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Azurin protein of Pseudomonas aeruginosa is an anti-tumor agent against breast cancer and mammaglobin-A (MAM-A) protein is a specific antigen on the surface of MCF-7 for induction of cellular immune. The purpose of the present study was to investigate the effects of simultaneous expression of azurin and human MAM-A genes on the mRNA expression level of apoptosis-related and cell cycle genes in MCF-7 breast cancer cell line. The recombinant or empty plasmids were separately transferred into MCF-7 cells using Lipofectamine reagent. Flow cytometry was done to detect cell death and apoptosis. The expression of azurin and MAM-A genes were evaluated by IF assay, RT-PCR and western blot methods. Finally, apoptosis-related and cell cycle genes expression was examined in transformed and non-transformed MCF-7 cells by qPCR method. The successful expression of azurin and MAM-A genes in the MCF-7 cell were confirmed by RT-PCR, IF and western blotting. The apoptosis assay was showed a statistically significant (p < 0.05) difference after transfection. The expression of BAK , FAS , and BAX genes in transformed cells compare with non-transformed and transformed MCF-7 by pBudCE4.1 were increased statistically significant (p < 0.05) increases. Although, the increase of SURVIVIN and P53 expressions in transformed cells were not statistically significant (p > 0.05). Co-expression of azurin and MAM-A genes could induce apoptosis and necrosis in human MCF-7 breast cancer cells by up-regulation of BAK , FAS, and BAX genes. In future researches, it must be better the immune stimulation of pBudCE4.1-azurin-MAM-A recombinant vector in animal models and therapeutic approaches will be evaluated. [ABSTRACT FROM AUTHOR]

Background and aims: Breast cancer is one of the most common types of malignancy in women with morbidity and mortality (15.0%) in the world. The antitumor activity of azurin protein produced by Pseudomonas aeruginosa has been described before. Mammaglobin-A (MAM-A) protein is especially expressed in 40%-80% of breast cancer types and this protein is a very specific molecular marker for stimulating the immune system. Accordingly, this study investigated the effects of pBudCE4.1-azurin-MAM-A recombinant vector on the induction of the immune system in laboratory mice by the real-time polymerase chain reaction (PCR) method. Methods: The pBudCE4.1-azurin-MAM-A recombinant and empty vectors were purchased and then separately transformed into Escherichia coli for multiplying. Next, each plasmid was extracted and the accuracy of transformation was confirmed by the PCR. These recombinant and empty (control) vectors were separately infused into the thigh muscle of the animals and the healthy group was infused with phosphatebuffered saline. The infusion sites, blood specimens, as well as the serum of the animals were collected and examined by serological and molecular tests. Results: Molecular and serological studies showed that the serum and expression levels of IL-2, IL-6, IL-7, and IL-10 in infused mice with pBudCE4.1-azurin-MAM-A recombinant vector significantly increased compared to healthy animals and injected mice with an empty vector (P < 0.05). Conclusion: In general, the findings revealed that the pBudCE4.1-azurin-MAM-A recombinant vector can stimulate the immune system of the mouse by an increase in the expression levels of IL-2, IL-6, IL-7, and IL-10. Thus, it would be better to examine the effects of this recombinant vector as a DNA vaccine on the prevention and treatment of breast cancer. [ABSTRACT FROM AUTHOR]