RESUMEN
The present studies were to determine whether the multi-kinase inhibitor sorafenib or its derivative regorafenib interacted with the ERBB1/ERBB2 inhibitor lapatinib to kill CNS tumor cells. In multiple CNS tumor cell types sorafenib and lapatinib interacted in a greater than additive fashion to cause tumor cell death. Tumor cells lacking PTEN, and anoikis or lapatinib resistant cells were as sensitive to the drug combination as cells expressing PTEN or parental cells, respectively. Similar data were obtained using regorafenib. Treatment of brain cancer cells with [sorafenib + lapatinib] enhanced radiation toxicity. The drug combination increased the numbers of LC3-GFP vesicles; this correlated with a reduction in endogenous LC3II, and p62 and LAMP2 degradation. Knock down of Beclin1 or ATG5 significantly suppressed drug combination lethality. Expression of c-FLIP-s, BCL-XL, or dominant negative caspase 9 reduced drug combination toxicity; knock down of FADD or CD95 was protective. Expression of both activated AKT and activated MEK1 or activated mTOR was required to strongly suppress drug combination lethality. As both lapatinib and sorafenib are FDA approved agents, our data argue for further determination as to whether lapatinib and sorafenib is a useful glioblastoma therapy.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Niacinamida/análogos & derivados , Compuestos de Fenilurea/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Quinazolinas/farmacología , Anoicis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Proteína 5 Relacionada con la Autofagia , Beclina-1 , Neoplasias Encefálicas/radioterapia , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/biosíntesis , Caspasa 9/biosíntesis , Línea Celular Tumoral , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Proteína de Dominio de Muerte Asociada a Fas/genética , Humanos , Lapatinib , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , MAP Quinasa Quinasa 1/biosíntesis , Proteínas de la Membrana/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Niacinamida/farmacología , Fosfohidrolasa PTEN/genética , Proteínas Proto-Oncogénicas c-akt/biosíntesis , Sorafenib , Serina-Treonina Quinasas TOR/biosíntesis , Respuesta de Proteína Desplegada/efectos de los fármacos , Proteína bcl-X/biosíntesis , Proteína bcl-X/metabolismo , Receptor fas/genéticaRESUMEN
We determined whether the multi-kinase inhibitor sorafenib or its derivative regorafenib interacted with phosphodiesterase 5 (PDE5) inhibitors such as Viagra (sildenafil) to kill tumor cells. PDE5 and PDGFRα/ß were over-expressed in liver tumors compared to normal liver tissue. In multiple cell types in vitro sorafenib/regorafenib and PDE5 inhibitors interacted in a greater than additive fashion to cause tumor cell death, regardless of whether cells were grown in 10 or 100% human serum. Knock down of PDE5 or of PDGFRα/ß recapitulated the effects of the individual drugs. The drug combination increased ROS/RNS levels that were causal in cell killing. Inhibition of CD95/FADD/caspase 8 signaling suppressed drug combination toxicity. Knock down of ULK-1, Beclin1, or ATG5 suppressed drug combination lethality. The drug combination inactivated ERK, AKT, p70 S6K, and mTOR and activated JNK. The drug combination also reduced mTOR protein expression. Activation of ERK or AKT was modestly protective whereas re-expression of an activated mTOR protein or inhibition of JNK signaling almost abolished drug combination toxicity. Sildenafil and sorafenib/regorafenib interacted in vivo to suppress xenograft tumor growth using liver and colon cancer cells. From multiplex assays on tumor tissue and plasma, we discovered that increased FGF levels and ERBB1 and AKT phosphorylation were biomarkers that were directly associated with lower levels of cell killing by 'rafenib + sildenafil. Our data are now being translated into the clinic for further determination as to whether this drug combination is a useful anti-tumor therapy for solid tumor patients.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/biosíntesis , Neoplasias/tratamiento farmacológico , Niacinamida/análogos & derivados , Compuestos de Fenilurea/administración & dosificación , Inhibidores de Fosfodiesterasa 5/administración & dosificación , Piperazinas/administración & dosificación , Sulfonamidas/administración & dosificación , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/genética , Sinergismo Farmacológico , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células Hep G2 , Humanos , Proteínas de Neoplasias/biosíntesis , Neoplasias/genética , Neoplasias/patología , Niacinamida/administración & dosificación , Purinas/administración & dosificación , Transducción de Señal/efectos de los fármacos , Citrato de Sildenafil , Sorafenib , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The present studies were undertaken to determine whether the multikinase inhibitors sorafenib/regorafenib cooperated with clinically relevant , phosphatidyl inositol 3 kinase (PI3K)-thymoma viral proto-oncogene (AKT) inhibitors to kill tumor cells. In liver, colorectal, lung, breast, kidney, and brain cancer cells, at clinically achievable doses, sorafenib/regorafenib