Suppression of arthritic bone destruction by adenovirus-mediated csk gene transfer to synoviocytes and osteoclasts

Rheumatoid arthritis (RA) is characterized by a chronic inflammation of the synovial joints resulting from hyperplasia of synovial fibroblasts and infiltration of lymphocytes, macrophages, and plasma cells, all of which manifest signs of activation. Recent studies have revealed the essential role of osteoclasts in joint destruction in RA. Src family tyrosine kinases are implicated in various intracellular signaling pathways, including mitogenic response to growth factors in fibroblasts, activation of lymphocytes, and osteoclastic bone resorption. Therefore, inhibiting Src activity can be a good therapeutic strategy to prevent joint inflammation and destruction in RA. We constructed an adenovirus vector carrying the csk gene, which negatively regulates Src family tyrosine kinases. Csk overexpression in cultured rheumatoid synoviocytes remarkably suppressed Src kinase activity and reduced their proliferation rate and IL-6 production. Bone-resorbing activity of osteoclasts was strongly inhibited by Csk overexpression. Furthermore, local injection of the virus into rat ankle joints with adjuvant arthritis not only ameliorated inflammation but suppressed bone destruction. In conclusion, adenovirus-mediated direct transfer of the csk gene is useful in repressing bone destruction and inflammatory reactions, suggesting the involvement of Src family tyrosine kinases in arthritic joint breakdown and demonstrating the feasibility of intervention in the kinases for gene therapy in RA. off     

A c-fms tyrosine kinase inhibitor, Ki20227, suppresses osteoclast differentiation and osteolytic bone destruction in a bone metastasis model

In bone metastatic lesions, osteoclasts play a key role in the development of osteolysis. Previous studies have shown that macrophage colony-stimulating factor (M-CSF) is important for the differentiation of osteoclasts. In this study, we investigated whether an inhibitor of M-CSF receptor (c-Fms) suppresses osteoclast-dependent osteolysis in bone metastatic lesions. We developed small molecule inhibitors against ligand-dependent phosphorylation of c-Fms and examined the effects of these compounds on osteolytic bone destruction in a bone metastasis model. We discovered a novel quinoline-urea derivative, Ki20227 (N-{4-[(6,7-dimethoxy-4-quinolyl)oxy]-2-methoxyphenyl}-N'-[1-(1,3-thiazole-2-yl)ethyl]urea), which is a c-Fms tyrosine kinase inhibitor. The IC(50)s of Ki20227 to inhibit c-Fms, vascular endothelial growth factor receptor-2 (KDR), stem cell factor receptor (c-Kit), and platelet-derived growth factor receptor beta were found to be 2, 12, 451, and 217 nmol/L, respectively. Ki20227 did not inhibit other kinases tested, such as fms-like tyrosine kinase-3, epidermal growth factor receptor, or c-Src (c-src proto-oncogene product). Ki20227 was also found to inhibit the M-CSF-dependent growth of M-NFS-60 cells but not the M-CSF-independent growth of A375 human melanoma cells in vitro. Furthermore, in an osteoclast-like cell formation assay using mouse bone marrow cells, Ki20227 inhibited the development of tartrate-resistant acid phosphatase-positive osteoclast-like cells in a dose-dependent manner. In in vivo studies, oral administration of Ki20227 suppressed osteoclast-like cell accumulation and bone resorption induced by metastatic tumor cells in nude rats following intracardiac injection of A375 cells. Moreover, Ki20227 decreased the number of tartrate-resistant acid phosphatase-positive osteoclast-like cells on bone surfaces in ovariectomized (ovx) rats. These findings suggest that Ki20227 inhibits osteolytic bone destruction through the suppression of M-CSF-induced osteoclast accumulation in vivo. Therefore, Ki20227 may be a useful therapeutic agent for osteolytic disease associated with bone metastasis and other bone diseases.     

