Osteolytic cancer cells induce vascular/axon guidance processes in the bone/bone marrow stroma.

Prostate and breast cancers frequently metastasize to bone. The physiological bone homeostasis is perturbed once cancer cells proliferate at the bone metastatic site. Tumors are complex structures consisting of cancer cells and numerous stroma cells. In this study, we show that osteolytic cancer cells (PC-3 and MDA-MB231) induce transcriptome changes in the bone/bone marrow microenvironment (stroma). This stroma transcriptome differs from the previously reported stroma transcriptome of osteoinductive cancer cells (VCaP). While the biological process "angiogenesis/vasculogenesis" is enriched in both transcriptomes, the "vascular/axon guidance" process is a unique process that characterizes the osteolytic stroma. In osteolytic bone metastasis, angiogenesis is denoted by vessel morphology and marker expression specific for arteries/arterioles. Interestingly, intra-tumoral neurite-like structures were in proximity to arteries. Additionally, we found that increased numbers of mesenchymal stem cells and vascular smooth muscle cells, expressing osteolytic cytokines and inhibitors of bone formation, contribute to the osteolytic bone phenotype. Osteoinductive and osteolytic cancer cells induce different types of vessels, representing functionally different hematopoietic stem cell niches. This finding suggests different growth requirements of osteolytic and osteoinductive cancer cells and the need for a differential anti-angiogenic strategy to inhibit tumor growth in osteolytic and osteoblastic bone metastasis.

Glutamate Receptor Agonists and Glutamate Transporter Antagonists Regulate Differentiation of Osteoblast Lineage Cells.

Development and function of osteoblast lineage cells are regulated by a complex microenvironment consisting of the bone extracellular matrix, cells, systemic hormones and cytokines, autocrine and paracrine factors, and mechanical load. Apart from receptors that transduce extracellular signals into the cell, molecular transporters play a crucial role in the cellular response to the microenvironment. Transporter molecules are responsible for cellular uptake of nutritional components, elimination of metabolites, ion transport, and cell-cell communication. In this report, the expression of molecular transporters in osteoblast lineage cells was investigated to assess their roles in cell development and activity. Low-density arrays, covering membrane and vesicular transport molecules, were used to assess gene expression in osteoblasts representing early and late differentiation states. Receptors and transporters for the amino acid glutamate were found to be differentially expressed during osteoblast development. Glutamate is a neurotransmitter in the central nervous system, and the mechanisms of its release, signal transduction, and cellular reabsorption in the synaptic cleft are well understood. Less clear, however, is the control of equivalent processes in peripheral tissues. In primary osteoblasts, inhibition of glutamate transporters with nonselective inhibitors leads to an increase in the concentration of extracellular glutamate. This change was accompanied by a decrease in osteoblast proliferation, stimulation of alkaline phosphatase, and the expression of transcripts encoding osteocalcin. Enzymatic removal of extracellular glutamate abolished these pro-differentiation effects, as did the inhibition of PKC- and Erk1/2-signaling pathways. These findings demonstrate that glutamate signaling promotes differentiation and activation of osteoblast lineage cells. Consequently, the glutamate system may represent a putative therapeutic target to induce an anabolic response in the skeletal system. Known antagonists of glutamate transporters will serve as lead compounds in developing new and specific bioactive molecules.

The molecular signature of the stroma response in prostate cancer-induced osteoblastic bone metastasis highlights expansion of hematopoietic and prostate epithelial stem cell niches.

