RANKL inhibition is an effective adjuvant for docetaxel in a prostate cancer bone metastases model

BACKGROUND: Docetaxel induces an anti-tumor response in men with advanced prostate cancer (PCa); however, the side effects associated with docetaxel treatment can be severe, resulting in discontinuation of therapy. Thus, identification of an effective adjuvant therapy to allow lower doses of docetaxel is needed. Advanced PCa is typically accompanied by skeletal metastasis. Receptor activator of NFkB ligand (RANKL) is a key pro-osteoclastic factor. Targeting RANKL decreases establishment and progression of PCa growth in bone in murine models. METHODS: The efficacy of inhibiting RANKL, using a recombinant soluble RANK extracellular domain fused with the immunoglobulin Fc domain (RANK-Fc), was tested as an adjuvant therapy with docetaxel for PCa bone metastasis in a murine intra-tibial model. RESULT: The combination of RANK-Fc and docetaxel reduced tumor burden in bone greater than either treatment alone. CONCLUSION: The combination of docetaxel with a RANKL-inhibiting agent merits further investigation for treatment of advance PCa.     

PTHrP increases RANKL expression by stromal cells from giant cell tumor of bone

Giant cell tumor of bone (GCT) presents with numerous osteoclast-like multinucleated giant cells that are principally responsible for the extensive bone resorption by the tumor. Although the precise etiology of GCT remains uncertain, the accumulation of giant cells is partially due to the high expression of the receptor activator of nuclear factor-κB ligand (RANKL) from the neoplastic stromal cells. Here, we have investigated whether parathyroid hormone-related protein (PTHrP) plays a role in the pathogenesis of GCT. Immunohistochemistry results revealed PTHrP expression in the stromal cells of the tumor, and that its receptor, the parathyroid hormone type 1 receptor (PTH1R), is expressed by both the stromal cells and giant cells. PCR and Western blot analyses confirmed the expression of PTHrP and PTH1R by isolated stromal cells from five patients presenting with GCT. Treatment of GCT stromal cells with varying concentrations of PTHrP (1-34) significantly increased both RANKL gene expression and the number of multinucleated cells formed from RAW 264.7 cells in co-culture experiments, whereas inhibition of PTHrP with a neutralizing antibody decreased RANKL gene expression. These results suggest that PTHrP is expressed within GCT by the stromal cells and can contribute to the abundant RANKL expression and giant cell formation within the tumor.     

Targeting the receptor activator of nuclear factor-kappaB (RANK) ligand in prostate cancer bone metastases

Newly formed bone in the typically osteoblastic bone metastases from prostate cancer shows characteristics of woven bone, e.g. marked defects in mineralization and microstructure. Adding to the reduced mechanical strength of prostate cancer bone metastasis is an increasingly recognized osteolytic component. The existence of osteoclasts in osteoblastic bone metastases and concomitant increases in urine or serum markers for bone resorption are reported in affected patients. Pathologically increased osteoclastic bone resorption is a key mediator of the clinical complications from bone metastases, among them fractures, spinal cord compression and bone pain. The receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) pathway has been identified as the main driving force for osteoclastogenesis and resulting bone resorption. Emerging data indicate that bone marrow-derived RANKL might also constitute a chemoattractant factor for RANK-expressing tumour cells that is likely to contribute to the pathogenesis of bone metastases, including those arising from prostate cancer. Cumulative evidence supports RANKL inhibition as a therapeutic goal for the treatment and prevention of bone metastases from prostate cancer.     

