μM Zoledronic Acid Research Articles

An invitro study on the effects of photobiomodulation by diode lasers (red, infrared, and red-infrared combination) on periodontal ligament mesenchymal stem cells treated with bisphosphonates.

This study evaluated the effect of photobiomodulation therapy (PBMT) using 660 and 808 nm diode lasers (individual and in combination) on periodontal ligament mesenchymal stem cells (PDLSCs) in the presence of zoledronic acid (ZA). PDLSCs were cultured for 48 h in DMEM complete medium containing 5 μM ZA. PBMT was done three times with a 24-h interval in groups 1 (660 nm, 5 J/cm2), 2 (880 nm, 3 J/cm2), and 3 (660 + 808 nm) either in normal or ZA-treated culture medium. Control groups did not receive PBMT. Twenty-four hours post-irradiation, cell proliferation and expression of RANKL and OPG were assessed using MTT and real-time PCR tests, respectively. The results showed a significant decrease in cell viability in ZA-treated cells (p < 0.001). Additionally, ZA induced the expression of OPG (p = 0.03) while reducing RANKL (p < 0.001). Cell proliferation was significantly increased in 808 and 660 + 808 nm groups. Moreover, all PBMT groups could significantly increase and decrease the RANKL and OPG, respectively, in the presence of ZA (all p < 0.001). A combination of 660 + 808 nm showed the highest effects on both genes. In conclusion, it seems that PBMT can modulate the effects of ZA by inducing PDLSC proliferation and increasing RANKL-to-OPG gene expression ratio.

Bisphosphonates attenuate age-related muscle decline in Caenorhabditis elegans.

Age-related muscle decline (sarcopenia) associates with numerous health risk factors and poor quality of life. Drugs that counter sarcopenia without harmful side effects are lacking, and repurposing existing pharmaceuticals could expedite realistic clinical options. Recent studies suggest bisphosphonates promote muscle health; however, the efficacy of bisphosphonates as an anti-sarcopenic therapy is currently unclear. Using Caenorhabditis elegans as a sarcopenia model, we treated animals with 100nM, 1, 10, 100 and 500μM zoledronic acid (ZA) and assessed lifespan and healthspan (movement rates) using a microfluidic chip device. The effects of ZA on sarcopenia were examined using GFP-tagged myofibres or mitochondria at days 0, 4 and 6 post-adulthood. Mechanisms of ZA-mediated healthspan extension were determined using combined ZA and targeted RNAi gene knockdown across the life-course. We found 100nM and 1μM ZA increased lifespan (P<0.001) and healthspan [954±53 (100nM) and 963±48 (1μM) vs. 834±59% (untreated) population activity AUC, P<0.05]. 10μM ZA shortened lifespan (P<0.0001) but not healthspan (758.9±37 vs. 834±59, P>0.05), whereas 100 and 500μM ZA were larval lethal. ZA (1μM) significantly improved myofibrillar structure on days 4 and 6 post-adulthood (83 and 71% well-organized myofibres, respectively, vs. 56 and 34% controls, P<0.0001) and increased well-networked mitochondria at day 6 (47 vs. 16% in controls, P<0.01). Genes required for ZA-mediated healthspan extension included fdps-1/FDPS-1 (278±9 vs. 894±17% population activity AUC in knockdown+1μM ZA vs. untreated controls, respectively, P<0.0001), daf-16/FOXO (680±16 vs. 894±17%, P<0.01) and agxt-2/BAIBA (531±23 vs. 552±8%, P>0.05). Life/healthspan was extended through knockdown of igdb-1/FNDC5 (635±10 vs. 523±10% population activity AUC in gene knockdown vs. untreated controls, P<0.01) and sir-2.3/SIRT-4 (586±10 vs. 523±10%, P<0.05), with no synergistic improvements in ZA co-treatment vs. knockdown alone [651±12 vs. 635±10% (igdb-1/FNDC5) and 583±9 vs. 586±10% (sir-2.3/SIRT-4), both P>0.05]. Conversely, let-756/FGF21 and sir-2.2/SIRT-4 were dispensable for ZA-induced healthspan [630±6 vs. 523±10% population activity AUC in knockdown+1μM ZA vs. untreated controls, P<0.01 (let-756/FGF21) and 568±9 vs. 523±10%, P<0.05 (sir-2.2/SIRT-4)]. Despite lacking an endoskeleton, ZA delays Caenorhabditis elegans sarcopenia, which translates to improved neuromuscular function across the life course. Bisphosphonates might, therefore, be an immediately exploitable anti-sarcopenia therapy.

Zoledronic acid affects the process of Porphyromonas gingivalis infecting oral mucosal epithelial barrier: An in-vivo and in-vitro study.

