The bisphosphonate zoledronic acid impairs Ras membrane [correction of impairs membrane] localisation and induces cytochrome c release in breast cancer cells

Abstract

Bisphosphonates are well established in the management of cancer-induced bone disease. Recent studies have indicated that these compounds have direct inhibitory effects on cultured human breast cancer cells. Nitrogen-containing bisphosphonates including zoledronic acid have been shown to induce apoptosis associated with PARP cleavage and DNA fragmentation. The aim of this study was to identify the signalling pathways involved. Forced expression of the anti-apoptotic protein bcl-2 attenuated bisphosphonate-induced loss of cell viability and induction of DNA fragmentation in MDA-MB-231 cells. Zoledronic acid-mediated apoptosis was associated with a time and dose-related release of mitochondrial cytochrome c into the cytosol in two cell lines. Rescue of cells by preincubation with a caspase-3 selective inhibitor and demonstration of pro-caspase-3 cleavage products by immunoblotting suggests that at least one of the caspases activated in response to zoledronic acid treatment is caspase-3. In both MDA-MB-231 and MCF-7 breast cancer cells, zoledronic acid impaired membrane localisation of Ras indicating reduced prenylation of this protein. These observations demonstrate that zoledronic acid-mediated apoptosis is associated with cytochrome c release and consequent caspase activation. This process may be initiated by inhibition of the enzymes in the mevalonate pathway leading to impaired prenylation of key intracellular proteins including Ras.

Figure 1

(A) Immunoblotting of bcl-2 protein in whole cell extracts of MDA-MB-231 clone-1 transfected with pUSEamp (+) plasmid containing wild-type mouse bcl-2 and a clone transfected with control vector. Levels of bcl-2 expression in transfected and control clones were compared relative to β actin protein. (B) Overexpression of bcl-2 suppresses ZOL effects on breast cancer cells. Both control vector and bcl-2 overexpressing MDA-MB-231 cells (clone 1) were incubated with 0, 50 and 100 μM ZOL for 3 days and cell viability was quantitated by MTS dye reduction assay. Clone 1 was shown to be significantly less sensitive to ZOL induced reduction in cell viability than cells transfected with control vector. Results are shown as mean±s.d. Significance was **P<0.0001 compared to bcl-2-transfected cells treated with 100 μM ZOL. (C) The induction of DNA fragmentation by ZOL on MDA-MB-231 cells was significantly inhibited in MDA-MB-231 clone 1, overexpressing bcl-2. Both control vector and clone 1 cells were treated with 100 μM of ZOL for 3 days. Results are shown as mean±s.d.. ZOL treatment significantly increased DNA fragmentation in control clone. **P<0.0001 (In bcl-2 transfected cells 100 μM of ZOL did not significantly increase DNA fragmentation. P=0.053).

Figure 2

(A) MDA-MB-231 cells were incubated in the presence of 100 μM ZOL and cell extracts were prepared on specified days. Extracts were twice centrifuged at 13 000 r.p.m. for 15 min and the post-mitochondrial supernatant (cytosol) was concentrated as described in Materials and Methods. Cytochrome c levels in samples having equivalent protein contents (15 μg) were determined by Western analysis. Lane order: 1, control cultures day 3; 2, ZOL treated, day 1; 3, ZOL treated, day 2; and 4, ZOL treated, day 3. (B) Corresponding densitometric analysis of cytochrome c levels in cytosol for each day of treatment. (C) MCF-7 cells were incubated in the presence of 10, 50 and 100 μM ZOL and cell extracts were prepared after 3 days of above treatments. Cytosol fractions were prepared as described above and the cytochrome c levels in samples having equivalent protein contents (20 μg) were determined using Western analysis. Lane order: 1, control cultures; 2, 10 μM ZOL; 3, 50 μM ZOL; and 4, 100 μM ZOL. (D) Corresponding densitometric analysis of cytochrome c levels in cytosol for each treatment.

