RESUMEN
SUMO (small ubiquitin like modifier) conjugated proteins have emerged as an important post translational modifier of cellular function. SUMOylation modulates several cellular processes involved in transcriptional regulation of genes, protein-protein interactions and DNA damage and repair. Since abnormalities in SUMOylation has been observed in neoplastic and neurodegenerative disorders, the SUMO pathway has become an attractive site for targeting of new therapies to regulate SUMOylation and reduce disease burden. Conjugation of SUMO to their respective substrates is orchestrated by an enzymatic cascade involving three main enzymes, E1, activation enzyme, E2, conjugating enzyme and E3, a protein ligase. Each of these enzymes are therefore potential "druggable" sites for future therapeutics. SUMOylation is a well-known mechanism by which the innate immune response is regulated in response to viral infections and in the adaptive immune response to tumor immunity. We have shown that small molecules which inhibit the SUMO activation pathway are also capable of inhibiting autoimmune response. TAK981 which forms adducts with SUMO and anacardic acid which inhibits the E1 enzyme of the SUMO pathway were effective in preventing the development of experimental allergic encephalitis (EAE), a mouse model of multiple sclerosis. Anacardic acid and TAK981 inhibited activation of TH17 cells and reduced clinical and pathological injury in IL-17 mediated myelin oligodendrocyte glycoprotein (MOG) induced EAE. Ginkgolic acid, another known inhibitor of SUMO pathway, was also shown to be effective in reducing the severity of inflammatory arthropathies which is also IL-17 mediated. In addition, the increase in the transcription of myelin genes with TAK981 and anacardic acid improved remyelination in experimental models of demyelination. In the present review paper, we examine the mechanism of action of inhibitors of the SUMO pathway on regulating the immune response and the possibility of the use of these agents as therapeutics for MS.
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Esclerosis Múltiple , Sumoilación , Animales , Humanos , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/terapia , Esclerosis Múltiple/metabolismo , Sumoilación/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismoRESUMEN
Acute myeloid leukemia (AML) presents a pressing medical need in that it is largely resistant to standard chemotherapy as well as modern therapeutics, such as targeted therapy and immunotherapy, including anti-programmed cell death protein (anti-PD) therapy. We demonstrate that programmed death-1 homolog (PD-1H), an immune coinhibitory molecule, is highly expressed in blasts from the bone marrow of AML patients, while normal myeloid cell subsets and T cells express PD-1H. In studies employing syngeneic and humanized AML mouse models, overexpression of PD-1H promoted the growth of AML cells, mainly by evading T cell-mediated immune responses. Importantly, ablation of AML cell-surface PD-1H by antibody blockade or genetic knockout significantly inhibited AML progression by promoting T cell activity. In addition, the genetic deletion of PD-1H from host normal myeloid cells inhibited AML progression, and the combination of PD-1H blockade with anti-PD therapy conferred a synergistic antileukemia effect. Our findings provide the basis for PD-1H as a potential therapeutic target for treating human AML.
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Evasión Inmune , Leucemia Mieloide Aguda , Animales , Humanos , Ratones , Médula Ósea , Inmunidad Celular , Inmunoterapia , Leucemia Mieloide Aguda/tratamiento farmacológicoRESUMEN
Gastrin-releasing peptide (GRP) is a proangiogenic ligand secreted by tumors and acts directly upon binding to GRP receptor in endothelial cells. Angiogenesis plays a critical role in the pathology of various diseases, including cancer, as the formation of new blood vessels potentiates the rate of tumor growth and dissemination. GRP increases the migration of endothelial cells, but much is unknown about its role on endothelial cell proliferation and survival, as well as the signaling pathways involved. In the present study, we showed that GRP increases endothelial cell proliferation and tubule formation. There was a time-dependent increase in the levels of phosphorylated AKT, mammalian target of rapamycin (mTOR), and S6R in human umbilical vein endothelial cells treated with GRP. Interestingly, GRP treatment decreased the expression of proautophagic factors, ATG5, BECN1, and LC3 proteins. GRP also attenuated rapamycin-induced formation of autophagosomes. Moreover, overexpression of ATG5 or BECN1 significantly decreased tubule formation induced by exogenous GRP, whereas siRNA against ATG5 or BECN1 resulted in increased tubule formation with GRP treatment. Our results show that GRP inhibits the process of autophagy in vascular endothelial cells, thereby increasing endothelial cell proliferation and tubule formation. Here, we describe a novel role of GRP in the regulation of autophagy of endothelial cells, thereby providing a potential new therapeutic strategy in targeting angiogenesis during cancer progression.
