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Bacteriology Laboratory, Department of P. G. Studies and Research in Biological Science, Rani Durgavati University, Madhya Pradesh, India
Cancer Pharmacology Division, Indian Institute of Integrative Medicine (IIIM-CSIR), Jammu & Kashmir, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 March 2015
Accepted 12 November 2015
Available online 17 November 2015
Asparaginase is an important antineoplastic drug extensively used for the treatment of acute lymphoblastic leukemia, but the intrinsic glutaminase activity of this enzymatic drug is responsible for several
life threatening side effects. This study describes the purication and characterization of glutaminase free
asparaginase from Pseudomonas otitidis. The puried enzyme exhibited molecular mass of
approximately 2053 kDa on native-PAGE and341 kDa on SDS-PAGE, revealing that the enzyme is
homohexamer. The isoelectric point of enzyme was 5.5, calculated by 2D-PAGE. Optimum activity of
asparaginase was achieved at 40 C and pH 7.5, which is close to the internal environment of the human
body. Monovalent cations (Na and K) and reducing agents (2-mercaptoethanol and glutathione) has
enhanced asparaginase activity. Whereas, divalent (Ca2, Mg2, Zn2 and Mn2), trivalent (Fe3) cations
and thiol group blocking agent (iodoacetamide) inhibited the enzyme activity signicantly. In vitro serum
and trypsin half life of asparaginase is almost 2 and 1.5 fold respectively, which is higher than commercial asparaginase. MTT assay results showed that the anticancer activity of puried asparaginase was
comparable or higher than commercial E. coli asparaginase. Microscopic studies and cell cycle analysis
suggested that puried enzyme induced apoptotic cell death in dose-dependent manner. Loss of mitochondrial membrane potential suggests that enzyme induces cell death via activation of intrinsic
apoptotic pathway. Puried asparaginase was found to be nontoxic for human noncancerous FR-2 cells
and human blood lymphocytes, which is a remarkable therapeutic feature.
te
Franaise de Biochimie et Biologie Mole
culaire (SFBBM). All rights
2015 Elsevier B.V. and Socie
reserved.
Keywords:
Asparaginase
Glutaminase
Purication
MTT assay
Cell cycle analysis
MMP loss
1. Introduction
Acute lymphoblastic leukemia is a malignant disorder of
lymphoid progenitor cells affecting a signicant segment of the
children and adults with peak prevalence between the age group of
2e5 years [1]. Bacterial asparaginase (L-Asparaginase amidohydrolase, E.C. 3.5.1.1) is an enzymatic drug used for the treatment of
children with acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML) and other lymphoid malignancies [2]. In the
recent past, scientists and clinicians have harnessed asparaginase
against sarcomas [3], ovarian cancer [4], brain cancer [5], and breast
cancer [6]. The anti-neoplastic action of asparaginase is explained
* Corresponding author.
E-mail address: anjoo1999@gmail.com (A. Sharma).
based on the fact that certain tumor cells, more specically ALL
tumor cells are decient in their ability to synthesize the nonessential amino acid asparagine de-novo due to absence of asparagine synthetase [7] and requires huge amount of asparagine to
keep up their rapid malignant growth. To fulll their nutritional
requirement they use serum asparagine. Asparaginase, as a
chemotherapeutic drug rapidly hydrolyzes serum asparagine into
aspartate and ammonia [8]. The nutritional stress induced by
asparaginase due to the depletion of serum asparagine inhibits
DNA, RNA and protein biosynthesis in ALL and other asparagine
dependent tumor cells, resulting in subsequent apoptosis due to
cell cycle arrest in G1 phase [9]. However, normal cells remains
unaffected due to presence of asparagine synthetase [10].
Asparaginase has been reported in several microbial species but
the biochemical, kinetic and anticancer activities of the enzyme
varies with genetic nature of the organisms [11]. Today
http://dx.doi.org/10.1016/j.biochi.2015.11.012
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culaire (SFBBM). All rights reserved.