The Role of RANK/RANKL/OPG Signalling Pathways in Osteoclastogenesis in Odontogenic Keratocysts, Radicular Cysts, and Ameloblastomas

The aim of this study was to evaluate the immunohistochemical expression of molecules involved in osteoclastogenesis, including the receptor activator of nuclear factor kappa B (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG) in odontogenic keratocysts (OKCs), which has been named as a keratocystic odontogenic tumour by the WHO, and compare their expression with radicular cysts and ameloblastomas. RANK is a member of tumour necrosis factor receptor family and it is activated by RANK ligand. OPG binds to RANKL and inactivates it. The imbalance of these factors could cause the differential bone resorption activity in some diseases and tumours. The expression of these molecules was evaluated in ameloblastomas (n = 20), OKCs (n = 20), and radicular cysts (n = 20) by immunohistochemistry. Immunohistochemical reactivity for RANK, RANKL, and OPG was detected in neoplastic and nonneoplastic epithelium and connective tissue cells. RANK showed the greatest expression in OKCs followed by ameloblastomas, with the lowest expression seen in radicular cysts. Expression of RANKL was detected in all lesions and no significant differences were observed between groups. OPG was expressed very low in all groups. In the stroma, the number of RANK positive cells was higher in OKCs when compared with ameloblastomas and radicular cysts but radicular cyst had higher numbers of RANKL positive cells in the stroma than ameloblastomas. The molecular system of RANK/RANKL/OPG is variably expressed in OKCs, radicular cysts, and ameloblastomas and this system may be involved in the osteoclastogenic mechanisms in OKCs and ameloblastomas. Advanced studies could further clarify the role of RANK, RANKL, and OPG in mediating tumour associated bone osteolysis.     

Establishment and characterization of osseous prostate cancer models: intra-tibial injection of human prostate cancer cells

BACKGROUND: To improve the therapy of advanced prostate cancer (CaP), it is critical to develop animal models that mimic CaP bone metastases. Unlike the human disease, CaP xenograft models rarely metastasize spontaneously to bone from the orthotopic site of primary tumor growth. METHODS: Single-cell suspensions of LNCaP, PC-3, LuCaP 35, and LuCaP 23.1 CaP cells were injected directly into tibia of SCID mice. Immunohistochemistry and bone histomorphometrical analyses were performed to characterize these osseous-CaP models. RESULTS: LuCaP 23.1 yields an osteoblastic response, LNCaP yields mixed lesions, and LuCaP 35 and PC-3 result in osteolytic responses. We have detected osteoprotegerin, RANK ligand, parathyroid hormone-related protein, and endothelin-1, proteins associated with bone growth and remodeling, in the CaP cells grown in the bone. CONCLUSIONS: These animal models can be used to study biological interactions, pathways, and potential therapeutic targets, and also to evaluate new agents for treatment and prevention of CaP bone metastasis.     

基于OPG/RANKL/RANK轴观察加味阳和汤及其拆方对去卵巢骨质疏松大鼠的影响

目的观察加味阳和汤及其拆方对OPG、RANKL、RANK含量的影响,探讨其防治绝经后骨质疏松症可能的作用机制及组方配伍的合理性。方法选取48只雌性SD大鼠,加味阳和汤按君臣佐使关系拆方,将大鼠等量随机分为假手术组(SHAM)、模型组(OVX)、君药+臣药组(A组)、君药+臣药+佐药组(B组)、君药+臣药+佐药+使药组(C组)、戊酸雌二醇组(E2V)。除SHAM组外,均采用去卵巢骨质疏松大鼠模型,干预给药后(灌胃90 d),处死动物后取右侧股骨及胫骨通过双能X射线骨密度仪检测骨密度(bone mineral density,BMD)及骨矿含量(bone mineral content,BMC),取左侧股骨行HE染色观察骨显微结构,检测血清中骨代谢指标ALP、Ca~(2+)、P~(3-)、E2及血清OPG、RANKL、RANK含量。结果与SHAM组相比,OVX组大鼠股骨及胫骨BMD、BMC降低(P0.05),骨小梁变细、间隙增大、结构缺失,血清Ca~(2+)、P~(3-)、E2、OPG水平下降(P0.05),血清ALP、RANKL、RANK水平上升(P0.05);与OVX组比较,除A组大鼠股骨及胫骨BMD、BMC、血清Ca~(2+)、P~(3-)、E2、OPG、RANKL及B组P~(3-)水平无显著差异外(P0.05),各给药组大鼠股骨及胫骨BMD、BMC均显著升高(P0.05),骨小梁增多、间隙减小、结构趋向完整,血清Ca~(2+)、P~(3-)、E2、OPG水平上升(P0.05),血清ALP、RANKL、RANK水平下降(P0.05)。结论加味阳和汤及其拆方通过提高去卵巢骨质疏松大鼠BMD、BMC,降低骨代谢,改善骨显微结构从而发挥治疗作用,调节OPG/RANKL/RANK轴是可能的机制。     