The reciprocal interaction between cancer cells and the tissue-specific stroma is critical for primary and metastatic tumor growth progression. Prostate cancer cells colonize preferentially bone (osteotropism), where they alter the physiological balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, and elicit prevalently an osteoblastic response (osteoinduction). The molecular cues provided by osteoblasts for the survival and growth of bone metastatic prostate cancer cells are largely unknown. We exploited the sufficient divergence between human and mouse RNA sequences together with redefinition of highly species-specific gene arrays by computer-aided and experimental exclusion of cross-hybridizing oligonucleotide probes. This strategy allowed the dissection of the stroma (mouse) from the cancer cell (human) transcriptome in bone metastasis xenograft models of human osteoinductive prostate cancer cells (VCaP and C4-2B). As a result, we generated the osteoblastic bone metastasis-associated stroma transcriptome (OB-BMST). Subtraction of genes shared by inflammation, wound healing and desmoplastic responses, and by the tissue type-independent stroma responses to a variety of non-osteotropic and osteotropic primary cancers generated a curated gene signature ("Core" OB-BMST) putatively representing the bone marrow/bone-specific stroma response to prostate cancer-induced, osteoblastic bone metastasis. The expression pattern of three representative Core OB-BMST genes (PTN, EPHA3 and FSCN1) seems to confirm the bone specificity of this response. A robust induction of genes involved in osteogenesis and angiogenesis dominates both the OB-BMST and Core OB-BMST. This translates in an amplification of hematopoietic and, remarkably, prostate epithelial stem cell niche components that may function as a self-reinforcing bone metastatic niche providing a growth support specific for osteoinductive prostate cancer cells. The induction of this combinatorial stem cell niche is a novel mechanism that may also explain cancer cell osteotropism and local interference with hematopoiesis (myelophthisis). Accordingly, these stem cell niche components may represent innovative therapeutic targets and/or serum biomarkers in osteoblastic bone metastasis.

Circulating fibronectin controls tumor growth.

Fibronectin is ubiquitously expressed in the extracellular matrix, and experimental evidence has shown that it modulates blood vessel formation. The relative contribution of local and circulating fibronectin to blood vessel formation in vivo remains unknown despite evidence for unexpected roles of circulating fibronectin in various diseases. Using transgenic mouse models, we established that circulating fibronectin facilitates the growth of bone metastases by enhancing blood vessel formation and maturation. This effect is more relevant than that of fibronectin produced by endothelial cells and pericytes, which only exert a small additive effect on vessel maturation. Circulating fibronectin enhances its local production in tumors through a positive feedback loop and increases the amount of vascular endothelial growth factor (VEGF) retained in the matrix. Both fibronectin and VEGF then cooperate to stimulate blood vessel formation. Fibronectin content in the tumor correlates with the number of blood vessels and tumor growth in the mouse models. Consistent with these results, examination of three separate arrays from patients with breast and prostate cancers revealed that a high staining intensity for fibronectin in tumors is associated with increased mortality. These results establish that circulating fibronectin modulates blood vessel formation and tumor growth by modifying the amount of and the response to VEGF. Furthermore, determination of the fibronectin content can serve as a prognostic biomarker for breast and prostate cancers and possibly other cancers.

Expression of antagonists of WNT and BMP signaling after non-rigid fixation of osteotomies.

Delayed fracture healing and non-unions represent rare but severe complications in orthopedic surgery. Further knowledge on the mechanisms of the bone repair process and of the development of a pseudoarthrosis is essential to predict and prevent impaired healing of fractures. The present study aimed at elucidating differences in gene expression during the repair of rigidly and non-rigidly fixed osteotomies. For this purpose, the MouseFix™ and the FlexiPlate™ systems (AO Development Institute, Davos, CH), allowing the creation of well defined osteotomies in mouse femora, were employed. A time course following the healing process of the osteotomy was performed and bones and periimplant tissues were analyzed by high-resolution X-ray, MicroCT and by histology. For the assessment of gene expression, Low Density Arrays (LDA) were done. In animals with rigid fixation, X-ray and MicroCT revealed healing of the osteotomy within 3 weeks. Using the FlexiPlate™ system, the osteotomy was still visible by X-ray after 3 weeks and a stabilizing cartilaginous callus was formed. After 4.5 weeks, the callus was remodeled and the osteotomy was, on a histological level, healed. Gene expression studies revealed levels of transcripts encoding proteins associated with inflammatory processes not to be altered in tissues from bones with rigid and non-rigid fixation, respectively. Levels of transcripts encoding proteins of the extracellular matrix and essential for bone cell functions were not increased in the rigidly fixed group when compared to controls without osteotomy. In the FlexiPlate™ group, levels of transcripts encoding the same set of genes were significantly increased 3 weeks after surgery. Expression of transcripts encoding BMPs and BMP antagonists was increased after 3 weeks in repair tissues from bones fixed with FlexiPlate™, as were inhibitors of the WNT signaling pathways. Little changes only were detected in transcript levels of tissues from rigidly fixed bones. The data of the present study suggest that rigid fixation enables accelerated healing of an experimental osteotomy as compared to non-rigid fixation. The changes in the healing process after non-rigid fixation are accompanied by an increase in the levels of transcripts encoding inhibitors of osteogenic pathways and, probably as a consequence, by temporal changes in bone matrix synthesis.