TGF-β signaling blockade inhibits PTHrP secretion by breast cancer cells and bone metastases development

Breast cancer frequently metastasizes to the skeleton, and the associated bone destruction is mediated by the osteoclast. Growth factors, including transforming growth factor-β (TGF-β), released from bone matrix by the action of osteoclasts, may foster metastatic growth. Because TGF-β inhibits growth of epithelial cells, and carcinoma cells are often defective in TGF-β responses, any role of TGF-β in metastasis is likely to be mediated by effects on the surrounding normal tissue. However, we present evidence that TGF-β promotes breast cancer metastasis by acting directly on the tumor cells. Expression of a dominant–negative mutant (TβRIIΔcyt) of the TGF-β type II receptor rendered the human breast cancer cell line MDA-MB-231 unresponsive to TGF-β. In a murine model of bone metastases, expression of TβRIIΔcyt by MDA-MB-231 resulted in less bone destruction, less tumor with fewer associated osteoclasts, and prolonged survival compared with controls. Reversal of the dominant–negative signaling blockade by expression of a constitutively active TGF-β type I receptor in the breast cancer cells increased tumor production of parathyroid hormone–related protein (PTHrP), enhanced osteolytic bone metastasis, and decreased survival. Transfection of MDA-MB-231 cells that expressed the dominant–negative TβRIIΔcyt with the cDNA for PTHrP resulted in constitutive tumor PTHrP production and accelerated bone metastases. These data demonstrate an important role for TGF-β in the development of breast cancer metastasis to bone, via the TGF-β receptor–mediated signaling pathway in tumor cells, and suggest that the bone destruction is mediated by PTHrP.     

过敏性哮喘患者高迁移率族蛋白1、Th1/Th2细胞亚群及其细胞因子表达分析

目的：探究剪切及多聚腺苷酸化特异因子6（CPSF6）在乳腺癌骨转移中的作用及机制。方法：通过实时荧光定量PCR（RT-qPCR）,免疫印迹（WB）,免疫组织化学染色（IHC）分析12例乳腺癌骨转移患者转移灶及20例乳腺癌患者原发灶CPSF6表达情况。通过细胞实验探索CPSF6对肿瘤细胞生长和转移能力的影响,通过体内转移模型探索CPSF6敲低对乳腺癌细胞骨转移能力的影响。进一步探索乳腺癌细胞通过CPSF6改造细胞外微环境的能力,并采用RT-qPCR, WB实验检验CPSF6对下游靶基因的调控作用。结果：CPSF6在乳腺癌患者转移灶中表达水平显著增高。CPSF6与乳腺癌细胞的增殖和迁移能力正相关。在231-BM细胞中敲低CPSF6后,其分泌的细胞上清促进破骨细胞前体细胞向成熟细胞分化的能力降低;体内实验中,CPSF6敲低的细胞在小鼠体内形成溶骨性病灶的能力显著降低,而且转移灶外周的破骨细胞数量显著下降。CPSF6缺失能够下调肿瘤细胞内Notch通路配体JAG1的表达水平,导致微环境中破骨前体细胞Notch通路失活和下游靶基因的抑制。我们推测肿瘤细胞在转移灶中通过高表达CPSF6,依赖Notch信号途径,促进转移灶周围破骨细胞的成熟,最终产生溶骨性病灶。结论：乳腺癌骨转移灶中存在CPSF6基因过度表达。CPSF6通过调控Notch信号通路,促进肿瘤组织周围破骨细胞的成熟和溶骨活性,从而导致乳腺癌的溶骨性骨转移。     

Platelet-derived lysophosphatidic acid supports the progression of osteolytic bone metastases in breast cancer

The role of lysophosphatidic acid (LPA) in cancer is poorly understood. Here we provide evidence for a role of LPA in the progression of breast cancer bone metastases. LPA receptors LPA(1), LPA(2), and LPA(3) were expressed in human primary breast tumors and a series of human breast cancer cell lines. The inducible overexpression of LPA(1) in MDA-BO2 breast cancer cells specifically sensitized these cells to the mitogenic action of LPA in vitro. In vivo, LPA(1) overexpression in MDA-BO2 cells enhanced the growth of subcutaneous tumor xenografts and promoted bone metastasis formation in mice by increasing both skeletal tumor growth and bone destruction. This suggested that endogenous LPA was produced in the tumor microenvironment. However, MDA-BO2 cells or transfectants did not produce LPA. Instead, they induced the release of LPA from activated platelets which, in turn, promoted tumor cell proliferation and the LPA(1)-dependent secretion of IL-6 and IL-8, 2 potent bone resorption stimulators. Moreover, platelet-derived LPA deprivation in mice, achieved by treatment with the platelet antagonist Integrilin, inhibited the progression of bone metastases caused by parental and LPA(1)-overexpressing MDA-BO2 cells and reduced the progression of osteolytic lesions in mice bearing CHO-beta3wt ovarian cancer cells. Overall, our data suggest that, at the bone metastatic site, tumor cells stimulate the production of LPA from activated platelets, which enhances both tumor growth and cytokine-mediated bone destruction.     