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Autofagia , Proliferación Celular , Células Endoteliales/fisiología , Péptido Liberador de Gastrina/fisiología , Neovascularización Patológica , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 5 Relacionada con la Autofagia , Beclina-1 , Técnicas de Cocultivo , Endotelio Vascular/crecimiento & desarrollo , Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Comunicación Paracrina , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Small ubiquitin like modifiers (SUMO) are reversible posttranslational modifiers of intracellular proteins. In the CNS, expression of myelin genes is regulated by state of SUMOylation of their respective transcription factors. In the immune system, deSUMOylation activates innate immune responses and promotes anti-viral immunity. However, the role played by SUMO in an adaptive immune response and in the development of T cell mediated autoimmune disease has not been previously described. TAK981 is a synthetic small molecule which by forming adducts with SUMO proteins prevents SUMOylation. We examined the expression of myelin genes and their transcription factors following culture with TAK981 in Oligodendrocyte Precursor Cells (OPC). We found that myelin basic protein (MBP), a key myelin protein, is upregulated in OPC in the presence of TAK981. We also found increased expression of transcription factors Sox10 and Myrf, which engage in the expression of MBP. In the Cuprizone model of demyelination/remyelination, animals which were treated with TAK981 showed increased remyelination in areas of demyelination and an increase in the number of maturing oligodendrocytes compared to vehicle treated controls. In in vitro cultures of lymphocytes, TAK981 reduced the expression of TH17 in T cells in mice immunized with MOGp35-55. Following in vivo treatment with TAK981, there was a significant reduction in the clinical and pathological severity in mice immunized to develop experimental allergic encephalitis (EAE). The dual effects of deSUMOylation on remyelination and in regulating an autoimmune adaptive response offers a novel approach to the management of human inflammatory demyelinating diseases such as multiple sclerosis.
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Enfermedades del Sistema Nervioso Central , Enfermedades Desmielinizantes , Remielinización , Ratones , Humanos , Animales , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/tratamiento farmacológico , Enfermedades Desmielinizantes/metabolismo , Remielinización/fisiología , Sumoilación , Interleucina-17 , Diferenciación Celular , Vaina de Mielina/patología , Oligodendroglía/metabolismo , Cuprizona/toxicidad , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Enfermedades del Sistema Nervioso Central/metabolismo , Factores de Transcripción/metabolismo , Ratones Endogámicos C57BL , Modelos Animales de EnfermedadRESUMEN
Effective eradication of leukemic stem cells (LSCs) remains the greatest challenge in treating acute myeloid leukemia (AML). The immune receptor LAIR-1 has been shown to regulate LSC survival; however, the therapeutic potential of this pathway remains unexplored. We developed a therapeutic LAIR-1 agonist antibody, NC525, that induced cell death of LSCs, but not healthy hematopoietic stem cells in vitro, and killed LSCs and AML blasts in both cell- and patient-derived xenograft models. We showed that LAIR-1 agonism drives a unique apoptotic signaling program in leukemic cells that was enhanced in the presence of collagen. NC525 also significantly improved the activity of azacitidine and venetoclax to establish LAIR-1 targeting as a therapeutic strategy for AML that may synergize with standard-of-care therapies.
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Leucemia Mieloide Aguda , Animales , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Células Madre Hematopoyéticas/metabolismo , Transducción de Señal , Modelos Animales de Enfermedad , Células Madre Neoplásicas/metabolismoRESUMEN
MicroRNA-145 (miR-145) plays a suppressive role in the process of tumorigenesis and an important role in induction of autophagy. However, the exact role of miR-145 in therapeutically resistant neuroblastoma cells remain elusive. Herein, we sought to evaluate the effects of miR-145 overexpression in chemo and radiation-resistant neuroblastoma cells. We hypothesized that miR-145 affects the aggressiveness of resistant cells by enhancing autophagy. We established Cisplatin-resistant (CDDP-R), Vincristine-resistant (Vin-R), and radiation-resistant (Rad-R) neuroblastoma cells and found that miR-145 expression was significantly decreased in the resistant cells compared to the parental cells. Exogenously expression of miR-145 inhibited oncogenic properties such as proliferation, clonogenicity, anchorage-independent growth, cell migration, and tubule formation in the resistant cells. In addition, we also found that an autophagy protein marker, LC3, was only minimally expressed in the resistant cells. In particular, when miR-145 was overexpressed in the resistant cells, LC3 I and II were expressed and an increased punctate fluorescence of LC3 protein was found indicating the induction of autophagy. Taken together, our data suggests that miR-145 inhibits tumorigenesis and aggressiveness via modulation of autophagy in neuroblastoma.
