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Clinical Cancer Research Vol. 12, 980-988, February 2006
© 2006 American Association for Cancer Research

Cancer Prevention

2-Methoxyestradiol Inhibits Prostate Tumor Development in Transgenic Adenocarcinoma of Mouse Prostate: Role of Tumor Necrosis Factor-{alpha}–Stimulated Gene 6

Gretchen E. Garcia7, Hans-Georg Wisniewski3, M. Scott Lucia4, Nicole Arevalo8, Thomas J. Slaga2, Susan L. Kraft5, Robert Strange6 and Addanki P. Kumar1

Authors' Affiliations: Departments of 1 Urology and 2 Pharmacology, University of Texas Health Science Center, San Antonio, Texas; 3 Department of Microbiology and Kaplan Cancer Center, New York University Medical Center, New York; 4 Department of Pathology, University of Colorado Health Sciences Center; 5 Department of Radiation Biology, Colorado State University, Fort Collins; 6 AMC Cancer Research Center; 7 National Jewish Research and Medical Center, Denver, Colorado; and 8 Affinity Bioreagents, Golden, Colorado

Requests for reprints: Addanki P. Kumar, Department of Urology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229. Phone: 210-567-5647; Fax: 210-567-6868; E-mail: kumara3{at}uthscsa.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results and Discussion
 References
 
Purpose: 2-Methoxyestradiol, an estrogenic metabolite, is in clinical trials for the treatment of hormone-refractory prostate cancer. However, neither the chemopreventive role nor the mechanism of 2-methoxyestradiol–induced biological activities is fully understood.

Experimental Design: Eight- and 24-week-old transgenic adenocarcinoma of mouse prostate (TRAMP) mice were fed a diet containing 50 mg 2-methoxyestradiol/kg body weight for 16 and 8 weeks, respectively. Chemopreventive efficacy was evaluated by magnetic resonance imaging, determining the prostate-seminal vesicle complex volume and histologic analysis of prostate tumor or tissue. Tumor invasion assays were used to show the role of tumor necrosis factor-{alpha}–stimulated gene (TSG-6), a 2-methoxyestradiol–up-regulated gene identified by DNA array analysis. Expression of TSG-6 was analyzed in a human tissue array containing different grades of prostate tumors.

Results: Dietary administration of 2-methoxyestradiol prevented the development of preneoplastic lesions independent of progression stage. TSG-6 was low or undetectable in prostate cancer cells (LNCaP, PC-3, and DU145) and TRAMP tumors but up-regulated in response to 2-methoxyestradiol. Immunohistochemistry of the human prostate tumor array showed a decrease in TSG-6–positive cells with increasing grade relative to normal prostate (P = 0.0001). Although overexpression of TSG-6 inhibited invasion of androgen-independent cells (P = 0.007), antisense TSG-6 reversed this effect.

Conclusions: To the best of our knowledge, this is the first report showing the potential of 2-methoxyestradiol as a chemopreventive agent. We have also identified TSG-6 as a potential marker that could be used for early diagnosis and prognosis of cancerous or precancerous lesions.

Prostate cancer is the second leading cause of cancer-related deaths in men and affects one man in nine over the age of 65 years (1). Although it is not clear whether appearance of prostatic intraepithelial neoplasia predicts the appearance of prostate cancer in men, preneoplastic lesions have been found in young men in the twenties and are fairly common in men in fifties (2). However, clinically detectable prostate cancer does not generally manifest until the age of 60 or 70 years. In addition, the occurrence of precancerous lesions is more prevalent (~1 in 3 men) than the incidence of carcinoma (~1 in 9 men; ref. 3). It is of utmost importance at this juncture to develop strategies for the prevention of early-stage prostate cancer to ensure quality of life for elderly men. Furthermore, although prostate-specific antigen is used as a marker to detect prostate cancer, its reliability has been found to be questionable in some cases (4, 5). Also presently, there is no molecular marker that can be used to detect a precancerous state or identify which premalignant lesions will develop into invasive prostate cancer.

Recent results from different groups, including ours, have shown that 2-methoxyestradiol inhibits the growth of androgen-responsive and androgen-independent human prostate cancer cells through induction of apoptosis (6, 7). However, the chemopreventive role of 2-methoxyestradiol has not been studied. We used the transgenic adenocarcinoma of mouse prostate (TRAMP) model of prostate cancer to test the chemopreventive ability of 2-methoxyestradiol. This model was developed by prostate-specific expression of SV40 large T antigen using the probasin (PB) promoter. TRAMP mice develop progressive forms of prostate cancer with lesions ranging from mild prostatic intraepithelial neoplasia to large multinodular malignant neoplasia over their lifetime (8). Because these animals are predisposed to develop prostate cancer, it served as a good model to test the chemopreventive ability of 2-methoxyestradiol (refs. 9–11 and references therein).

