In vitro cytotoxic activity of L-asparaginase extracted from pathogenic Escherichia coli against three leukemic cell lines

Abstract

Assessment in vitro cytotoxicity tests, has recently been become popular as a primary screening for the evaluation the anticancer activities of natural product. There is a continuous and urgent need to evaluate our purified L-asparaginase that was extracted from pathogenic E. coli isolated from Iraqi patients with UTI, as it will be further utilized on development of local therapeutic agent against cancer. In the present study, purified L-asparaginase was tested for anticancer effect on some Ieukemic cell lines. The cytotoxic activity of L-asparaginase was assessed by MTT assay. The cytotoxicity result was demonstrated as 1C50 value and selectivity index (SI) that was calculated from the 1C50 ratio m lymphocytes over the leukemic cells. Purified L-asparaginase showed cytotoxic effect and selectivity in all three leukemic cell lines after 24 hr treatment; Daudi (IC50=1.079±0.51, IS=3.4); Jurkat (IC50=1.380±0.28, IS=2.8) Molt4 (IC50= 1.079+0.51, S1=3.6) respectively. And the purified L-asparaginase showed better selectivity than the two chemotherapeutic drugs, melphalan and 5FU about 2 to 3 fold. Also these results indicate that Molt4 cell line have high sensitivity to L-asparaginase than the other leukemic cell lines which were used in this study. However, further biological test on the efficacy of the purified L-asparaginase is necessary to understand the mechanism of anticancer activity.

Full text

21
The 2'"1
Annual International Conference of Northeast Pharmacj Research Hi
February 13-14,2010 / Ordain.-.:o iiv S-'aailty of PliArmafy. Mah asajakliao ' inivnsny. Mataa S;u\i>:h.im. I'luulnai!
In vitro Cytotoxic Activity of L-asparaginase Extracted from Pathogenic
Escherichia coli Against Three Leukemic Cell Lines
Aljcwari H Nader M Alfaisal A 1, Wccrapreeyakul N % Barusrux S
' Genetic Engineering and Biotechnology Institute (GEBf), Baghdad University, Baghdad. Iraq
1 Center for Research and Development of Herbal Health Product, Faculty of Pharmaceutical Sciences. Khon Kaen University. Khon
Kaen. Thailand
■' Centre for Research and Development of Medical Diagnostic Laboratories (CMDL). Fat uity of Associated Medical Sciences, Khon
Kaen University. Khon Kaen. Thai land
Abstract
Assessment in vitro cytotoxicity tests has recently been become popular as a primary screening for the evaluation
the anticancer activities of natural product. There is a continuous and urgent need to evaluate our purified L-
asparaginase that was extracted from pathogenic Escherichia coli isolated from Iraqi patients with UTI, as it will be
further utilized on development of local therapeutic agent against cancer. Tn the present study, purified L-
asparaginase was tested for anticancer effect on some leukemic cell lines. The cytotoxic activity of L-asparaginase
was assessed by MTT assay. The cytotoxicity result was demonstrated as ICV1 value and selectivity index (SI) that
was calculated from the IC50ratio in lymphocytes over the leukemic cells. Purified L-asparaginase showed cytotoxic
effect and selectivity in all three leukemic cell lines after 24 lir treatment; Daudi (ICW1.156±0.3I, SI=3.4); Jurkat
(IC^ = 1.380±0.28, SI=2.8); Molt4 (IC^ = 1.079+0.51, SI=3.6) respectively. And the purified L-asparaginase showed
better selectivity than the two chcmotherapeutic drugs; melphalan and 5-FU about 2 to 3 fold. Also these results
indicate that Molt4 cell line have high sensitivity to L-asparaginase than the other leukemic cell lines which were
used in this study. However, further biological test on the efficacy of the purified L-asparaginase is necessary to
understand the mechanism of anticancer activity.
Keywords: L-asparaginase, Cytotoxic activity, Leukemic Cell Lines.
♦Corresponding author: Sahapal Barusrux. Facuhy of Associated Medical Scicnccs. Khon Kaen Univ ersity. Khon Kaen 40002 Thailand. Tel / Fax:
+66 43 202080, E-mail : sahapaK&kku.ac.U i
Introduction
L-asparaginase (L-asparagine amidohydrolase, EC
3.5.1.1) is an enzyme present in many animal tissues,
bacteria, plants, and in the serum of certain rodents,
but not in mankind. L-asparaginase with anticancer
activity is produced by large number of
microorganisms including Escherichia coli, Erwinia
cartovora, Enterobacter aero genes, corynebacterium
}>lutaniu tun. Candida utilis, staphylococcus aureus,
Thvnnus thermophilus, and Pisum sativum (lil-
Bessoumy et al., 2002). L-asparaginase is one of the
most frequently used in treatment of acute
lymphoblastic leukemia ALL (Roberts et al., 1972, and
Gutierrez et al., 2006). The antileukemic ef fect of L-
asparaginase is postulated to result from the rapid and
complete depletion of the circulating pool of
asparagine. Cytotoxicity is a result of the inhibition of
protein synthesis in the cells that are unable to
synthesize asparagine for their needs and rely on
circulating asparagine (Asselin et al., 1989 and Swain
el al., 1993). Interest in [.-asparaginase arose from the
striking effect capable to treatment of cancer. 1 lence,
this study
was planed to evaluate I he cytotoxic activity in
leukemic cell lines of L-asparaginase which was
extracted and purified from pathogenic Escherichia coli
isolated from Iraqi patients with urinary tract
infections.
Materials and Methods
Tested Materials
L-asparaginase (stock samples 100 IU/ml in
phosphate buffer) which was extracted and purified
from pathogenic Escherichia coli isolated from patients
with urinary tract infections in Baghdad, Iraq (Aljewari
et al.,2010). Melphalan and 5-fluorouracil (5-FU) was
purchased from Sigma (Germany). Stock samples of
melphalan and 5-FU were dissolved in DMSO
(dimethyl sulfoxide) at a concentration 100 mg/ml.
Cell culture
Three leukemia cell lines were obtained from the
Center for Research and Development of Medical
Diagnostic Laboratories (CMDL), Faculty of