AP23846, a novel and highly potent Src family kinase inhibitor, reduces vascular endothelial growth factor and interleukin-8 expression in human solid tumor cell lines and abrogates downstream angiogenic processes

c-Src is frequently activated in human malignancies, including colon, breast, and pancreatic carcinomas. Several recent studies have shown that activation of Src family kinases leads to tumor progression and metastasis by increasing cellular migration and invasion, promoting cell growth and survival, and deregulating expression of proangiogenic molecules. Therefore, selective inhibitors of Src are being developed for cancer therapy. In this study, we characterize the biological effects of the novel ATP-based Src family kinase inhibitor, AP23846, in tumor cells with high Src activity. As a lead compound, AP23846 is a potent c-Src kinase inhibitor (IC50 approximately 0.5 nmol/L in vitro, approximately 10-fold more potent than PP2, the most widely used commercially available Src family kinase inhibitor). At concentrations of 1 micromol/L, AP23846 led to complete Src inhibition for 48 hours in cells. No cytotoxicity was observed under these conditions, although proliferation rates were slower. Therefore, this was an excellent inhibitor to examine Src-regulated signaling pathways in tumor cells. AP23846 reduced cellular migration, vascular endothelial growth factor, and interleukin-8 in a dose-dependent fashion in pancreatic adenocarcinoma cells grown in vitro. Correspondingly, cell culture supernatants from L3.6pl pancreatic adenocarcinoma cells pretreated with AP23846 failed to promote migration of hepatic endothelial cells in vitro and failed to support angiogenesis into gel foams implanted s.c. in mice in vivo. These results suggest that Src inhibitors affect biological properties of tumor progression and may be useful as cancer therapeutic agents in more advanced disease.     

Targeted etanercept for treatment-refractory pain due to bone metastasis: two case reports

BACKGROUND: Parallel bodies of research suggest both a central role for osteoclasts in tumor-induced destruction of bone and the ability of biologic tumor necrosis factor-alpha (TNF-alpha) antagonists to attenuate the osteoclast-mediated bone destruction that accompanies a variety of nonmalignant disorders. Additional studies have implicated TNF-alpha in the promotion of osteoclast-mediated malignant osteolysis and the pathogenesis of neuropathic pain. TNF-alpha antagonists have the potential to interfere in both processes. OBJECTIVE: This article reviews the cases of 2 patients with treatment-refractory pain due to cancer metastases to bone who were given targeted injections of the biologic anti-TNF agent etanercept based on its potential to interfere directly with both malignant activation of osteoclasts and neuropathic pain. METHODS: One patient had a diagnosis of non-small cell lung cancer and the other had a diagnosis of breast cancer. Both presented with treatment-refractory pain due to bone metastases. The 2 patients received etanercept 25 mg by targeted SC injection in anatomic proximity to the site of spinal metastasis for relief of their treatment-refractory pain. RESULTS: Both patients experienced rapid, substantial, and sustained relief of chronic refractory pain at the treatment site after targeted administration of etanercept. Symptomatic improvement was correlated with objective measures of improvement, including weight gain in 1 patient and decreased uptake of radioactive tracer at the targeted site on positron emission tomography in the other. CONCLUSIONS: Etanercept delivered by targeted SC injection may be of clinical benefit in selected patients with treatment-refractory pain caused by bone metastases. Clinical trials are needed to define the potential benefit of biologic TNF-alpha antagonists in the treatment and prevention of malignant osteolysis.     