Results presented here show that dietary intervention with 2-methoxyestradiol prevents the development of prostate tumors with no indication of neoplasia irrespective of the stage of intervention. 2-Methoxyestradiol–induced cell proliferation inhibition involves up-regulation of tumor necrosis factor-{alpha} (TNF-{alpha})–stimulated gene 6 (TSG-6). TSG-6 was originally isolated from TNF-treated human fibroblasts and has been shown to be induced in response to cytokines, such as TNF-{alpha}, interleukin-1, and growth factors (12). Although TSG-6 was very well studied with respect to arthritis (inflammatory disease), its role in cancer remains largely unexplored (13). We analyzed the expression of TSG-6 in different grade prostate tumors and show lower expression of TSG-6 in high-grade human prostate tumors compared with normal prostate (P = 0.0001). Furthermore, TSG-6 inhibited the invasive ability of androgen-independent PC-3 cells (P = 0.007). These observations suggest that 2-methoxyestradiol inhibits the progression of preneoplastic lesions through up-regulation of anti-inflammatory molecule, such as TSG-6.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results and Discussion
 References
 
Transgenic mouse experiments. TRAMP mice in pure C57BL/6 background were bred at the AMC Cancer Research Center. All mice were maintained in a climate-controlled environment with a 12-hour light/12-hour dark cycle. Diet and water were supplied ad libitum. After weaning at 3 to 4 weeks of age, tail DNA from males was used to determine the presence of transgene by PCR as described previously (10). Eight-week-old transgenic males were fed AIN-96A pellets (a semipurified diet containing 2-methoxyestradiol, 50 mg/kg body weight; n = 9) or control diet (without 2-methoxyestradiol) for 16 weeks (group I). In another study, 24-week-old TRAMP mice were fed the same pelleted diet with or without 2-methoxyestradiol for 8 weeks (n = 5; group II). Development and progression of prostate cancer was assessed using magnetic resonance imaging in the second group of animals. Animals were imaged with a small-phased array transmit-receive coil of a 1.5-T General Electric LX MR instrument essentially as described in ref. (14). Each mouse was weighed once a week. Animal care and handling was conducted in accordance with established guidelines and protocols approved by the Institute's Animal Care and Use Committee.

Cell lines. Human prostate cancer cell lines, including androgen-responsive (LNCaP) and androgen-independent (PC-3 and DU145) cell lines, were grown as described earlier (6, 15). TRAMP cell lines were obtained from Dr. Norman Greenberg (Fred Hutchinson Cancer Research Center, Seattle, WA) and were grown as described earlier (15). PC-3 cells overexpressing TSG-6 and vector transfected (PC3-Neo) were generated using standard protocols.

Preparation and analysis of tissues. Necropsy was conducted on all animals to determine if there were any gross organ abnormalities in response to 2-methoxyestradiol. The prostate was dissected and weighed before additional processing for histopathologic evaluation. Prostate lesions were scored using the established grading for TRAMP mice (8).

Analysis of hormone levels in serum. The LAB-4000 testosterone assay and the LAB-4800 Ultra sensitive estradiol assay were done using the DSL-4000–coated RIA and DSL-4800 double antibody RIA for measuring the serum levels of testosterone and estradiol, respectively, as per manufacturer's recommendations (Diagnostic System Laboratories, Inc., Webster, TX). Values for the samples were derived by interpolating using standards available with the kit.

Tumor histology. Tumors were harvested and fixed in 10% neutral buffered formalin. Tumors were paraffin embedded, sectioned, placed on polylysine slides, and stained with H&E to visualize cell nuclei and cytoplasm. Images were recorded using a light microscope.

Immunochemical analysis. We used immunochemistry to examine the levels of TSG-6 using a rabbit polyclonal antibody generated against the COOH terminus in prostate cancer cell lines using standard protocols.

Tumor invasion assay. We used Cell Invasion Assay (Oncogene Research Products, San Diego, CA) to determine the direct role of TSG-6 in inhibiting tumor invasion. This assay uses an invasion chamber with an 8-µm pore size polycarbonate membrane. The upper surface of this membrane is coated with a uniform layer of basement membrane matrix solution that forms an effective extracellular matrix protein barrier and prevents noninvasive cells from going through the pores. In contrast, invasive cells are able to degrade the matrix proteins that occlude the pores and allow them to pass through. The ability to invade can be quantified by labeling the invaded cells with calcein-AM followed by fluorescence measurement. LNCaP and PC-3 cells were transfected with expression vectors containing TSG-6 cDNA in sense or antisense orientation or vector alone. The experiment was carried out according to the manufacturer's recommendations.

