Echinococcus granulosus: In Vitro Effects of Ivermectin and Praziquantel on hsp60 and hsp70 Levels

Experimental Parasitology 93, 171–180 (1999) Article ID expr.1999.4449, available online at http://www.idealibrary.com on

Echinococcus granulosus: In Vitro Effects of Ivermectin and Praziquantel on hsp60 and hsp70 Levels

J. Martinez,*,1 J. Perez-Serrano,* W. E. Bernadina,† and F. Rodriguez-Caabeiro* *Faculty of Pharmacy, Department of Microbiology and Parasitology, University of Alcala, 28871 Alcala de Henares, Madrid, Spain; †Institute of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, University of Utrecht, P.O. Box 80165, 3508 TD Utrecht, The Netherlands

Martinez, J., Perez-Serrano, J., Bernadina, W. E., Rodriguez-Caabeiro, F. 1999 Echinococcus granulosus: In vitro effects of ivermectin and praziquantel on hsp60 and hsp70 levels. Experimental Parasitology 93, 171–180. Organisms or cells exposed to injurious stresses such as heat shock or chemicals respond by increased (or altered) expression of heat-shock proteins (HSPs). Conversely, an earlier exposure to stress can prepare cells to cope with a subsequent more severe stress. In the present study, protoscolices of Echinococcus granulosus were subjected to several anthelmintic treatments, involving storage of the protoscolices for 18, 30, and 50 h with 0.1 mg/ml of ivermectin (IV), praziquantel (PZ), and a combination of each with albendazole (ALB). The organisms were analyzed for the effects of drug treatment on cell integrity and on levels of hsp60 and hsp70 production. Drug efficacy was evaluated by microscopy and by protein content measurement. Hsp60 and hsp70 were detected by Western blotting and incubation with antihsp60 and anti-hsp70 antibody, respectively, and quantitation of these proteins was obtained using image analysis. Incubation with IV alone produced the most damage to the protoscolices as indicated by viability loss, decreased protein content, and altered hsp60 and hsp70 levels; incubation with IV 1 ALB produced less damage as manifested by fewer changes in the aformentioned damage parameters but PZ and PZ 1 ALB, in this context, were poor anthelmintics. Exposure of protoscolices to thermal stress prior to anthelmintic treatment, in most cases, increased drug efficacy. It is concluded that in the E. granulosus model system drug efficacy is associated with decreased levels of hsp70 expression and increased levels of hsp60 expression. q 1999

INTRODUCTION

All organisms, including phylogenetically remote organisms, synthesize a number of highly conserved cellular proteins, called heat shock proteins, or HSPs (Lindquist 1986). Up to 5% of the cellular protein may be HSP (Feige and Polla 1994) and evidence is accumulating that HSPs have a role in many important functions in cells under both normal and abnormal conditions. HSP functions include protein synthesis (Ellis and Hemmingsen 1989), protein degradation (Terlecky 1994), stabilization of microfilaments (Liang and MacRae 1997), regulation of the immune response (JacquierSarlin et al. 1994), and, more recently, apoptosis involvement (Wei et al. 1995, Mehlen et al. 1996). Although it has been established from early studies (Ritossa 1962) that heat shock is a major element affecting expression of HSPs, a rapidly expanding body of knowledge gained from later experimental studies has implicated many other stressors in the induction of HSPs. These include, for example, reactive oxygen species, cytokines, and heavy metals (Lindquist 1986, Polla et al. 1993), parasites (Lindley et al. 1988, Himeno et al. 1993), and certain drugs (Salminen et al. 1997, Tosi et al. 1997). At our department of Microbiology and Parasitology, drug-efficacy studies on in vitro-cultured protoscolices from the hydatid organism Echinococcus granulosus have, over the past decade, been a subject of great interest. Thus, optimal formulations have been identified for (i) parasite culture (Casado et al. 1986), (ii) in vitro parasite

Academic Press

Index Descriptors and Abbreviations: Echinococcus granulosus; hsp70; hsp60; IV, ivermectin; PZ, praziquantel; ALB, albendazole.

