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Published ahead of print on April 3, 2008, doi:10.1164/rccm.200710-1513OC
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American Journal of Respiratory and Critical Care Medicine Vol 178. pp. 50-59, (2008)
© 2008 American Thoracic Society
doi: 10.1164/rccm.200710-1513OC

Original Article

Zoledronic Acid Is Effective against Experimental Malignant Pleural Effusion

Georgios T. Stathopoulos1, Charalampos Moschos1, Heleni Loutrari1, Androniki Kollintza1, Ioannis Psallidas1, Sophia Karabela1, Sophia Magkouta1, Zongmin Zhou1, Spyros A. Papiris2, Charis Roussos1 and Ioannis Kalomenidis1,2

1 Applied Biomedical Research and Training Center "Marianthi Simou" and "George P. Livanos" Laboratory, Department of Critical Care and Pulmonary Services, General Hospital "Evangelismos," School of Medicine, National and Kapodistrian University of Athens, Athens, Greece; and 2 Second Pulmonary Department, "Attikon" University Hospital, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece

Correspondence and requests for reprints should be addressed to Georgios T. Stathopoulos, M.D., Ph.D., 3 Ploutarhou Str., 10675 Athens, Greece. E-mail: gstathop{at}med.uoa.gr


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Aminobiphosphonates, such as zoledronic acid (ZA), exert potent indirect antitumor effects and are currently being tested against human solid tumors. The antitumor actions of aminobiphosphonates, including angiostasis, are relevant to the pathogenesis of malignant pleural effusion (MPE), but no study has addressed the efficacy of these compounds against malignant pleural disease.

Objectives: Here we hypothesized that treatment of immunocompetent mice with ZA would halt tumor progression in a mouse model of adenocarcinoma-induced MPE.

Methods: To induce MPE in mice, Lewis lung carcinoma cells were delivered directly into the pleural space. Subsequently, animals were treated with ZA in both a prevention and a regression protocol.

Measurements and Main Results: ZA treatment resulted in significant reductions in pleural fluid accumulation and tumor dissemination, while it significantly prolonged survival. These effects of ZA were linked to enhanced apoptosis of pleural tumor cells, decreased formation of new vessels in pleural tumors, and reduced pleural vascular permeability. In addition, ZA was able to inhibit the recruitment of mononuclear cells to pleural tumors, with concomitant reductions in matrix metalloproteinase-9 release into the pleural space. Finally, ZA limited the expression of proinflammatory and angiogenic mediators, as well as the activity of small GTP proteins Ras and RhoA, in tumor cells in vivo and in vitro.

Conclusions: ZA is effective against experimental MPE, suggesting that this intervention should be considered for testing in clinical trials.

Key Words: pleural disease • monocyte chemoattractant protein • vascular endothelial growth factor • angiogenesis • aminobiphosphonate



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
No specific treatment is available for malignant pleural effusion.

What This Study Adds to the Field
In this experimental study in mice, the aminobiphosphonate zoledronic acid was effective against malignant pleural effusion.

 
Malignant pleural effusions (MPEs) occur very frequently in the course of various malignancies (in up to 15% of patients with cancer), predominantly in adenocarcinomas of the lungs and other organs. The appearance of an MPE is ominous for patients with cancer, by rendering the tumor incurable and by negatively impacting survival and quality of life (1–4). Specific therapies for MPE are not available. Pleurodesis (chemically induced pleural fibrosis aimed at eliminating the pleural space), indwelling pleural catheters, and chemotherapy are currently used to block the reaccumulation of pleural fluid; however, these therapies are often ineffective and associated with morbidity (5, 6). To foster understanding of the pathobiology of MPE and to aid in the development of specific treatment methods, we have developed a model of MPE in immunocompetent mice that recapitulates salient features of the human disease, including tumor-associated inflammation, angiogenesis, and pleural vascular hyperpermeability (7, 8).

Newer aminobiphosphonates (NBPs), such as zoledronic acid (ZA), are currently used in the clinic against bone metastases from various solid tumors (9, 10). These compounds have been shown to exert potent antitumor effects that cannot be solely attributed to direct killing of tumor cells (11, 12). At the clinical level, NBPs are effective in the treatment and prevention of skeletally related events resulting from various solid tumors (9, 10). More recent clinical studies show that NBPs are capable of slowing down tumor growth and of preventing metastasis, properties that are likely going to lead to a more widespread use of the compounds in cancer therapeutics (13). At the tissue level, NBPs are potent inhibitors of tumor-associated new vessel formation (angiogenesis) (12–14). In addition, NBPs possess a unique ability to act as immunostimulatory molecules, and to promote the expansion of tumoricidal V{gamma}9V{delta}2 {gamma}{delta}TCR+ T cells in primates and humans (15, 16). At the cellular level, NBPs block the mevalonate pathway, thereby inhibiting the prenylation and activation of small membrane-associated GTP proteins like Ras and RhoA, which are involved in cell motility and regulation of gene expression (17–21).

The pathogenesis of MPE involves tumor-induced angiogenesis and associated vascular hyperpermeability, which could be prevented by the antiangiogenic effects of NBPs. To our knowledge, there have been no preclinical studies to address the effects of any NBP in malignant pleurisy to date. Here we hypothesized that ZA would be able to limit experimental MPE in both a preventive and a curative mode. We tested this hypothesis in a mouse model of MPE resulting from propagation of autologous lung adenocarcinoma cells in the pleural cavity of fully immunocompetent mice (7, 8). Some of the results of these studies have been previously reported in the form of an abstract (22).


   METHODS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Methods are described in greater detail in the online supplement.

Reagents
ZA (Novartis, Basel, Switzerland), Evans blue from Sigma (St. Louis, MO), neutralizing mouse anti–vascular endothelial growth factor (anti-VEGF) antibody (Ab) from R&D (Minneapolis, MN), purified mouse monocyte chemoattractant protein (MCP)-1 and neutralizing anti–MCP-1 Ab from Peprotech (London, UK) were used.

Cell Lines
Mouse lung adenocarcinoma (Lewis lung carcinoma [LLC]), human pleural mesothelial (Met5A), mouse mononuclear/macrophage (RAW 264.7) (American Type Culture Collection, Manassas, VA), and nuclear factor (NF)-{kappa}B reporter (pNGL) LLC cell culture are described elsewhere (References 7 and 8 and the online supplement).