Zoledronic acid (ZA), one of the commonly used bisphosphonates, is mainly used for bone-metabolic diseases. Studies proved that ZA has adverse effects on oral soft tissues. As the first line of innate immunity, the gingival epithelium could be infected by periodontal pathogens, which is a key process of the initiation of periodontal diseases. Yet, how ZA affects the periodontal pathogens infecting the epithelial barrier remains unclear. This study aimed to investigate the influences of ZA on the process of Porphyromonas gingivalis (P. gingivalis) infecting the gingival epithelial barrier via in-vitro and in-vivo experiments. In the in-vitro experiments, under the condition of different concentrations of ZA (0, 1, 10, and 100 μM), P. gingivalis was used to infect human gingival epithelial cells (HGECs). The infections were detected by transmission electron microscope and confocal laser scanning microscope. Besides, the internalization assay was applied to quantify the P. gingivalis, which infected the HGECs, in the different groups. To evaluate the expression levels of pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6, and IL-8, by infected HGECs, real-time quantitative reverse transcription-polymerase chain reactions were applied. In the in-vivo experiments, rats were given ZA solution (ZA group) or saline (control group) by tail intravenous injection for 8 weeks. Subsequently, we put ligatures around the maxillary second molars of all the rats and inoculated P. gingivalis to the gingiva every other day from day 1 to day 13. The rats were sacrificed on days 3, 7, and 14 for micro-CT and histological analyses. The in-vitro results manifested that the quantity of P. gingivalis that had infected HGECs increased with the ZA concentrations. Pro-inflammatory cytokines expression by HGECs were significantly increased by 100 μM ZA. In the in-vivo study, compared to the control group, more P. gingivalis was detected in the superficial layer of gingival epithelium in the ZA group. Besides, ZA significantly increased the expression level of IL-1β on day 14 and IL-6 on days 7 and 14 in gingival tissues. These findings suggest that the oral epithelial tissues of patients who receive high-dose ZA treatment may be more susceptible to periodontal infections, resulting in severe inflammatory conditions.

OPTIMIZED PROTOCOLS OF EXPANSION OF PERIPHERAL BLOOD-DERIVED δT-CELLS FOR THE ESTABLISHMENT OF AN OFF-THE-SHELF ADVANCED CELL THERAPY AGAINST CANCER

The use of autologous T cells expressing chimeric antigen receptors (CAR) has revolutionized the treatment of hematological malignancies. However, the use of allogeneic CAR-T cells offers many advantages over autologous therapies, including the immediate availability of cryopreserved batches for infusion and the use of healthy donor cells since it is possible to obtain high amounts of fully functional cells. Moreover, this strategy also allows for the scaling up of the production of CAR-T cells resulting in cost reduction. Cells that do not express TCR αβ, such as gamma delta (γδ) T, are an alternative source to allow widespread allogeneic therapy possible. Their ability to recognize targets in an MHC-independent manner and the expression of innate immunity receptors increases the potential of using these cells in immunotherapies. Aiming to improve the effectiveness of γδ T-cell-based immunotherapies against tumors, we tested various optimization strategies. The most widely used protocol for selectively expanding Vδ2T cells relies on zoledronic acid (ZOL) stimulation of PBMCs in combination with IL-2 or/and IL-15 cytokines. Here, we tested different concentrations of IL-2 (100 and 1,000 U/mL) and IL-15 (12.5 and 100 ng/mL) with 5 μM zoledronic acid. In addition, we compared cell expansion in three defined cell culture media, OpTmizer T Cell Expansion SFM, X-vivo10, and TexMACS (supplemented with 2% AB serum). A total of twenty-four different combinations of IL-2, IL-15, and media were tested in the present study.After twelve days of culture, cells were counted and labelled with anti-CD3, anti-CD8, anti-CD4, anti-Vδ2, anti-PD1, anti-CD69, and anti-NKG2D antibodies for flow cytometry analysis. Our results demonstrated that although TexMACS medium is capable of supporting T-cell activation and expansion, it was not optimal for maximal expansion of Vδ2T cells (fold expansion 5.99 ± 1.61). However, the fold expansion was similar in OpTmizer and X-vivo10 media (fold expansion 21.38 ± 5.13 and 18.37 ± 5.42, respectively). Among the medium supplementation strategies, cultivation in OpTmizer combined with 100 U/mL IL-2 + 12.5 ng/mL IL-15 led to highest expansion index (32.4-fold). As for the activation markers of Vδ2T cells, the frequency of NKG2D+ cells was similar for all conditions tested (above 85%). However, the percentage of CD69+ cells was higher in cells cultured in TexMACS medium compared to OpTmizer and X-vivo10 media (81.6±5.3, 69±9.9, 60.2±7.2, respectively). It is known that the Programmed Cell Death-1 receptor (PD-1) plays an important role in regulating immune cell function. We examined the expression of PD-1 after twelve days of expansion. We observed lower levels of PD-1 in cells expanded in X-vivo10 medium supplemented with 1,000 U/mL IL-2 + 100 ng/mL IL-15, whereas levels of expression were up-regulated significantly when cells were cultivated in the same conditions without IL-15 (28.7% versus 70.6%). Cells expanded in either OpTimizer or TexMACS demonstrated a similar frequency to PD-1 positive cells (41±13% and 49.7%±9.5). In conclusion, we reported an optimized protocol to improve the expansion and the quality of Vδ2 T cells. These findings set the groundwork for the establishment of an off-the-shelf advanced cellular immunotherapy using this alternative population of T cells.

