RESUMEN
An increasing number of studies have demonstrated the significant roles of the interplay between microenvironmental mechanics in tissues and biochemical-genetic activities in resident tumor cells at different stages of tumor progression. Mediated by molecular mechano-sensors or -transducers, biomechanical cues in tissue microenvironments are transmitted into the tumor cells and regulate biochemical responses and gene expression through mechanotransduction processes. However, the molecular interplay between the mechanotransduction processes and intracellular biochemical signaling pathways remains elusive. This paper reviews the recent advances in understanding the crosstalk between biomechanical cues and three critical biochemical effectors during tumor progression: calcium ions (Ca2+), yes-associated protein (YAP), and microRNAs (miRNAs). We address the molecular mechanisms underpinning the interplay between the mechanotransduction pathways and each of the three effectors. Furthermore, we discuss the functional interactions among the three effectors in the context of soft matter and mechanobiology. We conclude by proposing future directions on studying the tumor mechanobiology that can employ Ca2+, YAP, and miRNAs as novel strategies for cancer mechanotheraputics. This framework has the potential to bring insights into the development of novel next-generation cancer therapies to suppress and treat tumors.
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MicroARNs , Neoplasias , Biofisica , Calcio , Humanos , Mecanotransducción Celular , MicroARNs/genética , Neoplasias/genética , Microambiente TumoralRESUMEN
Many signaling pathways are dysregulated in cancer cells and the host tumor microenvironment. Aberrant receptor tyrosine kinase (RTK) pathways promote cancer development, progression, and metastasis. Hence, numerous therapeutic interventions targeting RTKs have been actively pursued. Axl is an RTK that belongs to the Tyro3, Axl, MerTK (TAM) subfamily. Axl binds to a high affinity ligand growth arrest specific 6 (Gas6) that belongs to the vitamin K-dependent family of proteins. The Gas6/Axl signaling pathway has been implicated to promote progression, metastasis, immune evasion, and therapeutic resistance in many cancer types. Therapeutic agents targeting Gas6 and Axl have been developed, and promising results have been observed in both preclinical and clinical settings when such agents are used alone or in combination therapy. This review examines the current state of therapeutics targeting the Gas6/Axl pathway in cancer and discusses Gas6- and Axl-targeting agents that have been evaluated preclinically and clinically.
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Péptidos y Proteínas de Señalización Intercelular/metabolismo , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Animales , Productos Biológicos/uso terapéutico , Humanos , Tirosina Quinasa del Receptor AxlRESUMEN
An imbalance in oxygen delivery to demand in solid tumors results in local areas of hypoxia leading to poor prognosis for the patient. We hypothesize that aerobic exercise increases tumor blood flow, recruits previously nonperfused tumor blood vessels, and thereby augments blood-tumor O2 transport and diminishes tumor hypoxia. When combined with conventional anticancer treatments, aerobic exercise can significantly improve the outcomes for several types of cancers.
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Ejercicio Físico/fisiología , Neoplasias/fisiopatología , Neoplasias/terapia , Microambiente Tumoral/fisiología , Humanos , Hipoxia/fisiopatología , Neoplasias/irrigación sanguínea , Consumo de Oxígeno/fisiologíaRESUMEN
It is estimated that approximately 90% of patients with advanced prostate cancer develop bone metastases; an occurrence that results in a substantial reduction in the quality of life and a drastic worsening of prognosis. The development of novel therapeutic strategies that impair the metastatic process and associated skeletal adversities is therefore critical to improving prostate cancer patient survival. Recognition of the importance of Cathepsin L (CTSL) to metastatic dissemination of cancer cells has led to the development of several CTSL inhibition strategies. The present investigation employed intra-cardiac injection of human PC-3ML prostate cancer cells into nude mice to examine tumor cell dissemination in a preclinical bone metastasis model. CTSL knockdown confirmed the validity of targeting this protease and subsequent intervention studies with the small molecule CTSL inhibitor KGP94 resulted in a significant reduction in metastatic tumor burden in the bone and an improvement in overall survival. CTSL inhibition by KGP94 also led to a significant impairment of tumor initiated angiogenesis. Furthermore, KGP94 treatment decreased osteoclast formation and bone resorptive function, thus, perturbing the reciprocal interactions between tumor cells and osteoclasts within the bone microenvironment which typically result in bone loss and aggressive growth of metastases. These functional effects were accompanied by a significant downregulation of NFκB signaling activity and expression of osteoclastogenesis related NFκB target genes. Collectively, these data indicate that the CTSL inhibitor KGP94 has the potential to alleviate metastatic disease progression and associated skeletal morbidities and hence may have utility in the treatment of advanced prostate cancer patients.