Animal Model
C57BL/6 mice (BSRC Al. Fleming and Hellenic Pasteur Institute, Vari and Athens, Greece) were inbred at the General Hospital "Evangelismos" (Athens, Greece). Experiments were approved by the Veterinary Administration Bureau, Prefecture of Athens, Greece. Mice were matched for sex, weight, and age. Intrapleural injections and killing (Day 14) were performed as described previously (7, 8). Briefly, after minimal skin, fascia, and muscle dissection over the left lateral chest wall, anesthetized mice received direct intrapleural injections of 1.5 x 105 LLC cells in 50 µl phosphate-buffered saline (PBS), and were observed until recovery. At 14 days, mice were killed using CO2, blood was drawn, pleural fluid was retrieved, and pleural tumors were enumerated and frozen or fixed in formalin. In pilot studies, MPEs occurred between 8 and 12 days and were largest at 14 days, after which significant mortality ensued. For survival studies, mice were killed when moribund. Mice received daily subcutaneous ZA (100 µg/kg, ~2.5 µg/mouse) in 100 µl sterile water in two protocols that have been reported as effective and nontoxic: prevention trial (ZA PT), Days 3–13; regression trial (ZA RT), Days 9–13 (14).

Vascular Permeability Assay
Animals received 200 µl, 4 mg/ml, Evans blue intravenously on Day 14 after LLC cells, and were killed 1 hour later. Pleural fluid Evans blue levels were determined as described elsewhere (7, 8).

Cytology
Cytocentrifugal specimens (cytospins) and blood smears were prepared and stained with May-Grünwald-Giemsa or immune-labeled with anti-F4/80 Ab (Abcam, Cambridge, UK) as described elsewhere (7, 8, 23).

Cytokine Determinations
Mouse and human VEGF, MCP-1, macrophage inflammatory protein (MIP)-2/IL-8, IL-6, and pro-matrix metalloproteinase (pro-MMP)-9 (detection limits: 5.1 and 2.0, 2.0 and 5.0, 1.5 and 2.0, 1.6 and 2.0, and 8.0 and 50.0 pg/ml, respectively) were determined using ELISA (Peprotech and R&D).

Histology
Mouse tissues were fixed, sectioned, and stained with hematoxylin and eosin or immune-labeled for proliferating cell nuclear antigen (PCNA) (Santa Cruz, Santa Cruz, CA), terminal deoxynucleotidyl nick-end labeling (TUNEL) (Roche, Penzberg, Germany), factor VIII–related antigen (Invitrogen, San Francisco, CA), and F4/80 (Abcam), as described previously (23–26). Quantification of immunoreactivity has been described elsewhere (24–26).

Biochemical and Cellular Assays
Protein concentration was determined using the Bio-Rad protein assay (Hercules, CA). MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulphophenyl]-2H-tetrazolium, inner salt) (Promega, Madison, WI) reduction and cytoplasmic histone 3 detection (Roche, Penzberg, Germany) assays were used to assess cell viability and death.

Western Immunoblotting
Cytoplasmic extracts of pleural tumors and LLC cells were run on 12.5% polyacrylamide gels containing sodium dodecyl sulfate. Alternatively, active RhoA was pulled down from cytoplasmic extracts of total protein using Rhotekin (Upstate, Lake Placid, NY), and active RhoA and total protein extracts were simultaneously run on separate gels (27, 28). For detection, we used rabbit polyclonal anti-Ras (Abcam, Cambridge, UK) and anti-RhoA (Santa Cruz) antibodies. Band intensities were determined using ImageJ software (Rasband 1997–2007; National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij).

Adenoviral Vectors
Adenoviral vectors (Ad) for transient expression of green fluorescent protein, wild-type, and dominant negative RhoA were produced with AdEasy (29) based on plasmids described previously (30). LLC cells were infected at multiplicity of infection (MOI) = 200 for 24 hours.

Statistical Analysis
All values represent mean ± SE. Survival is given as median (confidence interval). The Student's t test or one-way analysis of variance with least squares difference post hoc tests were used to test for differences in the means between two or multiple groups, respectively. Kaplan-Meier analysis with log-rank test was used for survival studies. All P values are two-tailed; P values less than 0.05 were considered significant. Statistical analyses were done using SPSS version 13.0.0 (SPSS, Inc., Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ZA Is Effective against Experimental MPE
In a first line of experiments, we tested our primary hypothesis that ZA would halt MPE in mice. For this, wild-type C57BL/6 mice received 1.5 x 105 LLC cells intrapleurally, were treated with ZA or water (control, n = 16) in a prevention and in a regression trial (respectively: ZA PT [n = 25] and ZA RT [n = 20]), and were killed after 14 days. Primary endpoints were the volume of pleural fluid and the number of pleural tumors. In both the PT and RT, ZA significantly reduced intrapleural fluid accumulation and tumor dissemination (Figures 1A and 1B). In addition, ZA limited the cachexia associated with the development of MPE: whereas mice that received sham (PBS) intrapleural injections instead of LLC cells gained approximately 10% body mass during the 2 weeks of the experiment, mice that received LLC cells developed cachexia and lost approximately 7% of their body mass. ZA-treated mice with an MPE showed an intermediate phenotype, preserving their body mass, but not growing further (Figure 1C). Finally, ZA was effective in significantly prolonging the survival of MPE-bearing mice in separate RTs (Figure 1D).


Figure 1
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  		Figure 1. Treatment with zoledronic acid (ZA) in both preventive (PT) and regression (RT) trials limits malignant pleural effusion (MPE) formation and intrapleural tumor dissemination, partially blocks the associated cachexia, and prolongs survival. (A–C) C57BL/6 mice received intrapleural Lewis lung carcinoma cells (n = 61) or phosphate-buffered saline (sham; n = 8), were treated daily with 100 µg/kg ZA or water subcutaneously (CTRL; n = 16) in a PT (Days 2–13; n = 25) and an RT (Days 9–13; n = 20), and were killed after 14 days. Pooled data are from three independent experiments. (D) Alternatively, mice were observed till moribund. (A) Reduced MPE volume in mice treated with ZA (CTRL, ZA PT, and ZA RT, respectively: n = 16, 25, and 20; analysis of variance [ANOVA] P = 0.003). (B) Reduced intrapleural tumor dissemination in mice treated with ZA (CTRL, ZA PT, and ZA RT, respectively: n = 16, 25, and 20; ANOVA P < 0.001). (C) Partial abrogation of cachexia associated with MPE by ZA treatment (sham, CTRL, ZA PT, and ZA RT, respectively: n = 8, 16, 25, and 20; ANOVA P = 0.0005). (D) Prolonged survival of mice treated with ZA in RTs (n = 20; 17 [15.6–18.4] d) compared with mice treated with water (n = 20; 14 [12.5–15.5] d) (log-rank P = 0.0008). Pooled data are from two independent experiments. ns = not significant. (A–C) Columns, mean; error bars, SE. (D) Lines, cumulative survival; gray zone, time period of MPE formation.