RESUMEN
Neuroblastoma remains one of the most difficult pediatric solid tumors to treat. In particular, the refractory and relapsing neuroblastomas are highly heterogeneous with diverse molecular profiles. We previously demonstrated that AKT2 plays critical roles in the regulation of neuroblastoma tumorigenesis. Here we hypothesize that targeting AKT2 could block the signal transduction pathways enhanced in chemo- and/or radiation-resistant neuroblastoma cancer stem-like cells. We found cell proliferation and survival signaling pathways AKT2/mTOR and MAPK were enhanced in cisplatin (CDDP)- and radiation-resistant neuroblastoma cells. Blocking these two pathways with specific inhibitors, CCT128930 (AKT2 inhibitor) and PD98059 (MEK inhibitor) decreased cell proliferation, angiogenesis, and cell migration in these resistant cells. We further demonstrated that the resistant cells had a higher sphere-forming capacity with increased expression of stem cell markers CD133, SOX2, ALDH, Nestin, Oct4, and Nanog. Importantly, the tumorsphere formation, which is a surrogate assay for self-renewal, was sensitive to the inhibitors of AKT2 and MAPK. Taken together, our findings suggest that CDDP- and radiation-resistant cancer stem-like neuroblastoma cells might serve as a useful tool to improve the understanding of molecular mechanisms of therapeutic resistance. This may aid in the development of more effective novel treatment strategies and better clinical outcomes in patients with neuroblastoma.
RESUMEN
The lost expression of α-catenin has been found in cancers, and reinstalling α-catenin inhibits tumor growth. Here we hypothesized that the α-N-catenin, a homologous member of α-catenin and neural-specific expressed, functions as a novel tumor suppressor in neural crest-derived tumor, neuroblastoma. We correlated CTNNA2 (encodes α-N-catenin) expression to neuroblastoma disease relapse-free survival probability using publicly accessible human neuroblastoma datasets in R2 platform. The result showed that it negatively correlated to relapse-free survival probability significantly in patients with neuroblastoma with non-MYCN amplified tumor. Conversely, overexpressing CTNNA2 suppressed the neuroblastoma cell proliferation as measuring by the clonogenesis, inhibited anchorage-independent growth with soft agar colony formation assay. Forced expression of CTNNA2 decreased cell migration and invasion. Further, overexpression of CTNNA2 reduced the secretion of angiogenic factor IL-8 and HUVEC tubule formation. Our results show, for the first time, that α-N-catenin is a tumor suppressor in neuroblastoma cells. These findings were further corroborated with in vivo tumor xenograft study, in which α-N-catenin inhibited tumor growth and reduced tumor blood vessel formation. Interestingly, this is only observed in SK-N-AS xenografts lacking MYCN expression, and not in BE(2)-C xenografts with MYCN amplification. Mechanistically, α-N-catenin attenuated NF-κB responsive genes by inhibiting NF-κB transcriptional activity. In conclusion, these data demonstrate that α-N-catenin is a tumor suppressor in non-MYCN-amplified neuroblastomas and it inhibits NF-κB signaling pathway to suppress tumor growth in human neuroblastomas. Therefore, restoring the expression of α-N-catenin can be a novel therapeutic approach for neuroblastoma patients who have the deletion of CTNNA2 and lack of MYCN amplification.
RESUMEN
PURPOSE: Poly(ADP-ribose) polymerase-1 (PARP-1) is the founding member of a family of enzymes that catalyze the addition of ADP-ribose units to proteins that mediate DNA repair pathways. Ionizing radiation induces DNA strand breaks, suggesting that PARP-1 inhibition may sensitize tumor cells to radiation. EXPERIMENTAL DESIGN: We investigated the combination of PARP-1 inhibition with radiation in lung cancer models. ABT-888, a novel potent PARP-1 inhibitor, was used to explore the effects of PARP-1 inhibition on irradiated tumors and tumor vasculature. RESULTS: ABT-888 reduced clonogenic survival in H460 lung cancer cells, and inhibited DNA repair as shown by enhanced expression of DNA strand break marker histone gamma-H2AX. Both apoptosis and autophagy contributed to the mechanism of increased cell death. Additionally, ABT-888 increased tumor growth delay at well-tolerated doses in murine models. For a 5-fold increase in tumor volume, tumor growth delay was 1 day for ABT-888 alone, 7 days for radiation alone, and 13.5 days for combination treatment. Immunohistochemical staining of tumor sections revealed an increase in terminal deoxyribonucleotide transferase-mediated nick-end labeling apoptotic staining, and a decrease in Ki-67 proliferative staining after combination treatment. Matrigel assay showed a decrease in in vitro endothelial tubule formation with ABT-888/radiation combination treatment, and von Willebrand factor staining of tumor sections revealed decreased vessel formation in vivo, suggesting that this strategy may also target tumor angiogenesis. CONCLUSIONS: We conclude that PARP-1 inhibition shows promise as an effective means of enhancing tumor sensitivity to radiation, and future clinical studies are needed to determine the potential of ABT-888 as a radiation enhancer.