1

To whom correspondence should be addressed. Fax: 8854663. E-mail: [email protected].

0014-4894/99 $30.00 Copyright q 1999 by Academic Press All rights of reproduction in any form reserved.

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172 treatment (Casado et al. 1989, Perez-Serrano et al. 1994), and (iii) measurement of anthelmitic-induced injury (Casado et al. 1994). We have employed these tested techniques to compare drug-mediated damage(s) with consequential production of HSPs. In this paper we study the effects of several anthelmintics, notably ivermectin (IV), praziquantel (PZ), and albendazole (ALB), on hsp60 and hsp70 levels in protoscolices of E. granulosus. We also present preliminary data indicating that in the system described herein, drug-induced destruction of the parasite and HSP expression by these are causally related. The effectiveness of the drugs used and the resulting levels of hsp60 and hsp70 have been assessed using protoscolice protein content and viability and Western blotting with subsequent image analysis, respectively, as parameters. In addition, these parameters have been compared with those obtained from protoscolices subjected to thermal stress prior to storage in the presence of anthelmintics. These studies appear to have led to the introduction of an approach using in vitro HSP production as a read-out system for appropriate anthelmintic selection.

MATERIALS AND METHODS

Reagents. The supportive membrane PVDF (polyvinylidene fluoride) was purchased from Millipore (Inmobilon P). Defatted milk powder was purchased from Nestle. PBS with 0.05% Tween 20 (PB-T) was routinely used. The blocking buffer consisted of PB-T 1 5% (w/v) defatted milk. Culture medium for protoscolices was medium 199 (Sigma) supplemented with 0.1 mg/ml streptomycin, 100 Ul/ml penicillin, and dimethyl sulfoxide (1/1000). Antibodies (Abs). The monoclonal antibodies (mAbs) anti-HSP60 (clone LK-2) and anti-HSP70 (clone BRM-22) were from Sigma. Peroxidase-conjugated goat anti-mouse serum was obtained from Sigma. Parasites and viability tests. The sheep strain of E. granulosus was used throughout the study. Hydatid cysts of sheep liver origin were obtained from the municipal abattoir in Alcala de Henares. Using sterile methodologies, protoscolices were removed from brood capsules, pooled, washed, and placed in Leighton tubes. Viability of larvae prior to testing always ranged between 98 and 100%. When needed, viability of protoscolices was assessed using the methylene blue exclusion test and microscopic examinations as described (Casado et al. 1986). Incubations with anthelmintics. Protoscolices (25,000/Leighton tube) were subjected to one of the following procedures in 10 ml of culture medium 199, that is, addition of 0.1 mg/ml of either IV, (IV 1 ALB), PZ, (PZ 1 ALB), or no anthelmintic (controls) and incubation at 378C for 18, 30, and 50 h (without periodic shaking). All experiments were performed in triplicate. In a parallel experiment, protoscolices were heat-shocked first at 408C for 2 h (hereafter termed

MARTINEZ ET AL.