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Neoplasias Óseas/genética , Catepsina L/genética , Osteoclastos/metabolismo , Neoplasias de la Próstata/genética , Animales , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Resorción Ósea/genética , Resorción Ósea/patología , Catepsina L/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Masculino , Ratones , Metástasis de la Neoplasia , Osteoclastos/patología , Neoplasias de la Próstata/patología , Tiosemicarbazonas/administración & dosificación , Tiourea/administración & dosificación , Tiourea/análogos & derivados , Carga Tumoral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
VEGF is a key regulator of normal and pathologic angiogenesis. Although many trans-activating factors of VEGF have been described, the transcriptional repression of VEGF remains much less understood. We have previously reported the identification of a SCAN domain-containing C2H2 zinc finger protein, ZNF24, that represses the transcription of VEGF. In the present study, we identify the mechanism by which ZNF24 represses VEGF transcription. Using reporter gene and electrophoretic mobility shift assays, we identify an 11-bp fragment of the proximal VEGF promoter as the ZNF24-binding site that is essential for ZNF24-mediated repression. We demonstrate in 2 in vivo models the potent inhibitory effect of ZNF24 on the vasculature. Expression of human ZNF24 induced in vivo vascular defects consistent with those induced by VEGF knockdown using a transgenic zebrafish model. These defects could be rescued by VEGF overexpression. Overexpression of ZNF24 in human breast cancer cells also inhibited tumor angiogenesis in an in vivo tumor model. Analyses of human breast cancer tissues showed that ZNF24 and VEGF levels were inversely correlated in malignant compared with normal tissues. These data demonstrate that ZNF24 represses VEGF transcription through direct binding to an 11-bp fragment of the VEGF proximal promoter and that it functions as a negative regulator of tumor growth by inhibiting angiogenesis.
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Vasos Sanguíneos/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas Represoras/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Sitios de Unión , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Regulación de la Expresión Génica , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Represoras/genética , Activación Transcripcional , Factor A de Crecimiento Endotelial Vascular/metabolismo , Pez CebraRESUMEN
Upregulation of cathepsin L in a variety of tumors and its ability to promote cancer cell invasion and migration through degradation of the extracellular matrix suggest that cathepsin L is a promising biological target for the development of anti-metastatic agents. Based on encouraging results from studies on benzophenone thiosemicarbazone cathepsin inhibitors, a series of fourteen benzoylbenzophenone thiosemicarbazone analogues were designed, synthesized, and evaluated for their inhibitory activity against cathepsins L and B. Thiosemicarbazone inhibitors 3-benzoylbenzophenone thiosemicarbazone 1, 1,3-bis(4-fluorobenzoyl)benzene thiosemicarbazone 8, and 1,3-bis(2-fluorobenzoyl)-5-bromobenzene thiosemicarbazone 32 displayed the greatest potency against cathepsin L with low IC50 values of 9.9 nM, 14.4 nM, and 8.1 nM, respectively. The benzoylbenzophenone thiosemicarbazone analogues evaluated were selective in their inhibition of cathepsin L compared to cathepsin B. Thiosemicarbazone analogue 32 inhibited invasion through Matrigel of MDA-MB-231 breast cancer cells by 70% at 10 µM. Thiosemicarbazone analogue 8 significantly inhibited the invasive potential of PC-3ML prostate cancer cells by 92% at 5 µM. The most active cathepsin L inhibitors from this benzoylbenzophenone thiosemicarbazone series (1, 8, and 32) displayed low cytotoxicity toward normal primary cells [in this case human umbilical vein endothelial cells (HUVECs)]. In an initial in vivo study, 3-benzoylbenzophenone thiosemicarbazone (1) was well-tolerated in a CDF1 mouse model bearing an implanted C3H mammary carcinoma, and showed efficacy in tumor growth delay. Low cytotoxicity, inhibition of cell invasion, and in vivo tolerability are desirable characteristics for anti-metastatic agents functioning through an inhibition of cathepsin L. Active members of this structurally diverse group of benzoylbenzophenone thiosemicarbazone cathepsin L inhibitors show promise as potential anti-metastatic, pre-clinical drug candidates.