Asunto(s)
Bencimidazoles/farmacología , Carcinoma de Pulmón de Células no Pequeñas/terapia , Neoplasias Pulmonares/terapia , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Tolerancia a Radiación/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Bencimidazoles/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Terapia Combinada , Reparación del ADN/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Antígeno Ki-67/análisis , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/radioterapia , Ratones , Ensayos Antitumor por Modelo de Xenoinjerto , Factor de von Willebrand/análisisRESUMEN
The phosphatidylinositol 3-kinase/Akt pathway plays a critical role in oncogenesis, and dysregulation of this pathway through loss of PTEN suppression is a particularly common phenomenon in aggressive prostate cancers. The mammalian target of rapamycin (mTOR) is a downstream signaling kinase in this pathway, exerting prosurvival influence on cells through the activation of factors involved in protein synthesis. The mTOR inhibitor rapamycin and its derivatives are cytotoxic to a number of cell lines. Recently, mTOR inhibition has also been shown to radiosensitize endothelial and breast cancer cells in vitro. Because radiation is an important modality in the treatment of prostate cancer, we tested the ability of the mTOR inhibitor RAD001 (everolimus) to enhance the cytotoxic effects of radiation on two prostate cancer cell lines, PC-3 and DU145. We found that both cell lines became more vulnerable to irradiation after treatment with RAD001, with the PTEN-deficient PC-3 cell line showing the greater sensitivity. This increased susceptibility to radiation is associated with induction of autophagy. Furthermore, we show that blocking apoptosis with caspase inhibition and Bax/Bak small interfering RNA in these cell lines enhances radiation-induced mortality and induces autophagy. Together, these data highlight the emerging importance of mTOR as a molecular target for therapeutic intervention, and lend support to the idea that nonapoptotic modes of cell death may play a crucial role in improving tumor cell kill.
Asunto(s)
Fosfohidrolasa PTEN/deficiencia , Neoplasias de la Próstata/radioterapia , Proteínas Quinasas/metabolismo , Fármacos Sensibilizantes a Radiaciones/farmacología , Sirolimus/análogos & derivados , Animales , Autofagia/efectos de los fármacos , Autofagia/efectos de la radiación , Línea Celular Tumoral , Everolimus , Humanos , Masculino , Ratones , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Inhibidores de Proteínas Quinasas/farmacología , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiación , Sirolimus/farmacología , Serina-Treonina Quinasas TOR , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína X Asociada a bcl-2/genéticaRESUMEN
PURPOSE: Survivin, a member of the inhibitor of apoptosis gene family, has also been shown to regulate mitosis. It binds Aurora B kinase and the inner centromere protein to form the chromosome passenger complex. Both Aurora B and survivin are overexpressed in many tumors. In this study, we examined whether irradiation affected survivin and Aurora B expression in mesothelioma cells, and how inhibition of these molecules affected radiosensitivity. METHODS AND MATERIALS: ZM447439 and survivin antisense oligonucleotides were used to inhibit survivin and Aurora B kinase respectively. Western blot was performed to determine the expression of survivin, Aurora B, phosphorylated-histone H3 (Ser 10), and caspase cleavage. Multinucleated cells were counted using flow cytometry, and cell survival after treatment was determined using clonogenic assay. RESULTS: At 3-Gy irradiation an increase was observed in levels of survivin and Aurora B as well as the kinase activity of Aurora B, with an increase in G2/M phase. The radiation-induced upregulation of these molecules was effectively attenuated by antisense oligonucleotides against survivin and a small-molecule inhibitor of Aurora B, ZM447439. Dual inhibition of survivin and Aurora B synergistically radiosensitized mesothelioma cells with a dose enhancement ratio of 2.55. This treatment resulted in increased formation of multinucleated cells after irradiation but did not increase levels of cleaved caspase 3. CONCLUSION: Inhibition of survivin and Aurora B induces mitotic cell arrest in mesothelioma cells after irradiation. These two proteins may be potential therapeutic targets for the enhancement of radiotherapy in malignant pleural mesothelioma.
