RESUMEN
Platelets are involved in tumor angiogenesis and cancer progression. Previous studies indicated that cancer could affect platelet content. In the current study, we investigated whether cancer-associated proteins can be discerned in the platelets of cancer patients, and whether antitumor treatment may affect the platelet proteome. Platelets were isolated from nine patients with different cancer types and ten healthy volunteers. From three patients, platelets were isolated before and after the start of antitumor treatment. Mass spectrometry-based proteomics of gel-fractionated platelet proteins were used to compare patients versus controls and before and after treatment initiation. A total of 4059 proteins were detected, of which 50 were significantly more abundant in patients, and 36 more in healthy volunteers. Eight of these proteins overlapped with our previous cancer platelet proteomics study. From these data, we selected potential biomarkers of cancer including six upregulated proteins (RNF213, CTSG, PGLYRP1, RPL8, S100A8, S100A9) and two downregulated proteins (GPX1, TNS1). Antitumor treatment resulted in increased levels of 432 proteins and decreased levels of 189 proteins. In conclusion, the platelet proteome may be affected in cancer patients and platelets are a potential source of cancer biomarkers. In addition, we found in a small group of patients that anticancer treatment significantly changes the platelet proteome.
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Plaquetas/metabolismo , Neoplasias del Sistema Digestivo/sangre , Proteoma/metabolismo , Anciano , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias del Sistema Digestivo/tratamiento farmacológico , Neoplasias del Sistema Digestivo/genética , Neoplasias del Sistema Digestivo/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteoma/genéticaRESUMEN
Mass spectrometry-based phosphoproteomics provides a unique unbiased approach to evaluate signaling network in cancer cells. The tyrosine kinase inhibitor sunitinib is registered as treatment for patients with renal cell cancer (RCC). We investigated the effect of sunitinib on tyrosine phosphorylation in RCC tumor cells to get more insight in its mechanism of action and thereby to find potential leads for combination treatment strategies. Sunitinib inhibitory concentrations of proliferation (IC50) of 786-O, 769-p and A498 RCC cells were determined by MTT-assays. Global tyrosine phosphorylation was measured by LC-MS/MS after immunoprecipitation with the antiphosphotyrosine antibody p-TYR-100. Phosphoproteomic profiling of 786-O cells yielded 1519 phosphopeptides, corresponding to 675 unique proteins including 57 different phosphorylated protein kinases. Compared to control, incubation with sunitinib at its IC50 of 2 µM resulted in downregulation of 86 phosphopeptides including CDK5, DYRK3, DYRK4, G6PD, PKM and LDH-A, while 94 phosphopeptides including Axl, FAK, EPHA2 and p38α were upregulated. Axl- (y702), FAK- (y576) and p38α (y182) upregulation was confirmed by Western Blot in 786-O and A498 cells. Subsequent proliferation assays revealed that inhibition of Axl with a small molecule inhibitor (R428) sensitized 786-O RCC cells and immortalized endothelial cells to sunitinib up to 3 fold. In conclusion, incubation with sunitinib of RCC cells causes significant upregulation of multiple phosphopeptides including Axl. Simultaneous inhibition of Axl improves the antitumor activity of sunitinib. We envision that evaluation of phosphoproteomic changes by TKI treatment enables identification of new targets for combination treatment strategies.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Células Renales/metabolismo , Indoles/farmacología , Neoplasias Renales/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Pirroles/farmacología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Carcinoma de Células Renales/tratamiento farmacológico , Línea Celular Tumoral , Supervivencia Celular , Ontología de Genes , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Concentración 50 Inhibidora , Neoplasias Renales/tratamiento farmacológico , Fosforilación , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transducción de Señal , Sunitinib , Tirosina Quinasa del Receptor AxlRESUMEN
BACKGROUND: Only a small proportion of patients respond to anti-VEGF therapy, pressing the need for a reliable biomarker that can identify patients who will benefit. We studied the biological activity of anti-VEGF antibodies in patients' blood during anti-VEGF therapy by using the Ba/F3-VEGFR2 cell line, which is dependent on VEGF for its growth. METHODS: Serum samples from 22 patients with cancer before and during treatment with bevacizumab were tested for their effect on proliferation of Ba/F3-VEGFR2 cells. Vascular endothelial growth factor as well as bevacizumab concentrations in serum samples from these patients were determined by enzyme linked immunosorbent assay (ELISA). RESULTS: The hVEGF-driven cell proliferation was effectively blocked by bevacizumab (IC50 3.7 µg ml-1; 95% CI 1.7-8.3 µg ml-1). Cell proliferation was significantly reduced when patients' serum during treatment with bevacizumab was added (22-103% inhibition compared with pre-treatment). Although bevacizumab levels were not related, on-treatment serum VEGF levels were correlated with Ba/F3-VEGFR2 cell proliferation. CONCLUSIONS: We found that the neutralising effect of anti-VEGF antibody therapy on the biological activity of circulating VEGF can be accurately determined with a Ba/F3-VEGFR2 bioassay. The value of this bioassay to predict clinical benefit of anti-VEGF antibody therapy needs further clinical evaluation in a larger randomised cohort.
