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

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

Fibrillar Human Serum Albumin Suppresses Breast Cancer Cell Growth and Metastasis 429

Fig. 3. ThT fluorescence assay of F-HSA. For fluorescence measurements, increasing

the corresponding measurements. A (1-42) was used as a positive control.

Fig. 4. Effect of F-HSA on viability of TS/A (A) and MDA-MB-231 cells (B).

**0 0.1 0.2 0.4 0.8 1**

**F-HSA (µM)**

Cell viability (%)

concentrations of proteins were incubated with 20 μM ThT for 1 h at room temperature, and fluorescence was measured in triplicate on a Wallac Victor2 1420 Multilabel Counter (Perkin Elmer Life Science, Waltham, MA, USA). Excitation and emission wavelengths were 430 nm and 486 nm, respectively. ThT background signal from buffer solution was subtracted from

Cell viability (%)

(a) (b)

To understand the effects of F-HSA on cell morphology and MET in TS/A cells and MDA-MB-231 cells, breast cancer cells were treated low concentrations of F-HSA and cell morphology was observed under light microscopy. F-HSA induced a morphological alteration in cells, from a fibroblast-like shape to a round shape (Fig. 5). We also examined whether F-HSA suppressed breast cancer-cell migration at non-cytotoxic concentrations by

**0 0.1 0.2 0.4 0.8 1**

**F-HSA (µM)**

cancer cell lines, TS/A and MDA-MB-231, using a 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-colorimetry assay to measure the cell viability (MERCK, Darmstadt, Germany). TS/A, a murine mammary adenocarcinoma cell line that is estrogen dependent, was cultured in Dulbecco's modified Eagle's medium (DMEM; GIBCO); and MDA-MB-231 (ATCC **HTB-26™**), a metastatic human breast cancer cell line that is estrogen independent, was cultured in DMEM/F12 medium (GIBCO). In brief, 2 104 breast cancer cells were incubated in serum-free medium and treated with serial dilutions of F-HSA. After incubation for 24 hours to allow the drug to take effect, 10 l MTT solution was added to each well. After incubation at 37°C in 5% CO2 for another 2 hours to allow the MTT solution to be metabolized, formazan (MTT metabolic product) was resuspended in 200 ul DMSO. Finally, the proportions of surviving cells were determined by optical density (570 nm test wavelength, 630 nm reference wavelength). The percentage of surviving cells was calculated as (O.D.treatment/O.D.control) 100%, and the percentage of growth inhibition was calculated as [1 - (O.D.treatment/O.D.control) ] 100%. IC50 value is the concentration at which the reagent produces 50% inhibition of cellular viability. F-HSA inhibited growth of the breast cancer cell lines TS/A and MDA-MB-231 in a dose dependent manner with IC50 values of 0.15 and 0.48 μM, respectively (Fig. 4). F-HSA at concentrations over 0.4 μM induced dose-dependent cytotoxicity in both TS/A cells and MDA-MB-231 cells, whereas concentrations of 0.1-0.2 μM did not affect cell viability significantly.

Fig. 2. Ultra-structures of F-HSA were observed by TEM. F-HSA was applied to a 300-mesh carbon-coated copper grid then the grid was air-dried. The F-HSA-bearing grid was negatively stained with 1% (w/v) phosphotungstic acid. Finally, transmission electron micrographs were observed at 20,000–150,000× magnification at 75 kV on a Hitachi H-7000 electron microscope. Arrows show F-HSA.