22
Associated Medical Sciences. Khon Kaen University.
Thailand. The human Daudi, Jurkat,, and Molt 4 are
nonadherent and were grown in RPMT 1640 medium
that had been supplemented with fetal bovine serum
(FBS, 10% v/v), Penicillin-streptomycin (1% v/v)
(Gibco. UK). The stock cultures were maintained at
37°C in a humidified atmosphere with
5%
CO. in 95 %
air in 24T flasks (Corning, USA). All the cells were
passaged twice to tliree time weekly.
Viable Cell counting
Counting cells were performed before each cytotoxicity
experiment. Cells were examined under phase contrast
microscope (lOxmagnify) to monitor cells at the
logarithmic growth phase. Then cells were collected by
centrifuge at 2,000 rpm for 5 min and resuspention with
5 ml complete RPMI medium. One microliter of
suspension cell was transferred to microtube to count
cell viability at density of 5xl05 cell/ml by using trypan
blue with 20 |iL cell suspension and 180 uL trypan blue
dye ratio. Ten uL of the above mixture was applied to
the hemocytometer and examined under light inverted
microscope. Only dead cells will uptake the trypan blue
dye. Dilution factor of cells stoke was calculated from
the average of cells count in the four large squares (N) in
the hemocytometer according to the equation as follow:
Dilution factor of cells stock = (Nx JO5) / 5x10s
Cytotoxic assay
L-asparaginase were tested for cytotoxicity against three
cancer cell lines and compared with two potent
chemotherapeutics drug (melphalan and 5-FU) by using
3-(4^-dimethyIthiazoI-2-yl)-2T5-diphenyltetrazolium
bromide (MTT) assay as described by Prayong ct al.
(2008) and Mossman (1983). Stock samples were diluted
with RPMI medium to desired concentrations of L-
asparaginase ranging from 0.01 to 5 IU/ml, and also for
melphalan and 5-FU ranging from 10 to 500 iig
/mJL.
One
hundred microliters of cells were seeded in 96 well at the
density 5x 10s cell/mL and incubated at 37°C in
5%
C02,95 % air for 24 hrs. Then, cells were treated with
various concentrations of samples in total volume (200
[it/well) for 24 hrs. At 21 hrs, cell were centrifuged at
2.000 for 10 min and resuspended with 180 uL RPMI
medium to rinse treated samples. Twenty uL of MTT
solution (5 mg/mL) was added to each well and
incubated ai 37°C for another 3h. Then the medium was
aspirated about 180 uL from each well. The formed
formazan crystals were dissolved with 180 uL of DMSO.
An optK .11 'iv u.iiv [i >!>> ol lormazan was detected by
a dual \vawli-iu\ili UV s|>ccirometer at 570 nm with 650
nni ivh-ii-iKV wavelength. The percentage of
cytotoxicity compared I lie untreated cell as a control
was determined as equation below.
^cytotoxicity - (OE> without tiv;itnicut - Of)
treatment) x 100 (Oh without
treatment)
The plot of ^cytotoxicity versus sample concentration
was used to calculate the concentration lethal to 50% of
the cells (IC^).
Lymphocyte isolated and selectivity index
Lymphocyte was isolated from blood provided from
blood center, Faculty of Medicine, Khon Kaen
University, Thailand, according to the method described
by Alhilli, (2004) with some modification. Human
lymphocyte was used to determine the nontoxic dose of
the tested sample. Selectivity index (SI) was calculated
from the IC..„ ratio in lymphocyte (Prayong et al., 2008)
as in equation below;
Selectivity index (SI) - »C50 of da.g in normal cell
(lymphocyte) ' IC50 of drug In Tested ccll
line
SI value indicates selectivity of samples to the cell lines
tested. The SI value higher than 3 will consider to be
high selectivity.
Results and Discussion
Cytotoxicity results of L-asparaginase and two
chemotherapcutic drugs (melphalan and 5-FU) in three
leukemic cell lines are summarized in Table 1.
L-asparaginase showed positive activity and high
selectivity to three leukemic cell lines Daudi (SI=3.4),
Jurkat (SI=2.8), and Mo!t4 (SI=3.6) respectively and
showed better effect than the chemotherapeutic drugs;
melphalan and 5-FU. Result showed that L-asparaginase
have higher activity and selectivity to Molt4 cell after 24
hr from treatment. Also the Jurkat cell line was sensitive
to 5-FU with 50 time higher selectivity over the
lymphocyte cells, but it was inactive against Daudi and
Molt4 ccll lines. Melphalan also showed positive activity
with less selectivity to cancer cells than the L-
asparaginase. Melphalan was found to be toxic to the
Jurkat and Molt4 coil lines compared to the normal
human lymphocyte. Our result showed the IC,(. of L-
asparaginase in Molt4 cell after 24 hr treatment
(1.08±0.51 IU/mL) higher than that of Irino ct al. (2004)
(<0.01 IU/mL) after 4 day from treatment. The
treatment time is the main factor for our explanation.

