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Keywords:

  • bone marrow stromal cells;
  • adhesion molecules;
  • zoledronic acid;
  • antitumor effect;
  • apoptosis

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

BACKGROUND

Myeloma plasma cells interact with the bone marrow microenvironment which, in turn, supports their growth and protects them from apoptosis. In vitro studies have demonstrated the antitumor potential of zoledronic acid (ZOL) on myeloma cell lines, but few data are available on its effects on bone marrow stromal cells (BMSCs). The aim of the current study was to evaluate the antiproliferative and apoptotic effect of ZOL on BMSCs, as well as its effect on the expression of adhesion molecules.

METHODS

BMSCs, obtained from bone marrow mononucleated cells of 8 patients with multiple myeloma, were treated with increasing concentrations of ZOL for 3 days. Cytotoxic effect was analyzed by 3-(4-5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide; thiazolyl blue (MTT) assay whereas the induction of apoptosis was evaluated by flow cytometric detection of fluorescein isothiocyanate-labeled annexin V, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, and nuclear changes. Moreover, expression of CD106, CD56, CD50, CD49d, CD44, and CD40 was analyzed by flow cytometry. Data were evaluated by the Friedman test.

RESULTS

After 3 days of exposure at concentrations of 10−4 to 10−5 M, ZOL induced a decrease in proliferation (P < 0.0001) and an increase in apoptosis (P < 0.002). Analysis of culture supernatants showed that myeloma BMSCs expressed interleukin (IL)-6, negligible levels of tumor necrosis factor-alpha, and no IL-1β. In vitro exposure to the lowest concentrations of ZOL decreased IL-6 production by BMSCs. Among the adhesion molecules, CD106, CD54, CD49d, and CD40, which were strongly expressed at baseline, showed a statistically significant reduction compared with controls after exposure to ZOL.

CONCLUSIONS

ZOL interfered with myeloma BMSCs by reducing proliferation, increasing apoptosis, and modifying the pattern of expression of adhesion molecules, especially those involved in plasma cell binding. These effects on BMSCs might explain the antitumor activity of ZOL. Cancer 2005;. © 2005 American Cancer Society.

Multiple myeloma (MM) is characterized by infiltration and growth of malignant plasma cells in the bone marrow (BM) microenvironment, with consequent osteoclast activation and extensive osteolysis. Tumor cells localize within the BM through the interaction of adhesion receptors with their ligands on BM stromal cells (BMSCs) and extracellular matrix (ECM) proteins.1 The adhesion of myeloma cells to the BM microenvironment is mediated by several plasma cell membrane receptors. These play an important role in growth regulation and migration of myeloma cells. In particular, the homing of myeloma cells into the BM is mediated by very late activation antigen (VLA)-1, VLA-4 (CD49d) VLA-5, and lymphocyte function-associated antigen-1 (LFA-1). CD44 (Homing-associated cell adhesion molecule [HCAM]) allows the attachment of plasma cells to laminin and/or fibronectin on the ECM.2, 3 MM cells increase their adhesion to BMSCs by binding VLA-4 and LFA-1 expressed on plasma cells to, respectively, vascular cell adhesion molecule (VCAM)-1 (CD106) and intercellular adhesion molecule (ICAM)-1 (CD54) expressed on BMSCs.4, 5.

Recent studies have shown the importance of the microenvironment in the pathophysiology of bone disease. Adhesive interactions of plasma cells play an important role in up-regulating the production of cytokines and growth factors by BMSCs, which consequently enhance tumor growth, bone destruction, and tumor survival.6 Binding of MM cells to the BM stroma maintains the plasma cells in the BM, and stimulates the production of osteoclast-activating factors by either myeloma or stromal cells.7 Among these factors, interleukin (IL)-6 seems primarily involved in myeloma osteolysis as well as in the growth and survival of malignant plasma cells. It is secreted by tumor plasma cells triggered via CD40,8 but there is strong evidence of a paracrine secretion by the BM microenvironment (BMSCs, osteoblasts, osteoclasts).1, 7, 9, 10 The hampering of the adhesion of MM cells to BMSCs by drugs such as thalidomide or proteasome inhibitors abrogates protection against apoptosis and blocks the increased secretion of the cytokines involved in the growth, survival, and migration of clonal cells.