cancer cell lines, TS/A and MDA-MB-231, using a 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-colorimetry assay to measure the cell viability (MERCK, Darmstadt, Germany). TS/A, a murine mammary adenocarcinoma cell line that is estrogen dependent, was cultured in Dulbecco's modified Eagle's medium (DMEM; GIBCO); and MDA-MB-231 (ATCC **HTB-26™**), a metastatic human breast cancer cell line that is estrogen independent, was cultured in DMEM/F12 medium (GIBCO). In brief, 2 104 breast cancer cells were incubated in serum-free medium and treated with serial dilutions of F-HSA. After incubation for 24 hours to allow the drug to take effect, 10 l MTT solution was added to each well. After incubation at 37°C in 5% CO2 for another 2 hours to allow the MTT solution to be metabolized, formazan (MTT metabolic product) was resuspended in 200 ul DMSO. Finally, the proportions of surviving cells were determined by optical density (570 nm test wavelength, 630 nm reference wavelength). The percentage of surviving cells was calculated as (O.D.treatment/O.D.control) 100%, and the percentage of growth inhibition was calculated as [1 - (O.D.treatment/O.D.control) ] 100%. IC50 value is the concentration at which the reagent produces 50% inhibition of cellular viability. F-HSA inhibited growth of the breast cancer cell lines TS/A and MDA-MB-231 in a dose dependent manner with IC50 values of 0.15 and 0.48 μM, respectively (Fig. 4). F-HSA at concentrations over 0.4 μM induced dose-dependent cytotoxicity in both TS/A cells and MDA-MB-231 cells, whereas concentrations of 0.1-0.2

**2 microns**

Fig. 2. Ultra-structures of F-HSA were observed by TEM. F-HSA was applied to a 300-mesh carbon-coated copper grid then the grid was air-dried. The F-HSA-bearing grid was negatively stained with 1% (w/v) phosphotungstic acid. Finally, transmission electron micrographs were observed at 20,000–150,000× magnification at 75 kV on a Hitachi H-7000

μM did not affect cell viability significantly.

electron microscope. Arrows show F-HSA.

Fig. 3. ThT fluorescence assay of F-HSA. For fluorescence measurements, increasing concentrations of proteins were incubated with 20 μM ThT for 1 h at room temperature, and fluorescence was measured in triplicate on a Wallac Victor2 1420 Multilabel Counter (Perkin Elmer Life Science, Waltham, MA, USA). Excitation and emission wavelengths were 430 nm and 486 nm, respectively. ThT background signal from buffer solution was subtracted from the corresponding measurements. A (1-42) was used as a positive control.

Fig. 4. Effect of F-HSA on viability of TS/A (A) and MDA-MB-231 cells (B).

To understand the effects of F-HSA on cell morphology and MET in TS/A cells and MDA-MB-231 cells, breast cancer cells were treated low concentrations of F-HSA and cell morphology was observed under light microscopy. F-HSA induced a morphological alteration in cells, from a fibroblast-like shape to a round shape (Fig. 5). We also examined whether F-HSA suppressed breast cancer-cell migration at non-cytotoxic concentrations by

Fibrillar Human Serum Albumin Suppresses Breast Cancer Cell Growth and Metastasis 431

Fig. 7. F-HSA suppressed MDA-MB-231 cell migration in a wound-healing assay. After 0.1 μM and 0.2 μM of F-HSA treatment at 37°C for 24 h, cell migration was observed under

**7. F-HSA suppresses breast cancer cell migration via 1 integrin signaling** 

cancer migration by F-HSA may be mediated by binding of 1 integrin.

