**5. Formation and purification of fibrillar human serum albumin**

Some diseases like Alzheimer's disease, transmissible spongiform encephalopathies, pancreatic islet amyloidosis, and familial amyloidosis are caused primarily by amyloid-like fibrils aggregation in organs and in the circulation (Jackson & Clarke, 2000). Recently, it has been documented that amyloid-like fibrils are cytotoxic to neuronal cells, BHK-21 cells, SKOV-3, and MCF-7 cancer cells (Gharibyan et al, 2007; Su & Chang, 2001; Zamotin et al, 2006). Whether the fibrillar proteins may be used as anti-cancer drugs in the cancer therapies is largely unclear. We have developed a novel process to convert globular proteins, bovine serum albumin and fibronectin, to fibrillar forms using detergent assisted refolding chromatography (Huang et al, 2009; Huang et al, 2010). This procedure is easier to perform than other methods reported to convert proteins to fibrillar structures such as glycation, sonication, or high temperature incubation (Azakami et al, 2005; Taboada et al, 2006). Fibrillar protein F-BSA induced apoptosis in human breast duct carcinoma cell line T47D, and F-FN induced apoptosis in human breast cancer cell line MCF-7 suggesting that fibrillar proteins may have therapeutic effect in human breast cancer cells. We thus further investigated the effects of the fibrillar form of human serum albumin (F-HSA) on the malignant breast cancer cell lines, TS/A and MDA-MB-231. We chose F-HSA for further study for two reasons: first, because F-HSA is less likely to provoke an immune response in the human body; and second, because HSA is easier to obtain and less costly than FN. We produced F-HSA using the same process as was used to produce F-BSA. In brief, 20 mg of HSA from human serum was dissolved in 10 ml of PBS with 1% SDS (w/v). The HSA solution was sonicated for 5 minutes and subsequently applied to a Superdex-200 column previously equilibrated with the eluting buffer (25 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.1 M NaCl, and 0.05% SDS). The fractions that contained HSA were pooled and dialysed against PBS to remove SDS. The yield of the F-HSA was about 67% (Fig. 1). The F-HSA produced was then tested for fibrillar structure by transmission electron microscopy (TEM). For TEM analysis of F-HSA, 2 mg/ml of protein was applied to a 300-mesh carbon-coated copper

against αvβ3 integrin (Brooks et al, 1994), CNTO 95 against both αvβ3 and αvβ5 integrins (Mullamitha et al, 2007), 17E6 against αvβ3, αvβ5, and αvβ1 integrins (Mitjans et al, 2000), LM609 against v3 integrin, and Tysabri (natalizumab) against α4 integrins (O'Connor, 2007). In addition, 1 integrins possess a RGD-binding region, therefore, based mainly on their RGD containing peptides and RGD peptidomimetics, some small molecule integrin antagonists have also shown potent inhibition of angiogenesis (Kumar et al, 2001). Both fibronectin and its receptor integrin α5β1 directly regulate angiogenesis (Kim et al., 2000). Thus, antagonist(s) of α5β1 integrin might be useful targets for the inhibition of angiogenesis associated with human tumor growth, and neovascular-related ocular and inflammatory diseases (Pasterkamp et al, 2003; Suzuki et al, 2007). Further, our own studies recently found that fibrillar bovine serum albumin (F-BSA) induced apoptosis in human breast duct carcinoma cell line T47D, and fibrillar fibronectin (F-FN) induced apoptosis in human breast cancer cell line MCF-7. F-BSA and F-FN induced BHK-21 cell (baby hamster kidney cell) apoptosis through negatively regulating the integrin/FAK/Akt/GSK-3β signaling pathway and activating SHP-2 and RhoA/ROCK (Huang et al, 2009; Huang et al, 2010). Together these results suggest that inhibition of the 1 integrin signaling pathway may provide a promising therapeutic approach to breast

**5. Formation and purification of fibrillar human serum albumin** 

Some diseases like Alzheimer's disease, transmissible spongiform encephalopathies, pancreatic islet amyloidosis, and familial amyloidosis are caused primarily by amyloid-like fibrils aggregation in organs and in the circulation (Jackson & Clarke, 2000). Recently, it has been documented that amyloid-like fibrils are cytotoxic to neuronal cells, BHK-21 cells, SKOV-3, and MCF-7 cancer cells (Gharibyan et al, 2007; Su & Chang, 2001; Zamotin et al, 2006). Whether the fibrillar proteins may be used as anti-cancer drugs in the cancer therapies is largely unclear. We have developed a novel process to convert globular proteins, bovine serum albumin and fibronectin, to fibrillar forms using detergent assisted refolding chromatography (Huang et al, 2009; Huang et al, 2010). This procedure is easier to perform than other methods reported to convert proteins to fibrillar structures such as glycation, sonication, or high temperature incubation (Azakami et al, 2005; Taboada et al, 2006). Fibrillar protein F-BSA induced apoptosis in human breast duct carcinoma cell line T47D, and F-FN induced apoptosis in human breast cancer cell line MCF-7 suggesting that fibrillar proteins may have therapeutic effect in human breast cancer cells. We thus further investigated the effects of the fibrillar form of human serum albumin (F-HSA) on the malignant breast cancer cell lines, TS/A and MDA-MB-231. We chose F-HSA for further study for two reasons: first, because F-HSA is less likely to provoke an immune response in the human body; and second, because HSA is easier to obtain and less costly than FN. We produced F-HSA using the same process as was used to produce F-BSA. In brief, 20 mg of HSA from human serum was dissolved in 10 ml of PBS with 1% SDS (w/v). The HSA solution was sonicated for 5 minutes and subsequently applied to a Superdex-200 column previously equilibrated with the eluting buffer (25 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.1 M NaCl, and 0.05% SDS). The fractions that contained HSA were pooled and dialysed against PBS to remove SDS. The yield of the F-HSA was about 67% (Fig. 1). The F-HSA produced was then tested for fibrillar structure by transmission electron microscopy (TEM). For TEM analysis of F-HSA, 2 mg/ml of protein was applied to a 300-mesh carbon-coated copper

cancer metastasis.

grid. Excess samples were removed, and the grid was air dried. The protein-bearing grid was negatively stained with 1% (w/v) phosphotungstic acid for 1 minute. Transmission electron micrographs were observed at 20,000–150,000× magnification at 75 kV on a Hitachi H-7000 electron microscope. TEM analysis showed that F-HSA did indeed have a fibril structure (Fig. 2).

Fig. 1. Elution profile of F-HSA from a Superdex-200 column. HSA (2 mg/ml dissolved in PBS containing 1% SDS) was applied to a Superdex-200 column and eluted at a rate of 1 ml/min with a buffer solution containing 0.05% SDS. Arrow shows F-HSA.

Specific binding to Thioflavin T (ThT) is one of the characteristics of amyloid-like proteins. ThT fluorescence assay was, thus, used to identify amyloid-like brils (LeVine, 1999). Like A (1-42), which is known to have fibrillar structure and was used as a positive control, F-HSA obtained from the Superdex-200 column exhibited a gradual dose-dependent increase in ThT fluorescence level (Fig. 3).
