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Carcinogenesis Advance Access originally published online on September 30, 2005
Carcinogenesis 2006 27(3):568-577; doi:10.1093/carcin/bgi233
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Carcinogenesis vol.27 no.3 © Oxford University Press 2005; all rights reserved.

N-(4-Hydroxyphenyl)retinamide and nitric oxide pro-drugs exhibit apoptotic and anti-invasive effects against bone metastatic breast cancer cells

Ann-Marie Simeone *, Stefano Colella 1, Ralf Krahe 1, Marcella M. Johnson 2, Edna Mora 3 and Ana M. Tari

Department of Experimental Therapeutics, 1 Department of Cancer Genetics and 2 Department of Biostatistics, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA and 3 Puerto Rico Cancer Center and Surgery Division, The University of Puerto Rico, Medical Sciences Campus, San Juan, PR 00936, USA

* To whom correspondence should be addressed. Email: amsimeon{at}mdanderson.org


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Breast cancer most frequently metastasizes to bone causing decreased quality of life and morbidity. Since current treatments are palliative, strategies to prevent bone metastases in breast cancer patients are required. There is substantial evidence indicating that high levels of nitric oxide (NO) suppress tumor growth and metastasis in vivo. We hypothesize that agents that produce high concentrations of NO could prevent the spread of breast cancer to bone. We previously demonstrated that the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) produces high levels of NO via the induction of NO synthases. NO pro-drugs are designed to produce large amounts of NO without inducing NO synthases but upon metabolism by their intracellular targets. The objective of this study was to determine the effectiveness of 4-HPR and an NO pro-drug, diethylamineNONOate/AM (NONO-AM), in inhibiting the growth and invasiveness of bone metastatic breast cancer cells. Parental MDA-MB-231 breast cancer cells were resistant to 4-HPR-induced apoptosis at clinically relevant doses, whereas 4-HPR-induced apoptosis in a dose-dependent manner in MDA-MB-231/F10 bone metastatic breast cancer cells. Unlike 4-HPR, NONO-AM induced apoptosis in a dose-dependent manner in both parental MDA-MB-231 cells and F10 cells. The bone metastatic F10 cells were more sensitive to the anti-invasive effects of 4-HPR and NONO-AM than were MDA-MB-231 cells. Although suppression of matrix metalloprotease-9 activity may be one mechanism by which 4-HPR decreases the invasion of F10 cells, it does not appear to be the anti-invasion mechanism of NONO-AM. These in vitro results suggest that 4-HPR and NO pro-drugs may be effective chemopreventive agents against bone metastatic breast cancer.

Abbreviations: DMEM/F12, Dulbecco's modified Eagle medium; FBS, fetal bovine serum; MMP, matrix metalloprotease; 4-HPR, N-(4-hydroxyphenyl)retinamide; NO, nitric oxide; NOS, nitric oxide synthase; NONO-AM, diethylamine NONOate/AM


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
As many as 85% of patients with advanced breast cancer develop bone metastases resulting in complications such as severe bone pain, fractures, hypercalcemia and spinal cord or nerve compression (1). The morbidity from bone metastases in patients with breast cancer remains a significant health problem, accounting for >70% of breast cancer deaths (2,3). Despite the prevalence of this problem, there are currently no satisfactory therapies available and most are administered with palliative intent. Chemotherapy and hormonal therapy are the preferred systemic treatments for bone metastatic breast cancer, but they are typically associated with only temporary control of symptomatic disease. Surgery, radiation, bisphosphonates and analgesics are also used to reduce bone pain and to prevent or repair fractures. Therefore, there is a need for the development of chemopreventive agents that specifically prevent the spread of breast cancer to bone.

The free radical nitric oxide (NO) plays an important role in regulating tumor growth and metastasis; however, it remains controversial whether NO positively or negatively impacts these processes. The cellular origin and the concentration of NO in the tumor microenvironment are crucial in determining whether NO exhibits tumor promoting or tumor suppressing effects. NO is synthesized by three nitric oxide synthase (NOS) isoforms. NOSI and NOSIII are expressed constitutively and produce trace amounts of NO (low pM for seconds to minutes). In contrast, NOSII is the inducible isoform and can generate large amounts of NO (µM for hours to days). Patient samples and animal models indicate that NOSII expression in tumor cells reduces, while NOSII expression in host cells enhances, breast cancer metastasis (4–7). Low concentrations of NO may protect some cell types from apoptosis induced by DNA-damaging agents (8) and may increase the invasiveness and metastatic potential of murine tumors (9,10). In contrast, high concentrations of NO are cytotoxic (11–16) and prevent murine tumor metastasis (17). Recently, there have been a number of studies demonstrating that NOSII-mediated NO suppresses tumorigenesis and metastasis in vivo (4,18,19). Therefore, molecules that can induce NOSII-mediated NO production in tumor cells could potentially be used as chemopreventive agents against bone metastatic breast cancer. One such molecule is the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR). We previously reported that 4-HPR at clinically relevant doses induces apoptosis in breast cancer cells by inducing NOSII-mediated NO production (15). 4-HPR has also been shown to inhibit the invasion of several cancer cell types, including ovarian cancer (20), prostate cancer (21–24) and Kaposi's sarcoma (25).

Unlike 4-HPR, NO donors are designed to spontaneously generate large amounts of NO in aqueous media at physiological pH and temperature. However, this indiscriminate release of NO can have deleterious effects on the cardiovascular system and induce severe hypotension. To maximize NO exposure to tumor cells while minimizing undesirable side effects elsewhere in the body, NO pro-drugs have been designed to generate large amounts of NO upon metabolism by intracellular enzymes. The NO pro-drug diethylamine NONOate/AM (NONO-AM), which releases NO upon activation by intracellular esterases, has been shown to induce apoptosis in leukemia cells (26).

