Effect of diethylcarbamazine, butylated hydroxy anisole and methyl substituted chalcone on filarial parasite Setaria cervi: Proteomic and biochemical approaches

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Effect of diethylcarbamazine, butylated hydroxy anisole and methyl substituted chalcone on filarial parasite Setaria cervi: Proteomic and biochemical approaches Sushma Rathaur⁎, Marshleen Yadav, Neetu Singh, Alka Singh Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi-221005 (U.P.), India

AR TIC LE I N FO Available online 4 May 2011

ABS TR ACT For survival, parasite exerts several lines of defense of which drug neutralization is one of the major phenomena. Lack of phase I cytochrome P450 in some of the nematode render

Keywords:

them depend on the phase II detoxification system involving GST as a major detoxifying

2D-electrophoresis

enzymes. In present study, the antifilarial DEC, phenolic compound BHA and methyl

Butylated hydroxy anisole

chalcone have been evaluated for proteomic and biochemical studies in Setaria cervi. BHA

Methyl chalcone

and methyl chalcone showed cytotoxic effect leading to irreversible inhibition in motility

Glutathione-S-transferase

and viability of parasites. These drugs showed marked alteration in proteomic profile of S.

Apoptosis

cervi at 100 μM concentration with 10.82, 8.52 and 6.75% downregulated (< 0.5) and 7.64, 31.78 and 24.32% upregulated (> 1.5) in DEC, BHA and methyl chalcone treatment respectively. Significant depletion in GSH level with increase in NO production was observed. Amongst these compounds, methyl chalcone demonstrated significant inhibitory effect (p < 0.05) on GST, PGHS and PTP activity leading to loss of metabolic homeostasis and parasite death. The cytotoxic response and altered expression profile of major enzymes under drug exposure suggested the oxidative stress induced apoptosis as a major cause of parasite killing which was further supported by DNA fragmentation in BHA and methyl chalcone. © 2011 Elsevier B.V. All rights reserved.

1.

Introduction

Worldwide, helminth parasites result in a combined disease burden of 8 million DALYs (Disability Adjusted Life Years) [1]. Lymphatic filariasis is one such tropical disease caused by filarial parasites Brugia malayi and Wuchereria bancrofti and transmitted to humans by mosquitoes. It afflicts approximately 120 million people in over 80 countries with more than 1.1 billion at risk of infection. Drug treatment for filariasis have not changed significantly in over 20 years, and with risk of resistance rising, there is an urgent need for the development of new anti-filarial drug therapies. The current treatment for filariasis is based on limited number of drugs such as diethylcarbamazine (DEC), albendazole and ivermectin [2]. These drugs are potential killer of larval stage of parasites

however; they are ineffective in adult parasite removal. Our previous study has demonstrated the inducible effect of DEC and butylated hydroxyl anisole (BHA) on antioxidant system of filarial parasites [3]. Additionally, the adulticidal effect of various substituted chalcones and combination of DEC plus aspirin in Setaria cervi have also been reported by us [4,5]. On the basis of these studies we have selected DEC, a known microfilaricidal, BHA, a phenolic antioxidant compound [6] and methyl chalcone (MC; 3-(4-methoxyphenyl)-1-(4-pyrrolidin-1-yl-phenyl) prop-2en-1-one) with potential antifilarial activity [4,7] for their effect on parasite survival, modulation of signaling molecules and proteome profile of filarial parasite. DEC exerts lethal effect on microfilariae however; its effect on adult parasite is not reported yet. BHA is a commonly used food preservative with broad biological activities, including protection against acute toxicity

⁎ Corresponding author. Tel.: +91 542 2307323; fax: + 91 0542 2368174. E-mail addresses: [email protected], [email protected] (S. Rathaur). 1874-3919/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2011.04.020

