Canine prostate stimulates osteoblast function using the endothelin receptors

BACKGROUND: Bone metastases are common in humans and dogs with late-stage prostate cancer. A unique feature of prostate cancer metastases is new bone formation at metastatic sites ("osteoblastic metastases"). Many carcinomas that metastasize to bone cause bone destruction, not new bone formation. The mechanisms by which prostate cancer induces bone formation at sites of bone metastasis are not well understood. We hypothesized that stimulation of osteoblasts by prostate tissue at metastatic sites was due to the paracrine actions of growth factors produced by prostate epithelial cells. METHODS: We have previously shown that normal canine prostate tissue induced new bone formation when implanted adjacent to the calvarium of nude mice. To complement this in vivo model, we developed an in vitro system of prostate-stimulated osteoblast function to investigate mechanisms of prostate-induced new bone formation. RESULTS: We found that treatment of cultured rat calvaria for 24 hr with proteins from normal dog prostate stimulated alkaline phosphatase activity in a dose-dependent manner 4-6 fold compared to controls. Stimulation began approximately 8 hr after treatment, and was diminished after 72 hr. Calvaria treated with homogenates of normal dog salivary gland, kidney, bladder, and muscle did not increase ALP activity. Pretreatment of the calvaria for 1 hr with endothelin antagonists, but not anti-parathyroid hormone-related protein (PTHrP) antibody or indomethacin, abrogated the stimulation of ALP. CONCLUSIONS: Our results indicated that osteoblast activation by canine prostate occurs via an endothelin-dependent mechanism, and that PTHrP or prostaglandin synthase-mediated pathways are likely not involved. This is a reliable, reproducible assay for determining the roles of molecules important in the activation of osteoblasts by the prostate.     

The effect of osteoprotegerin administration on the intra-tibial growth of the osteoblastic LuCaP 23.1 prostate cancer xenograft

Osteoprotegerin (OPG) plays a central role in controlling bone resorption. Exogenous administration of OPG has been shown to be effective in preventing osteolysis and limiting the growth of osteolytic metastasis. The objective of this study was to investigate the effects of OPG on osteoblastic prostate cancer (CaP) metastases in an animal model. LuCaP 23.1 cells were injected intra-tibially and Fc-OPG (6.0 mg/kg) was administered subcutaneously three times a week starting either 24 hours prior to cell injection (prevention regimen) or at 4 weeks post-injection (treatment regimen). Changes in bone mineral density at the tumor site were determined by dual x-ray absorptiometry. Tumor growth was monitored by evaluating serum prostate specific antigen (PSA). Fc-OPG did not inhibit establishment of osteoblastic bone lesions of LuCaP 23.1, but it decreased growth of the tumor cells, as determined by decreases in serum PSA levels of 73.0 ± 44.3% (P < 0.001) and 78.3 ± 25.3% (P < 0.001) under the treatment and prevention regimens, respectively, compared to the untreated tumor-bearing animals. Administration of Fc-OPG decreased the proliferative index by 35.0% (P = 0.1838) in the treatment group, and 75.2% (P = 0.0358) in the prevention group. The results of this study suggest a potential role for OPG in the treatment of established osteoblastic CaP bone metastases.     

Reduced gap junctional intercellular communication and altered biological effects in mouse osteoblast and rat liver oval cell lines transfected with dominant-negative connexin 43

Gap junctional intercellular communication (GJIC) maintains normal growth and differentiation of cells in a tissue. The intercellular molecules traversing gap junctions are largely unknown, but the molecular weight (MW) cutoff is normally 1200 Da. No differences in dye transfer were observed in normal or vector controls of WB-F344 rat liver epithelial or mouse osteoblastic MC3T3-E1 cells with either Lucifer Yellow (LY) with a MW of 457 Da (LY-457) or LY with a MW of 649 Da (LY-649). Transfection of a dominant negative-connexin 43 (Cx43) gene decreased GJIC (>50%) when LY-649 was used, however, normal GJIC was observed in both cell lines when LY-457 was used. Therefore, the MW cut off in these clones was considerably less than the wild type. The dominant negative clones of the MC3T3-E1 cells exhibited over 90% less alkaline phosphatase (ALPase) activity and calcium deposition after the induction of differentiation. Similarly, dominant negative Cx43 inhibited gene expression of ALPase and bone sialoprotein but not osteocalcin in MC3T3-E1. WB-F344 cells normally exhibit a biphasic response to 12-O-tetradecanoylphorbol-13-acetate (TPA) where inhibition of GJIC recovers after 2 h, but the dominant negative clones showed no recovery from inhibition of GJIC by TPA. Dominant negative Cx43 also inhibited the formation of network-like structures by WB-F344 cells on Matrigel. These results demonstrate that the dominant negative gene transfected into cell types containing the wild-type connexins result in diminished channel sizes, thus allowing the determination of whether distinct biological endpoints, i.e., differentiation, are dependent upon either small or high MW intercellular signals.     

