Terpenes increase the lipid dynamics in the Leishmania plasma membrane at concentrations similar to their IC50 values

Abstract

Although many terpenes have shown antitumor, antibacterial, antifungal, and antiparasitic activity, the mechanism of action is not well established. Electron paramagnetic resonance (EPR) spectroscopy of the spin-labeled 5-doxyl stearic acid revealed remarkable fluidity increases in the plasma membrane of terpene-treated Leishmania amazonensis promastigotes. For an antiproliferative activity assay using 5×10(6) parasites/mL, the sesquiterpene nerolidol and the monoterpenes (+)-limonene, α-terpineol and 1,8-cineole inhibited the growth of the parasites with IC50 values of 0.008, 0.549, 0.678 and 4.697 mM, respectively. The IC50 values of these terpenes increased as the parasite concentration used in the cytotoxicity assay increased, and this behavior was examined using a theoretical treatment of the experimental data. Cytotoxicity tests with the same parasite concentration as in the EPR experiments revealed a correlation between the IC50 values of the terpenes and the concentrations at which they altered the membrane fluidity. In addition, the terpenes induced small amounts of cell lysis (4-9%) at their respective IC50 values. For assays with high cell concentrations (2×10(9) parasites/mL), the incorporation of terpene into the cell membrane was very fast, and the IC50 values observed for 24 h and 5 min-incubation periods were not significantly different. Taken together, these results suggest that terpene cytotoxicity is associated with the attack on the plasma membrane of the parasite. The in vitro cytotoxicity of nerolidol was similar to that of miltefosine, and nerolidol has high hydrophobicity; thus, nerolidol might be used in drug delivery systems, such as lipid nanoparticles to treat leishmaniasis.

Figure 1. Chemical structures.

The four terpenes and the spin labels 5-DSA and 6-MSL used in this study.

Figure 2. EPR spectra of spin-labeled Leishmania plasma membrane.

Experimental (black lines) and best-fit (red lines) EPR spectra of 5-DSA in the plasma membranes of Leishmania amazonensis promastigotes from untreated and terpene-treated samples (4.8×10⁹ terpenes/cell). The best-fit spectra were obtained with the NLLS fitting, which provided the average rotational correlation time, τ_c, for each spectrum. Another parameter that is measured directly in the EPR spectrum, 2A_//, is indicated by the vertical lines in the figure. The mean and S.D. of τ_c and 2A_// are indicated. For both parameters, all terpene-treated samples were significantly different from the control sample (P<0.01).

Figure 3. Theoretical and experimental IC₅₀ dependencies on the cell concentration.

(A) Theoretical curves calculated from eq. 4 (see text for details) for the molar concentration ratio c_cal/c_w in comparison with the IC₅₀ values shown in Table 1 for different cell concentrations of nerolidol (square) and (+)-limonene (circle) as a function of V_w/V_m. (B) Theoretical curves calculated from eq. 8 for the ratio N/N_m compared with the IC₅₀ of nerolidol and (+)-limonene versus V_w/V_m.

Figure 4. EPR spectra of spin-labeled Leishmania plasma membrane.

Experimental (black lines) and best-fit (red lines) EPR spectra of 5-DSA in the plasma membranes of Leishmania amazonensis promastigotes for samples treated with terpenes at several concentrations around their IC₅₀ (two concentrations below and three above the IC₅₀). The EPR spectra are representative of three independent experiments and the means and S.D. of the rotational correlation time (τ_c) obtained from the NLLS fitting are indicated. The spectra of the control samples (without treatment) were similar to those already shown in Fig. 2 (τ_c = 8.1±0.2 ns).

Figure 5. Effect of terpenes on the dynamics of the Leishmania plasma membrane.

EPR parameters 2A_// (panel A) and rotational correlation time, τ_c, (panel B) of the spin label 5-DSA in the membranes of Leishmania amazonensis promastigotes as a function of the terpene concentration used in the treatment. The IC₅₀ values of the terpenes are represented by single points at the bottom of each curve and dotted lines. The estimated experimental errors for 2A_// and τ_c are 0.5 G and 0.2 ns, respectively.

Figure 6. EPR spectra of spin-labeled membrane proteins of Leishmania for untreated and terpene-treated samples.

The 2A_//-parameter values are indicated.

Figure 7. Percentage of cell lysis in samples of Leishmania amazonensis promastigotes treated with terpenes.

The IC₅₀ of each terpene is represented by its corresponding symbol and a vertical line.