The major mechanism of bisphosphonates (BPs) in reducing bone resorption is the induction of osteoclast apoptosis.11–13. However, in vitro studies have demonstrated a cytostatic and proapoptotic effect of BPs on myeloma and other human tumor cell lines (e.g., breast carcinoma and prostate carcinoma cell lines).14–19 In addition to the direct antitumor effects on myeloma cells, BPs may also affect the proliferation and survival of these cells by inhibiting the release of growth factors from osteoclasts and BMSCs.

The goals of the current study were to confirm the direct antiproliferative effect of zoledronic acid (ZOL) on plasma cells and stromal cells, and to evaluate the modulation of the adhesion molecules on BMSCs, particularly those involved in the interaction with plasma cells.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Reagents

ZOL [zometa:1-hydroxy-2-(1H-imidazole-1-yl)ethylidene] was kindly supplied as the hydrated disodium salt by Novartis Pharma AG (Basel, Switzerland). Stock solutions were prepared in phosphate-buffered saline (PBS), pH 7.4, filter sterilized using a 0.2-μm filter, and stored at −20 °C until use.

Cell Cultures

The human myeloma cell line RPMI-8226 was purchased from DSMZ (Braunschweig, Germany) and cultured according to the manufacturer's instruction in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin. Primary plasma cells were freshly isolated from three patients. Mononuclear cells from BM aspirates were isolated using Ficoll-Hypaque reagent. CD138-positive cells were enriched by the magnetic cell sorting system (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions. Only samples with a myeloma cell content ≥ 95% were cultured in RPMI-1640 supplemented with 20% fetal calf serum, 100 U/mL penicillin, 100 U/mL streptomycin, 1 mM sodium pyruvate, 2 mM glutamine, and 2 ng/mL IL-6 (Peprotech, London, England).20

BM mononucleated cells from eight patients with MM were separated by Ficoll-Hypaque density gradient centrifugation. Adherent cells were long-term cultured and expanded in MyeloCult H5100 (StemCell Technologies, Vancouver, British Columbia, Canada), 10−6 M hydrocortisone sodium succinate, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 °C with 5% CO2. Adherent cell monolayers were obtained usually after 2–3 weeks.21

Proliferation and Cytotoxicity Assays

RPMI-8226 cells were seeded in 96-well plates, each 1 containing 104 cells/0.1 mL of medium. Cells were cultured for 72 hours in medium alone (control) or medium containing ZOL (10−5 M, 5 × 10−5 M, 10−4 M, 5 × 10−4 M), recombinant human IL-6 (5 ng/mL), or a combination thereof. The effect of ZOL on RPMI cell proliferation was evaluated by 3H-thymidine assay. Cytotoxicity was evaluated by 3-(4-5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide; thiazolyl blue (MTT) assay as previously described.22 All assays were performed in triplicate and at least three times.

Adherent BMSCs (6 × 104 cells/mL) were seeded in 24-well plates with ZOL at concentrations of 10−5, 5 × 10−5, and 10−4 M for 3 days. Cells were incubated with 0.5 mg/mL of tetrazolium compound for 3 hours at 37 °C and 5% CO2 conditions. Finally, the MTT-containing medium was removed and the purple formazan crystals were dissolved with dimethylsulfoxide. The plates were gently stirred until the color reaction was uniform and 200 μL of solution from each well was transferred to 96-well plates.

Dye absorbance of RPMI-8226 cells and BMSCs was spectrophotometrically measured at 550 nm using a microplate reader.

