Indeed, these inhibitors have been shown to be antiproliferative agents against yeast, fungi and protists (Urbina et al., 1997; Rodrigues et al., 2002; Visbal et al., 2003; Song & Nes, 2007). One attractive feature of
these inhibitors for the treatment of a T. vaginalis infection is the Metformin absence of the inhibited enzyme in the sterol pathway of mammalian cells. The compounds 22,26 azasterol [20-piperidin-2-yl-5-pregnan-3β-20(R)-diol] (AZA) (Fig. 1a) and 24(R,S),25-epiminolanosterol (EIL) (Fig. 1b) are steroid compounds with a secondary amine in their side chain that have a potent inhibitory activity against 24-SMT, acting as analogues of the high-energy intermediates in the reaction catalysed by this enzyme (Song & Nes, 2007). In this work, we investigated the activity of AZA and EIL against T. vaginalis in vitro as an approach to the development of novel chemotherapeutic agents against this parasite. The JT strain of T. vaginalis was isolated at the Hospital
Universitário, Universidade Federal do Rio de Janeiro, Brazil, and has been maintained in culture for several years. Trophozoites were cultivated in TYM Diamond’s medium (Diamond, 1957) supplemented with 10% fetal calf serum (FCS). The cells were grown for 24 h at 36.5 °C. Madin–Darby canine kidney (MDCK) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco Invitrogen Corporation, NY) (Dulbecco & Freeman, 1959) supplemented with 10% heat-inactivated FCS and 50 μg mL−1 gentamicin at 37 °C in a 5% CO2/air Crizotinib in vitro mixture. The growth experiments with T. vaginalis trophozoites were initiated with 2 × 104 cells mL−1.
Appropriate volumes of the inhibitors of 24-SMT solutions from stocks prepared PTK6 in dimethyl-sulphoxide (DMSO) were added to the cultures at the desired final concentrations. The final concentration of DMSO in the growth medium never exceeded 1% (v/v) and had no effect on cell growth or morphology. The cell densities were determined in a haemocytometer with a light microscope. The experimental SMT inhibitors used for this study were AZA and EIL (Fig. 1) (Urbina, 1997; Rodrigues et al., 2002). AZA and EIL (Fig. 1) were synthesized and purified as described previously (Urbina et al., 1995; Atencio et al., 2001). Cells were adhered onto poly-l-lysine-coated glass coverslips and subsequently fixed in 2.5% glutaraldehyde in a 0.1 M cacodylate buffer, pH 7.2. Next, the cells were postfixed for 15 min in 1% OsO4, dehydrated in ethanol, and critical point dried with liquid CO2. The cells were then coated with a 15-nm-thick layer of gold–palladium and observed under a JEOL 5800 scanning electron microscope. The control and treated parasite cells were fixed for 24 h in 2.5% glutaraldehyde in a 0.1 M cacodylate buffer, pH 7.2. After fixation, the cells were postfixed for 40 min in a solution containing 1% OsO4 and 0.8% potassium ferrocyanide in a 0.1 M cacodylate buffer, washed in phosphate-buffered saline, dehydrated in acetone and embedded in Epon.
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