Bone cancer pain: the effects of the bisphosphonate alendronate on pain, skeletal remodeling, tumor growth and tumor necrosis

Patients with metastatic breast, lung or prostate cancer frequently have significant bone cancer pain. In the present report we address, in a single in vivo mouse model, the effects the bisphosphonate alendronate has on bone cancer pain, bone remodeling and tumor growth and necrosis. Following injection and confinement of green fluorescent protein-transfected murine osteolytic tumor cells into the marrow space of the femur of male C3H/HeJ mice, alendronate was administered chronically from the time the tumor was established until the bone cancer pain became severe. Alendronate therapy reduced ongoing and movement-evoked bone cancer pain, bone destruction and the destruction of sensory nerve fibers that innervate the bone. Whereas, alendronate treatment did not change viable tumor burden, both tumor growth and tumor necrosis increased. These data emphasize that it is essential to utilize a model where pain, skeletal remodeling and tumor growth can be simultaneously assessed, as each of these can significantly impact patient quality of life and survival.     

Peptidomimetic Src/Pretubulin Inhibitor KX-01 Alone and in Combination with Paclitaxel Suppresses Growth, Metastasis in Human ER/PR/HER2-Negative Tumor Xenografts

Src kinase is elevated in breast tumors that are ER/PR negative and do not overexpress HER2, but clinical trials with Src inhibitors have shown little activity. The present study evaluated preclinical efficacy of a novel peptidomimetic compound, KX-01 (KX2-391), that exhibits dual action as an Src and pretubulin inhibitor. KX-01 was evaluated as a single-agent and in combination with paclitaxel in MDA-MB-231, MDA-MB-157, and MDA-MB-468 human ER/PR/HER2-negative breast cancer cells. Treatments were evaluated by growth/apoptosis, isobologram analysis, migration/invasion assays, tumor xenograft volume, metastasis, and measurement of Src, focal adhesion kinase (FAK), microtubules, Ki67, and microvessel density. KX-01 inhibited cell growth in vitro and in combination with paclitaxel resulted in synergistic growth inhibition. KX-01 resulted in a dose-dependent inhibition of MDA-MB-231 and MDA-MB-157 tumor xenografts (1 and 5 mg/kg, twice daily). KX-01 inhibited activity of Src and downstream mediator FAK in tumors that was coincident with reduced proliferation and angiogenesis and increased apoptosis. KX01 also resulted in microtubule disruption in tumors. Combination of KX-01 with paclitaxel resulted in significant regression of MDA-MB-231 tumors and reduced metastasis to mouse lung and liver. KX-01 is a potently active Src/pretubulin inhibitor that inhibits breast tumor growth and metastasis. As ER/PR/HER2-negative patients are candidates for paclitaxel therapy, combination with KX-01 may potentiate antitumor efficacy in management of this aggressive breast cancer subtype. Mol Cancer Ther; 11(9); 1936-47. ?2012 AACR.     

The dual role of thymidine phosphorylase in cancer development and chemotherapy

Thymidine phosphorylase (TP), also known as “platelet‐derived endothelial cell growth factor” (PD‐ECGF), is an enzyme, which is upregulated in a wide variety of solid tumors including breast and colorectal cancers. TP promotes tumor growth and metastasis by preventing apoptosis and inducing angiogenesis. Elevated levels of TP are associated with tumor aggressiveness and poor prognosis. Therefore, TP inhibitors are synthesized in an attempt to prevent tumor angiogenesis and metastasis. TP is also indispensable for the activation of the extensively used 5‐fluorouracil prodrug capecitabine, which is clinically used for the treatment of colon and breast cancer. Clinical trials that combine capecitabine with TP‐inducing therapies (such as taxanes or radiotherapy) suggest that increasing TP expression is an adequate strategy to enhance the antitumoral efficacy of capecitabine. Thus, TP plays a dual role in cancer development and therapy: on the one hand, TP inhibitors can abrogate the tumorigenic and metastatic properties of TP; on the other, TP activity is necessary for the activation of several chemotherapeutic drugs. This duality illustrates the complexity of the role of TP in tumor progression and in the clinical response to fluoropyrimidine‐based chemotherapy. © 2009 Wiley Periodicals, Inc. Med Res Rev, 29, No. 6, 903–953, 2009     

Rationale for clinical trials of coagulation: reactive drugs in hepatocellular carcinoma