0300-9084/ 2015 Elsevier B.V. and Socie
asparaginases puried from Escherichia coli and Erwinia chrysanthemi are being used for clinical purposes [12]. Recent studies
have shown that asparaginase from both these sources have curative potential but are often associated with serious side effects that
can be life threatening. The intrinsic glutaminase activity is one of
the major drawbacks of both asparaginases (E. coli and
E. chrysanthemi) that cause serious side effects such as leucopenia,
immunosuppression, acute pancreatitis, thromboembolysis, hyperglycemia and neurological seizures [13]. Additionally, short
serum half life and low tolerance to proteolytic enzymes are some
other limitations that necessitate repetitive administration of
asparaginase [14]. However, multiple administrations of serologically same asparaginase in induction and consolidation chemotherapy has led to the formation of anti-asparaginase antibodies,
resulting in neutralizing asparaginase activity by the hostile
immunological system [15]. Therefore, for the event-free, longterm, survival rates of patients; glutaminase free asparaginase from
new sources is urgently required possessing similar or more
effective antitumor activity than the previously reported ones. In
our earlier reports, we had isolated glutaminase free asparaginase
producing indigenous bacterial strains [16] and fermentation process parameters were also standardized for maximum biosynthesis
of glutaminase free asparaginase from Enterobacter cloacae [17].
Subsequently, enzyme was puried and characterized from this
bacterium (Husain and Sharma, unpublished data). In the current
study, we have reported purication and characterization of a
glutaminase free asparaginase from Pseudomonas otitidis and
anticancer activity was evaluated against a panel of human cancer
cell lines. Furthermore, anticancer activity of the enzyme was
validated against human leukemia MOLT-4 cells.
2. Materials and methods
39
2.1. Reagents
Anhydrous L-asparagine, L-glutamine, D-glutamine, L-glutamic
acid, DL-aspartic acid, L-histidine, L-ornithine, BOC-L-asparagine, Na-acetyl-L-asparagine, urea, acrylamide, D-asparagine, DL-asparagine, EDTA, dithiothreitol, SDS, 2-mercaptoethanol, glutathione, Lcysteine, thiourea, trichloroacetic acid (TCA), dimethylsulphoxide
(DMSO) and Folin-Ciocalteu's phenol reagent, were purchased from
Himedia, Mumbai, India. Chromatographic matrix, RPMI-1640,
fetal bovine serum (FBS), 3-(4,5-dimethylthiazole-2-yl)-2, 5diphenyltetrazolium bromide (MTT), penicillin, streptomycin, propidium iodide, Rhodamine-123, 40 ,6-diamidino-2-phenylindole
dihydrochloride (DAPI), DNase-free RNase, proteinase-K, phenylmethanesulfonyl uoride (PMSF), eukaryotic protease inhibitor
cocktail, and triton X-100 were purchased from Sigma chemical Co.,
USA. IPG strips were purchased from Bio-Rad Lab., USA. All the
chemicals used were of analytical grade and purchased from
standard sources.
2.2. Bacterial strain, culture condition and enzyme production
The glutaminase free asparaginase producing strain P. otitidis
(NCBI accession no. KF607097) was obtained from Bacterial
Germplasm Collection Centre (BGCC no. 2388), Rani Durgavati
University, Jabalpur (M.P.), India, which was previously isolated in
our Lab [16]. The strain was maintained on Luria-Bertani (LB) slant
(pH 7) with regular sub-culturing after every four weeks and stored
at 4 C. Semi-synthetic broth medium [18] was used to produce
asparaginase. The primary inoculum was prepared by adding a
loopfull of 24 h old pure culture from slant into 20 ml of aforementioned medium. The ask was incubated overnight at 37 C and
180 rpm. The 2% inoculum (A600 0.6e0.8) of this culture was
40
41
42
Fig. 1. Purication of asparaginase from P. otitidis. (a) Cation exchange chromatography of asparaginase by DEAE-cellulose column (2 15 cm2, Sigma) pre-equilibrated with 0.05 M
TriseHCl buffer (pH 9.6) and absorbed protein was eluted with a linear gradient of KCl (0e200 mM). (b) The collected fraction of highest asparaginase activity were applied on gel
ltration chromatography with Sephadex G-100 column (2 20 cm2, Sigma), equilibrated with 0.05 M TriseHCl buffer (pH 8.6) and eluted with the same buffer at a ow rate of
0.2 ml min1 (c) Assessment of homogeneity and molecular weight analysis of puried extracellular asparaginase on Native-PAGE; Lane 1- Protein marker; Lane 2 and 3 Sephadex
G-100 puried asparaginase. (d) SDS-PAGE analysis of puried enzyme in 12% polyacrylamide gel; Lane 1- Protein marker; Lane 2-Sephadex G-100 puried asparaginase; Lane 3DEAE-cellulose puried asparaginase. (e) Two-dimensional electrophoretic resolution of puried asparaginase. Puried protein after was resolved by IEF and followed by SDS-PAGE.