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Antineoplásicos/síntesis química , Catepsina L/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/síntesis química , Tiosemicarbazonas/química , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Benzofenonas/química , Sitios de Unión , Catepsina B/antagonistas & inhibidores , Catepsina B/metabolismo , Catepsina L/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Inhibidores de Cisteína Proteinasa/uso terapéutico , Diseño de Fármacos , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Concentración 50 Inhibidora , Isomerismo , Cinética , Neoplasias Mamarias Animales/tratamiento farmacológico , Ratones , Simulación del Acoplamiento Molecular , Estructura Terciaria de Proteína , Tiosemicarbazonas/farmacología , Tiosemicarbazonas/uso terapéutico , Trasplante HeterólogoRESUMEN
Osteosarcoma is a highly fatal cancer, with most patients ultimately succumbing to metastatic disease. The purpose of this study was to evaluate the effects of the antirheumatoid drug aurothiomalate on canine and human osteosarcoma cells and on canine osteosarcoma growth and metastasis in a mouse xenograft model. We hypothesized that aurothiomalate would decrease osteosarcoma cell survival, tumor cellular proliferation, tumor growth, and metastasis. After performing clonogenic assays, aurothiomalate or a placebo was administered to 54 mice inoculated with canine osteosarcoma. Survival, tumor growth, embolization, metastasis, histopathology, cell proliferation marker Ki67, and apoptosis marker caspase-3 were compared between groups. Statistical analysis was carried out using the Kaplan-Meier method with the log-rank test and one-way analysis of variance with the Tukey's test or Dunn's method. Aurothiomalate caused dose-dependent inhibition of osteosarcoma cell survival (P<0.001) and decreased tumor growth (P<0.001). Pulmonary macrometastasis and Ki67 labeling were reduced with low-dose aurothiomalate (P=0.033 and 0.005, respectively), and tumor emboli and pulmonary micrometastases were decreased with high-dose aurothiomalate (P=0.010 and 0.011, respectively). There was no difference in survival, tumor development, ulceration, mitotic indices, tumor necrosis, nonpulmonary metastases, and caspase-3 labeling. Aurothiomalate treatment inhibited osteosarcoma cell survival and reduced tumor cell proliferation, growth, embolization, and pulmonary metastasis. Given aurothiomalate's established utility in canine and human medicine, our results suggest that this compound may hold promise as an adjunctive therapy for osteosarcoma. Further translational research is warranted to better characterize the dose response of canine and human osteosarcoma to aurothiomalate.
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Antineoplásicos/uso terapéutico , Neoplasias Óseas/tratamiento farmacológico , Tiomalato Sódico de Oro/uso terapéutico , Osteosarcoma/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias Óseas/patología , Caspasa 3/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Perros , Tiomalato Sódico de Oro/farmacología , Humanos , Antígeno Ki-67/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/secundario , Ratones , Osteosarcoma/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Angiogenesis is meticulously controlled by a fine balance between positive and negative regulatory activities. Vascular endothelial growth factor (VEGF) is a predominant angiogenic factor and its dosage is precisely regulated during normal vascular formation. In cancer, VEGF is commonly overproduced, resulting in abnormal neovascularization. VEGF is induced in response to various stimuli including hypoxia; however, very little is known about the mechanisms that confine its induction to ensure proper angiogenesis. Chromatin insulation is a key transcription mechanism that prevents promiscuous gene activation by interfering with the action of enhancers. Here we show that the chromatin insulator-binding factor CTCF binds to the proximal promoter of VEGF. Consistent with the enhancer-blocking mode of chromatin insulators, CTCF has little effect on basal expression of VEGF but specifically affects its activation by enhancers. CTCF knockdown cells are sensitized for induction of VEGF and exhibit elevated proangiogenic potential. Cancer-derived CTCF missense mutants are mostly defective in blocking enhancers at the VEGF locus. Moreover, during mouse retinal development, depletion of CTCF causes excess angiogenesis. Therefore, CTCF-mediated chromatin insulation acts as a crucial safeguard against hyperactivation of angiogenesis.
