentrations of 30 and 40 nM (67.30%,
p?=?0.01 and 54.87%, p?=?0.003, respectively) and Fe-bLf at concentrations of 20, 30 and 40 nM (74.86%,
p?=?0.04, 52.89%, p?=?0.003 and 46.40%, p?=?0.008) after 48 h. Furthermore, Apo and Fe-bLf reduced proliferation after 48 h
in MCF-7 cells at 40 nM (53.93%, p?=?0.03 and 51.35%, p?=?0.01 respectively) (Fig. 1d). No significant increase in proliferation was observed in any treatment condition
in either cell line.

Morphological images (Fig. 2a and b) indicated significant cell death after 48 h. Cells in treatment groups showed detachment
from culture surfaces, blebbing and rounded edges. Cell fragmentation and decrease
in number was also observed compared with untreated cells over the same time period.
These effects were observed more frequently with increasing concentration in MDA-MB-231
(Fig. 2a) and MCF-7 (Fig. 2b). These results indicate a time and dose dependant effect of Apo-bLf on MDA-MB-231
and MCF-7 cells in terms of increasing cell cytotoxicity and decreasing cell proliferation.
Furthermore, these results also highlight that Fe-bLf is less effective when compared
with Apo-bLf in these breast cancer cells.

Fig. 2. Cell morphology and internalization of bLf in MDA-MB-231 and MCF-7 cells. a MDA-MB-231 cells after 48 h treatments with both Apo-bLf and Fe-bLf at concentrations
of 20, 30 and 40 nM as well as untreated cells. b MCF-7 cells after 48 h treatments with both Apo-bLf and Fe-bLf at concentrations
of 20, 30 and 40 nM as well as untreated cells. All images at 400X magnification.
Confocal microscope images displaying internalisation of 40 nM Apo-bLf and Fe-bLf
in both MDA-MB-231 (c) and MCF-7 (d) cells after 4 h. Arrows indicate regions on the membrane, cytoplasm and nucleus
where bLf has internalised. Cells stained with DAPI nuclear stain (blue) and immunolabelled
with anti-bovine lactoferrin antibody followed by Alexa-Fluor TRITC conjugated secondary
antibody (red). Scale bars indicate 25 ?m

Apo-bLf and Fe-bLf show no cytotoxic effects in MCF-10-2A mammary epithelial cells

Both Apo-bLf and Fe-bLf were also tested in a normal, non-tumourigenic mammary epithelial
cell line MCF-10-2A. LDH cytotoxicity assays were performed on cells treated for 24
and 48 h at concentrations of 5, 10, 20, 30 and 40 nM. No significant increase in
cytotoxicity was observed after either 24 or 48 h with any of the Apo-bLf and Fe-bLf
treatments (Fig. 1e).

Apo-bLf and Fe-bLf efficiently internalizes into treated cells

BLf is known to internalize into normal and cancer cells through cell surface (membrane)
receptor mediated endocytosis 10], 30], 41], 42]. In order to determine the level of internalisation and localisation of bLf within
breast cancer cells, we performed immunofluorescence. Cells were treated with either
Apo-bLf or Fe-bLf at 40 nM for 4 h. A reduced time period was used to reduce cell
death so that cells could still be visualised by confocal microscopy. Both forms of
bLf internalised into MDA-MB-231 (Fig. 2c) cells with fragmentation displayed in cells treated with Fe?bLf at 40 nM. Both forms
of bLf were localised in the membrane, cytoplasm and nucleus of the cells. Untreated
MCF-7 cells displayed healthy, cluster formation (Fig. 2d). After treatment with both Apo-bLf and Fe-bLf, internalisation of bLf into the membrane,
cytoplasm and nucleus was observed (Fig. 2d). It is apparent that Apo-bLf induces mass cell fragmentation (Fig. 2d) which is due to the high cytotoxic potential of Apo-bLf. Cell clusters were also
much smaller in treated cells compared with untreated cells, indicating the effect
of treatment on cell viability following bLf internalisation.