Given their ability to produce large amounts of NO, 4-HPR and NONO-AM may hold promise for the prevention of bone metastases from breast cancer. The objective of this study was to determine the effects of 4-HPR and NONO-AM on the in vitro growth and invasiveness of bone metastatic breast cancer cells.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Reagents
4-HPR was purchased from Sigma Chemical (St Louis, MO). NONO-AM, GM6001, GM6001-negative and matrix metalloprotease (MMP)-2/MMP-9 inhibitor II {(2R)-[(4-biphenylsulfonyl)amino]-N-hydroxy-3-phenylpropionamide} were purchased from EMD Biosciences (La Jolla, CA). Matrigel was purchased from BD Biosciences (Bedford, MA). Hema-3 was purchased from Fisher Scientific (Middleton, VA). Casein and gelatin zymogram gels, non-reducing sample buffer, 10x zymogram renaturing buffer, 10x zymogram development buffer, Coomassie blue-R250 and Coomassie blue destaining solution were purchased from Bio-Rad Laboratories (Hercules, CA). Stock solutions of 4-HPR (10 mM), NONO-AM (10 mM), GM6001 (1 mM), GM6001-negative (1 mM) and MMP-2/MMP-9 inhibitor II (1 mM) were prepared in dimethyl sulfoxide and stored at –20°C. All reagents were diluted in the culture medium to the indicated final concentration.

Cell lines and culture conditions
The human MDA-MB-231 cell line was obtained from American Type Cell Culture (Manassas, VA). The MDA-MB-231 cell line is an estrogen independent highly metastatic human breast cancer cell line (27). MDA-MB-231 cells metastasize predominantly to bone, but may spread to the brain, ovary and adrenal glands after inoculation into the left ventricle of the heart in female nude mice (28). Since there are no commercially available bone metastatic breast cancer cell lines, we used a selective bone metastatic derivative of MDA-MB-231 cells, MDA-MB-231/F10. The MDA-MB-231/F10 cell line was established by inoculating MDA-MB-231 cells into the left ventricle of the heart of 6-week-old female nude mice. The tumor cells that metastasized to bone were isolated, grown in tissue culture and then reinoculated into the left ventricle of female nude mice. The cells that again metastasized to bone were isolated, expanded and reinoculated into the left ventricle of the heart. This process was repeated until no micrometastases were detected histologically or by X-ray in tissues other than bone within 4–5 weeks of injection. The MDA-MB-231/F10 cell line was established and kindly provided by Dr Yoneda (The University of Texas Health Science Center, San Antonio, Texas). F10 cells exclusively metastasize to bone after intracardiac inoculation in female nude mice (28). MDA-MB-231 and F10 breast cancer cells were cultured in Dulbecco's modified Eagles medium (DMEM/F12) supplemented with 5% heat-inactivated fetal bovine serum (FBS) at 37°C under 5% CO2 in a humidified incubator.

Cell growth and NO assay
MDA-MB-231 and F10 breast cancer cells were plated at 2.5 x 104 cells/well in 6-well plates in 2 ml of DMEM/F12 supplemented with 5% FBS. After 24 h, cells were treated with 4-HPR (1, 2.5 µM) or NONO-AM (0–100 µM). After 5 days of incubation, cell growth was determined by total live cell counts using trypan blue exclusion. Supernatants were collected from untreated and treated cells and stored at –80°C until NO determination. Total NO was determined by quantifying nitrite, the stable end product of NO oxidation, spectrophotometrically using a Colorimetric non-enzymatic Nitric Oxide Assay Kit (Oxford Biomedical Research, Oxford, MI) as described previously (15). Nitrite values were normalized for total cell counts and expressed as µM per one million cells. The values were reported as the mean (± SD) of experiments performed in triplicate.

Apoptosis assays
MDA-MB-231 and F10 cells were plated at 2.5 x 104 cells/well in 6-well plates in 2 ml of DMEM/F12 supplemented with 5% FBS. After 24 h, cells were treated with 4-HPR (1, 2.5 µM) or NONO-AM (40 µM). After 4 days of incubation, apoptosis was determined by using flow cytometry and immunohistochemistry. Cells were harvested and prepared for flow cytometry as described previously (15). For immunohistochemistry, cells were collected and suspended at 1 x 106 cells/ml in phosphate-buffered saline (PBS). Cytospins for each treatment were prepared using 100 µl of the appropriate cell suspension. Slides were quick-fixed in –20°C acetone and stored at –20°C until immunostaining. The FragELTM DNA Fragmentation Detection Kit (EMD Biosciences, La Jolla, CA) was used to detect apoptotic nuclei according to the manufacturer's instructions. This kit allows the recognition of DNA breaks in apoptotic nuclei. In this assay, terminal deoxynucleotidyl transferase binds to exposed 3'OH ends of DNA fragments and catalyzes the addition of biotin-labeled and unlabeled deoxynucleotides. Biotinylated nucleotides are detected using a streptavidin–horseradish peroxidase conjugate. Diaminobenzidine reacts with the labeled sample to generate an insoluble brown substrate at the site of DNA fragmentation. Samples were counterstained with methyl green to aid in distinguishing between normal and apoptotic cells.

