AAC Accepts, published online ahead of print on 27 October 2014 Antimicrob. Agents Chemother. doi:10.1128/AAC.03663-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Anti-malarial action of Artesunate involves DNA damage mediated by Reactive Oxygen Species
2 3 4
Anusha M Gopalakrishnan and Nirbhay Kumar
5 6
Department of Tropical Medicine and Vector-Borne Infectious Disease Research Center, Tulane
7
University School of Public Health and Tropical Medicine, New Orleans, LA, 70112, USA
8 9
Running Head: Artesuante and DNA damage in P. falciparum.
10 11
Address correspondence to: Nirbhay Kumar, [email protected]
12
Department of Tropical Medicine, Tulane University School of Public Health and Tropical
13
Medicine, New Orleans, 70112, LA
14 15 16 17 18 19
1
20
Abstract
21
Artemisinin-based combination therapy (ACT) is the recommended first line treatment
22
for Plasmodium falciparum malaria. It has been suggested that the cytotoxic effect of artemisinin
23
is mediated by free radicals followed by alkylation of P. falciparum proteins. The endoperoxide
24
bridge, the active moiety of artemisinin derivatives, is cleaved in the presence of ferrous iron
25
generating reactive oxygen species (ROS) and other free radicals. However, the emergence of
26
resistance to artemisinin in P. falciparum underscores the need for new insights into the
27
molecular mechanisms of anti-malarial activity of artemisinin. Here we show that artesunate
28
(ART) induces DNA double strand breaks in P. falciparum in a physiologically relevant dose
29
and time-dependent manner. DNA damage induced by ART was accompanied by an increase in
30
intracellular ROS in the parasites. Mannitol, a ROS scavenger, reversed the cytotoxic effect of
31
ART and reduced DNA damage and modulating glutathione (GSH) levels was found to impact
32
ROS and DNA damage induced by ART. Accumulation of ROS, increased DNA damage, and
33
resulting anti-parasite effect suggest a causal relationship between ROS, DNA damage and
34
parasite death. Finally, we also show that ART-induced ROS production involves a potential role
35
for NADPH oxidase, an enzyme involved in the production of superoxide anions. Our results in.
36
P. falciparum provide novel insights into previously unknown molecular mechanisms underlying
37
the anti-malarial activity of artemisinin derivatives, and may help in the design of new
38
generation anti-malarial drugs against the most virulent Plasmodium species.
39 40
Introduction.
41
Malaria accounts for hundreds of millions of clinical cases and nearly a million deaths annually
42
(1). Malaria parasites are transmitted by female Anopheles mosquitoes and infections caused by
2
43
Plasmodium falciparum and P. vivax are responsible for more than 90% of worldwide infections.
44
During its life cycle, parasites undergo a complex series of biological and biochemical
45
development that allow them to grow in the vertebrate hosts and to be successfully transmitted to
46
invertebrate mosquito vector. In the vertebrate host, actively proliferating intra-erythrocytic
47
asexual life cycle stages of the parasite are responsible for all the clinical symptoms, including
48
death and these stages are also the primary targets of anti-malarial drugs. Genetic diversity,
49
antigenic variation and parasite’s ability to adapt to new drugs continue to thwart control efforts.
50
Artesunate (ART) is the semi-synthetic derivative of artemisinin, developed from Artemisia
51
annua L which has been used in China as a traditional medicine for more than 2000 years.
52
Currently, artemisinin-based combination therapy (ACT) is recommended by the World Health
53
Organization as first line of treatment for P. falciparum malaria (2). These drugs act fast with
54
fewer side effects and are also active against P. falciparum that are resistant to other traditionally
55
used drugs, such as antifolates and quinolones. Artemisinin derivatives have been found to act
56
against various erythrocytic asexual stages as well as developing sexually differentiated
57
immature stages thus showing promise as an antimalarial drug with transmission blocking
58
potential (3-5). More recent studies have suggested that intra-erythrocytic ring stage parasites are
59
targets of artemisinin derivatives and ring stages of resistant parasites display longer survival as
60
compared to sensitive parasites(6, 7).
