The use of live bacteria in the treatment of cancer has a long and interesting history. We report the use of a purified bacterial redox protein, azurin, that enters human cancer (melanoma UISO-Mel-2) cells and induces apoptosis. The induction of apoptosis occurs readily in melanoma cells harboring a functional tumor suppressor protein p53, but much less efficiently in p53-null mutant melanoma (UISO-Mel-6) cells. A redox-negative mutant form of azurin (M44K͞ M64E) demonstrates much less cytotoxicity to the UISO-Mel-2 cells than the wild-type protein. Azurin has been shown to be internalized in UISO-Mel-2 cells and is localized predominantly in the cytosol and in the nuclear fraction. In the p53-null UISO-Mel-6 cells, azurin is localized only in the cytosol. Thus, intracellular trafficking of azurin to the nucleus is p53-dependent. Azurin forms a complex with p53, thereby stabilizing it and raising its intracellular level in cytosolic, mitochondrial, and nuclear fractions. Corresponding to an increasing level of p53, an inducer of apoptosis, the level of Bax also increases in mitochondria, allowing significant release of mitochondrial cytochrome c into the cytosol, thus initiating the onset of apoptosis. The M44K͞M64E mutant form of azurin, deficient in cytotoxicity, is also deficient in forming a complex with p53 and is less efficient in stabilizing p53 than wild-type azurin. Azurin has been shown to allow regression of human UISO-Mel-2 tumors xenotransplanted in nude mice and may potentially be used in cancer treatment.

Azurin, a member of the cupredoxin family of copper containing redox proteins, preferentially penetrates human cancer cells and exerts cytostatic and cytotoxic (apoptotic) effects with no apparent activity on normal cells. Amino acids 50 to 77 (p28) of azurin seem responsible for cellular penetration and at least part of the antiproliferative, proapoptotic activity of azurin against a number of solid tumor cell lines. We show by confocal microscopy and fluorescence-activated cell sorting that amino acids 50 to 67 (p18) are a minimal motif (protein transduction domain) responsible for the preferential entry of azurin into human cancer cells. A combination of inhibitors that interfere with discrete steps of the endocytotic process and antibodies for caveolae and Golgi-mediated transport revealed that these amphipathic, A-helical peptides are unique. Unlike the cationic cell-penetrating peptides, A-helical antennapedialike, or VP22 type peptides, p18 and p28 are not bound by cell membrane glycosaminoglycans and preferentially penetrate cancer cells via endocytotic, caveosome-directed, and caveosome-independent pathways. Once internalized, p28, but not p18, inhibits cancer cell proliferation initially through a cytostatic mechanism. These observations suggest the azurin fragments, p18 and p28, account for the preferential entry of azurin into human cancer cells and a significant amount of the antiproliferative activity of azurin on human cancer cells, respectively. [Cancer Res 2009;69(2):537-46]

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.

We report that amino acids 50 to 77 of azurin (p28) preferentially enter the human breast cancer cell lines MCF-7, ZR-75-1, and T47D through a caveolin-mediated pathway. Although p28 enters p53 wild-type MCF-7 and the isogenic p53 dominant-negative MDD2 breast cancer cell lines, p28 only induces a G 2 -M-phase cell cycle arrest and apoptosis in MCF-7 cells. p28 exerts its antiproliferative activity by reducing proteasomal degradation of p53 through formation of a p28:p53 complex within a hydrophobic DNA-binding domain (amino acids 80-276), increasing p53 levels and DNA-binding activity. Subsequent elevation of the cyclin-dependent kinase inhibitors p21 and p27 reduces cyclin-dependent kinase 2 and cyclin A levels in a time-dependent manner in MCF-7 cells but not in MDD2 cells. These results suggest that p28 and similar peptides that significantly reduce proteasomal degradation of p53 by a MDM2-independent pathway(s) may provide a unique series of cytostatic and cytotoxic (apoptotic) chemotherapeutic agents.