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. Author manuscript; available in PMC: 2014 Jun 27.
Published in final edited form as: Cancer Biol Ther. 2009 Nov 8;8(22):2153–2159. doi: 10.4161/cbt.8.22.9770

Dasatinib inhibits both osteoclast activation and prostate cancer PC-3 cell-induced osteoclast formation

John C Araujo 1,*, Ann Poblenz 2, Paul G Corn 1, Nila U Parikh 1, Michael W Starbuck 1, Jerry T Thompson 3, Francis Lee 4, Christopher J Logothetis 1, Bryant G Darnay 2
PMCID: PMC4073296  NIHMSID: NIHMS532682  PMID: 19855158

Abstract

Purpose

Therapies to target prostate cancer bone metastases have only limited effects. New treatments are focused on the interaction between cancer cells, bone marrow cells and the bone matrix. Osteoclasts play an important role in the development of bone tumors caused by prostate cancer. Since Src kinase has been shown to be necessary for osteoclast function, we hypothesized that dasatinib, a Src family kinase inhibitor, would reduce osteoclast activity and prostate cancer (PC-3) cell-induced osteoclast formation.

Results

Dasatinib inhibited RANKL-induced osteoclast differentiation of bone marrow-derived monocytes with an EC50 of 7.5 nM. PC-3 cells, a human prostate cancer cell line, were able to differentiate RAW 264.7 cells, a murine monocytic cell line, into osteoclasts and dasatinib inhibited this differentiation. In addition, conditioned medium from PC-3 cell cultures was able to differentiate RAW 264.7 cells into osteoclasts and this too, was inhibited by dasatinib. Even the lowest concentration of dasatinib, 1.25 nmol, inhibited osteoclast differentiation by 29%. Moreover, dasatinib inhibited osteoclast activity by 58% as measured by collagen 1 release.

Experimental design

We performed in vitro experiments utilizing the Src family kinase inhibitor dasatinib to target osteoclast activation as a means of inhibiting prostate cancer bone metastases.

Conclusion

Dasatinib inhibits osteoclast differentiation of mouse primary bone marrow-derived monocytes and PC-3 cell-induced osteoclast differentiation. Dasatinib also inhibits osteoclast degradation activity. Inhibiting osteoclast differentiation and activity may be an effective targeted therapy in patients with prostate cancer bone metastases.

Keywords: osteoclast, Src, prostate cancer, dasatinib, PC-3

Introduction

Bone metastases are the leading cause of morbidity in patients with prostate cancer. The American Cancer Society estimated that 186,320 new cases of prostate cancer would be diagnosed in 2008.1 An estimated 28,660 people will die of the disease in 2008, and 80% or more of them will have bone metastases. While those with disease confined to the prostate have an excellent predicted 10-y survival rate, those with bone metastases do not. In fact, prostate cancer metastasis to the bone is the major cause of mortality in these patients. Many factors have been implicated in the initiation of bone metastasis and progression of disease (reviewed in ref. 2). The dynamic interaction created between prostate cancer cells, bone marrow cells and bone matrix is being explored. Many therapies have targeted bone metastases to improve symptoms and overall survival. Such therapies include androgen ablation therapy, cytotoxic therapy, radioactive bone-targeting therapies and bone-strengthening therapies with bisphosphonates, all of which have only limited effects and cannot cure the disease (reviewed in ref. 3).

Although prostate cancer metastases are predominately osteoeoblastic, recent histomorphometric analysis has clearly shown that osteoclasts are present in prostate cancer bone metastases and possibly regulate the formation of the osteoblastic lesion.4–10 For instance, the prostate cancer-derived cell line LNCaP has been shown to induce the differentiation of RAW 264.7 cells into active osteoclasts.11 Furthermore, mice with increased bone turnover have a greater likelihood of developing prostate cancer bone metastasis,12 and men with prostate cancer who undergo the mainstay treatment (androgen blockade) have increased markers of bone turnover, even in the absence of prostate cancer metastasis.13

One pathway by which prostate cancer can induce osteoclast differentiation and activation is through activation of the receptor activator of nuclear factorκB ligand (RANKL).6 In addition, the regulation of the osteoclast podosomes, the bone reabsorptive structures, and osteoclast spreading has been shown to involve the non-receptor kinases c-Src and proline-rich tyrosine kinase-2 (PYK-2).14–18 Activation of RANK induces c-Src-dependent tyrosine phosphorylation and PYK-2 activation, leading to cytoskeletal rearrangement, migration, polarization and fusion.18,19 Further evidence that c-Src kinase is active in bone resorption is demonstrated by the fact that mice carrying a disrupted c-Src develop osteopetrosis.14,20–22 When c-Src is reintroduced into the c-Src-deficient mice, osteoclast function is recovered.21

