Acetylenic Synthetic Betulin Derivatives Inhibit Akt and Erk Kinases Activity, Trigger Apoptosis and Suppress Proliferation of Neuroblastoma and Rhabdomyosarcoma Cell Lines

Abstract

Neuroblastoma (NB) and rhabdomyosarcoma (RMS), the most common pediatric extracranial solid tumors, still represent an important clinical challenge since no effective treatment is available for metastatic and recurrent disease. Hence, there is an urgent need for the development of new chemotherapeutics to improve the outcome of patients. Betulin (Bet), a triterpenoid from the bark of birches, demonstrated interesting anti-cancer potential. The modification of natural phytochemicals with evidenced anti-tumor activity, including Bet, is one of the methods of receiving new compounds for potential implementation in oncological treatment. Here, we showed that two acetylenic synthetic Bet derivatives (ASBDs), EB5 and EB25/1, reduced the viability and proliferation of SK-N-AS and TE671 cells, as measured by MTT and BrdU tests, respectively. Moreover, ASBDs were also more cytotoxic than temozolomide (TMZ) and cisplatin (cis-diaminedichloroplatinum [II], CDDP) in vitro, and the combination of EB5 with CDDP enhanced anti-cancer effects. We also showed the slowdown of cell cycle progression at S/G₂ phases mediated by EB5 using FACS flow cytometry. The decreased viability and proliferation of pediatric cancers cells after treatment with ASBDs was linked to the reduced activity of kinases Akt, Erk1/2 and p38 and the induction of apoptosis, as investigated using Western blotting and FACS. In addition, in silico analyses of the ADMET profile found EB5 to be a promising anti-cancer drug candidate that would benefit from further investigation.

Keywords: ADMET; acetylenic synthetic betulin derivatives; betulin; chemotherapy; cisplatin; druglikeness; neuroblastoma; pediatric cancers; rhabdomyosarcoma; temozolomide.

Figure 1

Chemical structure of betulin (Bet) and its acetylenic synthetic derivatives (ASBDs). (A) Structure of Bet, (B) 28-O-propynoylbetulin (EB5) and (C) 28-O-propargyloxycarbonylbetulin (EB25/1).

Figure 2

ASBDs reduce viability and proliferation of pediatric cancer cells stronger than TMZ and CDDP in vitro, whereas they show moderate activity against normal cells. SK-N-AS, TE671 and HSF cell lines were treated with ASBDs for 96 h and then analyzed with MTT metabolism assay for cell viability/survival, or for 48 h with BrdU incorporation test for cell proliferation. (A) The line graph presents cell viability of SK-N-AS, TE671 and HSF cells after treatment with EB5 or EB25/1. (B) The line graph presents cell proliferation of SK-N-AS, TE671 and HSF cells after treatment with EB5 or EB25/1. (C) The line graph presents cell viability of SK-N-AS and TE671 cells after treatment with TMZ for 96 h and analyzed with MTT metabolism assay. (D) The line graph presents cell viability of SK-N-AS and TE671 cells after treatment with CDDP for 96 h and analyzed with MTT metabolism assay. The results were normalized to control cells (CTRL, treated with 0.1% dimethyl sulfoxide DMSO, as a solvent control) and represent the mean ± SEM of n = 32 from 4 independent experiments (MTT assay) or n = 24 from 3 independent experiments (BrdU test). Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test for multiple comparisons. p values were considered significant when p ≤ 0.05(*), p ≤ 0.01(**), p ≤ 0.001(***), p ≤ 0.0001(****).

Figure 3

Combination of ASBDs with CDDP enhances cytotoxicity of both CDDP and EB5 administered singly. SK-N-AS and TE671 cell lines were treated with CDDP and ASBDs (the doses of IC₅₀ values), and ASBDs in combination with CDDP (the doses of IC₅₀ value) for 96 h and then analyzed with MTT metabolism assay for cell viability/survival. (A) The bar graph presents cell viability of SK-N-AS cells after treatment with CDDP, EB5, EB25/1 and combination of CDDP with EB5 or EB25/1. (B) The bar graph presents cell viability of TE671 cells after treatment with CDDP, EB5, EB25/1 and combination of CDDP with EB5 or EB25/1. The results were normalized to control cells (CTRL, 0.1% DMSO as a solvent control) and represent the mean ± SEM n = 32 from 4 independent experiments. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons. p values were considered significant when p ≤ 0.0001 (****, vs. CTRL) and p ≤ 0.05 (#), p ≤ 0.01 (##), p ≤ 0.0001 (####).

