Fig. 4: Azurin-producing P. aeruginosa in human tumors.

The azurin-encoding gene (azu) was detected in tumors from patients with breast cancer and melanoma. It was amplified by PCR with P. aeruginosa azu-specific primers and confirmed by DNA sequencing. The amplified PCR product, as a single band, was sequenced and showed 100% identity to P. aeruginosa azu. In melanoma (a), 27.6% (8 of 29) of primary tumors and 5.9% (2 of 34) of metastatic tumors were azu positive (P < 0.05). In breast cancer (b), 22.7% (5 of 22) of primary tumors and 14.3% (2 of 14) of metastatic tumors were azu positive (P > 0.05). In addition, frozen tumor samples were processed for immunogold transmission electron microscopy (TEM) by using anti-P. aeruginosa and anti-azurin antibodies. TEM images of uranyl acetate-stained sections (arrowheads) showed the intracellular localization of P. aeruginosa (c) and its product azurin (d) in human melanoma sections that were azu positive by PCR. Magnification: ×3000. e, f Patients with azu-positive tumors displayed increased survival. Survival analysis of patients with azu-positive vs. azu-negative primary melanoma (e, P < 0.01) and primary breast cancer (f, P < 0.05) tumors. g Hemizygous MMTV-PyMT transgenic mice were injected with either PBS control or 2.5 mg/kg azurin (intraperitoneally, 3× weekly) for 2 months. The mean+SE values of tumor volume were calculated. h Tumor-free survival curve was generated with the PBS control and 2.5 mg/kg azurin groups of MMTV-PyMT transgenic mice. Median tumor-free survival of the PBS control and azurin-treatment groups was 67 and 72 days, respectively. P = 0.015.