Asunto(s)
Inhibidores de la Angiogénesis/sangre , Linfocitos B/efectos de los fármacos , Bevacizumab/sangre , Bioensayo , Ensayo de Inmunoadsorción Enzimática , Neoplasias/sangre , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Bevacizumab/farmacología , Bevacizumab/uso terapéutico , División Celular , Línea Celular , Interleucina-3/farmacología , Ratones , Neoplasias/tratamiento farmacológico , Receptores de Eritropoyetina/genética , Receptores de Interleucina-3/fisiología , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Reproducibilidad de los Resultados , Factor A de Crecimiento Endotelial Vascular/sangre , Factor A de Crecimiento Endotelial Vascular/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/inmunologíaRESUMEN
INTRODUCTION: VEGF-targeted therapy is currently the first line treatment for patients with metastatic clear cell renal cell carcinoma (ccRCC), but most patients either display primary (intrinsic) resistance or acquire drug resistance. In recent years multiple mechanisms of resistance to VEGF-targeted therapy emerged from preclinical research, but it is currently unknown to what extent these drug resistance modalities play a role in the clinic. Here we reviewed the current literature on biomarkers that predict treatment outcome in patients with ccRCC to gain insight in clinical drug resistance mechanisms. METHODS: A search syntax was compiled by combining different synonyms of "biomarker" AND "renal" AND "cancer". MEDLINE was accessed through PubMed, where this syntax was entered and used to search titles and abstracts of publications. Articles were selected based on three criteria: (1) description of patients with clear cell RCC, (2) treatment with VEGF targeted therapy and (3) discussion of biomarkers that were studied for potential association with treatment response. RESULTS: The literature search was performed on March 4th 2014 and yielded 1882 articles. After carefully reading the titles and abstracts based on the three previously mentioned criteria, 103 publications were evaluated. Backward citation screening was performed on all eligible studies and revealed another 24 articles. This search revealed that (1) High glucose uptake and low contrast enhancement on PET- and CT-imaging before start of treatment may correlate with poor response to therapy, (2) Low dose intensity due to treatment intolerance is related to shorter progression free survival. (3) Acquired resistance appears to be associated with rebound vascularization based on both longitudinal monitoring of contrast enhancement by CT and blood vessel counts in tumor tissue, and (4) Based on plasma cytokine and single nucleotide polymorphism (SNP) studies, interleukin-8, VEGFR-3, FGFR2 and HGF/MET emerged as potential clinical markers for chemoresistance. CONCLUSION: Low dose intensity, specific tumor-imaging techniques and potential biological biomarkers may be predictive for response to VEGF-targeted therapy in ccRCC. Some of these plausible biomarkers may also provide more insight into the underlying mechanisms of resistance such as altered glucose metabolism and rapid rebound vascularization.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/metabolismo , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/metabolismo , Carcinoma de Células Renales/irrigación sanguínea , Carcinoma de Células Renales/patología , Supervivencia sin Enfermedad , Resistencia a Antineoplásicos , Humanos , Neoplasias Renales/irrigación sanguínea , Neoplasias Renales/patología , Terapia Molecular Dirigida , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Resultado del Tratamiento , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors are effective agents in the treatment of metastatic renal cell cancer (mRCC). We here investigated whether inhibition of VEGFR signalin by sunitinib causes changes in plasma proteins associated with tumor endothelium. Forty-three patients with mRCC received sunitinib 50 mg/day in a 4-weeks on 2-weeks off schedule. Sequential plasma samples were obtained before treatment (C1D1), on C1D14, on C1D28, and on C2D1 before start of cycle 2. Plasma levels were assessed for VEGF, soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular cell adhesion molecule-1 (sICAM-1), von Willebrand factor (vWF), circulating angiopoietin-2 (Ang-2) and soluble Tie-2 (sTie-2). Total tumor burden was calculated at baseline and at first evaluation. Progression-free survival (PFS) and overall survival (OS) were determined. Tumor burden was positively associated with baseline circulating Ang-2 [Spearman's rho (ρ) = 0.378, p = 0.028] and vWF (ρ = 0.417, p = 0.008). During sunitinib treatment, circulating Ang-2 and sTie-2 significantly decreased (p < 0.001 for both), plasma levels of sVCAM-1 and VEGF significantly increased (p = 0.022 and p < 0.001), whereas those of sICAM-1 and vWF remained stable. These protein changes had recovered on C2D1. The reduction in circulating Ang-2 levels on C1D28 was positively correlated with the percentage decrease in tumor burden (ρ = 0.605; p = 0.002). Baseline protein levels and subsequent changes were not associated with PFS or OS. In conclusion, sunitinib-induced changes in Ang-2, sTie-2, sVCAM-1 and VEGF are related to the administration schedule, while reduction in Ang-2 is also associated with decrease in tumor burden.
