Effect of the antitubulin drug oryzalin on the encystation of Entamoeba invadens

Abstract

We have recently demonstrated that the antimicrotubule drug oryzalin inhibits the growth of Entamoeba invadens as well as E. histolytica, the former being more resistant to the drug than the latter, and that effective doses of oryzalin are higher for Entamoeba than for the other parasitic protozoa examined thus far. The aim of the present study was to examine the effect of oryzalin on the encystation of E. invadens using an axenic encystation system in vitro. Oryzalin inhibited the encystation of E. invadens strain IP-1 in a dose-dependent manner. The addition of oryzalin after the induction of encystation was also inhibitory for encystation and cyst maturation. Trophozoites incubated for 1 day in encystation medium with oryzalin did not encyst after removal of the drug. Although trophozoites grown in the presence of 300 microM oryzalin for 2 days did not encyst after their transfer to encystation medium containing the same concentration of drug, a number of trophozoites survived for at least 3 days. In contrast, trophozoites grown in the absence of oryzalin neither survived nor encysted after their transfer to encystation medium supplemented with the drug, which suggests that pretreatment of trophozoites with oryzalin contributes to their continued survival as trophozoites, i.e., without their transforming into cysts, in encystation medium. Trophozoites grown with oryzalin did encyst after their transfer to encystation medium without the drug. Accumulation of trophozoites in the mitotic phase was observed after culture with oryzalin. When cysts prepared at day 1 of encystation, most of which were mononucleate, were reincubated in the presence of oryzalin for an additional 2 days, inhibition of their maturation was observed. Thus, oryzalin is a potent mitotic-phase inhibitor of E. invadens and may become a useful tool for studies on the relationship between the cell cycle and encystation of this parasite.

Inhibition of encystation of Entamoeba invadens by wortmannin

“…In contrast, trophozoites exposed to the drug in growth medium could encyst in encystation medium without the drug, indicating that the wortmannin eect is reversible in growth medium. Similar results were obtained for several agents that we used previously, including oryzalin (Makioka et al 2000b), chelerythrine chloride (Makioka et al 2000c), and jasplakinolide (Makioka et al, 2001). The reason for the irreversible eects of these agents in encystation medium remains unresolved.…”

Effect of calcium antagonists, calcium channel blockers and calmodulin inhibitors on the growth and encystation of Entamoeba histolytica and E. invadens

“…The results presented here indicated that CaM inhibitors, W-7 and TFP, had an inhibitory eect on the growth of both Entamoeba species and the encystation of E. invadens was more inhibited, suggesting that the encystation process is also CaM-dependent. In this regard, CaM is known to regulate the assembly and disassembly of microtubules (Marcum et al 1978) and we have recently demonstrated that the antitubulin drug oryzalin inhibits the encystation of E. invadens (Makioka et al 2000b). Therefore CaM would contribute to the encystation of E. invadens through the regulation of microtubule assembly.…”

Heterogeneity in the sensitivity of microtubules of Giardia lamblia to the herbicide oryzalin

“…For some, as yet unclear reason, the sub-pellicular microtubules are more sensitive to oryzalin than other cytoplasmic microtubules. However, trophozoites of Entamoeba histolytica seem to be more resistant, since the accumulation of mitotic cells was observed with concentrations of oryzalin as high as 100 µM (Makioka et al 2000). In these parasites, oryzalin bound to a site found in α-tubulin.…”

“…(Chan et al 1991), Entamoeba spp. (Makioka et al 2000), Cryptosporidium parvum (Benbow et al 1998) and Toxoplasma gondii (Stokkermans et al 1996). Insights into the mode of action of oryzalin were recently obtained by the generation of α-tubulin mutants of T. gondii.…”