 
ZA Induces Apoptosis of Pleural Tumor and Host Cells
We then went on to determine the mechanism of action of ZA in mice with MPE. We first examined the proliferation and apoptosis of pleural tumor cells in vivo by PCNA immune-labeling and TUNEL of pleural tumor tissue sections. Tumor cell proliferation in vivo was not impaired (Figures 2A and 2B), but tumor cell apoptosis rates increased twofold by ZA treatment (Figures 2C and 2D). In vitro, ZA killed tumor (LLC) cells at concentrations exceeding 30 µM (Figures 2E and 2F). At similar concentrations, ZA exerted direct cytotoxic effects on human mesothelia (Met5A) and mouse macrophages (RAW264.7), cell types also present in mouse MPE (Figures 2G and 2H) (7, 8). To examine effects of the drug not dependent on direct cytotoxicity, subsequent in vitro experiments were designed to entail ZA doses below 3 µM.


Figure 2
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  		Figure 2. Zoledronic acid (ZA) kills pleural tumor and host cells in vivo and in vitro. (A–D) Mice were treated as in Figure 1. After 14 days, pleural tumors were examined for tumor cell proliferation and apoptosis using anti–proliferating cell nuclear antigen (anti-PCNA) immune-labeling and terminal deoxynucleotidyl nick end-labeling (TUNEL). (A) ZA treatment had no impact on tumor cell proliferation in vivo (n = 10/group; ANOVA P = ns). (B) Representative images of anti-PCNA immunohistochemistry (insets: isotype control; Å = 600; scale bars = 50 µm; brown = PCNA immunoreactivity; blue = hematoxylin nuclear counterstaining). (C) ZA treatment resulted in increased apoptosis rates of pleural tumor cells (n = 10/group; ANOVA P = 0.0045). (D) Representative images of pleural tumor tissue TUNEL (insets: isotype control; Å = 600; scale bars = 50 µm; arrows point at apoptotic cells; brown = TUNEL immunoreactivity; blue = hematoxylin nuclear counterstaining). (E–F) Lewis lung carcinoma cells were incubated in the presence of different concentrations of ZA. After varying time intervals, cell proliferation and death were determined using MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulphophenyl]-2H-tetrazolium, inner salt) reduction (E) (n = 4/data point; #P < 0.05, ##P < 0.01, and ###P < 0.001 compared with ZA 0 µM) and cytoplasmic histone 3 shedding (F) (n = 6/data point; ***P < 0.001 compared with ZA 0 µM). ZA was cytotoxic at concentrations exceeding 30 µM. (G–H) Human mesothelial cells (G) (Met5A) and mouse macrophages (H) (RAW264.7) were incubated with different concentrations of ZA. After 48 hours, cell proliferation was determined using MTS reduction (n = 6/data point; ###P < 0.001 compared with ZA 0 µM). ZA was cytotoxic at concentrations exceeding 30 µM. CTRL = control; ns = not significant; PT = prevention trial; RT = regression trial. Columns, points, mean; error bars, SE. A–D: Pooled data are from three independent experiments. E–H: Cell experiments were done three times.

 
ZA Limits MPE-associated Vascular Hyperpermeability and New Vessel Formation
We next studied the effect of ZA treatment on pleural vascular hyperpermeability, a main mechanism of malignant effusion formation. For this, control and ZA-treated animals received 0.8 mg Evans blue (an albumin-binding dye) intravenously 1 hour before being killed, as described previously (7, 8). The resulting levels of the dye in MPE from ZA-treated mice in both PT and RT modes were significantly lower compared with water-treated mice (Figure 3A), indicating a reduction of albumin leakage into the pleural space by ZA treatment. These results were in accord with those obtained using another marker of pleural vascular permeability established in human pleural effusions, the ratio of pleural fluid/serum protein (Figure 3B) (3). Because many albumin-leaky vessels in MPE are newly formed due to the presence of cancer (31), we examined the vascularity of pleural tumors from the three experimental groups. Neoangiogenesis determined by factor VIII–related antigen immune-labeling of pleural tumor tissue was markedly inhibited by ZA (Figures 3C and 3D). These results clearly showed that ZA limits the formation and the overall leakiness of pleural vessels.


Figure 3
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  		Figure 3. Zoledronic acid (ZA) limits vascular hyperpermeability and new vessel formation in the malignancy-affected pleura. Mice were treated as in Figure 1. (A) At Day 14, the mice received 0.8 mg albumin-binding Evans blue dye intravenously, were killed 1 hour later, and dye extravasation into the pleural fluid was determined. ZA-treated mice showed markedly reduced vascular leakiness of albumin (CTRL, ZA PT, and ZA RT, respectively: n = 13, 13, and 6; ANOVA P = 0.01). (B) Alternatively, protein content was determined in malignant pleural effusion (MPE) and matched serum. ZA treatment resulted in decreased protein leakage into the pleural space (CTRL, ZA PT, and ZA RT, respectively: n = 10, 7, and 6; ANOVA P = 0.005). (C) Pleural tumor examination using anti-factor VIII–related antigen (fVIIIra) immune-labeling showed markedly reduced new vessel formation in tumors from ZA-treated animals (CTRL, ZA PT, and ZA RT, respectively: n = 10, 12, and 10; ANOVA P = 0.000002). (D) Representative images of anti-fVIIIra immunohistochemistry (insets: isotype control; Å = 400; scale bars = 75 µm; arrows point at new vessels; brown = fVIIIra immunoreactivity; blue = hematoxylin nuclear counterstaining). CTRL = control; fVIIIra+ = factor VIII–related antigen positive; ns = not significant; PT = prevention trial; RT = regression trial. Columns, mean; error bars, SE. Pooled data are from three independent experiments.

 
ZA Inhibits Mononuclear Cell Recruitment to MPE
An important inflammatory cell population observed in lung cancer and MPE are mononuclear/macrophage cells (32, 33). In recent years, these cells have been ascribed protumor functions via the local release of cytokines, chemokines, and matrix-degrading enzymes (34, 35). We initially studied the numbers of macrophages infiltrating pleural tumors by immune-labeling for the F4/80 surface antigen. We observed a nonhomogenous, peripheral distribution of mononuclear cells in pleural tumor tissue. In fact, mononuclear infiltrates preferentially lined the pleural tumor surface facing the pleural space (the MPE), whereas the central areas of pleural tumors were relatively devoid of macrophages (images not shown), making quantification of the infiltrates difficult. We thus determined the content of MPE in mononuclear cells by immune-labeling for F4/80. ZA-treated mice had significantly fewer F4/80-expressing mononuclear cells than control animals, indicating impaired monocyte recruitment to the pleural space (Figures 4A and 4B). In addition, ZA-treated mice had fewer circulating monocytes in the blood (Figure 4C). We have previously determined increased local MCP-1 production in experimental MPE (7, 8). We postulated that ZA may limit local mononuclear recruitment via inhibition of local MCP-1 release. Indeed, whereas the MPE levels of the chemokine were equal between groups, we identified reduced MCP-1 expression in pleural tumors and serum from ZA-treated mice (Figure 4D). In cell experiments, we corroborated that ZA significantly limits MCP-1 expression by LLC (tumor), Met5A (mesothelial), and RAW264.7 (mononuclear/macrophage) cells (Figure 4E) at concentrations of 3 µM (Figures 2E–2H) that, as reported above (Figures 2F,2H), does not induce cell death. Collectively, these data indicated that ZA functioned to reduce the chemotactic recruitment of mononuclear cells to the malignancy-affected pleural space, rather than to decrease the local survival of recruited macrophages in MPE.


