ALBERT

Description: Enhanced angiogenesis is a hallmark of cancer. Pleiotrophin (PTN) is an angiogenic factor that is produced by many different human cancers and stimulates tumor blood vessel formation when it is expressed in malignant cancer cells. Recent studies show that monocytes may give rise to vascular endothelium. In these studies, we show that PTN combined with macrophage colony-stimulating factor (M-CSF) induces expression of vascular endothelial cell (VEC) genes and proteins in human monocyte cell lines and monocytes from human peripheral blood (PB). Monocytes induce VEC gene expression and develop tube-like structures when they are exposed to serum or cultured with bone marrow (BM) from patients with multiple myeloma (MM) that express PTN, effects specifically blocked with antiPTN antibodies. When coinjected with human MM cells into severe combined immunodeficient (SCID) mice, green fluorescent protein (GFP)–marked human monocytes were found incorporated into tumor blood vessels and expressed human VEC protein markers and genes that were blocked by anti-PTN antibody. Our results suggest that vasculogenesis in human MM may develop from tumoral production of PTN, which orchestrates the transdifferentiation of monocytes into VECs.

Description: Abstract 455 Functional B cell and plasma cell immune responses are dependent upon an exquisitely controlled process integrating signals from activating and inhibitory receptors present on the surface of these cells. These activating and inhibitory signaling pathways regulate both the quality and quantity of immunoglobulin (Ig) production. These signaling motifs, termed immunoreceptor tyrosine-based activation motif (ITAM) and ITIM provide the basis for two opposed signaling modules that duel for control of plasma cellular activation within the immune system. The inhibitory Fc receptor FcγRIIb is expressed on plasma cells and controls their persistence in the bone marrow and their ability to produce most serum Ig. Activation of FcγRIIb leads to the phosphorylation of ITIM and recruitment of SH2-containing protein tyrosine phosphatase-1 (SHP-1) SHP-2 and the SH2-containing inositol 5-phosphatase (SHIP) in plasma cells. FcγRIIb-mediated SHIP phosphorylation results in enhanced degradation of PtdIns (3,4,5)P, which is required for B-cell antigen receptor (BCR)-induced recruitment and activation of downstream ITAM signaling molecules. The inhibitory IgG Fc receptor FcγRIIB was the first discovered and remains the best studied example of an ITIM-containing receptor. In this study, we first investigated the IgG-binding ability of 18 MM patients and 10 normal donors to FcγRIIb using flow cytometric analysis. Each serum sample was incubated with MHC1 cells that only express FcγRIIb but do not express FcγRI and FcγRIIa. After washing three times with 1 × PBS, anti-human IgG antibody conjugated with FITC was added to the cells for another 30 minutes. The results showed MM patients' serum IgG have much lower FcγRIIb-binding ability than normal human IgG (P

Description: Abstract 4068 Several members of the tumor necrosis factor receptor-associated factor (TRAF) family, including TRAF1, TRAF2, TRAF3, TRAF5, and TRAF6 have been implicated in regulating signal transduction from various TRAF family members. However, the unique biological function of TRAF6 is largely determined by its TRAF-C domain, which does not interact with peptide motifs that are recognized by TRAF1, -2, -3 or -5. We have reported inhibition of MM cell proliferation and increase of apoptosis through regulation of the NF-κB and JNK pathways through silencing TRAF6 C-domain mRNA and the dominant negative peptide expression vector (Chen H. et al, Oncogene, 2006; Li M. et al, Blood 2009). TRAF6 have been recently found as a ligase for Akt ubiquitination (Yang WL et al, Science, 2009). Akt signaling plays a central role in many biological functions, such as cell proliferation and apoptosis. In this study, we first investigated whether TRAF6 is over-expressed in MM tumor cells. Twelve MM fresh bone marrow (BM) aspirates derived from MM patients were assessed using Western blot analysis and immunohistochemical staining with anti-TRAF6 antibody. We found that TRAF6 protein was highly expressed in tumor cells from MM patients compared to normal human BM samples. Based on TRAF6, CD40, and RANKL sequences and crystal structures, we targeted the TRAF6 C-domain binding residues. We found that TRAF6 dominant negative binding peptide (TRAF6dn) significantly inhibited MM cell proliferation maximally at 72 hours using the MTS cell proliferation assay whereas effects on inducing MM cell apoptosis were most prominent at 48 hours as assessed with Annexin V staining with flow cytometric analysis. The decrease in cell proliferation and increase in cell apoptosis occurred in