Limits...
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

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 μg) 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-1α, 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
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: 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 μg) 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-1α, 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

Mentions: 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


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)

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 μg) 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-1α, 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
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: 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 μg) 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-1α, 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
Mentions: 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

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