Matrigel invasion assay
The effect of 4-HPR and NONO-AM on the invasiveness of breast cancer cells was determined in vitro by determining the number of MDA-MB-231 and F10 cells that invaded through transwell inserts coated with the artificial basement membrane Matrigel. This assay was also used to determine the importance of MMP activity on the invasiveness of MDA-MB-231 and F10 cells. Briefly, 6-well plate transwell inserts with 8 µm pore-size polycarbonate filters (Fisher Scientific, Middleton, VA) were coated with Matrigel in cold serum-free DMEM/F12 at a final concentration of 0.7 mg/ml and placed at room temperature for 40 min. MDA-MB-231 and F10 cells were trypsinized, resuspended in serum-supplemented medium and counted. Cells were then washed three times with serum-free medium. Cells (3 x 105 in 500 µl) were added into each transwell insert and incubated for 72 h in the absence and presence of 4-HPR (1, 2.5 µM) or NONO-AM (20, 40 µM). Cells were also incubated in the presence of the broad-spectrum MMP inhibitor GM6001 (10 µM) or an identical amount of its specific product of negative control, GM6001-negative, or a specific MMP-2/MMP-9 inhibitor (10 µM) for 72 h. The lower chambers were filled with 2 ml of DMEM/F12 medium supplemented with 5% FBS. After incubation, non-invading cells on the upper surface of the filter were removed with cotton swabs. Cells that had passed through the pores onto the lower side of the filter were fixed, stained with Hema-3 and photographed. The migrated cells were counted in five fields for each filter under a light microscope at 40x magnification. The invasive ability of the cells was expressed as the mean number of cells that had invaded to the lower side of the filter. The experiments were performed in triplicate and repeated twice.

Zymographic analysis of MMP activity
MDA-MB-231 and F10 cells (3 x 105) were plated in T-25 flasks and incubated for 24 h in the absence or presence of 4-HPR (1, 2.5 µM) or NONO-AM (20, 40 µM). MDA-MB-231 and F10 cells were also incubated for 24 h with the MMP-2/MMP-9 inhibitor (10 µM) to verify the downregulation of MMP-9 activity. Cells were changed to serum-free medium the next day for 24 h. The medium was recovered and centrifuged for 5 min. The conditioned medium was collected and concentrated by using spin columns with 10-kDa-cutoff filters (Millipore, Bedford, MA). Concentrated conditioned medium (20 µl) was mixed (1:1) with non-reducing sample buffer, incubated at 37°C for 15 min, and then applied to gelatin and casein zymogram gels. After electrophoresis, the gels were incubated for 3 h in zymogram renaturing buffer at room temperature with gentle agitation, followed by an overnight incubation in zymogram development buffer at 37°C. The gels were stained with Coomassie blue-R250 for 3 h and then destained with Coomassie blue destaining solution. Gelatinase and caseinase activities were visible as clear bands against the dark blue background, indicating proteolysis of the substrate protein.

Statistical analyses
For statistical analysis of the invasion experiments, the Shapiro–Wilk test was first performed to assess the normality assumption of the data. Given that the data was normally distributed, two-sample t-tests were performed for each cell line to compare the number of invading cells for the untreated group with the number of invading cells for each of the 4-HPR doses. In addition, the number of invading cells was compared between the two 4-HPR doses. These analyses were also performed for the NONO-AM, GM6001 and MMP-2/MMP-9 inhibitor treatments used in the invasion experiments. The significance level for each individual comparison was adjusted by the Bonferroni method to account for multiple testing within each cell line to achieve an overall significance level of 0.05. All analyses were performed using SAS®.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
4-HPR's induction of apoptosis in F10 bone metastatic breast cancer cells was correlated with NO production
To assess the effect of 4-HPR on the growth of breast cancer cells, MDA-MB-231 and F10 cells were treated with 4-HPR. The two experimental doses 1 and 2.5 µM were chosen because they are clinically achievable (29). MDA-MB-231 cells were resistant to 4-HPR-induced growth inhibition (Table I). In contrast, 4-HPR at 1 and 2.5 µM inhibited the growth of F10 cells by 53 and 69%, respectively.


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  		Table I. 4-HPR selectively induced growth inhibition and NO production in bone metastatic breast cancer cells

 
NO production increased 3.4- and 5.5-fold in F10 cells treated with 1 and 2.5 µM 4-HPR, respectively (Table I). In contrast, NO production was not induced in MDA-MB-231 cells treated with 4-HPR. Thus, the growth inhibitory effects of 4-HPR were directly correlated with increases in NO production in F10 cells.

Flow cytometric analysis of propidium iodide-stained cells was used to determine whether apoptosis was related to the growth inhibition found in 4-HPR-treated bone metastatic breast cancer cells (Figure 1A). 4-HPR increased the percentage of apoptotic F10 cells in a dose-dependent manner (Figure 1A). The number of apoptotic F10 cells was increased from 1.1 to 26.7 and 47.3% by the 1 and 2.5 µM doses of 4-HPR, respectively (Figure 1A). The apoptotic effects of 4-HPR were confirmed by using DNA fragmentation (Figure 1B). These data indicate that F10 bone metastatic breast cancer cells were sensitive to pharmacologically achievable doses of 4-HPR.


Figure 1
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  		Fig. 1. 4-HPR selectively induced apoptosis in bone metastatic breast cancer cells. F10 cells (2.5 x 104 cells/well) were plated in 6-well plates in DMEM/F12 medium supplemented with 5% FBS. The next day, cells were treated with 4-HPR (1, 2.5 µM). After 4 days of incubation, cells were harvested and (A) flow cytometry and (B) immunohistochemical staining of DNA fragmentation were performed to determine the presence of apoptotic cells. Asterisk indicates the percentage of apoptotic cells (i.e. cells in the sub-G1 peak).