Gene array analysis. The Micromax microarray containing 281 human cancer–related genes, including oncogenes and tumor suppressor genes (Perkin-Elmer Life Sciences, Inc., Boston, MA), was used in this study. Total RNA from control and 2-methoxyestradiol–treated LNCaP cells was isolated using TRIZOL reagent (Ambion, Inc., Austin, TX). After assessing the integrity of the RNA by agarose gel electrophoresis, RNA from control cells was labeled with cyanine 5 and treated RNA with cyanine 3 as per manufacturer's recommendations. The combined cyanine 3– and cyanine 5–labeled mRNA was purified and used in hybridization. Data was analyzed using Custom Scanning and Data Processing service for Micromax system (Perkin-Elmer Life Sciences). Results are expressed as the ratio of the signals from cyanine 3 and cyanine 5 for each of the 281 cDNAs, and ratio of >2 is considered significantly differentially expressed. These were confirmed through independent approaches, including Western blot analysis or immunocytochemistry or immunohistochemistry.

Statistical analysis. Two-tailed statistical analyses were conducted at P = 0.05 (16).


   Results and Discussion
Top
 Abstract
 Materials and Methods
 Results and Discussion
References
 
TRAMP mice develop prostate tumors with 100% frequency in progressive stages exhibiting hyperplasia (8-12 weeks), neoplasia (15-18 weeks), and metastasis (24 weeks). We asked whether 2-methoxyestradiol could be used to prevent the progression of prostate tumors at various stages of development. To do this, one group of 8-week-old TRAMP males was shifted to 2-methoxyestradiol diet for 16 weeks (group I). Another group of 24-week-old TRAMP males was shifted to 2-methoxyestradiol diet for 8 weeks (group II). Necropsy was conducted at 24 weeks of age for group I and at 32 weeks for group II animals.

The dose of 50 mg/kg body weight was chosen from a dose escalation xenograft study (data not shown). The ability of 2-methoxyestradiol to inhibit neoplastic development was assessed by determining the weight of the prostate gland and histologic evaluation of the prostate gland or tumor. 2-Methoxyestradiol–fed group I animals displayed ~33% reduction in the weight of the prostate gland compared with mice on normal diet. There was no significant change in the body weight or weight of seminal vesicles (data not shown). Gross appearance of prostate and seminal vesicles from a representative 24-week-old untreated (i) and treated mouse (ii) is shown in Fig. 1A.


Figure 1
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  		Fig. 1. A, gross appearance of prostate and seminal vesicles of 24-week-old TRAMP mice (group I). Prostate gland from 24-week-old TRAMP mice on (i) normal diet and (ii) 2-methoxyestradiol diet. B, histologic analysis of prostate from untreated and treated TRAMP mice. Histologic sections of prostate tissues excised from TRAMP mice from group I on (i) control or (ii) 2-methoxyestradiol diet. Tissue sections were stained with H&E. Magnification, x200. C, magnetic resonance imaging of 32-week-old TRAMP mice (group II). Two groups of five 24-week-old TRAMP mice were on a diet with or without 2-methoxyestradiol (50 mg/kg body weight) for 8 weeks. At the end of this schedule, they were imaged with a small phased array transmit-receive coil of a 1.5-T General Electric LX MR instrument. Representative images show that the prostate of (i) control mice was unrecognizable due to a very large tumor that infiltrated into seminal vesicles; (ii) age-matched mice on 2-methoxyestradiol fed diet showed very small prostate with no evidence of cancer as confirmed by H&E analysis after necropsy. D, gross appearance of prostate 32-week-old TRAMP mice (group II). Prostate tumor along with vesicles from (i) control and (ii) 2-methoxyestradiol–fed TRAMP mouse at the time of sacrifice (32 weeks).

 
Histologic analysis of the prostate from a control TRAMP mice (i) displayed major epithelial proliferation in a characteristic cribriform pattern: hyperchromatic nuclei, mitotic figures, and apoptotic bodies (Fig. 1B). In contrast, prostate from TRAMP mice on 2-methoxyestradiol diet showed glands composed of columnar epithelium with round to oval nuclei with no indication of neoplasia (Fig. 1B, ii). Analysis of other tissues, including lungs, liver, and lymph nodes, showed normal morphology, indicating that 2-methoxyestradiol had no effect on any of these organs (data not shown). These data show that 2-methoxyestradiol feeding inhibits neoplastic progression in TRAMP mice.

