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, 50 (11), 3770-8

In Vitro Metacestodicidal Activities of Genistein and Other Isoflavones Against Echinococcus Multilocularis and Echinococcus Granulosus

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In Vitro Metacestodicidal Activities of Genistein and Other Isoflavones Against Echinococcus Multilocularis and Echinococcus Granulosus

Arunasalam Naguleswaran et al. Antimicrob Agents Chemother.

Abstract

Echinococcus multilocularis and Echinococcus granulosus metacestode infections in humans cause alveolar echinococcosis and cystic echinococcosis, respectively, in which metacestode development in visceral organs often results in particular organ failure. Further, cystic hydatidosis in farm animals causes severe economic losses. Although benzimidazole derivatives such as mebendazole and albendazole are being used as therapeutic agents, there is often no complete recovery after treatment. Hence, in searching for novel treatment options, we examined the in vitro efficacies of a number of isoflavones against Echinococcus metacestodes and protoscoleces. The most prominent isoflavone, genistein, exhibits significant metacestodicidal activity in vitro. However, genistein binds to the estrogen receptor and can thus induce estrogenic effects, which is a major concern during long-term chemotherapy. We have therefore investigated the activities of a number of synthetic genistein derivatives carrying a modified estrogen receptor binding site. One of these, Rm6423, induced dramatic breakdown of the structural integrity of the metacestode germinal layer of both species within 5 to 7 days of in vitro treatment. Further, examination of the culture medium revealed increased leakage of parasite proteins into the medium during treatment, but zymography demonstrated a decrease in the activity of metalloproteases. Moreover, two of the genistein derivatives, Rm6423 and Rm6426, induced considerable damage in E. granulosus protoscoleces, rendering them nonviable. These findings demonstrate that synthetic isoflavones exhibit distinct in vitro effects on Echinococcus metacestodes and protoscoleces, which could potentially be exploited further for the development of novel chemotherapeutical tools against larval-stage Echinococcus infection.

Figures

FIG. 1.
FIG. 1.
In vitro treatment of Echinococcus metacestodes with genistein induces distinct morphological and structural changes. (A and B) SEM. E. granulosus metacestodes were exposed to the solvent DMSO (A) or 10 μg/ml genistein (B), and the parasite tissue was visualized by SEM. Note the loss of cellular integrity of the germinal layer in panel B. Bars = 280 μm (for both panels A and B). Similar results were obtained for E. multilocularis (data not shown). (C through F) TEM of control and genistein-treated metacestodes. (C) Control tissue, showing a section through the E. granulosus vesicle wall with laminated layer (LL), tegument (Te) with microtriches (Mt), and germinal layer (Gl). (D through F) Treatment with genistein (10 μg/ml) for 7 days results in loss of microtriches, partial separation from tegument and laminated layer (arrows in panel D), formation of lipid droplets, and increased occurrence of small lipid vesicles (ld) in the laminated layer matrix (arrows in panel F). Bars = 2.4 μm (C), 2.8 μm (D), 1.9 μm (E), and 0.5 μm (F). Similar results were obtained for E. multilocularis (data not shown).
FIG. 2.
FIG. 2.
Assays for the detection of drug-induced metacestode damage. OD, optical density. (A) Results of an EmAP assay demonstrating the increased release of alkaline phosphatase activity from E. multilocularis metacestodes during in vitro treatment with nitazoxanide (positive control) and synthetic isoflavonoids Rm6423, Rm6424, Rm6426, and Rm6427. Note the increased efficacy of Rm6423. (B) Dose-response EmAP assay with Rm6423, showing a clear relationship between drug concentration and presence of EmAP activity in medium supernatant of E. multilocularis metacestode cultures. (C) Measurement of release of hydatid fluid compounds from E. granulosus metacestodes following treatment with Rm6423 by immunoblotting of medium supernatants after SDS-PAGE at different time points and labeling with a polyclonal antiserum directed against E. granulosus vesicle fluid. Note the time-dependent increase of signal. For a negative control, vesicles were incubated with equivalent concentrations of DMSO. Numbers in the center indicate the positions of molecular weight markers.
FIG. 3.
FIG. 3.
Gelatin zymography reveals differential protease expression patterns in culture supernatants of Rm6423-, TIZ-, and DMSO-treated E. granulosus metacestodes. (A) Both TIZ- and Rm6423-treated fractions exhibit profound differences compared to the DMSO-treated fraction. The protease bands marked by arrowheads are completely absent in Rm6423-treated medium supernatants. (B) Note the complete inhibition of all protease in the presence of phenanthroline, indicating that these are all metalloproteases. (C) No inhibition of protease activity in the presence of PMSF. Numbers to the right indicate the positions of molecular weight markers.
FIG. 4.
FIG. 4.
In vitro treatment of Echinococcus metacestodes with Rm6423. SEM (A and B) and TEM (C through E) showing the alterations induced by Rm6423 on E. multilocularis metacestodes. Similar findings were obtained with E. granulosus. Shown in panels A and C are control-treated parasites exhibiting intact parasite tissue. In panels B, D, and E, the damage induced by Rm6423 is shown. LL, laminated layer; Te, tegument; Gl, germinal layer. Arrows point to microtriches. Bars = 280 μm (A), 280 μm (B), 1.9 μm (C), 2 μm (D), and 2 μm (E).
FIG. 5.
FIG. 5.
Effects of synthetic isoflavones on E. granulosus protoscoleces. Protoscoleces of E. granulosus were exposed to Rm6423, Rm6426, NTZ, and TIZ for 7 days, and the viability of the parasites was assessed by trypan blue staining and light microscopic inspection. All drugs were added at 10 μg/ml (A) or 5 μg/ml (B). The percentages of still-viable protoscoleces are indicated at different time points. As shown in panel C, the effects of the different drug treatments (10 μg/ml) on the morphology and structural integrity of protoscoleces were visualized at day 3 of treatment.

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