Antiproliferative and Cytotoxic Properties of Propynoyl Betulin Derivatives against Human Ovarian Cancer Cells: In Vitro Studies

Abstract

Due to the incidence of ovarian cancer (OC) and the limitations of available therapeutic strategies, it is necessary to search for novel therapeutic solutions. The aim of this study was to evaluate the cytotoxic effect of betulin 1 and its propynoyl derivatives 2-6 against ovarian cancer cells (SK-OV-3, OVCAR-3) and normal myofibroblasts (18Co). Paclitaxel was used as the reference compound. The propynoyl derivatives 2-6 exhibited stronger antiproliferative and cytotoxic activities compared to betulin 1. In both ovarian cancer cell lines, the most potent compound was 28-propynoylbetulin 2. In the case of compound 2, the calculated IC₅₀ values were 0.2 µM for the SK-OV-3 cells and 0.19 µM for the OVCAR-3 cells. Under the same culture conditions, the calculated IC₅₀ values for compound 6 were 0.26 µM and 0.59 µM, respectively. It was observed that cells treated with compounds 2 and 6 caused a decrease in the potential of the mitochondrial membrane and a significant change in cell morphology. Betulin 1, a diol from the group of pentacyclic triterpenes, has a confirmed wide spectrum of biological effects, including a significant anticancer effect. It is characterized by low bioavailability, which can be improved by introducing changes to its structure. The results showed that chemical modifications of betulin 1 only at position C-28 with the propynoyl group (compound 2) and additionally at position C-3 with the phosphate group (compound 3) or at C-29 with the phosphonate group (compound 6) allowed us to obtain compounds with greater cytotoxic activity than their parent compounds, which could be used to develop novel therapeutic systems effective in the treatment of ovarian cancer.

Keywords: betulin derivatives; cytotoxicity; ovarian cancer; regulated cell death.

Figure 1

Chemical structure of betulin 1 and its propynoyl derivatives 2–6.

Figure 2

SK-OV-3 cell proliferation curves after exposition to betulin 1 and compounds 2–6 assessed using SRB assay. The series of curves represent SK-OV-3 cells’ proliferation measured in given time points: 1 day, 3 days, and 5 days of treatment with individual compounds at concentration range 0.1–30 µM. The plots are presented as the mean ± standard deviation.

Figure 3

OVCAR-3 cell proliferation curves after exposition to betulin 1 and compounds 2–6 assessed using SRB assay. The series of curves represent OVCAR-3 cells’ proliferation measured in given time points: 1 day, 3 days, and 5 days of treatment with individual compounds at concentration range 0.1–30 µM. The plots are presented as the mean ± standard deviation.

Figure 4

Cytotoxicity expressed as percentage of released LDH in (a) SK-OV-3 and (b) OVCAR-3 cells after 24 h incubation with betulin 1 and compounds 2–6. The results are presented as the means ± standard deviation. * p < 0.05 vs. control.

Figure 5

Determination of DNA fragmentation in (a) SK-OV-3 and (b) OVCAR-3 cells after 24 h incubation with betulin 1 and compounds 2–6. The results are expressed as fold change over control and presented as the means ± standard deviation; * p < 0.05 vs. control.

Figure 6

Effect of 24 h treatment with 30 µM betulins on morphology of SK-OV-3 cells. Cells were stained with acridine orange: (a) control cells; (b) betulin 1; (c) compound 2; (d) compound 3; (e) compound 5; (f) compound 6. White arrows indicate the apoptotic cells; arrowheads indicate pyknotic cell nuclei (magnification 100×). Scale bars equal 100 μm.

Figure 7

Effect of 3 h treatment with 30 µM betulins on the mitochondrial membrane potential in SK-OV-3 cells. Cells were stained with TMRM: (a) control cells; (b) betulin 1; (c) compound 2; (d) compound 3; (e) compound 5; (f) compound 6 (magnification 100×). Scale bars equal 100 μm.

Figure 8

Evaluation of (a) the DNA fragmentation and (b) the plasma membrane permeability in SK-OV-3 cells incubated with compound 6 for 24 h alone or with CsA. The results are expressed as fold change over control and presented as the means ± standard deviation; * p < 0.05 vs. control. Lines show significant differences between untreated and CsA-treated cells.