Cell surface receptors mediate cell-to-matrix and cell-to-cell interactions. Integrins are a large family of heterodimeric transmembrane receptors that mediate cell-ECM interactions. In eukayotic cells, integrins consist of 18 α subunits and 8 β subunits that form 24 different αβ integrins. The particular combination of and β subunits in integrin dimers determines their specificity for ligands, which include most of the ECM proteins such as FN and collagen (Plow et al, 2000). Upon activation by ECM proteins, integrins mediate cellular adhesion, migration, survival, and proliferation (Ginsberg et al, 2005). Integrin signaling is activated by ECM proteins or growth factors through focal adhesion kinase (FAK), PI3K, and Akt, a major downstream target of PI3K signaling, known to be involved in various cellular processes such as cell survival, cell cycle, metabolism, protein synthesis, and transcriptional regulation (Mitra & Schlaepfer, 2006). We showed that fibrillar proteins induced cellular apoptosis (Huang et al, 2009; Huang et al, 2010). The mechanism of the cytotoxic effects of F-BSA in BHK-21 cells (baby hamster kidney cell) was due to modulation of the 51 integrin/FAK/Akt/GSK-3β/caspase-3 signaling pathway. Furthermore, F-FN induced cytotoxicity via activating SHP-2 and RhoA/ROCK, and deactivation of Akt/GSK-3. Taken together these findings suggested that β1 integrin may play a critical role in mediating cancer growth and metastasis. Therefore, we measured the proportion of 5 integrin+ cells or 1 integrin+ cells in TS/A and MDA-MB-231 cells by flow cytometry. First, TS/A or MDA-MB-231 cells were collected and washed with 1× PBS three times. Then, specific monoclonal antibodies for 5 integrin-FITC and 1 integrin-FITC were added and co-incubated with cells (1 × 105/ml) at 4°C for 30 minutes. Cells were then washed three times using 1× PBS and finally stained with 5 μg/ml propidium iodide (PI) at 4°C for 10 minutes to exclude dead cells. Cell viability was determined using a flow cytometer (FACSCalibur; BD Bioscience) and CellQuest software. Data showed that 58.67% and 66.19% of TS/A cells were 5 integrin+ and 1 integrin+, respectively. 42.99% and 97.65% of MDA-MB-231 cells were 5 integrin+ and 1 integrin+, respectively (Table 1). Blocking 1 integrin signaling pathway with a specific mAb (mouse anti-human integrin beta1 monoclonal antibody; Millipore) could reverse F-HSA's effect on TS/A and MDA-MB-231 breast cancer cell migration (Fig. 8). Taken together, these results indicated that the suppression of breast

light microscopy.

**pathway** 

wound-healing assay. TS/A and MDA-MB-231 cells were plated onto six-well tissue culture dishes in complete tissue culture medium until they formed a confluent monolayer. The cell monolayer was scratched with a sterile pipette tip to generate a wound (width 2 mm). The remaining cells were washed three times with culture medium to remove cell debris. The medium was immediately replaced with serum-free medium with 0.1 or 0.2 µM of F-HSA, and cultured at 37°C for 24 hours. Spontaneous cellular migration was then monitored at 0 hours (immediately after wounding) and 24 hours (the end of F-HSA treatment) using and inverted microscope (Axiovert 200M; Zeiss) at 100× original magnification. The extent of wound healing was determined by the distance (migrating distance) traversed by cells migrating into the denuded area. F-HSA at concentrations of 0.1 to 0.2 µM suppressed cell migration of both TS/A and MDA-MB-231 cells (Figs. 6-7).

Fig. 5. F-HSA induced morphological alterations and mesenchymal-to-epithelial transition in breast cancer cells. After 0.1 μM and 0.2 μM of F-HSA treatment at 37°C for 24 h, cell morphology was observed under light microscopy. Scale bar, 5 μm

Fig. 6. F-HSA suppressed TS/A cell migration in a breast cancer cell wound-healing assay. After 0.1 μM and 0.2 μM of F-HSA treatment at 37°C for 24 h, cell migration was observed under light microscopy.

wound-healing assay. TS/A and MDA-MB-231 cells were plated onto six-well tissue culture dishes in complete tissue culture medium until they formed a confluent monolayer. The cell monolayer was scratched with a sterile pipette tip to generate a wound (width 2 mm). The remaining cells were washed three times with culture medium to remove cell debris. The medium was immediately replaced with serum-free medium with 0.1 or 0.2 µM of F-HSA, and cultured at 37°C for 24 hours. Spontaneous cellular migration was then monitored at 0 hours (immediately after wounding) and 24 hours (the end of F-HSA treatment) using and inverted microscope (Axiovert 200M; Zeiss) at 100× original magnification. The extent of wound healing was determined by the distance (migrating distance) traversed by cells migrating into the denuded area. F-HSA at concentrations of 0.1 to 0.2 µM suppressed cell

Fig. 5. F-HSA induced morphological alterations and mesenchymal-to-epithelial transition in breast cancer cells. After 0.1 μM and 0.2 μM of F-HSA treatment at 37°C for 24 h, cell

Fig. 6. F-HSA suppressed TS/A cell migration in a breast cancer cell wound-healing assay. After 0.1 μM and 0.2 μM of F-HSA treatment at 37°C for 24 h, cell migration was observed

morphology was observed under light microscopy. Scale bar, 5 μm

under light microscopy.

migration of both TS/A and MDA-MB-231 cells (Figs. 6-7).

