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[Cancer Research 65, 10464-10471, November 15, 2005]
© 2005 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Suppression of Urokinase Receptor Expression by Thalidomide Is Associated with Inhibition of Nuclear Factor {kappa}B Activation and Subsequently Suppressed Ovarian Cancer Dissemination

Hiroshi Kobayashi1, Tatsuo Yagyu2, Toshiharu Kondo3, Noriyuki Kurita4, Kiyokazu Inagaki2, Shoji Haruta5, Ryuji Kawaguchi5, Takashi Kitanaka5, Yoshiharu Sakamoto5, Yoshihiko Yamada5, Naohiro Kanayama1 and Toshihiko Terao1

1 Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Handayama, Hamamatsu, Shizuoka; 2 NetForce Co. Ltd., Taiko, Nakamura; 3 Computer Technology Integration, Co. Ltd., Meieki-minami, Nakamura, Nagoya, Aichi; 4 Department of Knowledge-Based Information Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi; and 5 Department of Obstetrics and Gynecology, Nara Medical University, Shijo-cho, Kashihara, Nara, Japan

Requests for reprints: Hiroshi Kobayashi, Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Handayama 1-20-1, Hamamatsu, Shizuoka 431-3192, Japan. Phone: 81-53-435-2309; Fax: 81-53-435-2308; E-mail: hirokoba{at}hama-med.ac.jp.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thalidomide has been used to treat a variety of diseases ranging from alleviation of autoimmune disorders to prevention of metastasis of cancers. It has been shown previously that increased levels of urokinase-type plasminogen activator receptor (uPAR) correlate well with higher invasive phenotype. We examined whether thalidomide is able to suppress the expression of uPAR mRNA and protein in human ovarian cancer cell line HRA and human chondrosarcoma cell line HCS-2/8. Here, we show that: (a) thalidomide suppresses the expression of constitutive and transforming growth factor-ß1 (TGF-ß1)–induced uPAR mRNA and protein; (b) a nuclear factor {kappa}B (NF-{kappa}B) activation system (phosphorylation of I{kappa}B-{alpha} and degradation of I{kappa}B-{alpha}) is necessary for the TGF-ß1-induced increase in uPAR expression, because L-1-tosylamido-2-phenylethyl chloromethyl ketone, a NF-{kappa}B inhibitor, reduced the uPAR production as well as mRNA expression; (c) thalidomide failed to further strengthen L-1-tosylamido-2-phenylethyl chloromethyl ketone's action; (d) the once-daily i.p. administration of thalidomide (400 µg/g body weight/d) decreased progressive growth of HRA tumors and ascites formation in an in vivo animal model; and (e) the once-daily i.p. administration of thalidomide in combination with paclitaxel (i.p., 100 µg/20 g at days 2 and 5) significantly decreased progressive growth of HRA cells in a synergistic fashion. We conclude that thalidomide down-regulates constitutive and TGF-ß1-stimulated uPAR mRNA and protein expression possibly through suppression of NF-{kappa}B activation. Furthermore, combination therapy with thalidomide plus paclitaxel may be an effective way to markedly reduce i.p. tumor growth and ascites in ovarian cancer dissemination.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Activation of receptor-bound urokinase-type plasminogen activator (uPA) on the cell surface seems to play an important role in cancer cell invasion and metastasis (1). uPA receptor (uPAR) binds uPA with high affinity with a Kd of ~0.5 nmol/L (2, 3). Most uPAR proteins are concentrated at the invasive foci (4); it accelerates plasmin formation at the cell surface. Overexpression of a human uPAR cDNA increased the ability of tumor cells to penetrate a barrier of basement membrane. Expression of both uPA and uPAR correlates with invasive cancer cell phenotype and poor prognosis (5–7). For colon cancer, a high uPAR level portends a low 5-year survival rate (8). Exposure of antibody to uPAR (9), soluble uPAR (10, 11), or stable transfection with antisense uPAR cDNA (12) rescues the invasiveness of tumor cells. The uPAR protein is inducible by epidermal growth factor (EGF), transforming growth factor-ß (TGF-ß; refs. 13, 14), hepatocyte growth factor (15), vascular endothelial growth factor (VEGF; ref. 16), IFN-{gamma}, tumor necrosis factor-{alpha} (17), and by the tumor promoter, phorbol ester (2, 18). Activation of the protein kinase C (PKC) pathway has been reported to increase uPAR mRNA in certain cell types (19).

Inhibition of specific target molecules in common signaling pathway(s) responsible for metastasis can have potential clinical relevance. Nuclear factor {kappa}B (NF-{kappa}B) and activator protein control the expression of uPA and uPAR (20). The inhibition of PKC and phosphatidylinositol 3-kinase signaling (through NF-{kappa}B and activator protein) suppressed the secretion of uPA, resulting in the inhibition of motility of highly invasive breast cancer cells (20).