Figure 4
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  		Figure 4. Zoledronic acid (ZA) treatment impairs mononuclear/macrophage cell recruitment to malignant pleural effusion (MPE). Mice were treated as in Figure 1. (A) At Day 14, pleural fluid cells were immune-labeled for the mononuclear/macrophage surface marker F4/80. ZA-treated mice had fewer F4/80+ mononuclear cells in the pleural fluid (CTRL, ZA PT, and ZA RT, respectively: n = 10, 9, and 7; ANOVA P = 0.0001). (B) Representative images of anti-F4/80 immunocytochemistry of pleural cells (insets: isotype control; Å = 600; scale bars = 50 µm; arrows point at F4/80+ pleural fluid macrophages; brown = F4/80 immunoreactivity; blue = hematoxylin nuclear counterstaining). (C) Fewer circulating monocytes were identified on peripheral blood smears (n = 10/group; ANOVA P < 10–7). (D) Although the levels of MCP-1 in MPE were equal between groups (left; n = 8/group, P = ns), expression of the chemokine was markedly reduced in pleural tumors (middle; n = 7/group, P = 0.014). Systemic MCP-1 release, as determined in the serum, was fourfold reduced in ZA-treated animals (right; n = 8/group, P = 0.0006). (E) Twenty-four hours of ZA treatment reduced MCP-1 expression by tumor (Lewis lung carcinoma [LLC]; left; n = 3, P = 0.008), as well as by mesothelial (Met5A; middle; n = 3, P < 0.0001) and mononuclear/macrophage (RAW264.7; right; n = 3, P = 0.005) cells. CTRL = control; MCP-1 = monocyte chemoattractant protein 1; ns = not significant; PT = prevention trial; RT = regression trial; WBC = white blood cells. Columns, mean; bars, SE. A–D: Pooled data are from three independent experiments. E: Cell experiments were done in triplicate.

 
ZA Reduces the Expression of Proinflammatory and Angiogenic Mediators in Pleural Tumors
We and others have previously identified several inflammation-, angiogenesis-, and vascular hyperpermeability–inducing mediators that are highly and locally expressed in human and mouse MPE, and may be implicated in its pathogenesis (7, 8, 31, 36–42). In subsequent experiments, we determined the levels of these mediators in MPE and tumor tissue, as well as in supernatants from tumor (LLC), mesothelial (Met5A), and mononuclear/macrophage (RAW264.7) cell cultures (Figure 5). In this regard, ZA limited the expression of VEGF, MIP-2, and IL-6 in pleural tumors in vivo (Figure 5A). In addition, ZA reduced the MPE levels of MIP-2 and pro–MMP-9 (Figure 5B). In cell experiments, ZA modestly but significantly knocked down the expression of VEGF and IL-6 by LLC (tumor) cells, of IL-6 by Met5A (mesothelial) cells, and of MIP-2 by RAW264.7 (macrophage) cells (Figures 5C–5E). These data collectively indicated that ZA inhibits the expression of proinflammatory and angiogenic cytokines by tumor and adjacent host cells in MPE.


Figure 5
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  		Figure 5. Zoledronic acid (ZA) limits proinflammatory and angiogenic mediator expression in vivo and in vitro. (A, B) Mice were treated as in Figure 1. At Day 14, malignant pleural effusion (MPE) and pleural tumor tissue were retrieved for mediator determination. (A) Reductions in VEGF (left; n = 8/group, P = 0.002), MIP-2 (second to left; n = 8/group, P = 0.036), and IL-6 (second to right; n = 8/group, P = 0.007), but not of pro–MMP-9 (right; n = 8/group, P = ns) expression in pleural tumor tissue. (B) Pro–MMP-9 (right; n = 9/group, P = 0.025) and MIP-2 (second to left; n = 8/group, P = 0.036), but not VEGF (left; n = 8/group, P = ns) and IL-6 (second to right; n = 8/group, P = ns) levels were significantly reduced in MPEs from ZA-treated mice. (C–E) Mouse tumor (Lewis lung carcinoma [LLC]), human mesothelial (Met5A), and mouse mononuclear/macrophage (RAW264.7) cells were incubated in the presence of different concentrations of ZA. After 24 hours, supernatants were assayed for mediator expression by ELISA. All experiments were done in triplicate (n = 3 for all graphs). (C) ZA treatment of LLC cells resulted in dose-dependent reductions in VEGF (left; P = 0.016) and IL-6 (second to right; P < 0.001), but not MIP-2 (second to left; P = ns) and pro–MMP-9 (right; P = ns) expression. (D) ZA treatment of Met5A cells resulted in dose-dependent reductions in IL-6 (second to right; P = 0.019), but not VEGF (left; P = ns), MIP-2 (second to left; P = ns), and pro–MMP-9 (right; P = ns) expression. (E) ZA treatment of RAW264.7cells resulted in dose-dependent reductions in MIP-2 (second to left; P < 0.001), but not VEGF (left; P = ns), IL-6 (second to right; P = ns), and pro–MMP-9 (right; P = ns) expression. Columns, mean; error bars, SE. CTRL = control; MIP = macrophage inflammatory protein; MMP = matrix metalloproteinase; nd = not detectable; ns = not significant; PT = prevention trial; RT = regression trial; VEGF = vascular endothelial growth factor.

 
ZA Blocks the Activity of GTP Proteins Ras and RhoA in Tumor Cells
We next sought to determine the underlying cancer cell molecular mechanisms that are inhibited by ZA, to explain its in vivo effects on experimental MPE. NBPs have been previously shown to inhibit the NF-{kappa}B pathway, as well as the post-translational lipid modification (prenylation) and the resulting activation of small membrane-associated GTP proteins, such as Ras and RhoA (17–21, 43). ZA treatment had no effect on luciferase expression by NF-{kappa}B reporter (pNGL) LLC cells (data not shown). On the contrary, using Western immunoblotting, we identified significant reductions in small GTP protein activity in vitro and in vivo after ZA treatment of LLC cells and MPE-bearing mice (Figure 6). In accord with previous studies, these data indicated that ZA inhibited GTP protein activation, which is central to tumor cell proliferation and gene expression (17–21).