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Cromatina/metabolismo , Elementos Aisladores/genética , Neovascularización Patológica/genética , Proteínas Represoras/metabolismo , Dedos de Zinc/genética , Animales , Factor de Unión a CCCTC , Línea Celular , Elementos de Facilitación Genéticos/genética , Genes Reporteros/genética , Humanos , Ratones , Neoplasias/irrigación sanguínea , Neoplasias/patología , Neovascularización Patológica/patología , Regiones Promotoras Genéticas/genética , Unión Proteica , Retina/crecimiento & desarrollo , Retina/patología , Transcripción Genética , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Background: Previous graduate students and postdoctoral associates from the University of Florida Health Cancer Center, in partnership with the University of Florida Student Science Training Program, implemented a cooperative learning curriculum, providing high school students with a broad overview of cancer topics over six weeks over the summer. However, the ongoing shift in education and training delivery initially necessitated by the COVID-19 pandemic has given rise to many discussions surrounding student autonomy and satisfaction. Furthermore, adapting hybrid and distance learning styles has notably influenced student-led collaboration and critical thinking skills. Here, we report on an update of experiences gleaned from the modified curriculum of this course to accommodate hybrid and cooperative teaching. Methods: This pre-post longitudinal observational study evaluated modifications to a cancer biology and therapeutics course. Student performance was assessed using surveys administered before and after the course to determine effectiveness. Results: Student performance tracked over a 7-year period indicated improved cumulative grade averages after modifying the previous curriculum. Post-assessment analysis revealed significant improvements in student benchmark understanding, notably in their ability to define cancer in one sentence (p = 0.0407), identify cancer therapies (p = 0.0040), and recognize cancer hallmarks (p < 0.0001). An increased trend in median response to the likelihood of pursuing cancer research (p = 0.8793) and the possibility of pursuing cancer research (p = 0.4874) were also observed, although not statistically significant. Moreover, feedback from participating students indicated that "the educational activities at the end of class (e.g., escape room, case studies)" and "learning about cancer and getting to work in groups " the curriculum fostered a positive educational learning environment. Conclusion: Students generally held a positive perception of the course. Post-assessment analysis revealed decreasing trends in students' perception of course difficulty compared to their expectations in the pre-assessment. Constructive feedback centered around fostering collaborative environments, with an observed increase in learner autonomy towards the end of the course, as evidenced by students' growing comfort in leading group case studies and conducting research on topics. We hope that future course directors engage actively and incorporate practical clinical examples for students, especially when introducing or discussing complex issues like cancer.
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Circulating tumor cells (CTCs) have gathered attention as a biomarker for carcinomas. However, CTCs in sarcomas have received little attention. In this work, we investigated cell surface proteins and antibody combinations for immunofluorescence detection of sarcoma CTCs. A microfluidic device that combines filtration and immunoaffinity using gangliosides 2 and cell surface vimentin (CSV) antibodies was employed to capture CTCs. For CTC detection, antibodies against cytokeratins 7 and 8 (CK), pan-cytokeratin (panCK), or a combination of panCK and CSV were used. Thirty-nine blood samples were collected from 21 patients of various sarcoma subtypes. In the independent samples study, samples were subjected to one of three antibody combination choices. Significant difference in CTC enumeration was found between CK and panCK + CSV, and between panCK and panCK + CSV. Upon stratification of CK+ samples, those of metastatic disease had a higher CTC number than those of localized disease. In the paired samples study involving cytokeratin-positive sarcoma subtypes, using panCK antibody detected more CTCs than CK. Similarly, for osteosarcoma, using panCK + CSV combination resulted in a higher CTC count than panCK. This study emphasized deliberate selection of cell surface proteins for sarcoma CTC detection and subtype stratification for studying cancers as heterogeneous as sarcomas.
