Enhanced Cytotoxicity and Antimelanoma Activity of Novel Semisynthetic Derivatives of Betulinic Acid with Indole Conjugation

Abstract

The prevalence and severity of skin cancer, specifically malignant melanoma, among Caucasians remains a significant concern. Natural compounds from plants have long been explored as potential anticancer agents. Betulinic acid (BI) has shown promise in its therapeutic properties, including its anticancer effects. However, its limited bioavailability has hindered its medicinal applications. To address this issue, two recently synthesized semisynthetic derivatives, N-(2,3-indolo-betulinoyl)diglycylglycine (BA1) and N-(2,3-indolo-betulinoyl)glycylglycine (BA2), were compared with previously reported compounds N-(2,3-indolo-betulinoyl)glycine (BA3), 2,3-indolo-betulinic acid (BA4), and BI. These compounds were evaluated for their effects on murine melanoma cells (B164A5) using various in vitro assays. The introduction of an indole framework at the C2 position of BI resulted in an increased cytotoxicity. Furthermore, the cytotoxicity of compound BA4 was enhanced by conjugating its carboxylic group with an amino acid residue. BA2 and BA3, with glycine and glycylglycine residues at C28, exhibited approximately 2.20-fold higher inhibitory activity compared to BA4. The safety assessment of the compounds on human keratinocytes (HaCaT) has revealed that concentrations up to 10 µM slightly reduced cell viability, while concentrations of 75 µM resulted in lower cell viability rates. LDH leakage assays confirmed cell membrane damage in B164A5 cells when exposed to the tested compounds. BA2 and BA3 exhibited the highest LDH release, indicating their strong cytotoxicity. The NR assay revealed dose-dependent lysosome disruption for BI and 2,3-indolo-betulinic acid derivatives, with BA1, BA2, and BA3 showing the most cytotoxic effects. Scratch assays demonstrated concentration-dependent inhibition of cell migration, with BA2 and BA3 being the most effective. Hoechst 3342 staining revealed that BA2 induced apoptosis, while BA3 induced necrosis at lower concentrations, confirming their anti-melanoma properties. In conclusion, the semisynthetic derivatives of BI, particularly BA2 and BA3, show promise as potential candidates for further research in developing effective anti-cancer therapies.

Keywords: 2,3-indolo-betulinic acid; B164A5 murine melanoma cells; glycine conjugates; melanoma.

Figure 1

Betulinic acid derivatives used in this study; for synthesis of BA1 and BA2, see [48]; for synthesis of BA3 and BA4, see [39]. Figure adapted from [48].

Figure 2

The cytotoxicity of compounds BI, BA1, BA2, BA3, and BA4 on the B164A5 cell line was assessed by conducting the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assessment after a 72-h treatment period. at varied concentrations (1, 10, 25, 50, and 75 µM) of the screened compounds. The groups were subjected to a one-way analysis of variance (ANOVA) and then analyzed using Dunnett’s post-test for multiple comparisons. A p-value below 0.05 was deemed to be statistically significant (**** p ≤ 0.0001 compared to the control group).

Figure 3

The cytotoxicity of compounds BI, BA1, BA2, BA3, and BA4 on the HaCaT cell line was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay after a 72 h treatment period. The compounds were tested at different concentrations (1, 10, 25, 50, and 75 µM). The groups underwent a one-way analysis of variance (ANOVA) and were then examined using Dunnett’s post-test for multiple comparisons. A p-value less than 0.05 was considered to have statistical significance (*** p ≤ 0.001; **** p ≤ 0.0001; ns (non-significant) compared to the control group).

Figure 4

The LDH release ratio of B164A5 cells after exposure to varying doses of BA1–BA4 and BI (1, 10, 25, 50, and 75 µM) over a duration of 72 h. The statistical distinctions were assessed by the use of one-way (ANOVA) complemented by Dunnett’s multiple comparisons test (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns (non-significant) against control cells).

Figure 5

Cytotoxic extent of murine melanoma cells (B164A5) when exposed to varying doses of BA1–BA4 and BI over a period of 72 h. The data are expressed in terms of mean ± SD. The statistically significant variations were determined using one-way ANOVA analysis, subsequently followed by Dunnett’s comparisons test (** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns (non-significant) against control cells).

Figure 6

The provided images illustrate the migratory capacity of B164A5 cells after a 24 h treatment with BA1–BA4 and BI. The presented bar graphs illustrate the proportional representation of wound closure in relation to the initial surface area after a 24 h period. The findings are presented as the average values ± SD of three distinct experiments conducted in triplicate. The study used the one-way ANOVA followed by Dunnett’s multiple comparisons post-test to assess the presence of significant differences between the control group and the treatment groups. Statistical significance was denoted by *** (p ≤ 0.001), and **** (p ≤ 0.0001) when compared to the control group.

Figure 7

Staining of cell nuclei of murine melanoma B164A5 cells using Hoechst 33342 reagent after 72 h exposure to three different concentrations (25, 50, 75 μM) of BA1-BA4 and BI. Staurosporine at concentration of 5 μM was used as positive control for apoptosis features and Triton X 100 at concentration of 0.5% was employed to observe the necrosis process. The apoptosis-related changes are marked by yellow arrows, whereas necrosis is highlighted by a red circle. The scale bar represents 50 µm.