and the PI3K inhibitor acetic acid (1S,4E,10R,11R,13S,14R)-[4-diallylaminomethylene-6-hydroxy-1-methoxymethyl-10,13-dimethyl-3,7,17-trioxo-1,3,4,7,10,11,12,13,14,15,16,17-dodecahydro-2-oxa-cyclopenta[a]phenanthren-11-yl ester (PX-866) cooperated in a greater than additive fashion to kill tumor cells. Cells lacking phosphatase and tensin homolog were as sensitive to the drug combination as cells expressing the protein. Similar data were obtained using the AKT inhibitors perifosine and 8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-1,2,4-triazolo[3,4-f] [1,6]naphthyridin-3(2H)-one hydrochloride (MK2206). PX-866 treatment abolished AKT/glycogen synthase kinase 3 (GSK3) phosphorylation, and cell killing correlated with reduced activity of AKT and mammalian target of rapamycin (mTOR). Expression of activated AKT and to a lesser extent activated mTOR reduced drug combination lethality. Expression of B-cell lymphoma-extra large or dominant negative caspase 9, but not cellular FLICE (FADD-like IL-1b-converting enzyme)-inhibitory protein short, protected cells from the drug combination. Treatment of cells with PX-866 increased protein levels of p62, lysosome-associated membrane protein 2 (LAMP2), and microtubule-associated protein light chain (LC) 3 and LC3II that correlated with a large increase in LC3-green fluorescent protein (GFP) vesicle numbers. Exposure of PX-866 treated cells to sorafenib reduced p62 and LAMP2 levels, decreased the ratio of LC3 to LC3II, and reduced LC3-GFP vesicle levels. Knockdown of Beclin1 or autophagy-related 5 suppressed drug toxicity by â¼40%. In vivo, sorafenib and PX-866 or regorafenib and MK2206 cooperated to suppress the growth of established HuH7 and HCT116 tumors, respectively. Collectively our data demonstrate that the combination of sorafenib family kinase inhibitors with inhibitors of the PI3K/AKT pathway kills tumor cells in vitro and in vivo.
Asunto(s)
Niacinamida/análogos & derivados , Compuestos de Fenilurea/administración & dosificación , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Piridinas/administración & dosificación , Timoma/tratamiento farmacológico , Timoma/patología , Neoplasias del Timo/tratamiento farmacológico , Neoplasias del Timo/patología , Animales , Comunicación Celular/efectos de los fármacos , Comunicación Celular/fisiología , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Línea Celular Tumoral , Sinergismo Farmacológico , Femenino , Gonanos/administración & dosificación , Células Hep G2 , Humanos , Ratones , Niacinamida/administración & dosificación , Fosfatidilinositol 3-Quinasas/metabolismo , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sorafenib , Timoma/metabolismo , Neoplasias del Timo/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We presently demonstrate that histone deacetylase inhibitors (HDACIs) enhance toxicity of melanoma differentiation-associated gene-7/interleukin 24 (mda-7/IL-24) in invasive primary human glioblastoma multiforme (GBM) cells. Additionally, a method is described to augment the efficacy of adenoviral delivery of mda-7/IL-24 in these cells. HDACIs synergized with melanoma differentiation-associated (MDA)-7/IL-24 killing GBM cells. Enhanced lethality correlated with increased autophagy that was dependent on the expression of ceramide synthase 6. HDACIs interacted with MDA-7/IL-24 prolonging generation of reactive oxygen species and Ca(2+). Quenching of reactive oxygen species and Ca(2+) blocked HDACI and MDA-7/IL-24 killing. In vivo MDA-7/IL-24 prolonged the survival of animals carrying orthotopic tumors, and HDACIs enhanced survival further. A serotype 5/3 adenovirus more effectively delivers mda-7/IL-24 to GBM tumors than a serotype 5 virus. Hence, we constructed a serotype 5/3 adenovirus that conditionally replicates in tumor cells expressing MDA-7/IL-24, in which the adenoviral early region 1A (E1A) gene was driven by the cancer-specific promoter progression elevated gene-3 [Ad.5/3 (INGN 241)-PEG-E1A-mda-7; also called Ad.5/3-CTV (cancer terminator virus)]. Ad.5/3-CTV increased the survival of mice carrying GBM tumors to a significantly greater extent than did a nonreplicative virus Ad.5/3-mda-7. Ad.5/3-CTV exhibited no toxicity in the brains of Syrian hamsters. Collectively our data demonstrate that HDACIs enhance MDA-7/IL-24 lethality, and adenoviral delivery of mda-7/IL-24 combined with tumor-specific viral replication is an effective preclinical GBM therapeutic.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/terapia , Glioblastoma/metabolismo , Glioblastoma/terapia , Inhibidores de Histona Desacetilasas/farmacología , Interleucinas/metabolismo , Adenoviridae/metabolismo , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/enzimología , Calcio/metabolismo , Línea Celular Tumoral , Cricetinae , Femenino , Terapia Genética/métodos , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Desnudos , Especies Reactivas de Oxígeno/metabolismo , Esfingosina N-Aciltransferasa/metabolismoRESUMEN