prestressed protoscolices) and then subjected to the same procedures described above. Processing of protoscolices. All procedures used to obtain protoscolice proteins for analysis of HSP production were carried out at 48C, including homogenization of the protoscolices (in 0.1ml of PBS, pH 7.2) by repeated sonication to disrupt all cells and centrifugation at 14,000 rpm for 20 min. The resulting supernatant was collected, analyzed for protein content by Bradford procedure (Bio-Rad), and stored in liquid nitrogen until needed. Electrophoretic analysis and Western blotting. Proteins from protoscolices were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) as described (Laemmli 1970). Gels were composed of a 10% acrylamide separating gel and a 4% acrylamide stacking gel. Samples, each with an equal amount of protein (0.09 mg), were loaded in the lanes and electrophoresis was performed in the cold room at a constant current of 40 mA. Following SDS–PAGE, electroblotting of the separated polypeptides was done according to a slightly modified procedure (Towbin et al. 1979) encompassing the use of PVDF (inmobilon P) instead of nitrocellulose as protein-supportive membrane. The PVDF membranes were then (and also between all the below incubations) washed with PB-T for 10 min. Blots were blocked using blocking buffer and incubation for 60 min. Appropriate dilutions of the Abs (1:5000 diluted anti-HSP70 and 1:1000 diluted anti-HSP60) were then added and blots were incubated for 90 min at room temperature. Following incubation with 1:6000 diluted peroxidase-conjugated anti-mouse serum, immunoreactions were visualized by incubation with a substrate comprising 0.06% 3,38 diaminobenzidine and concentrated H2O2 diluted to a 1:1000 final dilution. Statistical analysis. Immunoreactivity of the various triplicate samples on immunoblots was quantitated using an image analyzer (Program MIP 1.6., Microm, Spain). The values obtained at each testing time point were expressed as percentages of change vs means of experimental controls. Data were evaluated by ANOVA (analysis of variance) and post Dunnet’s multiple comparison test was used for statistical comparison within treatment groups vs controls for parameters of druginduced damage at testing time points. Linearity of Western blot test results. Prior to this study experiments were carried out to demonstrate linearity of immunoblot results under the same conditions used here. Recovery of each HSP by its HSPspecific Ab to be employed in the Western blot was assessed by assaying protoscolice specimens with varying amounts of proteins. Evaluation of test results on these samples by regression analysis, in all cases, revealed a very high correlation (r . 0.95) between observed immunoreactivity and amount of protein applied in the test.

RESULTS

The effects of anthelmintics or medium only on test protoscolice hsp70 levels are shown in Figs. 1–3. In protoscolices previously stressed or not and then stored in the presence of IV only or IV 1 ALB, hsp70 levels significantly decreased during the experiment (Figs. 1 and 3A). Note that maximally decreased hsp70 levels were detected at 50 or 18 h, these

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FIG. 1. Western blot analysis of hsp60 and hsp70 in E. granulosus larvae exposed to IV (cf. A/B) and IV 1 ALB (cf. C/D). HSP in protoscolices, whether not stressed (A/C) or stressed (B/D) thermally prior to drug exposure, compared with control specimens (i.e., not prestressed, not drug-treated).

levels, thereafter either persisting or increasing somewhat with time. On the other hand, no significantly altered hsp70 production was detected in protoscolices exposed to PZ only or PZ 1 ALB, except that here also hsp70 levels tended to increase with time (Figs. 2 and 3A). The levels of hsp60 detected in protoscolices stored in the presence of test anthelmintics are shown in Figs. 1, 2, and 3B. It was noted that hsp60 production significantly increased over control levels only when protoscolices were exposed, whether prestressed or not, to IV (Figs. 1 and 3B). In contrast, exposure to PZ 1 ALB produced a reversed

response, inasmuch that now significantly decreased levels of hsp60 were detected in prestressed protoscolices but not in non-prestressed ones (Figs. 2 and 3B). Viability scores in protoscolices stored in the presence and absence of test anthelmintics were determined. During the experiment, loss of viability and ultimately death of both prestressed and nonprestressed protoscolices correlated with exposure times to IV only or IV 1 ALB (Fig. 4A). In contrast, no appreciable change was detected in the viability scores of test protoscolices stored in PZ only or PZ 1 ALB. Figure 4B compares the levels of total protein found in

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FIG. 2. Western blot analysis of hsp60 and hsp70 in E. granulosus larvae exposed to PZ (cf. A/B) and PZ 1 ALB (cf. C/D). Hsp in larvae, whether not stressed (A/C) or stressed (B/D) thermally prior to drug exposure, compared with control specimens (i.e., not prestressed, not drug-treated).

protoscolices stored with and without anthelmintics. As can be seen, an increasing loss of protein, with time, was found in all test protoscolices exposed to IV only or IV 1 ALB but not in those exposed to either PZ alone or PZ 1 ALB (Fig. 4B). After exposure to PZ only or PZ 1 ALB protein levels significantly different from those in controls were detected only in prestressed organisms postexposure to PZ 1 ALB. Note that exposure of protoscolices to PZ only or PZ 1 ALB, but not to IV alone or IV 1 ALB, caused the production of big vesicle-like formations, termed “bladders” in all test organisms (data not shown).

