|Year : 2014 | Volume
| Issue : 4 | Page : 1004-1007
The comparison of antimutagenicity and anticancer activities of Echinophora platyloba DC on acute promyelocytic leukemia cancer cells
Maliheh Entezari1, Fataneh Hashem Dabaghian1, Mehrdad Hashemi2
1 Department of herbal medicine, Institue for Islamic and Complementary Medicine, Iran University of Medical Sciences, Tehran, Iran
2 Department of Genetics, Islamic Azad University, Tehran Medical Branch, Tehran, Iran
|Date of Web Publication||9-Jan-2015|
Department of Genetics, Islamic Azad University, Tehran Medical Branch, Tehran
Source of Support: Research Institute for Islamic and Complementary
Medicine, Iran University of Medical Sciences, Tehran, Iran, Conflict of Interest: None
Background: Cancer is one of the main causes of mortality in the world which is created by the effect of enviromental physico-chemical mutagen and carcinogen agents. In the last years, many studies have been performed on the anticancer effects of flavonoids. Echinophora platyloba DC plant (Khousharizeh) is one of the indigenous medicinal plants which is used as a food seasoning and medicine in Iran.
Materials and Methods: The extract was evaluated in terms of antimutagenicity properties by a standard reverse mutation assay (Ames Test). This was performed with histidine auxotroph strain of Salmonella typhimurium (TA100). Thus, it requires histidine from a foreign supply to ensure its growth. The afore mentioned strain gives rise to reverted colonies when expose to carcinogen substance (Sodium Azide). The other objective of this study was to examine the in vitro cytotoxic activity of cell death of crude methanolic extracts prepared from Echinophora platyloba on Acute Promyelocytic Leukemia cell line (NB4). Cytotoxicity and viability of methanolic extract was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dye exclusion assay.
Results: In Ames test the extract prevented the reverted mutations and the hindrance percent was 93.4% in antimutagenicity test. Data obtained from this assay indicated that the extract significantly reduced the viability of NB4 cells and inhibited cell growth in a dose dependent manner.
Conclusion: This study demonstrates the antimutagenicity effect of Echinophora Platyloba and suggests that it has a potential as an anticancer agent.
Echinophora platyloba DC对急性早幼粒细胞白血病癌细胞的抗突变、抗癌活性的比较
背景：癌症是世界上主要致死的原因之一，其发生归因于环境的物理化学诱变剂和致癌物介质。在过去的几年中，许多研究已经对黄酮类化合物的抗癌作用进行了（研究）。Echinophora platyloba DC植物（khousharizeh）是一种本土药用植物，在伊朗它被用作调味食品和药品。
关键词：急性早幼粒细胞白血病，Ames试验，echinophora platyloba DC，NB4细胞，MTTY试验
Keywords: Acute promyelocytic leukemia, Ames Test, Echinophora platyloba DC, NB4 cells, MTT assay
|How to cite this article:|
Entezari M, Dabaghian FH, Hashemi M. The comparison of antimutagenicity and anticancer activities of Echinophora platyloba DC on acute promyelocytic leukemia cancer cells. J Can Res Ther 2014;10:1004-7
|How to cite this URL:|
Entezari M, Dabaghian FH, Hashemi M. The comparison of antimutagenicity and anticancer activities of Echinophora platyloba DC on acute promyelocytic leukemia cancer cells. J Can Res Ther [serial online] 2014 [cited 2019 Nov 13];10:1004-7. Available from: http://www.cancerjournal.net/text.asp?2014/10/4/1004/137907
| > Introduction|| |
Cancer is the major cause of human's death because of high incidence and mortality.The identification of new cytotoxic drugs with low side effects on immune system has developed as important area in new studies of immunopharmacology. ,,
Many studies report that a high diet in fruits and vegetables lowers the incidence of cancer. , Some of fruits and vegetables are considered as the main anticancer foods, because of their abundant antioxidants such as phenols, vitamin C, vitamin E, beta-carotene and lipotene.  It has been reported that various fruit and vegetable extracts are capable of inhibiting the protoasome activity and this inhibition is associated with tumor cell apoptosis.  Echinophora is a ten-species genus of Apiaceae that contains four species native to Iran, including E. orientalis, E. sibthorpiana, E. cinerea, and E. platyloba.  Echinophora platyloba is widely used in western and central Iran as a food seasoning and edible vegetable. Localpeople add the plant to pickles and tomato pastes as an antifungal and antimicrobial preservative. 
