RESUMEN
The cellular energy sensor AMP-activated protein kinase (AMPK) is a metabolic hub regulating various pathways involved in tumor metabolism. Here we report that vacuolar H+-ATPase (V-ATPase) inhibition differentially affects regulation of AMPK in tumor and nontumor cells and that this differential regulation contributes to the selectivity of V-ATPase inhibitors for tumor cells. In nonmalignant cells, the V-ATPase inhibitor archazolid increased phosphorylation and lysosomal localization of AMPK. We noted that AMPK localization has a prosurvival role, as AMPK silencing decreased cellular growth rates. In contrast, in cancer cells, we found that AMPK is constitutively active and that archazolid does not affect its phosphorylation and localization. Moreover, V-ATPase-independent AMPK induction in tumor cells protected them from archazolid-induced cytotoxicity, further underlining the role of AMPK as a prosurvival mediator. These observations indicate that AMPK regulation is uncoupled from V-ATPase activity in cancer cells and that this makes them more susceptible to cell death induction by V-ATPase inhibitors. In both tumor and healthy cells, V-ATPase inhibition induced a distinct metabolic regulatory cascade downstream of AMPK, affecting ATP and NADPH levels, glucose uptake, and reactive oxygen species production. We could attribute the prosurvival effects to AMPK's ability to maintain redox homeostasis by inhibiting reactive oxygen species production and maintaining NADPH levels. In summary, the results of our work indicate that V-ATPase inhibition has differential effects on AMPK-mediated metabolic regulation in cancer and healthy cells and explain the tumor-specific cytotoxicity of V-ATPase inhibition.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Inhibidores Enzimáticos/farmacología , Macrólidos/farmacología , Neoplasias/tratamiento farmacológico , Tiazoles/farmacología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Humanos , Neoplasias/metabolismo , Fosforilación/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
BACKGROUND: The understanding of lysosomes has been expanded in recent research way beyond their view as cellular trash can. Lysosomes are pivotal in regulating metabolism, endocytosis and autophagy and are implicated in cancer. Recently it was discovered that the lysosomal V-ATPase, which is known to induce apoptosis, interferes with lipid metabolism in cancer, yet the interplay between these organelles is poorly understood. METHODS: LC-MS/MS analysis was performed to investigate lipid distribution in cells. Cell survival and signaling pathways were analyzed by means of cell biological methods (qPCR, Western Blot, flow cytometry, CellTiter-Blue). Mitochondrial structure was analyzed by confocal imaging and electron microscopy, their function was determined by flow cytometry and seahorse measurements. RESULTS: Our data reveal that interfering with lysosomal function changes composition and subcellular localization of triacylglycerids accompanied by an upregulation of PGC1α and PPARα expression, master regulators of energy and lipid metabolism. Furthermore, cardiolipin content is reduced driving mitochondria into fission, accompanied by a loss of membrane potential and reduction in oxidative capacity, which leads to a deregulation in cellular ROS and induction of mitochondria-driven apoptosis. Additionally, cells undergo a metabolic shift to glutamine dependency, correlated with the fission phenotype and sensitivity to lysosomal inhibition, most prominent in Ras mutated cells. CONCLUSION: This study sheds mechanistic light on a largely uninvestigated triangle between lysosomes, lipid metabolism and mitochondrial function. Insight into this organelle crosstalk increases our understanding of mitochondria-driven cell death. Our findings furthermore provide a first hint on a connection of Ras pathway mutations and sensitivity towards lysosomal inhibitors.