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Antineoplásicos/farmacología , Endotelio Vascular/efectos de los fármacos , Indoles/farmacología , Neoplasias Renales/tratamiento farmacológico , Pirroles/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/uso terapéutico , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Indoles/uso terapéutico , Neoplasias Renales/patología , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Pirroles/uso terapéutico , SunitinibRESUMEN
Anticancer drug resistance almost invariably emerges and poses major obstacles towards curative therapy of various human malignancies. In the current review we will distinguish between mechanisms of chemoresistance that are predominantly mediated by ATP-driven multidrug resistance (MDR) efflux transporters, typically of the ATP-binding cassette (ABC) superfamily, and those that are independent of such drug efflux pumps. In recent years, multiple nanoparticle (NP)-based therapeutic systems have been developed that were rationally designed to overcome drug resistance by neutralizing, evading or exploiting various drug efflux pumps and other resistance mechanisms. NPs are being exploited for selective drug delivery to tumor cells, to cancer stem/tumor initiating cells and/or to the supportive cancer cell microenvironment, i.e. stroma or tumor vasculature. Some of these NPs are currently undergoing preclinical in vivo studies as well as advanced stages of clinical evaluation with promising results. Nanovehicles harboring a payload of therapeutic drug combinations for the selective targeting and elimination of tumor cells as well as the simultaneous overcoming of mechanisms of drug resistance are a subject of intense research efforts, some of which are expected to enter clinical trials in the near future. In the present review we highlight novel approaches to selectively target cancer cells and overcome drug resistance phenomena, through the use of various nanometric drug delivery systems. In the near future, it is anticipated that innovative theragnostic nanovehicles will be developed which will harbor four major components: (1) a selective targeting moiety, (2) a diagnostic imaging aid for the localization of the malignant tumor and its micro- or macrometastases, (3) a cytotoxic, small molecule drug(s) or novel therapeutic biological(s), and (4) a chemosensitizing agent aimed at neutralizing a resistance mechanism, or exploiting a molecular "Achilles hill" of drug resistant cells. We propose to name these envisioned four element-containing nanovehicle platform, "quadrugnostic" nanomedicine. This targeted strategy holds promise in paving the way for the introduction of highly effective nanoscopic vehicles for cancer therapeutics while overcoming drug resistance.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Antineoplásicos/farmacología , Sistemas de Liberación de Medicamentos/métodos , Nanomedicina/métodos , Nanopartículas/uso terapéutico , Neoplasias/tratamiento farmacológico , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antineoplásicos/uso terapéutico , Transporte Biológico/efectos de los fármacos , Medios de Contraste , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Silenciador del Gen , Humanos , Imagen Molecular/métodos , Terapia Molecular Dirigida , Nanopartículas/química , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Transducción de Señal/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacosRESUMEN
Multiple molecular, cellular, micro-environmental and systemic causes of anticancer drug resistance have been identified during the last 25 years. At the same time, genome-wide analysis of human tumor tissues has made it possible in principle to assess the expression of critical genes or mutations that determine the response of an individual patient's tumor to drug treatment. Why then do we, with a few exceptions, such as mutation analysis of the EGFR to guide the use of EGFR inhibitors, have no predictive tests to assess a patient's drug sensitivity profile. The problem urges the more with the expanding choice of drugs, which may be beneficial for a fraction of patients only. In this review we discuss recent studies and insights on mechanisms of anticancer drug resistance and try to answer the question: do we understand why a patient responds or fails to respond to therapy? We focus on doxorubicin as example of a classical cytotoxic, DNA damaging agent and on sunitinib, as example of the new generation of (receptor) tyrosine kinase-targeted agents. For both drugs, classical tumor cell autonomous resistance mechanisms, such as drug efflux transporters and mutations in the tumor cell's survival signaling pathways, as well as micro-environment-related resistance mechanisms, such as changes in tumor stromal cell composition, matrix proteins, vascularity, oxygenation and energy metabolism may play a role. Novel agents that target specific mutations in the tumor cell's damage repair (e.g. PARP inhibitors) or that target tumor survival pathways, such as Akt inhibitors, glycolysis inhibitors or mTOR inhibitors, are of high interest. In order to increase the therapeutic index of treatments, fine-tuned synergistic combinations of new and/or classical cytotoxic agents will be designed. More quantitative assessment of potential resistance mechanisms in real tumors and in real time, such as by kinase profiling methodology, will be developed to allow more precise prediction of the optimal drug combination to treat each patient.
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Antineoplásicos/uso terapéutico , Doxorrubicina/uso terapéutico , Resistencia a Antineoplásicos , Indoles/uso terapéutico , Neoplasias/tratamiento farmacológico , Pirroles/uso terapéutico , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Doxorrubicina/farmacocinética , Doxorrubicina/farmacología , Resistencia a Múltiples Medicamentos/genética , Resistencia a Antineoplásicos/genética , Humanos , Indoles/farmacología , Neoplasias/genética , Neoplasias/metabolismo , Pirroles/farmacología , SunitinibRESUMEN
Mature circulating endothelial cell (CEC) as well as endothelial progenitor populations may reflect the activity of anti-angiogenic agents on tumor neovasculature or even constitute a target for anti-angiogenic therapy. We investigated the behavior of CECs in parallel with hematopoietic progenitor cells (HPCs) in the blood of renal cell cancer patients during sunitinib treatment. We analyzed the kinetics of a specific population of small VEGFR2-expressing CECs (CD45(neg)/CD34(bright)), HPCs (CD45(dim)/CD34(bright)), and monocytes in the blood of 24 renal cell cancer (RCC) patients receiving 50 mg/day of the multitargeted VEGF inhibitor sunitinib, on a 4-week-on/2-week-off schedule. Blood was taken before treatment (C1D1), on C1D14, C1D28, and on C2D1 before the start of cycle 2. Also plasma VEGF and erythropoietin (EPO) were determined. Remarkably, while CD34(bright) HPCs and monocytes decreased during treatment, CD34(bright) CECs increased from 69 cells/ml (C1D1) to 180 cells/ml (C1D14; P = 0.001) and remained high on C1D28. All cell populations recovered to near pre-treatment levels on C2D1. Plasma VEGF and EPO levels were increased on C1D14 and partly normalized to pre-treatment levels on C2D1. In conclusion, opposite kinetics of two circulating CD34(bright) cell populations, HPCs and small CECs, were observed in sunitinib-treated RCC patients. The increase in CECs is likely caused by sunitinib targeting of immature tumor vessels.
Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma de Células Renales/tratamiento farmacológico , Células Endoteliales/citología , Indoles/uso terapéutico , Neoplasias Renales/tratamiento farmacológico , Pirroles/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD34/metabolismo , Biomarcadores de Tumor/sangre , Recuento de Células Sanguíneas , Carcinoma de Células Renales/sangre , Células Endoteliales/efectos de los fármacos , Eritropoyetina/sangre , Femenino , Citometría de Flujo , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Indoles/farmacología , Neoplasias Renales/sangre , Cinética , Masculino , Persona de Mediana Edad , Pirroles/farmacología , Sunitinib , Resultado del Tratamiento , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/sangreRESUMEN
PURPOSE: A disturbed myeloid lineage development with abnormally abundant neutrophils and impaired dendritic cell (DC) differentiation may contribute to tumor immune escape. We investigated the effect of sunitinib, a tyrosine kinase inhibitor of fms-like tyrosine kinase-3, KIT, and vascular endothelial growth factor receptors, on myeloid differentiation in renal cell cancer (RCC) patients. EXPERIMENTAL DESIGN: Twenty-six advanced RCC patients were treated with sunitinib in a 4-week on/2-week off schedule. Enumeration and extensive phenotyping of myeloid subsets in the blood was done at baseline and at weeks 4 and 6 of the first treatment cycle. Baseline patient data were compared with sex- and age-matched healthy donor data. RESULTS: Baseline frequencies of DC subsets were lower in RCC patients than in healthy donors. After 4 weeks of sunitinib treatment, a generalized decrease in myeloid frequencies was observed. Whereas neutrophils and monocytes, which were both abnormally high at baseline, remained low during the 2-week off period, DC rates recovered, resulting in a normalized myeloid lineage distribution. Subsequent to sunitinib treatment, an increase to high levels of myeloid DC (MDC) subset frequencies relative to other myeloid subsets, was specifically observed in patients experiencing tumor regression. Moreover, high CD1c/BDCA-1(+) MDC frequencies were predictive for tumor regression and improved progression-free survival. CONCLUSION: The sunitinib-induced myeloid lineage redistribution observed in advanced RCC patients is consistent with an improved immune status. Immunologic recovery may contribute to clinical efficacy as suggested by the finding of highly increased MDC frequencies relative to other myeloid subsets in patients with tumor regression.
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Carcinoma de Células Renales/inmunología , Células Dendríticas/inmunología , Indoles/farmacología , Neoplasias Renales/inmunología , Células Mieloides/inmunología , Pirroles/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD1/metabolismo , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/patología , Linaje de la Célula , Supervivencia sin Enfermedad , Femenino , Glicoproteínas/metabolismo , Humanos , Indoles/uso terapéutico , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/patología , Masculino , Persona de Mediana Edad , Pirroles/uso terapéutico , SunitinibRESUMEN
Vascular homeostasis and regeneration in ischemic tissue relies on intrinsic competence of the tissue to rapidly recruit endothelial cells for vascularization. The mononuclear cell (MNC) fraction of blood contains circulating progenitors committed to endothelial lineage. These progenitors give rise to endothelial colony-forming cells (ECFCs) that actively participate in neovascularization of ischemic tissue. To evaluate if the initial clonal outgrowth of ECFCs from cord (CB) and peripheral blood (PB) was stimulated by hypoxic conditions, MNCs obtained from CB and PB were subjected to 20 and 1% O2 cell culture conditions. Clonal outgrowth was followed during a 30 day incubation period. Hypoxia impaired the initial outgrowth of ECFC colonies from CB and also reduced their number that were developing from PB MNCs. Three days of oxygenation (20% O2) prior to hypoxia could overcome the initial CB-ECFC outgrowth. Once proliferating and subcultured the CB-ECFCs growth was only modestly affected by hypoxia; proliferation of PB-ECFCs was reduced to a similar extent (18-30% reduction). Early passages of subcultured CB- and PB-ECFCs contained only viable cells and few if any senescent cells. Tube formation by subcultured PB-ECFCs was also markedly inhibited by continuous exposure to 1% O2. Gene expression profiles point to regulation of the cell cycle and metabolism as major altered gene clusters. Finally we discuss our counterintuitive observations in the context of the important role that hypoxia has in promoting neovascularization.