a concentration peptide-dependent fashion. Furthermore, phosphorylation of both AKT and NF-κB were also reduced using our human TRAF6dn or decoy peptides. We also examined the effect of the TRAF6dn peptide on the JNK pathway since this signaling pathway is also associated with cell cycle effects in MM. We measured JUN kinase kinase (JNKK), which activates the MAP kinase homologues SAPK and JNK in response to IL-1 receptor stimulation. The results showed that the phosphorylation of JNKK is markedly reduced after treatment with the TRAF6dn peptide. Furthermore, we examined c-Jun, a component of the transcription factor complex AP-1, which binds and activates transcription at TRE/AP-1 elements. We evaluated the effect of TRAF6dn peptide on osteoclast formation using cells from human monocytes isolated by anti-CD14 micro-bead affinity column from MM patients' BM or peripheral blood mononuclear cells. The monocytes were cultured on slide-culture dishes (2 × 105 cells/well).We found TRAF6dn markedly inhibited osteoclast cell formation from monocytes induced with RANKL and mCSF in a concentration- dependent fashion compared with a control group using tartrate resistant acid phosphatase staining. We further assessed whether TRAF6dn can reduce bone resorption using a dentin bone resorption assay. BM-derived monocytes were isolated as above and were cultured on dentin bone slides (4 × 105 cells/slide). The cells treated with a TRAF6dn peptide or the control peptide, were incubated with 50ng/ml RANKL and 10ng/ml MCSF. All cells were cultured for 21 days. It was found that TRAF6dn significantly inhibited lacunar resorption in a concentration-dependent fashion. These studies suggest that TRAF6 is over-expressed in MM and our TRAF6dn peptide inhibits many signaling pathways critical to the growth of MM and formation of osteoclasts resulting in marked anti-MM effects and reduction in osteoclast formation resulting in marked inhibition of bone resorption. Thus, this novel approach may offer a new therapeutic approach to both treat multiple myeloma and reduce the clinical consequences resulting from enhanced bone loss that commonly occur in these patients. Disclosures: No relevant conflicts of interest to declare.

Description: Objective and Rationale Yin-Yang 1 (YY1) is a multi-functional DNA-binding protein, which can activate, repress or initiate transcription depending on the context in which it binds. In addition, YY1 can modulate protein levels or activity through protein-protein interaction. YY1 has been identified as a potential repressor factor for several genes. We have reported that YY1 can act as a transcription repressor for both Fas and TRAIL DR5. In addition to YY1-mediated regulation of tumor cell resistance to cytotoxic immunotherapy, it also has been shown to regulate resistance to chemotherapy [Baritaki et al., J Immunol 80:6199,2008]. Overexpression of YY1 has been shown to be of prognostic significance in prostate cancer [Seligson et al., Int J Oncol 27:131,2005]. Hypothesis In this study, we have examined the expression of YY1 in MM (cell lines and patients’ bone marrow [BM]) by hypothesizing that the resistance of MM cells to various cytotoxic agents may be, in part, regulated by overexpression of YY1 and that YY1 may also be of prognostic significance. Designs and Methods MM cell lines and fresh BM samples from MM patients (n=21) were examined for YY1 expression, cytoplasmic and nuclear, by immunohistochemistry and by Western. The specificity of the anti-YY1 antibody was demonstrated by the competitive inhibition with a peptide used for immunization. Control isotype IgG did not show any staining on the cells. Results First, we found that various MM cell lines (RMPI 8226, IM-9, U266) overexpress YY1 both in the cytoplasm and the nucleus. Next, we examined the expression of YY1 in BM derived from MM patients by immunohistochemistry. BM from MM patients showed overexpression of YY1 as compared to normal bone marrow samples. Furthermore, analysis of both the intensity and frequency of cells expressing YY1 in both the cytoplasm and nucleus was shown to be significantly higher among patients with progressive disease as compared to patients with stable or responsive disease. Conclusions and Implications These findings show that the expression of YY1 among patients with MM may correlate with progression and also suggest the prognostic significance of YY1 in MM patients. Inhibition of YY1 by various agents (example: low-dose chemotherapy, proteasome inhibitors, NO donors, NF-kB inhibitors, etc.) all result in the reversal of resistance to various cytotoxic agents (eg CDDP, TRAIL) therefore, the findings also imply that agents that can inhibit YY1 expression in MM patients may be of therapeutic potential when used in combination with conventional therapeutics.