Asunto(s)
Muerte Celular , Metabolismo de los Lípidos , Mitocondrias/metabolismo , Muramidasa/metabolismo , Línea Celular Tumoral , Humanos , Estrés Oxidativo , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidoresRESUMEN
The vacuolar H(+)-ATPase (V-ATPase), a multisubunit proton pump, has come into focus as an attractive target in cancer invasion. However, little is known about the role of V-ATPase in cell death, and especially the underlying mechanisms remain mostly unknown. We used the myxobacterial macrolide archazolid B, a potent inhibitor of the V-ATPase, as an experimental drug as well as a chemical tool to decipher V-ATPase-related cell death signaling. We found that archazolid induced apoptosis in highly invasive tumor cells at nanomolar concentrations which was executed by the mitochondrial pathway. Prior to apoptosis induction archazolid led to the activation of a cellular stress response including activation of the hypoxia-inducible factor-1α (HIF1α) and autophagy. Autophagy, which was demonstrated by degradation of p62 or fusion of autophagosomes with lysosomes, was induced at low concentrations of archazolid that not yet increase pH in lysosomes. HIF1α was induced due to energy stress shown by a decline of the ATP level and followed by a shutdown of energy-consuming processes. As silencing HIF1α increases apoptosis, the cellular stress response was suggested to be a survival mechanism. We conclude that archazolid leads to energy stress which activates adaptive mechanisms like autophagy mediated by HIF1α and finally leads to apoptosis. We propose V-ATPase as a promising drugable target in cancer therapy caught up at the interplay of apoptosis, autophagy, and cellular/metabolic stress.
Asunto(s)
Muerte Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Autofagia , Línea Celular Tumoral , Proliferación Celular , Citocromos c/metabolismo , Humanos , Potencial de la Membrana Mitocondrial , Microscopía Confocal , Transducción de SeñalRESUMEN
The myxobacterial agent archazolid inhibits the vacuolar proton pump V-ATPase. V-ATPases are ubiquitously expressed ATP-dependent proton pumps, which are known to regulate the pH in endomembrane systems and thus play a crucial role in endo- and exocytotic processes of the cell. As cancer cells depend on a highly active secretion of proteolytic proteins in order to invade tissue and form metastases, inhibition of V-ATPase is proposed to affect the secretion profile of cancer cells and thus potentially abrogate their metastatic properties. Archazolid is a novel V-ATPase inhibitor. Here, we show that the secretion pattern of archazolid treated cancer cells includes various prometastatic lysosomal proteins like cathepsin A, B, C, D and Z. In particular, archazolid induced the secretion of the proforms of cathepsin B and D. Archazolid treatment abrogates the cathepsin B maturation process leading to reduced intracellular mature cathepsin B protein abundance and finally decreased cathepsin B activity, by inhibiting mannose-6-phoshate receptor-dependent trafficking. Importantly, in vivo reduced cathepsin B protein as well as a decreased proteolytic cathepsin B activity was detected in tumor tissue of archazolid-treated mice. Our results show that inhibition of V-ATPase by archazolid reduces the activity of prometastatic proteases like cathepsin B in vitro and in vivo.
Asunto(s)
Catepsina B/metabolismo , Macrólidos/farmacología , Tiazoles/farmacología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Animales , Western Blotting , Catepsina B/genética , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/enzimología , Células MCF-7 , Ratones , Ratones Endogámicos BALB C , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/prevención & control , Transporte de Proteínas/efectos de los fármacos , Interferencia de ARN , Receptor IGF Tipo 2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Carga Tumoral/efectos de los fármacos , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
PURPOSE: Poly(ADP-ribose) polymerase inhibitors (PARPi) have shown promising clinical results in the treatment of ovarian cancer. Analysis of biomarker subgroups consistently revealed higher benefits for patients with homologous recombination deficiency (HRD). The test that is most often used for the detection of HRD in clinical studies is the Myriad myChoice assay. However, other assays can also be used to assess biomarkers, which are indicative of HRD, genomic instability (GI), and BRCA1/2 mutation status. Many of these assays have high potential to be broadly applied in clinical routine diagnostics in a time-effective decentralized manner. Here, we compare the performance of a multitude of alternative assays in comparison with Myriad myChoice in high-grade serous ovarian cancer (HGSOC). METHODS: DNA from HGSOC samples was extracted from formalin-fixed paraffin-embedded tissue blocks of cases previously run with the Myriad myChoice assay, and GI was measured by multiple molecular assays (CytoSNP, AmoyDx, Illumina TSO500 HRD, OncoScan, NOGGO GISv1, QIAseq HRD Panel and whole genome sequencing), applying different bioinformatics algorithms. RESULTS: Application of different assays to assess GI, including Myriad myChoice, revealed high concordance of the generated scores ranging from very substantial to nearly perfect fit, depending on the assay and bioinformatics pipelines applied. Interlaboratory comparison of assays also showed high concordance of GI scores. CONCLUSION: Assays for GI assessment not only show a high concordance with each other but also in correlation with Myriad myChoice. Thus, almost all of the assays included here can be used effectively to assess HRD-associated GI in the clinical setting. This is important as PARPi treatment on the basis of these tests is compliant with European Medicines Agency approvals, which are methodologically not test-bound.