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of chemicals, modulation of macromolecule synthesis and immune response, induction of phase II detoxifying enzymes, and especially its potential tumor-promoting activities [8]. The metabolism of BHA leads to generation of O-demethylation to 2-tert-butyl (1, 4) hydroquinone (TBHQ) which subsequently peroxidized to 2-tert-butyl (1, 4) paraquinone and it is reported that these reactive metabolites could induce oxidative stress in cell by triggering caspases induced apoptosis [9]. The effect of methyl substituted chalcone has been shown to be exerted either by depleting GSH level or interacting with –SH group of GSH through its α, β-unsaturated carbonyl moiety leading to Michael type addition reaction [10]. GST, a phase II detoxification enzyme catalyzes the conjugation of glutathione (GSH) with xenobiotics and in some of the nematode it acts as a major defense system that protects the cell from toxic radicals generated from antioxidant reactions. It is known that many chemotherapeutic drugs may induce apoptosis by exhausting the intracellular thiol buffer system through depletion of GSH or redistribution of GSH [11] which may further leads to alteration in GST expression causing accumulation of free oxygen radicals in cells. PGHS, required for the conversion of arachidonic into prostaglandins is also shown to have role in parasite survival by participating in growth and development of parasite [12]. Figarella et al., (2005) have demonstrated that the metabolites of PGD2 induce apoptosis in T. brucei[13]. The reduced PGHS activity and high Nitric oxide (NO) with decreased protein tyrosine phosphatase activity is known to alter the mitochondrial membrane potential leading to release of cytochrome c [14,15]. In present study we have used Setaria cervi as a model organism for the evaluation of drugs since the parasite resembles human filarial parasite B. malayi and W. bancrofti in nocturnal periodicity and antigenic pattern [16]. The data presented here demonstrate the cellular cytotoxicity, reduction in GSH efflux and alteration in protein expression profile of aforementioned metabolic enzymes in S. cervi exposed to DEC, BHA and methyl chalcone compounds.

2.3.

The motility of parasites exposed to different compounds was assessed till 4 h and scored either positive or negative depending on their body movement. To check the effect of drugs on microfilariae (mf), adult female parasites were dissected longitudinally and released mf were collected in KRB buffer and visualized under microscope at 40× (Motic B1 series). MTT assay [18] was performed to check the viability of parasites as described earlier [4].

2.4.

Materials and methods

2.1.

Collection of parasites and preparation of homogenate

Adult, motile S. cervi worms were procured from the peritoneal fluid of freshly slaughtered Indian water buffaloes. Worms were washed with phosphate buffer saline (PBS) and maintained in Krebs’ Ringer bicarbonate buffer supplemented with streptomycin, penicillin, glutamine and 1% glucose (maintenance medium). Homogenate was prepared from adult parasites as described earlier [9] and protein content was estimated by Bradford method [17].

2.2.

Exposure of parasites to drugs

Equal number of adult female S. cervi (N = 16) were incubated in the maintenance medium containing 100 μM of DEC, BHA and methyl chalcone (kindly provided by Dr. S.K. Awasthi, Delhi University) for 4 h at 37 °C. Parasites incubated in medium alone served as control. After exposure to drugs parasites were recovered and washed with fresh PBS, homogenized, centrifuged and stored at −20 °C till further use.

Preparation of protein sample

The homogenate (prepared in Tris buffer pH 7.5) was treated with 10 volume ice chilled acetone and kept at − 20 °C for 3 h for protein precipitation followed by centrifugation at 7500 rpm for 10 min at 4 °C. Pellet was collected and air dried for 5 min and dissolved in 150 μl freshly prepared lysis buffer (20 mM Tris (pH 7.5), 7 M Urea, 2 M Thiourea, 4% CHAPS, 10 mM DTT, 1 mM EDTA, protease inhibitors cocktail (20 μg/μl) and 1 mM PMSF) and processed for 2D electrophoresis.

2.5.

Isoelectric focusing (IEF)

For IEF, the pre-cast Amersham immobilin Dry strips (IPG strip; 11 cm, pH range 4–7) were first rehydrated with rehydration stock solution (8 M urea, 0.5% (w/v) CHAPS, 0.2% (w/v) DTT 0.5% (v/v) IPG buffer and 0.002% bromophenol blue) containing 1 mg protein. Further, the strips were dipped in cover dry solution containing pharmalyte and left for 10 h at 25 °C. For IEF, rehydrated strips were processed in IPGphor IEF unit (BioRad) for 8–10 h. The program was set initially for 0.01 h at 300 V, followed by 1.30 h at 3500 V, 2 h at 5000 V, 7 h at 8000–13,000 V; and terminated after reaching up to14,000 V.

2.6.

2.