Despite its history as a human teratogen, thalidomide is emerging as a treatment for cancer and inflammatory diseases (21). It has shown great promise in aphthous and genital ulcers, cancer cachexia, HIV, tuberculosis, and chronic graft versus host disease. This compound is also being investigated for treatment of several malignancies such as multiple myeloma, renal cell carcinoma, and liver and thyroid cancers. A better understanding of its many mechanisms of action has provoked great interest in its potential use for treatment of various disorders (22). It has been reported that thalidomide suppressed NF-{kappa}B nuclear translocation, I{kappa}B degradation, and NF-{kappa}B-inducing kinase–induced NF-{kappa}B transcriptional activation. These results suggest that the molecular target of the effects of thalidomide may be I{kappa}B phosphorylation by I{kappa}B kinase, whose activation follows NF-{kappa}B-inducing kinase activation and precedes I{kappa}B degradation in the NF-{kappa}B pathway (23). We speculate that thalidomide inhibits lipopolysaccharide-induced up-regulation of cytokine expression possibly through suppression of p38 kinase–dependent NF-{kappa}B activation.6 This might inhibit tumor invasion and metastasis, possibly by suppression of the cell-associated plasminogen activation system. However, little is known concerning the potential role of thalidomide in the regulation of uPAR mRNA and its protein.

In this article, we report the positive modulation of uPAR mRNA and protein by TGF-ß1 and the negative regulatory effects by thalidomide on uPAR gene expression in human cancer cells (ovarian cancer cell line HRA and chondrosarcoma cell line HCS-2/8). We undertook the present study to determine the role of thalidomide on the regulation of NF-{kappa}B activation-dependent uPAR expression. Furthermore, we explored the possible effects of the interaction between thalidomide and paclitaxel by assessing tumor burden, ascites volume, and tumor dissemination.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials. Thalidomide was purchased from Calbiochem (La Jolla, CA). Stock solutions (20 mg/mL) of thalidomide were freshly prepared in DMSO (Sigma-Aldrich Japan, Tokyo, Japan) and diluted directly into cultured cells. The final concentration of DMSO in all experiments was <0.01%, and all treatment conditions were compared with vehicle controls. Anti-phospho-I{kappa}B-{alpha} antibodies were purchased from New England Biolabs, Inc. (Beverly, MA). Anti-p65 NF-{kappa}B and I{kappa}B-{alpha} antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The neutralizing uPA-specific antibody, anti-uPAR polyclonal rabbit antibody, and a specific ELISA for uPAR (IMUBIND 893) were obtained from American Diagnostica (Greenwich, CT). Nonimmune anti-mouse/rabbit IgG and anti-mouse/rabbit IgG conjugated with horseradish peroxidase were from Dako (Copenhagen, Denmark). [32P]dATP random prime labeling Mega Prime kit was purchased from Amersham Bioscience (Tokyo, Japan). l-1-Tosylamido-2-phenylethyl chloromethyl ketone (TPCK), a serine protease inhibitor that blocks I{kappa}B-{alpha} degradation, was purchased from Sigma. All reagents used were of analytic grade.

Cell culture. The human ovarian cancer cell line HRA (24) and human chondrosarcoma cell line HCS-2/8 (25) have been described previously. The HRA cells were provided by Dr. Y. Kikuchi and the HCS-2/8 cells were a gift from Dr. M. Takigawa (Okayama University, Okayama, Japan). Cells were maintained in RPMI 1640 (HRA) or DMEM (HCS-2/8) supplemented with penicillin (100 units/mL), streptomycin (100 µg/mL), and 10% heat-inactivated fetal bovine serum (Life Technologies, Inc., Rockville, MD) at 37°C in 5% CO2-air atmosphere. Before stimulation, cells were washed thrice with PBS and incubated overnight in complete medium containing 1% fetal bovine serum. The test drugs were added and incubation was continued for different time lapses. After culture, medium was aspirated and cells were harvested and washed extensively. Immediately before harvest, cell viability was consistently found to be >95%.

Nuclear NF-{kappa}B pull-down assay. Nuclear NF-{kappa}B pull-down assay was done as previously described (26). Cells were pelleted and resuspended in hypotonic lysis buffer. The nuclear pellet was extracted and the supernatants were diluted and incubated with agarose beads conjugated to a consensus NF-{kappa}B-binding oligonucleotide (Santa Cruz Biotechnology). The result was analyzed by SDS-PAGE and Western blotting using an anti-p65 NF-{kappa}B antibody.

Northern blot analysis. Northern blot analysis was done using standard methods. Ten micrograms of RNA were separated in 1.2% agarose gels and blotted onto Hybond N+ membranes. Prehybridization and hybridization were done in 50% formamide at 42°C with 5 x 106 cpm/mL uPAR cDNA probe as described previously (27), and filters were reprobed with the cDNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to correct for the amount of RNA loaded onto the filters. A 1.1 kb XbaI-EcoRI fragment of uPAR cDNA (28) was radiolabeled with 32P-dATP via random hexamer primer extension and used as hybridization probe. After each hybridization, the membranes were washed and exposed on Kodak BioMax MS-1 film at –70°C. Filters were quantitated by scanning densitometry using a Bio-Rad (Tokyo, Japan) model 620Video Densitometer with an Analyst software package.

