Anticancer effect of salina under stress and normal conditions against skin carcinoma cell line A431 in vitro

Item Type article

Authors Emtyazjoo, Mo; Moghadasi, Z.; Rabbani, M.; Emtyazjoo, Ma; Samadi, S.; Mossaffa, N.

Download date 25/09/2021 19:12:58

Link to Item http://hdl.handle.net/1834/37279 Iranian Journal of Fisheries Sciences 11( 2) 283-293 2012 Anticancer effect of Dunaliella salina under stress and normal conditions against skin carcinoma cell line A431 in vitro

Emtyazjoo Mo1; Moghadasi Z.*1; Rabbani M. 1; Emtyazjoo Maa; Samadi S.1; Mossaffa N.3 Received: March 2011 Accepted: November 2011

Abstract Dunaliella salina , a green microalga, has been consumed as food and medicine for a long time.The anti-oxidant and anticancer effects are related to more production of β- under stress conditions compared with normal circumstances.The scope of this study was survey of anticancer effect of the ethanol extract of Dunaliella salina under stress(EDSS) and normal (EDSN) conditions on death rate of skin carcinoma cell line A431 by MTT [3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide] method in vitro. Our results showed that the β-carotene amount in stress situation was 1.46 times more than in normal condition.The cytotoxic effect of EDSS exposed to 6.25, 12.5, 25, 50, 100 µg/ml in 6, 24, 48 h which was performed by MTT assay showed that there was a significant difference in the various time except in 6 h in both conditions.The results of compared death rate of A431cells exposed to various concentrations of EDSS and EDSN presented significant

difference in 6, 24, and 48 h after exposure. The LC 50 of EDSS in 24 and 48 h were 15.4 and 0.4 µg/ml , respectively and 478.6 and 46.4 µg/ml for EDSN.

Keywords: Dunaliella salina , Anticancer, A431 cell line, MTT assay, β-carotene

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1-Faculty of marine science and technology, Azad Islamic University North Tehran Branch, P.O.BOX: 19735-181, Tehran, Iran. 2 - Organ Biotechnology Department,USM University, Pinang, Malaysia. 3- Shahid Beheshti Medical Sciences Univercity, Department of Immunology, Tehran, Iran. * Corresponding author's email: [email protected]

284 Emtyazjoo Mo. et al., Anticancer effect of Dunaliella salina under stress and normal condition…

Introduction Pharmacological researches regarding al., 2002; Chidambara et al., 2005; Raja et natural materials of marine organisms al., 2007a; Raja et al., 2007b). Prevention which contain anticancer properties have of heart and chronic disease and malignant always been of concern to marine tumors , increase of cell division of scientists and other related fields have lymphocytes , extending the immunology always been concerned of pharmacological response and neoplastic transformations researches regarding natural materials of and growth control are the other marine organisms which contain characteristics of this substance (Wald et anticancer properties. Recently remarkable al.,1988; Stryker et al.,1990; Knekt et al., researches have been done on use of 1990; Challem, 1997; Buiatti, 1997; microalgae such as Dunaliella (Alejandaro Chidambara et al., 2005; Raja et al., et al., 2003). Algae and plants have some 2007a). Extracts of Dunaliella sp. showed curative of material for synthetic drug that its components can (Bechelli et al., 2011; Moghadasi et al., significantly inhibit induced fibrosarcoma 2011).The significant intracellular and lung cancer in mouse and lung cancer components of Dunaliella are , human cell line A549 and skin cancer, glycerol, protein and vitamins(Farouk et (Greenberg et al., 1990, Popple and al., 2002 ).The major part of these Goldbohm, 1995, Ming et al., 2008).This components has antioxidant properties research focused on the effect of EDSN (Gibbs and Duffus, 1975; Farouk et al., and EDSS on squamous cell carcinoma 2002). Dunaliella salina is extreme cell line A431.This cancer is related to halotolerant unicellular and motile green common skin cancer it is and almost algae (Chen and Jiang, 2011, Moghadasi et responsible for 20% of the malignant skin al., 2011).This alga has a unique property tumors.The significant cell characteristic of certain physiological responses. It has of this cancer is wide cells which look like Downloaded from jifro.ir at 13:50 +0330 on Tuesday February 13th 2018 produced more glycerol under stress scales of fish. This cancer is also called conditions such as high salinity, intensity Epidermoide. of light and limitation of nutrient especially phosphorus and nitrogen. A lot Materials and methods of researches were done for cartenoides Culture of alga especially β-carotene and also The initial sample of Dunaliella salina Tocopherol and Ascorbic acid (Farouk et was isolated and purified from Hoz-Soltan al., 2002; Takagi et al.; 2006, Raja et al., Lake located in the north-east of Qom in 2007b; Chen and Jiang, 2011; Moghadasi Iran. D.salina alga was cultured in et al., 2011). β-carotene was plentifully modified Janson medium.The condition of available in Dunaliella which has normal phase was done with a 15% significant effects on decreasing the risk of saltiness, pH 7.5, intensity glow light 100 lung, esophagus, pancreas, stomach, mol photons m -2s-1 (Takagi et al., 2006; breast, skin, colon , and ovary cancers Garcia et al., 2007) and a 35% stress (Poppel and Goldbohm, 1995; Farouk et condition of high saltiness, pH 8.2, and Iranian Journal of Fisheries Sciences, 11(2), 2012 285