Effect on parasite motility and viability

1D Electrophoresis

The focused strips were equilibrated in 10 ml equilibration solution (50 mM Tris–HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (v/v) SDS, 0.002% Bromophenol blue and 1% DTT) for 20 min at room temperature and washed twice with 1 × SDS gel running buffer. The strips were then loaded on 12.5% SDS-PAGE for second dimension separation using Hoefer gel system and gels were stained with silver nitrate.

2.7.

Image analysis

IMAGEMASTER 2D Platinum image analysis software (Amersham Biosciences) and MELANIE v 7.0 were used for quantitative analysis of spots. Experiment was conducted twice and resulted spots were normalized by using mean spot volume of three unchanging spots in two conditions. A Wilcoxon matched-pairs signed-ranks test was performed to analyze whether the observed differences were statistically significant. Spot intensity/volume detected above 1.5 and below 0.5 was considered as significantly upregulated or downregulated respectively.

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2.8.

MALDI-LC/MS

2.14.

DNA fragmentation analysis

The spots prominently regulated were excised from the silver stained gel and processed for MALDI-LC/MS analysis. This facility was availed from The Centre for Genomic Application (TCGA), New Delhi, India. Protein identification was performed by using MASCOT Peptide Mass Fingerprinting program (http:// www.matrixscience.com).

The pattern of DNA cleavage was analyzed by running it on 1.5% agarose gels, stained with ethidium bromide, and visualized under UV light.

2.9.

3.1. Cytotoxicity generated by DEC, BHA and methyl chalcone in filarial parasite

Estimation of glutathione level

To estimate the GSH level in adult worm extract, equal volumes of 5% perchloric acid and adult worm extract were mixed and centrifuged at 3000 rpm for 10 min at 4 °C. The reaction mixture (2 ml) contained 100 μl of supernatant, 1.88 ml 0.1 M Potassium phosphate buffer, pH 8.0 and 0.02 ml of 4% DTNB. Incubation of reaction mixture was carried out at room temperature for 3 min and absorbance of color developed was recorded at 412 nm [19]. Distilled water was used for blank.

2.10. Enzyme activity and protein expression profile of ScGST GST activity in fresh worm extract was assessed spectrophotometrically according to the method of Habig et al. [20] using GSH (1 mM) and CDNB/Ethacrynic acid/DCNB (1 mM) as substrate. Purification of ScGST was done as described earlier [3] from 10% crude homogenate of control and drug treated S. cervi prepared in 50 mM Tris buffer (pH 7.5) using GSH-agarose affinity column. Western blotting of purified ScGST was performed at 40 mA for 1.30 h and signal was detected using 1:100 dilution of anti-ScGST antibody previously raised in jirds [21].

2.11.

Assay of prostaglandin synthase (PGHS)

PGHS activity was assayed using arachidonic acid as substrate as described earlier by Singh and Rathaur (2010) [5]. In brief, the final concentration of reagents in the assay mixture was 50 mM Tris–HCl (pH 8.0), 0.05% tween-20, 100 mM adrenaline, 0.02 mM hemin, 0.15 mM arachidonic acid and 50 μl enzyme sample (0.5 μg/ml). The absorbance was monitored at 480 nm.

3.

Result and discussion

The major problem regarding the chemotherapy of filariasis is that no safe and effective drug is available which could combat the severity of the disease. Diethylcarbamazine (DEC) is a common drug used for the treatment of lymphatic filariasis [24]. In addition, treatment also involves administration of ivermectin in combination with albendazole for LF control mostly in regions (many parts of Africa) that are co endemic for onchocerciasis [25] however; these drugs have shown lack of macrofilaricidal effect [26]. Here in our present study, we have used DEC, BHA, a phenolic antioxidant and exhibits least side effects at lower concentration [27] and methyl chalcone (MC). In an initial experiment, the motility and viability of adult parasite was found significantly inhibited within 4 h of treatment with BHA and MC which was not recovered in fresh medium (Table 1A, Fig.1A) while DEC did not show any inhibitory effect on parasite motility. The assessment of protein level also demonstrated variation in treated groups. We observed at least more than two times increase in protein content in MC compared to control while no significant changes were seen in DEC (Table 1B). The increased protein content may suggest the release of stress proteins under drug exposure. These compounds also led to paralytic effect on mf release in test samples while in control, mf were found motile with coiled movement as shown in Fig. 1B. Coiling in body is a process generally observed in live mf from control group whilst, treated mf appeared needle shaped after exposure to inhibitor/drug. Microscopic visualization showed ruptured body wall along with the stiffness in mf treated with Table 1A – Effect of antifilarials on the motility of adult female Setaria cervi. Sample

2.12.