Occlusion of seminal vesicles increases sexual activity in a mouse model.

BACKGROUND: Little is known about the physiologic role of seminal vesicles beyond their fertility function. It has been suggested repeatedly that seminal vesicles have an impact on sexual activity. Although this has been investigated in various animal models, such a role has never been found. OBJECTIVE: To assess in a novel mouse model whether occlusion of seminal vesicles affects sexual activity. DESIGN, SETTING, AND PARTICIPANTS: Adult male CD1 mice (n=77) were assigned randomly to the experimental groups: (1) seminal vesicle occlusion (SVO) (n=24), (2) seminal vesicle resection (SVR) (n=23), and (3) sham operation (SO) (n=30). Adult females were brought into estrus by the Whitten effect. After recuperation, mouse pairs were observed during sessions of 3h each. Sexual activity was analyzed separately by three observers blinded to the experimental conditions. INTERVENTION: SVO, SVR, and SO. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary end point was percentage of sessions with intromission; secondary end points were number of intromissions and latency until first intromission. A logistic regression model and the Kruskal-Wallis test were used. RESULTS AND LIMITATIONS: A total of 141 sessions for a total of 423h were analyzed. Intromission was scored in 20 of 42 sessions (48%) with SVO mice, a significantly higher rate than the 8 of 39 sessions (21%) with SVR mice (p=0.001) and 18 of 60 sessions (30%) with SO mice (p=0.004). Secondary end points were comparable in all three groups (p=0.303 and 0.450, respectively). CONCLUSIONS: Males with SVO were significantly more often sexually active than males undergoing SVR or SO. This suggests that occluded, and thus engorged, seminal vesicles increase sex drive in male mice. Since the potential clinical benefit might be highly relevant, further studies should confirm these promising results and investigate the potential application in men.

Mechanisms of nanoparticle-mediated photomechanical cell damage.

Laser-assisted killing of gold nanoparticle targeted macrophages was investigated. Using pressure transient detection, flash photography and transmission electron microscopy (TEM) imaging, we studied the mechanism of single cell damage by vapor bubble formation around gold nanospheres induced by nanosecond laser pulses. The influence of the number of irradiating laser pulses and of particle size and concentration on the threshold for acute cell damage was determined. While the single pulse damage threshold is independent of the particle size, the threshold decreases with increasing particle size when using trains of pulses. The dependence of the cell damage threshold on the nanoparticle concentration during incubation reveals that particle accumulation and distribution inside the cell plays a key role in tissue imaging or cell damaging.

Stem-like cells with luminal progenitor phenotype survive castration in human prostate cancer.

Castration is the standard therapy for advanced prostate cancer (PC). Although this treatment is initially effective, tumors invariably relapse as incurable, castration-resistant PC (CRPC). Adaptation of androgen-dependent PC cells to an androgen-depleted environment or selection of pre-existing, CRPC cells have been proposed as mechanisms of CRPC development. Stem cell (SC)-like PC cells have been implicated not only as tumor initiating/maintaining in PC but also as tumor-reinitiating cells in CRPC. Recently, castration-resistant cells expressing the NK3 homeobox 1 (Nkx3-1) (CARNs), the other luminal markers cytokeratin 18 (CK18) and androgen receptor (AR), and possessing SC properties, have been found in castrated mouse prostate and proposed as the cell-of-origin of CRPC. However, the human counterpart of CARNs has not been identified yet. Here, we demonstrate that in the human PC xenograft BM18, pre-existing SC-like and neuroendocrine (NE) PC cells are selected by castration and survive as totally quiescent. SC-like BM18 cells, displaying the SC markers aldehyde dehydrogenase 1A1 or NANOG, coexpress the luminal markers NKX3-1, CK18, and a low level of AR (AR(low)) but not basal or NE markers. These CR luminal SC-like cells, but not NE cells, reinitiate BM18 tumor growth after androgen replacement. The AR(low) seems to mediate directly both castration survival and tumor reinitiation. This study identifies for the first time in human PC SC-/CARN-like cells that may represent the cell-of-origin of tumor reinitiation as CRPC. This finding will be fundamental for refining the hierarchy among human PC cancer cells and may have important clinical implications.