Apo-bLf and Fe-bLf treatments decrease the migration and invasion potential of breast
cancer cells

As migration and invasion are key properties of cancer cells leading to metastases
and secondary tumour sites within the body, assays were performed to determine the
effects of bLf in regards to these properties. Migration assays indicated a significant
reduction in the capacity of MDA-MB-231 and MCF-7 (Fig. 3a) cells to migrate through a porous membrane after treatment for 24 h with each Apo-bLf
and Fe-bLf at concentrations of 5 and 10 nM. The greatest reduction in migration was
observed with 10 nM Apo-bLf with 26.45% (p?=?0.001) of MDA-MB-231 and 38.78% (p?=?0.001) of MCF-7 cells migrating. Invasion assays (Fig. 3b and c) were performed with ECM. The same trend was observed in MDA-MB-231 cells with 29.85%
(p?=?0.001) of cells invading after treatment with 10 nM Apo-bLf. MCF-7 showed a large
reduction with 10 nM Apo-bLf (50.00%, p?=?0.003) however the greatest reduction in invasion was with 10 nM Fe-bLf treatments
with 26.25% (p?=?0.00008) invasion.

Fig. 3. Effect on migration and invasion capacity of MDA-MB-231 and MCF-7 cells after treatment
with bLf. Migration of MDA-MB-231 and MCF-7 (a) cells after bLf treatments for 24 h represented as a percentage of untreated (1%
FBS) control migration. Invasion of MDA-MB-231 and MCF-7 cells (b) after bLf treatments for 24 h represented as a percentage of untreated (1% FBS)
control invasion. *?=?p??0.05 compared with 1% FBS group. Representative images (250X magnification) of
invaded cells stained with crystal violet (c)

Both bLf forms induce significant apoptosis at a different rates in MDA-MB-231 and
MCF-7 cells

In order to determine if cell death was caused by apoptosis, Annexin-V-Fluos labelling
was performed on each MDA-MB-231 and MCF-7 cells (Fig. 4). Cell were treated with Apo-bLf or Fe-bLf at 20 and 40 nM for 24 h. Labelling was
detected via Flow cytometry and gating was performed on both dual (Fig. 4c) and single channel plots (Annexin-V and Propidium Iodide). Results indicated increases
in apoptotic cell death in MDA-MB-231 cells treated with bLf with significant increased
total apoptosis in cells treated with Apo-bLf at 40 nM (23.30%, p?=?0.03) (Fig. 4a). Results from MCF-7 cells showed increased apoptosis in all treatment groups with
significant apoptosis observed in cells treated with 40 nM Apo-bLf (46.85%, p?=?0.05) and Fe-bLf at 20 nM (53.60% p?=?0.02) and 40 nM (65.15%, p?=?0.04) (Fig. 4b). Furthermore, decreased viable cells were observed in MDA-MB-231 cells after Apo-bLf
at 40 nM and in both Fe-bLf treatment groups and in all MCF-7 treatment groups however
no significant increase in necrotic cells was observed in any of the treatment groups
(Table 1). This indicated that both Apo-bLf and Fe-bLf were inducing apoptosis in each cell
line.

Fig. 4. BLf-induced apoptosis in MDA-MB-231 and MCF-7 cells. Annexin-V-Fluos staining detected
apoptosis in MDA-MB-231 and MCF-7 cells following bLf treatments. Cells were treated
for 24 h with Apo-bLf and Fe-bLf at concentrations of 20 nM and 40 nM. a Total apoptotic MDA-MB-231 and b MCF-7 cells following bLf treatments. Flow cytometry plots of MDA-MB-231 and MCF-7
cells (c). Q1: Necrotic cells, Q2: Late apoptotic/Dead cells, Q3: Viable cells, Q4: Early
apoptotic cells. Total apoptotic cells were the sum of early and late apoptotic cell
populations. Data represented as means +SEM, student t-test used for statistical analysis