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Biomarcadores de Tumor , Células Neoplásicas Circulantes , Sarcoma , Humanos , Células Neoplásicas Circulantes/patología , Células Neoplásicas Circulantes/metabolismo , Sarcoma/patología , Sarcoma/sangre , Sarcoma/diagnóstico , Sarcoma/metabolismo , Biomarcadores de Tumor/sangre , Femenino , Masculino , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/inmunología , Queratinas/inmunología , Queratinas/metabolismo , Persona de Mediana Edad , Adulto , Vimentina/metabolismo , Vimentina/inmunología , Anciano , Anticuerpos/inmunología , Línea Celular TumoralRESUMEN
Biomimetic tumor microenvironment models bridge the gap between in vitro and in vivo systems and serve as a useful way to address the modeling challenge of how to recreate the cell and system complexity associated with real tissues. Our laboratory has developed an ex vivo rat mesentery culture model, which allows for simultaneous investigation of blood and lymphatic microvascular network remodeling in an intact tissue environment. Given that angiogenesis and lymphangiogenesis are key contributors to the progression of cancer, the objective of this study was to combine tissue and tumor spheroid culture methods to establish a novel ex vivo tumor spheroid-tissue model by verifying its use for evaluating the effects of cancer cell behavior on the local microvascular environment. H1299 or A549 tumor spheroids were formed via hanging drop culture and seeded onto rat mesenteric tissues harvested from adult male Wistar rats. Tissues with transplanted spheroids were cultured in serum-free media for 3 to 5 days. PECAM, NG2, CD11b, and αSMA labeling identified endothelial cells, pericytes, immune cells, and smooth muscle cells, respectively. Time-lapse imaging confirmed cancer cell type specific migration. In addition to increasing PECAM positive capillary sprouting and LYVE-1 positive endothelial cell extensions indicative of lymphangiogenesis, tumor spheroid presence induced the formation of lymphatic/blood vessel connections and the formation of hybrid, mosaic vessels that were characterized by discontinuous LYVE-1 labeling. The results support the application of a novel tumor spheroid microenvironment model for investigating cancer cell-microvascular interactions.
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Vasos Linfáticos , Ratas Wistar , Esferoides Celulares , Microambiente Tumoral , Animales , Esferoides Celulares/patología , Humanos , Masculino , Ratas , Vasos Linfáticos/patología , Vasos Linfáticos/fisiopatología , Linfangiogénesis , Línea Celular Tumoral , Neovascularización Patológica/patología , Remodelación Vascular , Microvasos/patología , Células A549RESUMEN
Cancer stem cells (CSC) represent a malignant subpopulation of cells in hierarchically organized tumors. They constitute a subpopulation of malignant cells within a tumor mass and possess the ability to self-renew giving rise to heterogeneous tumor cell populations with a complex set of differentiated tumor cells. CSC may be the cause of metastasis and therapeutic refractory disease. Because few markers exist to identify and isolate pure CSC, we used cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX) to create DNA aptamers that can identify novel molecular targets on the surfaces of live CSC. Out of 22 putative DNA sequences, 3 bound to ~90% and 5 bound to ~15% of DU145 prostate cancer cells. The 15% of cells that were positive for the second panel of aptamers expressed high levels of E-cadherin and CD44, had high aldehyde dehydrogenase 1 activity, grew as spheroids under nonadherent culture conditions, and initiated tumors in immune-compromised mice. The discovery of the molecular targets of these aptamers could reveal novel CSC biomarkers.
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Biomarcadores de Tumor/metabolismo , Sondas Moleculares , Células Madre Neoplásicas/patología , Neoplasias de la Próstata/metabolismo , Animales , Aptámeros de Nucleótidos , Citometría de Flujo , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunofenotipificación , Masculino , Ratones , Neoplasias de la Próstata/diagnóstico , Técnica SELEX de Producción de Aptámeros , Esferoides Celulares , Células Tumorales CultivadasRESUMEN
Genetically encoded calcium indicators (GECIs) and high-resolution confocal microscopy enable dynamic visualization of calcium signals in cells and tissues. Two-dimensional and 3D biocompatible materials mimic the mechanical microenvironments of tumor and healthy tissues in a programmable manner. Cancer xenograft models and ex vivo functional imaging of tumor slices reveal physiologically relevant functions of calcium dynamics in tumors at different progression stages. Integration of these powerful techniques allows us to quantify, diagnose, model, and understand cancer pathobiology. Here, we describe detailed materials and methods used to establish this integrated interrogation platform, from generating transduced cancer cell lines that stably express CaViar (GCaMP5G + QuasAr2) to in vitro and ex vivo calcium imaging of the cells in 2D/3D hydrogels and tumor tissues. These tools open the possibility for detailed explorations of mechano-electro-chemical network dynamics in living systems.