Description: Abstract 611 Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been implicated in regulating NF-κB and JNK signal transduction pathway resulting in inhibition of tumor cell proliferation and osteoclast formation. The unique biological function of TRAF6 is largely determined within its TRAF-C domain which does not interact with peptide motifs that are recognized by other TRAFs including 1, 2, 3 or 5. We have recently reported inhibition of cell proliferation and increased apoptosis of multiple myeloma (MM) cells through regulation of the NF-κB and JNK pathways through silencing the TRAF6 C-domain mRNA. In this study, we determined the effects of TRAF6 dominant negative peptides on MM cells, osteoclast formation and bone resorption. We cloned a 167 amino acid (in residues 333 to 508) fragment to produce a TRAF6 negative dominant (TRAF6dn) construct and synthesized an inhibitory decoy peptide of the TRAF6 interaction domain with CD40 and another peptide interacting with the TRAF6-RANK binding domain as well as a control peptide. All peptides were synthesized with a 16 amino acid permeable peptide. Using the MM1s, RPMI8226, and U266, we evaluated the effects of these peptides on MM tumor cell growth using an MTS assay and apoptosis with an Annexin V assay. We found that TRAF6dn peptides significantly inhibited MM cell proliferation maximally at 72 hours whereas effects on induction of apoptosis in MM cells were most prominent at 48 hours. The decrease in cell proliferation and increase in cell apoptosis occurred in a concentration-dependent fashion. We found that TRAF6dn also markedly inhibited osteoclast cell formation from freshly derived human monocytes induced by RANKL and M-CSF in a concentration-dependent fashion comparing with cells exposed to control peptide. We further examined the effects on MM cell apoptosis of the TRAF6 decoy or CD40 decoy peptides alone and in cells exposed to the combination of both peptides. The results showed either decoy peptide alone slightly induced apoptosis of MM tumor cells whereas the combination of both peptides demonstrated marked apoptosis of MM cells. We also showed that although melphalan alone induced apoptotic cell death, this effect was markedly enhanced when this alkylating agent was combined with the TRAF6 decoy peptide. Although the CD40 peptide alone did not inhibit osteoclast formation, TRAF6 decoy peptide alone and the combination of both decoy peptides markedly inhibited formation of these bone resorbing cells. We also examined the effects of TRAF6dn on NF-κB and JNK by measuring JUN kinase kinase (JNKK), which activates the MAP kinase homologues SAPK and JNK in response to IL-1 receptor stimulation. Phospho-NF-κB protein levels and phosphorylation of JNKK are both markedly reduced when MM cells are exposed to TRAF6dn fragment or TRAF6 decoy peptide. These studies suggest that TRAF6dn or the combination of TRAF6 decoy and CD40 decoy peptides may be excellent targets to block both myeloma cell and osteoclast cell formation. The study has been extended to assess the effects of these peptides in vivo using our SCID-hu murine model of human myeloma. Disclosures: No relevant conflicts of interest to declare.