Dasatinib has been known to induce cytogenetic remission in patients with chronic myeloid leukemia whose clones are resistant to imatinib.23–26 Dasatinib is a Src kinase family inhibitor and an Abl kinase inhibitor. Dasatinib has also been shown to inhibit the Src kinase family’s autophosphorylation of Src and Lyn as well as the downstream phosphorylation of focal adhesion kinase (FAK) and Crk-associated substrate (p-130CAS), both of which are involved in motility and invasion in the prostate cancer cell lines DU-145 and LNCaP.27 In addition, inhibition of Src family kinases has been shown to inhibit both tumor growth and lymph node metastasis of human prostate cancer cells in a nude mouse model.28 In this work, we demonstrate that dasatinib inhibits RANKL-induced osteoclast differentiation of mouse primary bone marrow-derived monocytes. In addition, dasatinib inhibits PC-3 cell-induced osteoclast differentiation. Dasatinib also inhibits osteoclast degradation activity. Taken together, our research suggests that dasatinib could be used as a multitargeted therapy, inhibiting prostate cancer cells and their ability metastasize as well as inhibit the cancers ability to induce osteoclast differentiation.

Results

Dasatinib treatment inhibits osteoclast differentiation of BMM

We first wanted to determine whether dasatinib (Fig. 1A) had the ability to inhibit the formation of TRAP-positive multinucleated osteoclasts differentiated from mouse primary BMMs. By incubating increasing concentrations of dasatinib with the BMMs cultured with MCSF and RANKL in vitro, we found that nanomolar concentrations of dasatinib inhibited osteoclast formation with an IC50 of approximately 7.5 nmol (Fig. 1B and C). Dasatinib demonstrated no cellular toxicity at doses tested (data not shown). Since dasatinib is a Src family kinase inhibitor and could have been exerting its effect on the MCSF receptor (cfms-1), MCSF and RANKL were added to the cells and allowed to activate their receptors, cfms-1 and RANK, respectively. Subsequently, at 0, 24 and 48 h, dasatinib was added, and its suppressive activity continued even after activation of both these receptors (Fig. 2A and B). However, because dasatinib’s suppressive activity was greater when incubation began at the zero time point than when it began at the later two time points, it probably suppressed both receptors.

Figure 1.

Figure 1

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Dasatinib is a potent inhibitor of osteoclastogenesis. (A) Structure of dasatinib (BMS-354825): N-(2-chloro-6-methylphenyl)-2-6-(4[-(2-hydroxyethyl) piperazin-1-yl]-2-methylpyrimidin-4-ylamino)thiazole-5-carboxamide monohydrate. (B) Primary mouse BMM cells were stimulated with MCSF (10 ng/mL) alone or with MCSF + RANKL (100 ng/mL) and with indicated concentrations of dasatinib. A dose dependant decrease in the number of osteoclasts formed was seen with the addition of dasatinib with statistical significance seen at 10 and 20 nM dasatinib (p < 0.05). (C) Photomicrographs of representative fields from BMM treated with MCSF and dasatinib or MCSF + RANKL and dasatinib using a 10x objective lens. Control MCSF alone or MCSF + RANKL are shown at the top. Data shown is representative of three independent experiments.

Figure 2.

Figure 2

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BMM cells respond to dasatinib in a time-dependent manner. (A) BMM cells were pretreated MCSF + RANKL (100 ng/mL) for the indicated times and then treated with 20 nmol of dasatinib (n = 4). Osteoclast formation is significantly reduced in all treatments compared to control with MCSF + RANKL alone. (B) Photomicrographs of representative fields from the different treatment times indicated using a x10 objective lens. For comparison purposes, cells treated with MCSF alone are shown in upper panel. Data shown is representative of three experiments.