Nitric oxide (NO) level

Nitric oxide (NO) level was measured as nitrite using Griess Reagent [22] and absorbance of the mixture was measured at 570 nm in a microplate reader. NO concentration was determined using a serial dilution (10 to 100 μM final concentrations) of NaNO2 as a standard.

2.13.

Assay of protein phosphatases

Method described by Taga et al., (1982) [23] was followed to assay hydrolysis of substrates P-L-Tyrosine, P-L-Serine, P-LThreonine by measuring free phosphate liberated. The free phosphate was measured by the quantitation of reduced phosphomolybdic acid at 700 nm using a molar extinction coefficient of 4 × 103 M−1 cm−1.

CON DEC BHA MC

Parasite mean motility score* 0h

1h

2h

3h

4h

++++ ++++ ++++ ++++

++++ ++++ +++ +++

+++ +++ ++ +

++ ++ +

++ + − −

Recovery†

+++ +++ − −

*Motility was visually checked at given time interval. Adult worms (N = 16) of equal size were incubated with each compound separately in 20 ml maintenance medium at 37 °C. Worms incubated in maintenance medium only served as control. Each value is the mean motility assessment (−, no movement; + + + +, motility equal to control; +, + +, and + + +, proportionate reductions in motility compared to control) of at least 4 parasites. Concentration of drug: 100 μM. † Worms were transferred into fresh medium (devoid of compounds) after 4 h and motility recovery in treated group was compared to control group. Results are from three independent experiments performed in duplicates.

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A

120

% Inhibition in viability

100

80

60

40

20

0 DEC

BHA

MC

B A

B

Immature egg

Mature egg Motile and coiled mf

C

D

Immotile Straight mf Immotile straight mf

E

Ruptured

egg sheath

Detached mf Sheath

Fig. 1 – A: Effect of antifilarials on viability of adult parasite. Adult worms (N = 16) of equal size were incubated with each compound (100 μM) separately in 20 ml maintenance medium at 37 °C and viability of each worm was assessed by MTT assay after 4 h of exposure. Worms incubated in maintenance medium only served as control. B: Effect of drugs on the motility of microfilariae released in excretory/secretory product (ES) of adult Setaria cervi and examined under MOTIC B1 compound microscope (40×). A–B: control; C: DEC; D: BHA and E: MC.

BHA and MC as compared to control. Nevertheless, effect of MC on eggs was also noticeable with shell outburst and exudation of internal fluid indicated the intensity of stress.

3.2.

Proteomic profile of parasite under drug exposure

2DE has revealed a differential protein expression profile of S. cervi exposed to 100 μM of DEC, BHA or MC which was

significantly altered compared to control expression map (Fig. 2A). A total of 190, 191, 210 and 180 protein spots were observed with significant intensity and volume (mean value 0.5) on SDS-PAGE of control, DEC, BHA and MC samples respectively (Table 2). Amongst these spots, 157 in DEC, 129 in BHA and 148 in MC were found overlapping with control gel. The Pearson's correlation between drug treated samples and untreated control was high (r = 0.934; 0.959 and 0.918 for DEC,

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Table 1B – Protein level in antifilarial treated S. cervi. Drugs

Protein (mg/ml)

CON DEC BHA MC

2.05 ± 0.123 2.30 ± 0.130 (−12.19) 3.6 ± 0.12 (+ 43) 4.65 ± 0.20 (+ 55.91)*

Protein quantification was done using Bradford (1976) method at 4 h. Results are representation of three independent experiments performed in duplicates. Values are expressed as mean ± S.D of absorbance. Values in parentheses indicate % decrease/ increase. * p < 0.05.