Zoledronic Acid Is Not Equally Potent on Osteoclasts Generated From Different Individuals.

ABSTRACTZoledronic acid is a bisphosphonate commonly used to treat bone diseases such as osteoporosis and cancer‐induced bone disease. Patients exhibit a variable sensitivity to zoledronic acid; the underlying explanation for this remains unclear. The objective of this study was to obtain more knowledge in this regard. We hypothesized that osteoclasts generated from different individuals would show a variable sensitivity to zoledronic acid in vitro. Osteoclasts were generated using monocytes from 46 healthy female blood donors (40 to 66 years). Matured osteoclasts were reseeded onto bone slices precoated with different concentrations of zoledronic acid. IC50 values were determined based on total eroded bone surface after 3 days of resorption. The IC50 for inhibition of osteoclastic bone resorption varied from 0.06 to 12.57μM zoledronic acid; thus, a more than 200‐fold difference in sensitivity to zoledronic acid among osteoclasts from different individuals was observed. Multiple linear regression analyses showed that the determined IC50 correlated with smoking status, and the average number of nuclei per osteoclast in vitro. Further analyses showed that: (i) increasing protein levels of mature cathepsin K in osteoclast cultures rendered the osteoclasts less sensitive to zoledronic acid; (ii) surprisingly, neither the gene nor the protein expression of farnesyl diphosphate synthase was found to correlate with the IC50; and (iii) trench‐forming osteoclasts were found to be more sensitive to zoledronic acid than pit‐forming osteoclasts within the same cell culture. Thus, we conclude that there indeed is a high degree of variation in the potency of zoledronic acid on osteoclasts when generated from different individuals. We propose that our findings can explain some of the varying clinical efficacy of zoledronic acid therapy observed in patients, and may therefore be of clinical importance, which should be investigated in a clinical trial combining in vitro and in vivo investigations. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Synthetic Hydroxyapatite Inhibits Bisphosphonate Toxicity to the Oral Mucosa In Vitro.

Medication-related osteonecrosis of the jaw (MRONJ) is a side effect of bisphosphonate therapy, characterised by exposed necrotic bone. The soft tissues of the oral mucosa no longer provide a protective barrier and MRONJ patients experience pain, infections and difficulties eating. We hypothesised that hydroxyapatite (Ca5(PO4)3(OH)) could reduce bisphosphonate concentrations and protect the oral mucosa by exploiting bisphosphonate’s calcium binding affinity. The effect of zoledronic acid (ZA) and pamidronic acid (PA) on the metabolism of oral fibroblasts, oral keratinocytes and three-dimensional oral mucosa models was investigated and then repeated in the presence of hydroxyapatite granules. Without hydroxyapatite, ZA and PA significantly reduced the metabolic activity of oral cells in a dose-dependent manner. Both drugs reduced epithelial thickness and 30 µM ZA resulted in loss of the epithelium. Hydroxyapatite granules had a protective effect on oral cells, with metabolic activity retained. Oral mucosa models retained their multi-layered epithelium when treated with ZA in the presence of hydroxyapatite granules and metabolic activity was comparable to controls. These results demonstrate hydroxyapatite granules protected oral soft tissues from damage caused by bisphosphonate exposure. Porous hydroxyapatite granules are currently used for socket preservation and this data suggests their potential to prevent MRONJ in at-risk patients.

Effect of photobiomodulation on the viability of osteoblasts and fibroblasts submitted to alendronate sodium or zoledronic acid

The goal of this study is to evaluate the effect of photobiomodulation therapy (PBMT) on the viability of osteoblasts and cultured fibroblasts in different concentrations of alendronate or zoledronic acid. Two cell lines: osteoblast-like mouse cells (OSTEO 1) and human buccal mucosa fibroblast (FMM1) were used. Cells were submitted to different concentrations of bisphosphonates (1 μM, 10 μM, and 100 μM sodium alendronate and 3 μM, 5 μM and 10 μM zoledronic acid) for 24 hours. Next, the cultures received PBMT. The irradiations were applied with a diode laser (InGaAIP, 660 nm, 30 mW, spot 0.028 cm2) in continuous, punctual and contact mode at two energy densities: 5 J/cm2 (4.5 s) or 10 Jcm2 (9s) with 6 hours-intervals. Cell viability was determined by mitochondrial activity assay (MTT) 24 h after the last irradiation. The data were compared by the one way- ANOVA, complemented by the Tukey’s test (p &lt; 0.05). Sodium alendronate at concentrations of 100 μM and 10 μM and zoledronic acid at 10 μM concentration showed higher long-term toxicity. The cellular viability of the PBMT treated group was significantly higher than that of the negative control group. The same occurred with the osteoblasts treated with the highest concentrations of the drug (5 and 10 μM), despite not reaching the cell viability of the positive control group, it presented greater viability than the negative control where the cells were not irradiated. In the groups submitted to zoledronic acid, positive controls presented greater cell viability. We concluded that under the parameters applied in this study, PBMT at an energy density of 5 J/cm2 was able to revert the toxicity of sodium alendronate applied at the higher concentrations in both cell types, whereas zoledronic acid toxicity, regardless of its concentrations, was not influenced by PBMT.

