Volume 28, Issue 6 (9-2021)
RJMS 2021, 28(6): 116-126 |
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Ethics code: IR.IAU.PS.REC.1399.254
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Saremi S, Naji T, Nasoohi N. Evaluation of the effects of phycocyanin on nitric oxide in fibrosarcoma HT1080 tumor cell line. RJMS 2021; 28 (6) :116-126
URL: http://rjms.iums.ac.ir/article-1-6774-en.html
URL: http://rjms.iums.ac.ir/article-1-6774-en.html
Tehran Medical Sciences, Islamic Azad University, Tehran, Iran , tnaji2002@gmail.com
Abstract: (2152 Views)
Background & Aims: The various studies show that cancer is currently one of the leading causes of death in the world. Its spread and high mortality rate is influenced by two factors, age and race. Spirulina platensis is a microalga with biological activities such as antioxidant, immunomodulatory, and anti-inflammatory which nowadays is used to produce nutritional supplements named Phycocyanin. Phycocyanine isolated from Spirulina algae is a non-toxic, water-soluble protein pigment that exhibits antioxidant, anti-inflammatory, hepatoprotective, and neuroprotective effects. In addition to these health benefits, this pigment has been used in dietary nutritional supplements and natural colorant applications in the food, cosmetic, and biotechnology industries. Fibrosarcoma is a rare example of soft tissue sarcoma that originates in fibrous tissue, fascia, and tendons in and between muscles. The purpose of this research was to investigated the effects of Phycocyanin on nitric oxide (NO) levels in the Fibrosarcoma HT1080 tumor cell line.
Methods: In this study, Spirulina platensis was obtained from the National Center for Genetic and Biological Resources of Iran, and for its cultivation, Zarok medium was used . The composition of the culture medium (g/l) includes [NaHCO3] 38.0 g, K2 [HPO4] 0.5 g, [NaNO3] 2.5 g, [ K2 SO4] 1.0 g,[ NaCl2] 1.0 g, [MgSO4 +7water ] 0.2 g, [FeSO4 +7 water] 0.05 g and urea 0.2 g in pH 8.5. After culturing the algae and exposing it to fluorescent light with an intensity of 3500 lux in period of 12 hours of darkness and 12 hours 0f light, and then for its cultivation the samples were placed at 29 ° C for 16 days. The specimens were shaken 3 times daily for good algal growth. After observing the algal crystal, cell masses were collected. This process was performed using 100 and 20λ filters which are used to separate larger particles and algal masses, respectively. The collected masses were washed 3 times to eliminate the existence of the culture medium, and for drying the algae it was put in a 45 ° C incubator for 48 hours. After extraction of Phycocyanin, destruction rate of cell with MTT test was used with various concentration of 125, 250, 500, 1000 µg/ml and evaluated on the cell line HT1080. For MTT Assay, cells were harvested from exponential-phase maintenance cultures . Single-cell suspensions were prepared, cells counted using a hemocytometer and then dispersed within replicate 96-well microliter plates to a total volume of 200 µl/well. Eight duplicate wells were used for each determination. Plates were maintained at 37°Cin a humidified atmosphere of 90% N2-5% CO2-5% O2. A 24-h pre incubation time was allowed prior to irradiation. To perform the MTT assay, culture medium was removed from the wells ensuring that the monolayer of cells was not disturbed. MTT solution (100 µl) at appropriate concentrations was then added to each well and the plates incubated at 37°C for 2-4 h, depending upon individual cell line requirements. Following incubation, cells were inspected using low power microscopy to confirm reduction of the tetrazolium and to assess confluency of the monolayer. The remaining MTT solution was then removed and 100 µl of DMSO was then added to each well to dissolve the formazan crystals. Plates were shaken for 5 min on a plate shaker to ensure adequate solubilization. Absorbance readings on each well were performed at 540 nm. A reference wavelength was not used in as much as this made little difference to the absorbance readings obtained. Control wells for absorbance readings contained no cells or medium but MTT solution was added as per experimental wells, and removed after incubation, then DMSO was added. All experiments were performed at least three times. The experimental groups were treated with 125, 250, 500, 1000 µg/ml of Phycocyanin keeping control group intact. HT1080 cells were exposed to 250 μg/ml of Phycocyanin for 24 hours. Then the supernatant was removed and the NO activity was evaluated HT1080 cells exposed to different concentrations of Phycocyanin using Grease method. In the Grease method, the amount of NO is measured indirectly in such a way that sulfanilic acid was made to react with nitrite in an acidic solution. This action forms an azo dye that can be measured at a light absorption of 520 to 590 nm. After measuring NO activity and RNA extraction, iNOS gene expression was evaluated using Real time PCR with designed primers. Keeping House gene was GAPDH. Data analysis was performed by SPSS18 software. One-way analysis of varience (ANOVA) was used to compare the data between the groups.
Results: Cell viability significantly decreased in HT1080 cells exposed to at least 250 μg / ml of Phycocyanin compared with control group. Nitric oxide assay showed that NO concentration significantly increased. iNOS gene expression level significantly increased in HT1080 cells exposed IC50 dose of Phycocyanin .
Conclusion: According to MTT analysis, it could be concluded that amount of NO after 24h treatments with phycocyanine showed significant discharge in concentration more than 250 μg/ml where toxicity initiated from this concentration. Extracted Phycocyanin from Spirulina algae with more than 250 μg/ml concentration could have anti-cancer effects. Also, the results of the NO release analysis confirmed the above claim. Because the amount of NO secretion after 24 hours of drug treatment showed a significant increase in NO secretion at concentrations above 250 μg / ml, which according to MTT analysis, the toxicity of this drug after this concentration, the drug from a concentration above 250 µg/ml can be effective in inducing anti-cancer effects.