Dasatinib treatment inhibits actin ring formation

RANKL has been shown to activate osteoclast differentiation and fusion through actin ring formation. Actin ring formation is correlated with, and may be required for, bone resorption.24,26 Therefore, to confirm that dasatinib was inhibiting this ring formation and inhibited osteoclast formation, BMMs were again incubated with either MCSF, RANKL, or both and then treated with dasatinib at 0, 24 and 48 h. Dasatinib inhibited actin ring formation as indicated by rhodamine-phalloidin staining (Fig. 3). Actin ring formation was inhibited two fold with the addition of 10 nmol of dasatinib to these cultures. Also, actin ring formation was inhibited even 48 h after the addition of RANKL. Using spectroscopy, we observed a 3.5-fold increase in actin ring formation in BMMs when RANKL was added to these cells.

Figure 3.

Figure 3

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Dasatinib inhibits actin ring formation and bone resorption of BMM cells. BMM cells were plated in 48-well plates (2 × 104 cells/well) and pretreated with MCSF (10 ng/mL) or MCSF + RANKL (100 ng/mL) for indicated times and then treated with 10 nmol of dasatinib (n = 4). MCSF was replenished on day 3. Cells were stained for rhodamine-phalloidin on day 5. Shown are photomicrographs of representative fields from the indicated treatments using a x10 objective lens. Data shown is representative of two experiments.

Dasatinib treatment inhibits osteoclast activity

To further demonstrate that dasatinib could affect bone resorption and not just osteoclast fusion, a collagen 1 release assay was performed. When BMM were treated with RANKL there was a 3.5-fold increase of collagen 1 released (Fig. 4). Addition of dasatinib (10 nM) suppressed this release by 58%. Indicating the osteoclasts retained an ability to reabsorp bone at a concentration of 10 nmol albeit lower than MCSF + RANKL.

Figure 4.

Figure 4

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Osteoclast activity was suppressed by dasatinib. BMM cells were treated with MCSF (10 ng/mL) or MCSF + RANKL (100 ng/mL) or MCSF + RANKL + dasatinib (10 nmol). Culture supernatants were analyzed for collagen 1 as described in the materials and methods. Absorbance was measured at 450 nm using the reading at 650 nm as reference and data shown is representative of four experiments. A significant increase in collagen 1 release is seen with addition of RANKL and this increase is significantly reduced by dasatinib.

Dasatinib treatment inhibits prostate cancer induced osteoclast differentiation

It is known that osteoclasts border prostate cancer-derived metastatic bone lesions and that prostate cancer PC-3 cells can induce osteoclast differentiation from both primary BMMs and RAW 264.7 cells, a murine monocytic cell line.8 Therefore, we tested whether PC-3 cell-induced osteoclast formation in RAW 264.7 cells could be inhibited by dasatinib. Increasing concentrations of PC-3 cells were incubated with RAW 264.7 cells to determine the optimal number of PC-3 cells that could induce osteoclast differentiation. The number of multinucleated, TRAP-positive cells induced by PC-3 cells increased slightly from 100–300 PC-3 cells however, at cell numbers greater than 300 the number of osteoclasts declined (data not shown). There were no statistically significant differences except compared to controls containing no PC-3 cells, which had 0 osteoclasts. As a positive control, 100 ng of purified RANKL differentiated RAW 264.7 cells into TRAP-positive multinucleated osteoclasts (data not shown).

To test the ability of dasatinib to inhibit PC-3-induced differentiation of RAW 264.7 cells into osteoclasts, 750 RAW 264.7 cells were incubated with 300 PC-3 cells. Dasatinib (10 or 20 nmol) was then added to the cell culture medium. Dasatinib suppressed the ability of PC-3 cells to induce differentiation of the RAW 264.7 cells into multinucleated osteoclasts (Fig. 5A). Dasatinib at a dose of 10 nmol was able to inhibit osteoclast formation by 50% while 20 nmol inhibited by 67%.

Figure 5.

Figure 5

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Dasatinib inhibits osteoclast differentiation induced by PC-3 cells or PC-3-conditioned media. (A) RAW 264.7 cells were co-cultured with PC-3 cells (300 cells/well). Increasing concentrations of dasatinib was added as indicated. Cells were stained for TRAP on day 5. Dasatinib could suppress osteoclast formation at nmol concentrations as indicated. (B) PC-3-conditioned medium was added to RAW 264.7 at a concentration of 20%. Dasatinib was then added at indicated concentrations at 24 h revealing a decrease in osteoclast formation at nmol concentrations. Data shown is representative of six experiments. Bars, statistically significant decreases were demonstrated (p < 0.05).

