Comparative Study of Cytotoxic and Membranotropic Properties of Betulinic Acid-F16 Conjugate on Breast Adenocarcinoma Cells (MCF-7) and Primary Human Fibroblasts

Abstract

The present study evaluates the cytotoxicity of a previously synthesized conjugate of betulinic acid (BA) with the penetrating cation F16 on breast adenocarcinoma (MCF-7) and human fibroblast (HF) cell lines, and also shows the mechanism underlying its membranotropic action. It was confirmed that the conjugate exhibits higher cytotoxicity compared to native BA at low doses also blocking the proliferation of both cell lines and causing cell cycle arrest in the G₀/G₁ phase. We show that the conjugate indeed has a high potential for accumulation in mitochondria, being visualized in these organelles, which is most pronounced in cancer cells. The effect of the conjugate was observed to be accompanied by ROS hyperproduction in both cancerous and healthy cells, despite the lower base level of ROS in the latter. Along with this, using artificial liposomes, we determined that the conjugate is able to influence the phase state of lipid membranes, make them more fluid, and induce nonspecific permeabilization contributing to the overall cytotoxicity of the tested agent. We conclude that the studied BA-F16 conjugate does not have significant selective cytotoxicity, at least against the studied breast cancer cell line MCF-7.

Keywords: BA; F16; breast cancer; cytotoxicity; delocalized lipophilic cation (DLC); oxidative stress.

Figure 1

Structure of BA (BA), F16 (E-4-(1H-indol-3-ylvinyl)-N-methylpyridinium iodide]), and BA–F16 conjugate [17].

Figure 2

Dose dependence of the effect of compounds BA, F16, and BA–F16 on the survival of MCF-7 (A) and HF (B) cells. Cytotoxicity was assessed 48 h after the addition of substances by crystal violet indicator. Data (mean ± SEM) of 4 independent experiments are shown.

Figure 3

MCF-7 (A) and HF (B) cell cycle analysis after 24 h incubation with BA (5 µM) and BA–F16 (1 µM). Data are mean ± SD of three independent experiments. (* indicates p < 0.05 when compared with control).

Figure 4

Evaluation of the mitochondrial localization of compound BA–F16 in MCF-7 breast adenocarcinoma cells (A–C) and human fibroblasts (D–F) by confocal microscopy. (A,D) MitoTracker Red fluorescence intensity image (200 nM, (λex/λem) = 638/650 nm), (B,E) F16 fluorescence intensity image (200 nM, (λex/λem) = 450/550 nm), (C,F) image obtained by overlaying images “A” and “B”, “D” and “E”. Scale bars: 10 µm.

Figure 5

Effect of 5 μM of BA and 1 μM of BA–F16 on the production of reactive oxygen species by MCF-7 and HF cells. (A–F) Typical distribution diagrams of MCF-7 (A–C) and HF (D–F) cell populations in the presence of BA (B,E) and BA–F16 (C,F). (G) Calculation of ROS-positive cells (%) in experimental groups. Means ± SD are shown (n = 4–5).

Figure 6

BA–F16 (1 μM) but not BA (50 μM) induces the release of the sulforodamine B dye from unilamellar lecithin liposomes. The incubation medium contained: 10 mM Tris-HCl (pH 7.5), 40 mM KCl, and 50 μM EGTA. The results are presented as means ± SEM (n = 5).

Figure 7

Effect of 10 µM of BA (A) and 2 µM of BA–F16 (B) on the phase state of DPPC liposomes. The transition from a solid- (G—gel state) to a liquid-crystalline state (F—fluid state) is defined as the point of maximum curvature. The incubation medium contained: 10 mM Tris-HCl (pH 7.5), 40 mM KCl, and 50 μM EGTA. Temperature measurements range from 25 to 54 °C. CTR—without additions. The results are presented as means ± SEM (n = 3). ** indicates p < 0.01 when compared with control curve.