The present studies were designed to compare and contrast the abilities of TRAIL (death receptor agonist) and obatoclax (BCL-2 family inhibitor) to enhance sorafenib + HDAC inhibitor toxicity in GI tumor cells. Sorafenib and HDAC inhibitor treatment required expression of CD95 to kill GI tumor cells in vitro and in vivo. In cells lacking CD95 expression, TRAIL treatment, and to a lesser extent obatoclax, enhanced the lethal effects of sorafenib + HDAC inhibitor exposure. In hepatoma cells expressing CD95 a similar data pattern emerged with respect to the actions of TRAIL. Downstream of the death receptor the ability of TRAIL to enhance cell killing correlated with reduced AKT, ERK1/2, p70 S6K, and mTOR activity and enhanced cleavage of pro-caspase 3 and reduced expression of MCL-1 and BCL-XL. Over-expression of BCL-XL or MCL-1 or expression of dominant negative pro-caspase 9 protected cells from drug toxicity. Expression of activated AKT, p70 S6K, mTOR, and to a lesser extent MEK1EE also protected cells that correlated with maintained c-FLIP-s expression, reduced BIM expression, and increased BAD phosphorylation. In vivo sorafenib + HDAC inhibitor toxicity against tumors was increased in a greater than additive fashion by TRAIL. Collectively, our data argue that TRAIL, rather than obatoclax, is the most efficacious agent at promoting sorafenib + HDAC inhibitor lethality.
Asunto(s)
Carcinoma Hepatocelular/tratamiento farmacológico , Inhibidores de Histona Desacetilasas/farmacología , Neoplasias Hepáticas/tratamiento farmacológico , Niacinamida/análogos & derivados , Compuestos de Fenilurea/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Animales , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Carcinoma Hepatocelular/metabolismo , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Línea Celular Tumoral , Sinergismo Farmacológico , Femenino , Células Hep G2 , Histona Desacetilasas/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Ratones , Ratones Desnudos , Niacinamida/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Sorafenib , Serina-Treonina Quinasas TOR/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína X Asociada a bcl-2/metabolismo , Proteína Letal Asociada a bcl/metabolismo , Receptor fas/metabolismoRESUMEN
Although primary and memory responses against bacteria and viruses have been studied extensively, T helper type 2 (T(H)2) effector mechanisms leading to host protection against helminthic parasites remain elusive. Examination of the intestinal epithelial submucosa of mice after primary and secondary infections by a natural gastrointestinal parasite revealed a distinct immune-cell infiltrate after challenge, featuring interleukin-4-expressing memory CD4(+) T cells that induced IL-4 receptor(hi) (IL-4R(hi)) CD206(+) alternatively activated macrophages. In turn, these alternatively activated macrophages (AAMacs) functioned as important effector cells of the protective memory response contributing to parasite elimination, demonstrating a previously unknown mechanism for host protection against intestinal helminths.
Asunto(s)
Macrófagos/inmunología , Nematodos/inmunología , Infecciones por Nematodos/inmunología , Infecciones por Strongylida/inmunología , Células Th2/inmunología , Traslado Adoptivo , Animales , Arginasa/antagonistas & inhibidores , Ácidos Borónicos/farmacología , Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD4-Positivos/trasplante , Complejo IV de Transporte de Electrones/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Interacciones Huésped-Parásitos , Inmunohistoquímica , Inyecciones Intravenosas , Interleucina-4/genética , Larva/enzimología , Larva/parasitología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Nematodos/fisiología , Infecciones por Nematodos/parasitología , Factor de Transcripción STAT6/genética , Infecciones por Strongylida/parasitologíaRESUMEN
Prior studies demonstrated that resistance to the ERBB1/2 inhibitor lapatinib could be overcome by the B cell CLL/lymphoma-2 (BCL-2) family antagonist obatoclax (GX15-070). Coadministration of lapatinib with obatoclax caused synergistic cell killing by eliciting autophagic cell death that was dependent upstream on mitochondrial reactive oxygen species generation and increased p62 levels and downstream on activation of p38 mitogen-activated protein kinase and inactivation of mammalian target of rapamycin. By immunohistochemical analysis, in drug combination-treated cells, microtubule-associated protein light chain 3 (LC3) associated with mitochondrial (cytochrome c oxidase), autophagosome (p62), and autolysosome (lysosomal associated membrane protein 2) proteins. Treatment of cells with 3-methyladenine or knockdown of beclin 1 was protective, whereas chloroquine treatment had no protective effect. Expression of myeloid cell leukemia-1 (MCL-1), compared with that of BCL-2 or BCL-2-related gene long isoform, protected against drug combination lethality. Lapatinib and obatoclax-initiated autophagy depended on NOXA-mediated displacement of the prosurvival BCL-2 family member, MCL-1, from beclin 1, which was essential for the initiation of autophagy. Taken together, our data argue that lapatinib and obatoclax-induced toxic autophagy is due to impaired autophagic degradation, and this disturbance of autophagic flux leads to an accumulation of toxic proteins and loss of mitochondrial function.