Annexin V Flow Cytometric Analysis of Apoptosis

To asses the effect of ZOL on apoptosis, BMSCs and primary plasma cells were analyzed by flow cytometric detection of fluorescein isothiocyanate (FITC)-labeled annexin V. Briefly, adherent cells of primary culture were plated at final concentrations of 3 × 105 cells. After 3 days of exposure to ZOL (10−5 M or 10−4 M), cells were detached with trypsin/ethylenediaminetetraacetic acid solution. Then, cells were collected and washed with PBS. In primary plasma cells, the presence of apoptotic death was evaluated daily for 1–4 days of exposure to ZOL at a concentration of 10−5 M or 10−4 M. Myeloma BMSCs and plasma cells (approximately 1 × 105 cells per sample) were stained with annexin V-FITC (Molecular Probes, Poortgebouw, The Netherlands) for 15 minutes at room temperature in the dark. To better detect BM plasma cells, 10 μL of CD38-TC and CD138-PE antibodies (Becton Dickinson, San Jose, CA) was tested in combination with annexin V-FITC to gate BM plasma cells on the basis of the expression of CD38 and CD138 and side scatter (SSC) laser light diffraction. For each sample, an autocontrol (sham buffer) was processed. Flow cytometric analysis was performed with a FACSCalibur flow cytometer (Becton Dickinson) and 5000–10,000 events were acquired for each sample. Data were collected and analyzed with CellQuest 3.1 software (Becton Dickinson).

In Situ Apoptosis Detection

DNA fragmentation was detected with the fluorescein-based TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay (In Situ Cell Detection Kit, Fluorescein, Roche, Basel, Switzerland).

Stromal cells were seeded at a density of 2 × 104 cells in chamber slides (Nunc, Roskilde, Denmark). Cells were cultured for 24 hours. Then, 5 × 10−5M of ZOL was added.19 After 3 days of exposure, the cell monolayer was fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 solution in PBS for 5 minutes. Finally, 50 μL TUNEL reaction mixture (terminal deoxynucleotidyl transferase and nucleotide mixture in reaction buffer) was added to each chamber. A negative and positive control was included in each experiment. To evaluate the cytoskeleton, cells were stained for 20 minutes with rhodamine-labeled phalloidin (Molecular Probes), which selectively binds to F-actin. After further rinsing with PBS, 1 μg/mL of Hoechst 33342 (Molecular Probes) was added to detect the chromatin condensation. After 15 minutes, the slides were washed, mounted in Gel Mount (Sigma, St. Louis, MO), and immediately evaluated using a universal microscope (Axioskop, Zeiss, Germany) with UV, FITC, and rhodamine illumination equipped with an MC100 automatic photomicrographic camera. Apoptotic cells were defined on the basis of characteristic changes in the nuclear morphology and for the their positive reaction to the TUNEL assay.

Determination of Interleukin-6, Interleukin-1β, and Tumor Necrosis Factor-Alpha Concentrations

Cytokine concentrations in BMSC culture supernatants were examined by enzyme-linked immunosorbent assays with commercially available kits (R&D Systems, Minneapolis, MN) and performed as previously described.23 Briefly, 6 × 104 BMSCs were seeded in MyeloCult 5100 for 24 hours in the presence of different concentrations of ZOL (10−11 to 10−6 M). Then, the culture medium was completely replaced with α-minimum essential medium (stem cell, supplemented with 2% FBS (stem cell), 2 mM glutamine, 100 U/mL penicillin, 100 U/mL streptomycin, and the different concentrations of ZOL. After an incubation of 48 hours, the supernatants were collected and frozen at − 80 °C until analysis.

Adhesion Molecule Expression on Bone Marrow Stromal Cells

BMSCs (3 × 105) were cultured in a T25 flask in medium alone or in the presence of ZOL (10−5 M or 10−4M) for 3 days. After enzymatic digestion, BMSCs were suspended in medium culture and washed with PBS.

