RESUMEN
Osteolytic bone disease is a hallmark of multiple myeloma (MM) mediated by MM cell proliferation, increased osteoclast activity, and suppressed osteoblast function. The proteasome inhibitor bortezomib targets MM cells and improves bone health in MM patients. Radium-223 dichloride (radium-223), the first targeted alpha therapy approved, specifically targets bone metastases, where it disrupts the activity of both tumor cells and tumor-supporting bone cells in mouse models of breast and prostate cancer bone metastasis. We hypothesized that radium-223 and bortezomib combination treatment would have additive effects on MM. In vitro experiments revealed that the combination treatment inhibited MM cell proliferation and demonstrated additive efficacy. In the systemic, syngeneic 5TGM1 mouse MM model, both bortezomib and radium-223 decreased the osteolytic lesion area, and their combination was more effective than either monotherapy alone. Bortezomib decreased the number of osteoclasts at the tumor-bone interface, and the combination therapy resulted in almost complete eradication of osteoclasts. Furthermore, the combination therapy improved the incorporation of radium-223 into MM-bearing bone. Importantly, the combination therapy decreased tumor burden and restored body weights in MM mice. These results suggest that the combination of radium-223 with bortezomib could constitute a novel, effective therapy for MM and, in particular, myeloma bone disease.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Mieloma Múltiple , Neoplasias Experimentales , Animales , Bortezomib/farmacología , Línea Celular Tumoral , Humanos , Ratones , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Radioisótopos/farmacología , Radio (Elemento)/farmacologíaRESUMEN
BACKGROUND: Valyl-prolyl-proline (VPP), a bioactive peptide formed during the fermentation with Lactobacillus helveticus LBK-16H (L. helveticus), has been shown to increase bone formation in vitro. The aim of the study was to determine whether VPP and L. helveticus fermented milk prevent bone loss in ovariectomized (OVX) rats. METHODS: During the 12-week intervention study, the OVX rats received VPP in water or L. helveticus fermented milk, containing VPP. Sham-operated rats receiving water acted as controls. The trabecular and cortical bone mineral density were determined by peripheral quantitative computed tomography before the operation and at 4 and 12 weeks. The mechanical testing and ash weight analysis as well as the static and dynamic histomorphometrical parameters were assessed at the end of the intervention. RESULTS: VPP given in water showed no clear effect on bone loss. L. helveticus fermented milk prevented bone loss by decreasing bone turnover and increasing the bone mineral density. Ovariectomy caused a 57% loss in the trabecular bone, which was attenuated by 16% in the L. helveticus group. CONCLUSIONS: VPP peptide did not prevent ovariectomy-induced bone loss, which could be due to the poor bioavailability of VPP from water solution. L. helveticus fermented milk prevented bone loss, whether this is due to the VPP peptide cannot be concluded.
Asunto(s)
Densidad Ósea/efectos de los fármacos , Productos Lácteos Cultivados , Oligopéptidos/farmacología , Osteoporosis/prevención & control , Probióticos/farmacología , Animales , Femenino , Lactobacillus helveticus , Osteoporosis/etiología , Ovariectomía/efectos adversos , RatasRESUMEN
Purpose: Radium-223 dichloride (radium-223, Xofigo), a targeted alpha therapy, is currently used for the treatment of patients with castration-resistant prostate cancer (CRPC) with bone metastases. This study examines the mode-of-action and antitumor efficacy of radium-223 in two prostate cancer xenograft models.Experimental Design: Mice bearing intratibial LNCaP or LuCaP 58 tumors were randomized into groups (n = 12-17) based on lesion grade and/or serum PSA level and administered radium-223 (300 kBq/kg) or vehicle, twice at 4-week intervals. X-rays and serum samples were obtained biweekly. Soft tissue tumors were observed macroscopically at sacrifice. Tibiae were analyzed by gamma counter, micro-CT, autoradiography and histology.Results: Radium-223 inhibited tumor-induced osteoblastic bone growth and protected normal bone architecture, leading to reduced bone volume in LNCaP and abiraterone-resistant LuCaP 58 models. Furthermore, radium-223 resulted in lower PSA values and reduced total tissue and tumor areas, indicating that treatment constrains prostate cancer growth in bone. In addition, radium-223 suppressed abnormal bone metabolic activity as evidenced by decreased number of osteoblasts and osteoclasts and reduced level of the bone formation marker PINP. Mode-of-action studies revealed that radium-223 was deposited in the intratumoral bone matrix. DNA double-strand breaks were induced in cancer cells within 24 hours after radium-223 treatment, and PSA levels were significantly lower 72 hours after treatment, providing further evidence of the antitumor effects.Conclusions: Taken together, radium-223 therapy exhibits a dual targeting mode-of-action that induces tumor cell death and suppresses tumor-induced pathologic bone formation in tumor microenvironment of osseous CRPC growth in mice. Clin Cancer Res; 23(15); 4335-46. ©2017 AACR.
