Apoptosis Induction of Armeniacae Semen Extractin Human Acute Leukemia (NALM-6 and KG-1) Cells

Background: Prunusarmeniaca is a member of the Rosacea family. The most important ingredient of this family is amygdalin that is believed to have anti-tumor and analgesic properties. The aim of this study was to evaluate the anti-proliferative effects of Armeniacae semen extract on the acute leukemia, NALM-6, and KG-1 cell lines, and investigate the effect of the extract on apoptosis of these cell lines and caspase-3 gene expression. Materials and Methods: We prepared aqueous, ethyl acetate, and hydro alcoholic extracts of the Armeniacae semen. The NALM-6 and KG-1 cell lines and mononuclear cells (PBMCs) of healthy controls were treated with different doses of the extracts for 48 hours, and then cell viability was investigated with the MTT test. High-Performance Liquid Chromatography was done for amygdalin identification. The percentage of apoptotic cells was determined using the Annexin V-FITC/PI flow cytometric kit, and caspase-3 gene expression was evaluated. Results: MTT test revealed that the strongest Inhibition Concentration (IC50) in KG-1 and NALM-6 cell lines was related to the ethyl acetate extract. This extract did not have toxic effects on PBMCs. Flow cytometric analysis showed that the ethyl acetate extract at its IC50 concentration led to almost 50% apoptosis in both cell lines after 48 hours. In the molecular examination, after treatment, a significant increase was seen in caspase-3 gene expression in NALM6 and KG1 cells compared to the control (P<0.001 and P <0.05, respectively). Conclusion: Our data confirmed that the ethyl acetate extract of Prunusarmeniaca could reduce the proliferation of KG-1 and NALM-6 cell lines probably by activating the apoptotic pathway.


INTRODUCTION
Acute leukemia refers to rapid and clonal proliferation of lymphoid and myeloid progenitor cells in the bone marrow 1,2 . Based on the type of stem cell involved, it is divided into two major groups: AML (acute myeloid leukemia) and ALL (acute lymphoid leukemia) 3 . AML is the most common cause of acute leukemia in the first few months of life, in middle-aged people, and in the International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir elderly, and has a prevalence of 10,000,000 per year in people over 60 years old 4 . ALL is the most common malignancy in childhood. ALL mostly occurs between the ages of 3 and 7 years 5 . There is a secondary increase in the incidence of ALL in patients older than 40 years 6 . Its specific treatment is chemotherapy. Dissatisfaction with conventional treatments and side effects of chemotherapy are the most important reasons for use of natural drugs 7,8 . Rosaceous plants, which are widely distributed, produce different economically important products, including many edible fruits, as bitter almonds, apricots, peaches, plums, etc 9 . The family has an important glycoside called amygdalin. This component decomposes under glycosidase reactions, releasing hydrocyanic acid and benzaldehyde. Hydrochloric acid is an anti-tumor compound and benzaldehyde has analgesic properties 10 .
Amygdalin has an antitumor effect by settling carcinogens in the body, inhibiting the nutritional source of cancer cells, and blocking the growth of the tumor cells. It can also improve the symptoms of patients in the last stages of cancer and increase their survival 9 13 . Because of these features and the lack of coherent studies on various types of leukemia, we decided to use the Armeniacae semen, a member of the Rosacea family, which contains large amount of the amygdalin, to evaluate its anti-proliferative effect on the acute leukemia, NALM-6 (ALL) and KG-1 (AML) cell lines. In addition, we investigated the effect of the Armeniacae semen on apoptosis of these cell lines and caspase-3 gene expression.

MATERIALS AND METHODS
Cell culture NALM-6 and KG-1 acute leukemia cell lines (ALL and AML, respectively), which were provided by the Pastor Institute of Iran, were grown and sub cultured in RPMI1640 containing 20mM HEPESbuffer and glutamax 1% (Biosera, France) supplemented with 10% heat-inactivated FBS (fetal bovine serum) (Gibco, USA) and 100µg/ml penicillin/streptomycin (Biosera). Mononuclear cells were isolated from the peripheral blood of healthy individuals using Ficoll-Paque. The cultures were incubated at 37 • C with 5% CO2 and 95% humidity. The medium was changed every 2-3 days.

