Volume 31, Issue 1 (3-2024)
RJMS 2024, 31(1): 1-12 |
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Research code: IR.IAU.PS.REC.1402.040
Ethics code: IR.IAU.PS.REC.1402.040
Clinical trials code: IR.IAU.PS.REC.1402.040
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Nori Derakhshan S, Montazeri M, Mohammadi M. Investigating the Effect of Cerasia marciscens Bacteria Pigment on the Survival of Ovarian Cancer SKOV3 Cell Line. RJMS 2024; 31 (1) :1-12
URL: http://rjms.iums.ac.ir/article-1-8296-en.html
URL: http://rjms.iums.ac.ir/article-1-8296-en.html
Assistant Professor, Department of Biology, Islamshahr Branch of Islamic Azad University, Islamshahr, Iran , mh_mohamadi@yahoo.com
Abstract: (761 Views)
Background & Aims: Ovarian cancer is the sixth most common cancer in women, which kills 152,000 people worldwide every year. Despite various efforts aimed at developing effective treatments, the overall survival rate of patients with ovarian cancer is still less than 50%, which reflects the fact that most patients are diagnosed at late stages and there is still no effective treatment for this disease. Recently, bacteria and substances produced by them have received serious attention as cancer treatment agents. However, the first use of bacteria and their products for the treatment of cancer was reported by William Coley, who used the supernatant of Streptococcus pyogenes and Ceracia marciscens (Coli toxins) to treat patients with unprotected tumors. Microbial infections cause the activation of macrophages and lymphocytes, which causes the production of cytotoxic substances such as TNF-α and helps to dissolve the tumor. Recently, it has been shown that bacteria and the substances they produce can have potential anti-cancer activities. Interestingly, bacteria alone can act as a potential antitumor agent by targeting the low-oxygen regions of solid tumors. In addition, by using different strategies such as the secretion of toxins, enzymes, proteases and lipases, bacteria effectively target cancer tissues. Salmonella, Serratia, Clostridium, Bifidiobacterium, Lactobacillus, Escherichia, Pseudomonas, Calobacter, Listeria, Proteus and Streptococcus are bacterial genera that have been previously reported as having anticancer potential. Studies show that the ability to produce pigments in microorganisms causes antibiotic resistance and resistance to heavy metals in the environment. In these bacteria, the pigment acts as a barrier against the penetration of antibiotics through the wall and cytoplasmic membrane of the bacteria. Pigment acts as an antioxidant and protects bacteria from oxygen free radicals. In the meantime, certain bacterial species are placed in this category as well-known bacterial models due to their ability to produce pigments and biotechnological management of bacterial cultivation, the process of extracting pigments, as well as their importance and practical features. They take among these; we can mention Cerasia marciscens species. Ceraschia marciscens is a bacterial species with the characteristic of producing red pigment prodigiosin. Prodigiosin is a tripyrrole, and recent studies have shown the anti-fungal and anti-cancer properties of this pigment and its importance in the pharmaceutical industry. Prodigiosin (C20H25N3O) and some of its analogs, such as undecyl prodigiosin (C25H35N3O), cycloprodigiosin (C20H23N3O) and metacycloprodigiosin (C25H33N3O), have important activities including antimicrobial, antifungal, antiprotozoal, antimalarial, They exhibit anti-cancer, immuno-suppressive and anti-viral effects. Prodigiosin is considered the most important candidate for cancer treatment due to its low toxic effect on normal cells and is used as a natural dye. Other possible applications that have recently been reported are the use of the molecule as a pH indicator, UV protector, autofluorescence product, and its use as a controlling factor in biofilm formation. Based on what has been said, the purpose of this research is to investigate the effect of the pigment of Ceraschia marciscens bacteria on the skov3 cell line of ovarian cancer.
Methods: Cerasia marciscens bacterium was purchased from Pasteur Institute microbial bank and its pigment was extracted with methanol solvent. The crude pigment was characterized by pure silica gel column chromatography and then by FT-IR. Investigating the apoptosis rate of SKOV3 cells that were treated with IC50 concentration of prodigiosin pigment was done using flow cytometry method. For this purpose, an average of 103 cells were treated in each well in a 24-well plate. A concentration of 0.01 mg/ml of prodigiosin pigment was added to the cells and after 72 hours, they were used to investigate apoptosis. After washing with PBS and one unit of trypsin enzyme, the cells were separated from the plate and centrifuged. Then the cell sediment was washed with 10% binding buffer solution and centrifuged at 15000 rpm for 15 minutes. The FITS solution attached to Annexin-V was incubated for 15 minutes at room temperature in the amount of 5 microliters. Then, using 5 microliters of PI solution, they were prepared for counting by flow cytometry. In this test, culture medium without pigment was used as a negative control. Statistical analysis The data were analyzed using SPSS version V.22 software. Considering the normality of the data distribution, the comparison of the treatment group and the control group was analyzed using the One Way Anova method and the p-value was calculated. The minimum level of significance was considered P<0.05. Tukey's method was used as post hoc test.