Figure 6
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  		Figure 6. Zoledronic acid (ZA) blocks Ras and RhoA activity in tumor cells in vivo and in vitro. (A) Lewis lung carcinoma (LLC) cells were incubated in the presence of 1 µM ZA. After 24 hours, cells were lysed and the lysates subjected to SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) for immunodetection of Ras and β-actin loading control. Alternatively, cell lysates were incubated with Rhotekin. After Rhotekin pull-down, total protein or active RhoA were separated on 12.5% polyacrylamide gels for immunodetection of RhoA. ZA treatment inhibited Ras and RhoA activation in LLC cells. (B) Summary of data obtained by densitometry of blot (A) (n = 3, #P < 0.05 compared with ZA 0 µM). (C) Mice were treated as in Figure 1. At Day 14, pleural tumor tissue was retrieved and homogenized, and protein was extracted and separated on 12.5% polyacrylamide gels for immunoblotting of RhoA, Ras, and β-actin loading control. Pleural tumor tissue from ZA-treated mice showed reduced Ras and RhoA activity. (D) Summary of data obtained by densitometry of blot (C) (n = 3, #P < 0.05 compared with water-treated animals). Shown is one of three similar experiments. A, C: Representative Western immunoblots; black vertical lines indicate cropping for demonstration purposes; 23-kD bands represent active and 21-kD bands inactive G protein. B, D: Columns, mean; error bars, SE. CTRL = control; PT = prevention trial; RT = regression trial.

 
ZA-Targeted GTP Proteins Likely Impact Paracrine Mediator Expression Rather than Cell Survival
We finally sought to determine the downstream effects of ZA-induced knockdown of proinflammatory/angiogenic mediator expression and of GTP protein activation in tumor (LLC) cells. Because ZA limited VEGF and MCP-1 release by LLC cells, we hypothesized that these molecules might exert autocrine growth effects on LLC cells. However, exogenous stimulation of serum-deprived or serum-supplemented LLC cells with recombinant mouse VEGF or MCP-1, as well as blockade of endogenous VEGF or MCP-1 with neutralizing antibodies, had no effects on LLC cell proliferation (Figures 7A and 7B). In addition, because ZA also limited Ras and RhoA activity in LLC cells, we hypothesized that these signal transducers impact LLC cell proliferation and paracrine gene expression. These functions are well established for Ras, a molecule we had no means of specifically modulating (18, 20, 21). However, using recombinant adenoviral vectors to specifically modulate RhoA expression in LLC cells (29, 30), we found that these manipulations did not affect cell proliferation (Figure 7D) but impacted VEGF and MCP-1 expression (Figures 7E and 7F). These results indicated that ZA-induced RhoA blockade mainly targets paracrine mediator expression and not growth of tumor (LLC) cells.


Figure 7
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  		Figure 7. The tumor cell pathways inhibited by zoledronic acid (ZA) affect paracrine mediator expression rather than cell growth. (A, B) Serum-deprived Lewis lung carcinoma (LLC) cells were cultured with or without VEGF, MCP-1, anti-VEGF, or anti–MCP-1 neutralizing antibodies at 2 nM concentration (~10 times the endogenous levels), and cell proliferation was determined by MTS reduction capacity. Exogenous and endogenous VEGF and MCP-1 did not affect LLC cell proliferation (n = 6, P = ns for all comparisons). (C–F) LLC cells were infected with adenoviral vectors (Ad) encoding green fluorescent protein (GFP) (control, Ad-GFP), wild-type RhoA (Ad-RhoA), or dominant negative RhoA (Ad-N19) at multiplicity of infection (MOI) = 200 for 24 hours. (C) Using Ad-GFP, the infection efficiency of LLC cells was estimated at approximately 70% (scale bar = 20 µm). (D) RhoA modulation did not alter LLC cell proliferation (n = 3, P = ns for all comparisons). (E, F) RhoA promoted VEGF and MCP-1 expression by LLC cells (n = 3, *P < 0.05 compared with Ad-GFP; #P < 0.05 compared with Ad-RhoA). Columns, mean; error bars, SE. MCP = monocyte chemoattractant protein; MTS = 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt; VEGF = vascular endothelial growth factor.

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In these studies, we tested the hypothesis that a potent NBP, ZA, would halt intrapleural fluid accumulation and tumor dissemination in experimentally induced MPE. This proved to be the case, because we found that ZA limits the formation and progression of adenocarcinoma-induced MPE in syngeneic C57BL/6 mice, thereby improving tumor-related cachexia and prolonging survival. Our results indicate that the effects of ZA are associated with increased tumor cell apoptosis, decreased pleural vascular permeability, and reduced new vessel formation in pleural tumors. We further show that ZA treatment limited the number of mononuclear/macrophage lineage cells in the pleural space, an effect that, at least in part, implicates reduced local and systemic release of MCP-1. Finally, our results indicate that ZA treatment reduces the elaboration of proinflammatory and angiogenic mediators by tumor and adjacent host cells, and knocks down the activity of small GTP proteins Ras and RhoA in tumor cells. Because these mediators and signal transducers have been implicated in enhanced tumor cell proliferation and survival, motility, gene expression, and tumor-induced angiogenesis, their inhibition by ZA may explain why this NBP blocks experimentally induced MPE.

This is the first study of the actions of any NBP on MPE. Our findings suggest that ZA affects MPE by targeting multiple biological events associated with the expansion of tumor cells and the accumulation of fluid in a pleural cavity invaded by cancer. First, the drug exerts a direct survival limiting effect on adenocarcinoma cells both in vitro and in vivo, an observation previously made with several cancer cell lines and believed to be secondary to small GTP protein inhibition (13, 17–20). Second, ZA indirectly affects MPE through blockage of macrophage accumulation in the tumor microenvironment, of tumor or host cell expression of certain proinflammatory and angiogenic cytokines, and of tumor angiogenesis and the associated pleural vascular hyperpermeability, all being of potential or proven importance for the pathogenesis of the disease (7, 8, 31, 33, 36–42).

Importantly, we did not examine the effects of ZA on MPE burden or survival after systemic or pulmonary delivery of cancer cells, but only after local injection directly into the pleural space. Therefore, it remains unknown whether the drug will be equally effective in tumor control in models of extrapleural (indirect) induction of MPE—for example, via the intravenous or intranasal routes—that more closely reflect human tumor spread to the pleura. On the other hand, the present studies were designed to elucidate the effects of ZA specifically on the ability of tumor cells to generate an MPE when sited in the pleural cavity. Our experimental mode of direct intrapleural delivery of tumor cells offers the advantage of dissecting out other aspects of the malignant phenotype, such as invasion and metastasis, and studying in isolation the capacity of tumor cells to lead to the development of an MPE. Importantly, our results indicate that ZA was equally effective whether given before the appearance of the MPE in a preventive mode (e.g., in mice with pleural tumors growing without yet having provoked an MPE), or when given after an MPE was established in a therapeutic mode. This fact, together with the marked reduction in vascular permeability induced by the drug, indicates that the effects of ZA may primarily target the malignant effusion-inducing phenotype of cancer cells, and not their growth in general.