Description: Multiple myeloma (MM) is a bone marrow-based malignancy characterized by expansion of plasma cells that produce monoclonal immunoglobulin. Although many new options are available to treat MM, patients develop resistance to these agents. Thus, new therapeutic options are necessary for patients. Vincristine has been used to treat MM but its neurotoxic side effects and low level of anti-MM activity have limited its clinical use. Pharmacokinetic studies on a different formulation of this vinca alkaloid, vincristine sulfate liposomes injection (VSLI, Marqibo®), have shown that the altered distribution and elimination phases with this drug may lead to increased exposure within the tumor versus traditional vincristine. Thus, this new agent offers the potential to both reduce the neurotoxicity and increase the anti-MM effects compared to standard vincristine. In the present study, we evaluated the anti-MM effects of Marqibo and vincristine using two severe combined immunodeficiency (SCID) murine models of human MM. These models were developed from intramuscular (i.m.) implantation of bone marrow biopsies from a MM patient before (LAGκ-1A) and following (LAGκ-1B) the development of both melphalan and bortezomib resistance clinically and have been successfully passaged. One week following implantation i.m., mice were treated with Marqibo, vincristine or vehicle alone. Marqibo was administered intravenously (i.v.) once weekly for three weeks at 0.5, 1, and 2.5 mg/kg and vincristine was given three times per week (M, W, F) i.v. at 0.3 mg/kg. Mice were bled in order to obtain sera to measure human immunoglobulin (Ig) G levels and tumor volume measured weekly. hIgG levels were measured by ELISA and tumor volume using standard calipers. Treatment of LAGk-1A–bearing mice with Marqibo at 0.5, 1 and 2.5 mg/kg inhibited paraprotein secretion as determined by hIgG levels (P = 0.0001; P = 0.0002; P = 0.0055, respectively) compared to mice receiving vehicle. A reduction in tumor volume, compared to control mice, was also observed in mice treated with Marqibo at the specified doses (P = 0.0001; P = 0.0001; P = 0.0001, respectively) and also when compared to vincristine (P = 0.0006; P = 0.0003; P = 0.0001, respectively). In fact, mice treated with vincristine showed no reduction in tumor volume compared to vehicle alone-treated mice. Marqibo at 0.5 mg/kg contained almost half the quantity of free vincristine compared to vincristine administered at 0.3 mg/kg, yet a significant inhibition of both human paraprotein secretion and reduction of tumor volume was observed in LAGκ-1A-bearing mice treated with this dose of Marqibo. The vincristine dose was administered three times more often than Marqibo, contained almost 100% more free vincristine compared to Marqibo at 0.5 mg/kg and most importantly was less effective. Mice bearing a slower growing MM tumor, LAGκ-1B, have recently begun treatment using the same doses and schedules as the LAGκ-1A study. This study is currently ongoing but preliminary data show inhibition of tumor volume growth and inhibition of IgG levels using Marqibo at 0.5, 1 and 2.5 mg/kg (hIgG; P = 0.011, P = 0.0045 and P = 0.0060, respectively). The results of this study show that Marqibo, a novel liposomal formulation of vincristine, produces anti-MM effects in this SCID-hu model of human MM whereas vincristine showed no anti-MM tumor volume growth activity. Further studies are currently being conducted to explore the optimal schedule and effects of this new agent in combination with other active MM agents. These studies should provide the rationale for further clinical development of Marqibo for the treatment of MM patients.

Description: We have recently shown that silencing of tumor necrosis factor receptor-associated factor 6 (TRAF6) with a C-terminal siRNA inhibits proliferation and increases apoptosis of multiple myeloma (MM) tumor cells. In addition, TRAF6 ubiquitin ligase is also essential for receptor activator of nuclear factor kappa B ligand (RANKL) signaling and osteoclast differentiation. Based on TRAF6, CD40, and RANKL sequences and the TRAF6 interaction domain with CD40 or RANKL resides between residues 333 to 508, we cloned a sequence representing a 167 amino acid sequence from this domain in order to produce a TRAF6 dominant negative fragment (TRAF6dn) from the NIH gene bank (U78798) into the PCRII-TOPO vector. Subsequently, we re-cloned this fragment into an expression vector (pLenti6.2-hTRAF6dn). Expression of the TRAF6 dominant negative peptide was confirmed by Western blot analysis. We used human MM monocytes isolated by anti-CD14 micro-bead affinity column from MM patients’ peripheral blood (PB) or bone marrow (BM). In order to quantify osteoclast formation, the cells were fixed and stained for tartrate resistant acid phosphatase following seven days of culture. The BM and PB CD14+ cells were cultured on slide-culture dishes at a density of 2 × 105 cells per well. The cells infected with the pLenti6.2-hTRAF6dn or with the control vector, pLenti6.2/GW/EmGFP, were treated with 50ng/ml RANKL and 10ng/ml mCSF at the beginning of the culture period, and these factors were added again during a medium change after three days of incubation. We found that the TRAF6dn vector significantly inhibited osteoclast cell formation of CD14+ cells induced by RANKL and mCSF in a concentration dependent fashion compared with the control group. Furthermore, we examined c-Jun, a component of the transcription factor complex AP-1, which binds and activates transcription at TRE/AP-1 elements. The results showed that total endogenous c-Jun is reduced after TRAF6dn blocks TRAF6 signaling whereas cells infected with the control vector showed no changes in c-Jun. We further examined the effects of TRAF6dn on MM cell growth and apoptosis. Both tumor cells from fresh MM BM and the U266 and MM1s cell lines showed decreased cell proliferation and increased apoptosis in the presence of the TRAF6dn vector at 72 hours whereas the control vector had no effect on MM tumor cell growth or apoptosis. Furthermore, the TRAF6dn vector led to marked decreases in phospho-NF-kB protein levels compared to the control vector. Thus, we have demonstrated that inhibition of TRAF6 with a dominant negative construct both inhibits MM cell growth as well as osteoclast formation, and also reduces NF-kB activation and c-Jun levels which likely results in decreased activation of TRE/AP-1 elements. These studies suggest that the inhibition of TRAF6 may be an excellent therapeutic target for multiple myeloma since its inhibition results in suppression of tumor growth as well as osteoclast formation.