The 2"'1 Annual International \ 'onference of Northeast Pharmacy Research 2010
February 1^-14. 2010.' Or;'.;iiii/x.-.l h\ |-«n .-'iv <»f Ph;irmacv. M:>li<>s;ir.iUi:iin Liiivasily. Maha
Sarakluim. Thailand
Conclusion
L-asparaginase showed cytotoxic activity and high selectivity
to all leukemic cell lines used in this study. A Molt4 cell line
showed the highest sensitivity to L-asparaginase. Further
biological testing on the efficacy of the L-asparaginase will be
conducted to gel better understand of the mechanism of
anticancer activity.
Acknowledgements
We gratefully acknowledge staff of Center for Research and
Development of Medical Diagnostic Laboratories (CMDL),
Associated Medical Sciences, Khon Kaen University (KKU)
for great support and Blood centre, Faculty of Medicine, KKU,
Thailand for provided blood to isolated lymphocyte. The
authors would like to express sincere thank to Mr. Chaiyachct
Nanok from Faculty of Associated Medical Sciences and Ms.
Sasipawan Machana from Faculty of Pharmaceutical Sciences,
KKU, Thailand for great laboratory assistance. This study was
supported by grants from the Scholarships &Cultural Affairs
Directorate, Ministry of Higher Education Scientific Research,
Iraq (No. 2798) and by a research grant from CHE-TRF
(MRG4680180). *
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IS
Table 1 In vitro cytotoxic activity and selectivity index of L-asparaginase in three leukemic cell lines.
Cell line IC^, ________________ _______________________ (Selective index)
Treatment
Lymphocvtc
Daudi
Jurkat
Molt4
L-asparaginasc
3.90+0.36
1.16+0.31
1.38+0.28
1.08+0.51
(IU/mL)
(3.4)
(2.8)
(3.6)
Mclphalan
42.5+5.24
28.19+4.19
153.46+18.65
125.88+16.86
(Hg/mL)
(1-5)
(0.3)
(0.3)
5-FU
Inactive 4
Inactive'
<10
Inactive'
(ug /ml)
(1)
(50)
(1)
• ICVJ > 5<K) ng/ml is considered to be inactive