1. 1. Fusion of the breakpoint cluster

1. Introduction
Chronic myeloid leukaemia (CML) is a hematopoietic stem cell disorder characterized by the reciprocal translocation between the long arms of chromosomes 9 and 22 1. Fusion of the breakpoint cluster region (BCR) on chromosome 22 with the Ableson murine leukemia(ABL) tyrosine of chromosome 9 results in a fusion gene called BCR-ABL. This gene is a tyrosine kinase signaling protein that is always on, causing the cell to divide uncontrollably 5. The K562 cell line was derived from a 53-yr-old female suffering from Chronic myeloid leukaemia at the terminal stage of blast crisis 2. Apoptosis is a process that occurs changes in cell include blebbing, cell rounding and shrinkage, nuclear fragmentation, chromatin condensation, shedding of small cellular fragments 3-4.
Thiosemicarbazones (R1R2C2=N3-N2(H)-C1(=S)N1R3R4)6 have been reported that they have many of bioactivities such as antibacterial, antifungal, antitumor, antiviral. Previous studies have been shown that the properties of this family are related to metal ion coordination and their metal complexes have more active than the free ligand.7 Brockman et al were reported the effect of antitumoral of pyridine-2-carboxaldehyde thiosemicarbazone on L1210 leukemia8 but was found this compound can be toxic7. The side effects of these compound can be decreased by comlex with metal7. Hosseini-Yazdi et al synthesized and characterization of methylthiosemicarbazone complex with Zn (II). They showed that this compound has cytotoxic effect on the KG1-a and K562 cell lines9.
In this study, we investigated the growth inhibitory and cytotoxicity effects of methylthiosemicarbazone complex with Zn2+ on human chronic myelogenous leukemia K562. Our results showed that methylthiosemicarbazone complex with Zn2+ inhibits the growth of K562 cells via the induction of apoptosis. Our data indicate that methylthiosemicarbazone complex with Zn2+ has potential role as a therapeutic factor to induction of apoptosis and can be considered for its further development as an antileukemia drug.

2. Materials and methods
2.1 Reagents
Methylthiosemicarbazone complex with Zn2+ was obtained from ……. RPMI-1640 medium and penicillin / streptomycin were purchased from Gibbon (Life Technologies, Paisley, Scotland). The culture plates were purchased from SPL (South Korea). Acridine orange / ethidium bromide (AO/EtBr) and proteinase K were obtained from Sigma Chemical Company (Germany). Annexin V FITC Apoptosis kit were bought from Roche (Mannheim, Germany). Cell Extraction was obtained from Invitrogen (life technologies, USA). Proteinase K, propidiumiodide(PI), dimethyl sulfoxide (DMSO),3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The K562 cell line was bought from Pasterur Institute (Tehran, Iran).
2.2 Cell culture conditions
Human chronic myelogenous leukaemia K562 cells were cultured in RPMI -1640 medium supplemented with 10% fetal bovine serum, 100 µg/Ml streptomycin and 100 µg/mL penicillin and then incubated at 37ºC in a humidified 5% CO2 containing incubate 15.
2.3 Preparation of stock drug
In brief,