Evidence for the regulation of cancer growth by components of the blood coagulation mechanism provides abundant opportunity for the development of novel hypotheses for the experimental treatment of malignancy. Information available on the heterogeneity in mechanisms of interaction between various cancer cell types, and procoagulant and fibrinolytic pathways, platelets, glycosaminoglycan-regulated growth factors and cell-adhesion molecules indicates that insightful clinical trial design may allow targeting of individual cancer cell types with agents capable of intercepting mechanisms of growth control that are relevant to specific tumor types. This paper reviews the evidence that the common anticoagulant, heparin, inhibits hepatocellular carcinoma cell proliferation and hepatocellular carcinoma tumor dissemination in experimental animals. Clinical trials of heparin performed to date have shown increased tumor response rates and survival in other tumor types. Expression of urokinase-type plasminogen activator by hepatocellular carcinoma cells enhances tumor cell proliferation, motility, invasiveness and metastatic dissemination. Inhibition of the urokinase-type plasminogen activator/plasmin system by protease inhibitors such as aprotinin (Trasylol, Bayer) have shown improvement in the clinical course of certain tumor types. These data suggest that drugs that are well-known in the field of vascular medicine may find a role in the treatment of hepatocellular carcinoma, a common tumor type that has resisted containment by other means.     

TGF-beta signaling pathway in pancreatic cancer cells

TGF-beta is a multifunctional cytokine which regulates cell growth, extracellular matrix deposition, cell differentiation, and immunosuppression etc. The signal is mainly mediated through Smad pathway to inhibit epithelial cell growth. In 50% of pancreatic cancer, Smad4/DPC4 gene is deleted or mutated, which might cause pancreatic carcinogenesis and be associated with highly invasive and metastatic character of the disease. On the other hand, TGF-beta signal itself has recently been shown to act in favor of cancer cells, especially in the late phase of tumor progression. In Smad4-inactivated pancreatic cancer cells, TGF-beta signal regulates a number of genes involved in tumor suppression and progression. The regulated genes and signaling pathways of TGF-beta signal should be investigated to obtain an effective therapeutic target molecule for pancreatic cancer.     

Antitumor and antimetastatic effect of warfarin and heparins.

Experimental and clinical studies have shown an anticancer effect of anticoagulant drugs. The aim of this study is to review the mechanisms by which the common types of anticoagulants influence the primary tumor and metastatic processes of solid tumors. The review evaluates the interference of unfractionated heparin (UFH), low molecular weight heparin (LMWH) and warfarin on the growth of primary tumors and on the development of metastases. The first part of the review evaluates the effect on the growth and development of primary tumors. Attention is paid to the interference with proliferation of cancer cells, tumor angiogenesis and to the interference with the immune system. The second part of the review describes the metastatic process and the effect of anticoagulants on the cell motility and cancer cell adhesion. The third part refers to the outcomes of clinical studies with anticoagulant treatment in patients with cancer. The problem of thromboembolic disease in patients with advanced cancer is also mentioned. The anticoagulants are more effective in inhibition of stages of the metastatic cascade than in the influence on primary tumors. They can interfere with tumor angiogenesis, immunity system, cancer cell motility and adhesion. The first clinical trials showed an effect on the development of primary tumors and survival of patients namely with lung cancer.     

Th17 functions as an osteoclastogenic helper T cell subset that links T cell activation and bone destruction

In autoimmune arthritis, traditionally classified as a T helper (Th) type 1 disease, the activation of T cells results in bone destruction mediated by osteoclasts, but how T cells enhance osteoclastogenesis despite the anti-osteoclastogenic effect of interferon (IFN)-gamma remains to be elucidated. Here, we examine the effect of various Th cell subsets on osteoclastogenesis and identify Th17, a specialized inflammatory subset, as an osteoclastogenic Th cell subset that links T cell activation and bone resorption. The interleukin (IL)-23-IL-17 axis, rather than the IL-12-IFN-gamma axis, is critical not only for the onset phase, but also for the bone destruction phase of autoimmune arthritis. Thus, Th17 is a powerful therapeutic target for the bone destruction associated with T cell activation.     