TNFα inhibits the development of osteoclasts through osteoblast-derived GM-CSF.

Inflammatory cytokines such as tumor necrosis factor-alpha (TNFα) are potent stimulators of osteoclast formation and bone resorption and are frequently associated with pathologic bone metabolism. The cytokine exerts specific effects on its target cells and constitutes a part of the cellular microenvironment. Previously, TNFα was demonstrated to inhibit the development of osteoclasts in vitro via an osteoblast-mediated pathway. In the present study, the molecular mechanisms of the inhibition of osteoclastogenesis were investigated in co-cultures of osteoblasts and bone marrow cells (BMC) and in cultures of macrophage-colony stimulating factor (M-CSF) dependent, non-adherent osteoclast progenitor cells (OPC) grown with M-CSF and receptor activator of NF-κB ligand (RANKL). Granulocyte-macrophage colony stimulating factor (GM-CSF), a known inhibitor of osteoclastogenesis was found to be induced in osteoblasts treated with TNFα and the secreted protein accumulated in the supernatant. Dexamethasone (Dex), an anti-inflammatory steroid, caused a decrease in GM-CSF expression, leading to partial recovery of osteoclast formation. Flow cytometry analysis revealed that in cultures of OPC, supplemented with 10% conditioned medium (CM) from osteoblasts treated with TNFα/1,25(OH)(2)D(3), expression of RANK and CD11c was suppressed. The decrease in RANK expression may be explained by the finding, that GM-CSF and the CM from wt osteoblasts were found to suppress the expression of c-Fos, Fra-1, and Nfatc-1. The failure of OPC to develop into CD11c(+) dendritic cells suggests that cell development is not deviated to an alternative differentiation pathway, but rather, that the monocytes are maintained in an undifferentiated, F4/80(+), state. The data further implies possible interactions among inflammatory cytokines. GM-CSF induced by TNFα acts on early hematopoietic precursors, inhibiting osteoclastogenesis while acting as the growth factor for M-CSF independent inflammatory macrophages. These in turn may condition a microenvironment enhancing osteoclast differentiation and bone resorption upon migration of the OPC from circulation to the bone/bone marrow compartment.

The role of the BMP signaling antagonist noggin in the development of prostate cancer osteolytic bone metastasis.

Members of the BMP and Wnt protein families play a relevant role in physiologic and pathologic bone turnover. Extracellular antagonists are crucial for the modulation of their activity. Lack of expression of the BMP antagonist noggin by osteoinductive, carcinoma-derived cell lines is a determinant of the osteoblast response induced by their bone metastases. In contrast, osteolytic, carcinoma-derived cell lines express noggin constitutively. We hypothesized that cancer cell-derived noggin may contribute to the pathogenesis of osteolytic bone metastasis of solid cancers by repressing bone formation. Intra-osseous xenografts of PC-3 prostate cancer cells induced osteolytic lesions characterized not only by enhanced osteoclast-mediated bone resorption, but also by decreased osteoblast-mediated bone formation. Therefore, in this model, uncoupling of the bone remodeling process contributes to osteolysis. Bone formation was preserved in the osteolytic lesions induced by noggin-silenced PC-3 cells, suggesting that cancer cell-derived noggin interferes with physiologic bone coupling. Furthermore, intra-osseous tumor growth of noggin-silenced PC-3 cells was limited, most probably as a result of the persisting osteoblast activity. This investigation provides new evidence for a model of osteolytic bone metastasis where constitutive secretion of noggin by cancer cells mediates inhibition of bone formation, thereby preventing repair of osteolytic lesions generated by an excess of osteoclast-mediated bone resorption. Therefore, noggin suppression may be a novel strategy for the treatment of osteolytic bone metastases.