 
NONO-AM induced apoptosis in MDA-MB-231 and F10 breast cancer cells
To assess the effect of NONO-AM on the growth of breast cancer cells, MDA-MB-231 and F10 cells were treated with increasing concentrations of NONO-AM (0–100 µM). In contrast to 4-HPR, NONO-AM inhibited the growth of both MDA-MB-231 and F10 cells in a dose-dependent manner (Figure 2A). The IC50 of NONO-AM was between 20 and 40 µM in both cell lines. Growth inhibition in NONO-AM–treated cells was correlated with dose-dependent increases in NO concentration (Figure 2B). To determine whether apoptosis was related to the growth inhibition found in the breast cancer cell lines treated with NONO-AM, MDA-MB-231 and F10 cells were treated with NONO-AM (40 µM) and examined by flow cytometry (Figure 2C). NONO-AM increased the number of apoptotic cells from 2.2 to 35% for MDA-MB-231 cells and from 1.7 to 46.8% for F10 cells (Figure 2C). The apoptotic effects of NONO-AM were confirmed by using DNA fragmentation (Figure 2D). These data indicate that both the parental and the F10 bone metastatic breast cancer cells were sensitive to the apoptotic effects of NONO-AM.


Figure 2
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  		Fig. 2. NONO-AM induced apoptosis in metastatic breast cancer cells. (A) MDA-MB-231 and F10 cells (2.5 x 104 cells/well) were plated in 6-well plates in DMEM/F12 medium supplemented with 5% FBS. The next day, cells were treated with NONO-AM (0–100 µM). After 5 days of incubation, cell growth was determined by total live cell counts using trypan blue exclusion. The values shown are the mean (± SD) of experiments performed in triplicate. (B) MDA-MB-231 and F10 cells (2.5 x 104 cells/well) were plated in 6-well plates in DMEM/F12 medium supplemented with 5% FBS. The next day, cells were treated with NONO-AM (0–100 µM). After 5 days of incubation, NO production was determined by measuring its stable end product nitrite using a Colorimetric Nitric Oxide Assay Kit. Nitrite values were normalized to cell number. The values shown are the mean (± SD) of experiments performed in triplicate. MDA-MB-231 and F10 cells (2.5 x 104 cells/well) were plated in 6-well plates in DMEM/F12 medium supplemented with 5% FBS. Cells were treated with NONO-AM (40 µM) 24 h later. After 4 days of incubation, cells were harvested and (C) flow cytometry and (D) immunohistochemical staining of DNA fragmentation were performed to detect the presence of apoptotic cells. Asterisk indicates the percentage of apoptotic cells (i.e. cells in the sub-G1 peak).

 
F10 bone metastatic breast cancer cells were more sensitive to the anti-invasive effects of 4-HPR and NONO-AM
We examined the effect of 4-HPR and NONO-AM on the invasive ability of MDA-MB-231 and F10 cells by using the Matrigel invasion assay. To avoid confounding any anti-invasive effects of 4-HPR and NONO-AM with their pro-apoptotic effects, these experiments were performed under conditions (initial plating density and treatment duration) in which <10% growth inhibition was induced (data not shown). Untreated MDA-MB-231 and F10 cells displayed a high invasive capacity on Matrigel (Figure 3A). 4-HPR significantly (P < 0.05) reduced the number of invasive cells in both cell lines; however, F10 cells were more sensitive to the anti-invasive effects of 4-HPR (Figure 3A). Significant inhibition (P < 0.05) of invasion was observed in F10 cells (74%) and a smaller but significant (P < 0.05) decrease in invasion (28%) was observed in MDA-MB-231 cells after treatment with 1 µM 4-HPR (Figure 3B). The 2.5 µM dose of 4-HPR had a similar anti-invasive effect in MDA-MB-231 (74%) and F10 cells (77%; Figure 3B). The two doses of 4-HPR did not have significantly different anti-invasive effects in F10 cells.


Figure 3
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  		Fig. 3. Bone metastatic breast cancer cells were more sensitive to the anti-invasive effects of 4-HPR than the parental metastatic breast cancer cells. (A) MDA-MB-231 and F10 cells (3 x 105 in 500 µl) were added into transwell inserts (8 µm pore-size) coated with Matrigel. Cells were incubated for 72 h in the absence and presence of 4-HPR (1, 2.5 µM). Cells that passed through the pores onto the lower side of the filter were fixed, stained with Hema-3 and photographed. (B) The migrated cells were counted in five fields for each filter under a light microscope at 40x magnification. The invasive ability of the cells was expressed as the mean number of cells that invaded to the lower side of the filter. Values shown are the mean (± SD) of experiments performed in triplicate. *, P < 0.05 when compared with untreated cells.

 
In contrast to 4-HPR, NONO-AM inhibited the invasion of F10 cells but not MDA-MB-231 cells (Figure 4A). At 20 and 40 µM, NONO-AM significantly (P < 0.05) reduced the number of invading F10 cells by 60 and 75%, respectively, compared with untreated cells (Figure 4B). The two doses of NONO-AM did not have significantly different anti-invasive effects in F10 cells (Figure 4B).


Figure 4
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  		Fig. 4. NONO-AM selectively inhibited the invasion of bone metastatic breast cancer cells. (A) MDA-MB-231 and F10 cells (3 x 105 in 500 µl) were added into transwell inserts (8 µm pore-size) coated with Matrigel. Cells were incubated for 72 h in the absence and presence of NONO-AM (20, 40 µM). Cells that passed through the pores onto the lower side of the filter were fixed, stained with Hema-3 and photographed. (B) The migrated cells were counted in five fields for each filter under a light microscope at 40x magnification. The invasive ability of the cells is expressed as the mean number of cells that invaded to the lower side of the filter. Values shown are the mean (± SD) of experiments performed in triplicate. *, P < 0.05 when compared with untreated cells.

 
Effects of 4-HPR and NONO-AM on MMP activities
MMPs, which are essential to the invasive process, are involved in the degradation of the extracellular matrix and basement membranes. To confirm that MMP activity was important for the invasiveness of MDA-MB-231 and F10 cells, the broad-spectrum MMP inhibitor GM6001 (10 µM) was used to block MMP activity in a Matrigel invasion assay. GM6001 at the dose used was not cytotoxic to either cell line (data not shown). Inhibition of MMP activity significantly (P < 0.05) decreased the invasiveness of MDA-MB-231 and F10 cells (Figure 5A). The number of invaded cells was decreased 40 and 54% in MDA-MB-231 and F10 cells, respectively, by treatment with GM6001 (Figure 5B). In contrast, GM6001-negative, the specific product of negative control for GM6001, did not influence the invasive ability of either cell line (Figure 5A and B).