Asunto(s)
Histonas/metabolismo , Mesotelioma/radioterapia , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Mitosis/efectos de los fármacos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Aurora Quinasa B , Aurora Quinasas , Benzamidas , Caspasa 3/metabolismo , Caspasa 3/efectos de la radiación , Supervivencia Celular , Fase G2/efectos de la radiación , Histonas/efectos de la radiación , Humanos , Proteínas Inhibidoras de la Apoptosis , Mesotelioma/metabolismo , Mesotelioma/patología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/efectos de la radiación , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/efectos de la radiación , Oligonucleótidos Antisentido/farmacología , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/efectos de la radiación , Quinazolinas , Tolerancia a Radiación/efectos de los fármacos , Survivin , Regulación hacia Arriba/efectos de la radiaciónRESUMEN
BAD, a proapoptotic molecule of the BCL2 family, is regulated by reversible phosphorylation. During survival, BAD is sequestered by 14-3-3 through serine 136 phosphorylation and is dissociated from BCL-X(L) through serine 155 phosphorylation. We report that phosphoserine 112 (pSer112) dephosphorylation functions as a gatekeeper for BAD-mediated apoptosis. During apoptosis, dephosphorylation of pSer112 preceded pSer136 dephosphorylation. Dephosphorylation of pSer112 accelerated dephosphorylation of pSer136, and inhibition of pSer112 dephosphorylation prevented pSer136 dephosphorylation, indicating that dephosphorylation of pSer112 is required for dephosphorylation of pSer136. Protein phosphatase 2A (PP2A) is the major pSer112 phosphatase. PP2A competed with 14-3-3 for BAD binding, and survival factor withdrawal enhanced PP2A association with BAD. Dephosphorylation of the critical residue, pSer136, could only be blocked by inhibition of all known subfamilies of serine/threonine phosphatases, suggesting that multiple phosphatases are involved in pSer136 dephosphorylation. Inhibition of PP2A rescued FL5.12 cells from apoptosis, demonstrating a physiologic role for PP2A-mediated pSer112 dephosphorylation. Thus, PP2A dephosphorylation of pSer112 is the key initiating event regulating the activation of BAD during interleukin-3 withdrawal-induced apoptosis.
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Apoptosis/fisiología , Proteínas Portadoras/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Fosfoserina/metabolismo , Proteínas 14-3-3 , Células 3T3 , Animales , Sitios de Unión , Unión Competitiva , Proteínas Portadoras/genética , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Humanos , Toxinas Marinas , Ratones , Mutación , Oxazoles/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/genética , Fosforilación , Proteína Fosfatasa 2 , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Proteína Letal Asociada a bcl , Proteína bcl-XRESUMEN
The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is known to be activated by radiation. The mammalian target of rapamycin (mTOR) is downstream of Akt, and we investigated the effects of radiation on Akt/mTOR signaling in breast cancer cell models. RAD001 (everolimus), a potent derivative of the mTOR inhibitor rapamycin, was used to study the effects of mTOR inhibition, as the role of mTOR inhibition in enhancing radiation remains unexplored. RAD001 decreased clonogenic cell survival in both breast cancer cell lines MDA-MB-231 and MCF-7, although the effect is greater in MDA-MB-231 cells. Irradiation induced Akt and mTOR signaling, and this signaling is attenuated by RAD001. The radiation-induced signaling activation is mediated by PI3K because inhibition of PI3K with LY294002 inhibited the increase in downstream mTOR signaling. Additionally, caspase-dependent apoptosis is an important mechanism of cell death when RAD001 is combined with 3 Gy radiation, as shown by induction of caspase-3 cleavage. An increase in G(2)-M cell cycle arrest was seen in the combination treatment group when compared with controls, suggesting that cell cycle arrest may have been a contributing factor in the increased radiosensitization seen in this study. We conclude that RAD001 attenuates radiation-induced prosurvival Akt/mTOR signaling and enhances the cytotoxic effects of radiation in breast cancer cell models, showing promise as a method of radiosensitization of breast cancer.