Finally, gene expression analysis was performed which showed that in the drug-treated sample iNOS gene expression was significantly increased compared to the control group (P≤0.001). The studies have shown the iNOS gene expresses a calcium / calmodulin-independent enzyme that can accelerate the production of nitric oxide, it can be concluded that Phycocyanin is involved in inhibiting cancer cells by inducing nitric oxide production.
During treatment with Phycocyanin, cell proliferation as well as the ability of cells to form colonies decreased. The other studies have shown that Phycocyanin induces apoptosis by increasing γ-H2AX levels and altering the Bcl2 / Bax ratio, followed by the release of cytochrome C and increasing Caspase 9 levels. It can be concluded that Phycocyanin can be a capable compound in the treatment of cancer.
Methods: In this study, Spirulina platensis was obtained from the National Center for Genetic and Biological Resources of Iran, and for its cultivation, Zarok medium was used . The composition of the culture medium (g/l) includes [NaHCO3] 38.0 g, K2 [HPO4] 0.5 g, [NaNO3] 2.5 g, [ K2 SO4] 1.0 g,[ NaCl2] 1.0 g, [MgSO4 +7water ] 0.2 g, [FeSO4 +7 water] 0.05 g and urea 0.2 g in pH 8.5. After culturing the algae and exposing it to fluorescent light with an intensity of 3500 lux in period of 12 hours of darkness and 12 hours 0f light, and then for its cultivation the samples were placed at 29 ° C for 16 days. The specimens were shaken 3 times daily for good algal growth. After observing the algal crystal, cell masses were collected. This process was performed using 100 and 20λ filters which are used to separate larger particles and algal masses, respectively. The collected masses were washed 3 times to eliminate the existence of the culture medium, and for drying the algae it was put in a 45 ° C incubator for 48 hours. After extraction of Phycocyanin, destruction rate of cell with MTT test was used with various concentration of 125, 250, 500, 1000 µg/ml and evaluated on the cell line HT1080. For MTT Assay, cells were harvested from exponential-phase maintenance cultures . Single-cell suspensions were prepared, cells counted using a hemocytometer and then dispersed within replicate 96-well microliter plates to a total volume of 200 µl/well. Eight duplicate wells were used for each determination. Plates were maintained at 37°Cin a humidified atmosphere of 90% N2-5% CO2-5% O2. A 24-h pre incubation time was allowed prior to irradiation. To perform the MTT assay, culture medium was removed from the wells ensuring that the monolayer of cells was not disturbed. MTT solution (100 µl) at appropriate concentrations was then added to each well and the plates incubated at 37°C for 2-4 h, depending upon individual cell line requirements. Following incubation, cells were inspected using low power microscopy to confirm reduction of the tetrazolium and to assess confluency of the monolayer. The remaining MTT solution was then removed and 100 µl of DMSO was then added to each well to dissolve the formazan crystals. Plates were shaken for 5 min on a plate shaker to ensure adequate solubilization. Absorbance readings on each well were performed at 540 nm. A reference wavelength was not used in as much as this made little difference to the absorbance readings obtained. Control wells for absorbance readings contained no cells or medium but MTT solution was added as per experimental wells, and removed after incubation, then DMSO was added. All experiments were performed at least three times. The experimental groups were treated with 125, 250, 500, 1000 µg/ml of Phycocyanin keeping control group intact. HT1080 cells were exposed to 250 μg/ml of Phycocyanin for 24 hours. Then the supernatant was removed and the NO activity was evaluated HT1080 cells exposed to different concentrations of Phycocyanin using Grease method. In the Grease method, the amount of NO is measured indirectly in such a way that sulfanilic acid was made to react with nitrite in an acidic solution. This action forms an azo dye that can be measured at a light absorption of 520 to 590 nm. After measuring NO activity and RNA extraction, iNOS gene expression was evaluated using Real time PCR with designed primers. Keeping House gene was GAPDH. Data analysis was performed by SPSS18 software. One-way analysis of varience (ANOVA) was used to compare the data between the groups.
Results: Cell viability significantly decreased in HT1080 cells exposed to at least 250 μg / ml of Phycocyanin compared with control group. Nitric oxide assay showed that NO concentration significantly increased. iNOS gene expression level significantly increased in HT1080 cells exposed IC50 dose of Phycocyanin .
Conclusion: According to MTT analysis, it could be concluded that amount of NO after 24h treatments with phycocyanine showed significant discharge in concentration more than 250 μg/ml where toxicity initiated from this concentration. Extracted Phycocyanin from Spirulina algae with more than 250 μg/ml concentration could have anti-cancer effects. Also, the results of the NO release analysis confirmed the above claim. Because the amount of NO secretion after 24 hours of drug treatment showed a significant increase in NO secretion at concentrations above 250 μg / ml, which according to MTT analysis, the toxicity of this drug after this concentration, the drug from a concentration above 250 µg/ml can be effective in inducing anti-cancer effects.
Finally, gene expression analysis was performed which showed that in the drug-treated sample iNOS gene expression was significantly increased compared to the control group (P≤0.001). The studies have shown the iNOS gene expresses a calcium / calmodulin-independent enzyme that can accelerate the production of nitric oxide, it can be concluded that Phycocyanin is involved in inhibiting cancer cells by inducing nitric oxide production.
During treatment with Phycocyanin, cell proliferation as well as the ability of cells to form colonies decreased. The other studies have shown that Phycocyanin induces apoptosis by increasing γ-H2AX levels and altering the Bcl2 / Bax ratio, followed by the release of cytochrome C and increasing Caspase 9 levels. It can be concluded that Phycocyanin can be a capable compound in the treatment of cancer.
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