Abstract
BackgroundAnti-resorptive bisphosphonates (BP) are used for the treatment of osteoporosis and bone metastases. Clinical studies indicated a benefit in survival and tumor relapse in subpopulations of breast cancer patients receiving zoledronic acid, thus stimulating the debate about its anti-tumor activity. Amino-bisphosphonates in nM concentrations inhibit farnesyl pyrophosphate synthase leading to accumulation of isopentenyl pyrophosphate (IPP) and the ATP/pyrophosphate adduct ApppI, which induces apoptosis in osteoclasts. For anti-tumor effects μM concentrations are needed and a sensitizer for bisphosphonate effects would be beneficial in clinical anti-tumor applications. We hypothesized that enhancing intracellular pyrophosphate accumulation via inhibition of probenecid-sensitive channels and transporters would sensitize tumor cells for bisphosphonates anti-tumor efficacy.MethodMDA-MB-231, T47D and MCF-7 breast cancer cells were treated with BP (zoledronic acid, risedronate, ibandronate, alendronate) and the pyrophosphate channel inhibitors probenecid and novobiocin. We determined cell viability and caspase 3/7 activity (apoptosis), accumulation of IPP and ApppI, expression of ANKH, PANX1, ABCC1, SLC22A11, and the zoledronic acid target gene and tumor-suppressor KLF2.ResultsTreatment of MDA-MB-231 with BP induced caspase 3/7 activity, with zoledronic acid being the most effective. In MCF-7 and T47D either BP markedly suppressed cell viability with only minor effects on apoptosis. Co-treatment with probenecid enhanced BP effects on cell viability, IPP/ApppI accumulation as measurable in MCF-7 and T47D cells, caspase 3/7 activity and target gene expression. Novobiocin co-treatment of MDA-MB-231 yielded identical results on viability and apoptosis compared to probenecid, rendering SLC22A family members as candidate modulators of BP effects, whereas no such evidence was found for ANKH, ABCC1 and PANX1.ConclusionsIn summary, we demonstrate effects of various bisphosphonates on caspase 3/7 activity, cell viability and expression of tumor suppressor genes in breast cancer cells. Blocking probenecid and novobiocin-sensitive channels and transporters enhances BP anti-tumor effects and renders SLC22A family members as good candidates as BP modulators. Further studies will have to unravel if treatment with such BP-sensitizers translates into preclinical and clinical efficacy.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-265) contains supplementary material, which is available to authorized users.
Highlights
Anti-resorptive bisphosphonates (BP) are used for the treatment of osteoporosis and bone metastases
It was reported for zoledronic acid (ZA) and to a lesser extend for ibandronate (IBN) and risedronate (RIS) as well as for alendronate (ALN) that treatment of cells led to the accumulation of isopentenyl pyrophosphate (IPP) and produced a new endogenous ATP analogue (triphosphoric acid 1-adenosin-5′-yl ester 3-(3-methylbut-3-enyl) ester (ApppI)), which caused apoptosis in osteoclasts by inhibiting the mitochondrial ADP/ATP translocase [5]
In MCF-7 cells we identified ZA target genes as KLF2, KLF6 and Ki-67 and we assumed that IPP/ApppI accumulation might mediate this effect in cell populations that are largely insensitive to apoptosis induction [15]
Summary
Anti-resorptive bisphosphonates (BP) are used for the treatment of osteoporosis and bone metastases. As a consequence protein prenylation of small GTP binding proteins like Rab, Ras or lamins, which are important for cytoskeleton organization and cellular polarization, is inhibited and may initiate apoptosis [4]. It was reported for zoledronic acid (ZA) and to a lesser extend for ibandronate (IBN) and risedronate (RIS) as well as for alendronate (ALN) that treatment of cells led to the accumulation of isopentenyl pyrophosphate (IPP) and produced a new endogenous ATP analogue (triphosphoric acid 1-adenosin-5′-yl ester 3-(3-methylbut-3-enyl) ester (ApppI)), which caused apoptosis in osteoclasts by inhibiting the mitochondrial ADP/ATP translocase [5]
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