Results: The results showed that with increasing pigment concentration, its toxicity against both SKOV3 cells and HEK-293 cells increases significantly and its effect depends on the concentration. On the other hand, the cytotoxicity of prodigiosin against cancer cells was higher than normal cells, so that the IC50 value of the pigment against cancer cells within 24 hours was 0.010 mg/ml (10 μg/ml) and against normal cells was 0.175 mg/ml (175 μg/ml). This means that only 10 micrograms per milliliter of pigment was enough to destroy half of cancer cells within 24 hours, but to destroy the same number of normal cells, a concentration of 175 micrograms per milliliter of pigment was enough. It was needed. After 48 hours, the survival of both studied cell lines showed a further decrease, which confirms the time-dependent effect of the pigment. Also, at 48 hours, a concentration-dependent effect of prodigiosin was observed, and with increasing concentration, the viability of both cell lines decreased and pigment toxicity increased. The toxicity of prodigiosin in 48 hours was also higher against cancer cells than normal cells, so that the IC50 value of the pigment against cancer cells during 48 hours was 0.002 mg/ml (2 μg/ml). and against normal cells was 0.040 mg/ml (40 μg/ml). This means that only 2 micrograms per milliliter of pigment was enough to destroy half of cancer cells within 48 hours, but to destroy the same number of normal cells, a concentration of 40 micrograms per milliliter of pigment was enough. It was needed. Induction of apoptosis in SKOV3 cells by prodigiosin pigment was investigated using flow cytometry method. The results showed that the number of living cells decreased by 41.39% in the presence of prodigiosin. On the other hand, primary apoptotic cells increased by 25% in the treatment group. Delayed apoptotic cells also increased by 18% in the treatment group. Necrotic cells did not change much and decreased from 3.54% in the control group to 1.59% in the treatment group, which was not statistically significant. Apoptosis has provided solutions for effective anti-cancer treatment and so far many compounds have been reported that have anti-cancer effects by inducing apoptosis. Therefore, nowadays, one of the interesting strategies that has been considered in cancer chemotherapy is drug interventions that can mediate the death of malignant cells by inducing apoptosis.
Conclusion: The results of this research showed that prodigiosin has this ability and can induce apoptosis in SKOV3 cells while not affecting necrosis. These observations, along with the specific cytotoxicity of prodigiosin against cancer cells, make it a potential anticancer compound. The efficacy of prodigiosin in ovarian cancer had not been studied until now, and it was discussed for the first time in the present study, but it needs more investigations.
Methods: Cerasia marciscens bacterium was purchased from Pasteur Institute microbial bank and its pigment was extracted with methanol solvent. The crude pigment was characterized by pure silica gel column chromatography and then by FT-IR. Investigating the apoptosis rate of SKOV3 cells that were treated with IC50 concentration of prodigiosin pigment was done using flow cytometry method. For this purpose, an average of 103 cells were treated in each well in a 24-well plate. A concentration of 0.01 mg/ml of prodigiosin pigment was added to the cells and after 72 hours, they were used to investigate apoptosis. After washing with PBS and one unit of trypsin enzyme, the cells were separated from the plate and centrifuged. Then the cell sediment was washed with 10% binding buffer solution and centrifuged at 15000 rpm for 15 minutes. The FITS solution attached to Annexin-V was incubated for 15 minutes at room temperature in the amount of 5 microliters. Then, using 5 microliters of PI solution, they were prepared for counting by flow cytometry. In this test, culture medium without pigment was used as a negative control. Statistical analysis The data were analyzed using SPSS version V.22 software. Considering the normality of the data distribution, the comparison of the treatment group and the control group was analyzed using the One Way Anova method and the p-value was calculated. The minimum level of significance was considered P<0.05. Tukey's method was used as post hoc test.
Results: The results showed that with increasing pigment concentration, its toxicity against both SKOV3 cells and HEK-293 cells increases significantly and its effect depends on the concentration. On the other hand, the cytotoxicity of prodigiosin against cancer cells was higher than normal cells, so that the IC50 value of the pigment against cancer cells within 24 hours was 0.010 mg/ml (10 μg/ml) and against normal cells was 0.175 mg/ml (175 μg/ml). This means that only 10 micrograms per milliliter of pigment was enough to destroy half of cancer cells within 24 hours, but to destroy the same number of normal cells, a concentration of 175 micrograms per milliliter of pigment was enough. It was needed. After 48 hours, the survival of both studied cell lines showed a further decrease, which confirms the time-dependent effect of the pigment. Also, at 48 hours, a concentration-dependent effect of prodigiosin was observed, and with increasing concentration, the viability of both cell lines decreased and pigment toxicity increased. The toxicity of prodigiosin in 48 hours was also higher against cancer cells than normal cells, so that the IC50 value of the pigment against cancer cells during 48 hours was 0.002 mg/ml (2 μg/ml). and against normal cells was 0.040 mg/ml (40 μg/ml). This means that only 2 micrograms per milliliter of pigment was enough to destroy half of cancer cells within 48 hours, but to destroy the same number of normal cells, a concentration of 40 micrograms per milliliter of pigment was enough. It was needed. Induction of apoptosis in SKOV3 cells by prodigiosin pigment was investigated using flow cytometry method. The results showed that the number of living cells decreased by 41.39% in the presence of prodigiosin. On the other hand, primary apoptotic cells increased by 25% in the treatment group. Delayed apoptotic cells also increased by 18% in the treatment group. Necrotic cells did not change much and decreased from 3.54% in the control group to 1.59% in the treatment group, which was not statistically significant. Apoptosis has provided solutions for effective anti-cancer treatment and so far many compounds have been reported that have anti-cancer effects by inducing apoptosis. Therefore, nowadays, one of the interesting strategies that has been considered in cancer chemotherapy is drug interventions that can mediate the death of malignant cells by inducing apoptosis.
Conclusion: The results of this research showed that prodigiosin has this ability and can induce apoptosis in SKOV3 cells while not affecting necrosis. These observations, along with the specific cytotoxicity of prodigiosin against cancer cells, make it a potential anticancer compound. The efficacy of prodigiosin in ovarian cancer had not been studied until now, and it was discussed for the first time in the present study, but it needs more investigations.
Type of Study: Research |
Subject:
Pharmacology
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