Prostate cancer induced differentiation is mediated by soluble factors and inhibited by dasatinib

To determine whether PC-3 cells induced RAW 264.7 cells to differentiate into osteoclasts by direct cell-to-cell contact or by an exogenous factor(s) secreted by PC-3 cells, conditioned medium from PC-3 cells was isolated. Increasing concentrations of this cell-free extract was incubated with RAW 264.7 cells. The maximum number of osteoclasts were formed using 5% conditioned medium (data not shown). In contrast to the number of osteoclasts formed with increasing numbers of PC-3 cells, osteoclast formation decreased as the percentage of conditioned medium increased. Adding dasatinib (10 or 20 nmol) to the cells inhibited the differentiation of RAW 264.7 cells into osteoclasts induced by PC-3 cell-free extracts (Fig. 5B). A concentration of 10 nmol was able to suppress osteoclast formation by 53% while 20 nmol inhibited differentiation by 79%, indicating a dose-dependent effect.

Osteoclasts border human prostate cancer

Mouse studies have demonstrated that osteoclasts border prostate cancer metastases.11 However this has not been shown in human prostate cancer metastases to bone. TRAP staining of human bone marrow samples was performed to identify if osteoclasts were bordering the bone near prostate cancer metastases. TRAP staining of five individual patients with prostate cancer bone metastasis reveal TRAP positive osteoclasts at the tumor-bone interface (Fig. 6A). In the bone marrow of a patient who had much less of a burden of prostate cancer cells TRAP positive osteoclasts were seen near the prostate cancer cells further supporting a paracrine factor from the prostate cancer cells is likely causing osteoclast proliferation or differentiation in this area. As well, Src and Src phosphorylation is present in the bone microenvironment of the metastatic prostate cancer epithelial cells located in the bone marrow as well as the osteoclasts which line the bone surface (Fig. 6B and C).

Figure 6.

Figure 6

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Bone Marrow from a representative prostate cancer patient reveals Src and pSrc within tumor cells and TRAP+ osteoclasts lining bone. TRAP positive osteoclasts line the bone marrow surface and are Src and pSrc positive.

Discussion

Prostate cancer metastasis to the bone is the major cause of mortality in these patients. Prostate cancer-derived cell lines have been shown to induce the differentiation of RAW 264.7 cells into active osteoclasts. Osteoclasts are specialized cells derived from the monocyte/macrophage haematopoietic lineage that develop and adhere to bone matrix, then secrete acid and lytic enzymes that degrade it in a specialized, extracellular compartment. Discovery of the RANK signaling pathway in the osteoclast has provided insight into the mechanisms of osteoclastogenesis and activation of bone resorption. The RANK signaling pathway acts through a series of secondary messengers, which include tumor necrosis factor receptor-associated factor 6, nuclear factor of activated T-cells c1, and c-fos, resulting in differentiation, fusion and activation of the mononuclear cells to form multinucleated osteoclasts. Osteoclast adhesion structures are podosomes, which consist of an F-actin core surrounded by cytoskeletal and signaling proteins and AvB3 integrin. C-Src may be activated by either the RANK pathway or by ligation of the AvB3 integrin.

The Src family of protein tyrosine kinases play an important role in the regulation of many important tumorigenic properties, including proliferation, angiogenesis, invasion and migration. Use of Src kinase inhibitors has been shown to inhibit growth of human prostate cancer cells in a nude mouse model as well as inhibit metastasis to the lymph node.28 In this study, we demonstrate for the first time Dasatinib, a Src family kinase inhibitor, can inhibit primary osteoclast differentiation from bone marrow derived monocytes. In addition, dasatinib can inhibit bone ring formation in vitro, a process related to bone absorption as well as osteoclast activity as measured by collagen I release. We also demonstrate that the human prostate cell line PC-3 can induce osteoclast differentiation of RAW 264.7 cells, a process which can be inhibited by dasatinib. This differentiation of RAW 264.7 is mediated by soluble factors produced by the cancer cells and this too, is inhibited by dasatinib. More importantly, the observation that dasatinib suppressed osteoclast formation in the absence of prostate cancer cells, means that dasatinib must exert some effect on the osteoclast precursors, not just cancer cells. The mechanism by which PC-3 cells induce activation of osteoclasts is unclear.