Zoledronic acid and bone cellular respiration

Phosphorescence O2 analyzer was used to measure calvarial bone cellular respiration (cellular mitochondrial O2 consumption) in Taylor Outbred mice in the presence and absence of zoledronic acid. This potent bisphosphonate inhibits osteoclast-mediated calcium resorption, and its effects on bone respiration have not been previously investigated. The change of O2 concentration with time was measured in closed vials containing phosphate-buffered saline (PBS), 5mM glucose and 5-25mg calvarial bone fragments, and it was complex for t=0-30h. Cyanide (specific inhibitor of cytochrome oxidase) halted O2 consumption, confirming the oxidation occurred in the respiratory chain. Initial rate of respiration was estimated from the zero-order plots d[O2]/dt for t=0-4h. For untreated specimens, the rate (mean±SD) was 2.0±1.2µM O2h-1mg-1 (n=6). This value was 7-10 times lower than that of other murine organs, but similar to that reported for rat and Guinea pig calvaria (averaging, 2.7nmolO2h-1mg-1). The corresponding rate in the presence of 10-100µM zoledronic acid was 2.7±0.7µM O2h-1mg-1 (n=11), p=0.216. The first-order plots ln ([O2] t ÷[O2] t=0) versus time for t=0-30h were also used to compare treated and untreated specimens. The rate (h-1mg-1103) for specimens incubated in PBS without glucose was 1.3±0.6 (n=3, p=0.007), in PBS+glucose it was 10.7±6.9 (n=10), in PBS+glucose+10µM zoledronic acid it was 12.1±6.7 (n=10, p=0.579), in PBS+glucose+20µM zoledronic acid it was 12.9±3.3 (n=9, p=0.356), and in PBS+glucose+100µM zoledronic acid it was 13.7±7.7 (n=9, p=0.447). Thus, exposure to high-doses of zoledronic acid over several hours imposed a statistically insignificant increase in calvarial bone cellular respiration.

ATP-Binding-Cassette A1 Regulates Extracellular Isopentenyl Pyrophosphate Release and Vγ9Vδ2 T-Cell Activation By Dendritic Cells

IntroductionPhosphorylated metabolites of mevalonate (Mev) pathway like isopentenyl-pyrophosphate (IPP) induce proliferation of Vγ9Vδ2 T cells. Endogenous IPP levels can be lowered or increased by aminobisphosphonates (NBPs) such as zoledronic acid (ZA). We have previously shown that ZA-treated dendritic cells (DC) produce and release high amounts of soluble IPP and are strong Vγ9Vδ2 T-cell activators. However, the mechanisms regulating IPP release in DC and other cells are still unknown.Methods"DC generation": purified CD14+ cells were cultured in standard culture medium at 0.5-1.5 x 106cells/ml supplemented with GM-CSF (1000 U/ml) and IL-4 (500 U/ml) in flat-bottomed 24-well plates for 24 h. After 24 h, DC were left untreated (ZA-) or treated for further 24 h with 1 µM ZA (ZA+). DC were washed after incubation with ZA and re-plated with fresh medium for an additional 24 and 48 h (washed DC [w-DC]). In selected experiments, supernatants were collected for quantification of extracellular IPP levels."Vγ9Vδ2 T-cell proliferation":on day 7, total counts of viable T cells were calculated with the trypan blue staining assay, and by flow cytometry. In selected experiments, PBMC from CTRL were stimulated for 7 days with supernatants obtained from autologous DCZA- and DCZA+and 10 IU/ml IL-2 to evaluate Vγ9Vδ2 T-cell proliferation."IPP efflux":cells were radiolabelled with 1 microCi/ml [3H]-acetate; after 24 h, cell supernatants were collected, [3H]-IPP was extracted and resolved by thin layer chromatography. After separation, the spot corresponding to IPP was cut and solubilized, and the radioactivity was quantified by liquid scintillation count."siRNA ABCA1": cells were incubated 96 h with a 20-25 nucleotide non targeting scrambled siRNA or specific ABCA1 siRNA (Accell Thermo Scientific), then lysed and subjected to the Western blot analysis of ABCA1 expression."Proximity ligation assay (PLA)":the ABCA1-BTN3A1 interaction was measured by the PLA method with the DuoLink In Situ kit (Sigma Chemicals Co.), using a mouse anti-human ABCA1 (Abcam) and a rabbit anti-human BTN3A1 (Sigma Chemicals Co.) antibody, respectively, as per the manufacturer's instructions. Nuclei were counterstained with 4',5-diamidino-2 phenylindole dyhydrochloride (DAPI). Cells were examined using a Leica TCS SP2 AOP confocal laser-scanning microscope (Leica Microsystem, Wetzlar, Germany).ResultsWe tested a variety of tumoral and non-tumoral cells for their ability to activate Vγ9Vδ2 T cells and correlated this ability with the release of extracellular IPP. We used samples from healthy donors included peripheral blood (PB) monocytes, monocyte-derived DCs, and bone marrow (BM) stromal cells; samples from cancer patients included myeloma cells, chronic lymphocytic leukemia (CLL) cells, and BM stromal cells from multiple myeloma (MM) and CLL patients; tumor cell lines included the monocytic THP1 and histiocytic U937 cells and the myeloma cell line SKMMI. We found that the activation of Vγ9Vδ2 T cells was proportional to the simultaneous efflux of IPP and to the activity/expression of ATP-binding cassette transporter A1 (ABCA1), a membrane transporter involved in the delivery of intracellular cholesterol to apolipoprotein A-I.We have demonstrated that the most potent IPP releaser cells had also the highest expression of ABCA1. Functional ABCA1 inhibition with probucol slightly increased intracellular IPP accumulation, but abrogated extracellular IPP release and Vγ9Vδ2 T-cell activation. Abca1 silencing experiments confirmed the key role played by ABCA1. ABCA1 is up-regulated by ZA viaLXRα transcriptional activation induced by intracellular IPP accumulation and inhibition of the PI3K/Akt/mTOR signaling pathway.Butyrophilin-3A1 (BTN3A1) is an immunoglobulin superfamily protein reported to be required for phosphoantigen presentation and target recognition by Vγ9Vδ2 T cells. We have also demonstrated that BTN3A1 is physically associated with ABCA1 and cooperates with ABCA1 in the activation of Vγ9Vδ2 T cells.ConclusionsThese results suggest that ABCA1 is involved in extracellular IPP release and Vγ9Vδ2 T-cell activation induced by ZA-treated DC and therefore play a crucial role as mediator of the immune response. DisclosuresBoccadoro:SANOFI: Honoraria, Research Funding; CELGENE: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Abbivie: Honoraria; Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Mundipharma: Research Funding; Amgen: Honoraria, Research Funding. Massaia:Roche: Other: advisory board, research support; Gilead: Other: advisory board; Janssen: Other: advisory board.