intensity glow light 4000 mol photons m - Institute of Iran . A431 cells extended 2s-1 (Farouk et al., 2002; Moghadasi et al., cultured in complete tissue culture medium 2011) 12 h in a dark and 12 h in a light contained RPMI 1640 (Royal Park place. To prepare the dried powder of Memorial Institute), FBS ( Fetal bovine D.salina the harvested samples were serum) 10% , Penicillin 100 u/ml, centrifuged (eppendorf model 5810R) at Streptomycin 100 µg/ml incubated at 37 º

3500 rpm for 10 minutes and after C, 5% CO 2 , and 80% humidity (Ming et desalination by the phosphate buffered al.,2008). saline (PBS) they were freeze dried via the dehydration mechanism (Farouk et al., Cytotoxic assay 2002; Fazeli et al., 2006; Raja et al., MTT assay was performed in the A431 2007a; Garcia et al., 2007). cells to measure the cytotoxicity of EDSS and EDSN. In living cells, the Extraction mitochondrial metabolism of 3-(4,5- Extraction was done using 30g of D.salina dimethylthiazol-2-y1)- powder, both conditions with pure ethanol 2,diphenyltetrazolium bromide (MTT) salt at room temperature in the dark place into formazan took place and the amount (Raja et al., 2007a; Ming et al., 2008). of produced formazan was correlated with After 48 h, the samples were strained and the number of viable cells present.The dried in rotary evaporation (Heidolph A431cell was seeded in the 96 well plates WB2000) at 40˚c and 90 rpm. Dried at 1х 10 4 cells/well in RPMI medium EDSN and EDSS were storage at -80˚c in supplemented with 10% FBS (Shokrgozar the dark (Raja et al., 2007a; Ming et al., et al., 2007; Ming et al., 2008). After 24 h, 2008). cells were washed with PBS and then exposed to 5 concentrations (6.25, 12.5, HPLC analysis 25, 50, 100 µg/ml) of EDSN and EDSS. Downloaded from jifro.ir at 13:50 +0330 on Tuesday February 13th 2018 The obtained extracts of EDSN and EDSS After 6, 24 and 48 h , the number of viable in both normal and stress conditions were cells was determined. RPMI alone was analyzed by high performance liquid applied for negative control and RPMI chromatography (HPLC) [young Lin medium with various concentrations of Acme 9000, South Korea, column: extract were added in wells without cells

Lichrosphare Rp 100 C 18 with particle size for estimating error. For every extract of 4µ ( 25 cm of 4.6 cm) at a wave length exposed toA431 cells 3 wells were of 40 nm via DAD/UV. The mobile phase applied.The assessment was performed at was a Aceto nitril: methanol: least in triplicates (Ming et al., 2008 ). dichloromethan (10:20:70,v/v/v) (Raquel MTT (5mg/ml in PBS) was added to each et al., 2006). well (10µl/90µl medium) and the plate was incubated at 37 ˚c for 2h. Cells were Cell culture then spun in a centrifuge at 300 rpm for 5

Human squamous cell carcinoma cell line min and the medium was carefully (A431) was purchased from Pasture aspirated. A 100 µl isopropanol alcohol 286 Emtyazjoo et al., Anticancer effect of Dunaliella salina under stress and normal condition…