Western blot. The cells treated with or without various agents for indicated times were washed with PBS. Cells (1 x 106) were lysed in 750 µL of lysis buffer (50 mmol/L HEPES, 0.5 mol/L NaCl, 0.05% Tween 20, 1% Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL E-64, 10 µg/mL leupeptin) at 4°C for 15 minutes and scraped with a rubber policeman. The protein concentrations in the supernatants of cell extracts were measured by the Bio-Rad protein assay. All samples were stored at –70°C until use. In parallel, cells treated in the same condition in different dishes were harvested and counted using a hemocytometer. Centrifuged lysates (50 µg) were analyzed by SDS-PAGE and transferred to a polyvinylidene difluoride membrane by semidry transfer (9). Membranes were blocked for 1 hour at room temperature in TBS containing 0.1% Tween 20 and 2% bovine serum albumin. Blots were probed overnight with the following primary antibodies at 4°C: phospho-I{kappa}B-{alpha}, I{kappa}B-{alpha}, and p65 NF-{kappa}B were detected by specific primary antibodies and horseradish peroxidase–conjugated secondary antibodies. The immunoblots were visualized by chemiluminescence with the enhanced chemiluminescence kit from Amersham Biosciences.

Invasion assay. Invasion assays were done essentially as described previously (29). The ability of cells to migrate across a Matrigel barrier (invasion) was determined by the modified Boyden chamber method. Briefly, HRA cells (105/chamber) were added to polyvinylpyrrolidone-free, 8 µm polycarbonate filters coated with 50 µL of 50 µg/mL Matrigel and incubated with complete medium containing 0.1% bovine serum albumin for 36 hours at 37°C. NIH3T3 fibroblast-conditioned medium was used as a chemoattractant in the lower chamber, and serum-free medium containing 0.1% bovine serum albumin was used as a negative control. Filters were removed from the chambers and stained with hematoxylin. Cells were counted at a magnification of x100, and the mean numbers of cells per field in five random fields were recorded. Duplicate filters were used, and the experiments were repeated thrice. The effects of agents that alter the activity of uPA/uPAR expression, including thalidomide or neutralizing monoclonal antibodies against uPA and uPAR, on the invasiveness of HRA cells were determined by measuring the ability of cells treated with these agents to pass through a layer of the extracellular matrix extract Matrigel coating a filter using chemoinvasion chambers.

Animals. Seven-week-old female nude mice (BALB-c nu/nu; SLC, Hamamatsu, Japan) were delivered to the Hamamatsu University School of Medicine, Laboratory Animal Center, housed in isolated conditions, maintained under specific pathogen-free conditions, fed autoclaved standard pellets and water, allowed to adapt to their new environment, and used according to institutional guidelines. All protocols were approved by the Committee on Animal Research, Hamamatsu University School of Medicine.

To produce tumors, HRA cells were harvested from subconfluent cultures by a brief exposure to 0.25% trypsin and EDTA. Trypsinization was stopped with medium containing 10% fetal bovine serum, and the cells were washed once in serum-free medium, and resuspended in PBS. Only single cell suspensions with >95% viability were used for the in vivo injections. The total number of viable cells was determined by trypan blue exclusion and lactate dehydrogenase assay. Mice were inoculated i.p. with HRA cells (5 x 106 cells per mouse in 200 µL of PBS). At the end of the experiment, mice underwent euthanasia with ether. The mice were killed at day 9. The mice were examined for the existence of ascites and the weight of tumor nodules formed in the peritoneum and on the diaphragm. The volume of ascites was measured, and tumor tissue was excised, weighted and fixed in 4% paraformaldehyde, and embedded in paraffin. Paraffin sections (5 µm) were used for histologic analysis. Histopathology confirmed the nature of the disease. For histology staining procedures, one part of the tumor tissue was fixed in formalin and embedded in paraffin. Another part of the tumor was snap-frozen in liquid nitrogen, and stored at –70°C. Frozen tissues were used for Western blotting. Body weights were measured twice weekly.

Experimental design. Mice were inoculated i.p. with HRA cells (n = 36). Two days after inoculation, mice were randomized into four groups (each group, n = 9) to receive once-daily i.p. injection of vehicle (DMSO, 0.1 mL/d; control group), or 100, 200, or 400 µg/g body weight of thalidomide for 7 days. Nine days after i.p. implantation of tumor cells, mice were killed to ascertain the presence and size of tumor lesions.

In the next set of studies, the mice were randomized into four treatment groups (each group, n = 9) to receive once-daily i.p. injection of vehicle (control), an i.p. injection of 100 µg/20 g body weight (5 µg/g) of paclitaxel at days 2 and 5, once-daily i.p. injection of 400 µg/g body weight of thalidomide, or once-daily i.p. injection of 400 µg/g body weight of thalidomide and an i.p. injection of 5 µg/g body weight of paclitaxel. The thalidomide was given i.p. daily. Paclitaxel (Sigma-Aldrich) was dissolved first in 50% Cremophor EL (Sigma-Aldrich) in ethanol, then diluted further in PBS.