Asunto(s)
Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Pirroles/farmacología , Quinazolinas/farmacología , Muerte Celular , Línea Celular Tumoral , Genes erbB-2 , Humanos , Indoles , LapatinibRESUMEN
The present studies sought to define whether checkpoint kinase 1 (CHK1) inhibitors and poly(ADP-ribose) polymerase 1 (PARP1) inhibitors interact in vitro and in vivo to kill breast cancer cells. PARP1 and CHK1 inhibitors interacted to kill estrogen receptor (ER)+, ER+ fulvestrant-resistant, HER2+, or triple-negative mammary carcinoma cells in a manner that was not apparently affected by phosphatase and tensin homolog deleted on chromosome 10 functional status. Expression of dominant-negative CHK1 enhanced and overexpression of wild-type CHK1 suppressed the toxicity of PARP1 inhibitors in a dose-dependent fashion. Knockdown of PARP1 enhanced the lethality of CHK1 inhibitors in a dose-dependent fashion. PARP1 and CHK1 inhibitors interacted in vivo both to suppress the growth of large established tumors and to suppress the growth of smaller developing tumors; the combination enhanced animal survival. PARP1 and CHK1 inhibitors profoundly radiosensitized cells in vitro and in vivo. In conclusion, our data demonstrate that the combination of PARP1 and CHK1 inhibitors has antitumor activity in vivo against multiple mammary tumor types and that translation of this approach could prove to be a useful anticancer therapeutic approach.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Animales , Bencimidazoles/farmacología , Neoplasias de la Mama/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Femenino , Humanos , Ratones , Ratones Desnudos , Poli(ADP-Ribosa) Polimerasa-1 , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Quinasas/genética , Proteínas Quinasas/fisiología , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Prior studies in breast cancer cells have shown that lapatinib and obatoclax interact in a greater than additive fashion to cause cell death and do so through a toxic form of autophagy. The present studies sought to extend our analyses to the central nervous system (CNS) tumor cells and to further define mechanisms of drug action. Lapatinib and obatoclax killed multiple CNS tumor isolates. Cells lacking PTEN (phosphatase and tensin homolog on chromosome 10) function were relatively resistant to drug combination lethality; expression of PTEN in PTEN-null cells restored drug sensitivity, and knockdown of PTEN promoted drug resistance. On the basis of knockdown of ERBB1-4 (erythroblastic leukemia viral oncogene homolog 1-4), we discovered that the inhibition of ERBB1/3/4 receptors were most important for enhancing obatoclax lethality rather than ERBB2. In parallel, we noted in CNS tumor cells that knockdown of BCL-xL (B-cell lymphoma-extra large)and MCL-1 (myeloid cell leukemia-1) interacted in an additive fashion to facilitate lapatinib lethality. Pretreatment of tumor cells with obatoclax enhanced the lethality of lapatinib to a greater extent than concomitant treatment. Treatment of animals carrying orthotopic CNS tumor isolates with lapatinib- and obatoclax-prolonged survival. Altogether, our data show that lapatinib and obatoclax therapy could be of use in the treatment of tumors located in the CNS.