Figure 5
Figure 5
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  		Fig. 5. Gelatinase and caseinase activities in MDA-MB-231 and F10 cells treated with 4-HPR or NONO-AM. (A) MDA-MB-231 and F10 cells (3 x 105 in 500 µl) were added into transwell inserts coated with Matrigel. Cells were incubated for 72 h in the absence and presence of broad-spectrum MMP inhibitor GM6001 (10 µM) or an identical amount of GM6001-negative for 72 h. GM6001-negative is the specific product of negative control for GM6001. Cells that passed through the pores onto the lower side of the filter were fixed, stained with Hema-3 and photographed. (B) The migrated MDA-MB-231 and F10 cells were counted in five fields for each filter under a light microscope at 40x magnification. The invasive ability of the cells was expressed as the mean number of cells that invaded to the lower side of the filter. Values shown are the means (± SD) of experiments performed in triplicate. *P < 0.05 when compared to untreated cells. (C) The effects of 4-HPR and NONO-AM on gelatinase activitiy were evaluated by zymography. MDA-MB-231 and F10 cells (3 x 105 cells) were plated in T-25 flasks. The next day, cells were treated for 24 h with 4-HPR (1, 2.5 µM) or NONO-AM (20, 40 µM). Cells were then changed to serum-free medium for 24 h. Concentrated conditioned medium (20 µl) was mixed (1:1) with non-reducing sample buffer and applied to gelatin substrate gels. The gels were incubated in renaturing buffer followed by an overnight incubation in development buffer at 37°C. The gel was stained with Coomassie blue-R250 and then destained. Gelatinase activities were visible as clear bands against the dark blue background, indicating proteolysis of the substrate protein. Lane 1, untreated; lane 2, 1 µM 4-HPR; lane 3, 2.5 µM 4-HPR; lane 4, 20 µM NONO-AM; and lane 5, 40 µM NONO-AM. (D) The effect of 4-HPR and NONO-AM on caseinase activity was evaluated by zymography. Casein zymography was performed in an identical manner to gelatin zymography except that concentrated conditioned medium was applied to casein substrate gels. Lanes were labeled as in (C). (E) Matrigel invasion assay of MDA-MB-231 and F10 cells treated with a specific MMP-9 inhibitor (10 µM) for 72 h. (F) The number of invaded MDA-MB-231 and F10 cells were counted in five fields for each filter under a light microscope at 40x magnification. The invasive ability of the cells was expressed as the mean number of cells that invaded to the lower side of the filter. Values shown are the mean (± SD) of experiments performed in triplicate. *, P < 0.05 when compared with untreated cells.

 
To determine whether 4-HPR and NONO-AM decreased the invasive potential of breast cancer cells by blocking MMP activities, casein and gelatin zymography was performed on untreated and treated MDA-MB-231 and F10 cells. MMP-9 and MMP-2 activities were detected by gelatin zymography in the supernatants of untreated MDA-MB-231 and F10 cells (Figure 5C). The activity of MMP-9 was greater than that of MMP-2 in both cell lines and was greater in F10 cells than in MDA-MB-231 cells. MMP-9 activity decreased in a dose-dependent manner in MDA-MB-231 cells treated with 4-HPR, whereas only the 2.5 µM dose of 4-HPR decreased MMP-9 activity in F10 cells. NONO-AM had no effect on MMP-9 activity in either cell line. MMP-2 activity was not affected by treatment with 4-HPR or NONO-AM in either cell line. Casein zymography revealed two protease bands with molecular weights of ~40–56 kDa in untreated F10 cells (Figure 5D). These bands may correspond to the stromelysins MMP-3 and MMP-10 or may represent an MMP-3 doublet (30). Caseinase activities were not altered in MDA-MB-231 and F10 cells by treatment with 4-HPR or NONO-AM.

Since 4-HPR decreased MMP-9 activity in MDA-MB-231 and F10 cells, the importance of MMP-9 in the invasiveness of these cell lines was determined. A specific MMP-9 inhibitor (10 µM) was used to block MMP-9 activity of the cell lines in a Matrigel invasion assay. The MMP-9 inhibitor at the dose used was not cytotoxic to either cell line (data not shown). The inhibitor suppressed MMP-9 activity as revealed by gelatin zymography (data not shown). Inhibition of MMP-9 activity significantly (P < 0.05) decreased the invasiveness of MDA-MB-231 and F10 cells (Figure 5E). The number of invaded cells was decreased 39 and 41% in MDA-MB-231 and F10 cells, respectively, by treatment with the MMP-9 inhibitor (Figure 5F). Therefore, suppression of MMP-9 activity may be one mechanism by which 4-HPR decreased the invasive behavior of the breast cancer cell lines.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
We found that the F10 bone metastatic breast cancer cells were sensitive to the apoptotic and anti-invasive effects of 4-HPR and NONO-AM. Both drugs produce high levels of NO but by different mechanisms. 4-HPR induces NO production by increasing NOSII expression in breast cancer cells (15), whereas NONO-AM releases NO when activated by intracellular esterases (26). We found that 4-HPR selectively induced apoptosis in F10 cells but not in MDA-MB-231 cells. On the other hand, NONO-AM was effective in inducing apoptosis in both cell lines. These data indicate that both the parental and the bone metastatic cells are sensitive to NO-mediated apoptosis. However, the parental cells may possess intrinsic resistance mechanisms that could prevent 4-HPR from inducing NO, probably by preventing 4-HPR from inducing NOSII expression, as is the case in HER2/neu-overexpressing breast cancer cells (31).