Asunto(s)
Antineoplásicos/administración & dosificación , Neoplasias Óseas/radioterapia , Neoplasias de la Próstata Resistentes a la Castración/radioterapia , Radio (Elemento)/administración & dosificación , Animales , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Huesos/patología , Huesos/efectos de la radiación , Línea Celular Tumoral , Proliferación Celular/efectos de la radiación , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Osteoclastos/efectos de la radiación , Neoplasias de la Próstata Resistentes a la Castración/patología , Radioisótopos/administración & dosificación , Microambiente Tumoral/efectos de la radiaciónRESUMEN
Human serum tartrate-resistant acid phosphatase exists as two enzyme isoforms (TRACP 5a and 5b), derived by differential, post-translational processing of a common gene product. Serum TRACP 5b is from bone-resorbing osteoclasts (OC) and becomes elevated in diseases of increased bone resorption. TRACP 5a is secreted by macrophages (MPhi) and dendritic cells (DC) and is increased in many patients with rheumatoid arthritis. Our purpose was to fully characterize the properties of human TRACP isoforms and to produce an antibody specific to TRACP 5a for use as a biomarker in chronic inflammatory diseases. Partially purified, natural serum TRACP isoforms and recombinant TRACP 5a (rTRACP 5a) were compared with respect to specific activity and subunit structure and presence of sialic acid. Mice were immunized with rTRACP 5a, and resulting hybridomas were screened for monoclonal antibody to serum TRACP 5a. One antibody, 220, was tested for its epitope specificity and use in various immunological techniques. rTRACP 5a had properties identical to serum TRACP 5a. Antibody 220 was specific for the trypsin-sensitive epitope in the loop peptide, present only in TRACP 5a. Antibody 220 was effective for specific immunoprecipitation, immunoassay, and immunoblot of TRACP 5a. Intact TRACP was present in MPhi, DC, and OC. TRACP 5a was the predominant isoform secreted by MPhi and DC, whereas TRACP 5b was the predominant isoform secreted by OC. TRACP isoforms 5a and 5b may have different functions inside and outside of monocyte-derived cells. Antibody 220 is an important resource for studies of the biosynthetic relationship among TRACP isoforms and of the significance of serum TRACP 5a as a marker in diseases of bone metabolism and inflammation.
Asunto(s)
Fosfatasa Ácida/química , Fosfatasa Ácida/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Monocitos/enzimología , Fosfatasa Ácida/genética , Animales , Anticuerpos Monoclonales/biosíntesis , Células Cultivadas , Células Dendríticas/enzimología , Epítopos/metabolismo , Femenino , Regulación Enzimológica de la Expresión Génica , Humanos , Isoenzimas/genética , Macrófagos/enzimología , Ratones , Ratones Endogámicos BALB C , Monocitos/citología , Osteoclastos/enzimología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sensibilidad y Especificidad , Fosfatasa Ácida TartratorresistenteRESUMEN
BACKGROUND: Bone metastases are associated with increased morbidity and poor prognosis in breast cancer patients. Radium-223 dichloride is a calcium mimetic that localizes to bone, providing targeted therapy for skeletal metastasis. METHODS: We investigated the mode of action of radium-223 dichloride using breast cancer cell, osteoclast, and osteoblast cultures as well as a mouse model of breast cancer bone metastasis. A single dose of radium-223 dichloride was used in three different settings mimicking the prevention or treatment of bone metastasis. Disease progression was monitored using fluorescence and radiographic imaging and histological analyses. The effect of radium-223 dichloride alone and in combination with doxorubicin or zoledronic acid on survival of mice was analyzed by Kaplan-Meier methods. All statistical tests used were two-sided. RESULTS: Radium-223 dichloride incorporated into bone matrix and inhibited proliferation of breast cancer cells and differentiation of osteoblasts and osteoclasts (all P values < .001) in vitro. In an established bone metastasis setting, radium-223 dichloride prevented tumor-induced cachexia (0/14 vs 7/14 control mice) and decreased osteolysis by 56% and tumor growth by 43% (all P values < .05). Radium-223 dichloride induced double-strand DNA breaks in cancer cells in vivo. Finally, radium-223 dichloride extended survival as a monotherapy (29.2 days, 95% confidence interval [CI] = 26.6 to 31.8 days, P = .039) and in combination with zoledronic acid (31.4 days, 95% CI = 28.8 to 34.0 days, P = .004) or doxorubicin (31.5 days, 95% CI = 29.5 to 33.5 days, P < .001) compared to the vehicle group (24.9 days, 95% CI = 23.4 to 26.4 days). Similar but even more pronounced effects were observed when radium-223 dichloride was administered in a preventive or micrometastatic setting. CONCLUSIONS: Our findings strongly support the development of radium-223 dichloride for the treatment of breast cancer patients with or at high risk of developing bone metastases.