61 62
Decreasing clinical efficacy and emerging resistance to artemisinin derivatives in Thailand and
63
Cambodia have raised serious concerns about future treatment options (8-10). Recent studies
64
have identified putative genes potentially involved in ART-resistance mechanisms (11-13) in P.
65
falciparum. These studies identified several point mutations in the kelch propeller domain (K-13
3
66
propeller) showing a strong correlation with slow parasite clearance (9, 11). However,
67
understanding how parasites acquire tolerance to ART has been difficult due to insufficient
68
knowledge of the molecular mechanisms underlying the anti-malarial functions of artemisinin.
69
The active moiety of artemisinin is a sesquiterpene lactone containing an endoperoxide bridge
70
whose cleavage in the presence of ferrous iron in a Fenton-type reaction results in the generation
71
of reactive oxygen species (ROS) such as hydroxyl radicals, superoxide anions and carbon-
72
centered free radicals (14-16). It has been suggested that free radicals are responsible for
73
mediating cytotoxic action of artemisinin derivatives in the parasites (17-19).
74 75
Previous studies have shown that artemisinin derivatives cause alkylation of heme and
76
proteins in the parasite (15-17), bind and inhibit P. falciparum translationally controlled tumor
77
protein (TCTP) homolog (20) and inhibit sarcoplasmic reticulum Ca2+ - transporting ATPases
78
(SERCA of malaria parasite identified as PfATP6) (21). Recently artemisinins have been shown
79
to be distributed to mitochondrial compartment in the parasite resulting in impaired
80
mitochondrial functions (22) and ROS-dependent depolarization of plasma and mitochondrial
81
membranes (23). Even though many cellular targets have been identified, the mechanism of
82
action of artemisinin derivatives still remains ambiguous. Free radicals and ROS have been
83
shown to cause DNA damage in cells, providing the premise for our hypothesis that in addition
84
to above reported effects, anti-malarial action of artemisinin derivatives involves direct DNA
85
damage leading to parasite death. Potential involvement of DNA damage and repair processes
86
during biological effects of artemisinin in P. falciparum is also supported by a recent study
87
identifying several single nucleotide polymorphisms (SNPs) in genes involved in mismatch
88
DNA repair pathways (11).
4
89
In this study we sought to investigate ART- induced DNA damage in P. falciparum. Our studies
90
indicate that ART- induced DNA damage results from oxidative stress via generation of free
91
radicals and ROS. We also demonstrate that reduced glutathione (GSH) plays an important role
92
in detoxifying ROS and oxidative stress in the parasites. Glutathione is well established for its
93
protective role against any form of oxidative damage. Erythrocytes contain high levels of
94
reduced GSH and other antioxidant enzymes such as catalase and superoxide dismutase. Thus
95
mechanisms interfering with reduced GSH availability will be expected to synergize with drugs
96
such as ART in inducing DNA double strand breaks (DSB) and possibly overall cytotoxicity.
97
The fact that erythrocytes carry oxygen bound to hemoglobin makes blood stage Plasmodium
98
organisms especially vulnerable to oxidative stress, under normal physiological development as
99
well as when exposed to anti-malarial drugs such as ART. Our studies suggest that anti-parasite
100
effect of ART is therefore an end result of DNA damage, and further studies are needed to
101
characterize the contribution of recombinational DNA repair processes in resistance to
102
artemisinin derivatives.
103 104
Materials and Methods.
105
Parasite culture.
106
supplemented with 25 mM Hepes, 0.37mM hypoxanthine at 4% hematocrit and 10% O+ normal
107
human serum (24). Parasites were synchronized using sorbitol method as described previously
108
(25). Briefly, cultured parasites were pelleted and then treated with 5% sorbitol for 10 min at
109
room temperature followed by three washes with RPMI-1640 medium and further maintenance
110
in culture. Synchronization was repeated two more times until >90 % of parasites were in a
111
specific stage. Synchronized intra-erythrocytic ‘ring’ stage parasites were cultured and employed
P. falciparum clone 3D7 was maintained in RPMI-1640 medium
5
112
for all the studies between 18 and 20 hours of synchronization. Parasitemia was determined after
113
Giemsa staining of thin smears and trophozoite stage parasites were used at 4% hematocrit and
114
1% starting parasitemia. The IC50 value (50% inhibitory concentration) of ART used was
115
determined by the SyBR Green method (26).