Fig. 7. F-HSA suppressed MDA-MB-231 cell migration in a wound-healing assay. After 0.1 μM and 0.2 μM of F-HSA treatment at 37°C for 24 h, cell migration was observed under light microscopy.

### **7. F-HSA suppresses breast cancer cell migration via 1 integrin signaling pathway**

Cell surface receptors mediate cell-to-matrix and cell-to-cell interactions. Integrins are a large family of heterodimeric transmembrane receptors that mediate cell-ECM interactions. In eukayotic cells, integrins consist of 18 α subunits and 8 β subunits that form 24 different αβ integrins. The particular combination of and β subunits in integrin dimers determines their specificity for ligands, which include most of the ECM proteins such as FN and collagen (Plow et al, 2000). Upon activation by ECM proteins, integrins mediate cellular adhesion, migration, survival, and proliferation (Ginsberg et al, 2005). Integrin signaling is activated by ECM proteins or growth factors through focal adhesion kinase (FAK), PI3K, and Akt, a major downstream target of PI3K signaling, known to be involved in various cellular processes such as cell survival, cell cycle, metabolism, protein synthesis, and transcriptional regulation (Mitra & Schlaepfer, 2006). We showed that fibrillar proteins induced cellular apoptosis (Huang et al, 2009; Huang et al, 2010). The mechanism of the cytotoxic effects of F-BSA in BHK-21 cells (baby hamster kidney cell) was due to modulation of the 51 integrin/FAK/Akt/GSK-3β/caspase-3 signaling pathway. Furthermore, F-FN induced cytotoxicity via activating SHP-2 and RhoA/ROCK, and deactivation of Akt/GSK-3. Taken together these findings suggested that β1 integrin may play a critical role in mediating cancer growth and metastasis. Therefore, we measured the proportion of 5 integrin+ cells or 1 integrin+ cells in TS/A and MDA-MB-231 cells by flow cytometry. First, TS/A or MDA-MB-231 cells were collected and washed with 1× PBS three times. Then, specific monoclonal antibodies for 5 integrin-FITC and 1 integrin-FITC were added and co-incubated with cells (1 × 105/ml) at 4°C for 30 minutes. Cells were then washed three times using 1× PBS and finally stained with 5 μg/ml propidium iodide (PI) at 4°C for 10 minutes to exclude dead cells. Cell viability was determined using a flow cytometer (FACSCalibur; BD Bioscience) and CellQuest software. Data showed that 58.67% and 66.19% of TS/A cells were 5 integrin+ and 1 integrin+, respectively. 42.99% and 97.65% of MDA-MB-231 cells were 5 integrin+ and 1 integrin+, respectively (Table 1). Blocking 1 integrin signaling pathway with a specific mAb (mouse anti-human integrin beta1 monoclonal antibody; Millipore) could reverse F-HSA's effect on TS/A and MDA-MB-231 breast cancer cell migration (Fig. 8). Taken together, these results indicated that the suppression of breast cancer migration by F-HSA may be mediated by binding of 1 integrin.

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Table 1. Percentages of 5 integrin+ cells and 1 integrin+ cells in TS/A and MDA-MB-231 cells.

Fig. 8. Blocking the 1 integrin signaling pathway with a specific mAb (mouse anti-human integrin beta1 monoclonal antibody) reversed the effect of 0.2 M F-HSA on TS/A and MDA-MB-231 breast cancer cell migration.