As effective drugs seemed to not clearly relate viability loss to protein loss and/or hsp60 and hsp70 production, it was important to establish statistical relevance between differences noted. Table I shows (i) the F and P values calculated by ANOVA and (ii) the P values for each damage parameter and anthelmintic regime by post Dunnet’s multiple comparision test approach at 18, 30, and 50 h. As can be seen, exposing larvae to IV alone for $ 18 h induced changes in viability, protein loss, and hsp70 and hsp60 production scores significantly different from pretreatment values. Combining IV with ALB caused less damage to test

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FIG. 3. Levels of hsp70 (A) and hsp60 (B) at the three time points indicated in E. granulosus larvae exposed to IV, IV 1 ALB, PZ, or PZ 1 ALB, compared with experimental controls. (h) Drug formulation given to larvae stressed thermally prior to the study.

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FIG. 4. Viability (A) and protein content (B) scores at the three indicated time points in test E. granulosus larvae exposed to IV, IV 1 ALB, PZ, or PZ 1 ALB, compared with experimental controls. (h) Drug formulation given to larvae stressed thermally prior to the study.

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EFFECT OF IVERMECTIN AND PRAZIQUANTEL ON E. granulosus

TABLE I Damage Analysis Scores of Protoscolices from Echinococcus granulosus Following Treatment with Anthelminthics Hsp60 b Treatment group a

18 h

IV S→

ns

S→

ns

S→

ns

S→

ns

S→

ns

S→

ns

S→

ns

S→

ns

IV(h) IV 1 ALB IV 1 ALB(h) PZ PZ(h) PZ 1 ALB PZ 1 ALB(h)

30 h F 5 10,62/** ns F 5 8,10/** ns F 5 1,28/ns ns F 5 1,87/ns ns F 5 0,94/ns ns F 5 1,53/ns ns F 5 2,43/ns ns F 5 4,47/* ns

Hsp70 b 50 h

18 h

*

**

*

**

ns

**

ns

*

ns

ns

ns

ns

ns

ns

*

ns

Protein b

30 h F 5 23,49/*** ** F 5 13,47/** ** F 5 21,10/*** ** F 5 10,68/** ** F 5 0,57/ns ns F 5 0,88/ns ns F 5 0,47/ns ns F 5 0,94/ns ns

50 h

18 h

**

ns

**

ns

**

ns

*

ns

ns

ns

ns

ns

ns

ns

ns

ns

30 h

Viability b 50 h

F 5 32,62/*** * ** F 5 =82,13/*** ** ** F 5 13,83/** ns ** F 5 19,17/*** ns ** F 5 0,58/ns ns ns F 5 2,00/ns ns ns F 5 1,58/ns ns ns F 5 4,46/* ns *

18 h

30 h

50 h

F 5 81,76/*** ** F 5 171,90/*** ** ** F 5 134,30/*** ns ** F 5 572,5/*** ns ** F 5 0,44/ns ns ns F 5 0,83/ns ns ns F 5 0,55/ns ns ns F 5 0,64/ns ns ns *

** ** ** ** ns ns ns ns

Note. F denotes the F scores plus corresponding significance as obtained by comparision of damage parameters evaluated, using ANOVA (analysis of variance). S denotes the significance of differences found, per treatment group, at 18, 30, and 50 h compared to control group (0 h) by post Dunnet’s multiple comparision test. *P , 0.05, **P , 0.01, ***P , 0.001, ns, not significantly different; (h), drug formulation given to larvae stressed thermally prior to the study. a Protoscolices (triplicate samples) exposed to indicated anthelminthic regimes at the start of the experiment (0 h). b Parameters of treatment-induced damage evaluated in statistic herein (cf. Materials and Methods).

protoscolices. In contrast, neither PZ nor PZ 1 ALB induced appreciable changes in the damage parameters used.

