Azurin, a copper-containing redox protein released by the pathogenic bacterium Pseudomonas aeruginosa, is highly cytotoxic to the human breast cancer cell line MCF-7, but is less cytotoxic toward p53-negative (MDA-MB-157) or nonfunctional p53 cell lines like MDD2 and MDA-MB-231. The purpose of this study was to investigate the underlying mechanism of the action of bacterial cupredoxin azurin in the regression of breast cancer and its potential chemotherapeutic efficacy. Azurin enters into the cytosol of MCF-7 cells and travels to the nucleus, enhancing the intracellular levels of p53 and Bax, thereby triggering the release of mitochondrial cytochrome c into the cytosol. This process activates the caspase cascade (including caspase-9 and caspase-7), thereby initiating the apoptotic process. Our results indicate that azurininduced cell death stimuli are amplified in the presence of p53. In vivo injection of azurin in immunodeficient mice harboring xenografted human breast cancer cells in the mammary fat pad leads to statistically significant regression (85%, P ¼ 0.0179, Kruskal-Wallis Test) of the tumor. In conclusion, azurin blocks breast cancer cell proliferation and induces apoptosis through the mitochondrial pathway both in vitro and in vivo, thereby suggesting a potential chemotherapeutic application of this bacterial cupredoxin for the treatment of breast cancer.

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Immunodeficient athymic mice with human tumor xenografts provide an important in vivo experimental model for cancer research. However, only a limited number of tumor types grow in these animals. For human breast carcinomas, the incidence of tumor-take is 6-15%. Recently, increased incidence of xenograft development in mice has been reported for various human tumors when the tumors were coinjected with Matrigel. We studied the development of human breast carcinoma xenografts in athymic mice with and without coinjection of Matrigel. Tumors developed in only 7.3% of enzyme-dispersed tumors injected subcutaneously in saline solution alone. None of these tumors metastasized to distant sites. On the other hand, 50% of enzyme-dispersed tumors coinjected with Matrigel developed xenografts; four out of five of these tumors metastasized to distant sites. Our data from the recent study suggest that, in athymic mice, Matrigel not only enhanced breast tumor growth but also facilitated tumor metastasis.

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Total and subcellular (cytosol and nuclear) concentrations of estrone (E1), estradiol (E2), and androstenedione were determined in non-malignant (n = 61) and malignant (n = 65) human breast tissues obtained from post-menopausal women. The 17 beta-hydroxysteroid dehydrogenase (17 beta-OH-SDH) activity was determined in 800g supernatant fraction. Total estrogens, E1 and E2 levels and 17 beta-OH-SDH activity were significantly (p less than 0.005, 0.0005, 0.001, respectively) higher in malignant than in non-malignant breast tissues. We failed to observe significant changes in subcellular steroid concentrations or enzyme activity associated with patients' obesity or tumor estrogen receptor status. When the steroid levels were analyzed in relation to clinical staging of the disease, nuclear contents of estradiol were significantly higher (p less than 0.005) in Stage-IV patients than in those with less advanced disease (Stages I to III). 17 beta-OH-SDH activity was significantly (p less than 0.001) lower in patients with advanced disease than in those with relatively less advanced (Stages I to III) disease and was positively correlated with tissue concentration of androstenedione. Our present data indicate that differential intracellular metabolism of steroid hormones may have some influence on availability of estradiol at nuclear sites. In postmenopausal women, local interconversion of estrogens may provide sufficient estrogenic stimulus to enhance the growth and progression of breast tumors.