Limits...
Albumin fibrillization induces apoptosis via integrin/FAK/Akt pathway.

Huang CY, Liang CM, Chu CL, Liang SM - BMC Biotechnol. (2009)

Bottom Line: We used globular bovine serum albumin (BSA) as a model protein to verify the properties of the fibrillar protein, investigated its cellular effects and studied the signaling cascade induced by the fibrillar protein.The column pore size was more important than column matrix composite in fibril formation.We report on a novel process for converting globular proteins into fibrillar form to cause apoptosis by modulating the integrin/FAK/Akt/GSK-3beta/caspase-3 signaling pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biotechnology, National Cheng Kung University, Tainan, Taiwan. l6892106@ccmail.ncku.edu.tw

ABSTRACT

Background: Numerous proteins can be converted to amyloid-like fibrils to increase cytotoxicity and induce apoptosis, but the methods generally require a high concentration of protein, vigorous shaking, or fibril seed. As well, the detailed mechanism of the cytotoxic effects is not well characterized. In this study, we have developed a novel process to convert native proteins into the fibrillar form. We used globular bovine serum albumin (BSA) as a model protein to verify the properties of the fibrillar protein, investigated its cellular effects and studied the signaling cascade induced by the fibrillar protein.

Results: We induced BSA, a non-cytotoxic globular protein, to become fibril by a novel process involving Superdex-200 column chromatography in the presence of anionic or zwittergenic detergent(s). The column pore size was more important than column matrix composite in fibril formation. The fibrillar BSA induced apoptosis in BHK-21 cell as well as breast cancer cell line T47D. Pre-treating cells with anti-integrin antibodies blocked the apoptotic effect. Fibrillar BSA, but not globular BSA, bound to integrin, dephosphorylated focal adhesion kinase (FAK), Akt and glycogen synthase kinase-3beta (GSK-3beta).

Conclusion: We report on a novel process for converting globular proteins into fibrillar form to cause apoptosis by modulating the integrin/FAK/Akt/GSK-3beta/caspase-3 signaling pathway. Our findings may be useful for understanding the pathogenesis of amyloid-like fibrils and applicable for the development of better therapeutic agents that target the underlying mechanism(s) of the etiologic agents.

Show MeSH

Related in: MedlinePlus

Evaluation of the apoptotic effect of fibrillar BSA. (A) BHK-21 cells were incubated with 1 μM G-BSA (BSA) or F-BSA (BSA-S200) for 3 h. The cells were observed under a fluorescence microscope, and their nuclei were stained with DAPI (magnification in all panels, ×400). (B) BHK-21 cells were incubated with 40 μM Aβ25–35 for 3 h. The cells were observed under a fluorescence microscope, and their nuclei were stained with DAPI (magnification in all panels, ×400). (C). BHK-21 cells were cultured with 0.8 μM G-BSA (BSA) or F-BSA (BSA-S200) for 15 h in serum-free medium, then underwent caspase-3 activity analysis measured by fluorogenic substrate as described under "Methods". Data are the mean ± SD of three experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2630307&req=5

Figure 4: Evaluation of the apoptotic effect of fibrillar BSA. (A) BHK-21 cells were incubated with 1 μM G-BSA (BSA) or F-BSA (BSA-S200) for 3 h. The cells were observed under a fluorescence microscope, and their nuclei were stained with DAPI (magnification in all panels, ×400). (B) BHK-21 cells were incubated with 40 μM Aβ25–35 for 3 h. The cells were observed under a fluorescence microscope, and their nuclei were stained with DAPI (magnification in all panels, ×400). (C). BHK-21 cells were cultured with 0.8 μM G-BSA (BSA) or F-BSA (BSA-S200) for 15 h in serum-free medium, then underwent caspase-3 activity analysis measured by fluorogenic substrate as described under "Methods". Data are the mean ± SD of three experiments.

Mentions: Since amyloid-like fibrils are cytotoxic to neuronal cells [20-23], we examined whether fibrillar BSAs induced by our method are cytotoxic to cells, we treated BHK-21 cells with various concentrations of BSA-S200, BSA-Zwit, or BSA-HW55S in serum-free medium for 8 h. BSA-S200, BSA-Zwit, and BSA-HW55S were all cytotoxic to cells in a dose-dependent manner (Fig. 3A). BSA-Zwit exhibited the strongest cytotoxicity among all tested. At 0.5 μM concentration, it induced almost 100% cytotoxicity, whereas BSA-S200 induced 35% and BSA-HW55S 10% cytotoxicity. The IC50 for BSA-Zwit, BSA-S200 and BSA-HW55S was 0.2, 0.75 and more than 10 μM, respectively. As controls, two globular proteins, BSA and BSA-S75 induced little, if any, cytotoxicity to cells (Fig. 3A). Interestingly, the cytotoxicity induced by all fibril BSAs (F-BSAs) in BHK-21 cells was stronger than amyloid Aβ25–35 which induced a mere 10% cytotoxicity at concentrations as high as 40 μM after 8 h incubation (Fig. 3B). Significant number of cell death induced by Aβ25–35 could be observed after 24 h incubation (Fig. 3C). Pre-treating BHK-21 cells with increasing concentrations of globular BSA (G-BSA) did not reverse the cytotoxicity induced by BSA-S200 (Fig. 3D). To examine whether F-BSA-induced cytotoxicity was related to cellular apoptosis, DAPI staining and measurement of caspase-3 activity were performed. Our results revealed that F-BSA induced nuclei condensation (Fig. 4A) and increased caspase-3 activity (Fig. 4C) as compared with G-BSA and Aβ25–35 (Fig. 4B). Taken together, these results suggest that treatment with F-BSA induces cytotoxicity and apoptosis of cells and this effect of F-BSA is not reversed by treatment with G-BSA.