Angiogenesis, the formation of new vessel networks within growing tumor tissues, and enhanced fluid extravasation from existing and newly formed vessels occur in the malignancy-affected pleural space of humans and mice and are believed to be central to the pathogenesis of MPE (3, 7, 8, 31, 36, 37, 44–47). Here we show how ZA blocks MPE-associated new vessel formation and overall fluid exudation from adjacent vessels into the pleural cavity, thereby inhibiting MPE formation and progression. The antiangiogenic effects of ZA may be explained by its Ras- and RhoA-blocking properties in tumor (as shown here) and host (as has been shown by other groups) cells. In this regard, ZA may directly target tumor-associated angiogenesis by inhibiting endothelial cell proliferation, adhesion, and migration, by inducing endothelial cell apoptosis, and by decreasing capillary-like tube formation, effects dependent on inhibition of small GTP protein function in endothelial cells (13, 17, 28, 48–51). However, our results are consistent with an additional paracrine antiangiogenic mechanism of action of ZA, which is dependent on blockade of Ras and RhoA in tumor cells: the drug seems to target MPE-associated angiogenesis indirectly, via reductions in angiogenic mediator elaboration by tumor cells (13, 14, 20, 43, 48–51). Indeed, in our hands, ZA reduced VEGF, MCP-1, and IL-6 secretion by LLC cells. This indirect antiangiogenic effect of ZA may not be restricted to tumor cells; ZA may knock down angiogenic mediator expression by bystander host cells other than endothelial cells, such as mesothelial and inflammatory cells, including macrophages. In this regard, ZA blocked angiogenic MMP-9 expression by tumor-infiltrating macrophages in a multistage cervical carcinogenesis model (14). In our hands, reduced in vivo expression of MIP-2 and pro–MMP-9 in pleural tumors and fluid from ZA-treated mice with MPE was not accounted for by concomitant reductions of gene expression in tumor (LLC) cells. Therefore, ZA may block the expression of MIP-2 and/or pro–MMP-9 by host cells in the proximity or within pleural tumors, as was the case for MIP-2 expression by RAW264.7 macrophages.

Pleural inflammation exists in a mutually dependent association with new vessel formation and hyperpermeability of the pleural vasculature and constitutes the sine qua non for the pathogenetic basis of the vast majority of exudative pleural effusions (1, 3). An inflammatory host response accompanies human solid tumors and malignant effusions (14, 21, 32–34, 37, 39). For many years, inflammation in tumor-involved tissues was regarded de facto as primarily functioning against tumor growth and spread (34). However, recent evidence has revealed that the inflammatory host response triggered by the presence of tumor may actually boost tumor growth, angiogenesis, and metastasis (7, 8, 21, 34–36). In this connection, pleural mononuclear cells from patients with MPE are found to be immunologically defective, but still capable of expressing various cytokines (52, 53). The above highlights the importance of using immune-competent animal models of cancer that recapitulate intact host–tumor interactions, including inflammation, in cancer research. In such an immune-intact mouse model of MPE, we have previously shown how the proinflammatory axis of NF-{kappa}B/tumor necrosis factor-{alpha} promotes MPE formation (7, 8). Here we show how an NBP compound favorably alters the course of experimental MPE, partly via the modulation of the inflammatory host component of MPE. Indeed, ZA inhibited MCP-1 release by tumor and host cells, reduced the number of monocytes in the pleural space, and blocked local proinflammatory and angiogenic mediator expression in pleural tumor tissues and exudates. As with angiogenesis, the mechanism(s) by which ZA limited proinflammatory gene expression in tumor and host cells may be reduced RhoA and Ras activity, because the role of these small GTP proteins (especially Ras) has been recently expanded from simple cell cycle networking to modulation of tumor cell gene expression with concomitant paracrine impact on the host milieu (20, 21). The data discussed above imply that pharmacologic blockage of the small GTP proteins may affect MPE not only by impairing cancer cell survival but also by down-regulating the expression of several inflammatory and angiogenic cytokines. These effects of ZA are most probably not restricted to tumor cells but also apply to host cells, such as inflammatory and mesothelial cells, whose significant contribution to mediator elaboration in an MPE possibly explains the observed discordance between the pleural fluid and tumor tissue levels of various mediators in the present studies. In addition, because the inflammation-modulating effects of ZA on experimental MPE were associated with marked reductions in MPE progression, our data add to the mounting evidence that the inflammatory response that accompanies cancerous involvement of the pleura may promote pleural fluid accumulation and intrapleural tumor dissemination.

However, a potential drawback of the present work is that ZA was shown to be an effective inhibitor of pleural metastases and MPE of a mouse lung tumor growing in mice; whether the drug will prove equally effective in the context of human tumors growing in the human pleural space remains to be determined by future clinical testing. In addition, although we postulate the latter, we did not determine whether the effects of ZA on MPE formation are specific, or applicable to other NBP compounds. We chose ZA based on its potency relative to other NBPs and its availability for patients with cancer-related skeletal events; whether other NBPs will be equally effective against MPE remains to be determined.

In conclusion, we showed that ZA, a relatively nontoxic drug currently marketed for cancer-related skeletal events, is effective against experimental MPE, the mouse analog of a very common, difficult to treat, and debilitating occurrence in patients with cancer. The effects of the drug on survival were modest, reflecting the likely palliative effects that would be anticipated in future clinical trials of ZA for MPE. The drug functioned by inducing tumor cell apoptosis and by limiting host inflammation, tumor angiogenesis, and pleural vascular permeability. Given the uniformly poor prognosis of patients with MPE, the findings provide a rationale by which future clinical trials can be contemplated.


   Acknowledgments
 
The authors thank Dr. A. Marantidou, Z. Kollia, and A. Apostolopoulou for professional veterinarian, animal care, and editorial assistance, respectively. The authors also thank Drs. C. Murphy (Laboratory of Biological Chemistry, Medical School, University of Ioannina, Greece) and A. Pintzas (Laboratory of Signal Mediated Gene Expression, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece) for generously providing the plasmids containing the wild-type and dominant negative mutants of RhoA, respectively.


   FOOTNOTES
 
Supported by the Thorax Foundation (Athens, Greece) and by a research grant by Novartis Hellas SA (to I.K.). I.K. received funding ({euro}15,000) from Novartis Hellas SA (marketer of zoledronic acid in Greece).

Novartis had no involvement in study design, collection, analysis, and interpretation of data, writing of the report, and in the decision to submit the report for publication. G.T.S. and I.K. had full control of all of the data in this study and take complete responsibility for the integrity of the data and the accuracy of the data analysis.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200710-1513OC on April 3, 2008

Conflict of Interest Statement: G.T.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. H.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. I.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Z.Z. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.A.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. I.K. has received an unrestricted educational grant from Novartis Hellas SA, the marketer of zoledronic acid in Greece.