In 24-week-old TRAMP mice that were fed 2-methoxyestradiol diet (50 mg/kg body weight) for 8 weeks, the efficacy of 2-methoxyestradiol was evaluated by volumetric analysis of the prostate-seminal vesicle complex using in vivo magnetic resonance imaging. Representative images in Fig. 1C show that the prostate from control group was indistinct due to a very large tumor that infiltrated into seminal vesicles (i), whereas treatment of animals showed very small prostate with no evidence of cancer (ii). Figure 1D shows that the prostate-seminal vesicle complex was larger in the control animal (i) compared with the animal on 2-methoxyestradiol diet (ii) at the time of necropsy.

Volumetric analysis at the time of magnetic resonance imaging showed that the average prostate-seminal vesicle complex volume was 3,263 ± 105 mm3 in the control animals (n = 5) compared with 235 ± 99 mm3 in the treated animals (n = 5; Fig. 2A). We have measured the levels of total testosterone and estradiol in serum collected from TRAMP animals that were on this study as described in Materials and Methods. Values for the sample analysis were derived by interpolation using standards available with the kit. Serum levels of estradiol were very low (<5 pg/mL). Dietary intervention with 2-methoxyestradiol did not affect the levels of estradiol. However, serum levels of total testosterone were reduced in 2-methoxyestradiol–treated animals (Fig. 2B). These data suggest that dietary intervention with 2-methoxyestradiol produced a regression of prostate tumors in TRAMP mice that is associated with a decline in serum testosterone levels with no effect on estradiol. In line with our findings, raloxifen, a selective estrogen receptor modulator, has been shown to regress prostate cancer in SV40Tag transgenic rats and male accessory sex organs in intact Sprague-Dawley rats associated with decline in serum testosterone levels (17). Studies to investigate whether 2-methoxyestradiol reduces testosterone levels by enhancing metabolic clearance or altering hepatic function to change the secretion of circulating proteins capable of binding steroids are currently in progress. In both experiments, feeding animals with 2-methoxyestradiol produced negligible body weight losses ranging from 1.2% to 2.8%.


Figure 2
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  		Fig. 2. A, volumetric analysis of prostate seminal vesicle complex volume (PSVC) from group II TRAMP mice. Experiment was conducted essentially as described in Fig. 1C, and prostate seminal vesicle complex volume was determined. Columns, average from five animals per group; bars, SD. B, serum levels of testosterone in TRAMP mice. Serum levels of total testosterone and estradiol were determined in control of 2-methoxyestradiol–fed (50 mg/kg body weight) TRAMP mice as described in Materials and Methods. Animals (n = 5) from each group were sacrificed at 24 weeks (group I) and 32 weeks (group II) of age. Serum was collected and analyzed for hormone levels. Values for the sample analysis were derived by interpolation using standards available with the kit. Since estradiol levels did not change in response to treatment, the data was not shown. C, immunohistochemical analysis of representative tumors or prostate tissue from 32-week-old control or treated TRAMP mice. Paraffin-embedded tissues sections were stained with PB-Tag (PBT) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The antibodies used were at a dilution of 1:100 in PBS and incubated overnight at 4°C. Immune complexes were revealed using a universal secondary antibody (100 µL for 30 minutes) followed by chromogen. Negative controls were included by omitting the primary antibody.

 
Because TRAMP mice were generated with SV40 large T antigen coupled with PB promoter (8), it is possible that 2-methoxyestradiol mediated effects are a consequence of down-regulation of the PB-Tag transgene by 2-methoxyestradiol. The PB promoter is androgen regulated, and 2-methoxyestradiol is an estrogenic metabolite; therefore, 2-methoxyestradiol may also exert its effects by influencing the expression of androgen-regulated genes. In view of this, we determined the expression of PB-Tag endogenous mouse PB and glyceraldehyde-3-phosphate dehydrogenase (non–androgen-regulated gene) using immunohistochemistry in prostate from age-matched control and treated animals. As shown in Fig. 2C, PB-Tag and glyceraldehyde-3-phosphate dehydrogenase expression was detected both in control tumor and treated tumor/tissue obtained from 32-week-old TRAMP mice. Glyceraldehyde-3-phosphate dehydrogenase levels did not change between control and treated prostatic tissues. These results show that our observations regarding 2-methoxyestradiol–induced biological effects are due to direct suppression of carcinogenesis and not due to the down-regulation of PB-Tag.