Albumin fibrillization induces apoptosis via integrin/FAK/Akt pathway.

Huang CY, Liang CM, Chu CL, Liang SM - BMC Biotechnol. (2009)

Evaluation of the apoptotic effect of fibrillar BSA. (A) BHK-21 cells were incubated with 1 μM G-BSA (BSA) or F-BSA (BSA-S200) for 3 h. The cells were observed under a fluorescence microscope, and their nuclei were stained with DAPI (magnification in all panels, ×400). (B) BHK-21 cells were incubated with 40 μM Aβ25–35 for 3 h. The cells were observed under a fluorescence microscope, and their nuclei were stained with DAPI (magnification in all panels, ×400). (C). BHK-21 cells were cultured with 0.8 μM G-BSA (BSA) or F-BSA (BSA-S200) for 15 h in serum-free medium, then underwent caspase-3 activity analysis measured by fluorogenic substrate as described under "Methods". Data are the mean ± SD of three experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2630307&req=5

Figure 4: Evaluation of the apoptotic effect of fibrillar BSA. (A) BHK-21 cells were incubated with 1 μM G-BSA (BSA) or F-BSA (BSA-S200) for 3 h. The cells were observed under a fluorescence microscope, and their nuclei were stained with DAPI (magnification in all panels, ×400). (B) BHK-21 cells were incubated with 40 μM Aβ25–35 for 3 h. The cells were observed under a fluorescence microscope, and their nuclei were stained with DAPI (magnification in all panels, ×400). (C). BHK-21 cells were cultured with 0.8 μM G-BSA (BSA) or F-BSA (BSA-S200) for 15 h in serum-free medium, then underwent caspase-3 activity analysis measured by fluorogenic substrate as described under "Methods". Data are the mean ± SD of three experiments.
Mentions: Since amyloid-like fibrils are cytotoxic to neuronal cells [20-23], we examined whether fibrillar BSAs induced by our method are cytotoxic to cells, we treated BHK-21 cells with various concentrations of BSA-S200, BSA-Zwit, or BSA-HW55S in serum-free medium for 8 h. BSA-S200, BSA-Zwit, and BSA-HW55S were all cytotoxic to cells in a dose-dependent manner (Fig. 3A). BSA-Zwit exhibited the strongest cytotoxicity among all tested. At 0.5 μM concentration, it induced almost 100% cytotoxicity, whereas BSA-S200 induced 35% and BSA-HW55S 10% cytotoxicity. The IC50 for BSA-Zwit, BSA-S200 and BSA-HW55S was 0.2, 0.75 and more than 10 μM, respectively. As controls, two globular proteins, BSA and BSA-S75 induced little, if any, cytotoxicity to cells (Fig. 3A). Interestingly, the cytotoxicity induced by all fibril BSAs (F-BSAs) in BHK-21 cells was stronger than amyloid Aβ25–35 which induced a mere 10% cytotoxicity at concentrations as high as 40 μM after 8 h incubation (Fig. 3B). Significant number of cell death induced by Aβ25–35 could be observed after 24 h incubation (Fig. 3C). Pre-treating BHK-21 cells with increasing concentrations of globular BSA (G-BSA) did not reverse the cytotoxicity induced by BSA-S200 (Fig. 3D). To examine whether F-BSA-induced cytotoxicity was related to cellular apoptosis, DAPI staining and measurement of caspase-3 activity were performed. Our results revealed that F-BSA induced nuclei condensation (Fig. 4A) and increased caspase-3 activity (Fig. 4C) as compared with G-BSA and Aβ25–35 (Fig. 4B). Taken together, these results suggest that treatment with F-BSA induces cytotoxicity and apoptosis of cells and this effect of F-BSA is not reversed by treatment with G-BSA.

Bottom Line: We used globular bovine serum albumin (BSA) as a model protein to verify the properties of the fibrillar protein, investigated its cellular effects and studied the signaling cascade induced by the fibrillar protein.The column pore size was more important than column matrix composite in fibril formation.We report on a novel process for converting globular proteins into fibrillar form to cause apoptosis by modulating the integrin/FAK/Akt/GSK-3beta/caspase-3 signaling pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biotechnology, National Cheng Kung University, Tainan, Taiwan. l6892106@ccmail.ncku.edu.tw

ABSTRACT

Background: Numerous proteins can be converted to amyloid-like fibrils to increase cytotoxicity and induce apoptosis, but the methods generally require a high concentration of protein, vigorous shaking, or fibril seed. As well, the detailed mechanism of the cytotoxic effects is not well characterized. In this study, we have developed a novel process to convert native proteins into the fibrillar form. We used globular bovine serum albumin (BSA) as a model protein to verify the properties of the fibrillar protein, investigated its cellular effects and studied the signaling cascade induced by the fibrillar protein.

Results: We induced BSA, a non-cytotoxic globular protein, to become fibril by a novel process involving Superdex-200 column chromatography in the presence of anionic or zwittergenic detergent(s). The column pore size was more important than column matrix composite in fibril formation. The fibrillar BSA induced apoptosis in BHK-21 cell as well as breast cancer cell line T47D. Pre-treating cells with anti-integrin antibodies blocked the apoptotic effect. Fibrillar BSA, but not globular BSA, bound to integrin, dephosphorylated focal adhesion kinase (FAK), Akt and glycogen synthase kinase-3beta (GSK-3beta).

Conclusion: We report on a novel process for converting globular proteins into fibrillar form to cause apoptosis by modulating the integrin/FAK/Akt/GSK-3beta/caspase-3 signaling pathway. Our findings may be useful for understanding the pathogenesis of amyloid-like fibrils and applicable for the development of better therapeutic agents that target the underlying mechanism(s) of the etiologic agents.

Show MeSH
Related in: MedlinePlus