116 117
Comet assay and DNA damage and parasite recovery.
118
falciparum (27) was used to assess the extent of DNA damage and recovery from DNA damage.
119
Briefly, parasites at different time points were collected by centrifugation lysed with 0.01%
120
saponin and washed 3-4 times in ice cold PBS and finally resuspended in PBS for single cell
121
electrophoresis (comet assay). Diluted cells (30 µl) were mixed with 300 µl 1% low-melting
122
point agarose (Comet LMAgarose, Trevigen) and the mixture added in the wells of Trevigen
123
slides. Slides were incubated with detergent lysis solution (Trevigen, Gaithersburg, MD, USA),
124
followed by alkaline lysis treatment and electrophoresis for 30 min at 1volt/cm in 1X TBE.
125
Slides were fixed in 70% ethanol (1 min), rinsed in distilled water, and dried overnight. Slide
126
wells were stained with 1X SyBR Green I and photographed at 200x magnification using Nikon
127
Eclipse 80i (Nikon) with Sensicam QE High Performance Camera (Cooke Corporation) and
128
scored using Comet assay IV software. Olive tail moment (OTM, product of the tail length and
129
the fraction of total DNA in the tail) was calculated to compare the extent of DNA damage in
130
each sample. To study the efficiency of parasite recovery P. falciparum parasites were treated
131
with ART for different time points (30, 45, 60, 90 and 120 min), washed and re-cultured in the
132
presence of fresh growth medium and uninfected erythrocytes (“return to growth” assay).
133
Cultures were harvested at various interval points (24 to 72 h) to prepare blood smears to check
134
parasite growth and DNA damage by comet assays.
6
Optimized comet assay for P.
135
Measurement of intracellular ROS. The ROS were measured using the dichloro-dihydro-
136
fluorescein diacetate (DCFH-DA) method (28) with slight modifications. Synchronized parasites
137
corresponding to 4% hematocrit at 1% infected red blood cells were collected and DCFH-DA
138
was added to trophozoite stage of P. falciparum culture at a final concentration of 20µM for 30
139
min before adding various concentrations of ART. Control samples were incubated with PBS
140
instead of ART. The fluorescence (excitation wavelength 485 nm and emission wavelength 530
141
nm) was measured using a SLM Aminco 8100 fluorescence spectrophotometer.
142 143
Glutathione assay. Total GSH and oxidized forms of glutathione (GSSG) were assayed using 5,
144
5’-dithiobis-2-nitrobenzoic acid method (29) with minor modifications to the manufacturer’s
145
protocol (Trevigen, MD). Sorbitol synchronized P. falciparum trophozoite stage parasites at 1%
146
parasitemia and 4% hematocrit were treated with ART for various time points and cells lysed in
147
4 volumes of ice-cold 5% metaphosphoric acid. Cell lysates were centrifuged at 12,000-14,000 x
148
g for 10 min at 4°C and supernatants used for GSH assay. For measuring oxidized glutathione
149
(GSSG), 50µl of diluted sample (20-fold dilution) in triplicates were treated with 1 µL of 2M 4-
150
vinyl-pyridine and incubated at room temperature for 60 min. For total GSH, 50µl of diluted
151
sample (20-fold dilution) was added to 96 well plates in triplicates. To each sample (GSH and
152
GSSG) and control, 150µl of freshly prepared glutathione reductase in reaction mixture was
153
added and absorbance read immediately at 405 nm using ELISA plate reader (VersaMax,
154
Molecular Devices) at 1 min intervals over 10 min time period. Absorbance values at various
155
time points from a set of standards were used to plot standard curves and the calculated net slope
156
was used to determine the pmole values of total and oxidized glutathione. Reduced GSH was
157
calculated by subtracting oxidized GSSG from total glutathione.