Evaluation of the frequency of putative prostate cancer stem cells in primary and metastatic prostate cancer.

BACKGROUND: Tumour cells with a stem cell-like phenotype have recently been identified in prostate tumors and it has been suggested that this population may be responsible for the diversity of cell types within tumors and also for the initiation of metastases. These cells carry a number of defined markers: they are cd133 and cd44+ve and express high levels of alpha2beta1 integrin. In this study we have, for the first time, assessed matched primary and bone marrow biopsies from prostate cancer patients for the distribution of cells carrying these and a number of other putative stem cell markers. METHODS: Eleven matched (primary and bone metastasis) specimens from prostate cancer patients were assessed for the presence of cd133, cd44, alpha2beta1 integrin, CXCR4, c-met, alpha6 integrin, and nestin using immunohistochemistry and stain intensity and distribution scored. RESULTS: In the bone metastases, tumor cells staining positively for cd133 were detected at low frequency in approximately 50% of samples. Staining for nestin was confined to endothelium. Positive staining of tumor cells for the other antigens was present at variable frequency in >70% of metastases with the exception of CXCR4 which was absent from all but 2 specimens. Where positive staining of tumor cells was present in the metastasis, cells staining for each antigen were present in the matched primary with the exception of cd44 which was absent in all but 2/11 matched primary tissues. CONCLUSIONS: In established metastases no single or combination of marker expression profiles identify the established metastatic phenotype, although cd44 expression was shown to be more frequent in metastases that in primary cancers.

In vitro propagation and characterization of neoplastic stem/progenitor-like cells from human prostate cancer tissue.

BACKGROUND: According to the cancer stem cell hypothesis, tumor growth is sustained by a subpopulation of cancer stem/progenitor-like cells. Self-renewal and high clonogenic potential are characteristics shared by normal stem and neoplastic stem/progenitor-like cells. We investigated whether human prostate cancer specimens contain cells with these properties. METHODS: Self-renewal and clonogenic potential were assessed by serial passaging of spheres and colony formation, respectively. Gene expression was analyzed by real time PCR. Protein expression was detected by immunocytochemistry. The neoplastic nature of the cells was verified by detection of the TMPRSS2/ERG gene fusion expression. RESULTS: The epithelial fraction isolated from surgical specimens generated colonies in 68% (19/28) of the patients. Laminin adhesion selected for cells with high clonogenic potential. The epithelial fraction from 85% (42/49) of the patients generated primary prostaspheres. Serial passaging of prostaspheres demonstrated their self-renewal capacity, which is also supported by their expression of the stem cell markers Oct-4, Nanog, Bmi-1, and Jagged-1 mRNA. Cells derived from prostaspheres were more clonogenic than the parental epithelial fraction. The pattern of mRNA expression in prostaspheres resembled that of the basal compartment of the prostate (CK5(+)/CK14(+)/CK19(high)/CK18(-/low)/c-met(+)/AR(-/low)/PSA(-/low)), but also included stem cell markers (CD49b(+)/CD49f(+)/CD44(+)/DeltaNp63(+)/Nestin(+)/CD133(+)). The distribution of marker expression in prostaspheres suggests their heterogeneous cell composition. Prostaspheres expressed significantly higher PSCA mRNA levels than the epithelial fraction. CONCLUSION: Human prostate cancer specimens contain neoplastic cells with self-renewal and clonogenic potential, which can be enriched and perpetuated in prostaspheres. Prostaspheres should prove valuable for the identification of prostate cancer stem/progenitor-like cells.

BMP7, a putative regulator of epithelial homeostasis in the human prostate, is a potent inhibitor of prostate cancer bone metastasis in vivo.

