Volume 28, Issue 6 (9-2021)                   RJMS 2021, 28(6): 116-126 | Back to browse issues page

Research code: --------
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
Tehran Medical Sciences, Islamic Azad University, Tehran, Iran , tnaji2002@gmail.com
Abstract:   (1781 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. 
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Type of Study: Research | Subject: Genetic

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