Description: Rituximab (chimeric anti-CD20 mAb) has been used for the treatment of Non Hodgkin B cell lymphomas (B-NHL), alone or in combination with CHOP. However, a subset of patients does not respond to treatment or develops refractoriness to further treatments. Therefore, there is an urgent need to develop new alternatives to treat these patients. We have reported that treatment of B-NHL cell lines with rituximab inhibits anti-apoptotic survival pathways and down regulates the expression of anti-apoptotic Bcl-2 family proteins (i.e. Bcl-2/Bcl-xl) resulting in sensitization to chemotherapeutic drugs. Further, rituximab-resistant clones showed over-expression of anti-apoptotic gene products (Jazirehi et al., Cancer Research1:1270–81, 2007). Therefore, we hypothesize that inhibitors of anti-apoptotic Bcl-2 family may reverse the resistance to apoptotic stimuli. We examined chemical inhibitors that mimic natural ligands of the anti-apoptotic BH3-only proteins. GX15–070 (Gemin X Biotechnologies, Inc., Canada) inhibits Bcl-2 protein-protein interactions resulting in Bak and Bax oligomerization, release of cytochrome C, and activation of caspases (Shore and Viallet, Hematology, 2005; ASH, 226–230). Treatment of B-NHL cell lines (Raji, Ramos, 2F7, DHL-4) with subtoxic concentrations of GX15–070 (20–50 uM) resulted in inhibition of cell proliferation and subsequently induction of apoptosis as determined by TUNEL. There was a time and concentration-dependent effect of GX15–070 on cytostasis and apoptosis. Analysis of cells treated with GX15–070 by western blotting revealed that Bcl-2, Bcl-xl, and Mcl-1 protein expressions were significantly inhibited as compared to controls. The protein inhibition by GX15–070 was not expected and needs further investigation. We also examined the effect of combination treatment of GX15–070 with the chemotherapeutic drug CDDP and there was an additive or synergistic cytotoxic effect. Treatment of rituximab-resistant clones (generated from 2F7, Raji and Ramos) treated with GX15–070 resulted in significant inhibition of cell growth and apoptosis. The cytotoxicity of GX15–070 in the B-NHL cell lines was tumor specific, because treatment of human peripheral blood leukocytes from different donors did not show any cytotoxic effect. Likewise, treatment of nude mice with different concentrations of GX15–070 did not show any detectable toxicity. These findings demonstrate that GX15–070 is cytotoxic to various drug/rituximab-resistant B-NHL cell lines and is not toxic to normal human leukocytes. This study suggests that combination of GX15–070 with subtoxic concentrations of chemotherapeutic drugs may have additive/synergistic effects. The present findings support the potential therapeutic application of GX15–070 in the treatment of patients with B-NHL that are resistant to current therapies.