Statistical analysis. Data are presented as mean ± SD. All statistical analyses were done using StatView. The Mann-Whitney U test was used for comparisons between different groups. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of uPAR mRNA by transforming growth factor-ß1 in HRA cells. Unstimulated HRA cells expressed different levels of 1.1 kb uPAR transcripts (Fig. 1). uPAR mRNA in HRA cells incubated with TGF-ß1 (10 ng/mL) for 6 hours was increased 5.5-fold as compared with the unstimulated cells, which appeared at 3 hours, peaked at 6 hours, and declined at 24 hours (Fig. 1A). The effect of TGF-ß1 on uPAR expression was dose-dependent at TGF-ß1 concentrations of 0.3 to 30 ng/mL, with a maximum increase seen after treatment of HRA cells with 10 ng/mL of TGF-ß1 (Fig. 1B). Similar TGF-ß1 effects on uPAR mRNA were found in the HCS-2/8 cells (data not shown).



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  		Figure 1. Stimulation of uPAR gene expression in HRA cells by Northern blot analysis. A, stimulation of uPAR gene expression in HRA cells by TGF-ß1 in a time-dependent manner. Cells were grown to 90% confluence and then stimulated with TGF-ß1 (10 ng/mL) for the indicated periods of time. B, TGF-ß1 stimulates uPAR gene expression in a dose-dependent manner. HRA cells were incubated for 6 hours with different doses of TGF-ß1. Total cellular RNA was extracted and separated on 1.2% agarose/formaldehyde gel and transferred to Hybond N+ membrane. Filters were hybridized with 32P-labeled uPAR cDNA or with 32P-labeled GAPDH cDNA probe. Top, representative autoradiograms; bottom, levels of uPAR mRNA expression as quantified by densitometric scanning. Columns, mean; bars, ± SD; different superscripts (a-c) indicate statistical difference (P < 0.05).

 
Suppression by thalidomide of uPAR mRNA accumulation by transforming growth factor-ß1. It is of interest to determine whether thalidomide affects the expression of unstimulated or TGF-ß1-induced uPAR mRNA (Fig. 2). When concentrations of thalidomide (0.04-5 µmol/L) were added in the presence of 10 ng/mL TGF-ß1 to HRA cells, there was a dose-dependent inhibition of 30% (lane 6) and 70% (lane 7) of the uPAR mRNA level at concentrations of 1 and 5 µmol/L, respectively, as determined by scanning densitometry. Similar effects of thalidomide inhibition were found in the HCS-2/8 cells (lane 8). After treatment with 5 µmol/L thalidomide alone (lane 12), a 50% reduction of uPAR mRNA was observed in HCS-2/8 cells.



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  		Figure 2. Thalidomide suppresses TGF-ß1-stimulated uPAR gene expression. Cells were incubated for 6 hours with or without 10 ng/mL TGF-ß1 in the presence or absence of thalidomide (0.04, 0.2, 1, or 5 µmol/L). Total cellular RNA was extracted and analyzed for uPAR mRNA expression by Northern blot analysis and compared with untreated control cells (vehicle). Top, representative autoradiograms; bottom, levels of uPAR mRNA expression as quantified by densitometric scanning. Columns, mean; bars, ± SD; different superscripts (a-e) indicate statistical difference (P < 0.05).

 
Suppression by thalidomide of unstimulated and transforming growth factor-ß1-induced uPAR protein expression. We measured the uPAR levels in HRA cells stimulated with or without TGF-ß1 (10 ng/mL) using a specific ELISA for uPAR (Fig. 3). The levels of uPAR protein in unstimulated and TGF-ß1-stimulated HRA cells were 9.4 ± 1.3 and 36.7 ± 4.0 ng/106 cells, respectively, demonstrating that, after stimulation, uPAR protein levels increased ~4-fold. The levels of uPAR protein in TGF-ß1-stimulated cells treated with thalidomide (1 and 5 µmol/L) were 26.1 ± 3.2 and 18.3 ± 2.4 ng/106 cells, respectively. Thus, the dose-dependent ability of thalidomide to inhibit expression of uPAR protein by cells was shown using ELISA. Similar effects of thalidomide inhibition were found in the HCS-2/8 cells.



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  		Figure 3. Effects of thalidomide on TGF-ß1-induced uPAR protein production. Confluent cells (90%) were preincubated without or with thalidomide (0.04, 0.2, 1, or 5 µmol/L, 2 hours), and then TGF-ß1 (10 ng/mL) was added and incubated for 24 hours. Concentrations of uPAR in the cell lysates were measured by ELISA. The minimum detectable level of uPAR was 10 pg/mL. Columns, mean; bars, ± SD; different superscripts (a-f) indicate statistical difference (P < 0.05).

 
Cell viability, monitored by lactate dehydrogenase leakage in the culture medium and trypan blue dye exclusion test, is not altered under the different exposure conditions (data not shown). These experiments showed that a marked decrease of uPAR expression is observed when thalidomide is added to the medium before stimulation by TGF-ß1.