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Neoplasias de la Mama/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fármacos Sensibilizantes a Radiaciones/farmacología , Transducción de Señal/efectos de la radiación , Sirolimus/análogos & derivados , Caspasa 3 , Caspasas/metabolismo , Muerte Celular , Supervivencia Celular/efectos de la radiación , Everolimus , Femenino , Humanos , Proteínas Quinasas/efectos de la radiación , Proteínas Proto-Oncogénicas c-akt/efectos de la radiación , Fármacos Sensibilizantes a Radiaciones/toxicidad , Transducción de Señal/efectos de los fármacos , Sirolimus/metabolismo , Sirolimus/farmacología , Sirolimus/toxicidad , Serina-Treonina Quinasas TOR , Células Tumorales CultivadasRESUMEN
p53 plays a critical role in cell cycle arrest and induction of apoptosis. Certain malignancies carry wild-type p53, which is frequently down-regulated by murine double minute 2 (MDM2) overexpression. Availability of a small-molecule inhibitor against MDM2, nutlin, has made it feasible to evaluate the anti-MDM2-based therapeutic strategies. The rationale for the current study is that functional p53 has been linked with improved responses to radiation treatment. Hence, this study evaluates the use of nutlin, a small-molecule inhibitor that blocks the interaction of p53 and MDM2, in sensitizing cancer cells to radiation. Expression of MDM2, p53, and p21 in both p53 wild-type and p53-defective lung cancer cell lines was examined. Clonogenic and 7-amino-actinomycin D studies were used to determine possible mechanisms of cell death. The combined effect of MDM2 inhibition and radiation on cell cycle was also studied. We found that radiosensitization by nutlin occurs in lung cancer cells with wild-type p53. There were increased apoptosis and cell cycle arrest following administration of nutlin and radiation. Furthermore, the combination of nutlin and radiation decreased the ability of endothelial cells to form vasculature, as shown by Matrigel assays. Our data suggest that nutlin is an effective radiosensitizer of p53 wild-type cells. The radiosensitizing effect seems to be at least partially due to induction of apoptosis and cell cycle arrest. In addition, nutlin may be an effective radiosensitizer of tumor vasculature.
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Imidazoles/farmacología , Neoplasias Pulmonares/metabolismo , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Fármacos Sensibilizantes a Radiaciones/farmacología , Ciclo Celular/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Humanos , Imidazoles/química , Neoplasias Pulmonares/enzimología , Piperazinas/química , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Tolerancia a Radiación/efectos de los fármacos , Estereoisomerismo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Signal transducer and activator of transcription 3 (Stat3) and Survivin are constitutively up-regulated in various human tumor cells. We previously found Survivin to be significantly reduced in response to radiation in human umbilical vein endothelial cells (HUVEC) but not in tumor cell lines. In this study, we examined the effect of Stat3 on Survivin expression in irradiated HUVECs and breast cancer cells. We also studied how inhibition of Stat3 and Survivin activity affects cell survival and angiogenesis following irradiation. We determined that Survivin was significantly increased by overexpression of an active Stat3 (Stat3-C). Following irradiation, the level of phospho-Stat3 Tyr(705), but not phospho-Stat3 Ser(727), was reduced in HUVECs, whereas it remained unchanged in irradiated breast cancer cells. Correspondingly, Stat3 DNA-binding activity following irradiation was specifically down-regulated in HUVECs but not in breast cancer cells. Mutation of Tyr(705) abolished radiation-induced down-regulation of Survivin. Clonogenic and endothelial cell morphogenesis assays suggested that DN-Stat3 and DN-Survivin together resulted in the greatest radiosensitization of MDA-MB-231, decreasing angiogenesis and cell survival. In summary, Stat3 modulates Survivin, and both are potential therapeutic targets for radiation sensitization in breast cancer.
