ABL-N-induced apoptosis in human breast cancer cells is partially mediated by c-Jun NH2-terminal kinase activation

Abstract

Introduction: The present study was designed to determine the possibility of acetylbritannilactone (ABL) derivative 5-(5-(ethylperoxy)pentan-2-yl)-6-methyl-3-methylene-2-oxo-2,3,3a,4,7,7a-hexahydrobenzofuran-4-yl 2-(6-methoxynaphthalen-2-yl)propanoate (ABL-N) as a novel therapeutic agent in human breast cancers.

Methods: We investigated the effects of ABL-N on the induction of apoptosis in human breast cancer cells and further examined the underlying mechanisms. Moreover, tumor growth inhibition of ABL-N was done in xenograft models.

Results: ABL-N induced the activation of caspase-3 in estrogen receptor (ER)-negative cell lines MDA-MB-231 and MDA-MB-468, as evidenced by the cleavage of endogenous substrate Poly (ADP-ribose) polymerase (PARP). Pretreatment of cells with pan-caspase inhibitor z-VAD-fmk or caspase-3-specific inhibitor z-DEVD-fmk inhibited ABL-N-induced apoptosis. ABL-N treatment also resulted in an increase in the expression of pro-apoptotic members (Bax and Bad) with a concomitant decrease in Bcl-2. Furthermore, c-Jun-NH2-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase (p38) were activated in the apoptosis induced by ABL-N and JNK-specific inhibitor SP600125 and JNK small interfering RNA (siRNA) antagonized ABL-N-mediated apoptosis. However, the p38-specific inhibitor SB203580 had no effect upon these processes. Moreover, neither of the caspase inhibitors prevented ABL-N-induced JNK activation, indicating that JNK is upstream of caspases in ABL-N-initiated apoptosis. Additionally, in a nude mice xenograft experiment, ABL-N significantly inhibited the tumor growth of MDA-MB-231 cells.

Conclusions: ABL-N induces apoptosis in breast cancer cells through the activation of caspases and JNK signaling pathways. Moreover, ABL-N treatment causes a significant inhibition of tumor growth in vivo. Therefore, it is thought that ABL-N might be a potential drug for use in breast cancer prevention and intervention.

Figure 1

Effect of ABL and ABL-N on cancer cell lines. (a) The chemical structures of ABL and ABL-N. (b) The differences of growth inhibition activity between ABL and ABL-N in MDA-MB-231 cells. (c) Effects of ABL-N on the viability of various cancer cell lines. Cells were treated with ABL-N for 24 hours and cell viability was determined by the MTT assay. The IC₅₀ is the concentration of ABL-N that reduced the cell viability by 50% under the experimental conditions (n = 6). (d) Time- and dose-dependent inhibition of cell viability by ABL-N treatment in MDA-MB-231, MCF-7, MDA-MB-468 and NBECs. Cells were treated with 5, 10, 20 or 40 μM ABL-N for 1, 3, 6, 12 and 24 hours and cell viability was assessed by MTT assay (n = 6). Results represent the means ± SE from three independent experiments.

Figure 2

Effect of ABL-N on cell cycle progression and apoptosis in breast cancer cells. After treatment with 20 μM ABL-N for indicated times, MDA-MB-231 cells were harvested. (a) The percentage of cell cycle distribution for cells done in triplicate with similar results. (b) Nuclear condensation was shown by DAPI-staining assay (magnification, ×100). (c) DNA fragmentation was evaluated using a Cell Death Detection ELISA^Plus kit. The data are expressed as means ± SE of three separate experiments.*P <0.05, **P <0.01, as compared with the group without ABL-N treatment. (d) The apoptotic status was determined by Annexin V/PI staining method. Percentages of negative (viable) cells, annexin V-positive (early apoptotic) cells, PI-positive (necrotic) cells, or annexin V and PI double-positive (late apoptotic) cells were shown (mean of three independent experiments) by a flow cytometric analysis.