The invasiveness of F10 cells was suppressed by 4-HPR and NONO-AM, suggesting that the bone metastatic breast cancer cells are highly sensitive to NO-mediated anti-invasive effects. In contrast, the invasiveness of the parental MDA-MB-231 cells appears to be insensitive to NO, since NONO-AM, which could generate large amounts of NO in MDA-MB-231 cells, did not decrease the invasive activity of these cells. Surprisingly, 4-HPR decreased the invasive activity of both MDA-MB-231 and F10 cells, albeit more effectively in F10 cells. In unpublished observations, cDNA microarray analysis of 4-HPR-modulated genes in MDA-MB-231 and F10 cells did not reveal any gene expression changes >2-fold on Affymetrix U133A and U133B GeneChips. In MDA-MB-231 cells, 4-HPR-modulated the expression of 19 genes by 1.5-fold on the Affymetrix U133A GeneChip and 15 genes on the Affymetrix U133B GeneChip. Some of these genes included thrombospondin-1, interleukin-8 and the C-X-C motif ligands 1, 2 and 3. In F10 cells, 4-HPR did not induce any gene expression changes >1.5-fold in either GeneChip. These data suggest that the induction of apoptotic and anti-invasive effects by 4-HPR in breast cancer cells does not occur at the transcriptional level.

MDA-MB-231 cells have been shown to secrete the gelatinase MMP-9 and the caseinase MMP-3 (32–34). In the present study, suppression of MMP-9 activity, but not MMP-2 or caseinase activities (MMP-3/MMP-10), was observed in MDA-MB-231 and F10 cells treated with 4-HPR. MMP-9 activity was important in the invasiveness of MDA-MB-231 and F10 cells since suppression of MMP-9 activity decreased the number of invaded cells. In MDA-MB-231 cells, 4-HPR decreased MMP-9 activity in a dose-dependent manner, which corresponded with the dose-dependent decrease in invasion. In F10 cells, invasion was inhibited to a similar degree by treatment with either 1 or 2.5 µM 4-HPR; however, decreased MMP-9 activity was observed only at the 2.5 µM dose. Suppressing the activity of MMP-9 may be one mechanism by which 4-HPR decreases the invasive behavior of MDA-MB-231 and F10 cells. However, it is apparent from these results that 4-HPR uses more than one mechanism to suppress the invasiveness of metastatic breast cancer cells. MDA-MB-231 cells have been shown to secrete the interstitial collagenases MMP-1 and MMP-13 (32). It is possible that 4-HPR may decrease the activity of these MMPs to suppress invasion. In addition, 4-HPR inhibited the invasion of transformed BALB/c 3T3 cells and prostate cancer cells by suppressing chemotactic motility (35) and by altering cellular adhesion to the extracellular matrix (21,35), so the drug may also use these mechanisms to inhibit the invasive capacity of metastatic breast cancer cells. Interestingly, NONO-AM did not have any effect on gelatinase or caseinase activities in F10 cells, indicating that the drug may have affected invasion by altering the activity of MMPs that we did not evaluate or by altering cellular adhesion.

Bone is the most frequent site of metastatic disease in patients with advanced breast cancer, and the prognosis in these patients is generally poor. According to National Cancer Institute, only 10–20% of women with metastatic breast cancer survive the disease and achieve permanent remission. Current treatments, including chemotherapy, hormonal therapy and radiotherapy, are generally administered with palliative intent; therefore, chemoprevention to prevent the formation of bone metastases could have a profound effect on the incidence of bone metastases and associated mortality rate. Bisphosphonates are an effective palliative treatment for bone metastatic breast cancer, but randomized clinical trials investigating the adjuvant use of bisphosphonates to prevent bone metastases have yielded conflicting results. Diel et al. (36) and Powles et al. (37) reported a reduction in the occurrence of bone metastases from breast cancer and an increase in overall survival in patients with primary breast cancer who received adjuvant clodronate for 2 years. However, a decrease (38) or lack of effect (37) of clodronate was evident at the 5-year follow-up. In contrast, Saarto et al. (39) found that 3 years of adjuvant clodronate had no effect on the occurrence of bone metastases and a negative effect on the occurrence of non-bone metastases as well as survival in breast cancer patients. Therefore, chemopreventive strategies that specifically prevent the occurrence of bone metastases from breast cancer are urgently needed. The results of the present in vitro studies indicate the potential of 4-HPR and NO pro-drugs as chemopreventive agents against bone metastases from breast cancer and warrant their further evaluation in in vivo chemoprevention studies.