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Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Tolerancia a Radiación , Factor de Transcripción STAT3/antagonistas & inhibidores , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/radioterapia , Ensayo de Cambio de Movilidad Electroforética , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Femenino , Humanos , Proteínas Inhibidoras de la Apoptosis , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/genética , Mutación , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Survivin , Células Tumorales CultivadasRESUMEN
PURPOSE: Clusterin plays important roles in cell survival and death. Inactivation of clusterin enhances the therapeutic efficacy of chemotherapy in lung cancer models. The purpose of this study was to determine whether inhibition of clusterin by an antisense-based investigative drug enhances radiation sensitization in a lung cancer model. METHODS AND MATERIALS: Cells were transfected with an antisense oligonucleotide (ASO) against clusterin (OGX-011). Apoptosis was determined by 7-aminoactinomycin D staining. Cell survival was examined by 3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) and clonogenic assay. Xenograft model was used to demonstrate tumor growth and tumor blood flow. RESULTS: OGX-011 specifically attenuated the expression of secreted clusterin (prosurvival), with no apparent effect on the expression of nuclear clusterin (proapoptotic). Apoptosis was significantly increased when H460 lung cancer cells were treated with OGX-011 plus radiation. Inhibition of clusterin followed by radiation greatly decreased cell survival. H460 xenografts that were treated with OGX-011 plus radiotherapy demonstrated growth delay beyond 17 days. Doppler studies showed that tumor blood flow was compromised when mice bearing H460 xenografts were treated with OGX-011 and radiation. CONCLUSION: A combination of radiotherapy and OGX-011 improved control of tumor growth and vascular regression in the H460 lung cancer model.
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Clusterina/antagonistas & inhibidores , Neoplasias Pulmonares/radioterapia , Proteínas de Neoplasias/antagonistas & inhibidores , Oligonucleótidos Antisentido/uso terapéutico , Tionucleótidos/uso terapéutico , Animales , Apoptosis , Línea Celular Tumoral , Femenino , Humanos , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/tratamiento farmacológico , Ratones , Ratones Desnudos , Transfección , Trasplante HeterólogoRESUMEN
BACKGROUND: MYCN amplification is a key molecular hallmark of high-risk neuroblastoma. Previously considered an "undruggable" target, MYCN transcription can be disrupted by inhibiting the bromodomain and the extraterminal (BET) domain family of proteins that regulates MYCN transcription epigenetically. JQ1 is a potent, small-molecule BET inhibitor that induces cell-cycle arrest and initiates apoptosis in neuroblastoma. Here, we sought to validate the antitumorigenic effects of JQ1 in neuroblastoma and to evaluate whether blocking N-myc expression with JQ1 promotes neural differentiation. METHODS: We determined the effects in vitro of JQ1 treatment on human neuroblastoma cell growth in both monolayer and sphere-forming conditions. Subcutaneous neuroblastoma xenografts were used for an in vivo study. Western blotting and immunohistochemistry were performed to evaluate the effects on neural differentiation and stem cell markers. RESULTS: JQ1 treatment blocked neuroblastoma cell growth in both monolayer and sphere-forming conditions; JQ1 also attenuated the growth of neuroblastoma xenograft in athymic nude mice. Neurofilament expression was enhanced with JQ1 treatment, indicating that JQ1 induces neuronal differentiation. Sphere forming conditions resulted in increased expression of multiple stem cell markers; these effects were reversed with JQ1 treatment. CONCLUSION: BET inhibition attenuates progression and promotes neural differentiation of neuroblastoma in vitro and in vivo in mice, providing insight into potential clinical applications of BET inhibitors in the treatment of patients with neuroblastoma.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Azepinas/farmacología , Neuroblastoma/tratamiento farmacológico , Neurogénesis/efectos de los fármacos , Triazoles/farmacología , Animales , Antineoplásicos/uso terapéutico , Azepinas/uso terapéutico , Biomarcadores de Tumor/metabolismo , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Desnudos , Proteína Proto-Oncogénica N-Myc , Neuroblastoma/genética , Neuroblastoma/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/metabolismo , Distribución Aleatoria , Triazoles/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
INTRODUCTION: Most drug resistant cancer cells also develop resistance to radiation therapy. In this study, we hypothesized that the dual inhibitor of phosphatidylinositol-3 kinase/mammalian target of rapamycin, NVP-BEZ-235, could potentially enhance radiosensitization in cisplatin-resistance (CDDP-R) non-small cell lung cancer (NSCLC) cells by disabling autophagy as a mechanism of self-preservation. METHODS: We used both in vitro and in vivo approaches, including clonogenic assays, Western blotting, molecular analyses of autophagy and apoptosis, a xenograft model of tumor growth, and immunohistochemical analysis. RESULTS: Basal p-Akt, p-mTOR and p-S6R proteins were enhanced in CDDP-R NSCLC cells. CDDP-R-resistant NSCLC cells are less radiation sensitive in comparison to parental cells (DER=0.82, p=0.02); BEZ-235 enhanced the radiosensitivity (DER=1.2, p=0.01). In addition, combining BEZ-235/RT showed a dramatic tumor growth delay in a mouse xenograft model. Immunohistochemistry showed that combination therapy yielded 50% decrease in caspase-3 activity. Moreover, cell proliferation was reduced by 87.8% and vascular density by 86.1%. These results were associated with a downregulation of PI3K/mTOR signaling pathway and an increase in autophagy. CONCLUSIONS: These findings may be utilized as a novel strategy to enhance the efficacy of radiation therapy in drug-selected non-small cell lung cancer exhibiting radioresistance.