Asunto(s)
Proteína BRCA1 , Neoplasias Ováricas , Humanos , Femenino , Proteína BRCA1/genética , Mutación , Proteína BRCA2/genética , Neoplasias Ováricas/diagnóstico , Neoplasias Ováricas/genética , Neoplasias Ováricas/tratamiento farmacológico , Inestabilidad Genómica/genética , Recombinación Homóloga/genéticaRESUMEN
Imatinib (IM) has been described to modulate the function of dendritic cells and T lymphocytes and to affect the expression of antigen in CML cells. In our study, we investigated the effect of the tyrosine kinase inhibitors IM and nilotinib (NI) on antigen presentation and processing by analyzing the proteasomal activity in CML cell lines and patient samples. We used a biotinylated active site-directed probe, which covalently binds to the proteasomally active beta-subunits in an activity-dependent fashion. Additionally, we analyzed the cleavage and processing of HLA-A3/11- and HLA-B8-binding peptides derived from BCR-ABL by IM- or NI-treated isolated 20S immunoproteasomes using mass spectrometry. We found that IM treatment leads to a reduction in MHC-class I expression which is in line with the inhibition of proteasomal activity. This process is independent of BCR-ABL or apoptosis induction. In vitro digestion experiments using purified proteasomes showed that generation of epitope-precursor peptides was significantly altered in the presence of NI and IM. Treatment of the immunoproteasome with these compounds resulted in an almost complete reduction in the generation of long precursor peptides for the HLA-A3/A11 and -B8 epitopes while processing of the short peptide sequences increased. Treatment of isolated 20S proteasomes with serine-/threonine- and tyrosine-specific phosphatases induced a significant downregulation of the proteasomal activity further indicating that phosphorylation of the proteasome regulates its function and antigen processing. Our results demonstrate that IM and NI can affect the immunogenicity of malignant cells by modulating proteasomal degradation and the repertoire of processed T cell epitopes.
Asunto(s)
Antígenos de Neoplasias/inmunología , Benzamidas/farmacología , Antígenos de Histocompatibilidad Clase I/inmunología , Fragmentos de Péptidos/inmunología , Piperazinas/farmacología , Complejo de la Endopetidasa Proteasomal/inmunología , Pirimidinas/farmacología , Secuencia de Aminoácidos , Presentación de Antígeno/efectos de los fármacos , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacología , Proteínas de Fusión bcr-abl/metabolismo , Antígenos HLA-A/inmunología , Antígenos HLA-B/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Mesilato de Imatinib , Células K562 , Leucemia/inmunología , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteína Fosfatasa 2/inmunología , Proteína Fosfatasa 2/metabolismoRESUMEN
It is widely known that most cancer cells display an increased reliance on glutaminolysis to sustain proliferation and survival. Combining glutamine deprivation with additional anti-cancer therapies is an intensively investigated approach to increase therapeutic effectiveness. In this study, we examined a combination of glutamine deprivation by starvation or pharmacological tools, with the anti-cancer agent archazolid, an inhibitor of the lysosomal V-ATPase. We show that glutamine deprivation leads to lysosomal acidification and induction of pro-survival autophagy, which could be prevented by archazolid. Surprisingly, a combination of glutamine deprivation with archazolid did not lead to synergistic induction of cell death or reduction in proliferation. Investigating the underlying mechanisms revealed elevated expression and activity of amino acid transporters SLC1A5, SLC38A1 upon starvation, whereas archazolid had no additional effect. Furthermore, we found that the export of lysosomal glutamine derived from exogenous sources plays no role in the phenotype as knock-down of SLC38A7, the lysosomal glutamine exporter, could not increase V-ATPase inhibition-induced cell death or reduce proliferation. Analysis of the cellular metabolic phenotype revealed that glutamine deprivation led to a significant increase in glycolytic activity, indicated by an elevated glycolytic capacity and reserve, when V-ATPase function was inhibited concomitantly. This was confirmed by increased glutamine uptake, augmented lactate production, and an increase in hexokinase activity. Our study, therefore, provides evidence, that glutamine deprivation induces autophagy, which can be prevented by simultaneous inhibition of V-ATPase function. However, this does not lead to a therapeutic benefit, as cells are able to circumvent cell death and growth inhibition by a metabolic shift toward glycolysis.