DISCUSSION

The response observed in protoscolices subjected to in vitro anthelmintic treatment provides a model system for studying drug function(s) and the parasite’s-regulatory mechanisms. Accordingly, this system has already been used to study viability and morpho(patho)logy in protoscolices (Casado et al. 1989, Perez-Serrano et al. 1994). In addition to employing a similar approach in the present study, we used the levels of hsp60 and hsp70 as bioindicators of druginduced injury and, by implication, of drug efficacy. As HSPs now are recognized as useful marker proteins of cellular stress, some authors suggest their further use for diagnostic purposes or for monitoring drug efficacy (Macario 1995). Monitoring HSP levels thus aids in predicting the progression or resolution of infection or disease as it answers the question whether cellular injury to the organism is reversible or progressing until apoptosis ensues. Thus changes in HSP

levels may indicate both resistance to the stressor (Wong et al. 1996) and proximity of cell death (Hamilton et al. 1995). A recent report has demonstrated the good use of HSP expression levels in chemotherapeutic investigations (Tosi et al. 1998). Our results demonstrate that some test anthelmintics exhibit clear HSP production changeover activity whereas others do not. For example, PZ and PZ 1 ALB caused no altered hsp70 production, although they induced some increased hsp70 levels in prestressed organisms over time. In the same parasites, however, hsp60 levels were either comparable to controls at all time points or significantly decreased (in nonprestressed organisms) at 50 h. Whether the apparent contrast in hsp70 and hsp60 activities of the two forms of protoscolices represents a difference in stresstackling ability remains to be established. In this regard, our observation may perhaps be surprising because of the requirement for stressful stimuli in altered HSP production (Hamilton et al. 1995, Wong et al. 1996). Indeed, our results using PZ or PZ 1 ALB support previous studies which have shown PZ to be inefficient against our test parasite (Gemmel and Parmenter 1983). Our study indicates that the same applies to the combination of PZ 1 ALB. Nevertheless,

178 treatment with these drugs caused parasites to produce bladders, these being the least abundant in prestressed organisms. The development of bladders in protoscolices following exposure to some drugs has previously been noticed in our laboratory (Casado et al. 1986). Using IV, we noted a significant decrease in the protoscolice hsp70 levels and an increase in their hsp60 levels at 50 h. Decreased hsp70 was associated with decreased protein content and loss of viability. However, despite this apparent causal relationship, no linear correlation was evident between decreased viability and either decreased hsp70 levels or total protein content. The lack of correlation notwithstanding, our data showed that whenever a drug was effective in our assay (i.e., producing significantly decreased viability scores) it would also significantly decrease hsp70 levels and protein contents. It should be stressed that almost all test larvae had hsp70 levels that either hardly decreased further or even increased somewhat after 18 h. We believe that the latter event may reflect the failed (through effective drug action) attempt of the protoscolice to restore cellular homeostasis. It has recently been suggested that hsp70 may protect cells by preventing or resisting apoptosis (Gordon et al. 1998). Our observations on hsp70 expression are supported by findings of others who also reported decreased hsp70 levels in cells entering the apoptotic stage (Tosi et al. 1998, Hamilton et al. 1995, Wei et al. 1995). Taken in conjunction with the relation to decreased viability criteria observed here, these results suggest that the parasite’s regulatory capacity for hsp70 expression is of considerable importance for drug effects. Although hsp60 involvement in apoptosis and related processes needs further study, the observed hsp60 levels after incubation with IV deserve mention in this regard. It was noted that, under such conditions, hsp60 levels in all larvae tend to increase after 18 h to reach levels significantly higher than control levels at 30 h; this was observed despite simultaneous occurrence of decreased total protein. The enhanced expression of hsp60 in association with a diminishing viability may point to its possible involvement in apoptotic processes here. This hypothesis is strengthened further by the finding of progressively decreased protein content with exposure time to IV, as it is known that such a phenomenon is typical of cells entering into apoptosis (Zhivotovsky et al. 1997, Poccia et al. 1996). A similarly altered hsp60 profile in dying organisms has been observed in our laboratory in Trichinella spiralis larvae exposed to lethal thermal stress (unpublished results). The apparent causal relationship between diminishing viability, decreased hsp70 levels, diminished protein content, and the tendency of hsp60 levels to increase strongly suggests that IV acts “rightly” as it causes protoscolices of E. granulosus to undergo apoptosis.