Taking into consideration, the potent antifungal and antibacterial activity suggested that the methanolic extract of Echinophora platyloba DC could be a potentialcandidate as a cytotoxic and growth inhibitory agent. , The present study was undertaken to evaluate the antimutagenicity and cytotoxicity effects of crude methanolic extract of E. platyloba in Salmonella typhimurium (TA100) and malignant cell line in vitro respectively. Ames test is one of the most current test to assay anticancer and antimutagenesis effects using bacteria with special mutants . Cell cytotoxicity and viability was examined by the 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dye exclusion assay.
| > Materials and methods|| |
Preparation of plant extract
The plant was collected in summer 2012 from Lashkarak hill located in eastern north of Tehran. Collected plat materials were dried under the shadow and its leaves removed from the stem and then they were powdered. Powdered samples of leaves were extracted by floating in methanol and then by hot water. Nearly 200 gr of powder for each sample solved in 600 ml of methanol and then left for about 48 h in the ambient temperature (25°) while shaking slowly. Each extract filtered by Watman paper filter No. 1. Filtered extracts were dried in the room temperature by air flow.  Extracts dried by UV ray were sterilized at night and their sterility was studied on nutrient agar medium by culturing the plant extract. All dried extracts stored in room temperature until examination.
NB4 cell line were obtained from the National Cell Bank of Iran (NCBI). The cells were cultured in RPMI-1640 containing 10% FBS, 2 mM glutamine, antibiotics (penicilin G, 60 mg/L; streptomycin, 100 mg/L; amphotericin B, 50 μL/L) under a humid atmosphere (37°C, 5% CO 2 , 95% air).
Upon reaching appropriate confluence, the cells were passaged The cells were incubated with 0,100,200,300,400 and 500 μg/mL concentrations of E. platyloba extract for a defined time.
Determination of cell viability
The assay detects the reduction of MTT [3- (4,5-dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide] (Sigma) (a colorimetric technique) by mitochondrial dehydrogenase to blue formazan product, which reflects the normal function of mitochondria and hence the measurement of cytotoxicity celland viability.
Briefly, l0 4 cells/well were treated with various concentrations of extract. After 24 hour incubation the cells were washed twice with phosphate buffered saline (PBS) and MTT (0.5 mg/mL PBS) was added to each well and incubated at 37°C for 3h. The formazan crystals that formed were dissolved by adding dimethyl sulfoxide (100 μL/well), and the absorbance was read at 570 nm using a microplate scanning spectrophotometer (ELISA reader, Organon Teknika, Netherlands). Toxicity level was calculated by the following formula:
To diminish test error level, MTT was added to some wells without cells and absorbance level was read and ultimately subtracted from whole the absorbance.
IC 50 determination
The 50% inhibition concentration (IC 50 ) values of extract on NB4 cells at 24h were determined. IC 50 was determined by probit analysis using the Pharm PCS (Pharmacologic Calculation System) statistical package (Springer- Verlag, USA)
Statistical significances of difference throughout this study were calculated by one-way variance analysis.
Salmonella typhimurium TA100 was used for Ames test. Fresh bacterial culture should be used for test and incubation time of bacterial culture in nutrient broth should not be more than 16 hours. Appropriate bacterial concentration was considered 1-2 × 10 9 cells/ml. After consideration of cytotoxicity effect of fruit juice on cancer cells, according to Ames, Extract was added to test tube containing 0.5ml of the overnight fresh bacterial culture, 0.5ml of histidine and biotin solution 0.5mM histidine/0.5mM biotin, 10 ml top agar (50 gr/L Agar + 50 gr/L NaCl), sodium azide as a carcinogene (1.5 μg/ml Sodium azide) and then content of this tube distributed on the surface of minimum medium of glucose agar (40% glucose), after 3 second shaking incubation was performed at 37°C for 48 hours. Each treatment was repeated 3 times. In the test after 48 h incubation at 37°C, reversed colonies were counted in control and test plates and after angular conversion, results were compared by analysis variance. Most materials in their original form are inactive in terms of carcinogenic effects and most materials to become metabolically are active to display mutagenesis properties. So it is necessary to add a microsomal sterile extract to mammalian tissue like rat. After 10 h starvation, livers of 10 male rats were separated. Starvation stimulates and enhances liver enzymes secretion. Livers homogenized in 0.15 M potassium chlorideand centrifuged for 10 min in 9000 rpm in at 4°C. Supernatant (S9 mixture) was removed and mixed with necessary cofactors including NADP, G6p (glucose 6 phosphate) and then 0.5 ml of the solution was added to Top agar in order to consider anticancer effect.
Also after the counting colonies in anticancer-antimutagenesis test, prevention percentage or antioxidant activity has been calculated as follows: 
T is reversed colonies in each Petri dish under carcinogen and extract and M shows reversed colonies in petri dishes related to positive control (mutagen).
| > Results|| |
The effects of methanolic extract of Echinophora platyloba on inhibition and proliferation of NB4 cell line
The effect of E. platyloba was studied as a dose-response experiment. Proliferation of NB4 cells was significantly inhibited by E. platyloba in a concentration-dependent manner in 24 h Different concentrations of extract at 24 h have cytotoxicity effects on NB4 cell line. Compared to the controls, different doses of extract (100 μg/ml)- 10.65%, (200 μg/ml)- 48.9.571%, (300 μg/ml)-61.334%, (400 μg/ml) -81.434 and (500 μg/ml) -87.35 decreased NB4 cells number (P < 0.05)
The IC 50 after 24h was calculated (P < 0.05).