RESUMEN
The total synthesis of the potent respiratory chain inhibitor ajudazol A was accomplished by a concise strategy in 17 steps (longest linear sequence). The modular approach was based on a direct oxazole functionalization strategy involving a halogen dance reaction for selective halogenation in combination with a challenging combination of sp2-sp2 and sp2-sp3 Negishi cross coupling reactions. The applicability of this strategy for analogue synthesis was demonstrated by the synthesis of a simplified as well as stabilized ajudazol analogue.
RESUMEN
Chronic myelogenous leukemia (CML) is a myeloproliferative disorder caused by excessive granulopoiesis due to the formation of the constitutively active tyrosine kinase BCR-ABL. An effective drug against CML is imatinib mesylate, a tyrosine kinase inhibitor acting on Abl kinases, c-KIT, and platelet-derived growth factor receptor. Recently, a study revealed that patients treated with imatinib showed impaired CTL responses compared with patients treated with IFN-alpha, which might be due to a treatment-induced reduction in immunogenicity of CML cells or immunosuppressive effects. In our study, we found that inhibition of BCR-ABL leads to a down-regulation of immunogenic antigens on the CML cells in response to imatinib treatment, which results in the inhibition of CML-directed immune responses. By treating CML cells with imatinib, we could show that the resulting inhibition of BCR-ABL leads to a decreased expression of tumor antigens, including survivin, adipophilin, hTERT, WT-1, Bcl-x(L), and Bcl-2 in correlation to a decreased development of CML-specific CTLs. In contrast, this reduction in immunogenicity was not observed when a CML cell line resistant to the inhibitory effects of imatinib was used, but could be confirmed by transfection with specific small interfering RNA against BCR-ABL or imatinib treatment of primary CML cells.
Asunto(s)
Antineoplásicos/farmacología , Proteínas de Fusión bcr-abl/inmunología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/inmunología , Piperazinas/farmacología , Pirimidinas/farmacología , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/inmunología , Benzamidas , Células Dendríticas/inmunología , Regulación hacia Abajo , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Activación de Linfocitos/efectos de los fármacos , ARN Interferente Pequeño/genética , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Hepatocellular carcinoma (HCC) is the fifth most frequent cancer worldwide and the third leading cause of cancer-related death. However, therapy options are limited leaving an urgent need to develop new strategies. Currently, targeting cancer cell lipid and cholesterol metabolism is gaining interest especially regarding HCC. High cholesterol levels support proliferation, membrane-related mitogenic signaling and increase cell softness, leading to tumor progression, malignancy and invasive potential. However, effective ways to target cholesterol metabolism for cancer therapy are still missing. The V-ATPase inhibitor archazolid was recently shown to interfere with cholesterol metabolism. In our study, we report a novel therapeutic potential of V-ATPase inhibition in HCC by altering the mechanical phenotype of cancer cells leading to reduced proliferative signaling. Archazolid causes cellular depletion of free cholesterol leading to an increase in cell stiffness and membrane polarity of cancer cells, while hepatocytes remain unaffected. The altered membrane composition decreases membrane fluidity and leads to an inhibition of membrane-related Ras signaling resulting decreased proliferation in vitro and in vivo. V-ATPase inhibition represents a novel link between cell biophysical properties and proliferative signaling selectively in malignant HCC cells, providing the basis for an attractive and innovative strategy against HCC.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Membrana Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Neoplasias Hepáticas/tratamiento farmacológico , Macrólidos/farmacología , Fluidez de la Membrana/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Tiazoles/farmacología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Proteínas ras/metabolismo , Animales , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/patología , Membrana Celular/enzimología , Membrana Celular/patología , Proliferación Celular/efectos de los fármacos , Colesterol/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Células Hep G2 , Humanos , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/patología , Lisosomas/efectos de los fármacos , Lisosomas/enzimología , Ratones SCID , Factores de Tiempo , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