The observation that c-Src knockout mice develop osteopetrosis led to further studies of the osteoclast. Studies from these mice revealed a decreased osteoclast activation and therefore caused decreased bone resorption.20,22 In addition, studies of the osteoclast have revealed downstream signaling of Src kinase through PYK-2, c-Cbl and FAK, resulting in increased fusion and migration. 29 Activation of osteoclasts by phosphorylation of PYK-2 and FAK could create an environment more conducive to the formation of bone metastases. PYK-2, a protein related to focal adhesion kinase, creates a sealing zone for osteoclast resorption of bone. Further, the proto-oncogene c-Cbl, which is activated by cell surface receptors, may also be involved in the complex with c-Src/PYK-2 and in osteoclast migration.16 Therefore, treatment with a Src kinase inhibitor like dasatinib might prove efficacious against the detrimental effects of prostate cancer bone marrow metastasis.

This study also demonstrates that osteoclasts are present near the prostate cancer metastasis in human bone samples from metastatic patients. Moreover, Src is highly present in the prostate cancer epithelial cells and osteoclasts. If this pathway could be inhibited in patients with metastatic disease, then disease progression in bone might be inhibited. In a mouse model of prostate cancer metastases, osteoclasts were found at the bone-prostate cancer interface where new metastatic lesions were formed.11 Perhaps through Src inhibition of osteoclast formation, prostate cancer-induced metastatic bone lesions could be inhibited. We are currently performing studies in a preclinical mouse model to determine if dasatinib is able to suppress PC-3 cell growth in mice injected intra-tibial with PC-3 cells. This would further identify dasatinib as a possible pharmaceutical agent for prostate cancer. Dasatinib, in combination with other agents, is currently being studied in the clinic for metastatic prostate cancer. As of yet, no information on the efficacy on pre-existing metastases is known, but the results of this study on osteoclast differentiation as well as others with prostate cancer itself, are encouraging.

Materials and Methods

Isolation of primary bone marrow monocytes

Black six mice (C57BL/6J), 8–12 w of age were cared for and euthanized following M.D. Anderson Cancer Center guidelines for animal care. Primary bone marrow monocytes (BMMs) were isolated from aseptically dissected tibiae and femora. Briefly, the bone marrow was collected into incomplete α-modified Eagle’s medium (α-MEM) and mixed vigorously by pipetting to create a cell suspension. Cells were collected by low speed centrifugation and re-suspended in 5 mL incomplete α-MEM. Bone marrow cells were incubated in 20 mL of red blood cell lysis buffer (8.3 g/L NH4Cl, 1 g/L sodium bicarbonate, 0.4 g/L EDTA) at room temperature for 1–2 min. α-MEM supplemented with 10% fetal bovine serum and 100 units/mL penicillin (complete α-MEM) was added, and cells were collected by low speed centrifugation. The cells were plated at 1.5–2 × 107 cells/10-cm dish with 10 mL of complete α-MEM and then cultured for 24 h. Non-adherent cells were collected by low speed centrifugation and plated at a concentration of 2.5 × 104 cells/well in 48-well plates or 5 × 103 cells/well in 96-well plates for osteoclast differentiation assays. Cells were cultured for 3 d in the presence of 10 ng/mL macrophage colony-stimulating factor (MCSF) before they were washed and used for further experiments.

Osteoclast differentiation of BMM

To stimulate/induce osteoclast differentiation, 30–100 ng/mL RANKL was added to the medium. Medium supplemented with MCSF and RANKL was changed every 3 d. Dasatinib was added with RANKL at time point zero or at specified times after RANKL administration. Cells were incubated in 5% CO2 at 37°C for 5 d, fixed and stained for tartrate-resistant alkaline phosphatase (TRAP) (Sigma-Aldrich Corp., St. Louis, MO). Eight wells were evaluated for each condition and four individual experiments performed. Cells that were TRAP positive and had ≥3 nuclei/cell were considered osteoclasts. To show the actin ring forming zone, treated cells were stained using rhodamine-phalloidin according to manufacturers instructions (Invitrogen Life Scinces, Carlsbad, CA).