Pretreatment with Bisphosphonate Enhances Osteogenesis of Bone Marrow Mesenchymal Stem Cells

Mesenchymal stem cell (MSC)-mediated bone regeneration is used to replace lost bone. However, methods to accelerate the process and stabilize regenerated bone remain limited. Therefore, we investigated the effect of bisphosphonates (BPs) on the function of bone marrow mesenchymal stem cells (BMMSCs) to determine if they might enhance MSC-mediated bone regeneration. We isolated and cultured BMMSCs from BALB/c mice and treated the cells with 0.1, 0.5, 1, 5, or 10 μM zoledronic acid (ZA; Zometa, a commercially available BP). ZA had a dose-dependent effect on BMMSCs proliferation and osteogenesis. ZA at concentrations of 5 and 10 μM inhibited the proliferation and osteogenic differentiation of BMMSCs. By contrast, in addition to inducing the proliferation and osteogenesis of BMMSCs, 0.5 μM ZA upregulated expressions of the osteogenesis-related genes Alp, osterix (Osx), and bone sialoprotein (Bsp) and enhanced osteogenesis in vivo when ZA-treated BMMSCs were implanted subcutaneously in nude mice. In addition, 0.5 μM ZA increased expression of Opg in BMMSCs, decreased the Rankl/Opg ratio, and decreased the number of osteoclasts. However, it was not associated with adverse effects on numbers of regulatory T cells or levels of Th17, transforming growth factor-β1 (TGF-β1), and interleukin-17a (IL-17a) when cocultured with T cells. In conclusion, 0.5 μM ZA pretreatment enhanced the proliferation and osteogenesis of BMMSCs in vitro and in vivo and decreased the number of osteoclasts without impairment of BMMSCs immunomodulatory properties. In vitro pretreatment of BMMSCs with BP and subsequent implantation may be a safe and effective way of enhancing MSC-mediated bone regeneration.

Effects of zoledronic acid on osteoblasts in three-dimensional culture

Background/purposeBisphosphonates (BPs) are synthetic drugs with antitumor and bone antiresorptive activities. The use of BPs is suspected to favor the emergence of osteonecrosis of the jaw (ONJ), a putative side effect whose pathogenesis remains unclear. Thus, we aimed to get insights about the impact of BPs on osteoblasts functions, using a three-dimensional (3D) culture model, which is suggested to be more similar to the in vivo tissues than monolayers. Materials and methodsEffects of low (0.1 μM) and high (10 μM) concentrations of zoledronic acid were investigated on osteoblasts (hFOB 1.19), cultured as multicellular spheroids (MCS). Proliferation, apoptosis, spheroid growth kinetics, and morphology were studied using the 3-(4,5-dimethyl-thiazoyl-2yl) 2,5-diphenyl-tetrazolium bromide (MTT) and acid phosphatase (APH) assays, caspase 3 Western-blotting, phase contrast imaging and scanning electron microscopy (SEM). ResultsProliferation, apoptosis, and spheroid morphology showed that 10 μM zoledronic acid (ZA) induced a significant reduction in the relative viable cell number, correlated with morphological alterations of spheroids, and induction of apoptosis. A lower ZA concentration (0.1 μM) promoted cell proliferation without affecting growth kinetics or spheroid morphology. ConclusionZA sensitivity of osteoblasts depends on concentration and experimental models. The dual dose-dependent effects of ZA on osteoblasts cultured as spheroids, thereby promoting or inhibiting cell proliferation, may provide opportunities in tissue engineering. At last, the hFOB spheroid culture system represents a valuable model for the exploration of the molecular basis of BPs action on osteoblasts and for the development and evaluation of implantable biomaterials in bone sites.

