A cupredoxin derived peptide inhibits angiogenesis and mammary end bud transformation in vitro.

A217

The cupredoxins are a family of small, single domain, copper containing redox proteins present in bacteria and plants. We have previously reported that azurin, a cupredoxin isolated from Pseudomonas arugenosum, preferentially penetrates cancer cells, alters cell cycle and induces apoptosis in several human cancer cell lines (Yamada et. al., Proc Natl Acad Sci U S A. 99:14098, 2002), including the human breast cancer cell line, MCF-7, in vitro and in vivo (Punj et. al. Oncogene. 23(:2367, 2004). A 28aa fragment of azurin (aa 50-77; p28) also inhibits tumor proliferation. Like azurin, p28 appears to exert its anti-proliferative activity through the tumor suppressor gene, p53. As p53 appears responsible for regulating a number of cellular processes, including differentiation, we explored whether azurin and could alter additional p53 mediated process i.e. cellular transformation, in mouse mammary organ culture (MMOC), an established model of cellular transformation and angiogenesis in a human umbilical vascular endothelial cell model. Azurin and p28 reproducibly induced a dose related inhibition in the incidence and multiplicity of mammary alveolar and ductal precancerous lesions. Confocal microscopy and FAC showed that azurin and p28 entered normal murine mammary epithelial cells (MM3MG) and mammary cancer cells (4T1). P28 also entered human umbilical vein endothelial cells (HUVEC) in a temperature, time and concentration dependent manner and induced a does related inhibition of capillary tube formation of HUVEC plated on Matrigel®. At 75uM, tube formation was completely inhibited. Cell migration was also inhibited (40-50%) in cell migration "scratch" and electro- wound assays in vitro The anti-angiogenic and anti-migratory effects of p28 were also evident in the presence of VEGF and FGF. Taken together, these observations suggest that azurin and p28 not only inhibit the proliferation of breast cancer cells, but angiogenesis and the transformation of relatively undifferentiated mammary gland structures believed to be involved in the genesis of mammary cancer. As such, these peptides appear to offer a unique approach to treating breast cancer.