acetic acid 4% (v/v) was added to each cells exposed to EDSN and EDSS in three well.The plates were placed at room periodic times and various concentrations. temperature for 10 minutes and the Mean percentage of cell death of A431 absorbance at visible region was measured cells by various concentrations of EDSN for A431 cells which were exposed to in 6, 24 and 48 h showed a significant extracts in 6, 24 and 48h on an Elisa difference (0.001, 0.001, 0.511) reader (Ming et al., 2008). The percentage respectively.Therefore, it could be of cell death was calculated according to indicated that between 24 and 48 h in the formula below (Ghazanfary et al., concentrations of 6.25, 12.5, 25, 50 and 2006; Shokrgozar et al., 2007): 100 g/ml a significant difference was observed. Also, the result of this effect in stress condition in the three time periods respectively showed significant p-value (0.001, 0001 and 0.332). Although in time of 6 h among various concentrations in Statistical analysis normal and stress conditions a significant The SPSS 16 software package was used to difference was not observed. By analyze the raw data.Therefore, the mean comparing the death rate of A431 between slope scale of results was calculated. The EDSN and EDSS a significant difference one way ANOVA test was performed to in various concentrations in time periods identify the significant differences between of 6, 24, and 48 h was found. As seen in data. Also, the LC 50 values of EDSN and Figure 1, although both extracts showed EDSS exposed to the A431 cell were significant differences in the percentage of determined by Probit value analysis from resulted death, in time period of 6 h there SPSS software (Shokrgozar et al., 2007, was a harmonized and equal increase in Ming et al., 2008). their death. Downloaded from jifro.ir at 13:50 +0330 on Tuesday February 13th 2018 A431 cells without exposure to Results ethanol extract D.salina showed marker Cytotoxic assessment viability very well. After evalouating the The effect of ethanol extracts of EDSN A431 cells growth , these cells contained and EDSS response that leads to producing suitable separation and natural morphology more β-carotene and accumulation was (Figure 2), A431 cells exposed to ethanol analyzed against A431cells. The results of extract of D.salina lost natural this survey in three time periods of 6, 24 morphology and showed marker death cell and 48 h and in 5 concentrations of so membrane cells unable were separated 6.25,12.5, 25, 50 and 100 g/ml from (Figure 3). EDSN and EDSS are shown in Table 1. Evaluation LC 50 The assay technique in two processes was The LC 50 was found from EDSN against based on evaluating the percentage of cell A431 cells for 48 h was 46.4µg/ml and for death and MTT method. Figure 1 shows EDSS at time of 24 h was 15.4 µg/ml. the mean percentage of cell death of A431 HPLC analysis Iranian Journal of Fisheries Sciences, 11(2), 2012 285

Following evaluation of the results obtained β-carotene components in EDSS was from analysis of EDSN and EDSS calculated 1.46 times compared to EDSN circumstances using HPLC, the amount of (Table 2).

Table 1: Effect of ethanol extract of EDSN and EDSS on the percentage of death rate of cell line A431 at different times

* Mean ± SD

Table 2: The average of β-carotene in EDSN and EDSS by HPLC Downloaded from jifro.ir at 13:50 +0330 on Tuesday February 13th 2018

*RetentionTime

288 Emtyazjoo et al., Anticancer effect of Dunaliella salina under stress and normal condition…

Normal condition stress condition

90 80 70 60 50 40 30 20 % death % death rate 10 0 -10

Integrated doses and times

Figur 1: Comparative effect of EDSN and EDSS on percentage death rate of A431 cells in different times and cocentrations D =Doses 6.25,12.5,25,50.100(g/ml) T =Times 6,24,48(h)

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Discussion In this research, for the first time we models also subsequent the used different amount of 6.25, 12.5, 25, epidemiological surveys about various 50 ,100 µg/ml ethanol extracts. In the cancers, the antioxidant and anticancer recent years, many researches have been of internal components especially done for using Dunalialla .This alga existent beta carotene in this alga have owing to have high reserve of β-carotene proved (Chidambara et al., 2005, Raja et is valuable from economical point of al., 2007a). In this research, HPLC views (Raja et al., 2007b, Moghadasi et analysis of extracted compounds showed al., 2011). Following several studies that the presence of β-carotene and revealed applied in vitro and in vivo on animal the comparison of the amount of this Iranian Journal of Fisheries Sciences, 11(2), 2012 289

component in alga extract in two neck epidermoid, stomach, intestinal, conditions (normal and stress). It and gland secretory of pancreas appeared that the amount of EDSN and (Stryker et al., 1990, Buiatti, 1997). β- EDSS was 1.46 times more than normal carotene is known as the perquisite in the condition. The obtained results the from production of vitamin A, correcting analysis of samples were similar to the controls of natural , research of Fazeli et al., 2006 , Raja et maintaining of epithelial tissue, and a al., 2007 , Moghadasi et al., 2011. By chemical material preventing epithelial