7
158
Statistical analysis. All experiments were performed in triplicates and most repeated a minimum
159
of three times. Graphpad Prism Software (Graphpad Software Inc., CA) was used for statistical
160
analyses of the data. Statistical significance of various treatments was compared to untreated
161
samples using Mann Whitney non parametric test. A p value of
1
Anti-malarial action of Artesunate involves DNA damage mediated by Reactive Oxygen Species
2 3 4
Anusha M Gopalakrishnan and Nirbhay Kumar
5 6
Department of Tropical Medicine and Vector-Borne Infectious Disease Research Center, Tulane
7
University School of Public Health and Tropical Medicine, New Orleans, LA, 70112, USA
8 9
Running Head: Artesuante and DNA damage in P. falciparum.
10 11
Address correspondence to: Nirbhay Kumar, [email protected]
12
Department of Tropical Medicine, Tulane University School of Public Health and Tropical
13
Medicine, New Orleans, 70112, LA
14 15 16 17 18 19
1
20
Abstract
21
Artemisinin-based combination therapy (ACT) is the recommended first line treatment
22
for Plasmodium falciparum malaria. It has been suggested that the cytotoxic effect of artemisinin
23
is mediated by free radicals followed by alkylation of P. falciparum proteins. The endoperoxide
24
bridge, the active moiety of artemisinin derivatives, is cleaved in the presence of ferrous iron
25
generating reactive oxygen species (ROS) and other free radicals. However, the emergence of
26
resistance to artemisinin in P. falciparum underscores the need for new insights into the
27
molecular mechanisms of anti-malarial activity of artemisinin. Here we show that artesunate
28
(ART) induces DNA double strand breaks in P. falciparum in a physiologically relevant dose
29
and time-dependent manner. DNA damage induced by ART was accompanied by an increase in
30
intracellular ROS in the parasites. Mannitol, a ROS scavenger, reversed the cytotoxic effect of
31
ART and reduced DNA damage and modulating glutathione (GSH) levels was found to impact
32
ROS and DNA damage induced by ART. Accumulation of ROS, increased DNA damage, and
33
resulting anti-parasite effect suggest a causal relationship between ROS, DNA damage and
34
parasite death. Finally, we also show that ART-induced ROS production involves a potential role
35
for NADPH oxidase, an enzyme involved in the production of superoxide anions. Our results in.
36
P. falciparum provide novel insights into previously unknown molecular mechanisms underlying
37
the anti-malarial activity of artemisinin derivatives, and may help in the design of new
38
generation anti-malarial drugs against the most virulent Plasmodium species.
39 40
Introduction.
41
Malaria accounts for hundreds of millions of clinical cases and nearly a million deaths annually
42
(1). Malaria parasites are transmitted by female Anopheles mosquitoes and infections caused by
2
43
Plasmodium falciparum and P. vivax are responsible for more than 90% of worldwide infections.
44
During its life cycle, parasites undergo a complex series of biological and biochemical
45
development that allow them to grow in the vertebrate hosts and to be successfully transmitted to
46
invertebrate mosquito vector. In the vertebrate host, actively proliferating intra-erythrocytic
47
asexual life cycle stages of the parasite are responsible for all the clinical symptoms, including
48
death and these stages are also the primary targets of anti-malarial drugs. Genetic diversity,
49
antigenic variation and parasite’s ability to adapt to new drugs continue to thwart control efforts.
50
Artesunate (ART) is the semi-synthetic derivative of artemisinin, developed from Artemisia
51
annua L which has been used in China as a traditional medicine for more than 2000 years.
52
Currently, artemisinin-based combination therapy (ACT) is recommended by the World Health
53
Organization as first line of treatment for P. falciparum malaria (2). These drugs act fast with
54
fewer side effects and are also active against P. falciparum that are resistant to other traditionally
55
used drugs, such as antifolates and quinolones. Artemisinin derivatives have been found to act
56
against various erythrocytic asexual stages as well as developing sexually differentiated
57
immature stages thus showing promise as an antimalarial drug with transmission blocking
58
potential (3-5). More recent studies have suggested that intra-erythrocytic ring stage parasites are
59
targets of artemisinin derivatives and ring stages of resistant parasites display longer survival as
60
compared to sensitive parasites(6, 7).
61 62
Decreasing clinical efficacy and emerging resistance to artemisinin derivatives in Thailand and
63
Cambodia have raised serious concerns about future treatment options (8-10). Recent studies
64
have identified putative genes potentially involved in ART-resistance mechanisms (11-13) in P.