Although several groups, including ours, have shown that 2-methoxyestradiol inhibits the growth of cancer cells through induction of apoptosis, the detailed molecular mechanisms involved in mediating its growth inhibitory activity is still unclear (6, 7, 18–22). To understand the molecular basis of 2-methoxyestradiol–mediated growth inhibition, we used cDNA array analysis of genes expression change by 2-methoxyestradiol treatment using LNCaP cells. Analysis of this data indicated genes involved in various cellular processes, including tumor suppression, cell cycle regulation, cytokine signaling, transcription control, and inflammation, were up-regulated. Genes showing >2-fold induction (up-regulated) and <0.5-fold (down-regulated) were considered significant and are shown in Table 1. We chose to conduct detailed studies on one such gene TSG-6 because of its known role in inflammation. TSG-6, is a secreted glycoprotein of 35 kDa, is associated with inflammation and fertility (reviewed in ref. 13). TSG-6 is a hyaluronan-binding protein, and its interaction with hyaluronan has been studied in detail (23–33). TSG-6 has shown anti-inflammatory activity in several models of acute inflammation and autoimmune arthritis (31–34). Inflammation has been thought to contribute to the pathogenesis of many cancers, including prostate. Consistent with this regular use of nonsteroidal anti-inflammatory drugs has been shown to be associated with reduced risk of prostate cancer (35). Although it is not clear whether inflammation is a cause or effect of hyperplastic growth in the prostate, it appears that chronic or recurrent prostatic inflammation may contribute to the development of prostate cancer (36). However, TSG-6 has not been studied in the context of cancer.


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  		Table 1. Genes showing >2-fold (up-regulation) or <0.5-fold (down-regulation) in response to 2-methoxyestradiol treatment in LNCaP cells

 
We used immunocytochemistry and Western blotting to detect the expression of TSG-6 in LNCaP, DU145, and PC-3 cells. As shown in Fig. 3A, a brown 3,3'-diaminobenzidine reaction product was formed in the cytoplasm of cells treated with 2-methoxyestradiol (3 µmol/L for 24 hours) but not in untreated controls of DU145 and PC-3 cells. Preimmune serum was negative for 3,3'-diaminobenzidine product in these samples (data not shown). TSG-6 protein was detected in LNCaP cells but not in PC-3 cells. However, the TSG-6 protein levels increased in response to 2-methoxyestradiol treatment both in LNCaP and PC-3 cells (Fig. 3B). Published reports show that constitutive expression of TSG-6 is either low or undetectable but becomes detectable in response to cytokines and growth factors in many cell types both in vitro and in vivo (12, 13). Our data showing low levels (basal expression) of TSG-6 in human prostate cancer cell lines are consistent with these observations.


Figure 3
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  		Fig. 3. A, TSG-6 in prostate cancer cells. Human prostate cancer cell lines (PC-3 and DU145) were either treated with 2-methoxyestradiol (2-ME; 3 µmol/L) for 24 hours or left untreated (solvent control). Following treatment, cells were harvested and fixed in 10% buffered formalin, and immunocytochemical analysis was done as described in Materials and Methods. Staining indicative of TSG-6 expression was detected only in cells treated with 2-methoxyestradiol. Untreated cells showed very little or no staining. B, Western blot analysis of TSG-6. Whole-cell extracts prepared from LNCaP cells treated with 2-methoxyestradiol (3 µmol/L) for different time points (2, 4, and 6 hours) or PC-3 cells treated with different concentrations of 2-methoxyestradiol for 24 hours (0.5, 1, and 3 µmol/L) was used in immunoblot analysis. Equal loading of protein was confirmed with ß-actin antibody.

 
These observations prompted us to determine the clinical application of TSG-6 for prostate cancer prognosis. Immunohistochemical staining for TSG-6 was conducted on a human prostate cancer tissue array containing 72 specimens of different Gleason grades with paired normal prostate. The array was prepared by the histology core Laboratory (University of Colorado Health Science Center, Denver, CO), the Prostate Diagnostic Laboratory, using a Beecher Instruments (Silver Springs, MD) tissue arrayer and 1.5-mm core diameter. Each specimen was analyzed for immunoreactivity using a 1 to 3+ scoring system for stain intensity and percentage of positive cells. Grading scale for intensity ranged from undetectable signal (1+) to strong signal (3+). ANOVA and bootstrap methods were used to determine the statistical significance of these observations. TSG-6 staining was predominantly localized in the cytoplasm of epithelial (basal and luminal) but not stromal cells. Membrane staining was observed in some specimens. There was a progressive loss of staining with increasing Gleason grade (P < 0.0001; Fig. 4); 80% to 100% of cells from normal prostate showed TSG-6 expression. A representative picture from normal and Gleason 5 tumors is shown in Fig. 4. Although these observations are based on a small sample size, they suggest that the loss of TSG-6 expression may be a significant predictor of prostate cancer progression.