Treating cancer is one of the big challenges of this century and it has become evident that single chemotherapeutic treatment is rarely effective. As tumors often carry multiple mutations using combination therapy which addresses different targets seems therefore more beneficial. One of the most frequently mutated genes in tumors is the tumor suppressor p53. Significant work has been put in the development of p53 activators, which are now in clinical studies against diverse cancers. Recently, we could show that inhibition of V-ATPase, a multisubunit proton pump, by archazolid induces p53 protein levels in cancer cells. In this study, we provide evidence that the combination of archazolid with the p53 activator nutlin-3a is synergistically inducing cell death in different p53 wild type tumor cell lines. Mechanistically, this effect could presumably be attributed to reduction of glycolysis as TIGAR mRNA levels were increased and glucose uptake and Glut1 protein levels were reduced. In addition, combination treatment highly activated pro-apoptotic pathways including IGFBP3 and Bax inducing caspase-9 and PARP cleavage. Remarkably, combination of archazolid and nutlin-3a was more efficient in reducing tumor growth compared to single dose treatment in a U87MG mouse model in vivo. Hence, our findings suggest the combination of archazolid and nutlin-3a as a highly promising strategy for the treatment of p53 wild type tumors.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Imidazoles/farmacología , Neoplasias/metabolismo , Neoplasias/patología , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Adenosina Trifosfatasas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Femenino , Glucólisis/efectos de los fármacos , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Ratones Endogámicos BALB C , Ratones SCID , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Transducción de Señal/efectos de los fármacos , Tiazoles/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Generalized strategies to improve breast cancer treatment remain of interest to develop. In this study, we offer preclinical evidence of an important metabolic mechanism underlying the antitumor activity of inhibitors of the vacuolar-type ATPase (V-ATPase), a heteromultimeric proton pump. Specifically, our investigations in the 4T1 model of metastatic breast cancer of the V-ATPase inhibitor archazolid suggested that its ability to trigger metabolic stress and apoptosis associated with tumor growth inhibition related to an interference with hypoxia-inducible factor-1α signaling pathways and iron metabolism. As a consequence of disturbed iron metabolism, archazolid caused S-phase arrest, double-stranded DNA breaks, and p53 stabilization, leading to apoptosis. Our findings link V-ATPase to cell-cycle progression and DNA synthesis in cancer cells, and highlight the basis for the clinical exploration of V-ATPase as a potentially generalizable therapy for breast cancer.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Hierro/metabolismo , Macrólidos/farmacología , Tiazoles/farmacología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/patología , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Células MCF-7 , Macrólidos/uso terapéutico , Redes y Vías Metabólicas/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Tiazoles/uso terapéutico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The HER2 oncogene targeting drug trastuzumab shows remarkable efficacy in patients overexpressing HER2. However acquired or primary resistance develops in most of the treated patients why alternative treatment strategies are strongly needed. As endosomal sorting and recycling are crucial steps for HER2 activity and the vacuolar Hâº-ATPase (V-ATPase) is an important regulator of endocytotic trafficking, we proposed that targeting V-ATPase opens a new therapeutic strategy against trastuzumab-resistant tumor cells in vitro and in vivo. V-ATPase inhibition with archazolid, a novel inhibitor of myxobacterial origin, results in growth inhibition, apoptosis and impaired HER2 pro-survival signaling of the