Received in original form October 12, 2007; accepted in final form April 3, 2008


   REFERENCES
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Sahn SA. Malignant pleural effusions. In: Fishman AP, Elias JA, Fishman JA, Grippi MA, Kaiser LR, Senior RM editors. Fishman's pulmonary diseases and disorders, 3rd ed. New York: McGraw-Hill; 1998. pp. 1429–1438.
  2. Antony VB, Loddenkemper R, Astoul P, Boutin C, Goldstraw P, Hott J, Rodriguez Panadero F, Sahn SA. Management of malignant pleural effusions. Am J Respir Crit Care Med 2000;162:1987–2001.[Free Full Text]
  3. Light RW, Lee YCG. Textbook of pleural diseases. Philadelphia: Lippincott, Williams, and Wilkins; 2001.
  4. Antunes G, Neville E, Duffy J, Ali N; for the BTS Pleural Disease Group, a subgroup of the BTS Standards of Care Committee. BTS guidelines for the management of malignant pleural effusions. Thorax 2003;58:ii29–ii38.[Free Full Text]
  5. Light RW. Talc for pleurodesis? Chest 2002;122:1506–1508.[CrossRef][Medline]
  6. Maskell NA, Lee YC, Gleeson FV, Hedley EL, Pengelly G, Davies RJ. Randomized trials describing lung inflammation after pleurodesis with talc of varying particle size. Am J Respir Crit Care Med 2004;170:377–382.[Abstract/Free Full Text]
  7. Stathopoulos GT, Zhu Z, Everhart MB, Kalomenidis I, Lawson WE, Bilaceroglu S, Peterson TE, Mitchell D, Yull FE, Light RW, et al. Nuclear factor-{kappa}B affects tumor progression in a mouse model of malignant pleural effusion. Am J Respir Cell Mol Biol 2006;34:142–150.[Abstract/Free Full Text]
  8. Stathopoulos GT, Kollintza A, Moschos C, Psallidas I, Sherrill TP, Pitsinos EN, Vassiliou S, Karatza M, Papiris SA, Graf D, et al. Tumor necrosis factor-{alpha} promotes malignant pleural effusion. Cancer Res 2007;67:9825–9834.[Abstract/Free Full Text]
  9. Gainford MC, Dranitsaris G, Clemons M. Recent developments in bisphosphonates for patients with metastatic breast cancer. BMJ 2005;330:769–773.[Free Full Text]
  10. Berenson JR, Rosen LS, Howell A, Porter L, Coleman RE, Morley W, Dreicer R, Kuross SA, Lipton A, Seaman JJ. Zoledronic acid reduces skeletal-related events in patients with osteolytic metastases. Cancer 2001;91:1191–1200.[CrossRef][Medline]
  11. Saad F. Clinical benefit of zoledronic acid for the prevention of skeletal complications in advanced prostate cancer. Clin Prostate Cancer 2005;4:31–37.[Medline]
  12. Clezardin P, Ebetino FH, Fournier PGJ. Bisphosphonates and cancer-induced bone disease: beyond their antiresorptive activity. Cancer Res 2005;65:4971–4974.[Abstract/Free Full Text]
  13. Santini D, Vespasiani Gentilucci U, Vincenzi B, Picardi A, Vasaturo F, La Cesa A, Onori N, Scarpa S, Tonini G. The antineoplastic role of bisphosphonates: from basic research to clinical evidence. Ann Oncol 2003;14:1468–1476.[Abstract/Free Full Text]
  14. Giraudo E, Inoue M, Hanahan D. An amino-bisphosphonate targets MMP-9–expressing macrophages and angiogenesis to impair cervical carcinogenesis. J Clin Invest 2004;114:623–633.[CrossRef][Medline]
  15. Kunzmann V, Bauer E, Feurle J, Weissinger F, Tony HP, Wilhelm M. Stimulation of gammadelta T cells by aminobisphosphonates and induction of antiplasma cell activity in multiple myeloma. Blood 2000;96:384–392.[Abstract/Free Full Text]
  16. Miyagawa F, Tanaka Y, Yamashita S, Minato N. Essential requirement of antigen presentation by monocyte lineage cells for the activation of primary human {gamma}{delta}T cells by aminobisphosphonate antigen. J Immunol 2001;166:5508.[Abstract/Free Full Text]
  17. van Beek E, Pieterman E, Cohen L, Lowik C, Papapoulos S. Farnesyl pyrophosphate synthase is the molecular target of nitrogen-containing bisphosphonates. Biochem Biophys Res Commun 1999;264:108–111.[CrossRef][Medline]
  18. Ohtsuka Y, Manabe A, Kawasaki H, Hasegawa D, Zaike Y, Watanabe S, Tanizawa T, Nakahata T, Tsuji K. RAS-blocking bisphosphonate zoledronic acid inhibits the abnormal proliferation and differentiation of juvenile myelomonocytic leukemia cells in vitro. Blood 2005;106:3134–3141.[Abstract/Free Full Text]
  19. Denoyelle C, Hong L, Vannier JP, Soria J, Soria C. New insights into the actions of bisphosphonate zoledronic acid in breast cancer cells by dual RhoA-dependent and -independent effects. Br J Cancer 2003;88:1631–1640.[CrossRef][Medline]
  20. Takai Y, Sasaki T, Matozaki T. Small GTP-binding proteins. Physiol Rev 2001;81:153–208.[Abstract/Free Full Text]
  21. Karin M. Inflammation and cancer: the long reach of Ras. Nat Med 2006;11:20–21.
  22. Stathopoulos GT, Moschos C, Kollintza A, Psallidas I, Loutrari H, Magkouta S, Petrocheilou H, Theodoropoulou P, Papiris SA, Roussos C, et al. Zoledronic acid limits experimental malignant pleural effusion [abstract]. Eur Respir J 2007;30:291s.
  23. Fingleton B, Carter KJ, Matrisian LM. Loss of functional Fas ligand enhances intestinal tumorigenesis in the Min mouse model. Cancer Res 2007;67:4800–4806.[Abstract/Free Full Text]
  24. Garcia RL, Coltrera MD, Gown AM. Analysis of proliferative grade using anti-PCNA/cyclin monoclonal antibodies in fixed, embedded tissues: comparison with flow cytometric analysis. Am J Pathol 1989;134:733–739.[Abstract]
  25. Cao Y, Bonizzi G, Seagroves TN, Greten FR, Johnson R, Schmidt EV, Karin M. IKK{alpha} provides an essential link between RANK signaling and cyclin D1 expression during mammary gland development. Cell 2001;107:763–775.[CrossRef][Medline]
  26. Lauk S, Zietman A, Skates S, Fabian R, Suit HD. Comparative morphometric study of tumor vasculature in human squamous cell carcinomas and their xenotransplants in athymic nude mice. Cancer Res 1989;49:4557–4561.[Abstract/Free Full Text]
  27. Reid T, Furuyashiki T, Ishizaki T, Watanabe G, Watanabe N, Fujisawa K, Morii N, Madaule P, Narumiya S. Rhotekin, a new putative target for Rho bearing homology to a serine/threonine kinase, PKN, and rhophilin in the rho-binding domain. J Biol Chem 1996;271:13556–13560.[Abstract/Free Full Text]