Antimutagenesis effect of methanolic extract of Echinophora platyloba.
The results of colony counting in Ames test under 200 μl/ml of the extract (with regard to the results of vital capacity test) showed that there was a significant difference between antimutagenesis effect on colony growth with controls (distilled water and sodium azide) (P < 0.01) [Figure 1].
|Figure 1: Results of colony counting in Ames test under 200 μg/ml of the extract in mutagenesis test|
Click here to view
The extract prevented the reverted mutations and the hindrance percent was 93.4% in antimutagenicity test.
| > Discussion|| |
This herb is one of the four species in Iran which is the only species in Iran , . This herb is used also as the food species.  Till now there are reports of scientific evidences about the anti-microbial effect of E. Platyloba. In recent studies on medicaltreatment application of E. platyloba, Entezari et al. showed that the antibacterial effect on the growth of two bacteria (Staphylococcus aureus and Pseudomonas aeroginosa) of E. platyloba methanolessences and they did not observed prevention in growth of Candida albicans, Aspergillus flavus and Aspergillus niger.  In study which is carried on the Echinophora it is cleared that this herb has elements such as Saponin, Alkaloids and Flavonoids. Different components antibacterial existing in this plant were previously studied and the most important ones include trans-β-ocimene (67/9%), 2-furanone (6.2%), myrcene (6%), linalool (3.1%) and cis-β-ocimene (2.3%). 
Zare et al. showed anticancer effect of E. platyloba on the growth of mouse fibrosarcoma cell line (WEHI-164). Observation proved that apoptosis was the major mechanism of cell death.  Since usual methods on cancer treatment (surgery, chemical treatment, radiotherapy) have an effect on natural dividing cells, in addition to tumor cell, and kill or arrest their cell division.  In recent years, herbals found widespread use in prevention and treatment of cancer which in this procedure, tumor cells are controlled while natural cells remain intact.  The effect of diverse antioxidant foods on cancer and cardiovascular disease has been proved and it has been revealed that these materials cause to enhance long life by 60%.  During laboratory researches on Tangeretin, it has been revealed that these materials have antioxidant and anticancer effects and preservative effect on hepatocytes.  Antimutagenesis effect of E. platyloba has not been reported, in the present study vital capacity test and Ames test were used to consider its anticancer effect with special emphasizes on application of Salmonella typhimurium to identify antimutagenesis and anticancer level of chemicals. In this research, the E. platyloba extract displayed anticancer and antimutagenesis effect. According to the Ames theory which presented in 1982, in case the number of colonies on positive control medium (contained carcinogen) is two times more than test sample, the substance will be considered as an antimutagenesis and when prevention percent ranges between 25-40%, mutagenesis effect in this test sample is assumed medium and when prevention percent is more than 40, mutagenesis effect of the test sample is strong and in case prevention percent is less than 25, mutagenesis effect is negative.  This was found in the E. platyloba extract (93.4%).
Acute myelocytic leukemia (AML) is a malignant neoplasm of hematopoietic cells, often associated with abnormal proliferation of myeloid precursor cells, and cessation of cell differentiation at several stages of hematopoietic precursors. AML is the most common type of leukemia often occurring in adults, with about 11,900 cases per year in the United States.  The incidence of AML increases with age, and the peak of onset is in the sixth decade of the human life. AML contains less than 1% of all cancers, and about 34% of all types of leukemia.Treatment phase of AML contains two general parts including remission induction and post-remission therapy. Cytarahine, Anthracycline, Daunoruhicin (DNR), and Thioguanine are commonly used for remission induction. And some studies suggest that Idarubicin or Mitoxantrone, and the highdose use of Cytarabine are effective too.Whether leukemia patients in the first remission should receive bone marrow transplant immediately or after relapse has been a controversial issue. Although the successful results of transplant especially in persons less than 20 years of age are notable, the most significant effects of bone marrow transplant has been seen in patients failing chemotherapy. 
Despite all progresses in the treatment of AML, patients with this cancer are faced with some complications and second malignancies. Since most patients with AML will experience the relapse, and long-term survival remains unresolved, there is a continued need to discover new strategies for treatment of leukemia. Recently, with the increase of laboratory proofs, researchers have begun targeted treatment of AML based on differentiation induction.  Researchers have so far evaluated many differentiation inducing agents which affect the proliferation and differentiation of tumor cells or can induce apoptotic cell death in cancer cells. Acute promyelocytic leukemia (APL) is one of the most malignant forms of acute leukemia with a fatal course of only weeks which represents 10-15% of AML in adults.  In vitro study on cancerous cell culture revealed that the E. platyloba extract severely repressed division of cancerous cells. Findings from the present study indicate that the E. platyloba extract is highly cytotoxic to human leukemia cells, supporting its use as an effective therapeutic agent in the management of acute promyelocytic leukemia.
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