含骨松宝的老龄大鼠血清对兔成骨细胞增殖作用的实验研究

目的：观察骨松宝对体外培养成骨细胞增殖和代谢的影响。方法：给18月龄老年大鼠灌胃骨松宝1．5g／kg体重,每日2次,连续3天,末次灌胃后1h取血分离血清,用D8900培养基分别配成含药鼠血清7．5％和15％的培养基,并用于培养成骨细胞24h,同时以加有7．5％和15％未用药老龄大鼠血清的D8900培养基、含20μmol／L氟化钠(NaF)的D8900无血清培养基及D8900无血清培养基作对照,用MTT法检测细胞增殖,并测定培养基上清液中Ca^2+浓度及碱性磷酸酶(ALP)的含量变化。结果：与加有相同浓度鼠血清的D8900培养基组、D8900培养基加NaF组和D8900培养基组比较,含药鼠血清组的成骨细胞增殖明显,差异有显著性(P<0．01),培养基中的Ca^2+消耗量明显增加(P<0．05,P<0．01),ALP浓度明显升高(P<0．05,P<0.01)。结论：骨松宝能促进成骨细胞DNA合成和提高对Ca2^2+的利用,促进成骨细胞的生长增殖。     

Overexpression of cadherins suppresses pulmonary metastasis of osteosarcoma in vivo

Osteosarcoma by nature shows aggressive pulmonary metastasis; however, the underlying molecular mechanisms remain unclear. We previously showed that N-cadherin and cadherin-11 (OB-cadherin), which are highly expressed in normal osteoblasts, are anomalously expressed in human osteosarcoma (Kashima et al., Am J Pathol 1999;155:1549-55). In the present study, we examined the role of cadherins in osteosarcoma metastasis using the mouse osteosarcoma cell line Dunn and its highly metastatic subline LM8. Oligonucleotide array and RT-PCR analyses demonstrated that Dunn and LM8 cells did not express appreciable levels of several members of the cadherin family, and Western blot analysis confirmed that Dunn and LM8 cells did not express P-cadherin, E-cadherin, N-cadherin or cadherin-11 protein. We therefore investigated the functional consequences of cadherin overexpression on cell migration and in vivo metastatic potential of LM8 cells. Several LM8 clones were isolated which expressed exogenous N-cadherin and cadherin-11 localized to the cell membrane and able to bind to beta-catenin. Overexpression of N-cadherin or cadherin-11 in LM8 cells did not affect cell proliferation but caused an inhibitory effect on cell migration in vitro. In vivo analysis showed that N-cadherin- and cadherin-11-overexpressing cells exhibited a marked reduction in their ability to form pulmonary metastases, with significant decreases in lung weight and the number and weight of metastatic lesions, as well as the size and weight of primary lesions at the s.c.-inoculated site. These observations demonstrate that disruption of N-cadherin- and cadherin-11-mediated cell-cell adhesion is critical in the pulmonary metastasis of osteosarcoma.     

红曲提取物对体外培养成骨细胞的作用研究

目的：研究红曲不同提取物对体外培养大鼠成骨细胞（ROB）增殖、分化的影响。方法：采用新生大鼠颅骨分离培养ROB,MTT法测定细胞增殖,碱性磷酸酶（ALP）试剂盒法测定ALP活性,观察红曲提取物对ROB增殖、分化的作用,同时考察了MTT法和ALP试剂盒法中细胞数和吸收值之间的线性关系。结果：10^-2和10^-3mg/ml的水提物能刺激ROB的增殖（P〈0.05）,10^-3mg/ml的水提物还能提高ROB的ALP活性（P〈0.05）;10^-2和10^-3mg/ml的95%乙醇提取物对ROB的增殖与分化均有明显的促进作用（P〈0.05或P〈0.01）。结论：红曲水提物和95%乙醇提取物均能促进ROB骨形成过程中增殖、分化,95%乙醇提取物作用效果显著。     

Smad蛋白在BMP-2诱导成骨分化过程中的作用

目的研究Smad蛋白在骨形态发生蛋白-2（BMP-2）诱导成骨分化过程中作用,探讨Smad蛋白在成骨分化期间信号传导的机制。方法将取自MDX大鼠颅盖骨组织分离获得成骨细胞进行培养,培养的成骨细胞分组,加入不同浓度的BMP-2或BMP-2与Trx2SARA混合物后,用反义PCR和Western Blot斑点杂交对各组诱导成骨分化的情况进行分析。体内实验中肌肉局部埋植BMP-2或BMP-2和Trx2SARA,取不同时间点处死动物,制备组织切片,用SO染色进行组织学观察。结果BMP-2处理的成骨细胞碱性磷酸酶增加,降钙素mRNA增加,Smad蛋白的表达水平呈剂量依赖性增加。Trx2SARA处理组出现碱性磷酸酶和降钙素mRNA下调。体内实验组织切片SO染色显示,BMP-2处理组大部分异位组织由增殖中、肥厚前和肥厚软骨细胞构成,而Trx2SARA处理组很少向软骨源细胞分化。结论Smad蛋白在BMP-2诱导成骨分化中发挥重要的信号传导作用。     

Emerging drugs for the treatment of bone metastasis

Introduction: Bone metastases are virtually incurable resulting in significant disease morbidity, reduced quality of life and mortality. Bone provides a unique microenvironment whose local interactions with tumor cells offer novel targets for therapeutic interventions. Increased understanding of the pathogenesis of bone disease has led to the discovery and clinical utility of bone-targeted agents other than bisphosphonates and denosumab, currently, the standard of care in this setting.

Areas covered: In this review, we present the recent advances in molecular targeted therapies focusing on therapies that inhibit bone resorption and/or stimulate bone formation and novel anti-tumoral agents that exerts significant effects on skeletal metastases, nowadays available in clinical practice or in phase of development.

Expert opinion: New emergent bone target therapies radium-223, mTOR inhibitors, anti-androgens have demonstrated the ability to increase overall survival in bone metastatic patients, other compounds, such as ET-1 and SRC inhibitors, up to now failed to clearly confirm in clinical trials their promising preclinical data.