considering the results of low LC50 in cancer(Raja et al., 2007a, Poppel and shorter time under stress condition Goldbohm, 1995, Ming et al., 2008). (production of greater β-carotene), it was Other inter cellular carotenoides of revealed that as an antioxidant β- Dunaliella alga are alfa carotene, carotene can repulse 1000 free radicals lycopene, lutein , zeaxanthin and and it could be caused by presence of cryptoxanthin which through many more β-carotene in the extract studies their antioxidant and anticancer (Challem,1997).The performed properties have been recognized epidemiological testimonies in USA by (Challem, 1997). Through extraction Peto et al., 1981 indicated that β- process, these carotenoides would be carotene with antioxidant properties extracted along by β-carotene (Ming et prevented cancer in various organs of the al., 2008). Existence of these body (Poppel and Goldbohm, 1995, components in cell structure of the micro Buiatti, 1997, Chidambara et al., 2005). alga involved in our study could cause In a study, scientists found out that β- its vast application in destroying cancer carotene via destroying the free radicals cells. By initiating 24 h an intense Downloaded from jifro.ir at 13:50 +0330 on Tuesday February 13th 2018 caused decrease of protuberance and β- difference between operations of these carotene reduce and erythema associated two extracts was always observed and in of with exposure to sun light (Stryker et low concentration of 25 g/ml for EDSS, al., 1990, Challem, 1997).The most a more significant peak was seen. In 48 h valid proof of its anti cancer more significant differences between the characteristics was resulted from two extracts was observed. Therefore, by researches on oral leukoplakia and via 5 increasing the concentration amounts of clinical tests it was proved that β- ethanol extracts of alga in EDSN and carotene supplement can transform this EDSS in three incubation times , the cell disease to the normal condition survival rates were decreased and effect (Challem, 1997).The curative substance of EDSS decreased more in cell survival of β-carotene has been proved to prevent rates compared to EDSN.The research of various tumors in human and animals Ming et al., 2008 showed that the such as melanoma skin cancer, head and ethanol extract DS could cause the cell 290 Emtyazjoo et al., Anticancer effect of Dunaliella salina under stress and normal condition…

death of human lung cancer A549 cell lower and acted in a shorter time line, by changing expression P21 and compared to normal condition also P53 proteins which are involved in the implying that alga extracts under stress regulation of the cell cycle then causing condition was more effective to destroy the cell cycle to stop. At the same A431 cells. Dunaliella can act as a strong research ethanol extracts of D.salina chemical inhibitor and maximum inhibited growth of Human Liukemeia accumulation of anticancer against as β- cell line (HL-60) with exposure to 10 carotene in Dunaliella salina occurred l/ml and biphenotypic B when it was grown under combined myelomonocytic leukemia cell line stress conditions (Farouk et al., 2002, (MV-4-11) with exposure to 8 l/ml Fazeli et al., 2006, Hosseini and Shariati, (Bechelli et al., 2011). In the current 2009). Our results suggest that research, we proved that the death performed and extended research on percentage of A431cells would increase molecular path way of cell dead by adding to the incubation time and signaling by these products and further raising various concentrations of EDSN study could be helpful for better use of and EDSS and the increased cell death Dunaliella extracts in both cell culture was shown further in EDSS that might system and another in vitro study. be owing to its high β-carotene (Ming et al., 2008). Besides, Raja et al., 2006 References demonstrated that Dunaliella salina Alejandaro, M. S., Mayer, Kirk, R., extract had anticancer effects against and Gustafson., 2003. Marine induced fibrosarcoma in Wistar rats pharmacology in 2000: Antitumor (Raja et al., 2007a). Also in the research and cytotoxic compounds. Downloaded from jifro.ir at 13:50 +0330 on Tuesday February 13th 2018 done by Fujii et al., 1993 the effect of International Journal Cancer, 105, Dunaliella bardawile in the natural 291-299. growth of udder cells and prevention of Buiatti, E., 1997. The role of tumor cells were proved (Fujii et al., chemoprevention in cancer control. 1993). Parallel with Fujii et al, another Salud Publica de Mexico, 39( 4), performed research by Xulex et al., 310-317. 1993 on Dunaliella bardawil extract Becheli, J., Coppage, M., Rossel, K., represented the significant prevention of and Liesveld. J., 2011 . Cytotoxicity NSAR induced cancer of proventriculus of Algae Extracts on Normal and in mice. Based on the obtained results Malignant Cells. Leukemia Research from this research the concentration of and Treatment, Volume 2011, 15.4 g/ml of EDSS against stress in 24 Article ID 373519, 7 pages. h could kill 50% of A431 cells, this Challem, J., 1997. Beta-carotene and

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