BHA and MC respectively) as shown in Fig. 2B. However, spots were profoundly altered in their expression as we have observed an upregulation (>1.5) in high molecular weight proteins with downregulation (<0.5) more in low molecular weight proteins depicting the functional differences in bigger and smaller proteins participated during stress. Nevertheless, we have observed three and one uniquely synthesized protein spots (not present in control) with complete disappearance of three and six protein spots in BHA and methyl chalcone (Fig. 2A, Table 3) suggested the transcriptional regulation of some of the proteins specifically under stress resulted from exposure with drugs. However, the effect of DEC was insignificant compared to other two compounds with slight alteration in protein range of 42–52 kDa. The gel analysis report based on mean intensity and volume demonstrated 12 upregulated (2.01–7.48 folds) proteins and 17 downregulated (2.01–5.44 fold) proteins in DEC exposed parasites. BHA and MC exposure resulted in upregulation of 41 and 36 (2.04–12.7 fold) and downregulation of 11 and 10 (2.04–5.20 fold) proteins respectively (Fig. 2C). The un-alteration in rest of the proteins with expression comparable to control could be due to the lack of observable transcriptional changes post drug treatment and these proteins might be either unresponsive to drugs or involve in housekeeping functions inside cells. Amongst these spots we have identified antioxidant enzymes such as Glutathione-S-transferase (GST; spot 169), Prostaglandin H Synthase (PGHS; spot 22), stress responsive Heat Shock Protein 70 (HSP70; spot 18) and a glycolytic enzyme Triose phosphate isomerase (TPI; spot 94) by MALDI-LC/MS (Fig.2D and Table 3). These proteins are generally involved in diverse biological functions including drug detoxification and redox regulation, cyclooxygenase pathway, stress response and molecular chaperone [28,29] and carbohydrate metabolism. Here, in our study the differential expression of these proteins in parasites exposed with abovementioned compounds proposed their specificity towards the chemical nature of compounds used. We observed more than two fold upregulation in GST, PGHS and HSP70 in BHA, downregulation of PGHS and HSP70 in DEC and PGHS in MC (Table 3). TPI, a glycolytic enzyme and most abundant protein in ES of B. malayi,[30] was found downregulated in BHA and increased both in DEC and methyl MC. The two fold upregulation of GST and PGHS has also been reported earlier in response to CdCl2 and suberonylanilide hydroxamic acid in tomato roots and HeLa cells respectively [31,32]. The drug induced altered expression of HSP70 in our study is in agreement of a previous report where HSP70 from

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Schistosoma mansoni has been identified as potential drug target [33].

3.3.

Cellular redox and GST expression

GSH is a known antioxidant and a primary regulator of intracellular redox which is maintained as balanced GSH/ GSSG ratio [34,35]. Depletion in intracellular GSH level before onset of apoptosis has been reported and demonstrated as inducer of cytochrome c release from mitochondria which render cells more sensitive to apoptotic agents through downstream caspase cascades[34,36]. Here, in our experiment, exposure of parasites with BHA and MC resulted in marked inhibition in GSH level however; DEC could not lead to any significant changes (Table 4). The observed depletion in GSH level under BHA and MC exposure could be correlated with our primary data on motility and viability inhibition in parasite under drug stress since depleted GSH level triggers the production of reactive oxygen radicals. Besides inducing oxidative stress and apoptosis, depletion in GSH could also alter the level of glutathione binding proteins expression. Thus, we have further examined the level of parasitic glutathione-s-transferase under drug exposure. GST is a major phase II detoxification enzyme and attributed with antioxidant property [37]. The upregulation in its activity under drug stress proposes it as a potential drug target. The expression of ScGST at 100 μM concentration of DEC and BHA was increased up to two folds and inhibited more than two fold in MC treatment (Table 3) agreeing with our previous studies where DEC and BHA has been shown as inducible agent for GST and methyl chalcone as inhibitory chalcone [3,4]. Similar results were observed in enzyme activity of GST where DEC and BHA induced the activity over 50% and methyl chalcone inhibited up to 54% with general substrate CDNB. However, with ethacrynic acid (π class substrate) activity increased by 73% and 50% in BHA and DEC respectively and decreased by 73% in methyl chalcone (Fig. 3A). Although, μ class GST activity was considerably low in control compared to π class and was affected insignificantly under drug stress. The data was also supported by differential expression of GST purified from drug treated S. cervi extract in western blotting (Fig. 3B) which showed that π class GST is a major form of GST in S. cervi and response towards drug exposure suggested its role in parasite survival. The protein expression profile of GST with gain/loss in activity under drug exposure suggested its alteration at transcriptional level. However, the possibility of structural or active site modification in GST in case of MC cannot be ruled out that might leads to loss in binding efficiency of GST with GSH matrix during purification. In our earlier study, we have shown that methyl chalcone can modify the active site of BmGST that may dissipate GSH from its proper binding site hence inhibit GST activity [7]. Nevertheless, an earlier report also shows the inhibitory effect of chalcone on GST activity by forming Michael addition complex with GSH [12]. To further confirm the involvement of π class GST in drug response in S. cervi, we have treated parasites with ethacrynic acid (EA) a specific inhibitor of π class GST [38,39]. As expected the significant inhibition in activity and expression of ScGST was observed with this compound which was comparable to MC results. The