Asunto(s)
Antineoplásicos/farmacología , Receptores ErbB/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Pirroles/farmacología , Quinazolinas/farmacología , Receptor ErbB-3/antagonistas & inhibidores , Proteína bcl-X/antagonistas & inhibidores , Autofagia , Línea Celular Tumoral , Humanos , Indoles , Lapatinib , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Neoplasias/patología , Fosfohidrolasa PTEN/fisiología , Receptor ErbB-4RESUMEN
Agents that generate reactive oxygen species (ROS) are recognized to enhance MDA-7/IL-24 lethality. The present studies focused on clarifying how such agents enhanced MDA-7/IL-24 toxicity in renal cell carcinoma cells (RCCs). Infection of RCCs with a tropism-modified serotype 5/3 adenovirus expressing MDA-7/IL-24 (Ad.5/3-mda-7) caused plasma membrane clustering of CD95 and CD95 association with pro-caspase 8, effects that were enhanced by combined exposure to 17-N-allylamino-17-demethoxygeldanamycin (17AAG), As(2)O(3), or fenretinide and that correlated with enhanced cell killing. Knockdown of CD95 or expression of cellular FADD (Fas-associated protein with death domain)-like interleukin-1ß-converting enzyme inhibitory protein, short form (c-FLIP-s) blocked enhanced killing. Inhibition of ROS generation, elevated cytosolic Ca(2+), or de novo ceramide synthesis blocked Ad.5/3-mda-7 ± agent-induced CD95 activation and the enhancement of apoptosis. Ad.5/3-mda-7 increased ceramide levels in a PERK-dependent fashion that were responsible for elevated cytosolic Ca(2+) levels that promoted ROS generation; 17AAG did not further enhance cytokine-induced ceramide generation. In vivo, infection of RCC tumors with Ad.5/3-mda-7 suppressed the growth of infected tumors that was enhanced by exposure to 17AAG. Our data indicate that in RCCs, Ad.5/3-mda-7-induced ceramide generation plays a central role in tumor cell killing and inhibition of multiple signaling pathways may have utility in promoting MDA-7/IL-24 lethality in renal cancer.
Asunto(s)
Adenoviridae/metabolismo , Carcinoma de Células Renales/virología , Ceramidas/metabolismo , Interleucinas/biosíntesis , Neoplasias Renales/virología , Especies Reactivas de Oxígeno/metabolismo , Adenoviridae/fisiología , Animales , Trióxido de Arsénico , Arsenicales/farmacología , Benzoquinonas/farmacología , Western Blotting , Carcinoma de Células Renales/química , Carcinoma de Células Renales/metabolismo , Línea Celular Tumoral , Ceramidas/análisis , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Femenino , Citometría de Flujo , Humanos , Interleucinas/metabolismo , Interleucinas/fisiología , Neoplasias Renales/química , Neoplasias Renales/metabolismo , Lactamas Macrocíclicas/farmacología , Ratones , Ratones Desnudos , Óxidos/farmacología , Especies Reactivas de Oxígeno/análisis , TransfecciónRESUMEN
We have explored the mechanism by which inhibition of multiple cytoprotective cell-signaling pathways enhance melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) toxicity toward invasive primary human glioblastoma multiforme (GBM) cells, and whether improving adenoviral infectivity/delivery of mda-7/IL-24 enhances therapeutic outcome in animals containing orthotopic xenografted GBM cells. The toxicity of a serotype 5 recombinant adenovirus to express MDA-7/IL-24 (Ad.5-mda-7) was enhanced by combined molecular or small molecule inhibition of mitogen-activated extracellular regulated kinase (MEK)1/2 and phosphatidyl inositol 3-kinase (PI3K) or AKT; inhibition of mammalian target of rapamycin (mTOR) and MEK1/2; and the HSP90 inhibitor 17AAG. Molecular inhibition of mTOR/PI3K/MEK1 signaling in vivo also enhanced Ad.5-mda-7 toxicity. In GBM cells of diverse genetic backgrounds, inhibition of cytoprotective cell-signaling pathways enhanced MDA-7/IL-24-induced autophagy, mitochondrial dysfunction and tumor cell death. Due partly to insufficient adenovirus serotype 5 gene delivery this therapeutic approach has shown limited success in GBM. To address this problem, we employed a recombinant adenovirus that comprises the tail and shaft domains of a serotype 5 virus and the knob domain of a serotype 3 virus expressing MDA-7/IL-24, Ad.5/3-mda-7. Ad.5/3-mda-7 more effectively infected and killed GBM cells in vitro and in vivo than Ad.5-mda-7. Future combinations of these approaches hold promise for developing an effective therapy for GBM.