Bone morphogenic protein 7 (BMP7) counteracts physiological epithelial-to-mesenchymal transition, a process that is indicative of epithelial plasticity. Because epithelial-to-mesenchymal transition is involved in cancer, we investigated whether BMP7 plays a role in prostate cancer growth and metastasis. BMP7 expression in laser-microdissected primary human prostate cancer tissue was strongly down-regulated compared with normal prostate luminal epithelium. Furthermore, BMP7 expression in prostate cancer cell lines was inversely related to tumorigenic and metastatic potential in vivo and significantly correlated to E-cadherin/vimentin ratios. Exogenous addition of BMP7 to human prostate cancer cells dose-dependently inhibited transforming growth factor beta-induced activation of nuclear Smad3/4 complexes via ALK5 and induced E-cadherin expression. Moreover, BMP7-induced activation of nuclear Smad1/4/5 signaling transduced via BMP type I receptors was synergistically stimulated in the presence of transforming growth factor beta, a growth factor that is enriched in the bone microenvironment. Daily BMP7 administration to nude mice inhibited the growth of cancer cells in bone. In contrast, no significant growth inhibitory effect of BMP7 was observed in intraprostatic xenografts. Collectively, our observations suggest that BMP7 controls and preserves the epithelial phenotype in the human prostate and underscore a decisive role of the tumor microenvironment in mediating the therapeutic response of BMP7. Thus, BMP7 can still counteract the epithelial-to-mesenchymal transition process in the metastatic tumor, positioning BMP7 as a novel therapeutic molecule for treatment of metastatic bone disease.

Lack of noggin expression by cancer cells is a determinant of the osteoblast response in bone metastases.

Prostate and mammary cancer bone metastases can be osteoblastic or osteolytic, but the mechanisms determining these features are unclear. Bone morphogenetic and Wnt proteins are osteoinductive molecules. Their activity is modulated by antagonists such as noggin and dickkopf-1. Differential expression analysis of bone morphogenetic and Wnt protein antagonists in human prostate and mammary cancer cell lines showed that osteolytic cell lines constitutively express in vitro noggin and dickkopf-1 and at least one of the osteolytic cytokines parathyroid hormone-related protein, colony-stimulating factor-1, and interleukin-8. In contrast, osteoinductive cell lines express neither noggin nor dickkopf-1 nor osteolytic cytokines in vitro. The noggin differential expression profile observed in vitro was confirmed in vivo in prostate cancer cell lines xenografted into bone and in clinical samples of bone metastasis. Forced noggin expression in an osteoinductive prostate cancer cell line abolished the osteoblast response induced in vivo by its intraosseous xenografts. Basal bone resorption and tumor growth kinetics were marginally affected. Lack of noggin and possibly dickkopf-1 expression by cancer cells may be a relevant mechanism contributing to the osteoblast response in bone metastases. Concomitant lack of osteolytic cytokines may be permissive of this effect. Noggin is a candidate drug for the adjuvant therapy of bone metastasis.

In vivo electroporation and ubiquitin promoter--a protocol for sustained gene expression in the lung.

BACKGROUND: Gene therapy applications require safe and efficient methods for gene transfer. Present methods are restricted by low efficiency and short duration of transgene expression. In vivo electroporation, a physical method of gene transfer, has evolved as an efficient method in recent years. We present a protocol involving electroporation combined with a long-acting promoter system for gene transfer to the lung. METHODS: The study was designed to evaluate electroporation-mediated gene transfer to the lung and to analyze a promoter system that allows prolonged transgene expression. A volume of 250 microl of purified plasmid DNA suspended in water was instilled into the left lung of anesthetized rats, followed by left thoracotomy and electroporation of the exposed left lung. Plasmids pCiKlux and pUblux expressing luciferase under the control of the cytomegalovirus immediate-early promoter/enhancer (CMV-IEPE) or human polyubiquitin c (Ubc) promoter were used. Electroporation conditions were optimized with four pulses (200 V/cm, 20 ms at 1 Hz) using flat plate electrodes. The animals were sacrificed at different time points up to day 40, after gene transfer. Gene expression was detected and quantified by bioluminescent reporter imaging (BLI) and relative light units per milligram of protein (RLU/mg) was measured by luminometer for p.Pyralis luciferase and immunohistochemistry, using an anti-luciferase antibody. RESULTS: Gene expression with the CMV-IEPE promoter was highest 24 h after gene transfer (2932+/-249.4 relative light units (RLU)/mg of total lung protein) and returned to baseline by day 3 (382+/-318 RLU/mg of total lung protein); at day 5 no expression was detected, whereas gene expression under the Ubc promoter was detected up to day 40 (1989+/-710 RLU/mg of total lung protein) with a peak at day 20 (2821+/-2092 RLU/mg of total lung protein). Arterial blood gas (PaO2), histological assessment and cytokine measurements showed no significant toxicity neither at day 1 nor at day 40. CONCLUSIONS: These results provide evidence that in vivo electroporation is a safe and effective tool for non-viral gene delivery to the lungs. If this method is used in combination with a long-acting promoter system, sustained transgene expression can be achieved.

