Apoptotic and Cell Cycle Effects of Triterpenes Isolated from Phoradendron wattii on Leukemia Cell Lines

Abstract

Current antineoplastic agents present multiple disadvantages, driving an ongoing search for new and better compounds. Four lupane-type triterpenes, 3α,24-dihydroxylup-20(29)-en-28-oic acid (1), 3α,23-dihydroxy-30-oxo-lup-20(29)-en-28-oic acid (2), 3α,23-O-isopropylidenyl-3α,23-dihydroxylup-20(29)-en-28-oic acid (3), and 3α,23-dihydroxylup-20(29)-en-28-oic acid (4), previously isolated from Phoradendron wattii, were evaluated on two cell lines of chronic (K562) and acute (HL60) myeloid leukemia. Compounds 1, 2, and 4 decreased cell viability and inhibit proliferation, mainly in K562, and exhibited an apoptotic effect from 24 h of treatment. Of particular interest is compound 2, which caused arrest in active phases (G2/M) of the cell cycle, as shown by in silico study of the CDK1/Cyclin B/Csk2 complex by molecular docking. This compound [3α,23-dihydroxy-30-oxo-lup-20(29)-en-28-oic acid] s a promising candidate for incorporation into cancer treatments and deserves further study.

Keywords: apoptosis; cell cycle; leukemia; lupane-type triterpene; molecular docking.

Figure 1

Chemical structures of compounds 1–4.

Figure 2

Effect of compounds 1–4 at different concentrations in K562 (A) and HL60 (B) cell and normal MNC (C). Normal and leukemia cells were cultured in presence of different compounds concentration by 48 h and the viability percentage was analyze using 7-AAD. Data are expressed as the percentage of 7-AAD ± SEM positive cells from at least three different experiments in triplicate. Statistical significance was determined by a one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc test. The differences were considered significant * p < 0.05, ** p < 0.01 vs. negative control (NC).

Figure 3

The data represent mean ± SEM of proliferation index from three different experiments K562 (A), HL60 (B) cell lines and normal MNC (C) at different concentrations. * p < 0.05 and ** p < 0.01 were compared to the negative control. ⁺ p < 0.05 and ⁺⁺ p < 0.01 were compared to the positive control. Statistical significance was determined by a one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc test.

Figure 4

The compounds 1, 2, and 4 induced apoptosis in K562 and compound 2 in HL60. The compounds 1 (50 µg/mL), 2 (25 µg/mL), and 4 (100 µg/mL) were evaluated in cell line K562 and normal MNC. The results in (A) represents mean ± SEM of at least three independent experiments in cell line K562 and normal MNC, and results in (B) represents mean ± SEM of at least three independent experiments in cell line HL60 and normal MNC of compound 2 (100 µg/mL). Statistical significance was determined by a one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc test. The differences were considered significant at * p < 0.05, ** p < 0.01 compared to negative control.

Figure 5

Results in cells K562 (A) and HL60 (B) of different compounds on the cell cycle status, represent mean ± SEM from three different experiments of cell cycle phase distribution in the different cells analyzed. Significance between cell cycle phase was determined using one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc test. Differences were considered significant at * p < 0.05, ** p< 0.01 vs. negative control (NC).

Figure 6

Binding modes of the compounds under study at the BCR-ABL binding site. (A) Binding site of nilotinib, ATP and compound 2. (B) Close-up of the binding site of nilotinib (Re-docking RMSD: 0.717 Å), ATP and compound 2. (C) Superimposition of compound 1, 2, 3, and 4 into ATP binding site of ABL kinase.

Figure 7

(A) Binding modes of the compounds under study into CDK1/Cyclin B/Csk2 binding site. (B) Close-up of the binding site of flavopiridol (Re-docking RMSD: 0.462 Å), ATP and compound 2. (C) Visualization of interactions between compounds 1–4 inside the binding site of CDK1. (D) Visualization of interactions between compound 2 inside the binding site of CDK1. (E) Visualization of interactions between flavopiridol inside the binding site of CDK1 in raw complex retrieved from PDB.