Figure 4
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  		Fig. 4. A, TSG-6 staining in human prostate. Human prostate tissue array containing different grade tumors with paired normal prostate was stained with TSG-6. TSG-6 was used at a dilution of 1:100 in PBS and incubated overnight at 4°C. Immune complexes were revealed using a universal secondary antibody (100 µL for 30 minutes) followed by chromogen as described in Materials and Methods. Left, little or no TSG-6 staining in the cancerous tissue; right, TSG-6 expression in normal prostate (P = 0.0001). Statistical significance of the data was determined using ANOVA and bootstrap methods as described in Materials and Methods. B, variability of TSG-6 expression in human prostate tumors. Tissue array containing human prostate tumors from different Gleason grade were incubated with a protein blocking serum-free reagent for 60 minutes to block nonspecific binding. TSG-6 was used at a dilution of 1:100 in PBS and incubated overnight at 4°C. Immune complexes were revealed using a universal secondary antibody (100 µL for 30 minutes) followed by chromogen. Negative controls were included by omitting the primary antibody (data not shown).

 
Based on the observed inverse correlation between TSG-6 expression and prostate cancer malignancy, we hypothesized that TSG-6 may have a role in tumor invasion. PC-3 cells were transfected with TSG-6 cDNA in the sense or antisense orientation or with vector alone and used in tumor invasion assays. These three comparisons (each condition to control) were regarded as a family. To control the family-wise error rate, Ps were multiplicity adjusted and derived from a bootstrap resampling of the residuals from a standard additive model for the estimates of the group means (16). As shown in Fig. 5, overexpression of TSG-6 inhibited the invasive ability of PC-3 cells significantly (P = 0.007 between control and TSG-6) when compared with vector-transfected control cells. This effect was also seen in LNCaP cells, but the data were not statistically significant (data not shown). It has been reported that LNCaP cells do not secrete matrix metalloproteinase-9 and are poorly invasive in Matrigel assays (37).


Figure 5
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  		Fig. 5. A, overexpression of TSG-6 inhibits tumor invasion. Effect of TSG-6 on tumor cell invasion was determined using Cell Invasion Assay (Oncogene Research Products) as described in Materials and Methods. Briefly, PC-3 cells were plated in 24-well plate coated with basement membrane matrix or transfected with sense TSG-6 cDNA either in the sense (TSG-6) or in antisense orientation (ASTSG-6). Cells that migrated to the lower chamber (invading cells) through the Matrigel were quantified by labeling with calcein-AM and measuring the fluorescence. Columns, mean % invasion with respect to untreated control set at 100% in three independent experiments; bars, SD. Statistical significance (P = 0.007 between control and TSG-6) was determined using bootstrap resampling of the data as described in Materials and Methods. B, tumor invasion ability in PC-3 cells stably expressing TSG-6. PC-3 cells stably expressing TSG-6 or control (PC-3Neo) cells were plated in 24-well plate coated with basement membrane matrix. Cells that migrated to the lower chamber (invading cells) through the Matrigel were quantified by labeling with calcein-AM and measuring the fluorescence as described above for (A). Columns, mean % invasion with respect to untreated control set at 100%; bars, SD.

 
To confirm that increased level of TSG-6 was required for 2-methoxyestradiol–mediated inhibition of tumor invasion, TSG-6 cDNA was stably transfected into PC-3 cells. Both PC-3 Neo and PC-3TSG-6 clones grew equally well.9 PC-3TSG-6 clones were less invasive than vector-transfected PC-3 Neo clones that were consistent with the transient transfection data discussed above (Fig. 5B). Although these data collectively indicate that TSG-6 inhibits tumor invasion in prostate cancer cells, the relationship between TSG-6 and prostate cancer development is unclear. We evaluated prostate tumor/tissue from control and 2-methoxyestradiol–fed TRAMP mice for TSG-6 expression to determine if the levels correlate with tumor development. A representative Western blot is shown in Fig. 6. The levels of TSG-6 was quantified and normalized to ß-actin as a loading control. A representative Western blot of tumor samples from a control animal and tissue from an animal on 2-methoxyestradiol diet is shown in Fig. 6 (inset). As shown in this figure, prostate from 2-methoxyestradiol–fed animals showed 1.5- to 2-fold induction of TSG-6. Results presented here show a strong link between the expression of TSG-6 and prostate cancer progression and therefore its potential use as a prognostic marker in 2-methoxyestradiol–induced prevention of prostate carcinogenesis.