Effect of monoterpenes on the formation and activation of osteoclasts in vitro.

UNLABELLED: Monoterpenes, present in aromatic plants, are known to inhibit bone resorption in vivo. In this in vitro study, they inhibited the activation of osteoclasts only at high concentrations but inhibited the formation at much lower concentrations. Therefore, monoterpenes may act in vivo directly on osteoclastogenesis. INTRODUCTION: Monoterpenes are the major components of essential oils, which are formed in many plants. Typically, they are found in herbs and certain fruits. When fed to rats, they inhibit bone resorption by an unknown mechanism. In this study, their effect on the activity and formation of osteoclasts in vitro was studied. MATERIALS AND METHODS: The effect of monoterpenes on the development of osteoclasts was studied in co-cultures of bone marrow cells and osteoblasts and in cultures of spleen cells grown with colony stimulating factor (CSF)-1 and RANKL. In cultures of primary osteoblasts, alkaline phosphatase activity and levels of mRNA encoding RANKL and osteoprotegerin (OPG) mRNA (RT-PCR), and in osteoblast and spleen cell cultures, lactate dehydrogenase activity, a measure of toxicity, were determined. The activity of isolated rat osteoclasts was determined by counting the osteoclasts with actin rings using histofluorometry. RESULTS: The monoterpenes inhibited the formation of osteoclasts more strongly in co-cultures (> or = 1 microM) than in cultures of spleen cells (> or = 10 microM). They had a minor effect on osteoblasts. Toxic effects were not observed. The inhibition of the formation of osteoclasts was not reversed by the addition of farnesol and geranylgeraniol, excluding an effect of the monoterpenes through the mevalonate pathway. A high concentration of 1 mM was required to inhibit the activation of osteoclasts. This effect, shown for menthol and borneol, was reversible. CONCLUSIONS: The results suggest that the monoterpenes inhibit bone resorption in vivo through a direct effect on the formation of osteoclasts acting mainly on the hemopoietic cells.

Tumor necrosis factor-alpha: alternative role as an inhibitor of osteoclast formation in vitro.

TNFalpha is known to stimulate the development and activity of osteoclasts and of bone resorption. The cytokine was found to mediate bone loss in conjunction with inflammatory diseases such as rheumatoid arthritis or chronic aseptic inflammation induced by wear particles from implants and was suggested to be a prerequisite for the loss of bone mass under estrogen deficiency. In the present study, the regulation of osteoclastogenesis by TNFalpha was investigated in co-cultures of osteoblasts and bone marrow or spleen cells and in cultures of bone marrow and spleen cells grown with CSF-1 and RANKL. Low concentrations of TNFalpha (1 ng/ml) caused a >90% decrease in the number of osteoclasts in co-cultures, but did not affect the development of osteoclasts from bone marrow cells. In cultures with p55TNFR(-/-) osteoblasts and wt BMC, the inhibitory effect was abrogated and TNFalpha induced an increase in the number of osteoclasts in a dose-dependent manner. Osteoblasts were found to release the inhibitory factor(s) into the culture supernatant after simultaneous treatment with 1,25(OH)(2)D(3) and TNFalpha, this activity, but not its release, being resistant to treatment with anti-TNFalpha antibodies. Dexamethasone blocked the secretion of the TNFalpha-dependent inhibitor by osteoblasts, while stimulating the development of osteoclasts. The data suggest that the effects of TNFalpha on the differentiation of osteoclast lineage cells and on bone metabolism may be more complex than hitherto assumed and that these effects may play a role in vivo during therapies for inflammatory diseases.

Differential expression of TGFbeta-stimulated clone 22 in normal prostate and prostate cancer.