Gel was visualized by coomassie brilliant blue R-250 staining. Molecular weights were calculated with standard molecular weight markers (PMWH, Bangalore Genei, India).
Table 1
Summary of various steps involved in purication of asparaginase from P. otitidis.
Purication step
Fold purication
Yield (%)
Crude extract
(NH4)2SO4 precipitation
DEAE-cellulose
Sephadex G-100
2370
1587
1269.6
922.04
3300
363
23.59
8.55
0.71
4.37
53.81
107.84
1
6.15
75.78
151.88
100
66.96
53.56
38.90
One unit of asparaginase (IU) is dened as the amount of enzyme that liberates 1 mmol of ammonia min1 at 37 C.
on SDS-PAGE gel. Thus, our result indicates that the pI of asparaginase is 5.5.
3.3. Effect of pH and temperature on activity and stability of
asparaginase
To optimize the pH and temperature, asparaginase activity was
evaluated from pH 4.5 to 10.5 and temperature in range of
20e70 C, using asparagine as a substrate. The puried asparaginase was more active at pH range 6e9. and maximum activity
was achieved at pH 7.5. However, enzyme activity decreased
abruptly when the pH was increased above 9. The enzyme showed
stability at slightly alkaline pH and retained more than 70% of its
original activity when incubated at pH 9 for 24 h (Fig. 2a and b). The
puried asparaginase exhibited maximum activity at 40 C and
decreased above 45 C. In thermal stability experiment, no significant activity was lost when the puried enzyme was pre-incubated
43
Fig. 2. Effect of physical conditions on asparaginase activity and stability (aeb) Effect of pH on activity and stability of puried asparaginase, enzyme activity was assayed at various
pH buffers. The buffer used as follows: pH 4.5e7.5, Potassium phosphate buffer, pH 8.0e10.5, TriseHCl buffer, (ced) effect of temperature on activity and thermal stability of puried
asparaginase. Effect of various metal ions (e) and modulators (f) on the asparaginase activity. Asparaginase was mixed with the corresponding metal salts (NaCl (50 mM), (KCl
(150 mM), (CaCl2 (150 mM), (MgCl2 (40 mM), (ZnCl2 (100 mM) (MnCl2 (100 mM), (FeCl3 (100 mM), (CdCl2 (10 mM), (NiCl2 (10 mM), (HgCl2 (100 mM) or modulators EDTA (5 mM),
DDT (5 mM), iodoacetamide (5 mM), SDS (2 mM), 2-mercaptoethanol (0.5 mM), glutathione (0.5 mM), L-cysteine (25 mM), thiourea (1 mM), and human serum (10%) in 50 mM
phosphate buffer (pH 7.5) for 30 min at 37 C and enzyme activity was measured by Nesslerization reaction. No addition was used as control. Each value represents the mean SD
for three determinations.
44
Table 2
Substrate specicities of puried asparaginase from P. otitidis.
Substrate
Concentration (mM)
L-Asparagine
10
10
10
10
10
10
10
10
10
10
10
10
10
100 1.6
1 0.2
3 0.4
N.D.
N.D.
N.D.
1 0.5
1.6 0.6
N.D.
N.D.
N.D.
N.D.
N.D.
D-Asparagine
DL-Asparagine
L-Glutamine
D-Glutamine
L-Glutamic
acid
DL-Aspartic acid
L-Histidine
L-Ornithine
BOC-L-Asparagine
N-a-Acetyl-L-Asparagine
Urea
Acrylamide
45
Fig. 4. Antiproliferative effects of puried P. otitidis and E. coli asparaginases on human leukemic cell line (MOLT-4 and K-562) and breast cancer (MDA-MB-231 and T47D) cell lines.