Figure 3

(A) Identification of caspase-3 activity in breast cancer cells. MDA-MB-231 cells were incubated in the presence of 100 μM ZOL for 3 days before cell extracts were prepared and Western blot analysis carried out. Two prominent cleaved products of pro-caspase-3 (indicating caspase activation) were seen at 20 and 11 kD. Lane order: 1, MDA-MB-231 cells treated with vehicle alone; 2, MDA-MB-231 treated with taxotere 10 nM for 2 days as a positive control; 3, MCF-7 treated with TNF-α 20 ng ml⁻¹ for 2 days, as a negative control; 4, MDA-MB-231 treated with pamidronate 100 μM and 5, MDA-MB-231 treated with ZOL 100 μM. (B) Identification of caspase-3 activation by ZOL in MCF-7 cells with forced expression of caspase-3. Both control vector and caspase-3 expressing MCF-7 cells were incubated with 100 μM ZOL for 3 days before cell extracts were prepared and Western blot analysis carried out. Lane order: 1, Control vector MCF-7 treated with vehicle alone; 2, Control vector MCF-7 treated with ZOL; 3, Caspase-3 expressing MCF-7 treated with vehicle alone and 4, caspase-3 expressing MCF-7 treated with ZOL. (C) Attenuation of ZOL effects by a caspase-3-selective inhibitor. MDA-MB-231 cells were treated with the caspase-3 inhibitor (0.5 μM) 3 h prior to addition of 35 μM of ZOL for 24 h. Medium was then replaced with medium containing either the caspase-3 inhibitor or vehicle (DMSO) alone. On day 3 cell viability was quantitated using MTS dye reduction assay. Results are shown as mean±s.d. Significance levels were ^a P<0.0005 vs untreated cells, ^b P<0.0005 vs ZOL treated cells.

Figure 4

(A) Identification of ZOL induced reduction in active Ras (membrane bound) in MDA-MB-231 cells. MDA-MB-231 cells were treated with 100 μM ZOL for 3 days. Level of expression of Ras protein in cytosol and membrane fractions was determined by Western analysis and equivalence of loading was determined by total protein staining as described in Materials and Methods. Lane order: 1, membrane fraction; 2, cytosol fraction. (B) MCF-7 cells were treated with 40 μM of FOH (mixed isomers) 3 h before addition of 50 μM ZOL for 24 h. Medium was then replaced with fresh medium containing 40 μM FOH (mixed isomers) or vehicle only for a further 2 days. Level of expression of Ras protein in cytosol and membrane fractions following each treatment was determined by Western analysis as described above. Equivalence of loading was determined by total protein staining as described in Materials and Methods. Lane order: 1, membrane fraction; 2, cytosol fraction.

Figure 5

(A) Effects of FOH (mixed isomers) on ZOL-induced inhibition of cell viability. MDA-MB-231 cells were treated with or without 40 μM of FOH (mixed isomers) 3 h before addition of 40 μM ZOL for 24 h. Medium was then replaced with fresh medium containing 40 μM FOH (mixed isomers) or vehicle (ethanol) only for a further 3 days. Results are shown as mean±s.d. Significance ^a P<0.0001 vs vehicle treated cells (control). ^b P<0.0001 vs ZOL alone treated cells. Control vs ZOL + FOH was not significant (P=0.86). (B) Effects of all trans-GGOH (40 μM) on ZOL induced inhibition of cell viability. MDA-MB-231 cells were treated with or without 40 μM all trans-GGOH for 3 h before exposing to 40 μM of ZOL for 24 h. Medium was then replaced with fresh medium containing 40 μM all trans-GGOH alone or vehicle only. Results are shown as mean±s.d. Significance ^a P<0.0001 vs vehicle treated cells (control). ^b P<0.0014 vs ZOL alone treated cells. Viability of cultures treated with ZOL+GGOH was significantly lower than control (P<0.01).