基于OPG/RANK/RANKL信号轴探讨中医“瘀证”与骨质疏松症之间的关系

近年来,随着中医学的不断发展,中医药在治疗骨质疏松症(osteoporosis,OP)中的应用愈加广泛,然而由于只有单纯的中医理论指导,而缺乏现代科学依据的支撑,故而广受争议。与此同时,OPG/RANK/RANKL信号轴的发现又是现代分子生物学的一大突破。因此,笔者立足于中西医结合的角度,基于中医脏腑亏虚立论,从OPG/RANK/RANKL信号调控机制探讨中医学从"瘀证"论治骨质疏松症的合理性,从而为中医从"瘀"辨治OP以及临床研究提供科学理论依据。     

葡萄糖对人牙周膜成纤维细胞OPG、RANKLmRNA表达的影响

目的：以体外培养的人牙周膜成纤维细胞（HPDLF）为研究对象,观察不同浓度的葡萄糖对HPDLF中骨保护素（OPG）、核因子KB受体活化剂配体（RANKL）mRNA表达的影响,探讨葡萄糖在牙槽骨吸收中的作用。方法：组织块法体外原代培养HPDLF并鉴定,选择4-8代的HPDLF作为实验的靶细胞,不同浓度的葡萄糖（0、5．5、11．1、16．7、22．2、33．3mmol／1）干预HPDLF,半定量逆转录聚合酶链反应（RT-PCR）检测HPDLF中OPG、RANKL mRNA的表达。结果：高浓度的葡萄糖可下调HPDLF中OPG mRNA的表达,呈剂量依赖性;可上调RANKL mRNA的表达,也呈剂量依赖性,且使RANKL／OPG mRNA的比值随着葡萄糖浓度的增高而呈现持续上升的趋势。结论：糖尿病时,牙周组织中高浓度的葡萄糖,可下调OPG的表达,上调RANKL的表达,使RANKL／OPG的比值升高,调节破骨细胞分化,刺激骨吸收,导致牙槽骨的破坏,加重牙周病的病情。     

Molecular mechanisms of metastasis in prostate cancer

Prostate cancer （PCa） preferentially metastasizes to the bone marrow stroma of the axial skeleton. This activity is the principal cause of PCa morbidity and mortality. The exact mechanism of PCa metastasis is currently unknown, although considerable progress has been made in determining the key players in this process. In this review, we present the current understanding of the molecular processes driving PCa metastasis to the bone.     

Plasma osteopontin in comparison with bone markers as indicator of bone metastasis and survival outcome in patients with prostate cancer

