Alendronate induces anti-migratory effects and inhibition of neutral phosphatases in UMR106 osteosarcoma cells

Abstract

Bisphosphonates are nonhydrolysable pyrophosphate analogues that prevent bone loss in several types of cancer. However, the mechanisms of anticancer action of bisphosphonates are not completely known. We have previously shown that nitrogen-containing bisphosphonates directly inhibit alkaline phosphatase of UMR106 rat osteosarcoma cells. In this study, we evaluated the effects of alendronate on the migration of UMR106 osteosarcoma using a model of multicellular cell spheroids, as well as the alendronate effect on neutral phosphatases. Alendronate significantly inhibited the migration of osteoblasts in a dose-dependent manner (10 − 6 -10 − 4 M). This effect was also dependent on calcium availability. The spheroid morphology and distribution of actin fibers were also affected by alendronate treatment. Alendronate dose-dependently inhibited neutral phosphatase activity in cell-free osteoblastic extracts as well as in osteoblasts in culture. Our results show that alendronate inhibits cell migration through mechanisms dependent on calcium, and that seem to involve inhibition of phosphotyrosine-neutral-phosphatases and disassembly of actin stress fibers.

Opposing effects of bisphosphonates and advanced glycation end-products on osteoblastic cells

“…On the other hand, both advanced glycation endproducts and lower doses of Alendronate (10 − 6 M) could be inhibiting mainly divalent cation-independent neutral phosphatases (such as tyrosine phosphatases). Interestingly, we have previously shown that osteoblastic neutral phosphatase activity decreases by 45% in the presence of Vanadate (an inhibitor of tyrosine phosphatases) (Molinuevo et al, 2007), which is a degree of inhibition similar to that which we have found for advanced glycation endproducts and for 10 − 6 M Alendronate in the present study.…”

“…Although we have not directly proven it, the same modulation of signal transduction mechanisms is probably operating under our experimental conditions. We have previously shown that different doses of bisphosphonates can inhibit both neutral and alkaline osteoblastic phosphatases (Vaisman et al, 2005;Molinuevo et al, 2007). In addition, other investigators have demonstrated that intracellular protein phosphorylation is essential for the process of osteoblastic differentiation (Xiao et al, 2002).…”

“…More recently, increasing lines of evidence have also implicated other bone cell types such as osteoblasts as possible targets for direct and indirect action of bisphosphonates (Tsuchimoto et al, 1994;Viereck et al, 2002), although the precise mechanisms have not been elucidated. In this line of work, our group has previously shown a biphasic effect of bisphosphonates on osteoblastic growth and differentiation, with an increase in both parameters at low concentrations of bisphosphonates, and a decrease at 10 − 4 -10 − 5 M (Vaisman neutral phosphatases (Molinuevo et al, 2007) and on alkaline phosphatase via chelation of divalent cations (Vaisman et al, 2005). In a series of elegant experiments, Plotkin et al (1999Plotkin et al ( , 2006 have demonstrated that bisphosphonates can prevent osteoblast and osteocyte apoptosis in vivo and in vitro, and have proposed that their anti-fracture actions could result in part from a preservation of the integrity of the osteocyte network, and/or from an increase in the working time of bone forming cells.…”

“…This assay was performed by incubating confluent UMR106 monolayers with 200 μg/ml of either bovine serum albumin or advanced glycation endproducts-modified bovine serum albumin plus different concentrations (10 were incubated with the fluorogenic substrate DiFMUP for 10 min as previously described (Molinuevo et al, 2007). The resulting fluorescent product was measured every minute at 360/450 nm (excitation/emission wavelengths) using an Aminco-Bowman SPF100 spectrofluorometer.…”

Evaluation and Comparison of In Vitro Biocompatibility of Poly (Glycolic Acid) and Poly (Lactide-Co-Glycolide Acid) on Mature Spheroids of Tumorigenic and Non-Tumorigenic Cell Lines

“…Ho we ver, only a few stu di es ha ve in vol ved tu mo ri ge nic cell li nes (i.e., ne u roblas to ma and gli ob las to ma cell li nes). [15][16][17][18][19][20] In the few exis ting stu di es, sing le tu mo ri ge nic and/or non-tumo ri ge nic cells we re co-cul tu red with the se bi opoly mers over the long term to de ter mi ne whet her they we re ab le to form a sphe ro id or not. 19,20 These cells in fact for med sphe ro ids with the bi o polymers and sub se qu ently con ti nu ed the co-cul tu re pro cess over the long term.…”