RESUMEN
Non-steroidal anti-inflammatory drugs (NSAIDs) and cyclo-oxygenase (COX) inhibitors are anti-inflammatory agents that have also shown to be useful in anticancer therapy. In the present study, we show that the specific COX-2 inhibitor celecoxib enhances the inhibitory effect of doxorubicin (dox) on human MDA-MB231 breast tumour growth in vivo and in vitro. We also found that celecoxib increased the intracellular accumulation and retention of dox in vitro. Since the NSAID indomethacin and the specific COX-2 inhibitor NS398 did not affect the in vitro actions of dox, these effects are likely to be mediated via a COX-independent mechanism. It has been suggested that some COX-inhibitors can enhance the actions of cytostatics by overcoming multidrug resistance through the inhibition of ABC-transporter proteins. However, we found that the three main ATP-binding cassette (ABC)-transporter proteins, implicated in dox transport, were inactive in MDA-MB231 cells. Therefore, the finding that the P-glycoprotein (P-gp) blocker PSC833 also increased cellular accumulation of dox was unexpected. In order to unravel the molecular mechanisms involved in dox accumulation, we examined the involvement of NF-kappaB, as this transcription factor has been implicated in celecoxib action as well as in chemoresistance. We found that celecoxib and PSC833, but not indomethacin or NS398, almost completely inhibited basal- and dox induced NF-kappaB gene-reporter activity and p65 subunit nuclear translocation. Furthermore, the NF-kappaB inhibitor PDTC mimicked the actions of celecoxib and PSC833 on cell growth and on intracellular accumulation of dox, suggesting that NF-kappaB is functionally involved in the actions of these compounds. In conclusion, we show that structurally different compounds, among which are celecoxib and PSC833, increase the intracellular accumulation of dox and enhance dox induced cytotoxicity in MDA-MB231 breast cancer cells most likely via the modulation of NF-kappaB activity.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Antibióticos Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Doxorrubicina/uso terapéutico , FN-kappa B/metabolismo , Pirazoles/farmacología , Sulfonamidas/farmacología , Transportadoras de Casetes de Unión a ATP/farmacología , Animales , Antibióticos Antineoplásicos/farmacocinética , Celecoxib , División Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Doxorrubicina/farmacocinética , Femenino , Humanos , Ratones , Ratones Endogámicos BALB CRESUMEN
Circulating cells of several lineages are thought to participate in angiogenesis and tumor growth. Experimental studies in tumor-bearing mice have pointed to the potential importance of VEGF-responding circulating (endothelial) progenitor cells in tumor growth. We have studied circulating CD31- and/or CD34-positive cell populations with a low to moderate VEGFR2 expression in human volunteers and cancer patients. We recognized four cell populations, which were further characterized by their content of major hematopoietic progenitor, monocytic, endothelial and platelet markers. After establishing the test-retest stability of the measurements in nine patients, we determined the frequencies of the various cell populations in a group of 20 volunteers and 14 cancer patients. Two populations were markedly increased in cancer patients. Small CD45(neg)/CD34(bright)/VEGFR2+ cells amounted to 12 and 64 cells/ml (P < 0.0001), respectively, and 246/ml and 578/ml VEGFR2+/CD45(bright) (/CD14+) monocytic cells were present in controls and cancer patients, respectively (P = 0.017). A third population of CD45(dim)/CD34(bright)/VEGFR2(low) cells amounted to 25 and 30 cells/ml (P = 0.38). Unexpectedly, a population of mainly anucleated CD45(low)/CD31(bright)/CD41(bright) cells was present in numbers of 9,076 and 16,697/ml (P = 0.04) in volunteers and cancer patients, which contained a VEGFR2(low) (compared to IgG isotype control) expressing population amounting to 1,142 and 1,642 cells/ml (P = 0.12). This fourth population probably reflects large platelets. The role of the herein identified VEGFR2+ circulating cell populations deserve further investigation in cancer patients treated with VEGF(R)-targeted therapies. Quantification of such cell populations in the blood of tumor patients may be valuable to monitor the efficacy of anti-angiogenic treatment.