Thalidomide inhibits transforming growth factor-ß1-induced NF-{kappa}B activation. TGF-ß1 treatment caused activation of NF-{kappa}B as shown by the measures of I{kappa}B-{alpha} phosphorylation and I{kappa}B-{alpha} degradation (Fig. 4A and B). NF-{kappa}B activation was determined by examining the phosphorylation of I{kappa}B because degradation of I{kappa}B via its phosphorylation is necessary for nuclear translocation of NF-{kappa}B and subsequent activation of target gene expression. Therefore, the level of the I{kappa}B-{alpha} protein decreased as the phosphorylation level of the I{kappa}B-{alpha} protein increased (lane 1 versus lane 2). Pretreatment of thalidomide (5 µmol/L, 2 hours; lane 3) blocked TGF-ß1-induced I{kappa}B-{alpha} phosphorylation by 57% and TGF-ß1-induced I{kappa}B-{alpha} degradation by 50%, respectively. Direct inhibition of NF-{kappa}B with TPCK (10 µmol/L, 1 hour; lane 4) blocked TGF-ß1-induced I{kappa}B-{alpha} phosphorylation by 74% and TGF-ß1-induced I{kappa}B-{alpha} degradation by 70%, respectively. This suggests that inhibition of TGF-ß1-induced NF-{kappa}B activation by thalidomide is responsible for the inhibition of uPAR expression.



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  		Figure 4. Effects of thalidomide on TGF-ß1-induced phosphorylation of I{kappa}B-{alpha} and degradation of I{kappa}B-{alpha} (A and B) and NF-{kappa}B nuclear translocation (C and D). A, 90% confluent cells pretreated with or without thalidomide (5 µmol/L, 2 hours) or TPCK (10 µmol/L, 1 hour) were exposed to medium alone or medium with TGF-ß1 (10 ng/mL) for 20 minutes. Expression levels of I{kappa}B-{alpha} and phosphorylated I{kappa}B-{alpha} (pI{kappa}B-{alpha}) were determined by Western blot analysis. B, blots of pI{kappa}B-{alpha} in (A) were scanned, and the band intensities were quantitated. The band intensity values were used to determine the relative amount of pI{kappa}B-{alpha}. C, 90% confluent cells pretreated with or without thalidomide (5 µmol/L, 2 hours) or TPCK (10 µmol/L, 1 hour) were exposed to medium alone or medium with TGF-ß1 (10 ng/mL) for 20 minutes. Expression levels of NF-{kappa}B were determined by Western blot analysis using p65 antibody. D, blots of NF-{kappa}B in (C) were scanned, and the band intensities were quantitated. The band intensity values were used to determine the relative amount of NF-{kappa}B. Columns, mean; bars, ± SD; different superscripts (a-d) indicate statistical difference (P < 0.05).

 
Transforming growth factor-ß1 induced nuclear translocation of NF-{kappa}B was inhibited by pretreatment with thalidomide. Thalidomide-dependent suppression of TGF-ß1-induced nuclear translocation of NF-{kappa}B was examined in HRA cells using nuclear NF-{kappa}B pull-down assays. Nuclear extracts prepared from control and TGF-ß1-treated HRA cells were incubated with agarose beads conjugated to consensus NF-{kappa}B oligomers, and nuclear NF-{kappa}B was assayed by Western blotting using anti-p65 antibody. As shown in Fig. 4C and D, TGF-ß1 activation induced increased nuclear NF-{kappa}B at 20 minutes of incubation (lane 3), which was inhibited by pretreatment with either thalidomide (5 µmol/L, 2 hours; lane 4) or TPCK (10 µmol/L, 1 hour; lane 5). Thalidomide did not enhance TPCK's action (lane 6). Both thalidomide and TPCK do not cause cell death (data not shown).

The effect of thalidomide as well as neutralizing antibodies against urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor on cell invasiveness. Our previous experiments showed that treatment with TGF-ß1 produced a significant stimulation of the invasiveness of HRA cells in a dose-dependent manner, with a maximum stimulation at 10 ng/mL TGF-ß1 (29). The uPA inhibitor amiloride (Sigma-Aldrich, Co.) was added to HRA cells in the upper chamber of the transwells at 100 µmol/L to study the effect of uPA inhibition on invasion. To confirm the results obtained with amiloride, invasion assay was done with specific blocking anti-uPA and anti-uPAR antibodies, using isotype-controlled, nonspecific IgG as a control. Amiloride led to a 67 ± 15% decrease in invasion of TGF-ß1-treated cells (P < 0.05 versus control; data not shown). Thus, TGF-ß1-mediated cell invasion is dependent on uPA. We next examined whether thalidomide and anti-uPA or anti-uPAR have the same effect on antiinvasive action. Figure 5 shows the effect of adding increasing concentrations of antibodies or thalidomide on the invasiveness of the TGF-ß1-stimulated cells. The TGF-ß1-stimulated cell invasion was specifically reversed by concurrent treatment with either neutralizing anti-uPA or anti-uPAR antibody, as well as with thalidomide. These data support the hypothesis that cancer cells leading to invasion are induced through up-regulation of the uPA/uPAR system. Thalidomide, which suppresses uPAR expression, could in turn modify the invasive behavior of these cells. However, thalidomide had no additive effect on antibody-mediated suppression of cell invasiveness.