  28. Loutrari H, Magkouta S, Pyriochou A, Koika V, Kolisis FN, Papapetropoulos A, Roussos C. Mastic oil from Pistacia lentiscus var. chia inhibits growth and survival of human K562 leukemia cells and attenuates angiogenesis. Nutr Cancer 2006;55:86–93.[CrossRef][Medline]
  29. He T-C, Zhou S, Da Costa LT, Yu J, Kinzler KW, Vogelstein B. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA 1998;95:2509–2514.[Abstract/Free Full Text]
  30. Qiu RG, Chen J, McCormick F, Symons M. A role for Rho in Ras transformation. Proc Natl Acad Sci USA 1995;92:11781–11785.[Abstract/Free Full Text]
  31. Yano S, Shinohara H, Herbst RS, Kuniyasu H, Bucana CD, Ellis LM, Fidler IJ. Production of experimental malignant pleural effusions is dependent on invasion of the pleura and expression of vascular endothelial growth factor/vascular permeability factor by human lung cancer cells. Am J Pathol 2000;157:1893–1903.[Abstract/Free Full Text]
  32. Olsen GN, Gangemi JD. Bronchoalveolar lavage and the immunology of primary lung cancer. Chest 1985;87:677–683.[CrossRef][Medline]
  33. Antony VB, Godbey SW, Kunkel SL, Hott JW, Hartman DL, Burdick MD, Strieter RM. Recruitment of inflammatory cells to the pleural space: chemotactic cytokines, IL-8, and monocyte chemotactic peptide-1 in human pleural fluids. J Immunol 1993;151:7216–7223.[Abstract]
  34. Pollard JW. Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer 2004;4:71–78.[CrossRef][Medline]
  35. Balkwill F, Coussens LM. Cancer: an inflammatory link. Nature 2004;431:405–406.[CrossRef][Medline]
  36. Yeh H-H, Lai W-W, Chen HHW, Liu H-S, Su W-C. Autocrine IL-6-induced Stat3 activation contributes to the pathogenesis of lung adenocarcinoma and malignant pleural effusion. Oncogene 2006;25:4300–4309.[CrossRef][Medline]
  37. Hamed EA, El-Noweihi AM, Mohamed AS, Mahmoud A. Vasoactive mediators (VEGF and TNF-a) in patients with malignant and tuberculous pleural effusions. Respirology 2004;9:81–86.[CrossRef][Medline]
  38. Eickelberg O, Sommerfeld CO, Wyser C, Tamm M, Reichenberger F, Bardin PG, Soler M, Roth M, Perruchoud AP. MMP and TIMP expression pattern in pleural effusions of different origins. Am J Respir Crit Care Med 1997;156:1987–1992.[Abstract/Free Full Text]
  39. Alexandrakis MG, Coulocheri SA, Bouros D, Mandalaki K, Karkavitsas N, Eliopoulos GD. Evaluation of inflammatory cytokines in malignant and benign pleural effusions. Oncol Rep 2000;7:1327–1332.[Medline]
  40. Jin HY, Lee KS, Jin SM, Lee YC. Vascular endothelial growth factor correlates with matrix metalloproteinase-9 in the pleural effusion. Respir Med 2004;98:115–122.[CrossRef][Medline]
  41. Belotti D, Paganoni P, Manenti L, Garofalo A, Marchini S, Taraboletti G, Giavazzi R. Matrix metalloproteinases (MMP9 and MMP2) induce the release of vascular endothelial growth factor (VEGF) by ovarian carcinoma cells: implications for ascites formation. Cancer Res 2003;63:5224–5229.[Abstract/Free Full Text]
  42. Fukumoto T, Matsukawa A, Yoshimura T, Edamitsu S, Ohkawara S, Takagi K, Yoshinaga M. IL-8 is an essential mediator of the increased delayed-phase vascular permeability in LPS-induced rabbit pleurisy. J Leukoc Biol 1998;63:584–590.[Abstract]
  43. Inoue R, Matsuki NA, Jing G, Kanematsu T, Abe K, Hirata M. The inhibitory effect of alendronate, a nitrogen-containing bisphosphonate on the PI3K-Akt-NFkappaB pathway in osteosarcoma cells. Br J Pharmacol 2005;146:633–641.[CrossRef][Medline]
  44. Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O'Shea KS, Powell-Braxton L, Hillan KJ, Moore MW. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 1996;380:439–442.[CrossRef][Medline]
  45. Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 1996;380:435–439.[CrossRef][Medline]
  46. Herbst RS, Fidler IJ. Angiogenesis and lung cancer: potential for therapy. Clin Cancer Res 2000;6:4604–4606.[Free Full Text]
  47. Zebrowski BK, Yano S, Liu W, Shaheen RM, Hicklin DJ, Putnam JB Jr, Ellis LM. Vascular endothelial growth factor levels and induction of permeability in malignant pleural effusions. Clin Cancer Res 1999;5:3364–3368.[Abstract/Free Full Text]
  48. Hasmim M, Bieler G, Ruegg C. Zoledronate inhibits endothelial cell adhesion, migration and survival through the suppression of multiple, prenylation-dependent signaling pathways. J Thromb Haemost 2007;5:166–173.[CrossRef][Medline]
  49. Wood J, Bonjean K, Ruetz S, Bellahcène A, Devy L, Foidart JM, Castronovo V, Green JR. Novel antiangiogenic effects of the bisphosphonate compound zoledronic acid. J Pharmacol Exp Ther 2002;302:1055–1061.[Abstract/Free Full Text]
  50. Li X, Liu L, Tupper JC, Bannerman DD, Winn RK, Sebti SM, Hamilton AD, Harlan JM. Inhibition of protein geranylgeranylation and rhoa/rhoa kinase pathway induces apoptosis in human endothelial cells. J Biol Chem 2002;18:15309–15316.
  51. Tang Y, Kim M, Carrasco D, Kung AL, Chin L, Weissleder R. In vivo assessment of RAS-dependent maintenance of tumor angiogenesis by real-time magnetic resonance imaging. Cancer Res 2005;65:8324–8330.[Abstract/Free Full Text]
  52. Mantovani G, Maccio A, Pisano M, Versace R, Lai P, Esu S, Massa E, Ghiani M, Dessi D, Melis GB, et al. Tumor-associated lympho-monocytes from neoplastic effusions are immunologically defective in comparison with patient autologous PBMCs but are capable of releasing high amounts of various cytokines. Int J Cancer 1997;71:724–731.[CrossRef][Medline]
  53. Gjomarkaj M, Pace E, Melis M, Spatafora M, Toews GB. Mononuclear cells in exudative malignant pleural effusions: characterization of pleural phagocytic cells. Chest 1994;106:1042–1049.[CrossRef][Medline]

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