Genistein is the most abundant isoflavone and is implicated in prevention of numerous types of cancer and cardiovascular disease. Genistein and a number of other isoflavones have been shown to mediate antiplasmodial and anticoccidial activities (5
). The compound exhibits micromolar potency in inhibiting protein tyrosine kinases, which probably accounts for most of its effects (2
Our results show that in vitro treatment with genistein induces a number of significant alterations in E. multilocularis
and E. granulosus
metacestodes that could eventually impair parasite viability and lead to parasite death (Fig. ). Genistein, like other isoflavones, exhibits the basic structure of estrogen and, thus, can exert estrogenic effects through binding to the estrogen receptor. This represents a serious health concern in terms of the use of isoflavones as therapeutic agents, especially for long-term treatments, such as for treatment of echinococcosis (4
). Crystal structure analysis of ligand-receptor complexes involving estrogen receptor-β and genistein showed that the phenolic C-ring interacts with estrogen receptor-β (27
). Thus, it was demonstrated that the C-ring and, more precisely, the respective 4′-OH group, are responsible for the estrogenic effects.
In this study, we used synthetic isoflavones in which the crucial 4′-OH group on the C-ring was removed, and other functional steric groups were added at different positions. Of the drugs tested (see reference 10
), only Rm6423 exhibited a profound antiparasitic effect towards metacestodes of both E. multilocularis
and E. granulosus
, leading to leakage of parasite proteins into the medium supernatants (Fig. ). Rm6423 is almost identical to genistein but lacks the OH group on the 4′ position of the C-ring, and a bromo-group is added at position 2′. Interestingly, if the bromo-group is added to position 3′ of the C-ring, as in Rm6424 (10
), the efficacy of the compound is completely lost. As evidenced by SEM and TEM, the damage induced by Rm6423 (Fig. ) is comparable to what had been previously observed for NTZ and TIZ, with no retraction of microtriches but efficient and complete disintegration and necrosis of the germinal layer (37
), but this was clearly different from the changes identified in genistein-treated metacestodes (Fig. ). However, a similar enrichment of small vesicles within the matrix of the laminated layer has also been previously observed in NTZ-treated E. multilocularis
), and retraction of microtriches had been previously observed in E. multilocularis
metacestodes treated with albendazole sulfoxide or albendazole sulfone (17
All these features probably reflect the different mechanisms of action of these drugs. Benzimidazoles, such as albendazole, have been shown to bind to tubulin and inhibit its polymerization into microtubules (21
). In contrast, the mode of action of thiazolides, such as NTZ and TIZ, in helminths has not been elucidated so far. Possibly, the drugs interfere in the functional activity of enzymes that are similar to pyruvate ferredoxin oxidoreductase in anaerobic bacteria, but other mechanisms of action are currently being discussed (9
Gelatin zymography clearly demonstrated that the activities of some metalloprotease bands were impaired in medium supernatants of Rm6423-treated E. granulosus
metacestodes, despite the fact that the overall protein concentration of hydatid fluid components was increased by drug treatment. This suggests that Rm6423 has a negative influence on metalloprotease expression in Echinococcus
metacestodes. In mammalian cells, metalloproteinase expression is regulated through selective activation or inhibition of a number of signaling systems, including the EGF receptor-regulated p38 MAP kinase in cancer cells (16
). Whether any member of the recently discovered Echinococcus
EGF signaling pathway and MAP kinase cascade (reviewed in reference 3
) is affected by Rm6423 needs to be investigated in future studies.
Treatment of freshly isolated E. granulosus protoscoleces with the small panel of isoflavones used in this study resulted in the identification of Rm6423 and Rm6426 as two compounds with limited protoscolicidal activity. This effect was dose dependent and was not evident anymore at 1 μg/ml. Neither isoflavone was as efficient as NTZ or TIZ. In addition, the isoflavones had basically lost their efficacies after 4 days, in contrast to NTZ and TIZ, which continued to exert their antiparasitic activities until day 7, when all protoscoleces were nonviable (Fig. ). However, when the media containing Rm6423 and Rm6426 were replaced with fresh drug-containing media after 7 days, all protoscoleces lost viability within the next 24 h. It is therefore possible that, in contrast to the thiazolides, during the first 3 to 4 days, isoflavones are metabolized or converted into inactive compounds and lose antiparasitic efficacy.
Taken together, our results show that genistein and the genistein derivative Rm6423 exhibit profound activities against Echinococcus metacestodes. Rm6423 is an extremely interesting compound, as it lacks a functional estrogen receptor binding domain and the expected toxicity of the drug is low. Therefore, animal experimentation will be required to provide the proof of the concept that Rm6423 could be useful for in vivo treatment of Echinococcus infection.