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Células Endoteliales/citología , Neoplasias/sangre , Células Madre/química , Receptor 2 de Factores de Crecimiento Endotelial Vascular/análisis , Antígenos CD34/análisis , Células Sanguíneas/citología , Circulación Sanguínea , Estudios de Casos y Controles , Células Endoteliales/química , Citometría de Flujo , Humanos , Antígenos Comunes de Leucocito/análisis , Células Madre/citologíaRESUMEN
PURPOSE: On the basis of the finding of marked overexpression in angiogenic microvessels, aminopeptidase N/CD13 has recently been suggested to play a prominent role in tumor angiogenesis. A soluble form of CD13 (sCD13) is present in human plasma, but its role in cancer has not been addressed. We hypothesized that sCD13 would be shed by tumor cells and/or endothelial cells lining tumor vessels, giving high levels of sCD13 in intratumoral fluid (TF) deposits and in malignant effusions. If so, sCD13 could be a convenient potential marker for tumor load and/or activated tumor endothelium. EXPERIMENTAL DESIGN: We have measured the specific sCD13 activity in effusions from 90 cancer patients and 12 patients with a nonmalignant condition, and studied its relationship with other major (anti-)angiogenic factors. In a separate group of patients (n = 41), the relationship of sCD13 activity in plasma with tumor load was studied. RESULTS: The sCD13 activity was highest in plasma from cancer patients 71.9 (fmol/ml/s hydrolyzed substrate) versus 42.4 for healthy subjects. In TF, malignant effusions, and nonmalignant effusions, the activities were 52.8, 33.5, and 18.6, respectively. We further studied the relationship of sCD13 with tumor load as well as with vascular endothelial growth factor (VEGF), endostatin, matrix metalloproteinase (MMP)-2, MMP-9, urokinase-type plasminogen activator, and plasmin. A significant correlation of sCD13 activity in plasma was found with tumor load (r = 0.68; P = 0.01), suggesting that plasma sCD13 is, at least, partly originating from tumor(-endothelium). The concentrations of VEGF and endostatin and the activities of urokinase-type plasminogen activator and MMP-9, but not MMP-2, were significantly higher in TF compared with all other effusions. In TF, a correlation between sCD13 and VEGF was found (r = 0.67; P = 0.03). No correlation of sCD13 with the other protease activities was found. CONCLUSION: The sCD13 activity is elevated in plasma and effusions of cancer patients. A strong correlation of plasma sCD13 with tumor load was found. On the basis of these results, the potential of sCD13 activity as a tumor and/or angiogenesis marker warrants further investigation.
Asunto(s)
Líquido Ascítico/metabolismo , Biomarcadores de Tumor/metabolismo , Antígenos CD13/metabolismo , Neoplasias/metabolismo , Derrame Pleural/metabolismo , Adulto , Colágeno/metabolismo , Endostatinas , Factores de Crecimiento Endotelial/metabolismo , Femenino , Fibrinolisina/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Humanos , Immunoblotting , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Linfocinas/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Persona de Mediana Edad , Neoplasias/patología , Fragmentos de Péptidos/metabolismo , Derrame Pleural/patología , Pronóstico , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
PURPOSE: Expression of aminopeptidase N/CD13 can be detected in several solid tumor types. Thus far, the role of CD13 in ovarian cancer has not been studied. We have investigated the expression pattern and biological function of CD13 in ovarian cancer. EXPERIMENTAL DESIGN: First, we studied the expression of CD13 in ovarian cancer tissue of 15 patients representing three different histological types (5 patients each) by immunohistochemistry. We then stably transfected the IGROV-1 human ovarian cancer cell line with a CD13 expression vector and examined the biological effect of CD13 in vitro and in vivo. RESULTS: The expression of CD13 in ovarian cancer was associated with the histological subtype: CD13 expression in tumor cells was observed in 80-100% of the patients with a serous or mucinous carcinoma and in only 20% of the clear cell carcinoma patients. In all patients' tumor samples, CD13-positive blood vessels were present. CD13 overexpression in IGROV-1 cells did not affect in vitro cell growth and sensitivity to doxorubicin, cisplatin, or gemcitabine. CD13 overexpression reduced invasion in Matrigel, which appeared to be independent of the aminopeptidase activity of CD13. Furthermore, the growth rate of IGROV-1/CD13 xenografts was reduced. The area of the vessel lumens was enlarged in a small percentage of vessels in the CD13-overexpressing xenografts. In addition, the CD13-overexpressing tumors were less sensitive to cisplatin. CONCLUSIONS: CD13 is expressed in tumor as well as endothelial cells in human ovarian cancer. Our results suggest that CD13 overexpression affects ovarian cancer growth, vascular architecture, and response to chemotherapy. Further elucidation of the mechanism of the observed effects of CD13 is warranted to better understand its role in the pathophysiology of ovarian cancer.
Asunto(s)
Antígenos CD13/biosíntesis , Cisplatino/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Adenocarcinoma de Células Claras/metabolismo , Adenocarcinoma Mucinoso/metabolismo , Aminopeptidasas/metabolismo , Animales , Apoptosis , Adhesión Celular , División Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Colágeno/farmacología , Cistadenocarcinoma Seroso/metabolismo , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Femenino , Humanos , Inmunohistoquímica , Laminina/farmacología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Plásmidos/metabolismo , Proteoglicanos/farmacología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fase S , Factores de Tiempo , TransfecciónRESUMEN
Tumor cells may become resistant to conventional anticancer drugs through the occurrence of transmembrane transporter proteins such as P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), or members of the multidrug resistance-associated protein family (MRP1-MRP5; ABCC1-ABCC5). In this report, we studied whether tumor cells that are cytostatic drug resistant because of overexpression of one of the above mentioned proteins are sensitive to a new anticancer agent, interleukin-4 toxin (IL-4 toxin). IL-4 toxin is a fusion protein composed of circularly permuted IL-4 and a truncated form of Pseudomonas exotoxin (PE) [IL-4(38-37)-PE38KDEL]. Ninety-six-h cytotoxicity assays and 10-day clonogenic assays showed that drug-selected multidrug resistant (MDR) tumor cells that overexpress P-glycoprotein or breast cancer resistance proteins are still sensitive to IL-4 toxin. Also, tumor cells transfected with cDNA for MRP2-5 showed no resistance, or marginal resistance, only to the toxin as compared with the parent cells. In contrast, MRP1-overexpressing cells, both drug selected and MRP1 transfected, are clearly resistant to IL-4 toxin with resistance factors of 4.3 to 8.4. MRP1-overexpressing cells were not resistant to PE itself. IL-4 toxin resistance in MRP1-overexpressing cells could be reversed by the MRP1 inhibitors probenecid or MK571 and were not affected by glutathione depletion by DL-buthionine-S,R-sulfoximine. In a transport assay using plasma membrane vesicles prepared from MRP1-overexpressing cells, IL-4 toxin and IL-4, but not PE, inhibited the translocation of the known MRP1 substrate 17beta-estradiol 17-(beta-D-glucuronide) (E(2)17betaG). These data suggest that MRP1-overexpressing cells are resistant to IL-4 toxin because of extrusion of this agent by MRP1. Still, the results of this study demonstrate that IL-4 toxin effectively kills most MDR tumor cells and, therefore, represents a promising anticancer drug.
