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Iron-free and iron-saturated bovine lactoferrin inhibit survivin expression and differentially modulate apoptosis in breast cancer.

Gibbons JA, Kanwar JR, Kanwar RK - BMC Cancer (2015)

Bottom Line: Iron binding, naturally occurring protein bovine lactoferrin (bLf) has attracted attention as a safe anti-cancer agent capable of inducing apoptosis.Apo-bLf induced significantly greater cytotoxicity and reduction in cell proliferation in both cancer cells showing a time and dose dependent effect.Key apoptotic molecules including p53, Bcl-2 family proteins, IAP members and their inhibitors were significantly modulated by both forms of bLf, though differentially in each cell line.

View Article: PubMed Central - PubMed

Affiliation: Nanomedicine - Laboratory for Immunology and Molecular Biomedical Research, Molecular and Medical Research Facility, School of Medicine, Faculty of Health, Deakin University, Geelong, Victoria, Australia. jgib@deakin.edu.au.

ABSTRACT

Background: Iron binding, naturally occurring protein bovine lactoferrin (bLf) has attracted attention as a safe anti-cancer agent capable of inducing apoptosis. Naturally, bLf exists partially saturated (15-20%) with Fe(3+) however, it has been demonstrated that manipulating the saturation state can enhance bLf's anti-cancer activities.

Methods: Apo-bLf (Fe(3+) free) and Fe-bLf (>90% Fe(3+) Saturated) were therefore, tested in MDA-MB-231 and MCF-7 human breast cancer cells in terms of cytotoxicity, proliferation, migration and invasion. Annexin-V Fluos staining was also employed in addition to apoptotic protein arrays and Western blotting to determine the specific mechanism of bLf-induced apoptosis with a key focus on p53 and inhibitor of apoptosis proteins (IAP), specifically survivin.

Results: Apo-bLf induced significantly greater cytotoxicity and reduction in cell proliferation in both cancer cells showing a time and dose dependent effect. Importantly, no cytotoxicity was detected in normal MCF-10-2A cells. Both forms of bLf significantly reduced cell invasion in cancer cells. Key apoptotic molecules including p53, Bcl-2 family proteins, IAP members and their inhibitors were significantly modulated by both forms of bLf, though differentially in each cell line. Most interestingly, both Apo-bLf and Fe-bLf completely inhibited the expression of survivin protein (key IAP), after 48 h at 30 and 40 nM in cancer cells.

Conclusions: The capacity of these forms of bLf to target survivin expression and modulation of apoptosis demonstrates an exciting potential for bLf as an anti-cancer therapeutic in the existing void of survivin inhibitors, with a lack of successful inhibitors in the clinical management of cancer.

No MeSH data available.


Related in: MedlinePlus

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 μg 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
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Fig8: 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 μg 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

Mentions: 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


Iron-free and iron-saturated bovine lactoferrin inhibit survivin expression and differentially modulate apoptosis in breast cancer.

Gibbons JA, Kanwar JR, Kanwar RK - BMC Cancer (2015)

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 μg 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
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4440599&req=5

Fig8: 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 μg 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
Mentions: 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

Bottom Line: Iron binding, naturally occurring protein bovine lactoferrin (bLf) has attracted attention as a safe anti-cancer agent capable of inducing apoptosis.Apo-bLf induced significantly greater cytotoxicity and reduction in cell proliferation in both cancer cells showing a time and dose dependent effect.Key apoptotic molecules including p53, Bcl-2 family proteins, IAP members and their inhibitors were significantly modulated by both forms of bLf, though differentially in each cell line.

View Article: PubMed Central - PubMed

Affiliation: Nanomedicine - Laboratory for Immunology and Molecular Biomedical Research, Molecular and Medical Research Facility, School of Medicine, Faculty of Health, Deakin University, Geelong, Victoria, Australia. jgib@deakin.edu.au.

ABSTRACT

Background: Iron binding, naturally occurring protein bovine lactoferrin (bLf) has attracted attention as a safe anti-cancer agent capable of inducing apoptosis. Naturally, bLf exists partially saturated (15-20%) with Fe(3+) however, it has been demonstrated that manipulating the saturation state can enhance bLf's anti-cancer activities.

Methods: Apo-bLf (Fe(3+) free) and Fe-bLf (>90% Fe(3+) Saturated) were therefore, tested in MDA-MB-231 and MCF-7 human breast cancer cells in terms of cytotoxicity, proliferation, migration and invasion. Annexin-V Fluos staining was also employed in addition to apoptotic protein arrays and Western blotting to determine the specific mechanism of bLf-induced apoptosis with a key focus on p53 and inhibitor of apoptosis proteins (IAP), specifically survivin.

Results: Apo-bLf induced significantly greater cytotoxicity and reduction in cell proliferation in both cancer cells showing a time and dose dependent effect. Importantly, no cytotoxicity was detected in normal MCF-10-2A cells. Both forms of bLf significantly reduced cell invasion in cancer cells. Key apoptotic molecules including p53, Bcl-2 family proteins, IAP members and their inhibitors were significantly modulated by both forms of bLf, though differentially in each cell line. Most interestingly, both Apo-bLf and Fe-bLf completely inhibited the expression of survivin protein (key IAP), after 48 h at 30 and 40 nM in cancer cells.

Conclusions: The capacity of these forms of bLf to target survivin expression and modulation of apoptosis demonstrates an exciting potential for bLf as an anti-cancer therapeutic in the existing void of survivin inhibitors, with a lack of successful inhibitors in the clinical management of cancer.

No MeSH data available.


Related in: MedlinePlus