Figure 6
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  		Fig. 6. A, TSG-6 staining in TRAMP tissue and TRAMP cell lines. Paraffin-embedded tissues sections were deparaffinized with xylene and rehydrated using graded ethanol and used in immunohistochemistry as described in Materials and Methods. i, no TSG-6 staining in the prostate from animal on normal diet. Magnification, x40. ii, TSG-6 expression in prostate from a representative animal on 2-methoxyestradiol diet that showed positive staining. B, TSG-6 in TRAMP mouse prostate cancer cells. TRAMP C2, mouse prostate cells were either treated with 2-methoxyestradiol (3 µmol/L) for 24 hours or left untreated (solvent control). Following treatment, cells were harvested and fixed in 10% buffered formalin, and immunocytochemical analysis was done as described in Materials and Methods. Staining indicative of TSG-6 expression was detected only in cells treated with 2-methoxyestradiol. Untreated cells showed very little or no staining. C, Western blot analysis of TSG-6. TSG-6 expression was determined in TRAMP tumors (control) or prostate tissue (2-methoxyestradiol–fed animals) depending on the case using Western blot analysis. Data was quantified using Scion image analysis program.

 
2-Methoxyestradiol is an endogenous nontoxic metabolic byproduct of estrogens that is present in human urine and blood. 2-Methoxyestradiol has been shown to (a) inhibit endothelial cell proliferation implicating it in angiogenesis; (b) inhibit the growth of different cancer cells, including lung, breast, pancreatic, hepatocellular carcinoma, neuroblastoma, medulloblastoma, melanoma, and gastric cancer (6, 7, 18–22). Oral administration of 2-methoxyestradiol (75 mg/kg body weight) for 4 weeks inhibited tumor growth by about 60% with no evidence of toxicity in a breast cancer model, reduced the number of metastases in a lung tumor model by 59%, and reduced tumor size in mice with angiosarcoma by 68%. Furthermore, efficacy of 2-methoxyestradiol was evaluated by administering it orally to hormone-refractory prostate cancer patients who had failed other treatments, including hormonal therapy. This study concluded that (a) 2-methoxyestradiol was safe and well tolerated; (b) prostate-specific antigen levels declined or stabilized in some patients (37). Work from many laboratories and our own shows that growth inhibitory effect of 2-methoxyestradiol is specific for tumor cells and does not affect normal cells. Therefore, our novel finding presented here showing inhibition of precancerous cells is noteworthy.

Although the role of TSG-6 in prostate carcinogenesis is unexplored, TSG-6 has been shown to be up-regulated by various cytokines and growth factors and activated in pathologic conditions, such as rheumatoid arthritis and osteoarthritis. It has also been shown that TSG-6 forms a stable complex with inter-{alpha}-inhibitor, a Kunitz-type serine protease inhibitor in plasma. This combination of TSG-6 and inter-{alpha}-inhibitor inhibits plasmin, a serine protease that activates matrix metalloproteinases that degrade extracellular matrix during inflammatory reactions. Based on these observations, we speculate that treatment of prostate cancer cells with 2-methoxyestradiol leads to activation of TSG-6 and complex formation with inter-{alpha}-inhibitor that in turn inhibits activation of serine proteases and consequently inhibits prostate cancer progression. It is also plausible that TSG-6 exert its anti-inflammatory effects in an inter-{alpha}-inhibitor–independent manner (38). Alternatively, 2-methoxyestradiol could exert its effects through TSG-6 involving other matrix degrading enzymes, such as cysteine proteases or matrix metalloproteinases.