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2.4 Determination of cytotoxic activity
Cytotoxicity of this compound was measured using the 3-(4,5-dimethy-2-thiazoyl)-2,5-diphenyltetrazolium bromide (MTT) assay.The K562 cells (5×104 cells/mL) were cultured in 96-well plates and then exposure to various concentration of metylthiosemicarbazon complex with Zn2+. For IC50 values determination, dimethyl sulfoxide (DMSO) was added to each well and then incubated 4 hours at 37ºc. Cell viability was calculated by measuring the absorbance at 570nm by a multi-well plate reader (Quant Bio-tektruments, USA).
2.5 DNA laddering assay
DNA fragmentation is a feature of apoptosis so that the K562 cells were treated with 100µM (at IC50 value) of metylthiosemicarbazon complex with Zn2+ . After 72h, the cells were collected and washed with phosphate buffered saline (PBS).
2.6 Morphological changes of the apoptotic cells
The K562 cells were treated first with 100µM ( at IC50 value) of the anticancer drug to induce apoptosis for 24-72h. The cells were collected by centrifugation and washed with cold PBS. Subsequently, the cells were incubated with the fluorescent dyes, 100µg/mL of acridine orange and 100µg/mL of ethidium bromide (AO/EtBr). 5µL of cell suspension was placed on a slide and then was analyzed by fluorescence microscopy (Olympus BX 41, Germany).
2.7 Cell cycle studies
Briefly, the K562 cells were cultured in 96-well plates for various time (24-72h). The cells (1×104 cells/well) were treated with IC50 value of metylthiosemicarbazon complex with Zn2+. The cells were harvested and washed twice with cold PBS and then fix cells by adding 70% (V/V) cold ethanol and stored at -20ºC for several weeks until analysis. Afterwards, the control (untreaded) and treated cells incubated with 50 µg/mL propidium iodide (PI) containing 20µg/mL RNase A in the darkroom at 37ºC for 2 h. The stained cells were analyzed by flow cytometry (BD FACSCalibur TM, BD Biosciences, CA, USA).
2.8 cell apoptosis analysis
The K562 cells, seeded on a 96 well plate for 24, 48, 72 hours. The cells were untreated (control) and treated with metylthiosemicarbazon complex with Zn2+ at the indicated concentration (at IC50 value). The total cells harvested and then washed twice with PBS. The Cells were stained with Annexin-v-FITC and PI (eBioscience, CA, USA) for 15 min at room temperature in a dark area then analyzed by a flow cytometry (BD FACSCalibur TM, BD Biosciences, CA, USA).
2.9 statistical analysis
All data of experiments were reported as means ± S.D. Significant differences between the groups were expressed by the unpaired Student´s t-test. P- value less than 0.05 were considered as statistically significant.
3. Results
3.1 Cell viability
The MTT assay is based on the conversion of MTT into formazan crystals by living cells, that determines number of viable cells. The treatment of K562 cells with different concentrations of metylthiosemicarbazon complex with Zn2+ (30, 50, 80, 100, 130, 150 and 200µM) at 24, 48 and 72 h. As showen Fig, metylthiosemicarbazon complex with Zn2+ has cytotoxic activity on the K562 cell line and was able to inhibit the proliferation of the K562 cell line. The IC50 is a measure of the effectiveness of metylthiosemicarbazon complex with Zn2+ in inhibiting of proliferation of K562 cells. The IC50 value of this compound is 100µM in 72 h.

Figure 1: Effect of metylthiosemicarbazon complex with Zn2+ on K562 cells. The K562 cells were treated with various concentrations (30-150µM) of metylthiosemicarbazon complex with Zn2+ for 24, 48, 72 h and then were investigated by MTT assay. Data are shown as mean ± SD.

3.2 Morphological study of K562 cells
In order to evaluate the effects of metylthiosemicarbazon complex with Zn2+ on K562 cells, were studied by fluorescent microscope. The K562 cells were treated 100µM (IC50) of compound for 24, 48 and 72 h to induce apoptosis, then were stained with Acridine Orange/Ethidium Bromide (AO/EB). Metylthiosemicarbazon complex with Zn2+ induced apoptosis in K562 cells. AS shown Fig, the control cells are uniformly green because the plasma membrane of normal cells incorporated just AO. The apoptotic cells are bright green and orange that their nuclei are indicating condensed and DNA fragmentation. Fig shows The number of normal cells decreased and apoptotic cells increased in a time dependent.
3.3 DNA fragmentation assay
Apoptosis is associated with the fragmentation of chromosomal DNA into approximately 180 bp nucleosome fragments. In apoptosis, after activation of the 3 caspase, CAD in nuli actives and casused DNA fragmentation. In normal cells CAD anzyme is inhabited by ICAD enzyme but in apoptotic cells, ICAD enzyme Detection of fragmented DNA following extraction can be performed via a DNA fragmentation assay involving gel electrophoresis.This experiment showed apoptosis in the K562 treated cells. Fig.

3.4 analysis of cell cycle
For more investigated, the K562 cells were treated with 100µM concentration of compound metylthiosemicarbazon complex with Zn2+ for 24, 48, 72 h. The cell cycle distribution in the K562 cells were analyzed via flow cytometry. As displayed in Fig. The cell cycle has two major phases: interphase and the mitotic phase include G0 / G1, S, G2, M. During the cell cycle phases, DNA levels change, DNA dyes such as PI to generate characteristic cellular DNA content profiles. The results demonstrated that metylthiosemicarbazon complex with Zn2+ induced apoptosis in a time-dependent manner. The rate of sub-G1 was increased from 5.01 in the control cells to 3.62, 18.93 and 22.72.


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