Table 1. Annexin-V results following bLf treatments in breast cancer cells

To determine the mechanism of action of apoptosis induced by bLf, apoptotic protein
arrays were performed on each MDA-MB-231 (Fig. 5) and MCF-7 (Fig. 6) cells following treatments with Apo-bLf and Fe-bLf for 24 h. Results indicated different
mechanisms of action between the two forms of bLf and between the cells. MDA-MB-231
showed significant reduction in survivin expression along with increased expression
of HTRA2 with each Apo-bLf and Fe-bLf (Fig. 5b). In addition, Apo-bLf reduced cIAP2 and SMAC was increased with Fe-bLf (Fig. 5b). In MCF-7 cells, HTRA2 and SMAC were both significantly increased with both forms
of bLf, with a stronger effect observed with Apo-bLf (Fig. 6b). These results indicate that bLf is having an impact on the IAP mechanism, allowing
apoptosis to progress by activating SMAC and HTRA2 which subsequently bind IAP proteins,
preventing their inhibition of the caspase cascade.

Fig. 5. Apoptosis protein array MDA-MB-231 breast cancer cells. Apoptosis protein array results
following incubation of MDA-MB-231 cell lysate after treatments with Apo-bLf and Fe-bLf
at 40 nM for 24 h. Cell lysate (250 ?g) incubated with nitrocellulose membrane pre-labelled
with capture antibodies (duplicate spots) and detected via chemiluminescence. a Bcl-2 family proteins Bad, Bax, Bcl-2 and Bcl-xL. b Inhibitor of apoptosis (IAP) proteins cIAP-1 and 2, Livin, Survivin and XIAP, and
inhibitors SMAC and HTRA2. c Extrinsic pathway proteins and receptors TRAIL 1 and 2, FADD, Fas and TNF R1. d Pro-apoptotic proteins Pro-caspase-3, Cleaved Caspase-3 and Cytochrome C, anti-apoptotic
proteins Catalase, PON2 and Clusterin. e Cell stress proteins HIF-1?, HMOX1, HMOX2, HSP27, HSP60 and HSP70. f Claspin, p21, p27, phospho-p53 (S15), phospho-p53 (S46), phospho-p53 (S392) and phospho-Rap17
(S635). Average density determined using ImageJ software and fold change calculated
compared with untreated. Data represented as means with?+?SEM. *?=?p??0.05, **?=?p??0.01, ***?=?p??0.001 compared with the untreated group. Statistical analysis was performed via
Student’s t-test

Fig. 6. Apoptosis protein array MCF-7 breast cancer cells. Apoptosis protein array results
following incubation of MCF-7 cell lysate after treatments with Apo-bLf and Fe-bLf
at 40 nM for 24 h. Cell lysate (250 ?g) incubated with nitrocellulose membrane pre-labelled
with capture antibodies (duplicate spots) and detected via chemiluminescence. a Bcl-2 family proteins Bad, Bax, Bcl-2 and Bcl-xL. b Inhibitor of apoptosis (IAP) proteins cIAP-1 and 2, Livin, Survivin and XIAP, and
inhibitors SMAC and HTRA2. c Extrinsic pathway proteins and receptors TRAIL 1 and 2, FADD, Fas and TNF R1. d Pro-apoptotic proteins Pro-caspase-3, Cleaved Caspase-3 and Cytochrome C, anti-apoptotic
proteins Catalase, PON2 and Clusterin. e Cell stress proteins HIF-1?, HMOX1, HMOX2, HSP27, HSP60 and HSP70. f Claspin, p21, p27, phospho-p53 (S15), phospho-p53 (S46), phospho-p53 (S392) and phospho-Rap17
(S635). Average density determined using ImageJ software and fold change calculated
compared with untreated. Data represented as means with?+?SEM. *?=?p??0.05, **?=?p??0.01, ***?=?p??0.001 compared with untreated group. Statistical analysis was performed via Student’s
t-test

Furthermore, results indicate apoptosis in MDA-MB-231 cells occurring via the intrinsic
pathway with a significant reduction in expression of anti-apoptotic proteins in Bcl-2
(Apo-bLf) and PON2 (both forms of bLf) (Fig. 5a). A reduction in extrinsic proteins FADD and Fas (Fig. 5c) was also observed however the opposite occurring in MCF-7 (Fig. 6c) with extrinsic proteins FADD and TNF R1 each upregulated with both forms of bLf.
Furthermore, MCF-7 cell also show significant increases of pro-apoptotic proteins
Bad and cytochrome C (Fig. 6a d).