BMSC samples (approximately 1 × 105 cells per sample) were incubated (10 minutes at room temperature, lyse no wash technique) in the presence of 10 μL of each monoclonal antibody (MoAb), according to the recommendations of the manufacturer. For the flow cytometric analysis of adhesion molecule expression, the following MoAbs were tested: CD40-phycoerythrin (PE), CD44-FITC (HCAM), CD49d-PE (VLA-4), CD50-PE (ICAM-3), CD54-PE (ICAM-1), and CD106-PE (VCAM-1) (Ancell, Bayport, MN). After adding 2 mL per tube of PBS, 4-parameter, 2-color flow cytometry analysis was performed with a FACSCalibur flow cytometer (Becton Dickinson). Approximately 10,000 events were acquired for each sample. For each sample, a sham buffer (negative control) and an isotypic control reagent were stained to analyze flow cytometric data. Data were collected and analyzed with the CellQuest 3.1 program (Becton Dickinson). Immunophenotypic data were expressed as the percentage of cells positive for ≥ 1 antigen.

Statistical Analysis

Data were evaluated by the Friedman test and a nonparametric analysis of variance, in view of the small sample size. Moreover, the Wilcoxon test was used to compare untreated and treated cells. The STATISTICA computer program was used. (Stat Soft, Inc., Tulsa, OK). Significance was set at P < 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Zoledronic Acid Induces Cytotoxicity to RPMI-8226 Cells

Due to the few primary plasma cells selected from each patient, it was not possible to evaluate simultaneously the cytotoxic and apoptotic effect of ZOL, and, therefore, the cytotoxicity was studied only on RPMI-8226 cells. Proliferation and cytotoxicity were analyzed using 3H-thymidine and the MTT assay. As expected, exposure for 72 hours with 10−5, 5 × 10−5, 10−4, and 5 × 10−4 M ZOL induced a significant progressive inhibition of the proliferation (P < 0.02) and an increase of cytotoxicity of the MM cell line (P < 0.007) (Fig. 1).

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Figure 1. Cytoreductive effects of zoledronic acid (ZOL) on RPMI-8226 cells. Tumor cells were cultured for 72 hours with different concentrations of ZOL and then assayed by incorporation of 3H-thymidine to determine percentage of proliferation or by 3-(4-5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide; thiazolyl blue (MTT) assay to determine the percentage of cytotoxicity with respect to untreated control cells. The LD50 value calculated for the MTT assay was 62 μM. Results are the mean ± the standard deviation of three independent experiments. Filled diamonds: cytotoxicity; open squares: proliferation.

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To ensure that the decrease of MM cell growth was not related to a reduction in the production of IL-6, 5 ng/mL recombinat human IL-6 was added to the cultures. Our results showed that the addition of this cytokine was unable to reverse the antiproliferative effect of ZOL.

Zoledronic Acid Induces Apoptosis to Multiple Myeloma Cells Isolated from Patients

Tumor plasma cells were isolated from BM aspirates of 3 patients with myeloma and cultured with 10−5 M or 10−4 M of ZOL for 4 days. All 3 fresh MM cell suspensions contained > 95% CD138-positive plasma cells as detected by flow cytometry analysis. The percentage of apoptotic cells was evaluated daily by flow cytometry detection of fluorescein-labeled annexin V, which recognized inverted phosphatidylserine on the exterior part of the plasma membrane as an early-stage apoptotic marker. As shown in Figure 2, ZOL apparently induced dose-dependent and time-dependent apoptosis, but the statistical analysis showed that the apoptotic effect reached statistical significance (P < 0.05) only at the highest concentration (10−4 M).

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Figure 2. Dose-dependent apoptotic effect of zoledronic acid on primary plasma cells at concentrations of 10 μM and 100 μM for 4 days. Data are expressed as the mean of 3 independent experiments (P < 0.04). Filled diamonds: control; filled triangles: 10 μM; filled squares: 100 μM.

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Zoledronic Acid Decreases Cell Viability in Bone Marrow Stromal Cells by Apoptotic Cell Death

The effect of ZOL on BMSC viability was analyzed in 10 samples of BMSCs derived from patients with MM using the MTT cytotoxic assay, in the presence or absence of the drug. After a 96-hour incubation, cell survival was determined by the ability of viable cells to reduce the MTT dye to formazan. ZOL induced a significant (P < 0.0002) concentration-dependent cell death in adherent cells when exposed to 10−5, 5 × 10−5, and 10−4 M of drug for 3 days (Fig. 3). The cytotoxic effect becomes more evident after exposure to a concentration > 10−5 M.