Rationale for clinical trials of coagulation: reactive drugs in hepatocellular carcinoma

Evidence for the regulation of cancer growth by components of the blood coagulation mechanism provides abundant opportunity for the development of novel hypotheses for the experimental treatment of malignancy. Information available on the heterogeneity in mechanisms of interaction between various cancer cell types, and procoagulant and fibrinolytic pathways, platelets, glycosaminoglycan-regulated growth factors and cell-adhesion molecules indicates that insightful clinical trial design may allow targeting of individual cancer cell types with agents capable of intercepting mechanisms of growth control that are relevant to specific tumor types. This paper reviews the evidence that the common anticoagulant, heparin, inhibits hepatocellular carcinoma cell proliferation and hepatocellular carcinoma tumor dissemination in experimental animals. Clinical trials of heparin performed to date have shown increased tumor response rates and survival in other tumor types. Expression of urokinase-type plasminogen activator by hepatocellular carcinoma cells enhances tumor cell proliferation, motility, invasiveness and metastatic dissemination. Inhibition of the urokinase-type plasminogen activator/plasmin system by protease inhibitors such as aprotinin (Trasylol^®, Bayer) have shown improvement in the clinical course of certain tumor types. These data suggest that drugs that are well-known in the field of vascular medicine may find a role in the treatment of hepatocellular carcinoma, a common tumor type that has resisted containment by other means.     

Cyclooxygenase-2 in oncogenesis

Compelling experimental and clinical evidence supports the notion that cyclooxygenase-2, the inducible isoform of cyclooxygenase, plays a crucial role in oncogenesis. Clinical and epidemiological data indicate that aberrant regulation of cyclooxygenase-2 in certain solid tumors and hematological malignancies is associated with adverse clinical outcome. Moreover, findings extrapolated from experimental studies in cultured tumor cells and animal tumor models indicate that cyclooxygenase-2 critically influences all stages of tumor development from tumor initiation to tumor progression. Cyclooxygenase-2 elicits cell-autonomous effects on tumor cells resulting in stimulation of growth, increased cell survival, enhanced tumor cell invasiveness, stimulation of neovascularization, and tumor evasion from the host immune system. Additionally, the oncogenic effects of cyclooxygenase-2 stem from its unique ability to impact tumor cell surroundings and create a proinflammatory environment conducive for tumor development, growth and progression. The initial enthusiasm generated by the availability of cyclooxygenase-2 selective inhibitors for cancer prevention and therapy has been lessened by the severe cardiovascular adverse side effects associated with their long-term use, as well as by the mixed results of recent clinical trials evaluating the efficacy of cyclooxygenase-2 inhibitors in adjuvant chemotherapy. Therefore, our ability to efficiently target the oncogenic effects of cyclooxygenase-2 for therapeutic and preventive purposes strictly depends on a better understanding of the spatial and temporal aspects of its activation in tumor cells along with a clearer elucidation of the signaling networks whereby cyclooxygenase-2 affects tumor cells and their interactions with the tumor microenvironment. This knowledge has the potential of leading to the identification of novel cyclooxygenase-2-dependent molecular and signaling networks that can be exploited to improve cancer prevention and therapy.     

恩度在中晚期非小细胞肺癌治疗中的应用现状及进展

恩度为世界首例重组人血管内皮抑制素,是一种多靶点的血管内皮抑制剂.其作用于微血管的内皮细胞,抑制其迁移,诱导其凋亡,从而抑制血管生成和肿瘤生长.已经完成的Ⅰ期和Ⅱ期临床试验表明恩度有一定的抗肿瘤活性,安全性好;Ⅲ期和Ⅳ期临床试验表明恩度联合化疗可成为NSCLC的一线方案.本文综述了恩度治疗非小细胞肺癌的临床研究和进展.     

Bisphosphonates in breast cancer: antitumor effects

Bone metastases add to the burden of breast cancer, with patients experiencing severe bone pain, pathologic fractures, spinal cord compression, and hypercalcemia of malignancy. Nitrogen-containing bisphosphonates have become the standard treatment for skeletal-related events and bone pain, as well as for bone loss associated with chemotherapy and aromatase inhibitors. Emerging preclinical and clinical evidence indicates that bisphosphonates negatively affect multiple processes that support tumor growth and proliferation and formation of metastases. Several small clinical trials suggest that bisphosphonates can modify angiogenic factors, immune surveillance, and disseminated tumor cells detected in bone marrow. Emerging data suggest that bisphosphonates used for osteoporosis prevention may inhibit breast cancer development. Three large prospective studies have shown improved outcomes with the addition of zoledronic acid to conventional neoadjuvant or adjuvant therapy. This article focuses on current clinical trials examining the use of bisphosphonates in patients with breast cancer.