Fig 1.
Molecular structure of 28-O-α-l-rhamnopyranosylbetulin 3β-O-α-l-rhamnopyranoside (Bi-L-RhamBet).
Fig 2.
Bi-L-RhamBet induces growth inhibition of healthy and cancer cell lines.
Survival of human healthy lung cell lines (MRC-5; HEL299), mouse Lewis lung cancer cells (LLC1), and human non-small cell lung cancer cells of different stages including: A549, NCI-H23, NCI-H2087 (stage 1), NCI-H522 (stage 2), NCI-H1993 (stage 3a), and NCI-H1755 (stage 4) all decrease with increased concentrations of Bi-L-RhamBet. The values in parentheses correspond to the concentrations inhibiting fifty percent of the cell growth (IC50). They represent mean values ± standard deviation of triplicates (n = 3) and are representative of three independent experiments.
Fig 3.
Tumor growth inhibition induced by Bi-L-RhamBet.
Lewis lung tumor-bearing mice were untreated (control) or given doses of 25 or 50 mg/kg of Bi-L-RhamBet from days 1 to 4. The results are expressed as tumor volume in mm3 recorded between days 10 and 18. Data represent mean values ± standard deviation for ten mice (n = 10). *Values are significantly different from those of untreated (control) mice; Kruskal-Wallis One Way test followed by post-hoc Student-Newman-Keuls’ test.
Fig 4.
Hematoxylin and eosin-stained sections of Lewis lung tumor-bearing mice.
Images of cells from the control (A) or treatment with 50 mg/kg of Bi-L-RhamBet (B). Red arrows indicate the presence of condensed chromatin (pyknosis) suggesting apoptotic cell death. Magnification at 400×. The section is representative of three different mice.
Fig 5.
Effect of Bi-L-RhamBet on the cell cycle of A549 cancer cell lines.
(A) untreated cells (control), A549 cells treated 24 h with 3.12 μM (B), 6.25 μM (C), and 12.5 μM (D) of Bi-L-RhamBet. DNA was stained with propidium iodide and analyzed by flow cytometry. This analysis is representative of three independent samples.
Fig 6.
Morphological changes in A549 cells stained with acridine orange (AO) and ethidium bromide (EB).
Cells were untreated (A) or treated for 3 h with 1 μM staurosporine (B), 10 μM β-hederin (C), 5 μM Bi-L-RhamBet (D), 10 μM Bi-L-RhamBet (E), and 20 μM Bi-L-RhamBet (F). A549 cells were stained with AO/EB for 5 min and then visualized using fluorescence microscopy. Arrows indicate apoptotic blebbing. This analysis is representative of three independent experiments.
Table 1.
Bi-L-RhamBet induces delayed cytotoxicity in A549 cells.
Fig 7.
Kinetics of pro-apoptotic caspase activation induced by Bi-L-RhamBet.
A549 cells were treated with 1 μM of Staurosporine (STS), 10 μM of x-hederin (Hed) and 5, 10, and 20 μM of Bi-L-RhamBet. The activity of caspase 8 (A), caspase 9 (B) and caspase 3/7 (C) was measured after 2, 4, 6, 8, and 10 h. *Values are significantly different from untreated (control) cells (n = 3); Kruskal-Wallis One Way test followed by post-hoc Student-Newman-Keuls’ test. This analysis is representative of three independent experiments.
Fig 8.
DNA electrophoretic profiles of treated A549 cells.
Cells were either untreated (CTR) or treated for 24 h with either 5, 10, and 20 μM of Bi-L-RhamBet or 1 μM of staurosporine (STS). M: DNA size marker. This analysis is representative of three independent experiments.
Fig 9.
Mitochondrial ROS induced by Bi-L-RhamBet in A549 cells using DHR123.
A549 cells were (A) untreated (CTR); or treated with 5 μM Bi-L-RhamBet (B), 10 μM rotenone (ROT) (C), 5 μM Bi-L-RhamBet + 10 μM ROT (D), 10 mM malonate (MAL) (E), 5 μM Bi-L-RhamBet + 10 mM MAL (F), 10 μM antimycin A (AMA) (G), and 5 μM Bi-L-RhamBet + 10 μM AMA (H). The fluorescence intensity of each image was quantified using Image J software and results are presented as histogram. This analysis is representative of three independent experiments.
Fig 10.
Mitochondrial chain transfer inhibitors prevent the activation of caspases induced by Bi-L-RhamBet in A549 cells.
The cells were treated with 20 μM Bi-L-RhamBet and either 10 μM rotenone (ROT), 10 mM malonate (MAL), or 10 μM antimycine A (AMA). *Values are significantly different from Bi-L-RhamBet-only treated cells (n = 3); Kruskal-Wallis One Way test followed by post-hoc Student-Newman-Keuls’ test. This analysis is representative of three independent experiments.
Fig 11.
Mitochondrial membrane depolarization of A549 cells induced by Bi-L-RhamBet.
A549 cells were (A) untreated (control) or (B) treated for 6 h with 5 μM of Bi-L-RhamBet. Mitochondrial membrane depolarization of A549 cells was assessed using the fluorescent dye TMRM. The intensity of TMRM fluorescence is proportional to the mitochondrial membrane potential. The fluorescence intensity was quantified using Image J software. *Values are significantly different from control (n = 3); Kruskal-Wallis One Way test followed by post-hoc Student-Newman-Keuls’ test. This analysis is representative of three independent experiments.
Fig 12.
Dichloroacetate (DCA) potentiates the cytotoxicity of Bi-L-RhamBet in A549 cells.
The cells were treated with 0.8 or1.6 μM of Bi-L-RhamBet combined with cytotoxic concentrations of 10, 20, and 30 mM of DCA. *Values are significantly different from Bi-L-RhamBet-only (0 mM of DCA) samples (n = 3); Kruskal-Wallis One Way test followed by post-hoc Student-Newman-Keuls’ test. This analysis is representative of three independent experiments.