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
Islamic Azad University , mh_mohamadi@yahoo.com
Abstract: (357 Views)
Background and purpose: Cancer is the second cause of death after heart diseases in developed societies. Cancer treatment is much more difficult than other diseases due to its limitations and fundamental challenges)1). One of the most important limitations in cancer treatment is to destroy tumor cells in the presence of normal body cells, without causing toxicity to normal cells. Therefore, the targeted treatment of cancer cells or the use of compounds with side effects are less than the most important concerns of researchers in order to provide therapeutic solutions(2,3). Ovarian cancer is the sixth most common cancer in women, which kills 152,000 people worldwide every year(4). Despite various efforts aimed at developing effective treatments, the overall survival rate of ovarian cancer patients 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(5). Recently, bacteria and substances produced by them have received serious attention as cancer treatment agents. 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, Cerasia marciscens (Coli toxins) to treat patients with unprotected tumors(6). 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 already been reported as having anti-cancer potential(7). Studies show that the ability to produce pigments in microorganisms causes antibiotic resistance and resistance to heavy metals in the environment(8). 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 free oxygen radicals(9). Among these, 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 culture, the process of extracting pigments, as well as their importance and practical features(10). They take Among these, we can mention Ceraschia marciscens species. Prodigiosin red pigment is produced by Ceraschia marciscens bacteria and has antimicrobial, anticancer and antifouling activities(11). In the current research, the anticancer effect of this pigment on ovarian cancer cells was evaluated.
Methodology: Cerasia marciscens bacterium was purchased from the microbial bank of Pasteur Institute and its pigment was extracted with methanol solvent. The crude pigment was purified by silica gel column chromatography and then characterized by FT-IR. Then, the cytotoxicity of the pigment against ovarian cancer SKOV3 and normal HEK-293 cells was evaluated by MTT test. Also, the pigment's ability to induce apoptosis in cancer cells was studied by flow cytometry. 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 as P0.05. Tukey's method was used as post hoc test.
Results: Cytotoxicity of prodigiosin was evaluated within 24 and 48 hours against SKOV3 cancer cells and normal HEK-293 cells at different concentrations using MTT test. The results after 24 hours showed that with the increase in 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). 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). Induction of apoptosis in SKOV3 cells by prodigiosin pigment was investigated using flow cytometry method. The amount of live, apoptotic and necrotic cells in the control and treated groups with a concentration of 0.01 mg/ml prodigiosin was compared. The number of living cells in the presence of prodigiosin decreased from 95.08% to 53.69% (P<0.001). On the other hand, primary apoptotic cells increased from 0.58% in the control group to 25.62% in the treatment group (P<0.001). Delayed apoptotic cells also increased from 0.79% in the control group to 19.10% in the treatment group (P<0.001). 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 (P>0.05).
Conclusion: In general, the results of this research showed the cytotoxic effect of prodigiosin isolated from Ceraschia marciscens bacteria against ovarian cancer SKOV3 cells. According to the database searches, the effect of prodigiosin on ovarian cancer has not been investigated so far, and this research reports for the first time that this pigment is able to inhibit the growth of SKOV3 cancer cells. In this research, it was also observed that the toxicity of prodigiosin against normal cells is significantly lower than its toxicity against cancer cells. This problem strengthens the potential of prodigiosin as an anticancer candidate because one of the important features of anticancer agents is specific toxicity against cancer cells, which prodigiosin has this feature. In another part of the research, the induction of apoptosis by prodigiosin was observed in SKOV3 cancer cells. Our results showed that prodigiosin decreases the number of living cells and increases the number of apoptotic cells, while the number of necrotic cells did not change much. Inducing apoptosis and not causing necrosis is also one of the important features of an anticancer compound. Necrosis causes inflammation and a combination that does not cause necrosis while inducing apoptosis can be promising. Therefore, prodigiosin can be considered as a potential compound for the treatment of ovarian cancer, however, more research is required in this field.
Methodology: Cerasia marciscens bacterium was purchased from the microbial bank of Pasteur Institute and its pigment was extracted with methanol solvent. The crude pigment was purified by silica gel column chromatography and then characterized by FT-IR. Then, the cytotoxicity of the pigment against ovarian cancer SKOV3 and normal HEK-293 cells was evaluated by MTT test. Also, the pigment's ability to induce apoptosis in cancer cells was studied by flow cytometry. 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 as P0.05. Tukey's method was used as post hoc test.
Results: Cytotoxicity of prodigiosin was evaluated within 24 and 48 hours against SKOV3 cancer cells and normal HEK-293 cells at different concentrations using MTT test. The results after 24 hours showed that with the increase in 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). 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). Induction of apoptosis in SKOV3 cells by prodigiosin pigment was investigated using flow cytometry method. The amount of live, apoptotic and necrotic cells in the control and treated groups with a concentration of 0.01 mg/ml prodigiosin was compared. The number of living cells in the presence of prodigiosin decreased from 95.08% to 53.69% (P<0.001). On the other hand, primary apoptotic cells increased from 0.58% in the control group to 25.62% in the treatment group (P<0.001). Delayed apoptotic cells also increased from 0.79% in the control group to 19.10% in the treatment group (P<0.001). 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 (P>0.05).
Conclusion: In general, the results of this research showed the cytotoxic effect of prodigiosin isolated from Ceraschia marciscens bacteria against ovarian cancer SKOV3 cells. According to the database searches, the effect of prodigiosin on ovarian cancer has not been investigated so far, and this research reports for the first time that this pigment is able to inhibit the growth of SKOV3 cancer cells. In this research, it was also observed that the toxicity of prodigiosin against normal cells is significantly lower than its toxicity against cancer cells. This problem strengthens the potential of prodigiosin as an anticancer candidate because one of the important features of anticancer agents is specific toxicity against cancer cells, which prodigiosin has this feature. In another part of the research, the induction of apoptosis by prodigiosin was observed in SKOV3 cancer cells. Our results showed that prodigiosin decreases the number of living cells and increases the number of apoptotic cells, while the number of necrotic cells did not change much. Inducing apoptosis and not causing necrosis is also one of the important features of an anticancer compound. Necrosis causes inflammation and a combination that does not cause necrosis while inducing apoptosis can be promising. Therefore, prodigiosin can be considered as a potential compound for the treatment of ovarian cancer, however, more research is required in this field.
Type of Study: Research |
Subject:
Pharmacology
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