RESUMEN
PURPOSE: Aurora kinase A (AURKA) overexpression is associated with poor prognosis in neuroblastoma and has been described to upregulate VEGF in gastric cancer cells. However, the exact role of AURKA in the regulation of neuroblastoma tumorigenesis remains unknown. We hypothesize that AURKA-mediated stabilization of N-Myc may affect VEGF expression and angiogenesis in neuroblastoma. Therefore, we sought to determine whether inhibition of AURKA modulates neuroblastoma angiogenesis. METHODS: Cell viability and anchorage-independent growth were determined after silencing AURKA or after treatment with MLN8237, AURKA inhibitor. Immunofluorescence was used to determine N-Myc localization. Human umbilical vein endothelial cells (HUVECs) were used to assess angiogenesis in vitro. Real time-PCR and ELISA were performed to determine VEGF transcription and secretion, respectively. RESULTS: Knockdown of AURKA significantly reduced cell proliferation and inhibited anchorage-independent growth. It also decreased N-Myc protein levels and nuclear localization. AURKA inhibition also decreased HUVECs tubule formation along with VEGF transcription and secretion. Similarly, MLN8237 treatment decreased neuroblastoma tumorigenicity in vitro. CONCLUSIONS: Our findings demonstrate that AURKA plays a critical role in neuroblastoma angiogenesis. AURKA regulates nuclear translocation of N-Myc in neuroblastoma cells, thus potentially affecting cell proliferation, anchorage-independent cell growth, and angiogenesis. Targeting AURKA might provide a novel therapeutic strategy in treating aggressive neuroblastomas.
Asunto(s)
Antineoplásicos/farmacología , Aurora Quinasa A/antagonistas & inhibidores , Azepinas/farmacología , Terapia Molecular Dirigida , Proteínas de Neoplasias/antagonistas & inhibidores , Neovascularización Patológica/tratamiento farmacológico , Neuroblastoma/patología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , ARN Interferente Pequeño/farmacología , Aurora Quinasa A/fisiología , Adhesión Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Técnicas In Vitro , Proteínas de Neoplasias/fisiología , Neovascularización Patológica/enzimología , Neovascularización Patológica/etiología , Neuroblastoma/irrigación sanguínea , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/enzimología , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/fisiología , ARN Neoplásico/biosíntesis , Ensayo de Tumor de Célula Madre , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Neuroblastoma is characterized by florid vascularization leading to rapid tumor dissemination to distant organs; angiogenesis contributes to tumor progression and poor clinical outcomes. We have previously demonstrated an increased expression of gastrin-releasing peptide (GRP) and its receptor, GRPR, in neuroblastoma and that GRP activates the PI3K-AKT pathway as a proangiogenic factor during tumor progression. Interestingly, AKT activation phosphorylates MTOR, a critical negative regulator of autophagy, a cellular process involved in the degradation of key proteins. We hypothesize that inhibition of GRPR enhances autophagy-mediated degradation of GRP and subsequent inhibition of angiogenesis in neuroblastoma. Here, we demonstrated a novel phenomenon where targeting GRPR using shRNA or a specific antagonist, RC-3095, decreased GRP secretion by neuroblastoma cells and tubule formation by endothelial cells in vitro. Furthermore, shGRPR or RC-3095 treatment enhanced expression of proautophagic proteins in human neuroblastoma cell lines, BE(2)-C, and BE(2)-M17. Interestingly, rapamycin, an inhibitor of MTOR, enhanced the expression of the autophagosomal marker LC3-II and GRP was localized within LC3-II-marked autophagosomes in vitro as well as in vivo, indicating autophagy-mediated degradation of GRP. Moreover, overexpression of ATG5 or BECN1 attenuated GRP secretion and tubule formation, whereas opposite effects were observed with siRNA silencing of ATG5 and BECN1. Our data supported the role of autophagy in the degradation of GRP and subsequent inhibition of angiogenesis. Therefore, activation of autophagy may lead to novel antivascular therapeutic strategies in the treatment of highly vascular neuroblastomas.