Background

Prostate cancer is the second leading cause of cancer death among men in the United States, and there is clinical evidence of bone metastases in approximately 80 % of those who have died [1, 2]. A comprehensive understanding of signaling interactions between invading epithelial-derived prostate cancer cells and the host bone stromal environment that promote bone metastasis is crucial to the development of effective therapeutic strategies.

Although markers of bone production and resorption may be present in patients, prostate carcinoma bone metastases are generally characterized by new bone formation initiated by the differentiation of mesenchymal progenitor cells into osteoblasts [3–5]. We have previously demonstrated that human prostate cancer cells, which express high levels of Sonic hedgehog (Shh), activate the signaling pathway in MC3T3 pre-osteoblasts and induce osteoblast differentiation [6].

Shh is a secreted glycopeptide that plays critical functions in the normal development of many organs including the prostate; and, deregulation of the Shh pathway has been linked to human cancer [7–9]. Expression of Shh and other members of the signaling pathway have been reported in human primary prostate carcinomas and metastases, including bone [10–39, 40]. Further studies to determine the role of Shh-expressing prostate cancer cells in regulating the structural properties of bone matrix collagen, particularly under in vivo conditions, will increase our understanding of the significance of the Shh pathway in sha** the bone stromal microenvironment to support metastasis.

The role of matrix collagen on cell functions has been investigated mostly through use of scaffolds formed by either commercially-available, laboratory-prepared native type 1 collagen extracted from rat tail tendons, or collagen formed by osteoblasts. In these studies, cells are either added onto the gellified matrix or mixed with the matrix as it solidifies. Thus, matrix density and collagen fibril structural characteristics are largely pre-determined for the inoculated cells. Our mixed cell culture system enables both cancer cells and osteoblasts to interact during the process of AA-dependent collagen matrix formation. This allows investigations into reciprocal in situ interactions among cancer cells, osteoblasts, and osteoblast-synthesized matrix proteins that should provide significant insights into signaling processes relevant to bone metastasis.

Conclusions

AA-induced formation of collagen matrix potentiates paracrine Shh signaling between prostate cancer cells and osteoblasts, and synergistically enhances Shh-induced osteoblast differentiation, an early and defining event in prostate carcinoma bone metastasis. Co-targeting of the Shh pathway and processes that regulate collagen matrix formation in bone presents a viable therapeutic approach against bone metastasis.

Methods

Cells and plasmid transfections

Parental LNCaP human prostate cancer cells and mouse calvaria-derived non-transformed pre-osteoblast cells MC3T3-E1 (subclone 4; designated as MC3T3 cells) were commercially obtained (ATCC, Rockville, MD).

LNCaP cells have been previously stably transfected with a 1.44 kb human Shh cDNA cloned into a pIRES2-EGFP mammalian cell expression vector (designated LNShh cells) or with pIRES2-EGFP vector alone as controls (designated LNCaP cells) [10]. We have previously confirmed the increased expression of Shh at the gene and protein levels in LNShh cells compared to LNCaP cells [10]. The morphology of LNCaP and LNShh cells appears identical and these cells exhibit similar growth properties in culture [10, 41]. LNCaP and LNShh cells were maintained at 37 C, 5 % CO2 in complete culture medium consisting of RPMI-1640 supplemented with 10 % fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco Invitrogen). Shh gene and protein expression were routinely determined by quantitative real time RT-PCR and western blot analysis, respectively, and GFP expression was monitored by fluorescence microscopy.

MC3T3 cells have been previously stably transfected with pCMV-GLI(−)TAD: a human GLI1 cDNA lacking a t ransa ctivation d omain and cloned into pcDNA3 plasmid (designated M-TAD cells) [6]. We have previously shown that both M-TAD cells and parental MC3T3 cells (used as controls) express endogenous mouse Gli1 message; but, only the M-TAD cells express the message for the human GLI1(−TAD) transgene whose GLI1 translated product is expected to bind to the consensus DNA GLI binding site but not activate the pathway; thus, acting as a dominant negative transcription factor [6]. MC3T3 and M-TAD cells were maintained at 37 C, 5 % CO2 in non-differentiation complete culture medium consisting of ascorbic acid (AA)-free α-MEM supplemented with 10 % FBS, 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco Invitrogen).

Mixed culture of cells

LNCaP or LNShh cells (5 × 104) and MC3T3 or M-TAD cells (0.5 × 104) were mixed in AA-free α-MEM complete culture medium and seeded per well of 6-well tissue culture plates. When grown in chamber slides, pre-osteoblasts and prostate cancer cells were mixed at equal concentrations of 1 × 104 cells per cell line. Cultures were maintained for the length of time specified in the experiments with media changes every 2–3 days.

Effect of ascorbic acid

L-ascorbic acid (AA; Aldrich) was dissolved in AA-free α-MEM complete culture medium at 50 μg/ml final concentration. Mixed cultures were maintained in complete culture medium with AA or in complete culture medium only as controls. The AA concentration used in these experiments is below pharmacologic concentrations that may be cytotoxic to prostate cancer cells including LNCaP [42, 43]. To determine the direct effect of AA, MC3T3 cells were seeded alone onto 6-well culture plates at 1 × 105 cells per well and treated with AA as above.

Effect of Shh peptide

Shh-N, a modified active N-terminal peptide of human Shh (kindly provided by Curis Inc., Cambridge, MA), was prepared in serum-free AA-free α-MEM culture medium at 1 μg/ml final concentration. MC3T3 cells were seeded onto 6-well tissue culture plates at 1 × 105 cells per well in AA-free α-MEM complete culture medium. Following overnight incubation, cells were maintained for 24 h in serum-free culture medium with Shh-N or in serum-free culture medium only as controls.

