Angiocidin inhibits breast cancer proliferation through activation of epidermal growth factor receptor and nuclear factor kappa (NF-ĸB)

Abstract

Angiocidin, a tumor-associated peptide, has been previously shown to inhibit tumor progression by blocking angiogenesis. We now show that angiocidin has a direct inhibitory effect on tumor cell proliferation. MDA-MB-231 breast cancer cells were inhibited from proliferating in the presence of epidermal growth factor (EGF) and angiocidin. Angiocidin transfected breast cancer cells also displayed growth inhibition in vitro and failed to develop significant tumors in mice as compared to vector controls. The anti-proliferative effect of angiocidin was reversed by treating the cells with the epidermal growth factor receptor (EGFR) inhibitor 4557W, a potent tyrosine kinase inhibitor. Consistent with these results, we found that treatment of breast cancer cells with angiocidin induced a 2.3 fold increase in EGFR tyrosine 845 phosphorylation while no change in phosphorylation was observed in the remaining 16 phosphorylation sites of EGFR and those of its family members as measured by a human EGFR phosphorylation array. Treatment of breast cancer cells with angiocidin also resulted in the activation of nuclear factor ĸB (Nf-ĸB) and the de novo up-regulation of many down-stream genes transcribed by Nf-ĸB, including cytokines, inflammatory mediators and the cell cycle inhibitor p21(waf1). Therefore, angiocidin is a peptide that not only inhibits tumor angiogenesis but also directly induces inhibition of tumor growth progression through the activation of EGFR and down-stream genes transcribed by Nf-ĸB.

Fig.1

Morphological appearance and growth of angiocidin-treated MB-231 cells. MDA-MB-231 cells were seeded in 0.1% BSA DMEM without recombinant angiocidin and incubated at 37° C for sixteen hours. After sixteen hours the cells were photographed. The number of adherent, spread cells resembling the morphology of normal MB-231 cells in four representative fields was counted. The percentage of adherent, spindle-shaped cells was determined to be 36 out of 225, or 16% (panel A). Cell proliferation was determined in a 96 well plate using the Alamar blue proliferation assay under the same culture conditions (Panel B). Experiments were repeated three times and the results of a representative experiment are shown in the figure.

Fig. 2

Angiocidin-transfected MDA-MB-231 tumor cells develop smaller tumors in mice and smaller colonies in soft agar. MDA-MB-231 breast cancer cells were transfected with angiocidin using the pcDNA3.1 transfection system as described by Invitrogen. At least two angiocidin over-expressing clones were selected sense 1 and 2 as well as the vector control. The panels marked sense show the sense 1 clone. Similar results were obtained with sense 2 clones. Panel A show paraformaldehyde-fixed cells grown in culture and stained with anti-angiocidin antibody and developed with DAB (100×). Panel B shows Western blot of cells grown in panel A. Panel C shows colonies grown in soft agar as seen by phase contrast (50x mag). Panel D shows tumor volumes of cells grown in mice (n=6 per group ± std). In vitro experiments were repeated three times and the results of a representative experiment are shown in the figure.

Fig. 3

Angiocidin inhibits proliferation of MB-231 breast cancer cells through activation of EGFR and up-regulation of p21^waf1. Panel A- Cells were grown in DMEM media containing 10% serum and 10 ng/ml EGF either alone or treated with 10 μg/ml or 50 μg/ml angiocidin in the presence or absence of 10 μg/ml EGFR inhibitor 4557W and proliferation was measured after 24 hours using the Alamar blue proliferation assay. Panel B- Cells were grown in DMEM media containing 2% serum for 24 hours in six well plates and then treated with various concentrations of angiocidin for an additional 24 hours, harvested and blotted for p21^waf1. Cell lysate from A-431 was used as the negative control and cell lysate from HEK-293 cells was the positive control. Panel C- p21^waf1 positive bands in Panel B quantitated by densitometry using Image J software. Panel D- EGFR phosphoarray results (top-stained array, bottom- spots encircled in the stained array were quantitated by densitometric analysis of the spots using Image J software ). Experiments were repeated two times and the results of a representative experiment are shown in the figure.

Fig. 4

Angiocidin promotes the activation of NFκB and phosphorylation of IκBα. Panel A-NFκB p65 Western Blot of Nuclear and cytoplasmic extracts of MB-231 cells treated with 10 μg/ml of angiocidin in 2% FBS DMEM for a period of time ranging from 5 minutes to 90 minutes. Panel B-Phospho-IKBα Western Blot of MB-231 cells treated for twenty-four hours with 0 μg/ml, 10 μg/ml, or 20 μg/ml of angiocidin in DMEM containing 0.1% BSA. Experiments were repeated three times and the results of a representative experiment are shown in the figure.

Fig. 5

Angiocidin up-regulates cytokines, immune and inflammatory mediators in MB-231 cells. Panel A-Genes up-regulated 4 fold or greater in angiocidin-treated cells as detected with a PCR Toll- like receptor pathway array. Panel B- Cytokines detected in conditioned media from untreated and angiocidin-treated MB-231 using a cytokine array. Details of the array analysis are described in Material and Methods.