Osteoclast differentiation of RAW 264.7 cells

RAW 264.7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium supplemented with 10% fetal calf serum (FCS) (Life Technologies, Inc., Gaithersburg, MD). PC-3 cells were maintained in RPMI 1640 supplemented with 10% FCS. Both RAW 264.7 cells and PC-3 cells were maintained at subconfluence and passaged every 3 d. For co-culture experiments, RAW 264.7 cells (500 cells/well) were plated in 96-well plates and allowed to adhere for 12 h. Increasing numbers of PC-3 cells were then resuspended in DMEM/F12 supplemented with 10% FCS and introduced into the RAW 264.7 cultures. Increasing concentrations of dasatinib in DMEM/F12 were added to the co-cultures 12 h after the plating of PC-3 cells. Cells were incubated in 5% CO2 at 37°C for 5 d, fixed and stained for tartrate-resistant alkaline phosphatase (TRAP) (Sigma-Aldrich Corp.). Eight wells were evaluated for each condition and 4 individual experiments performed. Cells that were TRAP positive and had ≥3 nuclei/cell were considered osteoclasts. Osteoclasts were counted by direct visualization at 400x magnification. For conditioned medium experiments, culture supernatant was obtained from PC-3 cells that had been plated at 1 × 106 cells in a 10-cm plate and grown for 36 h. The medium was removed and cellular material removed by low speed centrifugation. RAW 264.7 cells (750 cells/well) in DMEM/F12 medium supplemented with 10% FCS were plated in 96-well plates and allowed to adhere for 12 h. Increasing concentrations of conditioned medium was added into the RAW 264.7 cultures. Increasing concentrations of dasatinib in DMEM/F12 were added to the cells 12 h after the addition of the conditioned medium. Cells were incubated in 5% CO2 at 37°C for 5 d. Osteoclasts were counted for each condition by direct visualization at x400 magnification as described above.

Collagen I release assay

BMM cells (5 × 103 cells/well) were seeded on the OsteoAssay plate from Cambrex (Rockland, ME) and initially treated with 100 ng/mL RANKL and 10 ng/mL MCSF with or without 300 nM dasatinib. MCSF was replenished on day 3. A 20 μL aliquot of the supernatant was evaluated by measuring collagen I release using the CrossLaps for Culture ELISA kit (Nordic Bioscience Diagnostics, Portsmouth, VA). Absorbance was measured at 450 nm using the reading at 650 nm as reference.

Immunohistochemistry of bone marrow biopsy samples

Paraffin-embedded tumor tissues were sectioned 8–10 μm thick and mounted on positively charged gold plus microscope slides. Tissue slides were pre-heated at 60°C for 16 h and dewaxed by immersion in xylene followed by successively diluted solutions of ethanol. Antigen retrieval was accomplished by heating the slides at 70°C for 4 h, immersed in Borg decloaker solution (Biocare Medical Inc., Concord, CA) for Src, autophosphorylated SFKs and total Src staining. Endogenous peroxidase activity was blocked by incubating in 3% H2O2 in PBS for 12 min. After rinsing with PBS three times for three minutes each, non-specific tissue binding was blocked for one hour in protein block solution (Cyto Q immuno-diluent buffer; Innovex, Richmond, CA). Primary antibody was diluted in protein block solution and incubated overnight at 4°C. Dilution of primary antibodies are as follows: anti-Src antibody (1:100, Cell Signaling Technology Inc., Beverly, MA), and anti-phospho-[Y416]-Src antibody 1:100, Cell Signaling Technology Inc., Beverly, MA. Slides were washed with PBS three times for three minutes each followed by Mach 4 Universal HRP polymer (Biocare Medical Inc., Concord, CA) application for 20 min as a secondary antibody. The stain was visualized by incubation in 3,3′-diaminobenzidine (DAB) and counterstained with Gill’s No. 3 Hematoxylin. Internal negative control samples were exposed to protein block solution instead of the primary antibodies and demonstrated no specific signaling. Slides were dried and mounted with Universal Mount solution (Research Genetics, Invitrogen Co., Carlsbad, CA).

TRAP staining of bone marrow samples

Deparaffinized, 5 um sections were preincubated in warmed sodium acetate buffer with L-(+) tartaric acid (50 mM), pH 5.0 for 30 min at 37°C. Naphthol AS-BI substrate solution was added and slides incubated at 37°C for 30 min. Slides were added to acetate buffer containing, sodium nitrite solution and pararosaniline dye (Sigma-Aldrich Corp.), incubated at room temp 2–8 min, rinsed in distilled water and counter stained with Carrazi’s hematoxylin for 1 min. Slides were subsequently rinsed with tap water, coverslips placed with aquatex and dried overnight.

Statistics

Statistical analysis of the data was performed using GraphPad Prism Software, (La Jolla, CA). Basic t tests were used to compare the test data to the control data. An asterisk is used to identify values significantly different (p value of 0.05) from control.

Acknowledgments

This work supported in part by The University of Texas SPORE in Prostate Cancer P50-CA90270 and T32 CA009666 Training Grant.

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