   Acknowledgments
 
The authors thank Drs Douglas Boyd and Lingegowda Mangala for their advice with the invasion assay and Dr Arturo Chavez-Reyes for his help with the DNA fragmentation assay and photographs. The authors thank Karen Ramirez and The University of Texas M. D. Anderson Cancer Center Department of Immunology Flow Cytometry Core Lab for the technical assistance. This research was supported in part by the IDO1-014 U54 RFA CA 096300 (A.M.T. and E.M.) and the institutions NCI Core Grant (CA16672).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Coleman,R.E. and Rubens,R.D. (1987) The clinical course of bone metastases from breast cancer. Br. J. Cancer, 55, 61–66.[Web of Science][Medline]
  2. Aaron,A.D. (1994) The management of cancer metastatic to bone. JAMA, 272, 1206–1209.[Abstract/Free Full Text]
  3. Weiss,L. (1992) Comments on hematogenous metastatic patterns in humans as revealed by autopsy. Clin. Exp. Metastasis, 10, 191–199.[CrossRef][Medline]
  4. Gauthier,N., Lohm,S., Touzery,C., Chantome,A., Perette,B., Reveneau,S., Brunotte,F., Juillerat-Jeanneret,L. and Jeannin,J.-F. (2004) Tumour-derived and host-derived nitric oxide differentially regulate breast carcinoma metastasis to the lungs. Carcinogenesis, 25, 1559–1565.[Abstract/Free Full Text]
  5. Thomsen,L.L., Miles,D.W., Happerfield,L., Bobrow,L.G., Knowles,R.G. and Moncada,S. (1995) Nitric oxide synthase activity in human breast cancer. Br. J. Cancer, 72, 41–44.[Web of Science][Medline]
  6. Tschugguel,W., Schneeberger,C., Unfried,G., Czerwenka,K., Weninger,W., Mildner,M., Gruber,D.M., Sator,M.O., Waldhor,T. and Huber,J.C. (1999) Expression of inducible nitric oxide synthase in human breast cancer depends on tumor grade. Breast Cancer Res. Treat., 56, 145–151.[CrossRef][Web of Science][Medline]
  7. Vakkala,M., Kahlos,K., Lakari,E., Paakko,P., Kinnula,V. and Soini,Y. Inducible nitric oxide synthase expression, apoptosis, and angiogenesis in in situ and invasive breast carcinomas. Clin. Cancer Res., 6, 2408–2416.
  8. Xu,W., Liu,L., Smith,G.C. and Charles,G. (2000) Nitric oxide upregulates expression of DNA-PKcs to protect cells from DNA-damaging anti-tumour agents. Nat. Cell Biol., 2, 339–345.[CrossRef][Web of Science][Medline]
  9. Orucevic,A., Bechberger,J., Green,A.M., Shapiro,R.A., Billiar,T.R. and Lala,P.K. (1999) Nitric-oxide production by murine mammary adenocarcinoma cells promotes tumor-cell invasiveness. Int. J. Cancer, 81, 889–896.[CrossRef][Web of Science][Medline]
  10. Jadeski,L.C., Hum,K.O., Chakraborty,C. and Lala,P.K. (2000) Nitric oxide promotes mammary tumor growth and metastasis by stimulating tumour cell migration, invasiveness and angiogenesis. Int. J. Cancer, 86, 30–39.[CrossRef][Web of Science][Medline]
  11. Bani,D., Mansini,E., Bello,M.G., Bigazzi,M. and Sacchi,T.B. (1995) Relaxin activates the L-arginine-nitric oxide pathway in human breast cancer cells. Cancer Res., 55, 5272–5275.[Abstract/Free Full Text]
  12. Adami,A., Crivellente,F., De Prati,A.C., Cabalieri,E., Cuzzolin,L., Tommasi,M., Suzuki,H. and Benoni,G. (1998) Biotransformation and cytotoxic properties of NO-donors on MCF7 and U251 cell lines. Life Sci., 63, 2097–2105.[CrossRef][Web of Science][Medline]
  13. Binder,C., Schulz,M., Hiddemann,W. and Oellerich,M. (1999) Induction of inducible nitric oxide synthase is an essential part of tumor necrosis factor-(-induced apoptosis in MCF-7 and other epithelial tumor cells. Lab. Invest., 79, 1703–1712.[Web of Science][Medline]
  14. Umansky,V., Ushmorov,A., Ratter,F., Chlichlia,K., Bucur,M., Lichtenauer,A. and Rocha,M. (2000) Nitric oxide-mediated apoptosis in human breast cancer cells requires changes in mitochondrial functions and is independent of CD95(APO-1/Fas). Int. J. Oncol., 16, 109–117.[Web of Science][Medline]
  15. Simeone,A., Ekmekcioglu,S., Broemeling,L.D., Grimm,E.A. and Tari,A.M. (2002) A novel mechanism by which N-(4-hydroxyphenyl)retinamide inhibits breast cancer cell growth: the production of nitric oxide. Mol. Cancer Ther., 1, 1009–1017.[Abstract/Free Full Text]
  16. Hussain,S.P., Hofseth,L.J. and Harris,C.C. (2003) Radical causes of cancer. Nat. Rev. Cancer, 3, 276–285.[CrossRef][Web of Science][Medline]
  17. Dong,Z., Staroselsky,A.H., Qi,X., Xie,K. and Fidler,I.J. (1994) Inverse correlation between expression of inducible nitric oxide synthase activity and production metastasis in K-1735 murine melanoma cells. Cancer Res., 54, 789–793.[Abstract/Free Full Text]
  18. Dhar,A., Brindley,J.M., Stark,C., Citro,M.L., Keefer,L.K. and Colburn,N.H. (2003) Nitric oxide does not mediate but inhibits transformation and tumor phenotype. Mol. Cancer Ther., 2, 1285–1293.[Abstract/Free Full Text]
  19. Le,X., Wei,D., Huang,S., Lancaster,J.R. and Xie,K. (2005) Nitric oxide synthase II suppresses the growth and metastasis of human cancer regardless of its upregulation of protumor factors. Proc. Natl Acad. Sci. USA, 102, 8758–8763.[Abstract/Free Full Text]