E. coli asparaginase procured from Sigma Chemical Co. St. Louis, was used as a reference preparation. The cells were treated in triplicate with different concentration (2, 5, 10 and
15 IU ml1) of puried and reference asparaginase preparation for 48 h and the cell viability was determined by the MTT assay.
Fig. 5. P. otitidis asparaginase inhibits the colony formation of MOLT-4 cells. Cells
treated with indicated concentration of puried asparaginase and colony forming
assay was performed. Data are Mean S.D. of calculated colonies percentages from
three similar experiments.
further suggest that the puried enzyme affect the cell viability and
induces apoptotic cell death (Fig. 6).
46
Fig. 6. Asparaginase induced cell death in MOLT-4 cell line. Cells were treated with the indicated concentration of asparaginase puried from P. otitidis for 24 h and observed for
morphological changes under microscope (1 81, Olympus).
Fig. 7. Alteration in nuclear morphology by treatment with puried P. otitidis asparaginase. MOLT-4 cells were incubated with indicated concentration of asparaginase, collected
after centrifugation at 1600 rpm, washed once with PBS and then stained with DAPI for 10 min. The procedure is discussed in Materials and Methods.
asparaginase severely damaged MOLT-4 cells by DNA fragmentation which leads to apoptosis.
3.12. Cell cycle phase distribution
MTT results showed that puried enzyme affects the viability of
cells. Microscopic studies suggested that enzyme induced
apoptosis. Further, FACS analysis was performed to analyze the
effect of asparaginase on cell cycle progression. For this, MOLT4 cells were harvested after 24 h of treatment with puried
asparaginase, stained with propidium iodide and subjected to ow
cytometry. According to the results presented in Fig. 9, the histogram of control (untreated) cells showed standard cell cycle
pattern, in which G1 and G2 peaks were separated by S phase. It is
very interesting to note that at all the three (1, 2, and 5 IU ml1)
concentration of enzyme we could not nd any cell cycle arrest at
G0/G1, S, and G2/M phase. In contrast, on comparison with control
(untreated) cells, 4.97%, 16.92% and 35.80% apoptotic cell death
were found at 1, 2, and 5 IU ml1 treatment of enzyme, respectively.
Hence, the results of this experiment suggest that P. otitidis asparaginase induced apoptotic cell death without G0/G1, S, and G2/M
phase arrest, which could be transient arrest that comes before
47
Fig. 8. P. otitidis asparaginase induced DNA fragmentation in human leukemia cell line
MOLT-4. Genomic DNA was extracted from cells treated with different concentrations
of puried asparaginase for 24 h. Other conditions are described in Materials and
Methods.
48
Fig. 9. Effect of puried asparaginase on cell cycle progression in leukemia cells. MOLT-4 cells (1 106) were seeded in 12-well plates and treated with different concentration (1, 2
and 5 IU ml1) of asparaginase for 24 h. After incubation, cells were collected at 1600 rpm, washed once with PBS, and xed in 70% ethanol overnight. Cells were then washed once
with PBS and stained with 100 mg of propidium iodide for 30 min. Modt software was used to differentiate between phases and to determine the amount of apoptotic population.
Fig. 10. Asparaginase induced concentration dependent mitochondrial membrane potential (MMP or Djm) loss in human leukemia cells. MOLT-4 cells (1 106) were treated with
indicated concentration of puried asparaginase for 24 h, washed once with PBS and stained with Rhodamine-123. MMP was measured as discussed in Materials and Methods.
49
Fig. 11. Toxicological evaluation of asparaginase puried from P. otitidis against human normal cells. (a) Cytotoxic effects of asparaginase on noncancerous human breast epithelial
cell line FR-2 and (b) blood lymphocytes. Cells were treated in triplicate with different concentration (2, 5, 10 and 15 IU ml1) of puried asparaginase for 48 h and the cell viability
was determined by the MTT assay. (c) Hemolytic effect of puried and crude asparaginase on human erythrocytes. (d) Quantitative measurement of the hemolytic activity. A: blank
(Sodium phosphate buffer); B: positive control (without asparaginase); C: crude asparaginase; D: 15 IU ml1; E: 7.5 IU ml1; F: 3.75 IU ml1 concentration of asparaginase.
50
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