Introduction: Breast Cancer:
In recent years, breast cancer has become the most common cancer in the world, accounting for approximately 12% of all cancer cases globally. It is the most prevalent cancer among women and the leading cause of death in females.
According to the World Health Organization's statistics, lung cancer was the most common cancer in 2020, but now breast cancer incidence has surpassed that of lung cancer.
Global statistics indicate that around twenty million people are affected by breast cancer. Given the daily rise in breast cancer cases in various communities, it is expected that the number of patients will increase by 47% over the next two decades.
The World Health Organization predicts that the incidence of breast cancer in Iran will reach 22,828 cases per year by 2040.
What is MCF7?
MCF7 is a breast cancer cell line that was isolated in 1970 from a 79-year-old Kazakh woman. MCF7 stands for Michigan Cancer Foundation 7, referring to the institute in Detroit, USA, where Dr. Herbert Soule and his colleagues first isolated this cell line.
Actinomycetes:
Actinomycetes are some of the most valuable microorganisms for producing and synthesizing therapeutic compounds and important economic antibiotics. They are the source of over 50% of the discovered bioactive compounds, including antitumor agents, antibiotics, enzymes, and immunosuppressive agents. Many of these bioactive secondary metabolites have been isolated from terrestrial actinomycetes. However, in recent years, the discovery rate of new bioactive compounds has decreased. Therefore, it is crucial to explore new groups of actinomycetes from unexplored or underexploited environments to obtain new bioactive secondary metabolites. Unknown areas such as saline regions, conditions near marine aquatic environments, high pH zones, and low oxygen areas are suitable for isolating these actinomycetes (Valan Arasu, 2016).
Salinosporamide A is an anticancer compound isolated from the marine actinomycete Salinispora tropica. Its chemical structure is a beta-lactone gamma-lactam bicyclic compound. Salinosporamide A acts by inhibiting the proteasome, leading to apoptosis in myeloma cells. Nereus Pharmaceuticals developed Salinosporamide A, named NPI-0052, for cancer treatment in humans. This drug is the first anticancer agent derived from marine actinomycetes (Karthikeyan et al., 2022). In the same year, Silva and colleagues conducted a study among actinobacteria in polar regions to find species that produce secondary metabolites with antitumor properties. The diversity of actinobacterial species associated with Antarctic regions was analyzed, focusing on s rRNA16, and the produced metabolites were examined. It was observed that the Actinomycetales order included the majority of secondary metabolite producers. Among these metabolites, 17 samples exhibited antitumor activity against cancer cell lines. Further tests revealed a specific anti-proliferative activity for crude extracts obtained from Streptomyces sp. cells. This study indicates that the rhizosphere is a prominent reservoir of bioactive actinobacterial strains, making it a significant source for discovering potential antitumor agents (Silva et al., 2020).
Methods: Collection of Bacteria and Extraction:
In this stage, actinomycete bacteria were isolated from soil and cultured in Starch Agar medium. After allowing sufficient growth, the culture medium along with the bacteria was transferred into a beaker, ground with a mortar, and taken under a fume hood. Ethyl acetate, a toxic solvent, was added, and the mixture was placed on a shaker for 2 hours. Afterward, the remaining liquid was filtered and poured into a petri dish. We waited for the ethyl acetate to evaporate from the extract. The extract in the petri dish was then washed with DMSO and transferred to a vial, allowing the DMSO to separate from the extract. After several hours, the extract precipitated in the vial.
Cell Culture:
In the experimental study, the MCF7 cell line (human breast cancer cells) was purchased from the Medical School of Tehran University of Medical Sciences (Tehran, Iran) and cultured in 25 mL flasks in DMEM-F12 medium containing 10% FBS (fetal bovine serum) and 1% penicillin-streptomycin antibiotics at 37°C in a humidified environment with 5% CO2. The cells were examined under an inverted microscope every 24 hours, and the culture medium was replaced as needed. Three to four days after initial seeding, cell density was assessed under the microscope, and when cell growth reached 80%, passaging was performed.
Assessment of Cytotoxicity using the MTT Method:
The effect of actinomycete bacterial extract on the proliferation and viability of MCF7 breast cancer cells was evaluated using the MTT (Micro-culture Tetrazolium Test) assay compared to a control group. This assay is based on the activity of succinate dehydrogenase in the mitochondria of viable cells, converting the yellow MTT solution into insoluble purple formazan crystals, which can be measured after dissolving in dimethyl sulfoxide (DMSO) using an ELISA plate reader (BioTech, Germany).
After several passages and reaching the required cell density, the cells were transferred to a 96-well plate, with 15,000 cells per well in DMEM-F12 medium containing 10% FBS. The cells were exposed to various concentrations of the extract (0, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg).
The microplates containing the extract and cells were incubated under identical conditions for 24 hours. On the reading day, 200 µL of DMSO replaced the culture medium containing MTT, gently pipetting to dissolve the purple formazan crystals. Absorbance was measured at a wavelength of 570 nm using the ELISA plate reader. Each concentration was evaluated in triplicate, with 100% cell viability defined for the negative control group. The percentage of cytotoxicity was calculated as follows:
Cytotoxicity %=(Mean Negative ControlMean Negative Control−Mean Dose)×100
The concentration of the tested compounds that reduced cell viability by half was considered the IC50 (Inhibition Concentration 50), determined from the graph generated using GraphPad Prism software.
Results: The extract of actinomycete bacteria has shown a positive effect on MCF7 cancer cells; for example, at a dose of 1000 mg, the viability of MCF7 cells significantly decreased. Therefore, we can conclude that actinomycete bacteria may contribute to the improvement or treatment of breast cancer.
Conclusion: Based on the data and results presented in Figures 3 and 4, it was observed that the extract of actinomycete bacteria significantly reduced the viability of human breast cancer cells over a 24-hour period. At a concentration of 1000 µg, it exhibited a 20% reduction in cell viability compared to the control group, highlighting the extract's significant potential in inducing cell death.
Chemotherapy drugs typically cause various side effects, including fatigue, hair loss, easy bruising and bleeding, infections, anemia (low red blood cell count), nausea and vomiting, appetite changes, constipation, diarrhea, oral and throat issues like sores and pain during swallowing, peripheral neuropathy or other neurological problems such as numbness, tingling, and pain, skin and nail changes like dryness and discoloration, urinary and bladder changes, kidney issues, weight fluctuations, concentration difficulties, mood changes, and fertility problems. These side effects can have diverse negative medical, economic, and social impacts on the lives of cancer survivors. Moreover, existing drugs do not demonstrate uniform efficacy across all cancer types.
Given the adverse effects of chemotherapy, innovative methods for inhibiting cancer cells are being explored, one of which involves the use of bacteria. The advantage of this study is that it may eliminate the side effects associated with chemotherapy. Notably, there have been no similar studies conducted in the country, and literature in this area is scarce. The research utilized a strain of actinomycete from Syria, which has not been previously studied. Therefore, it is crucial to continue investigations aimed at discovering new and effective natural pharmaceutical compounds for cancer treatment. Furthermore, the characteristics and historical research on other strains of this bacteria suggest a hopeful future for its use in cancer therapy.
Keywords: Breast cancer cell،Viability،MTT،Actinomycete،Extraction،Bacteria