Cytotoxic Effects of Zoledronic Acid on Human Epithelial Cells and Gingival Fibroblasts

Bisphosphonate-induced osteonecrosis has been related to the cytotoxicity of these drugs on oral mucosa cells. A previous study showed that 5 µM of zoledronic acid (ZA), a nitrogen-containing bisphosphonate, is the highest concentration of this drug found in the oral cavity of patients under treatment. Therefore, in order to simulate an osteonecrosis clinical condition, the aim of this study was to evaluate the highest concentration of ZA applied on human epithelial cells (HaCaT) and gingival fibroblasts. For this purpose, cells (3 × 10(4) cells/cm2) were seeded in wells for 48 h using complete culture medium (cDMEM). After 48 h incubation, the cDMEM was replaced by fresh serum-free culture medium (DMEM-FBS) in which the cells were maintained for additional 24 h. Then, 5 µM ZA were added to the DMEM-FBS and the cells incubated in contact with the drug for 48 h. After this period, the number of viable cells (trypan blue), cell viability (MTT assay), total protein (TP) production and cell morphology (SEM analysis) were assessed. Data were analyzed statistically by Mann-Whitney, ANOVA and Tukey's test (α=0.05). ZA caused a significant reduction in the number of viable cells and decreased the metabolic activity of both cell lines. However, decrease of TP production occurred only in the epithelial cell cultures. Morphological alterations were observed in both cell types treated with ZA. In conclusion, ZA (5 µM) was cytotoxic to human epithelial cells and gingival fibroblast cultures, which could be associated, clinically, with the development of bisphosphonate-induced osteonecrosis.

Exposing human epithelial cells to zoledronic acid can mediate osteonecrosis of jaw: an in vitro model

Osteonecrosis of the jaw (ONJ) is a chronic complication of bisphosphonate therapy, mainly when intravenous, in cancer patients with bone metastases and myeloma. Its pathophysiology is not yet fully elucidated; in particular, the molecular/cellular events triggering ONJ remain unclear. This complication could result from the effect of bisphosphonates released from bone into the soft-tissues, or from osteolysis induced by soft-tissues directly exposed to bisphosphonates. This research investigated the possibility that ONJ may be evocated by changes induced in osteoblast activity by factors released by soft-tissue cells exposed to zoledronic acid. An 'in vitro' model was used, in which human osteoblast-like MG-63 cells were grown in medium conditioned by human keratinocytes NCTC 2544, exposed or not to zoledronic acid (5 or 50 μM); 5 μM zoledronic acid was also directly administered to MG-63 cells. In NCTC 2544 cells, zoledronic acid decreased proliferation via decreased hydroxy-3-methyl-glutaryl-CoA reductase, suggesting that a decrease in healing capability can occur in case of injury. An increased pro-inflammatory potential was also observed. Osteoblasts grown in medium conditioned in the presence of zoledronic acid showed decreased proliferation and osteogenic properties, and increased ability to induce osteoclast differentiation and inflammatory process. Zoledronic acid directly administered to MG-63 modulated only some parameters and in a lesser extent. The research evidenced, for the first time, the direct involvement of epithelial cells in zoledronic acid-triggered molecular mechanisms leading to osteonecrosis of the jaw, by modulating both osteoblast and osteoclast properties.

P1-01-04: Immunological Effects of Bisphosphonates on gdT Cells in Breast Cancer.