Cell stress proteins in MDA-MB-231 cells, HMOX1 and HSP60 were both reduced with Apo-bLf
(Fig. 5e) as were cell cycle regulators claspin, p21, p27, phospho-p53 (S15, S46 and S392)
and phospho-Rad17 (S635) (Fig. 5?f). Claspin was also significantly reduced with both Apo-bLf and Fe-bLf (Fig. 5?f). In MCF-7 cells, cell stress proteins were increased by both Apo-bLf and Fe-bLf
including HIF1?, HMOX2, HSP27, HSP60 and HSP70 (Fig. 6e). Finally, cell cycle regulator p21 was increased in MCF-7 cells with Apo-bLf, Claspin
with Fe-bLf and phospho-p53 S15 and S46 were increased with both Apo-bLf and Fe-bLf
(Fig. 6?f).

Apo-bLf and Fe-bLf forms induce increase in caspase cleavage

Following the observations form the Apoptotic array that bLf modulated aspects of
both the intrinsic and extrinsic pathways, as well as increases in cleaved caspase-3,
the effect of bLf on cleaved initiator caspase-8 (extrinsic) and caspase-9 (intrinsic)
were assed via Western blotting. In addition, pro and cleaved effector caspase-3 were
also analysed. MDA-MB-231 and MCF-7 cells were both treated with 40 nM Apo-bLf and
Fe-bLf for 24 h. Following treatments, cells were lysed with RIPA buffer and lysates
were separated via SDS-PAGE followed by Western blotting. Identical gels were run
and Western blotting performed for GAPDH. Fold change was calculated by determining
band densities via ImageJ software, normalizing proteins with GAPDH and calculating
fold change relative to untreated cells.

Cleaved caspases-8 and ?9 were increased with 40 nM Apo-bLf in MDA-MB-231 cells (Fig. 7a). Fe-bLf however did not show increases in either cleaved caspase-8 or ?9. Apo-bLf
increased the active fragment (18 kDa) of cleaved caspase-8 by 2.3 fold compared with
untreated cells. Most notably however, cleaved caspase-9 was increased by 8.9 fold
with Apo-bLf.

Fig. 7. BLf induces caspases-3, ?8 and ?9 cleavage in breast cancer cell lines. Western blot
analysis of cleaved caspases-8 and ?9 in MDA-MB-231 (a) and MCF-7 (b) cells following treatment with 40 nM Apo-bLf and Fe-bLf for 24 h. Western blot analysis
of pro and cleaved caspase-3 in MDA-MB-231 (c) and MCF-7 (d) cells following treatment with 40 nM Apo-bLf and Fe-bLf for 24 h. Lysates were collected
and 100 ?g loaded and run on standard SDS-PAGE followed by transfer to PVDF membranes.
Membranes were then blocked with 1% skim milk followed by incubation with anti-cleaved
caspases-3, ?8 and ?9 primary antibodies (Cell Signalling) and anti-mouse secondary
antibody (Sigma-Aldrich). Membranes were viewed via XRS camera. Band densities determined
by ImageJ software and compared with untreated (0 nM). Separate, identical gels were
run for GAPDH (Santa Cruz) which were used to normalize band densities. Band density
analysis was performed using ImageJ software. Fold change was calculated relative
to untreated cells. Relative fold change values per band are indicated below blots
as well as plotted on graphs

