Figure 1.
Non-small cell lung cancer cell lines express MRP1/ABCC1 activity.
For P-gp/ABCB1 activity, A549 or H460 cells were incubated for 30 min with medium (AF) or 400 nM Rhodamine 123 in the absence (Rho) or presence of 50 µM verapamil (VP), For MRP1/ABCC1, cells were incubated with 5 µM CFDA in the absence (CF) or presence of 50 µM MK-571 (MK). After washing, cellular fluorescence was measured by flow cytometry. Histograms are representative of three independent experiments performed in duplicate.
Figure 2.
Oleanolic acid (OA) decreases cell viability and induces apoptosis of non-small cell lung cancer cell lines.
A549 or H460 cells were incubated with various OA concentrations (10, 25, 40 or 50 µg/mL or 21.9, 54.7, 87.6 or 109.5 µM) or cisplatin for 48 h. (A) Cell viability was assessed by MTT and plotted as percentage of cell viability inhibition. Results represent mean ± SD of 4 experiments performed in triplicate. (B) In parallel, cells were treated with a hypotonic solution containing propidium iodide (PI) and DNA-content was analyzed by flow cytometry. Upper panel: representative histogram of the cell cycle. Lower panel: percentage of apoptotic sub-G1 cells. Values represent mean ± SD of 3 experiments performed in triplicate. (C) Caspase-3 activated cells was determined by flow cytometry using a CaspGlow kit as described in M&M. Histograms are representative of two independent experiments performed in duplicate.
Figure 3.
Effect of Oleanolic acid on MRP1/ABCC1 activity and expression.
(A) To determine MRP1 activity, A549 or H460 cells were incubated for 30 min with medium (AF), 5 µM CFDA in the absence (CF) or presence of 50 µM MK-571 (MK-571), or with CFDA in the presence of various concentrations of OA (6.25, 12.5, 25 or 50 µg/mL). Cellular fluorescence was measured by flow cytometry. Results are expressed as the mean ± S.D of the mean fluorescence intensity obtained in 3 different experiments performed in triplicate. * and ** indicate p<0.05 and p<0.01 respectively, in relation to the control (CF). (B) To determine MRP1 expression, cells were treated with medium or OA (25 or 50 µg/mL) for 24 h before being harvested, permeabilized and incubated with PE-labeled anti-MRP1 for 30 minutes at 4°C. After two PBS washings, the cells were re-suspended in FACS solution and the fluorescence was evaluated. Histograms are representative of three independent experiments. (C) To determine MRP1 by western blot, whole-cell extracts were obtained from cells treated as described in B. Proteins were separated by sodium dodecyl sulphate (SDS)-gels, transferred to PDF membranes and probed with an antibody to MRP1 as described in the M&M section. Numbers represent band intensity in relation to α-tubulin.
Figure 4.
Effect of Oleanolic acid (OA) on proteins involved on apoptotic and metastatic pathways.
(Upper panel) Immunofluorescence of A549 cells stained for Bcl2 (A–B), Bax (C–D), survivin (E–F) and VEGF (G–H). A549 cells were treated with medium (A,C,E,G) or with 50 µg/mL OA (B, D, F, H) for 24 h, fixed, stained with the primary antibodies, revealed with biotinilated IgG followed by streptavidin-Cy3 and counter stained with DAPI, as described in M&M. Representative immunofluorescence micrographs of three experiments. Bar: 100 µm. (Lower panel) Graphical representation of the histomorphometrical results of the immunocytochemical assay. The percentage of immunoreactive cells was calculated from the DAPI positive cells. Bcl-2 was not modulated by OA (p>0.05), but Bax was significantly increased (* p<0.005), while survivin and VEGF were significantly decreased after treatment (**p<0.0001 and ***p<0.05, respectively).
Figure 5.
A549 cells were treated with medium or with 50 µg/mL Oleanolic acid for 24 h and whole cell extracts were obtained as described in M&M. Proteins were separated by SDS-PAGE, transferred to PDF membranes, probed with antibodies to Bcl-2, Bax, Survivin, VEGF or α-tubulin and developed with ECL, as described in M&M. Numbers represent band intensity in relation to α-tubulin.
Figure 6.
Oleanolic acid (OA) inhibits the development of lung metastasis.
Groups of mice, injected into the tail veins with B16F10 melanoma cells, were treated with saline, DMSO, OA (5 mg kg−1 day−1) or OA (10 mg kg−1 day−1), as described in the M&M section and in the upper panel; the number of lung metastasis was counted on day 18 (lower panel). Results are expressed as mean ± SD of the number of metastasis. * indicates p<0.001 in relation to the control (DMSO).