Figure 3

Induction of caspase activities by ABL-N. MDA-MB-231 cells were treated with 20 μM ABL-N for indicated times. (a) Whole cell protein lysates were prepared and subjected to Western blot analysis for detection of cleavage of caspase-3 and caspase-9. (b) Induction of caspase activities by ABL-N in MDA-MB-231 cells. (c) Cleavage of PARP was induced by ABL-N. (d) Cells were pretreated with 50 μM of either z-VAD-fmk or z-DEVD-fmk for one hour, followed by 20 μM ABL-N for 24 hours, and caspase-3 activity, DNA fragmentation and sub-G₁ DNA contents were determined. The data are expressed as means ± SE of three separate experiments. (e) Abrogation of PARP cleavage by caspase inhibitors.

Figure 4

Effects of ABL-N on the expression of Bcl-2 family. (a) Treatment of ABL-N decreased the level of the anti-apoptotic protein Bcl-2, but increased the expression of the pro-apoptotic proteins Bax and Bad in MDA-MB-231 cells. (b) Effect of ABL-N on the Bax/Bcl-2 ratio in MDA-MB-231 cells. The data obtained from the Western blot analysis of Bax and Bcl-2 were used to evaluate the effect of ABL-N on the Bax/Bcl-2 ratio. The densitometric analysis of Bax and Bcl-2 bands was performed using TotalLab TL120 software, and the data (relative density normalized to β-actin) were plotted as Bax/Bcl-2 ratio. Data are expressed as means ± SE of three separate experiments with similar results. *P <0.05, **P <0.01, as compared with the group without ABL-N treatment.

Figure 5

Time course of MAP kinase activation by ABL-N. MDA-MB-231 cells were treated with vehicle (0.5% ethanol) or 20 μM of ABL-N and harvested at the indicated times. (a) JNK; (b) p38; and (e) ERK activation was determined by Western blot analysis using antibodies that recognize the phosphorylated form of the respective active MAP kinase. (c) JNK activity was determined by an in vitro kinase assay as described in Materials and methods. Phosphorylation of c-Jun, which represents the intrinsic activity of JNK, was visualized by Western blotting. (d) ABL-N (20 μM) and GST-JNK1 fusion proteins (1 μg) were incubated for 12 hours or cells were treated with ABL-N (20 μM) for 12 hours and lysates were prepared, then JNK activity was measured as described above. Phosphorylation of c-Jun was visualized by Western blotting. The same blot was stripped and reprobed with a c-Jun antibody to monitor equal loading of proteins. Data are representative of three independent experiments.

Figure 6

Role of JNK in ABL-N-induced apoptosis. MDA-MB-231 cells were incubated with ABL-N (20 μM) and either SB203580 (20 μM) or SP600125 (30 μM), or a combination of both for 24 hours. (a) MDA-MB-231 cells were transfected with JNK siRNA (25 nM) or control siRNA (25 nM) for 48 hours, and cell lysates were subjected to Western blot with antibodies to JNK. (b) ABL-N-induced cell death was abrogated by inhibition of JNK using MTT assay. (c) Sub G₁ DNA content was analyzed by flow cytometry. (d) Caspase-3 activity was determined by Caspase-Glo assay and the cleavage of PARP was analyzed via Western blotting. Data are expressed as the means ± SE of three independent experiments. (e) Effect of MAP kinase inhibitors on ABL-N-induced JNK activation. (f) Effect of caspase inhibitors on JNK activation. Cells pretreated with or without z-VAD-fmk (50 μM) or z-DEVD-fmk (50 μM, one hour) were further incubated with vehicle or 20 μM ABL-N for 24 hours. Data are representative of three independent experiments.

Figure 7

The effect of ABL-N on the growth of MDA-MB-231 xenografts. Female nude mice bearing MDA-MB-231 tumor xenografts were treated with ABL-N (15 mg/kg), or vehicle (10% DMSO, 10% ethanol in water), and tumors were measured with calipers on alternate days. Points = mean tumor volume in each experimental group containing six mice; bars = SD; *, P < 0.05; **, P < 0.01, compared with vehicle treated group.