Asunto(s)
Adenoviridae/genética , Neoplasias Encefálicas/terapia , Vectores Genéticos , Glioblastoma/terapia , Interleucinas/uso terapéutico , Transducción de Señal , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Humanos , Interleucinas/administración & dosificación , Interleucinas/genética , Resultado del TratamientoRESUMEN
Melanoma differentiation associated gene-7/interleukin 24 (mda-7/IL-24) is a unique interleukin (IL)-10 family cytokine displaying selective apoptosis-inducing activity in transformed cells without harming normal cells. The present studies focused on defining the mechanism(s) by which recombinant adenoviral delivery of MDA-7/IL-24 inhibits cell survival of human ovarian carcinoma cells. Expression of MDA-7/IL-24 induced phosphorylation of protein kinase R-like endoplasmic reticulum kinase (PERK) and eukaryotic initiation factor2alpha (eIF2alpha). In a PERK-dependent fashion, MDA-7/IL-24 reduced ERK1/2 and AKT phosphorylation and activated c-Jun NH(2)-terminal kinase (JNK) 1/2 and p38 mitogen-activated protein kinase (MAPK). MDA-7/IL-24 reduced MCL-1 and BCL-XL and increased BAX levels via PERK signaling; cell-killing was mediated via the intrinsic pathway, and cell killing was primarily necrotic as judged using Annexin V/propidium iodide staining. Inhibition of p38 MAPK and JNK1/2 abolished MDA-7/IL-24 toxicity and blocked BAX and BAK activation, whereas activation of mitogen-activated extracellular-regulated kinase (MEK) 1/2 or AKT suppressed enhanced killing and JNK1/2 activation. MEK1/2 signaling increased expression of the MDA-7/IL-24 and PERK chaperone BiP/78-kDa glucose regulated protein (GRP78), and overexpression of BiP/GRP78 suppressed MDA-7/IL-24 toxicity. MDA-7/IL-24-induced LC3-green fluorescent protein vesicularization and processing of LC3; and knockdown of ATG5 suppressed MDA-7/IL-24-mediated toxicity. MDA-7/IL-24 and cisplatin interacted in a greater than additive fashion to kill tumor cells that was dependent on a further elevation of JNK1/2 activity and recruitment of the extrinsic CD95 pathway. MDA-7/IL-24 toxicity was enhanced in a weak additive fashion by paclitaxel; paclitaxel enhanced MDA-7/IL-24 + cisplatin lethality in a greater than additive fashion via BAX. Collectively, our data demonstrate that MDA-7/IL-24 induces an endoplasmic reticulum stress response that activates multiple proapoptotic pathways, culminating in decreased ovarian tumor cell survival.
Asunto(s)
Diferenciación Celular/fisiología , Cisplatino/farmacología , Interleucinas/fisiología , Neoplasias Ováricas/metabolismo , eIF-2 Quinasa/metabolismo , Receptor fas/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Cisplatino/uso terapéutico , Sinergismo Farmacológico , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Femenino , Técnicas de Transferencia de Gen , Humanos , Interleucinas/administración & dosificación , Interleucinas/genética , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/enzimologíaRESUMEN
The novel cytokine melanoma differentiation associated gene-7 (mda-7) was identified by subtractive hybridization in the mid-1990s as a protein whose expression increased during the induction of terminal differentiation, and that was either not expressed or was present at low levels in tumor cells compared with non-transformed cells. On the basis of conserved structure, chromosomal location and cytokine-like properties, MDA-7, has now been classified as a member of the expanding interleukin (IL)-10 gene family and designated as MDA-7/IL-24. Multiple studies have shown that the expression of MDA-7/IL-24 in a wide variety of tumor cell types, but not in the corresponding equivalent non-transformed cells, causes their growth arrest and ultimately cell death. In addition, MDA-7/IL-24 has been noted to be a radiosensitizing cytokine, which is partly because of the generation of reactive oxygen species and ceramide that cause endoplasmic reticulum stress. Phase I clinical trial data has shown that a recombinant adenovirus expressing MDA-7/IL-24 [Ad.mda-7 (INGN-241)] was safe and had measurable tumoricidal effects in over 40% of patients, which strongly argues that MDA-7/IL-24 may have significant therapeutic value. This review describes what is known about the impact of MDA-7/IL-24 on tumor cell biology and its potential therapeutic applications.
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Apoptosis , Interleucinas/uso terapéutico , Neoplasias/tratamiento farmacológico , Tolerancia a Radiación , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Citocinas/metabolismo , Femenino , Genes Supresores de Tumor , Terapia Genética , Humanos , Interleucinas/administración & dosificación , Interleucinas/genética , Interleucinas/metabolismo , Masculino , Persona de Mediana Edad , Neoplasias/diagnóstico por imagen , Neoplasias/metabolismo , Neoplasias/patología , Cintigrafía , Transducción de SeñalRESUMEN
The present studies were to determine whether the multi-kinase inhibitor pazopanib interacted with histone deacetylase inhibitors (HDACI: valproate, vorinostat) to kill sarcoma cells. In multiple sarcoma cell lines, at clinically achievable doses, pazopanib and HDACI interacted in an additive to greater than additive fashion to cause tumor cell death. The drug combination increased the numbers of LC3-GFP and LC3-RFP vesicles. Knockdown of Beclin1 or ATG5 significantly suppressed drug combination lethality. Expression of c-FLIP-s, and to a lesser extent BCL-XL or dominant negative caspase 9 reduced drug combination toxicity; knock down of FADD or CD95 was protective. Expression of both activated AKT and activated MEK1 was required to strongly suppress drug combination lethality. The drug combination inactivated mTOR and expression of activated mTOR strongly suppressed drug combination lethality. Treatment of animals carrying sarcoma tumors with pazopanib and valproate resulted in a greater than additive reduction in tumor volume compared with either drug individually. As both pazopanib and HDACIs are FDA-approved agents, our data argue for further determination as to whether this drug combination is a useful sarcoma therapy in the clinic.
