Nitrogen-containing bisphosphonates (N-BPs) are effective antiosteolytic agents in patients with multiple myeloma. Preclinical studies have also demonstrated that these agents have direct antitumor effects in vitro and can reduce tumor burden in a variety of animal models, although it is not clear whether such effects are caused by direct actions on tumor cells or by inhibition of bone resorption. N-BPs prevent bone destruction in myeloma by inhibiting the enzyme farnesyl pyrophosphate synthase in osteoclasts, thereby preventing the prenylation of small GTPase signaling proteins. In this study, utilizing a plasmacytoma xenograft model without complicating skeletal lesions, treatment with zoledronic acid (ZOL) led to significant prolongation of survival in severe combined immunodeficiency mice inoculated with human INA-6 plasma cells. Following treatment with a clinically relevant dose of ZOL, histological analysis of INA-6 tumors from the peritoneal cavity revealed extensive areas of apoptosis associated with poly (ADP-ribose) polymerase cleavage. Furthermore, Western blot analysis of tumor homogenates demonstrated the accumulation of unprenylated Rap1A, indicative of the uptake of ZOL by nonskeletal tumors and inhibition of farnesyl pyrophosphate synthase. These studies provide, for the first time, clear evidence that N-BPs have direct antitumor effects in plasma cell tumors in vivo and this is executed by a molecular mechanism similar to that observed in osteoclasts.

Zoledronate (ZOL) is the most potent nitrogen-containing bisphosphonate and is effective at preventing osteolytic bone disease in patients with multiple myeloma (MM) and solid tumors. ZOL inhibits the enzyme farnesylpyrophosphate synthase and thus blocks the prenylation of small GTPases. In vitro studies have demonstrated that ZOL can also directly affect the growth and viability of myeloma cells, however, the molecular mechanisms underlying this activity have not been fully elucidated. The goal of our study was to investigate direct antimyeloma effects of ZOL in vitro and in vivo. In five myeloma cell lines (RPMI8226, L363, U266, JK-6L, and the IL-6 dependent INA-6), growth was inhibited and apoptosis induced by ZOL in a dose-dependent manner (IC50's between 30 μM and 285 μM). Similar results were obtained in the presence of bone marrow stromal cells, IL-6 (20 ng/mL), IGF-1 (200 ng/mL), or a combination of both cytokines. The potential antitumor effect of ZOL on myeloma cells in vivo was studied in the INA-6 SCID model, in which mice are injected intraperitoneally with INA-6 cells and subsequently develop plasmacytomas. Mice treated with ZOL had reduced tumor burden and a significant survival benefit compared to the control group (p=0.002). Histological examination of plasmacytomas explanted 72 hours after a single injection of 8 μg ZOL revealed extensive apoptotic/necrotic areas while no such areas were found in tumors of untreated animals. Induction of apoptosis was confirmed by Western blot analysis of tumor lysates, which revealed increased levels of cleaved poly (ADP-ribose) polymerase (PARP) in tumors of ZOL treated vs. untreated animals. This correlated with an accumulation of the unprenylated form of the small GTPase Rap1A, which was virtually absent in tumors of untreated mice. Our findings demonstrate a direct and specific effect of ZOL in plasmacytomas in vitro and in vivo and point to a therapeutic potential in MM beyond the prevention of osteolytic lesions.

Bisphosphonates such as zoledronic acid (ZOL) are effective at preventing osteolytic bone disease in patients with multiple myeloma. ZOL inhibits bone resorption by inhibiting FPP synthase and preventing the prenylation of small GTPases in osteoclasts. In vitro studies have demonstrated previously that ZOL can also directly affect the growth and viability of myeloma cells by inhibiting protein prenylation and therefore could, potentially, have a direct anti-tumour effect in vivo in addition to effects on osteoclasts. To examine this further, the effect of ZOL on six myeloma cell lines, including the IL-6 dependent INA-6 line, was investigated. ZOL caused cell cycle arrest and concentration-dependent growth inhibition in all six cell lines, with varying sensitivity (IC50= 30–285 μM). The potential anti-tumour effect of ZOL on INA-6 cells in vivo was studied in a SCID mouse xenograft model, in which mice injected intraperitoneally with INA-6 cells develop plasmacytomas but do not develop significant osteolytic lesions. In several experiments involving more than 50 mice, ZOL was administered subcutaneously (sc) or intravenously (iv). ZOL, at a dose of 8 μg or 2 μg three times per week for two weeks after inoculation of INA-6 cells, significantly reduced tumour burden and increased survival of the mice (p= 0.002). The effect of ZOL on protein prenylation in plasmacytomas dissected from the mice was measured by western blotting to specifically detect the unprenylated form of the small GTPase Rap1A. Unprenylated Rap1A was virtually absent from tumour samples of untreated animals, while a single iv injection of 8 μg ZOL induced a marked accumulation of unprenylated Rap1A in the tumours after 24–72 hours. These studies are the first to demonstrate that ZOL can inhibit protein prenylation in plasmacytomas in vivo. Together with the decreased tumour burden and increased survival, the data suggest that ZOL may have direct anti-tumour effects in this animal model. Thus, nitrogen-containing bisphosphonates such as ZOL may have therapeutic potential in multiple myeloma beyond the prevention of osteolytic lesions.

Summary. Anti‐resorptive bisphosphonates, such as pamidronate, are an effective treatment for osteolytic disease and hypercalcaemia in patients with multiple myeloma, but have also been shown to cause apoptosis of myeloma cell lines in vitro. In this study, we found that a single infusion of pamidronate, in 16 newly diagnosed patients with multiple myeloma, caused a marked increase in apoptosis of plasma cells in vivo in 10 patients and a minimal increase in four patients (P < 0·05). The nitrogen‐containing bisphosphonates pamidronate and zoledronic acid also induced apoptosis of authentic, human bone marrow‐derived plasma cells in vitro. Apoptosis of plasma cells in vitro was probably caused by inhibition of the mevalonate pathway and loss of prenylated small GTPases, as even low concentrations (≥ 1 µmol/l) of zoledronic acid caused accumulation of unprenylated Rap1A in cultures of bone marrow mononuclear cells in vitro. GGTI‐298, a specific inhibitor of geranylgeranyl transferase I, also induced apoptosis in human plasma cells in vitro, suggesting that geranylgeranylated proteins play a role in signalling pathways that prevent plasma cell death. Our results suggest that pamidronate may have direct and/or indirect anti‐tumour effects in patients with multiple myeloma, which has important implications for the further development of the more potent nitrogen‐containing bisphosphonates, such as zoledronic acid, in the treatment of myeloma.