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Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Exotoxinas/fisiología , Interleucina-4/metabolismo , Interleucina-4/fisiología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Recombinantes/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antimetabolitos Antineoplásicos/farmacología , Antineoplásicos/farmacología , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Butionina Sulfoximina/farmacología , Línea Celular Tumoral , Membrana Celular/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Exotoxinas/química , Exotoxinas/metabolismo , Humanos , Interleucina-4/química , Antagonistas de Leucotrieno/farmacología , Probenecid/farmacología , Propionatos/farmacología , Quinolinas/farmacología , Transfección , Uricosúricos/farmacologíaRESUMEN
The 91st Annual Meeting of the American Association for Cancer Research (AACR) provided the latest developments in cancer research. We highlight here presentations on resistance mechanisms (tumor microenvironment, efflux pumps, apoptosis), new targets and drugs in development (signal transduction, cell cycle, apoptosis, microtubules, topoisomerases) and new technologies (the cancer genome anatomy project, the human genome project, proteomics). The importance of signaling pathways or networks ('cell context') in defining the role of particular proteins was brought up in several presentations. A summary of specific drugs in development is also included in this report. Copyright 2000 Harcourt Publishers Ltd.
RESUMEN
The realization that cellular homeostasis is dependent on the continuous integration of survival and death signals picked up from the environment and the recent advances in identifying the molecular players involved in these networks may increase our ability to manipulate apoptosis for therapeutic purposes. A recent paper by Buckley et al. in Nature(1)brings this goal one step closer by identifying peptides, containing the motif arginine-glycine-aspartate (RGD), that induce apoptosis by direct activation of caspase-3. We put this finding in the context of what is known about the RGD motif in the light of cancer treatment and suggest the possibility of a synergistic action with anticancer drugs. Copyright 1999 Harcourt Publishers Ltd.
RESUMEN
The design, synthesis, and initial biological evaluation of a doxorubicin prodrug that contains a dual tumor specific moiety, which allows enhanced tumor recognition potential, is reported. Both a tumor-specific recognition site and a tumor selective enzymatic activation sequence are incorporated in the prodrug. The first tumor-specific sequence is the bicyclic CDCRGDCFC (RGD-4C) peptide that selectively binds alpha v beta 3 and alpha v beta 5 integrins. These integrins are highly overexpressed on invading tumor endothelial cells. The second tumor-specific sequence is a D-Ala-Phe-Lys tripeptide that is selectively recognized by the tumor-associated protease plasmin, which is involved in tumor invasion and metastasis. An aminocaproyl residue was incorporated as a spacer between the two peptide sequences, whereas a self-eliminating 4-aminobenzyl alcohol spacer was inserted between the plasmin substrate and doxorubicin. Although the prodrug showed a decreased binding affinity as compared with the unconjugated reference peptide, it was still a potent ligand for alpha v beta 3 and alpha v beta 5 integrin receptors. The synthesized construct also possessed plasmin substrate properties as demonstrated by doxorubicin release from 1 upon incubation with plasmin. The release of doxorubicin from 1 was not complete, possibly related to low prodrug solubility. In vitro prodrug 1 showed plasmin-dependent cytotoxicity for endothelial cells and HT1080 fibrosarcoma cells. On the basis of these in vitro results, derivatives of 1 with improved water solubility are considered good candidates for additional development and in vivo evaluation of this dual targeting concept.