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Antineoplásicos/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Pirimidinas/farmacología , Sarcoma/tratamiento farmacológico , Sulfonamidas/farmacología , Animales , Antineoplásicos/uso terapéutico , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia/efectos de los fármacos , Proteína 5 Relacionada con la Autofagia , Beclina-1 , Línea Celular Tumoral , Sinergismo Farmacológico , Femenino , Técnicas de Silenciamiento del Gen , Inhibidores de Histona Desacetilasas/uso terapéutico , Indazoles , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Desnudos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Pirimidinas/uso terapéutico , Receptores de Muerte Celular/metabolismo , Sarcoma/metabolismo , Sarcoma/patología , Transducción de Señal/efectos de los fármacos , Sulfonamidas/uso terapéutico , Ácido Valproico/uso terapéuticoRESUMEN
Not surprisingly, the death of a cell is a complex and well controlled process. For several decades, apoptosis, the first genetically programmed death process to be identified has taken centre stage as the principal mechanism of programmed cell death (type I cell death) in mammalian tissues. Apoptosis has been extensively studied and its contribution to the pathogenesis of disease well documented. However, apoptosis does not function alone in determining the fate of a cell. More recently, autophagy, a process in which de novo formed membrane enclosed vesicles engulf and consume cellular components, has been shown to engage in complex interplay with apoptosis. As a result, cell death has been subdivided into the categories apoptosis (Type I), autophagic cell death (Type II), and necrosis (Type III). The boundary between Type I and II cell death is not completely clear and as we will discuss in this review and perhaps a discrete difference does not exist, due to intrinsic factors among different cell types and crosstalk among organelles within each cell type. Apoptosis may begin with autophagy and autophagy can often end with apoptosis, inhibition or a blockade of caspase activity may lead a cell to default into Type II cell death from Type I.
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Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/fisiología , Autofagia/fisiología , Caspasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis/genética , Autofagia/genética , Beclina-1 , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Membranas Mitocondriales/metabolismo , Necrosis/genética , Proteína Sequestosoma-1 , Transducción de SeñalRESUMEN
The present studies focused on defining the mechanisms by which anoikis-resistant (AR) mammary carcinoma cells can be reverted to a therapy-sensitive phenotype. AR mammary carcinoma cells had reduced expression of the toxic BH3 domain proteins BAX, BAK, NOXA, and PUMA. In AR cells expression of the protective BCL-2 family proteins BCL-XL and MCL-1 was increased. AR cells were resistant to cell killing by multiple anti-tumor cell therapies, including ERBB1/2 inhibitor + MCL-1 inhibitor treatment, and had a reduced autophagic flux response to these therapies, despite similarly exhibiting increased levels of LC3II processing. Knockdown of MCL-1 and BCL-XL caused necro-apoptosis in AR cells to a greater extent than in parental cells. Pre-treatment of anoikis-resistant cells with histone deacetylase inhibitors (HDACIs) for 24 h increased the levels of toxic BH3 domain proteins, reduced MCL-1 levels, and restored/re-sensitized the cell death response of AR tumor cells to multiple toxic therapies. In vivo, pre-treatment of AR breast tumors in the brain with valproate restored the chemo-sensitivity of the tumors and prolonged animal survival. These data argue that one mechanism to enhance the anti-tumor effect of chemotherapy could be HDACI pre-treatment.
