Iranian Journal of Medical Microbiology

Materials and Methods

Table 1. Specifications of breast, prostate, and bladder cancer patients

Separation of PBMCs from Patients with Cancer and Healthy Controls

Separation of PBMCs from patients with cancer and healthy control samples was performed using the ficoll density gradient method (18). The blood sample was transferred to a 50 mL tube and diluted 1:1 ratio with PBS (Merck, Germany). Then, diluted blood samples were slowly added to the Ficoll (Inno-Train, Germany). The samples were centrifuged at 400 rpm for 20 minutes at 20°C (Hettich, Germany). The PBMCs (cloudy layer) were collected from the diluted plasma/ficoll interface using a serological pipette and the cells were placed into a sterile 50 mL tube. Again, the cells were washed by adding PBS and centrifuged at 330 rpm for 10 minutes (19).

Cultures of PBMCs Isolated from Patients with Cancer and Healthy Controls

After the separation was performed, 100 µL PBMCs isolated from the patients with cancer and the healthy control group was transferred to a 25 cm² flask (Jetbiofil, Canada) containing RPMI 1640 medium (Biosera, France) and 20% fetal bovine serum (FBS) (Biosera, France). The flask was placed in a 5% CO₂ incubator (Lab Tech, South Korea) at 37°C and 98% humidity. After 48 hours, the culture medium was replaced and the cells were passaged (20).
In order to harvest the cells attached to the bottom of the flask, the medium was washed thoroughly and slowly with PBS. Then, added the trypsin enzyme (Merck, Germany) to the flask and placed in the incubator at 37°C for 2 to 5 minutes. After pipetting, the cells isolated from the flask floor were transferred to a 15 mL sterile falcon tube. The tube containing the cells was centrifuged for about 5 minutes at 1200 rpm. The supernatant was evacuated to the cells and the new medium was added. The cell suspension was split into several new flasks. 20 μL cell suspension was mix-ed with 20 μL trypan blue (Sigma, USA). Then, live cells (cells that do not penetrate the color) were counted in the 25th hemocytometer (HBG, Germany) (20).

Effect of Pyocyanine on the Secretion of IL-2 from PBMCs of Patient and Control Groups

Half of the PBMCs of cancer patients and healthy controls were treated with pyocyanine (0.5 μg/mL), and the rest of them remained without treatment. Then, the supernatants were removed and the concentration of IL-2 was determined by ELISA using a commercial human IL-2 ELISA kit (Thermo Fisher Scientific, Australia), according to the manufacturer’s instructions.

Statistical Analysis

The secretion of IL-2 from the treated and untreated cells with pyocyanine was compared by paired sample's T-test. Also, statistical analysis of the samples was done using SPSS version 22 (SPSS Inc., Chicago, IL., USA). All results were expressed as mean ± standard error (SE). A p-value of 0.05 or less was considered statistically significant.

Results

Discussion

The bacterial pigments have immunostimulatory effects on the immune system by the release of cytokines in cancer patients (21). Despite the importance of the immune system in response to cancer treatment, there has been no report on how to stimulate the immune system with the pyocyanine pigment of Pseudomonas aeruginosa.
Based on the results of this study, a low concen-tration of pyocyanine stimulates the IL-2 secretion by PBMCs in cancer patients in vitro. Imm-unotherapy using IL-2 is the most effective anti-cancer treatment (12). The mechanism of the action of pyocyanine on IL-2 production is still unclear. Pigment probably stimulates the patient’s immune system (22). In 2011, research conducted by Rada and colleagues found that pyocyanine can induce the tran-scription of inflammatory cytokines and epider-mal growth factor receptors, as well as IL-1, IL-6, IL-8 cytokines, and TNFα (alpha tumor necrosis factor) (22). Perhaps certain bacterial pigments (such as pyoc-yanine) activate signal transduction pathways through Toll-like receptors (TLR) in PBMC, thereby stimulating the secretion of cytokines including IL-2 (23). The immunostimulatory effect of pyocyanine depends on the concentration of pyocyanine tested (24).
In one study, the inhibitory and stimulating activity of pyocyanine on IL-2 secretion was tested (10). The results were different and the cellular response was related to the concentration of pyocyanine (10). Low concentrations (0.1, 0.2, 0.5 μg/mL) of pyocyanine will increase the response of T lymphocytes (IL-2 production) and B lymphocytes (differentiation of immunoglobulin-producing cells), while high concen-trations of this pigment (1 μg/mL) has an inhibitory effect (10). Similarly, in this study, it was observed that after treatment with a low concentration of pyoc-yanine (0.5 μg/mL) in vitro, the IL-2 secreted by the PBMC of breast cancer, prostate cancer and bladder cancer patients increased.
IL-2 is an activator of cytotoxic T cells (CTL) and natural killer cells (NK cells) (10). NK cells destroy the target cell by secreting their granules (enzymes and proteins) and designing apoptosis (25). NK cells are transformed into lymphokine-activated killer cells (LAK cells) under the action of IL-2 (25). LAK cells have higher apoptotic activity and can destroy different tumor cells (25).
In studies on the role of IL-2 in cancer treatment, similar studies have been conducted on the positive effects of IL-2 in the recovery and treatment of patients with kidney, breast, prostate, and bladder cancer (26-30). Metastatic melanoma and renal cell carcinoma treated with high-dose Interleukin-2 (30). In advanced melanoma, high-dose IL-2 injection is effective in 15% to 20% of patients (30). The patients with metastatic breast cancer were treated with IL-2 and this cytokine increased the number of NK cells and cell lysis function in the body (29). Freytag and colleagues demonstrated the IL-2 gene therapy strategy for local and metastatic prostate cancer in 2007 (28). The result was a 50% reduction in tumors in patients after IL-2 treatment (28). Askeland and colleagues in 2012, showed the function of IL-2 in increasing the NK cells in patients with bladder cancer, and the systemic result of IL-2 in reducing the size of the tumor, tumor growth, and survival time of cancer patients (27). These results indicate that IL-2 has an important role in reducing inflammation in the tumor environment and can have a very positive effect during regular periods, which can indicate a reduction in tumor volume growth. It is recommended in future research, increase the size of the statistical community as much as possible.

Conclusion

The results of this study indicate that treatment with pyocyanine pigment can increase the IL-2 concen-tration of PBMCs in patients with breast, prostate, bladder cancer, and healthy controls. Low concen-trations of pyocyanine may activate the host's imm-une response. This pigment stimulates the secretion of IL-2 by activating the TLR signal transduction pathway in PBMC and inducing the transcription of cytokines. With the unique properties of pyocyanine and the human desire for biology and low-risk treatments, we hope that it can broaden our understanding of immunobiology and recognize the immunostimulatory effects of various bacterial pigments on cancer patients. Although this study is the first step to identify the immunostimulatory effects of pigments in vitro, the anti-tumor effects of pyocyanine in experimental animals should be further studied. Also, more efforts are needed to determine the mode of action and the potential of using the pigment in the stimulation of PBMCs in cancer patients for the anti-cancer IL-2 secretion.

Acknowledgements

This article is the result of a part of the master's thesis of microbiology from the Islamic Azad University, Kazerun Branch. The project was approved by the committee for ethics in biomedical research of Islamic Azad University, Kazerun Branch (IR.IAU.KAU.-REC.1399.031). The authors thank all those who helped them writing this article.

Conflicts of Interest

The authors declared no conflict of interests.