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/secundario , Neoplasias de la Mama/patología , Caquexia/prevención & control , ADN de Neoplasias/efectos de los fármacos , Radio (Elemento)/farmacología , Animales , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Conservadores de la Densidad Ósea/administración & dosificación , Neoplasias Óseas/complicaciones , Neoplasias Óseas/metabolismo , Neoplasias Óseas/prevención & control , Caquexia/diagnóstico , Caquexia/etiología , Proliferación Celular/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de los fármacos , Difosfonatos/administración & dosificación , Modelos Animales de Enfermedad , Doxorrubicina/administración & dosificación , Femenino , Imidazoles/administración & dosificación , Estimación de Kaplan-Meier , Ratones , Osteoblastos/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Radioisótopos/farmacología , Radioisótopos/uso terapéutico , Radio (Elemento)/uso terapéutico , Células Madre/efectos de los fármacos , Células Tumorales Cultivadas , Ácido ZoledrónicoRESUMEN
Cancer progression has been associated with the presence of tumor-associated M2-macrophages (M2-TAMs) able to inhibit anti-tumor immune responses. It is also often associated with metastasis-induced bone destruction mediated by osteoclasts. Both cell types are controlled by the CD115 (CSF-1R)/colony-stimulating factor-1 (CSF-1, M-CSF) pathway, making CD115 a promising target for cancer therapy. Anti-human CD115 monoclonal antibodies (mAbs) that inhibit the receptor function have been generated in a number of laboratories. These mAbs compete with CSF-1 binding to CD115, dramatically affecting monocyte survival and preventing osteoclast and macrophage differentiation, but they also block CD115/CSF-1 internalization and degradation, which could lead to potent rebound CSF-1 effects in patients after mAb treatment has ended. We thus generated and selected a non-ligand competitive anti-CD115 mAb that exerts only partial inhibitory effects on CD115 signaling without blocking the internalization or the degradation of the CD115/CSF-1 complex. This mAb, H27K15, affects monocyte survival only minimally, but downregulates osteoclast differentiation and activity. Importantly, it inhibits monocyte differentiation to CD163(+)CD64(+) M2-polarized suppressor macrophages, skewing their differentiation toward CD14(-)CD1a(+) dendritic cells (DCs). In line with this observation, H27K15 also drastically inhibits monocyte chemotactic protein-1 secretion and reduces interleukin-6 production; these two molecules are known to be involved in M2-macrophage recruitment. Thus, the non-depleting mAb H27K15 is a promising anti-tumor candidate, able to inhibit osteoclast differentiation, likely decreasing metastasis-induced osteolysis, and able to prevent M2 polarization of TAMs while inducing DCs, hence contributing to the creation of more efficient anti-tumor immune responses.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Diferenciación Celular/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Macrófagos/efectos de los fármacos , Monocitos/efectos de los fármacos , Osteólisis/prevención & control , Animales , Anticuerpos Monoclonales/inmunología , Diferenciación Celular/inmunología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Células Cultivadas , Quimiocina CCL2/inmunología , Quimiocina CCL2/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Citometría de Flujo , Células HL-60 , Humanos , Interleucina-6/inmunología , Interleucina-6/metabolismo , Factor Estimulante de Colonias de Macrófagos/inmunología , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Monocitos/inmunología , Monocitos/metabolismo , Células 3T3 NIH , Osteoclastos/efectos de los fármacos , Osteoclastos/inmunología , Osteoclastos/metabolismo , Osteólisis/inmunología , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Unión Proteica/inmunología , Receptor de Factor Estimulante de Colonias de Macrófagos/inmunología , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunologíaRESUMEN
TGF-ß regulates several steps in cancer metastasis, including the establishment of bone metastatic lesions. TGF-ß is released from bone during osteoclastic bone resorption and it stimulates breast cancer cells to produce osteolytic factors such as interleukin 11 (IL-11). We conducted a cell-based siRNA screen and identified heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) as a critical gene for TGF-ß-induced IL-11 production in highly bone metastatic MDA-MB-231(SA) breast cancer cells. HS6ST2 attaches sulfate groups to glucosamine residues in heparan sulfate glycosaminoglycans. We subsequently showed how heparin and a high-molecular-weight Escherichia coli K5-derived heparin-like polysaccharide (K5-NSOS) inhibited TGF-ß-induced IL-11 production in MDA-MB-231(SA) cells. In addition, K5-NSOS inhibited bone resorption activity of human osteoclasts in vitro. We evaluated the therapeutic potential of K5-NSOS and fragmin in a mouse model of breast cancer bone metastasis. MDA-MB-231(SA) cells were inoculated into the left cardiac ventricle of athymic nude mice which were treated with fragmin, K5-NSOS, or vehicle once a day for four weeks. Both heparin-like glycosaminoglycans inhibited weight reduction, decreased osteolytic lesion area, and reduced tumor burden in bone. In conclusion, our data imply novel mechanisms involved in TGF-ß induction and support the critical role of heparan sulfate glycosaminoglycans in cancer metastasis as well as indicate that K5-NSOS is a potential antimetastatic and antiresorptive agent for cancer therapy. This study illustrates the potential to translate in vitro siRNA screening results toward in vivo therapeutic concepts.
Asunto(s)
Cápsulas Bacterianas , Heparina , Osteoclastos/efectos de los fármacos , Sulfotransferasas , Animales , Cápsulas Bacterianas/química , Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Resorción Ósea/genética , Resorción Ósea/patología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Heparina/química , Heparina/farmacología , Humanos , Interleucina-11/antagonistas & inhibidores , Interleucina-11/metabolismo , Ratones , Ratones Desnudos , ARN Interferente Pequeño , Sulfotransferasas/antagonistas & inhibidores , Sulfotransferasas/genética , Sulfotransferasas/metabolismo , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
IMPORTANCE OF THE FIELD: Osteoporosis affects nearly 100 million people in Europe, Japan and the US, and the number is increasing due to aging of the population. Preclinical efficacy studies performed according to regulatory guidelines are large, long and expensive, and there is a need for guidance and recommendations on how to perform preliminary studies prior to the regulatory studies. AREAS COVERED IN THIS REVIEW: We review research models that can be used for preclinical efficacy testing of new drug candidates for osteoporosis. Our focus is on testing compounds targeted to directly decrease osteoclastic bone resorption or increase osteoblastic bone formation. WHAT THE READER WILL GAIN: We provide an overview of in vitro bone cell culture systems and osteoporosis animal models useful for preclinical efficacy studies and a step-by-step approach on how the most interesting compound can be selected from thousands of drug candidates. Different approaches for testing anti-catabolic and anabolic compounds are provided. TAKE HOME MESSAGE: Efficacy of new osteoporosis drug candidates can be first proven conveniently using in vitro bone cell cultures and then confirmed in short-term animal studies, followed by more extensive animal studies, and finally a regulatory study performed according to the guidelines of regulatory authorities.
RESUMEN
We cultured human bone marrow-derived stem cells on bovine bone slices in 96-well plates in the presence of M-CSF and RANKL, allowing them to differentiate into osteoclasts. Secreted TRACP 5b was a useful endpoint measurement to demonstrate effects of inhibitors of osteoclast differentiation in the culture system, reflecting accurately the number of formed osteoclasts. Inhibitors of osteoclast activity were added into the cultures after the differentiation period, and the cultures were continued to allow the formed osteoclasts to resorb bone. CTX values obtained after the resorption period were normalized with TRACP 5b values obtained after the differentiation period, before adding the inhibitors. This normalization prevents false results that could be obtained from the presence of different amounts of osteoclasts in different wells before adding the inhibitors. These results demonstrate that the use of TRACP 5b and CTX allows rapid and reliable testing of antiresorptive compounds in human osteoclast cultures.