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Antineoplásicos Hormonales/farmacología , Resistencia a Antineoplásicos , Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Células Madre Neoplásicas/metabolismo , Ácido Valproico/farmacología , Animales , Anoicis , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Supervivencia Celular , Epigénesis Genética/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Indoles , Lapatinib , Ratones , Ratones Desnudos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Pirroles/farmacología , Quinazolinas/farmacología , ARN Interferente Pequeño/genética , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
In the present study we show that histone deacetylase inhibitors (HDACIs) enhance the anti-tumor effects of melanoma differentiation associated gene-7/interleukin 24 (mda- 7/IL-24) in human renal carcinoma cells. Similar data were obtained in other GU tumor cells. Combination of these two agents resulted in increased autophagy that was dependent on expression of ceramide synthase 6, with HDACIs enhancing MDA-7/IL-24 toxicity by increasing generation of ROS and Ca (2+). Knock down of CD95 protected cells from HDACI and MDA-7/IL-24 lethality. Sorafenib treatment further enhanced (HDACI + MDA-7/IL-24) lethality. Anoikis resistant renal carcinoma cells were more sensitive to MDA-7/IL-24 that correlated with elevated SRC activity and tyrosine phosphorylation of CD95. We employed a recently constructed serotype 5/3 adenovirus, which is more effective than a serotype 5 virus in delivering mda- 7/IL-24 to renal carcinoma cells and which conditionally replicates (CR) in tumor cells expressing MDA-7/IL-24 by virtue of placing the adenoviral E1A gene under the control of the cancer-specific promoter progression elevated gene-3 (Ad.5/3-PEG-E1A-mda-7; CRAd.5/3-mda-7, Ad.5/3-CTV), to define efficacy in renal carcinoma cells. Ad.5/3-CTV decreased the growth of renal carcinoma tumors to a significantly greater extent than did a non-replicative virus Ad.5/3-mda-7. In contralateral uninfected renal carcinoma tumors Ad.5/3-CTV also decreased the growth of tumors to a greater extent than did Ad.5/3-mda-7. In summation, our data demonstrates that HDACIs enhance MDA-7/IL-24-mediated toxicity and tumor specific adenoviral delivery and viral replication of mda-7/IL-24 is an effective pre-clinical renal carcinoma therapeutic.
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Adenoviridae/genética , Antineoplásicos/farmacología , Carcinoma de Células Renales/terapia , Inhibidores de Histona Desacetilasas/farmacología , Interleucinas/farmacología , Neoplasias Renales/terapia , Animales , Antineoplásicos/uso terapéutico , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/metabolismo , Línea Celular Tumoral , Interacciones Farmacológicas , Femenino , Terapia Genética , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Interleucinas/genética , Interleucinas/metabolismo , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/metabolismo , Ratones Desnudos , Proteínas Recombinantes/farmacología , Transducción de SeñalRESUMEN
The present studies were designed to determine whether the multi-kinase inhibitor sorafenib (Nexavar) interacted with histone deacetylase inhibitors to kill glioblastoma and medulloblastoma cells. In a dose-dependent fashion sorafenib lethality was enhanced in multiple genetically disparate primary human glioblastoma isolates by the HDAC inhibitor sodium valproate (Depakote). Drug exposure reduced phosphorylation of p70 S6K and of mTOR. Similar data to that with valproate were also obtained using the HDAC inhibitor vorinostat (Zolinza). Sorafenib and valproate also interacted to kill medulloblastoma and PNET cell lines. Treatment with sorafenib and HDAC inhibitors radio-sensitized both GBM and medulloblastoma cell lines. Knock down of death receptor (CD95) expression protected GBM cells from the drug combination, as did overexpression of c-FLIP-s, BCL-XL and dominant negative caspase 9. Knock down of PDGFRα recapitulated the effect of sorafenib in combination with HDAC inhibitors. Collectively, our data demonstrate that the combination of sorafenib and HDAC inhibitors kills through activation of the extrinsic pathway, and could represent a useful approach to treat CNS-derived tumors.
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Antineoplásicos/farmacología , Bencenosulfonatos/farmacología , Neoplasias del Sistema Nervioso Central/enzimología , Inhibidores de Histona Desacetilasas/farmacología , Piridinas/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Neoplasias del Sistema Nervioso Central/genética , Sinergismo Farmacológico , Humanos , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Inhibidores de Proteínas Quinasas/farmacología , Sorafenib , Receptor fas/genética , Receptor fas/metabolismoRESUMEN
We have further defined mechanism(s) by which the drug OSU-03012 (OSU) kills tumor cells. OSU lethality was suppressed by knock down of PERK and enhanced by knock down of ATF6 and IRE1α. OSU treatment suppressed expression of the chaperone, BiP/GRP78, and did so through reduced stability of the protein. Knock down of BiP/GRP78 further enhanced OSU lethality. Overexpression of BiP/GRP78 abolished OSU toxicity. Pre-treatment of cells with OSU enhanced radiosensitivity to a greater extent than concomitant or sequential drug treatment with radiation exposure. Expression of a mutant active p110 PI3K, or mutant active forms of the EGFR in GBM cells did not differentially suppress OSU killing. In contrast loss of PTEN function reduced OSU lethality, without altering AKT, p70 S6K or mTOR activity, or the drug's ability to radiosensitize GBM cells. Knock down of PTEN protected cells from OSU and radiation treatment whereas re-expression of PTEN facilitated drug lethality and radiosensitization. In a dose-dependent fashion OSU prolonged the survival of mice carrying GBM tumors and interacted with radiotherapy to further prolong survival. Collectively, our data show that reduced BiP/GRP78 levels play a key role in OSU-3012 toxicity in GBM cells, and that this drug has in vivo activity against an invasive primary human GBM isolate.