Description: There have been significant advances in the treatment of patients with B-NHL using combination of rituximab and CHOP. However, a subset of patients does not initially respond or develop resistance to further treatments; hence, the need for alternative therapies to overcome resistance. TRAIL and agonist DR4/DR5 monoclonal antibodies have been examined clinically against a variety of tumors in Phase I/II. However, the majority of B-NHL derived from patients and cell lines are resistant to TRAIL-induced apoptosis. Recent findings demonstrated that treatment of TRAIL-resistant-B-NHL with rituximab sensitizes the tumor cells to TRAIL apoptosis. The underlying mechanism of rituximab-induced sensitization to TRAIL, however, is not clear. We have recently reported that treatment of tumor cells with sensitizing agents (example CDDP, proteasome inhibitors) resulted in the reversal of resistance to TRAIL via induction of Raf-1 kinase inhibitor protein (RKIP) and demonstrated the pivotal role of RKIP in the regulation of tumor cell sensitivity to TRAIL. Hence, since rituximab induces the expression of RKIP in B-NHL, we determined the role of RKIP induction by rituximab in the sensitization of B-NHL to TRAIL apoptosis. Various B-NHL cell lines were used as models for study. Treatment of B-NHL cells with rituximab (20 ng/ml) and TRAIL (5–10 ng/ml) resulted in significant potentiation of apoptosis and synergy was achieved. Rituximab induced the expression of RKIP as determined by RT-PCR and western concomitantly with inhibition of NF-kB. The inhibition of NF-kB resulted in upregulation of RKIP expression and was mediated, in large part, by inhibition of the transcription repressor Snail (downstream of NF-kB). Further, RKIP-induced inhibition of NF-kB by rituximab resulted in downstream inhibition of the DR5 transcription repressor Yin Yang 1 (YY1) and concomitantly with the upregulation of DR5 expression. The role of RKIP induction by rituximab in the upregulation of DR5 and sensitization to TRAIL apoptosis was corroborated by the use of cells over expressing RKIP which were sensitive to TRAIL apoptosis in the absence of rituximab. Our findings reveal a novel mechanism of rituximab-induced sensitization of B-NHL to TRAIL apoptosis via inhibition of NF-kB and Snail and upregulation of RKIP and DR-5. The combination of rituximab and TRAIL may be effective in the treatment of B-NHL. Further, our studies suggest that agents other than rituximab that can induce RKIP can reverse resistance to TRAIL in B-NHL that are unresponsive to rituximab treatment.

Description: Recent studies suggest that zoledronic acid (ZOL) and other nitrogen-containing bisphosphonates (BPs) inhibit angiogenesis by reducing angiogenic factor production and signaling by these factors. However, few studies have addressed the potential role of BPs in blocking the formation of new vasculature or so-called vasculogenesis. Thus, we determined whether ZOL could impact this process using a chorioallantoic membrane (CAM) vasculogenesis model. First, fertilized chick eggs were incubated horizontally at 37.5°C in a humidified incubator and windowed on day 8. Another set of E8 chicken embryonic skins were cultured in insert dishes with different concentrations of ZOL (1.0 or 10 uM) or without drug treatment for 24 hours. The embryonic skins were transferred to CAM. Endothelial cells of CAM normally started to proliferate and migrate into feather buds after two days. Blood vessel formation was determined after four days of culturing under the microscope. We found that endothelial cell proliferation and migration of CAM was completely inhibited and development of embryonic skin buds was blocked by ZOL at 10 uM. At a lower concentration of ZOL (1.0 uM), the vasculogenesis was also decreased and embryonic skin buds were also reduced in size (but to a lesser extent than at the higher drug concentration) compared with a control group without ZOL. We also examined whether zoledronic acid affected development of embryonic feather buds (epithelial cell) directly. The results showed that embryonic feather bud growth was unaffected when buds were cultured on insert dishes with 10uM ZOL compared to buds without this drug. This suggests that ZOL blocks endothelial cell proliferation in CAM and migration but does not effect epithelial cell development. We further examined endothelial cell gene expression of the bud cells on CAM treated with and without zoledronic acid. We measured vascular endothelial growth factor receptor-2 (Flk-1) expression with Western blot analysis. The results showed that Flk-1 is markedly reduced after buds were treated with ZOL at 10 uM and there was also some reduction in the expression of Flk-1 at the lower concentration (1.0 um) of ZOL. To determine which signal transduction pathway(s) may be involved in blocking endothelial cell proliferation and migration by ZOL, we determined gene expression of β-catenin, Runx2, and smad7 by RT-PCR in the embryonic buds. Runx2 is a target of β-catenin /TCF1 for the stimulation of bone formation, and Smad7 gene expression is increased in human hematopoietic stem cells (HSCs) and is required for TGF-β-induced expression of β-catenin. The results showed that expression of β-catenin was down-regulated by ZOL whereas expression of Runx2 and smad7 was up-regulated by this bisphosphonate. These results suggest that ZOL blocks the β-catenin pathway, and also add to other studies suggesting the important role of this pathway in blood vessel development. We have also shown that zoledronic acid profoundly suppresses vasculogenesis, and this effect adds another potential mechanism by which this bisphosphonate may possess anti-tumor effects.