**4. Surface membrane integrins as potential drug-discovery targets**

It is well known that cell activation, migration, proliferation, and differentiation require direct contact between cells and the extracellular matrix (ECM). Cell-to-cell and cell-tomatrix interactions are mediated by the integrin, selectin, cadherin and/or immunoglobulin families and several studies have focused on investigating cancer therapies based on the integrin superfamily. Integrin expression on cancer cells is frequently associated with cancer progression and metastasis; therefore, targeting small-molecule antagonists of the integrin superfamily provides an opportunity to suppress cancer development and metastasis (Mullamitha et al, 2007). β1 integrin, which frequently aberrantly expressed in human breast carcinomas, has been verified to play a central role in metastasis and contribute to growth factor receptor signaling. Inhibition of the 1 integrin signaling pathway has been shown to abolish the formation of metastasis in breast and gastric cancer models. Additionally, the 1 integrin signaling pathway also plays a significant role in mediating resistance to cytotoxic chemotherapies by enhancing cell survival in hematologic malignancies, lung, and breast cancers (Lu et al, 2008). Recent studies have shown that α1β1, α2β1, and α3β1 integrins regulate hepatocarcinoma cell invasion, angiogenesis of human squamous cell carcinoma, and increase migration and invasion of malignant glioma, melanoma and mammary adenocarcinoma cells, respectively. Expression of α5β1 integrin in colon cancer cells decreases HER-2-mediated proliferation (Kuwada et al, 2005). Loss of the α7β1 integrin in melanoma increases highly tumorigenic and metastatic phenotypes (Ziober et al, 1999). Several preclinical and clinical trials have shown that some integrin targeting antibodies can effectively block tumor growth and metastasis. These antibodies include MEDI-522 (vitaxin)

Fibrillar Human Serum Albumin Suppresses Breast Cancer Cell Growth and Metastasis 427

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

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

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

Previously, we demonstrated that F-BSA and F-FN induced apoptosis in the less malignant T47D and MCF-7 breast cancer cell lines, respectively (Huang et al, 2009; Huang et al, 2010). In this study, we examine whether F-HSA induced cytotoxicity in the more malignant breast

ml/min with a buffer solution containing 0.05% SDS. Arrow shows F-HSA.

in ThT fluorescence level (Fig. 3).

**6. Effects of F-HSA on cell viability** 

structure (Fig. 2).

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 cancer metastasis.