No detailed studies of the effects of 2-methoxyestradiol on the regulation of TSG-6 transcription or TSG-6 mRNA stability have been conducted. However, some conclusions can be drawn from studies of the transcriptional regulation of TSG-6 expression in fibroblasts and studies of the signaling pathways affected by 2-methoxyestradiol in the induction of apoptosis in LNCaP cells. TSG-6 regulation in response to inflammatory cytokines has been studied in primary human fibroblasts. Three closely arranged cis-acting elements located in the proximal TSG-6 promoter [one activator protein (AP-1) site and two nuclear factor/interleukin-6 sites] are responsible for the induction of TSG-6 by TNF-{alpha} and interleukin-1ß (39, 40). In a different line of research, 2-methoxyestradiol has been found to activate the transcription factor AP-1 in LNCaP cells (7). We found Fra-1 and Fra-2 (Fos-related antigen) mRNA was up-regulated by 2-methoxyestradiol treatment in LNCaP cells (Table 1). Fra-1 and Fra-2 are members of transcription factor AP-1 complex. These data implicate AP-1 in the induction of TSG-6 transcription in LNCaP cells. This could lead to the up-regulation of TSG-6 mRNA steady-state levels that we observed. Furthermore, it is known that binding of TNF-{alpha} to its receptor causes activation of transcription factor AP-1 and nuclear factor-{kappa}B that in turn induce genes involved in inflammatory response (41). It is noteworthy to mention that 2-methoxyestradiol has been shown to activate both AP-1 and nuclear factor-{kappa}B (7). Based on this, we speculate that 2-methoxyestradiol may exert its biological effects through TNF-{alpha} signaling pathway. TSG-6 expression was not detected in p53-deficient (WS1-E6) cells, indicating that it is under the control of p53 (42). The observed low level expression of TSG-6 in LNCaP cells (containing wild-type p53) is consistent with this finding. However, the importance of p53 status for biological functions of TSG-6 in prostate cancer will be the subject of future studies.

One potential mechanism for the indicated effect of TSG-6 on prostate cancer is suggested by the recent determination that TSG-6 interacts with thrombospondin-1 (TSP-1) and TSP-2 (43). TSP-1 and TSP-2 have antitumor effects that are mediated by several mechanisms (reviewed in ref. 44). TSP-1 has a prominent antiangiogenic effect that entails inhibition of endothelial cell migration, induction of endothelial cell apoptosis, tumor cell apoptosis, and the inhibition of growth factor mobilization (44, 45). It has been shown that TSP-1 binding to TSG-6 does not interfere with the binding of TSG-6 to hyaluronan (43). This opens the possibility that TSG-6 acts as a docking protein that mediates binding of TSP to hyaluronan. Because TSG-6 has been shown to form stable complexes with hyaluronan (27), this may result in the virtual immobilization of TSP in the extracellular matrix of tumors, and in particular in the tumor stroma, where high concentrations of accumulated hyaluronan have been detected (46).

2-Methoxyestradiol may act through modulation of expression of androgen-regulated genes. Reduced levels of TSG-6 immunoreactivity in high-grade human prostate tumors compared with normal prostate strongly support the notion that TSG-6 could potentially be developed as a molecular marker to detect a precancerous state or identify which premalignant lesions will develop into invasive prostate cancer. Loss of expression of TSG-6 in high-grade prostate tumors is somewhat similar to the published data showing loss of expression of homeobox gene Nkx 3.1 and transcription factor AP-2 in high-grade prostate tumors (47, 48). Because TSG-6 is a secretory protein, it has an additional advantage of being developed as a marker in urine or serum samples. At this point, it is not known whether TSG-6 is specific to prostate or expressed in other tissues. A large number of samples from different grade prostate tumors as well as other tissues need to be analyzed to elucidate the precise role of TSG-6 in carcinogenesis.


   Acknowledgments
 
We thank Dr. Norman Greenberg for providing us with the TRAMP colony and cell lines and for his comments on the article; Drs. Ghosh, Rao, and Thompson (University of Texas Health Science Center, San Antonio) for their comments on the article; Pamela Wolfe for statistical analysis; Ashima Gupta for technical assistance; Laura Chubb (Colorado State University) and Elizabeth Genova (Prostate Diagnostic Laboratory, University of Colorado Health Sciences Center) for their assistance with magnetic resonance imaging and TSG-6 staining, respectively; and the University of Colorado Comprehensive Cancer Center Histology core facility for assistance with tissue sections.


   Footnotes
 
Grant support: American Cancer Society grants RSG-04-169-01 and R21 CA 98744 (A.P. Kumar) and San Antonio Cancer Institute/National Cancer Institute's Barbara Bowman Cancer Prevention award P30 CA-054174-16 (A.P. Kumar).

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.

Note: This work was conducted in the Center for Cancer Causation and Prevention at AMC Cancer Research Center, Denver, Colorado.

9 A.P. Kumar and G. E. Garcia, unpublished data. Back

Received 9/21/05; revised 11/ 7/05; accepted 11/29/05.


   References
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 Abstract
 Materials and Methods
 Results and Discussion
 References
 

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