Asunto(s)
Conservadores de la Densidad Ósea/farmacología , Osteoclastos/efectos de los fármacos , Fosfatasa Ácida/metabolismo , Animales , Conservadores de la Densidad Ósea/uso terapéutico , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/fisiopatología , Bovinos , Células Cultivadas , Colágeno Tipo I/metabolismo , Humanos , Isoenzimas/metabolismo , Osteoclastos/citología , Osteoclastos/metabolismo , Fosfatasa Ácida TartratorresistenteRESUMEN
PURPOSE: To study the effects of estrogen withdrawal on osteoclast number and osteoclast activity in the rat ovariectomy (OVX) model. METHODS: We first cultured human CD34+ osteoclast precursor cells on bovine bone slices, allowing them to differentiate into mature resorbing osteoclasts. Secreted tartrate-resistant acid phosphatase 5b (TRACP 5b) and C-terminal cross-linked telopeptides of type I collagen (CTX) were determined from the culture medium. TRACP 5b correlated strongly with osteoclast number and CTX with osteoclast activity, facilitating their subsequent use in the rat OVX model. An 8 week OVX study was then performed including sham-operated rats receiving vehicle, OVX rats receiving vehicle, and OVX rats receiving 10 microg/kg/day 17 beta-estradiol (E2). Trabecular bone parameters were determined from the tibial metaphysis using peripheral quantitative computed tomography and histomorphometry. Osteoclast number was normalized with bone perimeter (N.Oc/B.Pm) and tissue area (N.Oc/T.Ar, indicating absolute number of osteoclasts). TRACP 5b and CTX were determined from fasting serum samples. RESULTS: Trabecular bone parameters indicated substantial bone loss after OVX that was prevented by E2. N.Oc/B.Pm increased after OVX, while N.Oc/T.Ar and TRACP 5b decreased, and TRACP 5b correlated strongly with N.Oc/T.Ar. However, CTX values increased after OVX, and the "resorption index" CTX/TRACP 5b showed more substantial changes than either CTX or TRACP 5b alone. CONCLUSION: These results show that TRACP 5b is a reliable marker of osteoclast number, and the index CTX/TRACP 5b is a useful parameter in rat OVX model. The high elevation of CTX/TRACP 5b values by OVX demonstrates that estrogen withdrawal generates high activity of osteoclasts in the rat OVX model.
Asunto(s)
Fosfatasa Ácida/sangre , Biomarcadores/sangre , Resorción Ósea , Isoenzimas/sangre , Osteoclastos/citología , Osteoclastos/metabolismo , Ovariectomía , Animales , Bovinos , Recuento de Células , Células Cultivadas , Modelos Animales de Enfermedad , Estradiol/farmacología , Femenino , Humanos , Osteoclastos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Fosfatasa Ácida TartratorresistenteRESUMEN
Serum procollagen I N-terminal propeptide (PINP) is a sensitive bone formation marker in humans. We have developed a nonradioactive immunoassay for rat PINP and studied PINP as a bone formation marker in the rat ovariectomy (OVX) model. Two OVX studies were performed with 3-month-old rats, both including measurement of PINP, C-terminal cross-linked telopeptide of type I collagen (CTX), and N-terminal mid-fragment of osteocalcin. A pilot 14-day study contained a sham-operated control group and an OVX group, and an extensive 8-week study contained a sham-operated control group and OVX groups receiving vehicle and 17 beta-estradiol (E2, 10 microg/kg/day s.c.). The bone markers were measured before the operation and at days 2, 4, 7, 10, and 14 in the pilot study and before the operations and at 2 and 8 weeks in the extensive study. Trabecular bone parameters were determined by peripheral quantitative computed tomography and histomorphometry from tibial metaphysis in the extensive study. The rat PINP immunoassay had the following characteristics: intra-assay coefficient of variation (CV) 2.8%, interassay CV 7.5%, dilution linearity 95%, and recovery 107%. PINP increased significantly during the first 2 weeks after OVX and returned to sham level at 8 weeks. E2 prevented the increase caused by OVX. Changes in PINP at 2 weeks correlated strongly with changes in CTX and osteocalcin at 2 weeks and with trabecular bone parameters at 8 weeks. As a conclusion, short-term changes in PINP predict long-term changes in trabecular bone parameters, suggesting that PINP is a reliable marker of bone formation in the rat OVX model.