BACKGROUND: The study was undertaken to evaluate the diagnostic and prognostic value of plasma osteopontin (OPN) in comparison to bone markers as well as the relationships between the markers and clinico-pathological factors in prostate cancer (PCa) patients. METHODS: OPN and the bone markers carboxyterminal-telopeptide of type I collagen, bone-specific alkaline phosphatase (bALP), and aminoterminal-propeptide of type I procollagen (PINP) were measured in 90 PCa patients with and without bone metastases, 35 patients with benign prostatic hyperplasia, and 29 healthy men. RESULTS: OPN and bone markers were significantly elevated in patients with bone metastases compared to the other groups. Significant correlations were found between all four-bone markers (r(s) = 0.43-0.79, all P < 0.01). OPN correlated with tumor grade (r(s) = 0.23, P < 0.05). In receiver-operating characteristics (ROC) analyses, OPN and bone markers were effective in distinguishing PCa patients with and without bone metastases showing areas under the curve (AUC) between 0.80 and 0.88 (all P < 0.001). OPN had an AUC of 0.85 that increased in combination with bALP up to 0.93 providing at the point with the highest diagnostic accuracy both a sensitivity and specificity of about 90%. Kaplan-Meier analyses and Cox proportional hazards regression models showed decreased survival of patients with high OPN and bone marker levels, while only high OPN and PINP were independent negative prognostic factors for PCa-related death. CONCLUSIONS: OPN alone or in combination with bone markers is useful as diagnostic marker in the detection of bone metastases and as prognosticator in the survival prediction in PCa patients.     

人成骨样细胞和前破骨样细胞ER亚型、OPG/RANKL/RANK系统、IL-6及其受体以及破骨细胞标志基因表达的研究

目的分析比较两种人成骨样细胞系(U2-OS、MG-63)和两种人前破骨样细胞系(U937、HL-60)ER亚型、OPG/RANKL/RANK系统、IL-6及其受体以及破骨细胞标志基因表达的差异,为今后研究雌激素与IL-6等细胞因子在骨组织中相互关系提供适宜的细胞模型。方法RT-PCR和免疫印迹法检测ERα、ERβ的表达,ELISA法测定IL-6的分泌,OPG、RANKL、RANK及IL-6受体的表达采用免疫印迹法进行检测,TRAP、MMP-9则采用RT-PCR技术进行分析。结果(1)转录水平及蛋白水平证实四种细胞均表达ERα和ERβ;(2)四种细胞不同程度地表达OPG和RANKL,而RANK则仅见于U937细胞表达;(3)四种细胞均表达IL-6受体(IL-6Rα、gp130),除U2-OS细胞外其余三种细胞均组成型分泌IL-6;(4)破骨细胞标志基因TRAP在两种人前破骨样细胞U937、HL-60中均表达,且表达水平相近;而MMP-9仅在U937细胞中弱表达;两种人成骨样细胞U2-OS、MG-63未见有这两种基因表达。结论筛选出用于研究雌激素与IL-6等细胞因子在骨组织中相互关系的细胞模型,同时也为今后深入阐明骨靶向和其他新型抗骨质疏松雌激素的分子机制研究奠定基础。     

慢性间歇性缺氧对大鼠骨代谢及OPG、RANKL基因表达的影响

目的通过建立wistar大鼠慢性间歇性缺氧(chronic intermittent hypoxia,CIH)模型探讨CIH对骨代谢及骨组织骨保护素(osteoprotegerin,OPG)、核因子κB受体活化因子配体(receptor activator for nuclear factor-κB ligand,RANKL)基因表达的影响。方法 20只健康wistar大鼠随机分为正常对照组及CIH组,每组10只。对照组置于空气循环舱内,CIH组于置于间歇性缺氧舱内,每日8 h,共8周。应用双能X线骨密度仪测定大鼠全身、股骨、腰椎骨密度(bone mineral density,BMD),采用ELISA法检测大鼠血清骨特异性碱性磷酸酶(bone specific alkaline phosphatase,BALP)、骨钙素(bone glaprotein,BGP)、Ⅰ型胶原N端前肽(type Ⅰ collagen N terminal peptide,PINP)、Ⅰ型胶原羧基端肽β降解产物(β-C-terminal telopeptide of type Ⅰ collagen,β-CTX)、抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)水平,应用Real-time PCR技术测定股骨OPG、RANKL mRNA相对表达量。结果与对照组相比,CIH组全身和股骨BMD下降[(0.141±0.028)vs(0.122±0.027)和(0.143±0.026)vs(0.125±0.034)]、血清BGP降低[(26.42±3.71)vs(22.05±2.16)]、血清β-CTX和TRAP升高[(132.24±26.25)vs(173.82±22.16)和(20.12±2.43)vs(23.58±2.36)]、股骨组织OPG mRNA相对表达量降低[(1.031±0.125)vs(0.924±0.141)]、RANKL mRNA相对表达量升高[(0.856±0.068)vs(1.113±0.134)],差异均具有统计学意义(P0.05)。结论慢性间歇性缺氧可使骨组织OPG mRNA表达减少、RNAKL mRNA表达增加,影响骨代谢,降低骨密度,增加了骨质疏松的发生风险。     

