title: Probenecid as a sensitizer of bisphosphonate-mediated effects in breast cancer cells.
abstract: BACKGROUND
  Anti-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.
  
  
  METHODS
  MDA-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.
  
  
  RESULTS
  Treatment 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.
  
  
  CONCLUSIONS
  In 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.
authors.forename: Regina
authors.initials: R
authors.lastname: Ebert
authors.forename: Jutta
authors.initials: J
authors.lastname: Meissner-Weigl
authors.forename: Sabine
authors.initials: S
authors.lastname: Zeck
authors.forename: Jorma
authors.initials: J
authors.lastname: Määttä
authors.forename: Seppo
authors.initials: S
authors.lastname: Auriola
authors.forename: Sofia
authors.initials: S
authors.lastname: Coimbra de Sousa
authors.forename: Birgit
authors.initials: B
authors.lastname: Mentrup
authors.forename: Stephanie
authors.initials: S
authors.lastname: Graser
authors.forename: Tilman D
authors.initials: TD
authors.lastname: Rachner
authors.forename: Lorenz C
authors.initials: LC
authors.lastname: Hofbauer
authors.affiliation: Orthopedic Center for Musculoskeletal Research, University of Würzburg, Brettreichstrasse 11, 97074 Würzburg, Germany. f-jakob.klh@uni-wuerzburg.de.
authors.forename: Franz
authors.initials: F
authors.lastname: Jakob
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001859:Bone Neoplasms / Q000188:drug therapy / Q000378:metabolism; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D017630:Connexins / Q000378:metabolism; D004164:Diphosphonates / Q000494:pharmacology*; D012968:Etidronic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005260:Female; D045782:Hemiterpenes / Q000494:pharmacology; D006801:Humans; D000077557:Ibandronic Acid; D007093:Imidazoles / Q000494:pharmacology; D061986:MCF-7 Cells; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000494:pharmacology; D010010:Osteoclasts / Q000187:drug effects / Q000378:metabolism; D028061:Phosphate Transport Proteins / Q000378:metabolism; D011339:Probenecid / Q000494:pharmacology*; D000068296:Risedronic Acid; D000077211:Zoledronic Acid
chemical_list: D000970:Antineoplastic Agents; D053148:Caspase 3; D053179:Caspase 7; D017630:Connexins; D004164:Diphosphonates; D012968:Etidronic Acid; D045782:Hemiterpenes; D000077557:Ibandronic Acid; D007093:Imidazoles; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009419:Nerve Tissue Proteins; D009943:Organophosphorus Compounds; D028061:Phosphate Transport Proteins; D011339:Probenecid; D000068296:Risedronic Acid; D000077211:Zoledronic Acid; C498770:ANKH protein, human; C492764:PANX1 protein, human; C004809:isopentenyl pyrophosphate; C421109:multidrug resistance-associated protein 1