Extracts preparation
Two hundred grams of the Armeniacae semen was hatched from the shell and dried in the shade for a week. The seeds were then crushed by a pounder. At first, the dry powder was macerated in a petroleum ether-solvent to remove oils to not disturb the cytotoxic test. After oil extraction, the seed powder was allowed to dry completely and the solvent evaporate. Then, 50 g of the powder was weighed to prepare an aqueous extract. For this purpose, the seed powder was macerated in 90 ° C water for 30-45 minutes. Then, 500 ml of ethyl acetate, was added to the rest of the powder. After 48 hours, the solvent was removed and a new ethyl acetate solvent was added again. The procedure was repeated 3 times. The above processes were also repeated for 60% methanol. The extracts from each step were filtered with filter papers, transferred to a rotary balloon (Heidolph, Germany) and concentrated at 100 rpm at 40 ° C.

SPE method
A solid-phase extraction cartridge (Chromafix-Germany) was used to separate and concentrate the target material of the extract, including a mixture of different materials. At first, the cartridge was washed with 5 ml of methanol and activated with 5ml of water. Then, 5 ml of the extract at a concentration of 20 mg/ml was passed through the cartridge. After washing with 10% methanol aqueous solution, to remove co-adsorbents, the remaining analytes were collected on SPE with 5 ml of pure methanol.

Amygdalin identification by reverse phase HPLC
In order to Amygdalin identification in the extract that had the best IC50 on the MTT test, the gradient method was used to elute other components quickly and reduce the analytical time after amygdalin peak observation. For this purpose, the mobile phase was adjusted to 15: 85 methanolwater for 30 minutes and pure methanol after 30 minutes. The mobile phase was filtered before use with a vacuum filter system containing a 0.45μm filter (Millipore-Germany). Separation was conducted on a C18 column (250×4.6mm). The column temperature was 30°C, and the detection wavelength was set at 215 nm. At first, the amygdalin standard was injected to the Agilent 1260 chromatographic system, including a UV detector and ChemStation data system at a concentration of 100μg / ml and a volume of 20 μl, and then the exact location of the peak was determined. This action was repeated 3 times. Then, 20 μl of the specimen (isolated by SPE) was injected and after peak observation, the test was repeated 3 times. By comparing the peak area of the standard and sample, the percentage of amygdalin in the extract was obtained.

Flow cytometry
In order to determine the amount of primary and secondary apoptosis induced under the influence of the extract in each cell line, the cells were grown at a concentration of 1×10 6 cell/ well on a 6-well plate. Then, the concentration of (the best) IC50 was added to each well. After 48 hours, flow cytometry was performed by the BD FACS Calibur Flow Cytometry Machine (BD Biosciences_ USA), and the results were analyzed using the FlowJo.7.6.1 software. We conducted flow cytometry measurements based on the protocols of eBioscience.

RT-PCR
RT-PCR was performed to identify caspase-3 gene expressions. For this purpose, the cells were collected after 48 hours of treatment with the extract that had the best IC50 on the MTT test, and their total RNA was extracted using the Trizol (Qiagen, Germany) method. Then, 1µg total RNA was converted to cDNA using the PrimeScript 1st strand cDNA Synthesis kit (Takara, Japan) according to kit manufacturer's instructions. RQ-PCR assay was performed to investigate the level of caspase-3 gene expression in drug-treated and control groups using primers in Table 1. β-2 microglobulin (B2M) was used as a housekeeping gene for normalization of RT-qPCR data .It should be noted that all tests were performed in triplicate. The fold change Casp3 mRNA in treated cells in comparison with untreated cells was computed by the 2-ΔΔCT method.

Analysis of apoptosis by double staining KG-1 and NALM-6 cells with annexin V-FITC and PI
KG-1 and NALM-6 cells became apoptotic after they were treated with the ethyl acetate extract of Armeniacae semen. The distribution of apoptotic cells measured by flow cytometry showed that the percentage of early (Q3) and late-apoptotic cells (Q2) was 63±1.48% and 32.7±2.31% in KG-1 and 59.22±1.43% and 30.61±1.94% in NALM-6 cell lines, respectively.