trastuzumab-resistant cell line JIMT-1. This is accompanied by a decreased expression on the plasma membrane and accumulation of HER2 in the cytosol, where it colocalizes with endosomes, lysosomes and autophagosomes. Importantly, microscopic analysis of JIMT-1 xenograft tumor tissue of archazolid treated mice confirms the defect in HER2-recycling which leads to reduced tumor growth. These results suggest that V-ATPase inhibition by archazolid induces apoptosis and inhibits growth of trastuzumab-resistant tumor cells by retaining HER2 in dysfunctional vesicles of the recycling pathway and consequently abrogates HER2-signaling in vitro as well as in vivo. V-ATPase inhibition is thus suggested as a promising strategy for treatment of trastuzumab-resistant tumors.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Receptor ErbB-2/genética , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos/uso terapéutico , Mama/efectos de los fármacos , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Inhibidores Enzimáticos/uso terapéutico , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones SCID , Receptor ErbB-2/metabolismo , Trastuzumab , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
Fighting metastasis is a major challenge in cancer therapy and novel therapeutic targets and drugs are highly appreciated. Resistance of invasive cells to anoikis, a particular type of apoptosis induced by loss of cell-matrix contact, is a major prerequisite for their metastatic spread. Inducing anoikis in metastatic cancer cells is therefore a promising therapeutic approach. The vacuolar-ATPase (V-ATPase), a proton pump located at the membrane of acidic organelles, has recently come to focus as an antimetastatic cancer target. As V-ATPase inhibitors have shown to prevent invasion of tumor cells and are able to induce apoptosis, we proposed that V-ATPase inhibition induces anoikis-related pathways in invasive cancer cells. We used the V-ATPase inhibitor archazolid to investigate the mechanism of anoikis induction in various metastatic cancer cells (T24, MDA-MB-231, 4T1, 5637) in vitro. Anoikis induction by archazolid was characterized by decreased c-FLIP expression and caspase-8 activation as well as reduction of active integrin-ß1 and an early increase of the proapoptotic protein BIM. However, we observed that archazolid also induces mechanisms opposing anoikis such as degradation of BIM mediated by extracellular signal-regulated kinase (ERK), Akt and Src kinases at later time points and induction of reactive oxygen species. Still, intravenous injection of archazolid-treated 4T1-Luc2 mouse breast cancer cells resulted in reduced metastasis in mouse lungs. Thus, V-ATPase inhibition is not only an interesting option to reduce cancer metastasis, but also to better understand anoikis resistance and to find choices to fight against it.
Asunto(s)
Anoicis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Macrólidos/farmacología , Invasividad Neoplásica/genética , Tiazoles/farmacología , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Animales , Anoicis/genética , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Hepáticas Experimentales/secundario , Macrólidos/uso terapéutico , Masculino , Ratones , Ratones Endogámicos BALB C , Transducción de Señal/efectos de los fármacos , Tiazoles/uso terapéutico , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Natural compounds derived from marine organisms have shown a wide variety of anti-tumor effects and a lot of attention has been drawn to further development of the isolated compounds. A vast quantity of individual chemical structures from different organisms has shown a variety of apoptosis inducing mechanisms in a variety of tumor cells. The bis-steroidal cephalostatin 1 for example, induces apoptosis via activation of caspases whereas the polyketide discodermolide inhibits cell growth by binding to and stabilizing microtubule and salisporamide A, the product of an actinobacterial strain, is an inhibitor of the proteasome. This great variety of mechanisms of action can help to overcome the multitude of resistances exhibited by different tumor specimens. Products from marine organisms and their synthetic derivates are therefore an important source for new therapeutics for single agent or combined therapy with other chemotherapeutics to support the struggle against cancer.