Figure 4

ASBDs inhibit cell cycle progression of pediatric cancer cells by affecting S phase. SK-N-AS and TE671 cell lines were treated with selected concentrations of ASBDs for 24 h, stained with PI, and then analyzed with FACS flow cytometry for cell cycle distribution. (A) The bar graphs present percentage of cells in phase G₁, S and G₂ in SK-N-AS cells after treatment with EB5 (upper graph) and EB25/1 (bottom graph). (B) The bar graphs present percentage of cells in phase G₁, S and G₂ in TE671 cells after treatment with EB5 (upper graph) and EB25/1 (bottom graph). Control cells (CTRL) were treated with 0.1% DMSO as a solvent control. Nocodazole (NOC, 5 µg/mL) was used as a reference drug for induction of G₂/M cell cycle arrest. The results represent the mean ± SEM of n = 10 from 5 independent experiments. Statistical significance was determined by two-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test for multiple comparisons. p values were considered significant when p ≤ 0.05 (*, vs. CTRL), p ≤ 0.01 (**, vs. CTRL), p ≤ 0.001 (***, vs. CTRL).

Figure 5

ASBDs inhibit phosphorylation of kinases crucial for growth and proliferation of cancer cells. SK-N-AS and TE671 cell lines were treated with selected concentrations of ASBDs for 3 h and then analyzed with Western blotting for kinases activation. (A) Representative immunoblots show the level of phosphorylated Akt and MAP (Erk1/2 and p38) kinases in SK-N-AS cell line after treatment with ASBDs. (B) Representative immunoblots show the level of phosphorylated Akt and MAP (Erk1/2 and p38) kinases in TE671 cell line after treatment with ASBDs. Total: Akt1, Erk2, p38 and β-actin were used as the loading controls for an equal amount of protein. Control cells (CTRL) were treated with 0.1% DMSO as a solvent control.

Figure 6

EB5 induces apoptosis of pediatric cancer cells in vitro in a concentration- and time-dependent manner. SK-N-AS and TE671 cell lines were treated with selected concentrations of ASBDs (1–20 µM of EB5, and 5–20 µM of EB25/1) for 24 or 48 h and then analyzed with FACS flow cytometry and Western blotting for induction of apoptotic cell death. (A) Representative dot plots show gating of cell subpopulation with activated caspase 3 in SK-N-AS cells. (B) The bar graphs present quantification of caspase 3-positive cells percentage in SK-N-AS cell line after treatment with EB5 (for 24 h and 48 h, upper graph) and EB25/1 (for 24 h, bottom graph). The results represent the mean ± SEM of n = 6 from 3 independent experiments. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test for multiple comparisons (bottom graph) or two-way ANOVA followed by Sidak’s post hoc test for multiple comparisons (upper graph 24 h vs. 48 h). p values were considered significant when p ≤ 0.0001 (****, vs. CTRL), and p ≤ 0.01 (##), p ≤ 0.0001 (####). (C) Representative immunoblots show the level of cell death markers (cleaved PARP1 and cleaved caspase 3) in SK-N-AS cell line. (D) Representative dot plots show the gating of cell subpopulation with activated caspase 3 in TE671 cells. (E) The bar graphs present the quantification of caspase 3-positive cell percentage in TE671 cell line after treatment with EB5 (for 24 h and 48 h, upper graph) and EB25/1 (for 24 h, bottom graph). The results represent the mean ± SEM of n = 6 from 3 independent experiments. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test for multiple comparisons (bottom graph) and two-way ANOVA followed by Sidak’s post hoc test for multiple comparisons (upper graph 24 h vs. 48 h). p values were considered significant when p ≤ 0.05 (*, vs. CTRL), p ≤ 0.0001 (****, vs. CTRL), and p ≤ 0.0001 (####). (F) Representative immunoblots show the level of cell death markers (cleaved PARP1 and cleaved caspase 3) in TE671 cell line. β-actin was used as a loading control for an equal amount of protein. Control cells (CTRL) were treated with 0.1% DMSO as a solvent control. Camptothecin (CPT, 20 µM) was used as a reference drug for induction of apoptosis.

Figure 7

Synthesis of ASBDs. Reagents and reaction conditions: i—propynoic acid, DDC (N,N′-dicyclohexylcarbodiimide), DMAP (4-dimethylaminopyridine), dichloromethane, from −10 °C to room temperature; ii—propargyl chloroformate, pyridine, benzene, from −5 °C to room temperature [25].