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mechanism of EA cytotoxicity is attributed to the drug's known capacity to inhibit glutathione-s-transferase (GST), causing increased cellular oxidative stress. Here, the depleted GSH and GST activity in drug exposed parasite could be involved in ROS generated oxidative stress or induction of

A

redox sensitive JNK pathway which may leads to initiation of apoptosis [40]. One of the mechanisms of GST (specifically of pi class) induced inhibition of apoptosis is inactivation of JNK by forming GST-JNK complex and blocking JNK dimerization. Thus, here we can speculate that inhibition in GST activity and

CON

DEC

135 95 PGHS

PGHS HSP70

HSP70

72

52 Actin

Actin

42 34

GST

TPI

GST

TPI

26

17

BHA

MC

135 95

PGHS

PGHS

72 HSP70

HSP70

52 Actin

Actin

42

34

TPI

GST

TPI

26

GST

17 4.0

4.5

5.0

5.5

6.0

6.5

7.0

4.0

4.5

5.0

5.5

6.0

6.5

7.0

Fig. 2 – A: 2D electrophoresis profile of Silver stained crude extracts of S. cervi treated with 1: CON; 2: DEC; 3: BHA and 4: MC. The first dimension was run on pH 4–7 IEF strips followed second dimension on 12.5% SDS-PAGE. Red circle: upregulated proteins; blue circle: downregulated proteins; light blue circle: protein completely suppressed and green arrow: protein observed only in drug exposure. B: Correlation graph of spot matching. C: Differential expression of S. cervi proteins under drug treatment. Analysis of protein spots in adult female parasites under drug exposure. Light gray region: downregulated spots and Dark gray region: upregulated spots. Values shown in brackets represent % upregulation/downregulation. D: Highlighted portion of 2D-gels showing expression profile of actin; PGHS and GST, HSP70 and TPI.

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B

CON 6

CON 6

5

5

4

4

3

3

2

2

1

1

0

0 0

1

2

3

4

5

1

0

6 DEC

2

3

4

5

6 BHA

CON 6

5

4

3

2

1

0 0

C

1

2

3

5

4

6 MC

60

Protein spot numbers

50 11 10

40 30 20

17

41

36

BHA

MC

10 12

0 DEC

Fig. 2 (continued). its expression in case of MC could be due to drug induced ROS production which further leads to GST pi oligomerization [41] and may initiate JNK activation hence could trigger apoptosis in parasites.

3.4.

Drug induced alteration in signaling molecules

The mechanism of parasite killing under DEC, BHA and MC exposure was seen by checking the levels of few signaling

molecules such as PGHS, nitric oxide and protein phosphatase involved in apoptosis. PGHS activity was found increased (54%) in BHA exposed parasites and decreased considerably in MC (73%) and DEC (39%) as shown in Fig.4A. The upregulated PGHS in BHA might be involved in its metabolism since it has been reported to convert hydroquinone (TBHQ) of BHA into corresponding quinine (TBQ) by redox cycling [9]. However, this reaction results into production of ROS which might therefore be responsible for increased GST activity and

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D

CON

MC

BHA

DEC

Actin

PGHS

GST

HSP70

TPI

Fig. 2 (continued).