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Calcio , Neoplasias , Humanos , Calcio/metabolismo , Línea Celular , Indicadores y Reactivos , Colorantes , Microscopía Fluorescente/métodos , Neoplasias/genética , Señalización del Calcio/fisiología , Microambiente TumoralRESUMEN
Although solid tumors continuously shed cells, only a small fraction of the neoplastic cells that enter the blood stream are capable of establishing metastases. In order to be successful, these cells must attach, extravasate, proliferate and induce angiogenesis. Preclinical studies have shown that small-molecule ATP-competitive Src kinase inhibitors can effectively impair metastasis-associated tumor cell functions in vitro. However, the impact of these agents on the metastatic cascade in vivo is less well understood. In the present studies, we have examined the ability of saracatinib, a dual-specific, orally available inhibitor of Src and Abl protein tyrosine kinases, to interfere with the establishment of lung metastases in mice by tumor cells introduced into the blood stream. The results demonstrate that Src inhibition most effectively interferes with the establishment of secondary tumor deposits when treatments are administered while tumor cells are in the initial phases of dissemination.
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Benzodioxoles/uso terapéutico , Neoplasias Pulmonares/secundario , Células Neoplásicas Circulantes/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinazolinas/uso terapéutico , Familia-src Quinasas/antagonistas & inhibidores , Animales , Benzodioxoles/farmacología , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/prevención & control , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Transducción de Señal/efectos de los fármacos , Familia-src Quinasas/metabolismoRESUMEN
Acute myelogenous leukemias (AMLs) and endothelial cells depend on each other for survival and proliferation. Monotherapy antivascular strategies such as targeting vascular endothelial growth factor (VEGF) has limited efficacy in treating AML. Thus, in search of a multitarget antivascular treatment strategy for AML, we tested a novel vascular disrupting agent, OXi4503, alone and in combination with the anti-VEGF antibody, bevacizumab. Using xenotransplant animal models, OXi4503 treatment of human AML chloromas led to vascular disruption in leukemia cores that displayed increased leukemia cell apoptosis. However, viable rims of leukemia cells remained and were richly vascular with increased VEGF-A expression. To target this peripheral reactive angiogenesis, bevacizumab was combined with OXi4503 and abrogated viable vascular rims, thereby leading to enhanced leukemia regression. In a systemic model of primary human AML, OXi4503 regressed leukemia engraftment alone and in combination with bevacizumab. Differences in blood vessel density alone could not account for the observed regression, suggesting that OXi4503 also exhibited direct cytotoxic effects on leukemia cells. In vitro analyses confirmed this targeted effect, which was mediated by the production of reactive oxygen species and resulted in apoptosis. Together, these data show that OXi4503 alone is capable of regressing AML by a multitargeted mechanism and that the addition of bevacizumab mitigates reactive angiogenesis.
