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.

Conjugation of Triterpenic Acids of Ursane and Oleanane Types with Mitochondria-Targeting Cation F16 Synergistically Enhanced Their Cytotoxicity against Tumor Cells

“…One of the approaches that can be applied to overcome this problem is the chemical modification of triterpene molecules by their conjugation with delocalized lipophilic cations (DLCs) and, in particular, F16. This DLC has the ability to pass through membranes and selectively accumulate in mitochondria due to the presence of a potential gradient across their inner membrane, which was confirmed in experiments on the visualization of the F16 conjugate and betulinic acid in cells [ 23 ].…”

“…At the same time, BT474 cells were the most resistant to all conjugates. Based on the results of this work and our previous data [ 23 ], we also calculated the Chou–Talalay combination index (CI), which allows evaluation of the synergism of the action of two substances, in particular, in a conjugated form. The CI quantitatively depicts synergism (CI < 1), additive effect (CI = 1), and antagonism (CI > 1) [ 36 , 37 ].…”

“…The CI quantitatively depicts synergism (CI < 1), additive effect (CI = 1), and antagonism (CI > 1) [ 36 , 37 ]. The CI for OA–F16 calculated for MCF7 cells based on the IC 50 values for OA, F16 [ 23 ], and OA–F16 is 0.03, indicating a pronounced synergism. The CI in the case of UA–F16 is 0.05, which also indicates a synergistic effect.…”

“…The hybrid compounds we obtained demonstrated a significant synergistic enhancement of the toxic effect on human leukemia cells (Jurkat, U937, and K562) with acceptable selectivity for the non-malignant human fibroblast cell line (HPDF). A significant toxic effect of chemically binding triterpenic acids with F16 was also confirmed by studying one of the most active conjugates of betulinic acid and F16 with a triethylene glycol spacer on the MCF-7 breast adenocarcinoma cell line [ 23 ]. Confocal fluorescence visualization revealed the high potential of the investigated F16-derived betulinic acid to accumulate in mitochondria.…”

F16 Hybrids Derived from Steviol or Isosteviol Are Accumulated in the Mitochondria of Tumor Cells and Overcome Drug Resistance

“…Currently, hybrids of pentacyclic triterpene carboxylic acids with a distal-linked, substituted N -methylpyridinium radical, so-called F16 derivatives [ 7 , 8 ], can also be prepared, which exhibit low EC 50 values with good selectivity [ 9 , 10 , 11 ]. A decrease in the intensity of ADP phosphorylation was also observed for these derivatives.…”