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis , Organismos Acuáticos/química , Productos Biológicos/farmacología , Neoplasias/patología , Alcaloides/farmacología , Alcanos/farmacología , Animales , Antibacterianos/farmacología , Brioestatinas/farmacología , Carbamatos/farmacología , Proliferación Celular , Depsipéptidos/farmacología , Dioxoles/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Lactonas/farmacología , Macrólidos/farmacología , Microtúbulos/metabolismo , Modelos Químicos , Neoplasias/metabolismo , Fenantrolinas/farmacología , Fenazinas/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Pironas/farmacología , Pirroles/farmacología , Quinolinas/farmacología , Compuestos de Espiro/farmacología , Esteroides/farmacología , Tetrahidroisoquinolinas/farmacología , Tiazoles/farmacología , TrabectedinaRESUMEN
The abundance of the multimeric vacuolar ATP-dependent proton pump, V-ATPase, on the plasma membrane of tumor cells correlates with the invasiveness of the tumor cell, suggesting the involvement of V-ATPase in tumor metastasis. V-ATPase is hypothesized to create a proton efflux leading to an acidic pericellular microenvironment that promotes the activity of proinvasive proteases. An alternative, not yet explored possibility is that V-ATPase regulates the signaling machinery responsible for tumor cell migration. Here, we show that pharmacologic or genetic reduction of V-ATPase activity significantly reduces migration of invasive tumor cells in vitro. Importantly, the V-ATPase inhibitor archazolid abrogates tumor dissemination in a syngeneic mouse 4T1 breast tumor metastasis model. Pretreatment of cancer cells with archazolid impairs directional motility by preventing spatially restricted, leading edge localization of epidermal growth factor receptor (EGFR) as well as of phosphorylated Akt. Archazolid treatment or silencing of V-ATPase inhibited Rac1 activation, as well as Rac1-dependent dorsal and peripheral ruffles by inhibiting Rab5-mediated endocytotic/exocytotic trafficking of Rac1. The results indicate that archazolid effectively decreases metastatic dissemination of breast tumors by impairing the trafficking and spatially restricted activation of EGFR and Rho-GTPase Rac1, which are pivotal for directed movement of cells. Thus, our data reveals a novel mechanism underlying the role of V-ATPase in tumor dissemination.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Macrólidos/farmacología , Tiazoles/farmacología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Animales , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Regulación hacia Abajo , Receptores ErbB/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas de Unión al GTP rab5/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Evasion of cell death by overexpression of anti-apoptotic proteins, such as Bcl-2, is commonly observed in cancer cells leading to a lack of response to chemotherapy. Hence, there is a need to find new chemotherapeutic agents that are able to overcome chemoresistance mediated by Bcl-2 and to understand their mechanisms of action. Helenalin, a sesquiterpene lactone (STL), induces cell death and abrogates clonal survival in a highly apoptosis-resistant Bcl-2 overexpressing Jurkat cell line as well as in two other Bcl-2 overexpressing solid tumor cell lines (mammary MCF-7; pancreatic L6.3pl). This effect is not achieved by directly affecting the mitochondria-protective function of Bcl-2 in the intrinsic pathway of apoptosis since Bcl-2 overexpressing Jurkat cells do not show cytochrome c release and dissipation of mitochondrial membrane potential upon helenalin treatment. Moreover, helenalin induces an atypical form of cell death with necrotic features in Bcl-2 overexpressing cells, neither activating classical mediators of apoptosis (caspases, AIF, Omi/HtrA2, Apaf/apoptosome) nor ER-stress mediators (BiP/GRP78 and CHOP/GADD153), nor autophagy pathways (LC3 conversion). In contrast, helenalin was found to inhibit NF-κB activation that was considerably increased in Bcl-2 overexpressing Jurkat cells and promotes cell survival. Moreover, we identified reactive oxygen species (ROS) and free intracellular iron as mediators of helenalin-induced cell death whereas activation of JNK and abrogation of Akt activity did not contribute to helenalin-elicited cell death. Our results highlight the NF-κB inhibitor helenalin as a promising chemotherapeutic agent to overcome Bcl-2-induced cell death resistance.