Effect of collagen synthesis inhibitor

The collagen synthesis inhibitor 3,4-dehydro-L-proline (DHP; Sigma) was dissolved in AA-free α-MEM complete culture medium. Mixed cultures were maintained in complete culture medium with 50 μg/ml AA and varying concentrations of DHP or in complete culture medium only (i.e., without both AA and DHP) as controls. In some experiments, single cultures of MC3T3 cells were used.

RNA isolation and real time quantitative RT-PCR

Total RNA was extracted using Trizol (Invitrogen), purified using the RNeasy Mini Kit (Qiagen) and subjected to DNase treatment with RQ1 RNase-free DNase (Promega) to remove contaminating genomic DNA. The TaqMan® Gold PCR Core Reagent Kit along with MuLV Reverse Transcriptase and RNase Inhibitor (Applied Biosystems) were used for cDNA synthesis. PCR primers (Invitrogen) and FAM-QSY7 probes (MegaBases, Inc.) for genes of interest and the housekee** gene glyceraldehyde-3-phosphate dehydrogenase were designed using the Primer Express 3.0 software program. Mouse species-specific primer sequences, which amplified genes of interest in mouse MC3T3 but not in human prostate cancer cells, have been published [6]. mRNA expression was measured in duplicate or triplicate per sample using 40 cycles of amplification in the 7500 Fast Real-Time PCR System (Applied Biosystems). Reactions were routinely performed without Reverse Transcriptase to demonstrate RNA dependence of the reaction products. Results were analyzed using the comparative Ct method as described previously [6]. Data are expressed as relative fold change in gene expression.

Cell proliferation assay

MC3T3 cells were seeded onto 24-well tissue culture plates at 0.2 × 104 cells per well and maintained in AA-free α-MEM complete culture medium. Proliferation was determined using the Cell Counting Kit-8 (Do**do Laboratories, Japan) which is based on the formation of a water-soluble formazan dye through the activity of dehydrogenases in living cells. Absorbance measurements at 450 nm are in direct proportion to the number of living cells.

Immunocytochemistry

Cells were maintained as mixed cultures in Lab-TekII CC2-treated chamber slides (Nunc) for the length of time specified in the experiments with media changes every 2–3 days. Cells were fixed in 10 % neutral buffered formalin for 10 minutes and processed for von Gieson staining for collagen. For positive control, a section of aorta was similarly processed and stained. Slides were viewed in a Leica DMR-HC Upright Microscope and images were captured with imaging software (Improvision Openlab).

Alkaline phosphatase activity

Quantitative determination of ALP activity was done using the p-Nitrophenyl Phosphate (pNPP) Liquid Substrate System (Sigma Aldrich) as previously described [6]. Absorbance at 405 nm was measured using a microplate reader, and ALP activity was calculated according to manufacturer’s instructions. Protein determination was done using the Bio-Rad DC Protein Microplate Assay according to manufacturer’s protocol.

Staining for ALP activity was performed on mixed cultures which were fixed with 10 % neutral buffered formalin for 10 minutes and incubated with alkaline phosphatase substrate solution (Sigma-Aldrich) for at least 30 minutes at room temperature in the dark as previously described [6].

Transmission electron microscopy

Mixed cultures of MC3T3 pre-osteoblasts and LNCaP or LNShh cells were maintained in the presence of AA (50 μg/ml) on Thermanox cover slips (13 mm diameter; Electron Microscopy Services) kept in 24-well tissue culture plates for 7, 14, and 21 days. At end of culture, samples were fixed overnight at 4 C in 0.1 M sodium cacodylate buffer pH7.3 containing 2 % paraformaldehyde and 2.5 % glutaraldhyde, post-fixed with 2 % osmium tetroxide in 0.1 M sodium cacodylate buffer, and rinsed with distilled water. Following, samples were stained en bloc with 3 % uranyl acetate, rinsed in distilled water, dehydrated in ascending grades of ethanol, embedded in resin mixture of Embed 812 and Araldite, and cured in oven at 60 C. Samples were sectioned on a Leica Ultracut UC6 ultramicrotome, and 70 nm thin sections were collected on 200 mesh copper grids and post-stained with 3 % uranyl acetate and Reynolds lead citrate. Samples were sectioned either parallel to cell layers to reveal fibril orientation or perpendicular to cell layers to allow morphometric measurement of fibril diameters [23, 39]. Samples were examined on FEI Tecnai Spirit G2 TEM, and digital images were captured on an FEI Eagle camera at magnifications ranging from 1900x to 49000x. Two to three grids per sample were examined and images from several sections per grid were taken.

Analysis of collagen fibril diameter

Diameters of collagen fibrils were measured, using the analysis measurement tool of the Adobe Photoshop CS3 Extended software, on TEM images of samples sectioned perpendicular to cell layers and examined at magnification of 49000×. Fibril diameters were measured from 500 randomly selected collagen fibrils from 8–14 TEM images from different grid sections from each of 2 samples per group.

Data analysis

Data were analyzed by ANOVA and pair-wise multiple comparisons were done using the Bonferroni t-test at P < 0.05. Comparison between two groups was done by Student’s t-test. Fibril diameter size distributions were compared using the asymptotic Kolmogorov-Smirnov two-sample test [21].