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  		Figure 5. Suppression of invasiveness in TGF-ß1-stimulated HRA cells by treatment with antibodies and thalidomide HRA cells (left) and HCS-2/8 cells (right; 5 x 104 cells) were placed in the top wells of a chemoinvasion chamber apparatus with the neutralizing antibodies against uPA (10 µg/mL) and uPAR (10 µg/mL), or thalidomide (5 µmol/L) in the presence or absence of 10 ng/mL TGF-ß1. Columns, mean of measurements made on three independent wells. Different superscripts (a-d) indicate statistical difference (P < 0.05).

 
In vivo treatment of HRA tumors by once-daily injection of thalidomide. We finally evaluated the therapeutic efficacy of once-daily i.p. injection of different doses of thalidomide against human ovarian cancer HRA cells growing in the peritoneum of nude mice. Two days after tumor cell inoculation, groups of mice (n = 9) received once-daily i.p. injections of DMSO vehicle (control) or thalidomide at doses of 100, 200, or 400 µg/g body weight/d for 7 days. Nine days after i.p. inoculation of HRA cells, 100% of control mice developed tumor nodules on the peritoneal surface. Tumors were also found on the surface of the diaphragm, intestines, mesentery, and liver in the control group. Tumor implants were nodular and of different sizes (diameter, 2-5 mm) and were so numerous that in places they had merged together to coat the diaphragmatic surface. Some tumor nodules could be detected on all peritoneal surfaces, e.g., mesentery and abdominal wall, resembling the anatomic pattern of the transperitoneal metastasis of human ovarian cancer. Control tumors aggressively invaded the peritoneum and liver and penetrated the diaphragm, whereas thalidomide-treated (400 µg) tumors neither invaded the peritoneum and liver nor penetrated the diaphragm, and macroscopically, the tumors had a smooth surface. Histologic examination showed significant differences in 9-day-old nodules (data not shown). In mice treated with vehicle, the tumor deposits were very invasive, infiltrating into the underlying liver parenchyma as well as into the underlying submesothelial extracellular matrix, adipose tissue, and diaphragmatic muscle. In contrast, in animals treated with thalidomide, there were few or no remarkable invasion of tumor cells into liver parenchyma or subdiaphragmatic tissue.

The effect of thalidomide on peritoneal dissemination was assessed by counting the number and weight of metastatic nodules in the mesentery and peritoneal wall. The mean tumor burden was 2.71 ± 0.50, 2.64 ± 0.18, 2.49 ± 0.21, or 1.91 ± 0.11 g in the control and thalidomide-treated groups (100, 200, or 400 µg), respectively (Fig. 6, open columns). The weight of solid tumors was significantly decreased in treatment groups (thalidomide, 400 µg/g). Thalidomide at a dose of 200 and 400 µg/g body weight/d significantly inhibited the formation of ascites (P < 0.05; Fig. 6, filled columns). Thalidomide treatment resulted in a 32% decrease in the weight of metastatic nodules in the mesentery, diaphragm, and peritoneum, suggesting that thalidomide had a statistically significant effect on the size of the subdiaphragmatic metastatic tumors as well as the i.p. disseminated metastasis. Furthermore, we found that thalidomide treatment neither enhances apoptosis nor necrosis in the tumors isolated from the animals (data not shown). No effect on food intake was observed in either the treated or control groups. Thalidomide had no significant effects on the general well-being of the animals (data not shown). The weight (g) of the tumor was significantly correlated with the volume (mL) of ascites (P = 0.011); the correlation coefficient was 0.302.



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  		Figure 6. Effects of thalidomide on tumor burden (open columns) and ascites formation (filled columns) in mice inoculated with HRA cells. Four groups of nude mice (each group, n = 9) were used. Thalidomide (0, 100, 200, or 400 µg/g body weight) was given i.p. once daily for 7 days. At autopsy, ascites fluid was quantified and tumors were excised and weighed. Columns, mean; bars, ± SD; *, P < 0.05 versus control (thalidomide = 0 µg/g body weight).

 
Western blot analysis of the frozen tumors showed the intensive expression of uPA and uPAR in the tumors isolated from the control animals.7 In contrast, faint staining bands for uPA and uPAR were seen in the tumors isolated from the animals treated with 400 µg/mL thalidomide. Thus, the expression of uPA and uPAR in the tumors isolated from the animals treated with thalidomide was significantly lower than the control animals.

