ICANCER RESEARCH57, 3890-3894. September 15, 1997] Advances in Brief Bisphosphonates Inhibit Prostate and Breast Carcinoma Cell Adhesion to Unmineralized and Mineralized Bone Extracellular Matrices1 Sandrine Boissier, Sandrine Magnetto, Lucien Frappart, Beatrice Cuzin, Frank H. Ebetino, Pierre D. Delmas, and Philippe Clezardin2 Institut National de Ia Sante et de Ia Recherche MEdicale Research Unit 403, Pavilion F (S. B., S. M., P. D. D., P. C.), Service d'Anatomopathologie IL F.), and Pavilion V (B. C.), HOpital Edouard Herriot, 69437 Lyon, France: and Procter & Gamble Pharmaceuticals, Mason@Ohio (F. H. El. Abstract The molecular mechanisms by which tumor cells induce osteolytic metastases are likely to Involve tumor cell adhesion to bone as well as the release of soluble mediators from tumor cells that stimulate osteoclast mediated bone resorption. Bisphosphonates (BPs) are powerful Inhibitors of the osteoclast activity and are, therefore, used In the treatment of cancer-associated osteolytic metastases. Here, we Investigated the effect of BPs on breast and prostate carcinoma cell adhesion to unmineralized and mineralized bone extracellular matrices. BP pretreatment of tumor cells inhibited tumor cell adhesion to unmlnerallzed and mineralized osteoblas tic extracellular matrices in a dose-dependent manner. In contrast, HP did not affect adhesion of normal cells (fibroblasts) to extracellular matrices. The order of potency for four BPs In InhibIting tumor cell adhesion to extracellular matrices was found to be: ibandronate > NE.10244 (antire sorptlve active pyrldinium analogue ofrisedronate) > pamidronate > do dronate. HP did not affect [3H]thymldineIncorporation by tumor cells, as assessed by a mitogenesls assay, indicating that HP did not exert any cytotoxlc effect at concentrations used to Inhibit tumor cell adhesion. NE-58051, the Inactive pyrldylpropylldene analogue of risedronate, had no Inhibitory effect on tumor cell adhesion compared to that observed with Its active counterpart NE-10244, suggesting that the mechanism of action of BP on tumor cells involved a stereospecific recognition step. Although Integrins mediate cell-matrix Interactions, HP recognition by tumor cells did not mOdUlatecell surface Integrinexpression. In conclu slon, our results provide evidence for a direct cellular effect of BP In preventing tumor cell adhesion to bone, suggesting that HPs may be useful agents for the prophylactic treatment of patients with cancer that Is known to preferentially metastasize to bone. Introduction A very common metastatic site for prostate and breast carcinomas is bone (1, 2). In bone metastases, tumor cells reside in the bone marrow, and the bone marrow tumor nest is frequently surrounded by numerous osteoclasts that are resorbing bone (2). Tumor cells are also found adjacent to the resorbed endosteal bone surface where there is no evidence of resorbing osteoclasts, suggesting that tumor cells could directly cause bone destruction (I, 2). Although the molecular mech anisms by which tumor cells induce osteolytic metastases are not fully understood, some of these mechanisms are likely to involve tumor cell adhesion to bone extracellular matrix molecules as well as the release of soluble mediators from tumor cells that stimulate osteoclast-medi ated bone resorption (2). Thus, any agent that could interfere with Received 9/6/96; accepted 7/30/97. The costs of publicationof this article were defrayedin partby the paymentof page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact. I This study was supported by grants from the Institut National de la Sante et de la RechercheMédicele(INSERM; to P.C.),Rhbne-Poulenc RORERLaboratories (toP.C.), and the Association pour la Recherche sur Las Tumeurs de la Prostate (to P. C.). 2 To whom requests for reprints should be addressed, at INSERM Research Unit 403, Pavillon F, Hbpital Edouard Herriott, Place d'Arsonval, 69437 Lyon Cédex03, France. these mechanisms would be a major therapeutic advance in the treatment of bone metastases. BPs3 are analogues of the naturally occurring compound pyrophos phate (3). They bind avidly to the bone mineral and are powerful inhibitors of bone resorption (3). They act directly on osteoclasts by preventing their recruitment from bone marrow, inhibiting their ca pacity to resorb bone, and shortening their life span (3). This provided the rationale for the use of BPs in the treatment of patients with osteolytic metastases. BPs (pamidronate and clodronate) reduce the occurrence of fractures and decrease the formation of newly osteolytic lesions in breast cancer patients (4, 5). Animal studies also demon strate that pretreatment of nude mice with a potent antiresorptive BP, risedronate [2-(3-pyridyl)-l-hydroxyethylidene bisphosphomc acid], before breast carcinoma cell inoculation shows a marked reduction of the osteolytic lesions and a marked decrease in tumor burden in bone (6). BP pretreatment of corticalboneslicesalsopartiallyinhibited breast carcinoma cell adhesion in vitro (7). Moreover, it has been recently shown that adjuvant treatment of breast cancer patients with the BP clodronate reduces the incidence and number of bone and nonbone metastases (8). These very important observations (6—8) suggest, therefore, that BPs not only act on osteoclasts but may also affect the invasive behavior of metastatic cells in bone. Here, we investigated the effect of BPs on tumor cell adhesion to bone. We have found that BPs inhibit tumor cell adhesion to unmineralized and mineralized bone extracellular matrices through a specific action on tumor cells. These results suggest that BPs may be useful agents for the prophylactic treatment of patients with cancer that is known to preferentially metastasize to bone. Materials and Methods HPs. Five BPs were used. Clodronate[dichloromethylenebisphosphonic acid] was obtained from RhOne-Poulenc RORER Laboratories (France). Pam idronate [3-amino-l-hydroxypropylidene bisphosphomc acidi was obtained fromCiba-Geigy(Germany).Ibandronate[l-hydroxy-3(methylpentylamino)- propylidenebisphosphomcacid] was a gift from Dr. F. Bauss (Bochringer Mannheim, Germany). Disodium 2-(N-methyl-3-pyridinyl)-l-hydmxyethyli dene bisphosphonic acid (NE-l0244), a potent antiresorptiveanalogue of risedronate, and 3-(3-pyridyl)-l-hydroxypropylidsne bisphosphomc acid (NE 58051), an inactive analogue of risedronate, were obtained from Procter & Gamble Pharmaceuticals. CellLInes. Humanbreastcarcinoma(MCF-7andMDA-MB-231),prostate carcinoma (PC3), and osteosarcoma (MG-63) celllines were obtained from the American Type Culture Collection. Cell line PmPC3 is a highly metastatic variant ofhuman prostate carcinoma cell line PC3. Human foreskin fibroblasts were kindly providedby Dr. D. Schmitt (HOpitalEdouardHerriot).Human osteoblastswere isolatedfrom trabecularbone andculturedusing methodsas described previously (9). 3 The abbreviation used is: BP, bisphosphonate. 3890 Research. on October 11, 2021. © 1997 American Association for Cancer cancerres.aacrjournals.org Downloaded from