cytotoxicity in parasite. Further, it has been known that chalcones are potent inhibitor of tyrosine of protein as they are involved in hydrolysis of aromatic rings of tyrosine [42]. Tyrosine plays an important role in catalytic mechanism of PGHS [43]. Thus the observed downregulation in PGHS in MC treatment could be due to the modification of active site of this enzyme as abovementioned. On the contrary, these drugs triggered the release of Nitric oxide in parasites was increased by 47% and 58% in DEC and BHA treatment respectively and significantly 76% in MC exposure (Fig. 4B). It is reported that decreased PGHS and increased NO level induce free radicals production and NO is also believed to stimulate the activity of phospholipase A2 to release arachidonic acid [44] which in turn causes accumulation of arachidonic acid and increase in

ceramide production leading towards apoptosis [45]. BHA has been reported to alter the mitochondrial membrane potential triggering the release of cytochrome c which further initiates the caspase cascade induced apoptosis [8]. Here, we can speculate that the intermediate form during BHA metabolism could be involved in cytotoxicity in parasites or there might be some direct interaction of this drug with mitochondrial membrane or through some other molecules. Yu et al. (2000) have shown that BHA may itself induce apoptosis independent of reactive metabolites in rat hepatocytes [8] however; the exact mechanism of action of BHA on mitochondria is yet to be explored. The high NO production in parasite under BHA and MC treatment might be involved in triggering mitochondrial changes since NO is also known to alter the mitochondrial

Table 2 – Protein spot detection and alteration in their levels by drug treatment. Drug

Control DEC BHA MC

Total spot* 190 191 210 180

Overlap with control

Spot Regulated† 100

75

42–52

157 129 148

+ + +

+ + +

− + +

*Mean count of spots observed in gels run in three different experiments. † Protein molecular weight range in kDa. (+): Upregulated (> 1.5); (−): downregulated (< 0.5); (+/−): both up and down regulated.

20–17

Newly expressed spots

Completely suppressed spots

+/− +/− −

ND 3 1

ND 3 5

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Table 3 – 2-DE based identified proteins of S. cervi by MALDI-LC/MS. Spota

Protein (Accession code†)

169

Mw /pI Expected

GST(P46427.1)

Identified Peptide

Experimental

26/ 6.96

25.8/6.4

22 18

PGHS(P27607) HSP70(O96541)

70/6.89 70.2/5.63

69.11/6.5 68/5.4

94

TPI(A8XZ64)

26.59/5.99

25.12/6.0

K.LTYFSIR.G R.GLAEPIR.L PEWDDEQLFQTT.R K.DSGAIAGLNVLR.I R. IINPTAAAIAYGLD.K DVDGFLVGGASLKPEFIDIINA.K FFVGGNWKMNGD.K

m/z ratio

MASCOT

Fold change*

Score

DEC

BHA

MC

899.5250 1094.600 1512.778 1185.63 1660.57 2417.289 1514.846

55

1.90

2.20

−2.50

55 56

–1.28 –0.74

2.24 2.42

−2.32 2.0

66

1.24

−1.05

1.4

a

Spot number obtained from control gel taken as reference for drug treated groups. † Protein matched obtained from UniProt database (http//:www.uniprot.org) with spots. *Fold change calculated from mean intensity and volume of spots observed in treated and untreated control groups run on three different sets of experiments. NE: no effect.

Table 4 – Glutathione level in antifilarial treated adult female S. cervi. Drugs

GSH (nmol/mg protein)

% Decrease of GSH level

CON DEC BHA MC

7.80 ± 0.134 7.5 ± 0.109 3.50 ± 0.129 ND

3.8 55.1 100

GSH level was estimated by Ellman method 1959 as described in glutathione estimation section of materials and methods. ND-not detectable. Results are from three independent experiments performed in duplicates. Values are expressed as mean ± S.D of absorbance.

membrane potential leading to cyt c release [14]. The increased NO level is also hypothesized as inhibitor of Protein Tyrosine Phosphatase (PTP) [46]. Here in our study, drug stress led to

A 0.05 CDNB EA DCNB

*

GST activity (u/mg)

0.04

0.03

0.02 ** 0.01

0.00 CON

DEC

BHA

MC

EA

B

CON DEC

BHA

MC

EA

Fig. 3 – A: Enzyme activity of GST in adult female S. cervi under drug exposure. B: Western blot analysis of ScGST purified from crude extract of adult female parasites exposed to drugs. Anti-ScGST antibody was used as primary antibody at 1:100 dilution. CON: control; DEC: diethylcarbamazine; BHA: butylated hydroxyanisole and MC: methyl chalcone and EA: ethacrynic acid.