MARTINEZ ET AL.

In all test protoscolices subjected to the IV protocol, we obtained comparable hsp70 levels; however, viability in the prestressed forms was far lower than in the nonprestressed organisms at any time during the experiment. It should be stressed that the thermal stress per se did not affect cell viability, this being 98–100% for all test larvae at the time of study; protein content in the prestressed group, however, was significantly lower than that in controls (i.e., not prestressed, not treated with anthelmintics). Despite the evidence that thermal stress increases the efficacy of IV, we caution against discarding the protective activity of HSPs, as it is conceivable that the IV concentration used could influence the apparent values obtained herein. The 100 mg/ ml IV used in the present experiments may have been too high to permit homeostatic mechanisms to function properly (in already stressed cells), thus leading to cell death. Lowering the stressor’s concentration has, in a system using cytotoxic agents other than those in this study, shown the (re)induction of HSPs of a protective nature (Gordon 1998, Samali and Cotter 1996). Our results demonstrated similar responses in test protoscolices treated with IV 1 ALB or IV alone, although decreases in viability and protein content were less pronounced in the former. That hsp60 levels in IV 1 ALB-treated larvae tended not to increase was yet another point confirming the lesser efficacy of IV 1 ALB compared to IV alone. Two apparent explanations for this phenomenon arise here: (i) IV and ALB have antagonistic effects on test larvae, and/ or (ii) only IV is really effective. Although increased drug efficacy occurred in some instances in which protoscolices were heat-shocked first and then exposed to drug treatment, the absence of such a response in other instances remains to be explained. Several possibilities exist including (the already mentioned) drug concentration and thermally induced apoptosis (Gordon et al. 1998). Follow-up studies in this area should address whether a weak thermal stress would produce parasite HSPs that prepare them to better cope with subsequent exposure to otherwise lethal anthelmintic concentrations. Such studies should also test lower concentrations of anthelmintics and allow for an adequate resting period between thermal stressing of parasites and exposure to anthelmintics as also reported earlier (Samali and Cotter 1996). In conclusion, the effective drug (i.e., IV) exposure data obtained with our E. granulosus test system have indicated that induced loss of viability is related to decreased hsp70, decreased protein content, and increased hsp60 levels. No linear correlation, though, was evident when viability was compared with hsp70 levels or protein contents in test larvae. In the latter regard, it was of note that a temperature factor

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may influence correlation values obtained, as prestressed vs nonprestressed larvae exhibited greater protein loss when exposed to the same anthelmintic regime. Further similar studies are required to allow HSPs to be appropriately used as indicators of drug efficacy in the future. Such studies and subsequent studies may aid (i) to elucidate the role(s) of HSPs in both the promotion and the prevention of cellular death and (ii) to determine possible therapeutic uses.

Jacquier-Sarlin, M. R., Fuller, K., Dinh-Xuan, A. T., Richard, M. J. and Polla, B. S. 1994. Protective effects of hsp70 in inflammation. Experientia 11/12, 1031–1038.

ACKNOWLEDGMENTS

This work was supported by U.A.H. project E035/98 and DGICYT project no. PM96-0014.

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Zhivotovsky, B., Burgess, D. H., Vanags, D. M. and Orrenius, S. 1997. Involvement of cellular proteolytic machinery in apoptosis. Biochemical and Biophysical Research Communications 230, 481–488. Received 5 April 1999; accepted with revision 28 July 1999