cell

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, antittumoral, 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 leukemia[8] but was found this compound can be toxic[7]. The si de effects of
these compound can be decreased by comlex with metal[7]. Hosseini-Yazdi et al synthesized  and characterizantion of methylthiosemicarbazone
complex with Zn (II). They showed that this compound has cytotoxic effect on
the KG1-a and K562 cell lines[9].

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). 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).

Preparation of stock drug

In brief,

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 incubator.????

2.3 Determination of cytotoxic activity

cytotoxicity of this compound was measured using the 3-(4,5-dimethy-2-thiazoyl)-2,5-diphenyltetrazolium
bromide (MTT) assay.we cultured
K562 cells (5×104 cells/mL) 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).

2.4 DNA laddering assay

DNA fragmentation is a feature of apoptosis……..the K562 cells were
treated with 100µM (at IC50 value) of thiosemicarbazon complex with Zn2+ .
After 72h, the cells were collected and washed with phosphate buffered saline (PBS).

2.5 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 thiosemicarbazon 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

The K562 cells, seeded on a 96 well plate for 24, 48, 72 hours. The
cells were untreated (control) and treated with thiosemicarbazon 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

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). M 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.

 

  Figure 4: Cell cycle distribution
in K562 cells after incubation with metylthiosemicarbazon complex with Zn2+.
Cells were analyzed via flow cytometry and then and the percentage of cells was
determined in each phase of cell cycle. The K562 cell population percent reported in sub-G1
phase in untreated and treated (24, 48, 72 h) K562 cells were 1.03, 0.98, 8.14
and 9.79 , respectively.

 

 

3.5

 In previous studies, it has
been showed that the phosphatidylserine of plasma membrane plays a role in
apoptosis process. In normal cells, the PS places outer leaflet of plasma
membrane but location of PS in apoptotic cells is inner leaflet [10]. Annexin
V/PI assay was detected the phosphatidylserine localization in cell surface
membrane. As shown in Fig,
when the k562 were exposed to metylthiosemicarbazon complex with Zn2+,
the early (Annexin V+/PI-) and late (Annexin V+/PI+)
apoptotic cells increased but normal cells (Annexin V-/PI-)
decreased. Therefore, these findings suggested that the induces apoptosis in
K562 cells.

 

 

Figure: Effect of metylthiosemicarbazon complex with Zn2+
on apoptosis of K562 cells were studied by Annexin-V/PI double staining assay. Flow
cytometric analysis of the K562 cells showed that metylthiosemicarbazon complex
with Zn2+ induced apoptosis in K562 cells.

Discussion

Leave a Reply

Your email address will not be published. Required fields are marked *