65
falciparum. These studies identified several point mutations in the kelch propeller domain (K-13
3
66
propeller) showing a strong correlation with slow parasite clearance (9, 11). However,
67
understanding how parasites acquire tolerance to ART has been difficult due to insufficient
68
knowledge of the molecular mechanisms underlying the anti-malarial functions of artemisinin.
69
The active moiety of artemisinin is a sesquiterpene lactone containing an endoperoxide bridge
70
whose cleavage in the presence of ferrous iron in a Fenton-type reaction results in the generation
71
of reactive oxygen species (ROS) such as hydroxyl radicals, superoxide anions and carbon-
72
centered free radicals (14-16). It has been suggested that free radicals are responsible for
73
mediating cytotoxic action of artemisinin derivatives in the parasites (17-19).
74 75
Previous studies have shown that artemisinin derivatives cause alkylation of heme and
76
proteins in the parasite (15-17), bind and inhibit P. falciparum translationally controlled tumor
77
protein (TCTP) homolog (20) and inhibit sarcoplasmic reticulum Ca2+ - transporting ATPases
78
(SERCA of malaria parasite identified as PfATP6) (21). Recently artemisinins have been shown
79
to be distributed to mitochondrial compartment in the parasite resulting in impaired
80
mitochondrial functions (22) and ROS-dependent depolarization of plasma and mitochondrial
81
membranes (23). Even though many cellular targets have been identified, the mechanism of
82
action of artemisinin derivatives still remains ambiguous. Free radicals and ROS have been
83
shown to cause DNA damage in cells, providing the premise for our hypothesis that in addition
84
to above reported effects, anti-malarial action of artemisinin derivatives involves direct DNA
85
damage leading to parasite death. Potential involvement of DNA damage and repair processes
86
during biological effects of artemisinin in P. falciparum is also supported by a recent study
87
identifying several single nucleotide polymorphisms (SNPs) in genes involved in mismatch
88
DNA repair pathways (11).
4
89
In this study we sought to investigate ART- induced DNA damage in P. falciparum. Our studies
90
indicate that ART- induced DNA damage results from oxidative stress via generation of free
91
radicals and ROS. We also demonstrate that reduced glutathione (GSH) plays an important role
92
in detoxifying ROS and oxidative stress in the parasites. Glutathione is well established for its
93
protective role against any form of oxidative damage. Erythrocytes contain high levels of
94
reduced GSH and other antioxidant enzymes such as catalase and superoxide dismutase. Thus
95
mechanisms interfering with reduced GSH availability will be expected to synergize with drugs
96
such as ART in inducing DNA double strand breaks (DSB) and possibly overall cytotoxicity.
97
The fact that erythrocytes carry oxygen bound to hemoglobin makes blood stage Plasmodium
98
organisms especially vulnerable to oxidative stress, under normal physiological development as
99
well as when exposed to anti-malarial drugs such as ART. Our studies suggest that anti-parasite
100
effect of ART is therefore an end result of DNA damage, and further studies are needed to
101
characterize the contribution of recombinational DNA repair processes in resistance to
102
artemisinin derivatives.
103 104
Materials and Methods.
105
Parasite culture.
106
supplemented with 25 mM Hepes, 0.37mM hypoxanthine at 4% hematocrit and 10% O+ normal
107
human serum (24). Parasites were synchronized using sorbitol method as described previously
108
(25). Briefly, cultured parasites were pelleted and then treated with 5% sorbitol for 10 min at
109
room temperature followed by three washes with RPMI-1640 medium and further maintenance
110
in culture. Synchronization was repeated two more times until >90 % of parasites were in a
111
specific stage. Synchronized intra-erythrocytic ‘ring’ stage parasites were cultured and employed
P. falciparum clone 3D7 was maintained in RPMI-1640 medium
5
112
for all the studies between 18 and 20 hours of synchronization. Parasitemia was determined after
113
Giemsa staining of thin smears and trophozoite stage parasites were used at 4% hematocrit and
114
1% starting parasitemia. The IC50 value (50% inhibitory concentration) of ART used was
115
determined by the SyBR Green method (26).