In MCF-7 cells, both cleaved caspases-8 and ?9 were increased with 40 nM Apo-bLf and
Fe-bLf (Fig. 7b). The effects of Apo-bLf and Fe-bLf were similar in their effects on cleaved caspases-8
and ?9. Active caspase-8 (18 kDa fragment) was increased to 1.5 and 1.3 fold with
Apo-bLf and Fe-bLf respectively while the remaining, unused fragments (41 and 43 kDa)
from the pro-caspase was increased to 7.7 and 7.5 fold respectively indicating large
levels of cleavage. Furthermore, cleaved caspase-9 was increased by 2.6 and 2.5 fold
with Apo-bLf and Fe-bLf respectively. Analysis of effector caspase-3 was then performed
in MDA-MB-231 (Fig. 7c) and MCF-7 (Fig. 7d) cells. In MDA-MB-231 cells, a large decrease in pro-caspase-3 was observed with
both Apo-bLf and Fe-bLf with expression reduced to 0.3 and 0.4 fold respectively of
untreated cells. This was taken to indicate caspase cleavage and this was further
confirmed by Western blot analysis of cleaved caspase-3 levels in MDA-MB-231 cells.
Cleaved caspase-3 levels increased by 2.1 fold in cells treated with Apo-bLf and slight
increase by 1.3 fold in cells treated with Fe-bLf. In MCF-7 cells, pro-caspase-3 increased
with both Apo-bLf (2.1 fold) and Fe-bLf (2.2 fold). Cleaved caspase-3 was relatively
unchanged in Fe-bLf and a slight increase observed with Apo-bLf (1.3 fold) (Fig. 7d).

BLf treatment leads to time and dose dependent down-regulation of survivin protein
expression in both MDA-MB-231 and MCF-7 cells

As survivin is a key IAP protein in cancer and significant reduction was observed
in MDA-MB-231 cells in apoptotic arrays following 24 h treatments (Fig. 5b), Western blotting was performed on a greater concentration range for 48 h in each
cell line. Cells were treated with Apo-bLf and Fe-bLf at 20, 30 and 40 nM and cell
lysates were blotted for survivin. Survivin was detected in both untreated lysates
(Fig. 8). Survivin was greatly reduced in all treatment groups with reduction in MDA-MB-231
cells to 0.4 and 0.1 fold with 20 nM Apo-bLf and Fe-bLf respectively. Survivin expression
in MCF-7 cells was reduced to 0. 5 and 0.3 fold with 20 nM Apo-bLf and Fe-bLf and
to 0.1 fold that of untreated cells with 30 nM Apo-bLf. No survivin was detected in
MDA-MB-231 cells with either form of bLf at concentrations of 30 and 40 nM. Survivin
was also not detected in MCF-7 cells after treatment with Fe-bLf at 30 nM and both
forms at 40 nM. Western blotting confirmed findings from the apoptotic array and provided
important evidence for the mechanism of bLf in terms of its apoptosis-inducing potential.

Fig. 8. BLf reduces survivin expression in breast cancer cell lines. Western blotting for
survivin of MDA-MB-231 and MCF-7 cell lysates following treatments for 48 h with Apo
and Fe-bLf at 20, 30 and 40 nM. Lysates were collected and 100 ?g loaded and run on
standard SDS-PAGE followed by transfer to PVDF membranes. Membranes were then blocked
with 1% skim milk followed by incubation with anti-survivin primary antibody (Santa
Cruz) and anti-mouse secondary antibody (Sigma-Aldrich). Membranes were viewed via
XRS camera. Band densities determined by ImageJ software and compared with untreated
(0 nM). Separate, identical gels were run for Tubulin which were used to normalize
band densities. Band densities determined by ImageJ software and compared with untreated
(0 nM). Separate, identical gels were run for Tubulin (Santa Cruz) which were used
to normalize band densities. Band density analysis was performed using ImageJ software.
Fold change was calculated relative to untreated cells. Relative fold change values
per band are indicated below blots as well as plotted on graphs