  20. Um,S.-J., Lee,S.-Y., Kim,E.-J., Han,H.-S., Koh,Y.-M., Hong,K.-J., Sin,H.-S. and Park,J.-S. (2001) Antiproliferative mechanism of retinoid derivatives in ovarian cancer cells. Cancer Lett., 174, 127–134.[CrossRef][Web of Science][Medline]
  21. Kim,J.H., Tanabe,T., Chodak,G.W. and Rukstalis,D.B. (1995) In vitro anti-invasive effects of N-(4-hydroxyphenyl)-retinamide on human prostatic adenocarcinoma. Anticancer Res., 15, 1429–1434.[Medline]
  22. Webber,M.M., Bello-DeOcampo,D., Quader,S., Deocampo,N.D., Metcalfe,W.S. and Sharp,R.M. (1999) Modulation of the malignant phenotype of human prostate cancer cells by N-(4-hydroxyphenyl)retinamide (4-HPR). Clin. Exp. Metastasis, 17, 255–263.[CrossRef][Web of Science][Medline]
  23. Quader,S.T.A., Bello-DeOcampo,D., Williams,D.E., Kleinman,H.K. and Webber,M.M. (2001) Evaluation of the chemopreventive potential of retinoids using a novel in vitro human prostate carcinogenesis model. Mutat. Res., 496, 153–161.[Medline]
  24. Sharp,R.M., Bello-DeOcampo,D., Quader,S.T.A. and Webber,M.M. (2001) N-(4-hydroxyphenyl)retinamide (4-HPR) decreases neoplastic properties of human prostate cells: an agent for prevention. Mutat. Res., 496, 163–170.[Medline]
  25. Ferrari,N., Morini,M., Pfeffer,U., Minghelli,S., Noonan,D.M. and Albini,A. (2003) Inhibition of Kaposi's sarcoma in vivo by fenretinide. Clin. Cancer Res., 9, 6020–6029.[Abstract/Free Full Text]
  26. Saavedra,J.E., Shami,P.J., Wang,L.Y., Davies,K.M., Booth,M.N., Citro,M.L. and Keefer,L.K. (2000) Esterase-sensitive nitric oxide donors of the diazeniumdiolate family. In vitro antileukemic activity. J. Med. Chem., 43, 261–269.[CrossRef][Web of Science][Medline]
  27. Brunner,N., Thompson,E.W., Spang-Thomsem,M., Rygaard,J., Dano,K. and Zwiebel,J.A. (1992) lacZ transduced human breast cancer xenografts as an in vivo model for the study of invasion and metastasis. Eur. J. Cancer, 28A, 1989–1995.
  28. Yoneda,T., Williams,P.J., Hiraga,T., Niewolna,M. and Nishimura,R. (2001) A bone-seeking clone exhibits different biological properties from the MDA-MB-231 parental human breast cancer cells and a brain-seeking clone in vivo and in vitro. J. Bone Min. Res., 16, 1486–1495.[CrossRef][Web of Science][Medline]
  29. Formelli,F., Clerici,M., Campa,T., Di Mauro,M., Magni,A., Mascotti,G., Moglia,D., De Palo,G., Costa,A. and Veronesi,U. (1993) Five-year administration of fenretinide: pharmacokinetics and effects on plasma retinol concentrations. J. Clin. Oncol., 11, 2036–2042.[Abstract/Free Full Text]
  30. Hagemann,T., Robinson,S.C., Schulz,M., Trumper,L., Balkwill,F.R. and Binder,C. (2004) Enhanced invasiveness of breast cancer cell lines upon co-cultivation with macrophages is due to TNF-alpha dependent upregulation of matrix metalloproteases. Carcinogenesis, 25, 1543–1549.[Abstract/Free Full Text]
  31. Simeone,A., Broemeling,L.D., Rosenblum,J. and Tari,A.M. (2003) HER2/neu reduces the apoptotic effects of N-(4-hydroxyphenyl)retinamide (4-HPR) in breast cancer cells by decreasing nitric oxide production. Oncogene, 22, 6739–6747.[CrossRef][Web of Science][Medline]
  32. Balduyck,M., Zerimach,F., Gouyer,V. et al. (2000) Specific expression of matrix metalloproteases 1, 3, 9 and 13 associated with invasiveness of breast cancer cells in vitro. Clin. Exp. Metastasis, 18, 171–178.[CrossRef][Web of Science][Medline]
  33. Morini,M., Mottolese,M., Ferrari,N., Ghiorzo,F., Buglioni,S., Mortarini,R., Noonan,D.M., Natali,P.G. and Albini,A. (2000) The alpha 3 beta 1 integrin is associated with mammary carcinoma cell metastasis, invasion, and gelatinase B (MMP-9) activity. Int. J. Cancer, 87, 336–342.[CrossRef][Web of Science][Medline]
  34. Weber,M.H., Lee,J. and Orr,F.W. (2002) The effect of Neovastat (AE-941) on an experimental metastatic bone tumor model. Int. J. Oncol., 20, 299–303.[Web of Science][Medline]
  35. Vaccari,M., Silingardi,P., Argnani,A., Horn,W., Giungi,M., Mascolo,M.G., Grilli,S. and Colacci,A. (2000) In vitro effects of fenretinide on cell-matrix interactions. Anticancer Res., 20, 3059–3066.[Web of Science][Medline]
  36. Diel,I.J., Solomayer,E.F., Costa,S.D., Gollan,C., Goerner,R., Wallwiener,D., Kaufmann,M. and Bastert,G. (1998) Reduction in new metastases in breast cancer with adjuvant clodronate treatment. N. Engl. J. Med., 339, 357–363.[Abstract/Free Full Text]
  37. Powles,T., Paterson,S., Kanis,J.A. et al. (2002) Randomized, placebo-controlled trial of clodronate in patients with primary operable breast cancer. J. Clin. Oncol., 20, 3219–3224.[Abstract/Free Full Text]
  38. Diel,I.J., Solomyer,E.F. and Bastert,G. (2000) Bisphosphonates and the prevention of metstases: first evidences from preclinical and clinical studies. Cancer, 88, 3080–3088.[CrossRef][Medline]
  39. Saarto,T., Blomqvist,C., Virkkunen,P. and Elomaa,I. (2001) Adjuvant clodronate treatment does not reduce the frequency of skeletal metastases in node-positive breast cancer patients: 5-year results of a randomized controlled trial. J. Clin. Oncol., 19, 10–17.[Abstract/Free Full Text]
Received March 28, 2005; revised September 5, 2005; accepted September 24, 2005.


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