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Figure 3. Apoptotic effect versus cell viability in bone marrow stromal cells (BMSCs). BMSCs were exposed to increasing concentrations of zoledronic acid for 3 days and then analyzed by annexin V assay to determine the percentage of apoptotic cells and by 3-(4-5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide; thiazolyl blue (MTT) assay to determine the loss of cell viability. The two effects were significant at all the concentrations tested. Data are expressed as the mean of eight independent experiments. Filled diamonds: annexin V; filled squares: MTT assay.

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To determine if the reduction of cell viability observed with ZOL treatment was due to apoptotic death, BMSCs were analyzed by cytometric detection of annexin V. After 3 days of exposure, BMSCs showed a significant (P < 0.001) dose-dependent increase in the percentage of apoptotic cells, which was greater than that observed with the plasma cells at baseline (Fig. 3). Apoptosis was also evaluated through the typical nuclear changes, including chromatin condensation (Hoechst staining) and DNA fragmentation detected by the TUNEL assay. ZOL at the dose of 5 × 10−5 M for 3 days caused these nuclear changes in BMSCs. Beyond the morphologic changes of BMSCs, the exposure to ZOL provoked the loss of cytoskeletal integrity, characterized by cell rounding and loss of stress fibers (Fig. 4).

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Figure 4. Morphologic changes in bone marrow stromal cells induced by zoledronic acid (ZOL) after 3 days of treatment. Analysis was performed by fluorescent microscopy. Nuclei were stained with Hoechst 33342 and cytoskeleton with rhodamine-labeled phalloidin. The micrographs were obtained after double exposure using an MC100 automatic photomicrographic camera. (A) Untreated cells. (B, D, E) Cells were treated with 50 μM of ZOL. In contrast to normal cells, the nuclei of apoptotic cells (arrow) have highly condensed chromatin and show a typical change in their morphology (B–D). (E) After the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, only the nuclei of apoptotic cells were labeled with fluorescein (green). Original magnification × 100 (A, B); × 400 (C–E).

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Effect of Zoledronate on Cytokine Secretion of Bone Marrow Stromal Cells

To determinate whether small concentrations of ZOL (10−11 to 10−6 M) could decrease cytokine secretion, IL-6, IL-1β, and tumor necrosis factor-alpha (TNF-α) were investigated in the supernatants of 8 different samples of BMSCs. IL-6 secretion in media alone was 210 ± 148 (mean ± standard deviation) pg/mL, whereas IL-1β and TNF-α were undetectable apart from 2 samples in which the TNF-α concentration was 23 ± 6 pg/mL. After exposure to 10−6 to 10−7 M of ZOL for 3 days, IL-6 secretion was significantly decreased (P < 0.05) with respect to untreated controls. No significant effect was observed for concentrations < 10−8 M.

Effect of Zoledronic Acid on Molecular Adhesion Expression in Bone Marrow Stromal Cells

The BMSCs of eight patients with MM were analyzed to assess the capacity of ZOL to modulate their expression of the adhesion molecules. After exposure to the highest concentration of ZOL (10−4 M), a statistically significant down-regulation of CD40, CD49d, CD54, and CD106 was observed with respect to the baseline condition (P < 0.05). In contrast, no statistical differences were observed after exposure to the lower concentration of ZOL (10−5 M) neither between ZOL 10−5 M and ZOL 10−4 M concentrations. As well, no statistically significant variations were observed regarding the expression of CD44 and CD50 (Fig. 5).