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Inhibidores de la Angiogénesis/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Difosfatos/uso terapéutico , Leucemia Mieloide Aguda/prevención & control , Neovascularización Patológica/prevención & control , Sarcoma Mieloide/prevención & control , Estilbenos/uso terapéutico , Anciano , Animales , Anticuerpos Monoclonales Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptosis , Bevacizumab , Western Blotting , Proliferación Celular , Humanos , Técnicas para Inmunoenzimas , Subunidad gamma Común de Receptores de Interleucina/fisiología , Leucemia Mieloide Aguda/clasificación , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Persona de Mediana Edad , ARN Mensajero/genética , Especies Reactivas de Oxígeno/metabolismo , Inducción de Remisión , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sarcoma Mieloide/patología , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A tumor's dependence on angiogenesis for survival and growth has led to the advancement of a variety of blood vessel directed anticancer treatment strategies. Overexpression of angiopoietin-2 (Ang-2) in tumor vasculature and its crucial role in angiogenesis, i.e. the destabilization of endothelial/peri-endothelial cell interactions, now raises the possibility of additional novel anti-angiogenic therapeutics. The present study utilized a co-culture sphere model to (i) demonstrate the destabilizing effect of Ang-2 on endothelial/smooth muscle cell contact and (ii) evaluate the impact of the investigational Ang-2 antibody MEDI3617 on endothelial/smooth muscle cell dissociation. Real time imaging of spheres showed both exogenous Ang-2 and PMA induced endogenous Ang-2 secretion resulted in sphere destabilization (loss of endothelial cells from smooth muscle cell core). The presence of MEDI3617 inhibited this process. To assess the anti-angiogenic potential of MEDI3617 in vivo, nude mice were injected intradermally with human renal cell carcinoma cells (Caki-1, Caki-2) and the number of blood vessels induced over a 3 day period was scored. MEDI3617 (2, 10, 20 mg/kg) significantly reduced the initiation of blood vessels for both tumor models at all doses investigated. These data indicate that MEDI3617 treatment significantly impairs the initiation of angiogenesis by inhibiting the Ang-2 mediated disruption of endothelial/muscle cell interaction associated with blood vessel destabilization and thereby reduces tumor cell induced angiogenesis. The results support the notion that targeting the angiopoietin/Tie2 axis may offer novel anti-angiogenic strategies for cancer treatment.
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Angiopoyetina 2/química , Anticuerpos Monoclonales/farmacología , Anticuerpos/química , Carcinoma de Células Renales/metabolismo , Microcirculación , Inhibidores de la Angiogénesis/farmacología , Angiopoyetina 2/biosíntesis , Animales , Anticuerpos Monoclonales Humanizados , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática/métodos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Desnudos , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Trasplante de Neoplasias , Neoplasias/tratamiento farmacológico , Neovascularización PatológicaRESUMEN
BACKGROUND: The c-Met receptor tyrosine kinase is aberrantly activated in many solid tumors. In a prior study we showed that prostate cancer PC-3 cells exhibit constitutively activated c-Met without exogenous hepatocyte growth factor (HGF); however whether this characteristic is due to an endogenous HGF/c-Met autocrine loop remains controversial. In the current study we examined the response of PC-3 cells to an anti-HGF neutralizing antibody or a small molecule Met kinase inhibitor (BMS-777607). METHODS: Cell scattering was tested by monitoring cell morphology after HGF stimulation. Cell migration was examined by both "wound-healing" and transwell assasy and invasion was detected by Matrigel-coated transwell assay. Proliferation, survival and anoikis were determined by MTT, colony formation and trypan blue exclusion assay, respectively. Gene and protein expression were assessed by real-time PCR and Western blot, respectively. RESULTS: Although HGF mRNA could be detected in PC-3 cells, the molecular weight of secreted "HGF" protein was inconsistent with the functional recombinant HGF. Furthermore, conditioned medium from PC-3 cell cultures was ineffective at triggering either motogenic behavior or c-Met signaling in DU145, another prostate cancer cell line expressing c-Met but lacking basal c-Met activation. PC-3 cells also were not responsive to the anti-HGF neutralizing antibody in experiments assessing proliferation, migration, or c-Met signaling. BMS-777607 treatment with micromolar doses nonetheless led to significant inhibition of multiple PC-3 cell functions including proliferation, clonogenicity, migration and invasion. At the molecular level, BMS-777607 suppressed autophosphorylated c-Met and downstream c-Src and Akt pathways. CONCLUSIONS: These results suggest that the constitutive c-Met activation in PC-3 is independent of autocrine stimulation. Because PC-3 cells were responsive to BMS-777607 but not the anti-HGF antibody, the findings also indicate that under circumstances where c-Met is constitutively hyperactive in the absence of functional HGF, targeting the c-Met receptor remains a viable therapeutic option to impede cancer progression.