Potential interactions between thalidomide and paclitaxel. In the final set of experiments, we determined whether administration of the optimal biological dose of thalidomide (400 µg/g body weight/d) combined with paclitaxel (5 µg/g body weight i.p. injection at days 2 and 5) would produce additive or synergistic therapeutic effects in the control of ovarian tumor growth and ascites formation. At day 9, all mice were necropsied. All mice (nine of nine) in the control group, in the group treated with paclitaxel alone, or in the group treated with thalidomide alone had peritoneal disease (albeit to different degrees). In contrast, the incidence of disease in mice receiving both thalidomide and paclitaxel was reduced to five of nine (P < 0.05), suggesting synergistic effects. HRA tumors were highly sensitive to thalidomide treatment, with a dramatic delay of tumor growth and a reduction in ascitic volume (P < 0.05). The weight of peritoneal tumors was significantly reduced from a mean weight of 2.65 ± 0.27 g in DMSO-treated mice, to 2.01 ± 0.22 g in mice treated with thalidomide alone, to 0.82 ± 0.10 g in mice treated with paclitaxel alone, and to 0.20 ± 0.01 g in mice treated with 400 µg of thalidomide plus 5 µg of paclitaxel (Fig. 7, open columns). We have shown that paclitaxel is more effective than thalidomide in suppressing growth of i.p. HRA tumors and a reduction in ascitic volume (P < 0.05). Simultaneous treatment with thalidomide and paclitaxel significantly reduced tumor growth and ascitic volume compared to paclitaxel alone (P < 0.05). Figure 7 (filled column) also shows the results of the study of the control of ascites formation by thalidomide and paclitaxel treatment, singly and in combination. The mean volume of ascites in the control group was 2.72 mL. In contrast, virtually no or very little ascites developed in the thalidomide plus paclitaxel-treated group or in the group that was treated with paclitaxel alone. Thalidomide alone reduced ascites formation by ~35%. The weight (g) of the tumor was significantly correlated with the volume (mL) of ascites (P = 0.001); the correlation coefficient was 0.658.



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  		Figure 7. Effects of thalidomide plus paclitaxel on tumor burden (open columns) and ascites formation (filled columns) in mice inoculated with HRA cell. Four groups of nude (each group, n = 9) mice were used. Treatment groups consist of control (vehicle alone), once-daily i.p. administration of thalidomide (400 µg/g body weight), thalidomide plus paclitaxel (100 µg i.p. at days 2 and 5), and paclitaxel alone. Columns, mean; bars, ± SD; *, P < 0.01 versus control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The serine protease uPA and its receptor uPAR play a key role in the invasive and metastatic capacity of tumor cells (5, 6, 30). The uPA system is causally involved at multiple steps in cancer progression. In particular, the uPA/uPAR system has been implicated in remodeling of the extracellular matrix, enhancing both cell proliferation and invasion and modulating cell adhesion. Tumor dormancy is induced by down-regulation of uPAR (31): 70% reduction in the uPAR level in human carcinoma HEp3 cells induced a protracted state of tumor dormancy in vivo. Therefore, treatment of uPAR-rich cells with reagents interfering with the NF-{kappa}B activation-mediated uPAR signal, may mimic the in vivo dormancy induced by down-regulation of uPAR. With these in mind, we investigated the regulation by thalidomide of NF-{kappa}B activation as a signaling molecule in TGF-ß1-induced uPAR overexpression in the highly invasive human ovarian cancer cell line and in the human chondrosarcoma cell line.

In the in vitro experiments, the present results clearly show that thalidomide specifically inhibits expression of uPAR mRNA and protein, possibly through suppression of NF-{kappa}B activation, which results in the inhibition of cell invasiveness. We found a relatively similar half-life of uPAR mRNA of 10 to 12 hours, irrespective of whether cells were treated with thalidomide (data not shown). Thus, it is unlikely that thalidomide reduces uPAR mRNA stability. Again, we found that thalidomide had no measurable effects on cell viability or on the yield of the total RNA.

A recent report showed that binding of uPA to uPAR activates extracellular signal-regulated kinase (ERK)-1/2, which is required for increased cellular motility in breast cancer cells (32). A variety of growth factors including TGF-ß, EGF, fibroblast growth factor, and VEGF, which up-regulate uPAR synthesis, also stimulate ERK activity (14). However, the signaling mechanism by which growth factors modulate uPAR expression is not completely understood. We have focused on TGF-ß1, because both HRA and HCS-2/8 cells exhibited TGF-ß1-dependent invasion. It is most likely that the TGF-ß1-induced uPAR expression shares the characteristics of that of uPA expression, because the increased uPAR expression is affected by PKC inhibitors (calphostin C and staurosporin), and the uPA expression is also affected by PKC and ERK inhibition (33). The effect of TGF-ß on the expression of other genes has been ascribed to activation of the classical pathway (Ras->c-Raf-1->ERK signaling cascade; ref. 34). An alternative pathway consists of the sequential activation of Rac1, MEKK1, c-Jun amino-terminal kinase kinase, and the c-Jun amino-terminal kinase subset of mitogen-activated protein kinases (35). Thus, some of the uPA and uPAR signalings overlap or cross-talk.