116 117
Comet assay and DNA damage and parasite recovery.
118
falciparum (27) was used to assess the extent of DNA damage and recovery from DNA damage.
119
Briefly, parasites at different time points were collected by centrifugation lysed with 0.01%
120
saponin and washed 3-4 times in ice cold PBS and finally resuspended in PBS for single cell
121
electrophoresis (comet assay). Diluted cells (30 µl) were mixed with 300 µl 1% low-melting
122
point agarose (Comet LMAgarose, Trevigen) and the mixture added in the wells of Trevigen
123
slides. Slides were incubated with detergent lysis solution (Trevigen, Gaithersburg, MD, USA),
124
followed by alkaline lysis treatment and electrophoresis for 30 min at 1volt/cm in 1X TBE.
125
Slides were fixed in 70% ethanol (1 min), rinsed in distilled water, and dried overnight. Slide
126
wells were stained with 1X SyBR Green I and photographed at 200x magnification using Nikon
127
Eclipse 80i (Nikon) with Sensicam QE High Performance Camera (Cooke Corporation) and
128
scored using Comet assay IV software. Olive tail moment (OTM, product of the tail length and
129
the fraction of total DNA in the tail) was calculated to compare the extent of DNA damage in
130
each sample. To study the efficiency of parasite recovery P. falciparum parasites were treated
131
with ART for different time points (30, 45, 60, 90 and 120 min), washed and re-cultured in the
132
presence of fresh growth medium and uninfected erythrocytes (“return to growth” assay).
133
Cultures were harvested at various interval points (24 to 72 h) to prepare blood smears to check
134
parasite growth and DNA damage by comet assays.
6
Optimized comet assay for P.
135
Measurement of intracellular ROS. The ROS were measured using the dichloro-dihydro-
136
fluorescein diacetate (DCFH-DA) method (28) with slight modifications. Synchronized parasites
137
corresponding to 4% hematocrit at 1% infected red blood cells were collected and DCFH-DA
138
was added to trophozoite stage of P. falciparum culture at a final concentration of 20µM for 30
139
min before adding various concentrations of ART. Control samples were incubated with PBS
140
instead of ART. The fluorescence (excitation wavelength 485 nm and emission wavelength 530
141
nm) was measured using a SLM Aminco 8100 fluorescence spectrophotometer.
142 143
Glutathione assay. Total GSH and oxidized forms of glutathione (GSSG) were assayed using 5,
144
5’-dithiobis-2-nitrobenzoic acid method (29) with minor modifications to the manufacturer’s
145
protocol (Trevigen, MD). Sorbitol synchronized P. falciparum trophozoite stage parasites at 1%
146
parasitemia and 4% hematocrit were treated with ART for various time points and cells lysed in
147
4 volumes of ice-cold 5% metaphosphoric acid. Cell lysates were centrifuged at 12,000-14,000 x
148
g for 10 min at 4°C and supernatants used for GSH assay. For measuring oxidized glutathione
149
(GSSG), 50µl of diluted sample (20-fold dilution) in triplicates were treated with 1 µL of 2M 4-
150
vinyl-pyridine and incubated at room temperature for 60 min. For total GSH, 50µl of diluted
151
sample (20-fold dilution) was added to 96 well plates in triplicates. To each sample (GSH and
152
GSSG) and control, 150µl of freshly prepared glutathione reductase in reaction mixture was
153
added and absorbance read immediately at 405 nm using ELISA plate reader (VersaMax,
154
Molecular Devices) at 1 min intervals over 10 min time period. Absorbance values at various
155
time points from a set of standards were used to plot standard curves and the calculated net slope
156
was used to determine the pmole values of total and oxidized glutathione. Reduced GSH was
157
calculated by subtracting oxidized GSSG from total glutathione.
7
158
Statistical analysis. All experiments were performed in triplicates and most repeated a minimum
159
of three times. Graphpad Prism Software (Graphpad Software Inc., CA) was used for statistical
160
analyses of the data. Statistical significance of various treatments was compared to untreated
161
samples using Mann Whitney non parametric test. A p value of