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Aminopiridinas/farmacología , Comunicación Autocrina/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/metabolismo , Piridonas/farmacología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/inmunología , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Masculino , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Electrically excitable cells such as neurons transmit long-distance calcium or electrical signals to regulate their physiological functions. While the molecular underpinnings and down-stream effects of these intercellular communications in excitable cells have been well appreciated, little is known about whether and how non-excitable cancer cells spontaneously initiate and transmit long-distance intercellular signals. Here we report that non-excitable human colon and prostate cancer cells spontaneously initiate and spread intercellular calcium waves, in vitro and ex vivo. Xenograft model studies suggest that these calcium signals promote the growth rate of tumors in mice. Pharmacological studies elucidated that the inositol-trisphosphate-receptor (IP3R)-regulated calcium release from endoplasmic reticulum (ER), which is activated by the Gq-PLC-IP3R pathway, is a major cause for the initiation of spontaneous calcium transients. Further, the spatial-temporal characteristics of calcium dynamics can be tuned by the culture substrates of different mechanical stiffnesses. Our results provide evidence that calcium dynamics enables long-distance functional communication in non-excitable cancer cells and offer the potential to modulate calcium signaling for new cancer therapies.
Asunto(s)
Calcio , Neoplasias , Masculino , Humanos , Ratones , Animales , Calcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/farmacología , Señalización del Calcio , Retículo Endoplásmico/metabolismo , Neoplasias/metabolismoRESUMEN
Although full-length fibroblast growth factor 7 (FGF7) blocks cyclophosphamide-induced urothelial apoptosis in mice, limitations include high production costs because of its large size. We previously identified a small peptide derived from FGF2 that mitigated acute radiation syndrome as well as full-length FGF2. Based on the sequence of the FGF2 peptide, we synthesized a corresponding 19 amino acid FGF7 peptide (FGF7p). Our objectives were to determine if systemic FGF7p triggered the downstream targets and protected against cyclophosphamide bladder injury similar to full-length FGF7. We administered FGF7p or vehicle subcutaneously (SQ) to mice subjected to no injury or intraperitoneal (IP) cyclophosphamide and harvested bladders 1 day after injury. We then performed hematoxylin and eosin, TUNEL and immunofluorescence (IF) staining. In uninjured mice, a 20 mg/kg threshold FGF7p dose induced expression of phosphorylated (activated) FRS2α (pFRS2α), and pAKT in urothelium (consistent with cytoprotective effects of FGF7). We then gave FGF7p (20 mg/kg) or vehicle at 72 and 48 h prior to cyclophosphamide. One day after injury, TUNEL staining revealed many more apoptotic urothelial cells with vehicle treatment versus FGF7p treatment. IF for pAKT and readouts of two anti-apoptotic AKT targets (BAD and mTORC1) revealed minimal staining with vehicle treatment, but strong urothelial expression for all markers with FGF7p treatment. In conclusion, FGF7p appears to block bladder urothelial apoptosis via AKT and its targets, similar to FGF7. FGF7p is much more inexpensive to make and has a longer shelf life and higher purity than FGF7.
Asunto(s)
Vejiga Urinaria , Urotelio , Animales , Ciclofosfamida/farmacología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 7 de Crecimiento de Fibroblastos/farmacología , Ratones , Péptidos/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Vejiga Urinaria/metabolismo , Urotelio/metabolismoRESUMEN
Adult bone marrow (BM) contributes to neovascularization in some but not all settings, and reasons for these discordant results have remained unexplored. We conducted novel comparative studies in which multiple neovascularization models were established in single mice to reduce variations in experimental methodology. In different combinations, BM contribution was detected in ischemic retinas and, to a lesser extent, Lewis lung carcinoma cells, whereas B16 melanomas showed little to no BM contribution. Using this spectrum of BM contribution, we demonstrate the necessity for site-specific expression of stromal-derived factor-1alpha (SDF-1alpha) and its mobilizing effects on BM. Blocking SDF-1alpha activity with neutralizing antibodies abrogated BM-derived neovascularization in lung cancer and retinopathy. Furthermore, secondary transplantation of single hematopoietic stem cells (HSCs) showed that HSCs are a long-term source of neovasculogenesis and that CD133(+)CXCR4(+) myeloid progenitor cells directly participate in new blood vessel formation in response to SDF-1alpha. The varied BM contribution seen in different model systems is suggestive of redundant mechanisms governing postnatal neovasculogenesis and provides an explanation for contradictory results observed in the field.