Recently, the role of NF-{kappa}B-Rel A–related proteins in plasminogen activator system synthesis has been precisely investigated in human ovarian cancer cells by inhibiting their expression using the antisense oligodeoxynucleotide technology (36). They reported that exposure of cells to antisense-oligodeoxynucleotide directed to Rel A lead to a decrease of uPA protein and mRNA levels. Antisense-oligodeoxynucleotide directed to NF-{kappa}B1 (p50) or c-rel had no effect on uPA protein expression. Antisense-oligodeoxynucleotide directed to I{kappa}B-{alpha} expression increased uPA. uPAR production and synthesis of plasminogen activator inhibitor type-1 were not altered by either antisense-oligodeoxynucleotides applied. Thus, the accumulating data on NF-{kappa}B activation on plasminogen system activation is somewhat controversial in different cell types. Notwithstanding these contradictions, these proteins seem to be implicated in plasminogen system regulation and may thereby contribute to tumor invasion and metastasis.

The antimetastatic therapy of advanced cancer is currently under active investigation, with a number of protease inhibitors including matrix metalloproteinase (MMP) inhibitors (see http://www.cancer.gov/clinicaltrials/developments/anti-angio-table) being studied in the laboratory and in clinical trials. Recent studies have indicated that treatment with an antiangiogenic agent might be useful. It has been reported that the antimetastatic efficacy against colon cancer and gastric cancer were augmented by the combination therapy of MMI-166, an orally active MMP inhibitor, with CPT-11 (19). This combination therapy exhibited potent antimetastatic efficacy without increased hematotoxicity (19). On the other hand, batimastat was the first synthetic MMP inhibitor studied in humans with advanced malignancies, but its usefulness has been limited (37). Recently, a number of antiangiogenetics including thalidomide have been developed and have reached different stages in preclinical evaluation and clinical testing.

A growing body of evidence has accumulated that thalidomide significantly reduced the tumor volume, the mitotic index and cell proliferation of xenograft tumor (38–42), whereas some authors showed that this compound failed to inhibit tumor growth (43). Concurrent with its evaluation in various clinical trials for cancer, thalidomide's mechanism of action is sought and new analogues with improved efficacy and pharmacologic profile are emerging (44). For example, thalidomide analogues (CC-7034 and CC-9088) were identified that had enhanced antiangiogenic activity compared with parental thalidomide (45).

Antiangiogenic effects, direct activity in tumor cells such as the induction of apoptosis or G1 arrest of the cell cycle, the inhibition of growth factor production, the regulation of interactions between tumor and stromal cells, and the modulation of tumor immunity have been considered as possible mechanisms of thalidomide (46). The present data allow us to speculate that thalidomide inhibits tumor cell invasion by mechanisms other than antiangiogenesis. Many other agents modulating uPA/uPAR system will be able to replace thalidomide in this assay, yet give the same results.

In the second set of experiments, the present studies indicated that the combination of thalidomide and the conventional chemotherapeutic agent, paclitaxel, could significantly inhibit tumor growth and dissemination as well as malignant ascites formation. The antimetastatic, antitumor, and antiascites effects of thalidomide plus paclitaxel were markedly greater than those of paclitaxel alone or thalidomide alone. These results suggest that combining thalidomide with a chemotherapeutic agent such as paclitaxel is an effective way to control the growth of ovarian carcinoma with fewer side effects than either agent alone. We confirmed the previously published results that coadministration of thalidomide and anticancer drugs, including paclitaxel and cyclophosphamide, gave markedly greater activity against tumor compared with either drug alone (38–42). Paclitaxel inhibits cell division in the G2-M phase of the cell cycle (47). In addition, it can inhibit angiogenesis by suppressing VEGF expression (27). Tumor growth might be affected not only by direct cytotoxicity but also by inhibition of new vessel formation, and the neutralizing antibody to VEGF could enhance the antiangiogenic effects of paclitaxel, as well as decreasing development of drug resistance to paclitaxel (48). Therefore, the antiangiogenic effect of paclitaxel on ovarian cancer may be markedly enhanced by combination with thalidomide.

The present study, for the first time, provides new insight on how thalidomide may be involved in the modulation of the metastatic phenotype in solid tumors: suppression of uPAR-dependent increased cell invasion by thalidomide, at least, via suppression of transcriptional factor NF-{kappa}B activation. Therefore, reagents which modulate NF-{kappa}B activation may offer fresh insights into the prevention of uPA/uPAR-dependent tumor invasion and metastasis in certain types of tumor cell lines. New insights into the biology of the disease suggests that antimetastatic agents including thalidomide may work via a number of other mechanisms and the advent of these compounds with their differential effects on survival and proliferation pathways has opened up a new era in the understanding and treatment of the disease.


   Acknowledgments
 
Grant support: Grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan, the Fuji Foundation for Protein Research, the Kanzawa Medical Foundation, Sagawa Cancer Research Foundation, and the Aichi Cancer Research Foundation (H. Kobayashi).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
6 Unpublished data. Back

7 Unpublished data. Back

Received 3/ 2/05. Revised 7/20/05. Accepted 9/ 9/05.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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