Abstract Background: Human γδT cells play an important role in the first-line of defence. Accumulating data demonstrate that γδ T cells recognize nonpeptide antigens such as IPP and HMBPP and activated γδT cells have potent cytotoxic activity against tumor cells. Nitrogen-containing bisphosphonates like zoledronic acid inhibit farnesyl diphosphate synthetase, resulting in the accumulation of upstream metabolites such as IPP. Previous studies on the adoptive transfer of ex-vivo amplified γδ T cells demonstrated the safety and feasibility of γδT cell-based immunotherapy for metastatic cancer. According to ABCSG-12 clinical trial, zoldedoric acid improved DFS of patients with early-stage breast cancer under adjuvant endocrine therapy. Whereas a precise mechanism remained unclear, it is likely that γδT cells are involved in this anti-tumor effect in breast cancer. Materials and Methods: Peripheral blood mononuclear cells derived from 63 (54 untreated non-metastatic and 9 metastatic) patients with breast cancer were examined for the baseline population of γδT cells and their proliferative responses to zoledronic acid in vitro. Tumor-infiltrating lymphocytes were isolated from 20 tumor biopsies and 2 pleural effusions. Peripheral blood γδT cells were stimulated with 5 μM zoledronic acid and cultured in the presence of 100 IU/ml IL-2 for 10 days. Amplified γδ T cells were analyzed for cytokine production and cytotoxic activity. Results: The proportion of γδ (TCRVδ2 -bearing) T cells among peripheral blood CD3+ cells was 4.2 ± 5.0%. Although it was equivalent to the previously reported value of 4.3 ± 2.2% in healthy volunteers, 14% (9/63) of patients exhibited an increase in the number of peripheral blood γδT cells at baseline. The level of TCRVδ2 T cells with potent anti-tumor activity was significantly higher than that of TCRVδ1 T cells (4.2± 5.0% vs. 1.5 ± 1.8%, p&amp;lt;0.001), whereas they were present in tumor-infiltrating cells at essentially the same levels (4.4± 9.6% vs. 3.7 ± 9.7%, p=0.434). Percentage of TCR Vδ2+ CD3+ cells among amplified γδ T cells in vitro and cytokine (IFN-γ and TNF-α) production by them were significantly proportional to the initial level of TCRVδ2+/CD3+ ratio in peripheral blood. Upon treatment with zoledronic acid, breast cancer cell lines became susceptible to γδ T cells. Conclusions: γδ (TCR Vδ2-bearing) T cells increased in some patients with breast cancer and the TCRVδ2+/CD3+ ratio at baseline was correlated with the efficiency of proliferation and cytotoxic activity when treated with zoledronic acid. Zoledronic acid efficiently sensitized breast tumor cells to be lyzed by γδ T cells. The present results suggest that γδ T cells stimulated with zoledronic acid can be utilized for immunotherapy against breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-01-04.

Differential Effect Of Zoledronic Acid On Quiescent Vs. Proliferating Vascular Smooth Muscle Cells

Introduction: Restenosis following arterial interventions is primarily due to phenotypic alteration vascular smooth-muscle cells. Zoledronic Acid (ZA), the most potent member of the nitrogen containing Bisphosphonates (BP), has proven to be effective in treating bone resorption and bone malignancies in humans. BP are known to exert their effects by inhibiting the post-translational modification of the Ras-related GTP binding proteins (e.g. Rap1A) by an isoprenoid lipid (prenylation) which are part of the mevalonate pathway. These experiments were designed to compare the relative sensitivity and the dose response of actively proliferating vs. quiescent cultured human vascular smooth cells to ZA treatment. Finally, to test whether ZA will modulate the prenylation of Rap-1A, an important mediator of multiple different cellular activities. Methods: Human aortic smooth muscle cells (HASMC) were cultured under growth condition until they reached ∼60% confluence. Cells were switched to quiescent media for 48 hours to achieve growth arrest. After which, cells were divided into two groups; proliferating cells group which was fed with growth media, or the quiescent cells group which continued to be fed with quiescent media. Both groups were supplemented with 0, 1, 10, 100μM ZA. Relative metabolic activity and cytotoxicity were measured by WST-1 and Cyto-tox fluor assays respectively at 48 hours. The effect of ZA on proliferating vs. quiescent HASMC migration was assessed by modified Boyden chamber. The relative prenylation of Rap-1A protein (index of mevolanate pathway activity) was evaluated by western blotting. Data was expressed as percent of the untreated control cells (0 μM ZA) from the same group. Results: Proliferating HASMC treated with 1μMZA did not differ in their metabolic activity or cytotoxicity index compared to untreated cells (Metabolic activity: p=0.99, Cytotoxicity: p=0.19). In contrast at 10 and 100μMZA treatment remarkably decreased metabolic activity (10μM p=0.03, 100μM p=0.03) and increased cytotoxicity in proliferating cells compared to quiescent cells (10μM p=0.007, 100μM p=0.03). The decreased metabolic activity and increased cytotoxicity correlated with the appearance of unprenylated Rap-1A protein. Further more ZA was more effective in inhibiting cellular migration in actively proliferating cells compared to quiescent cells at all ZA concentrations (1μM: p<0.002, 10μM: p<0.002, 100μM: p<0.002). Conclusions: These data suggest that ZA is more effective in inhibiting migration and promoting cytotoxicity in proliferating rather than quiescent HASMC. Since the expression of unprenylated RAP-1A correlated with inhibition of migration and increased cellular toxicity and reduced metabolic activity, this suggest that mevolante pathway may play a role in the inhibition of these crucial cellular functions. ZA might have a favorable therapeutic profile as a candidate drug to prevent intimal hyperplasia.