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Antineoplásicos/síntesis química , Doxorrubicina/farmacología , Diseño de Fármacos , Profármacos/farmacología , Secuencia de Aminoácidos , Antineoplásicos/farmacología , Alcoholes Bencílicos/farmacología , Sitios de Unión , Adhesión Celular , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Endotelio/citología , Endotelio Vascular/citología , Fibrinolisina/química , Fibrinolisina/metabolismo , Humanos , Concentración 50 Inhibidora , Integrina alfaVbeta3/metabolismo , Integrinas/química , Integrinas/metabolismo , Ligandos , Modelos Químicos , Datos de Secuencia Molecular , Invasividad Neoplásica , Metástasis de la Neoplasia , Oligopéptidos , Péptidos/química , Profármacos/síntesis química , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Vitronectina/metabolismoRESUMEN
BACKGROUND: Mass spectrometry based phosphoproteomics emerged as advantageous approach for the analysis of tyrosine phosphorylation on proteins and tyrosine kinase signaling. Immunoaffinity purification is required for comprehensive analysis. Here we compared the performance of two antibodies for label-free phosphotyrosine-based phosphoproteomics. METHODS: Phosphopeptide immunoprecipitation of six technical replicates corresponding to 10mg protein from HCT116 cells was performed using agarose bead-coupled phosphotyrosine antibodies P-Tyr-1000 (N=3) and 4G10 (N=3). NanoLC-MS/MS was performed using a Q Exactive mass spectrometer. For relative quantitation of protein phosphorylation, spectral counts of phosphoproteins and ion intensities of phosphopeptides were determined using MaxQuant. RESULTS: From the 3 samples incubated with P-Tyr-1000 a total of 689 phosphopeptides were identified with 60% ID reproducibility. The phosphopeptide capture using 4G10 resulted in a total of 421 at 46% ID reproducibility. The P-Tyr-1000 was applied to EGFR mutated U87 cells. Erlotinib reduced EGFR phosphorylation with 59% at y978, y1125, y1138, y1172, and y1197. EGFR inhibition was accompanied by enhanced phosphorylation of FYN, MET, PTK2, DYRK1A, MAPK1 and EPHA2. CONCLUSION: The P-Tyr-1000 phosphotyrosine antibody performs superiorly when compared to 4G10 antibody for label-free phosphotyrosine-based phosphoproteomics. This workflow allows evaluation of drug target phosphorylation and may give insights in the pharmacodynamic effects of tyrosine kinase inhibitors. CLINICAL SIGNIFICANCE: In the past decade multiple tyrosine kinase inhibitors (TKIs) have been implemented in standard treatment regimens for patients with cancer. Unfortunately the majority of patients develops resistance to these drugs. Reliable tools for analysis of pharmacodynamic effects and drug resistance mechanisms are therefore warranted. Phosphoproteomic analyses have meanwhile emerged as a sophisticated approach for the determination of protein phosphorylation. These analyses rely on antibodies for enrichment of tyrosine-phosphorylated peptides. Here we compared two commercially available phosphotyrosine antibodies and show that P-Tyr-1000 yields 64% more phosphopeptides than 4G10 antibody, while including almost all 4G10 captured phosphopeptides. The workflow can be reproducibly performed at intermediate protein input levels of 10mg. Furthermore, application of the P-Tyr-1000 antibody in a standardized phosphoproteomics workflow allows relative quantitation of drug target inhibition and provides insights in alternative signaling pathways in cancer cells. This article is part of a Special Issue entitled: HUPO 2014.
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Anticuerpos Antineoplásicos/química , Fosfoproteínas/análisis , Fosfotirosina/análisis , Línea Celular Tumoral , Receptores ErbB/análisis , Receptores ErbB/metabolismo , Clorhidrato de Erlotinib/farmacología , Humanos , Espectrometría de Masas/métodos , Fosfoproteínas/metabolismo , Fosfotirosina/metabolismoRESUMEN
PURPOSE: When during cancer treatment resistance to a tyrosine kinase inhibitor (TKI) occurs, switching to another TKI is often considered as a reasonable option. Previously, we reported that resistance to sunitinib may be caused by increased lysosomal sequestration, leading to increased intracellular lysosomal storage and, thereby, inactivity. Here, we studied the effect of several other TKIs on the development of (cross-) resistance. METHODS: TKI resistance was induced by continuous exposure of cancer cell lines to increasing TKI concentrations for 3-4 months. (Cross-) resistance was evaluated using MTT cell proliferation assays. Intracellular TKI concentrations were measured using LC-MS/MS. Western blotting was used to detect lysosome-associated membrane protein-1 and -2 (LAMP1/2) expression. RESULTS: The previously generated sunitinib-resistant (SUN) renal cancer cells (786-O) and colorectal cancer cells (HT-29) were found to be cross-resistant to pazopanib, erlotinib and lapatinib, but not sorafenib. Exposure of 786-O and HT-29 cells to sorafenib, pazopanib or erlotinib for 3-4 months induced drug resistance to pazopanib and erlotinib, but not sorafenib. Intracellular drug accumulation was found to be increased in pazopanib- and erlotinib-, but not in sorafenib-exposed cells. Lysosomal capacity, reflected by LAMP1/2 expression, was found to be increased in resistant cells and, in addition, to be transient. No cross-resistance to the mTOR inhibitor everolimus was detected. CONCLUSIONS: Our data indicate that tumor cells can develop (cross-) resistance to TKIs, and that such resistance includes increased intracellular drug accumulation accompanied by increased lysosomal storage. Transient (cross-) resistance was found to occur for several of the TKIs tested, but not for everolimus, indicating that switching from a TKI to a mTOR inhibitor may be an attractive therapeutic option.