Assessment of Bishosphonate Activity In Vitro

L.      M. Mitrofan; S.      Auriola; H.      Monkkonen; J.      Monkkonen

Abstract

Bisphosphonates are a class of drugs developed over the past three decades for the treatment of metabolic bone diseases with high bone turnover, such as Pagets disease, tumor associated osteolysis and osteoporosis. The exceptional pharmacokinetic profile of bisphosphonates makes them very suitable and safe drugs for the treatment of bone diseases, because, by conventional administration, osseous tissue and bone resorbing osteoclasts are the targets for these drugs as a result of the very high affinity of bisphosphonates for bone mineral. Several recent studies have demonstrated; however, that bisphosphonates decrease tumor burden in bone in rodent models of myeloma and metastatic bone disease, with suggestions of antitumor effects also in patients. Although, decreased tumor burden could be a consequence of inhibition of bone resorption, there is increasing evidence that bisphosphonates might also have direct effects on tumor cell in vivo, since effects on tumors outside of skeleton or at doses not inhibiting bone resorption have been reported. Recent studies also suggest that bisphosphonates have inhibitory effect also on endothelial cell function and angiogenesis in tumor tissue. These findings suggest that the target cells for bisphosphonates as well as their molecular mechanism of action may be more diverse and complex than realized so far. This review highlights the main methodologies used to monitor the action of BPs in vitro cell models, with a special emphasis on the detection of BP-induced ATP-analoques by mass spectrometry. In addition, cell death monitoring, immunomodulatory effects and inhibition of growth/proliferation are described.

Keywords: BPs, ATP-analogs, mass spectrometry, cell death, cell growth, uptake, immunomodulation, flow cytometry, Bisphosphonates, Paget's disease, angiogenesis, zoledronic acid, farnesyl pyrophosphate synthase (FPPS), geranylgeranyl pyrophosphate (GGPP), AppCp-type, protein isoprenylation, co-eluting isomers, BPs treatment, UnRap1A, apoptosis, zol-induced apoptosis, [14C]mevalonolactone, malignant diseases, growth factors, vicious cycle, matrix-bound tumor, tumor cell migration, cellu-lar death process, G-proteins, geranylgeranylation, farnesyl-farnesylation, drug-induced cytotox-icity, lactate dehydrogenase (LDH), Cell Death Program, cellular shrinkage, chromatin condensation, DNA fragmenta-tion, Permeability Transition Pore, Complex (PTPC), Lysosomes, Wunderwaffe for cancer therapy, phosphatidylserine (PS), pamidronate, ibandro-nate, alendronate, risedronate, intrinsic stimulatory activity, Nucleotidic anti-gens, cytotoxic effects, fluorescent protein (GFP), over-express luciferase