inhibition in PTP activity by 45–52% in BHA and MC respectively however, DEC led only up to 10% inhibition insignificant compared to control and other two drugs (Fig. 4C). Decrease in PTP leads to Bcl-2 protein phosphorylation and loss of its anti-apoptotic activity causing cell death in the hypoxic brain [46]. Further, phosphorylated JNK is reported to antagonize Bcl2 anti-apoptotic action thus could enhance Bax activity and may promote apoptosis [47]. On the contrary, Ser/Thr Phosphatase activity was increased in all parasite treated with all three drugs with highest activity of serine phosphatase in BHA (4.3 fold) followed by DEC (3.02 fold) and methyl chalcone (1.06). While Threonine phosphatase activity was highest in DEC (10.8 fold) followed by BHA and methyl chalcone (9.4 and 7.7 fold respectively). The upregulated activity of Ser/Thr phosphatase in DEC treated worms may indicate towards the dephosphorylation of downstream Bad, thereby preventing apoptosis by not allowing its dimerization with Bcl2. On the other hand, some Ser/Thr phosphatases like PP1, induces apoptosis by enhancing the expression of proapototic bcl-x, which could support the induced phosphatases activity in BHA and MC treated worms [48]. On the basis of response of signaling molecules we can propose that decrease in PGHS activity leads to enhance NO level which inactivates the tyrosine phosphatase and in turn may affects the expression of Bcl-2 leading to mitochondrial mediated apoptosis in parasite [5]. It is also reported that excess synthesis of NO can increase Bax level and decrease Bcl-2 expression [45]. The significant decrease in PTP level in MC affected parasite also provided a hint towards enhanced kinase activity and GST-JNK regulated apoptotic pathway where oxidation of GST leads to release of JNK and further phosphorylation of JNK in scarcity of PTP and could mediate ROS induced JNK mediated apoptosis. The overall results suggested the

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C 80

0.16

60

0.14

40

PTP Activity (U/ml)

% Increase/ decrease in PGHS activity

A

20 0 -20 -40 -60

0.10 0.08 0.06 0.04 0.02

-80 -100

0.00 DEC

BHA

MC

B

CON

D Ser/Thr Phosphatase Activity (U/ml)

100

% Increase in NO level

0.12

80

60

40

20

0 DEC

BHA

DEC

BHA

MC

120 Ser Phosphatase Thr Phosphatase

100 80 60 40 20 0

MC

CON

DEC

BHA

MC

Fig. 4 – Effect of drug on A: Prostaglandin H synthase activity; B: Nitric oxide level and C: Protein tyrosine phosphatase activity and D: Ser/Thr Phosphatase activity. DEC: diethylcarbamazine; BHA: butylated hydroxyanisole and MC: methyl chalcone.

apoptosis as a major killing mechanism in parasites under drug exposure. This was further confirmed by DNA fragmentation observed in case of BHA and MC. To rule out the occurrence of necrosis in parasites, effect of ethacrynic acid on DNA was also checked which showed intact DNA comparable to control group. EA is a known necrotic agent which leads to outburst of cell membrane and releases cytosolic contents to neighbor cells causing inflammation [49]. On the contrary, EA is also reported to cause apoptosis in tumor cells at high concentration by an unknown mechanism [37]. However, here in our study we observed a sudden arrest in motility in parasites exposed to EA in initial first hour which did not revive in recovery medium demonstrated the necrotic effect of EA on parasites. In fragmentation analysis, DNA was also found intact in EA exposed parasite (Fig. 5).

4.

Kb 10

1 0.5

Conclusion

In conclusion, BHA an antioxidant and methyl chalcone induced imbalance is a major detoxification pathway in filarial parasite leading to generation of cytotoxicity and parasite death. Further studies using proteomics and biochemical assays of GSH-GST system and signaling molecules suggested the BHA and MC at 100 μM as potent adulticide and oxidative

L

1

2

3

4

5

Fig. 5 – DNA fragmentation analysis. L: DNA ladder 1Kb, 1-Control, 2-DEC, 3- BHA, 4- EA (ethacrynic acid) and 5-MC.

J O U R NA L OF PR O TE O MI CS 7 4 ( 2 01 1 ) 1 5 9 5–1 6 0 6

stress induced apoptosis as a major killing mechanism of these compounds.

Acknowledgement Authors gratefully acknowledge University Grant Commission, New Delhi for financial assistance. Authors also acknowledge Dr. M.P. Singh, Indian Institute of Toxicology Research, Lucknow, India for providing 2D gel electrophoresis facility.

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