Analysis of endogenous ATP analogs and mevalonate pathway metabolites in cancer cell cultures using liquid chromatography–electrospray ionization mass spectrometry

Nitrogen-containing bisphosphonates (N-BPs) are shown to inhibit a key enzyme of intracellular mevalonate pathway, FPP synthase, leading to intracellular accumulation of pathway metabolites isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). In our previous studies we have shown that a new type of ATP analog, ApppI (triphosphoric acid 1-adenosin-5′-yl ester 3-(3-methylbut-3-enyl) ester), is also formed in addition to IPP and DMAPP accumulation. ApppI has cytotoxic effects leading to direct apoptosis of various cancer cells. In this study we present a validated method based on ion-pair LC–MS 2 for the analysis of isomeric mevalonate pathway metabolites and ATP analogs in cell culture samples. Limit of quantitation for IPP and DMAPP was 0.030 μM (1.35 fmol on-column) and for ApppI and ApppD 0.020 μM (0.9 fmol on-column). Acceptable accuracies and precision were also obtained for quality control samples in low and high concentrations of the calibration curve. In addition, we present a new method for quantitation of each coeluting isomer utilizing the peak intensity ratios of two characteristic fragment ions of each compound. For IPP and DMAPP, fragment ions m/ z 177 and m/ z 159 in the MS 2 were monitored, whereas for ATP analogs, ApppI and ApppD (triphosphoric acid 1-adenosin-5′-yl ester 3-(3-methylbut-2-enyl) ester), the same fragments in the MS 3 spectra were followed. IPP and DMAPP accumulation as well as ApppI and ApppD formation was demonstrated using MCF-7 breast cancer cells. Cells were treated with 25 μM zoledronic acid (an N-BP) for 24 h, conditions found to induce significant production of the metabolites. We found that the total amount of IPP and DMAPP was 2.4 nmol/mg of protein and amount of ApppI and ApppD was 1.1 nmol/mg protein. Relative portions of the isomers were approximately 1:4 IPP:DMAPP and 3:7 ApppI:ApppD. Untreated control samples did not contain IPP, DMAPP, ApppI or ApppD.

Zoledronic acid determines S-phase arrest but fails to induce apoptosis in cholangiocarcinoma cells

Cholangiocarcinoma is the second most common primary hepatic neoplasia and the only curative therapy is surgical resection or liver transplantation. Biphosphonates (BPs) are an emerging class of drugs widely used to treat bone diseases and also appear to possess direct antitumor activity. In two human cholangiocarcinoma cell lines (TFK-1 and EGI-1) we investigated, for the first time, the activity of zoledronic acid by determining proliferation, cell cycle analysis and apoptosis. The results obtained indicate that zoledronic acid induces cell-narrowing and growth inhibition, both reversed by 25 μM GGOH, and significantly affects the colony-forming ability of these cells. The inhibition by zoledronic acid of Rap1A prenylation was reversed in cell co-treated with GGOH. At 10–50 μM zoledronic acid exerted an S-phase cell cycle arrest which was confirmed by changes in the level of cyclins and of regulators p27 KIP1 and pRb. Interestingly, the expression level of cyclin A (putative S-phase marker) shows a dose-dependent increment in contrast to the decrement of cyclin D1 (putative G1 phase marker). However, neither hypodiploid cells nor cleaved PARP or caspase-3 was detected. The lack of TP53 or loss of its function, the large constitutive expressions of anti-apoptotic proteins Bcl-xL and HSP27 together with the low level of the pro-apoptotic Bax are the likely factors which protect cells from apoptosis. In conclusion, our study indicates that zoledronic acid induces S-phase arrest and cell-narrowing, both reversed by GGOH and, by changing the delicate balance between pro- and anti-apoptotic proteins, allows survival of cholangiocarcinoma cells.

Differential Inhibition of Invasion and Proliferation by Bisphosphonates: Anti Metastatic Potential of Zoledronic Acid in Prostate Cancer

Objectives: To determine the mode of action of Zoledronic acid in the inhibition of metastasis in prostate cancer and the reduction of prostate cancer bone metastases. Methods: Benign and malignant primary prostatic epithelial cells (PEC) and the PC-3 prostate cancer cell line were studied in co-culture using human bone marrow stroma in the presence of escalating doses of EDTA, Clodronate, Pamidronate and Zoledronic acid. PEC binding and colony growth in bone marrow stroma was measured using standardised quantitative techniques. PEC cellular invasion through Matrigel and an endothelial monolayer was measured either in invasion chambers or by the measurement of endothelial monolayer permeability to fluorescent dextran. Co-culture supernatants were assayed for specific cytokine levels. Bone marrow cellular toxicity was assessed using a standard Mix assay. Results: Treatment of PEC with up to 100 μM bisphosphonate did not affect their ability to bind to bone marrow endothelium or stroma. Bone marrow endothelial permeability was reduced by 100 μM Zoledronic acid by 3.8% ( p =0.03856). Both Pamidronate (40% at 100 μM, p ≤0.05) and Zoledronic acid inhibited PEC invasion, with Zoledronic acid being the most potent (40% at 10 μM, p ≤0.05 rising to 91% at 100 μM, p ≤0.001). Zoledronic acid inhibits malignant PEC proliferation in bone marrow stroma co-culture (26.5% at 10 μM rising to 66.5% at 40 μM). This was accompanied by changes within the cytokine milieu with a >800% rise in TIMP-2. Conclusion: Zoledronic acid is a potent inhibitor of PEC invasion across bone marrow endothelium and colony formation with the bone marrow stroma, affecting the MMP: TIMP-2 balance to favour MMP inhibition.