The transforming growth factor-beta (TGFbeta) superfamily and its downstream effector genes are key regulators of epithelial homeostasis. Altered expression of these genes may be associated with malignant transformation of the prostate gland. The cDNA array analysis of differential expression of the TGFbeta superfamily and functionally related genes between patient-matched noncancerous prostate (NP) and prostate cancer (PC) bulk tissue specimens highlighted two genes, namely TGFbeta-stimulated clone-22 (TSC-22) and Id4. Verification of their mRNA expression by real-time PCR in patient-matched NP and PC bulk tissue, in laser-captured pure epithelial and cancer cells and in NP and PC cell lines confirmed TSC-22 underexpression, but not Id4 overexpression, in PC and in human PC cell lines. Immunohistochemical analysis showed that TSC-22 protein expression in NP is restricted to the basal cells and colocalizes with the basal cell marker cytokeratin 5. In contrast, all matched PC samples lack TSC-22 immunoreactivity. Likewise, PC cell lines do not show detectable TSC-22 protein expression as shown by immunoblotting. TSC-22 should be considered as a novel basal cell marker, potentially useful for studying lineage determination within the epithelial compartment of the prostate. Conversely, lack of TSC-22 seems to be a hallmark of malignant transformation of the prostate epithelium. Accordingly, TSC-22 immunohistochemistry may prove to be a diagnostic tool for discriminating benign lesions from malignant ones of the prostate. The suggested tumour suppressor function of TSC-22 warrants further investigation on its role in prostate carcinogenesis and on the TSC-22 pathway as a candidate therapeutic target in PC.

Interference with the microenvironmental support impairs the de novo formation of bone metastases in vivo.

Interference with the microenvironmental growth support is an attractive therapeutic strategy for repressing metastatic tumor growth. Bone is a highly dynamic tissue that is continuously remodeled by bone resorption and subsequent bone formation. Growth factors supporting bone metastatic growth are released especially during bone resorption. Differently from most other tissues, drugs that can limit local turnover, such as bisphosphonates, are available for bone. In the present study, we tested the hypothesis that inhibition of bone turnover can affect development and growth progression of experimental bone metastasis. Whole-body bioluminescent reporter imaging was used for the detection, monitoring, and quantification in vivo of the growth progression of bone metastases induced by intracardiac or intraosseous injection of luciferase-transfected breast cancer cells (MDA-231-B/luc+) to nude mice. Suppression of bone turnover by bisphosphonates, before bone colonization by cancer cells, inhibited by a great extent the number of developing bone metastasis. Tumor growth in the few, but still developing, bone metastases was affected only transiently. Reduction of bone turnover had no effect on growth progression of bone metastases, which were already established when bisphosphonate treatment was initiated, despite a substantial reduction in osteolysis. Therefore, cancer cells metastatic to bone, after an initial growth phase that depends on the interaction with the local stroma, become independent of microenvironmental growth factor support and progress autonomously. Inhibition of bone turnover may represent a useful adjuvant therapy especially for cancer patients at risk to develop bone metastasis.

Electroporation enhances transfection efficiency in murine cutaneous wounds.

Transfection of wounds with DNA-encoding growth factors has the potential to improve healing, but current means of nonviral gene delivery are inefficient. Repeated high doses of DNA, necessary to achieve reliable gene expression, are detrimental to healing. We assessed the ability of in vivo electroporation to enhance gene expression. Full-thickness cutaneous excisional wounds were created on the dorsum of female mice. A luciferase- encoding plasmid driven by a CMV promoter was injected at the wound border. Following plasmid administration, electroporative pulses were applied to injection sites. Pulse parameters were varied over a range of voltage, duration, and number. Animals were euthanized at intervals after transfection and the luciferase activity measured. Application of electric pulses consistently increased luciferase expression. The electroporative effect was most marked at a plasmid dose of 50 micro g, where an approximate tenfold increase was seen. Six 100- micro s-duration pulses of 1750 V/cm were found to be the most effective in increasing luciferase activity. High numbers of pulses tended to be less effective than smaller numbers. This optimal electroporation regimen had no detrimental effect on wound healing. We conclude that electroporation increases the efficiency of transgene expression and may have a role in gene therapy to enhance wound healing.