DISCUSSION
The main purpose of this study was to evaluate whether Prunus armeniaca extract causes apoptosis in NALM-6 (acute lymphatic leukemia) and KG-1 (acute myeloid leukemia) cell lines. In the present study, based on the evaluation of cell survival by the MTT assay, the cell viability in both KG-1 and NALM-6 cell lines was dose dependent and the survival rate of the cells reduced, with an increase in the concentration of the mixture. Moreover, the best IC50 was related to the ethyl acetate extract in both cell lines. The effect of the extract on the cell viability seems to be dependent on the cell type. Our results are in line with other studies, showing cytotoxic effect of amygdalin on various cancer cell types 10,11,14,15 . In 2005, Park HJ et al. reported that the extract from the amygdalin has a dose-dependent cytotoxic effect on human colon cancer (SNU-C4) 11 . They also reported that treatment of human chronic myeloid leukemia (K562 cell line) with amygdalin at different concentrations resulted in decrease in cell viability in a dose-dependent manner 14 . In other studies, cytotoxic effect of amygdalin was shown on prostate cell lines (DU145 and LNCaP) and human cervical cancer HeLa cells 10,15 . Since most anticancer and cytotoxic compounds induce apoptosis in tumor cells, a quantitative study of apoptosis was performed in both KG-1 and NALM-6 cell lines treated with the ethyl acetate extract of Armeniacae semen. Analysis of flow cytometric results in both cell lines indicated an almost 50% apoptosis at IC50. Moreover, investigation of caspase-3 (executive pathway) gene expression at IC50 showed a significant increase in both cell lines compared to controls. The other studies involving other cancerous cell lines also confirmed apoptosis induction and increased caspase-3 activity by amygdalin 7,10,15,16 . Evaluation of the treatment of UMUC-3, TCCSUP, and RT-112 bladder cancer cell lines with amygdalin at concentrations of 1, 10, 25 mg/ml using the Annexin-V-FITC/PI kit showed a dose-dependent relationship between amygdalin concentration and Annexin V positive cells 7 .
The key role of Caspase-3 in apoptotic pathways is well established. Therefore, the mRNA expression level of Casp3 was assessed to evaluate the effect of the amygdalin extract on the induction of cell death. Our gene expression study showed that the ethyl acetate extract of Armeniacae semen increases the Casp3 at mRNA level. Chang et al. showed that amygdalin increased caspase-3 activity significantly in both DU145 and LNCaP prostate cancer cell lines 10 . Lee et al. showed that amygdalin induces apoptosis in Hs578T breast cancer cells through the caspase-3 pathway 16 . The effect of the ethyl acetate extract of Armeniacae semen on PBMCs isolated from normal blood at different concentrations did not show significant cytotoxic effects compared to the negative control. There are hypotheses about the mechanisms of the differences in the response of normal and cancerous cells to amygdalin. Some studies suggest that the cancer cells are rich in βglucosidase, which is capable of digesting amygdalin to produce cyanide, leading to toxic effects on cancer cells 17,18 . Some other studies suggest that rhodanase, which is capable of eliminating cyanide toxicity, exist in normal tissues, but is ineffective in cancer cells. Combined activity of the two enzymes may be responsible for inducing toxic effects in amygdalin-treated cancer cells, while normal cells remain unaffected 11,19 .

CONCLUSION
Taken together, the results of the present in vitro study (which never was done before on these types of leukemia) suggest that the ethyl acetate extract of Armeniacae semen have anticancer effects on AML and ALL. However, future studies should focus on the isolation of the effective ingredients of the ethyl acetate extract and evaluation of their effects International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir on the KG-1 and NALM-6 cell lines in human and animal models as well as the assessment of the effect of the extract on the cell cycle. In addition to measuring the expression of caspase-3 gene, the activity of this enzyme should be assessed through the caspase-3 activity measurement kit.