橙皮苷对去卵巢骨质疏松症大鼠骨质流失和机制的影响

目的研究橙皮苷对去卵巢骨质疏松大鼠骨质流失和骨保护素(osteoprotegerin,OPG)/核因子kappa B配体的受体激活物(receptor activator of nuclear factor kappa B ligand,RANKL)/RANK通路的影响。方法将48只雌性Sprague-Dawley(SD)大鼠随机分为假手术组、去卵巢骨质疏松组、橙皮苷低剂量组、橙皮苷中剂量、橙皮苷高剂量组和雌激素组。检测骨组织骨体积分数(bone volume fraction,BV/TV)、骨小梁厚度(trabecular thickness,Tb.Th)、骨密度(bone mineral density,BMD)、骨小梁数量(trabecular number,Tb.N)、骨小梁分离度(trabecular separation,Tb.Sp),通过HE染色观察骨组织病理损伤,采用酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)检测人Ⅰ型胶原C端肽(C-terminal peptide collagen typeⅠ,CTX-Ⅰ)和血清骨钙素(bone glaprotein,BGP)的表达量。运用qRT-PCR和Western bolt分别检测骨组织OPG、RANKL、RANK的mRNA和蛋白表达。结果在橙皮苷治疗后,与去卵巢骨质疏松组相比,骨质疏松标志物BV/TV、BMD、Tb.Th和Tb.N水平升高,Tb.Sp水平降低;大鼠骨小梁数量明显增加,且骨小梁连接更为紧密;骨吸收标志物CTX-Ⅰ水平降低;骨形成标志物BGP水平升高。OPG mRNA相对表达量和蛋白的表达量升高,RANKL和RANK mRNA相对表达量和蛋白的表达量降低。结论橙皮苷能缓解去卵巢骨质疏松大鼠骨质流失并调节OPG/RANKL/RANK信号通路。     

MiR-622 acts as a tumor suppressor to induce cell apoptosis and inhibit metastasis in human prostate cancer

Growing evidence indicating the critical modulator roles of microRNAs (miRNAs) involved in prostate cancer (PCa) metastasis that holds great promise as therapeutic targets. Herein, we transfected the miR-622 mimic into PC3 cells and evaluated the effects of this interference on these tumour cells' growth and the expression of specific metastatic genes. Transfecting of miR-622 mimic and inhibitor, negative control (NC) inhibitor and NC was established using Lipofectamine 2000. The mRNA levels of miR-622 and metastatic genes were evaluated using the qRT-PCR and Western blot. Cytotoxic effects of miR-622 were assessed by MTT. Apoptosis was detected using an ELISA cell death assay kit. miR-622 is down-regulated in PC3 cells. As expected, cell viability effects after transfection were described as miR-622 inhibitor >NC and NC inhibitor >miR-622 mimic (p < .01). Importantly, we showed that transfected miR-622 mimic could enhance the apoptosis of PC3 cells, while transfected miR-622 inhibitor could decrease cell apoptosis (p < .01). Furthermore, miR-622 overexpression could increase significantly down-regulated the MMP2, MMP9, CXCR-4, c-Myc and K-Ras expression levels. Findings demonstrate a novel mechanism by which miR-622 modulates PCa cells' metastasis by targeting metastatic genes. These data confirm the tumour-suppressive function of miR-622 in PCa cells by enhancing apoptosis and reducing metastasis.