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Figure 5. Effect of zoledronic acid (ZOL) on adhesion molecule expression of bone marrow stromal cells after exposure for 3 days to different concentrations of ZOL. Data are the means of eight independent experiments ± the standard deviation. The expression of CD40, CD49d, CD54, and CD106 was significantly reduced after treatment with the highest dosage of ZOL. Bars with heavy stippling: control; bars with light stippling: 10 μM; bars with stripes: 100 μM.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

The myeloma microenvironment is characterized by the presence of clonal plasma cells, stromal cells (e.g., osteoclasts, osteoblasts, fibroblasts, and endothelial cells), ECM protein, and several growth factors. It has been shown that BMSCs support MM cell growth and survival, protect them from apoptosis, enhance cytokine production, and induce development and activation of osteoclasts. All these events are primed by the interaction between MM and stromal cells through the expression of cell adhesion molecules. Several studies have demonstrated that the abrogation of this contact inhibits tumor cell growth and suppresses osteoclastic activation.24

BPs are a class of nonhydrolyzable analogs of pyrophosphate which have shown, besides the strong antiosteoclastic activity, a direct antitumor effect on several cancer cells, including myeloma cells.

In the current study, we investigated the antitumor effect of ZOL with particular attention to the modulation of adhesion molecules expressed by BMSCs. As previously demonstrated, we observed antiproliferative and apoptotic effects on myeloma cell lines, primary plasma cells, stromal cells, and a reduction in IL-6 production.

These data are in accordance with the majority of in vitro studies on myeloma cell lines. In our study, however, in contrast to that of Derenne et al.,23 who showed antiproliferative and proapoptotic effects of ZOL on BMSCs only at the highest concentration, we observed that at all concentrations (10−5 and 10−4 M) had a dose-dependent pattern.

Apoptosis induced by ZOL was associated with DNA fragmentation and morphologic changes in BMSCs (Fig. 4). These peculiar features have been shown to be related to the BP down-regulation of bcl-2, which, in turn, causes a release of cytochrome C from mitochondria and caspase-3 activation.25 Other authors have also hypothesized that nitrogen-containing (N)-BPs mediate this effect through the inhibition of the mevalonate pathway,26–28 which purports the block of the synthesis of isoprenoid compounds, including Ras, Rho, and Rac, involved in important cellular pathways.

To evaluate if the apoptosis of the BMSCs induced by ZOL was correlated to an inhibition of cell adhesion, we studied the expression of the following adhesion molecules on BMSCs: CD40, CD44 (HCAM), CD49d (VLA-4), CD50 (ICAM-3), CD54 (ICAM-1), and CD106 (VCAM-1).

We found that ZOL down-regulates the expression of some of these molecules, in particular those involved in the cell-to-cell contact with plasma cells. To the best of our knowledge, no other studies have focused on this effect of ZOL. We observed that the exposure to the highest concentration of ZOL reduced significantly the expression of CD40, CD49d, CD54, and CD106. The strong modulation of integrin CD49d (P < 0.005) may indicate a possible effect of ZOL on cell anchorage. Conversely, the down-regulation of CD54 (ICAM-1) and CD106 (VCAM-1), which mediate the cell-to-cell-contact with plasma cells, can affect tumor cell growth, survival, and local secretion of IL-6. Actually, the evaluation of IL-6 levels in the supernatant of BMSC long-term culture after exposure to ZOL, even at very low concentrations, produced a significant down-regulation of IL-6 production. This observation could be consistent with the central role of IL-6 even in the modulation of adhesion molecules. This hypothesis is also supported by recent evidence supporting the reduced expression of VCAM-1 in an IL-6–deficient stroma with respect to wild-type stroma.29 Moreover, other authors have demonstrated that the contact of mouse myeloma plasma cells with BMSCs via α4β1 integrin/VCAM-1 interactions enhances the production of osteoclastogenesis factors and that disruption of this cell-to-cell contact reduces their production24 and can suppress the development of MM and associated osteoclastic osteolysis.30

In conclusion, ZOL shows in vitro antitumor activity on myeloma BMSCs and plasma cells, inducing apoptosis and reducing cell proliferation. It interferes also with the BMSC down-regulation of cytokine secretion and modifies the expression of adhesion molecules, in particular those involved in cell-to-cell-contact. The modulation of the adhesion molecules could represent the main pathogenetic mechanism of the antitumor activity of ZOL.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES