RESUMO
The topic of cancer stem cells (CSCs) is of significant importance due to its implications in our understanding of the tumor biology as well as the development of novel cancer therapeutics. However, the question of whether targeting CSCs can hamper the growth of tumors remains mainly unanswered due to the lack of specific agents for this purpose. To address this issue, we have developed the first mutated version of herpes simplex virus-1 that is transcriptionally targeted against CD133+ cells. CD133 has been portrayed as one of the most important markers in CSCs involved in the biology of a number of human cancers, including liver, brain, colon, skin, and pancreas. The virus developed in this work, Signal-Smart 2, showed specificity against CD133+ cells in three different models (hepatocellular carcinoma, colorectal cancer, and melanoma) resulting in a loss of viability and invasiveness of cancer cells. Additionally, the virus showed robust inhibitory activity against in vivo tumor growth in both preventive and therapeutic mouse models as well as orthotopic model highly relevant to potential clinical application of this virus. Therefore, we conclude that targeting CD133+ CSCs has the potential to be pursued as a novel strategy against cancer. Stem Cells 2018;36:1154-1169.
Assuntos
Antígeno AC133/genética , Herpesvirus Humano 1/fisiologia , Neoplasias/genética , Neoplasias/terapia , Vírus Oncolíticos/fisiologia , Transcrição Gênica , Antígeno AC133/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Modelos Animais de Doenças , Humanos , Masculino , Camundongos Nus , Invasividade Neoplásica , Neoplasias/patologia , Especificidade de Órgãos , Fenótipo , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Medulloblastoma (MDB) represents a major form of malignant brain tumors in the pediatric population. A vast spectrum of research on MDB has advanced our understanding of the underlying mechanism, however, a significant need still exists to develop novel therapeutics on the basis of gaining new knowledge about the characteristics of cell signaling networks involved. The Ras signaling pathway, one of the most important proto-oncogenic pathways involved in human cancers, has been shown to be involved in the development of neurological malignancies. We have studied an important effector down-stream of Ras, namely RalA (Ras-Like), for the first time and revealed overactivation of RalA in MDB. Affinity precipitation analysis of active RalA (RalA-GTP) in eight MDB cell lines (DAOY, RES256, RES262, UW228-1, UW426, UW473, D283 and D425) revealed that the majority contained elevated levels of active RalA (RalA-GTP) as compared with fetal cerebellar tissue as a normal control. Additionally, total RalA levels were shown to be elevated in 20 MDB patient samples as compared to normal brain tissue. The overall expression of RalA, however, was comparable in cancerous and normal samples. Other important effectors of RalA pathway including RalA binding protein-1 (RalBP1) and protein phosphatase A (PP2A) down-stream of Ral and Aurora kinase A (AKA) as an upstream RalA activator were also investigated in MDB. Considering the lack of specific inhibitors for RalA, we used gene specific silencing in order to inhibit RalA expression. Using a lentivirus expressing anti-RalA shRNA we successfully inhibited RalA expression in MDB and observed a significant reduction in proliferation and invasiveness. Similar results were observed using inhibitors of AKA and geranyl-geranyl transferase (non-specific inhibitors of RalA signaling) in terms of loss of in vivo tumorigenicity in heterotopic nude mouse model. Finally, once tested in cells expressing CD133 (a marker for MDB cancer stem cells), higher levels of RalA activation was observed. These data not only bring RalA to light as an important contributor to the malignant phenotype of MDB but introduces this pathway as a novel target in the treatment of this malignancy.
Assuntos
Neoplasias Encefálicas/metabolismo , Cerebelo/metabolismo , Meduloblastoma/metabolismo , Proteínas ral de Ligação ao GTP/metabolismo , Animais , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Cerebelo/patologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Feminino , Feto , Regulação Neoplásica da Expressão Gênica/fisiologia , Inativação Gênica , Humanos , Masculino , Meduloblastoma/patologia , Camundongos , Camundongos Nus , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/fisiologia , Transdução GenéticaRESUMO
Cancer stem cells (CSCs) are believed to be the regenerative pool of cells responsible for repopulating tumors. Gaining knowledge about the signaling characteristics of CSCs is important for understanding the biology of tumors and developing novel anti-cancer therapies. We have identified a subpopulation of cells positive for CD133 (a CSC marker) from human primary malignant peripheral nerve sheath tumor (MPNST) cells which were absent in non-malignant Schwann cells. CD133 was also found to be expressed in human tissue samples and mouse MPNST cells. CD133+ cells were capable of forming spheres in non-adherent/serum-free conditions. The activation levels of Ras and its downstream effectors such as ERK, JNK, PI3K, p38K, and RalA were significantly increased in this population. Moreover, the CD133+ cells showed enhanced invasiveness which was linked to the increased expression of ß-Catenin and Snail, two important proteins involved in the epithelial to mesenchymal transition, and Paxilin, a focal adhesion protein. Among other important characteristics of the CD133+ population, endoplasmic reticulum stress marker IRE1α was decreased, implying the potential sensitivity of CD133+ to the accumulation of unfolded proteins. Apoptotic indicators seemed to be unchanged in CD133+ cells when compared to the wild (unsorted) cells. Finally, in order to test the possibility of targeting CD133+ MPNST cells with Ras pathway pharmacological inhibitors, we exposed these cells to an ERK inhibitor. The wild population was more sensitive to inhibition of proliferation by this inhibitor as compared with the CD133+ cells supporting previous studies observing enhanced chemoresistance of these cells.
Assuntos
Antígenos CD/metabolismo , Glicoproteínas/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias de Bainha Neural/metabolismo , Neoplasias de Bainha Neural/patologia , Peptídeos/metabolismo , Transdução de Sinais , Proteínas ras/metabolismo , Antígeno AC133 , Animais , Apoptose , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Estresse do Retículo Endoplasmático , Citometria de Fluxo , Humanos , Imunofenotipagem , Camundongos , Invasividade Neoplásica , Células-Tronco Neoplásicas/patologiaRESUMO
MT477 is a novel thiopyrano[2,3-c]quinoline with anti-cancer activity. The purpose of the present study was to evaluate different doses and treatment schedules of MT477 in an in vivo xenograft model of non-Ras-mutated cancer, as well as determine its biological effects and mechanism of action via the four conventional PKC isoforms: α, ßI, ßII, and γ. Here, we show that MT477 inhibits the activity of PKC-α and its downstream targets, ERK1/2 and Akt, before it has an effect on Ras activity. MT477 treatment of cultured H226 cells induced apoptosis and increased focal cell adhesion and formation of actin stress fibers. H226 tumor size in mice continuously treated with intraperitoneal MT477 (1 mg/kg) was 62.1 ± 15.3% smaller than the average tumor size in control mice. Blood serum chemistry revealed minimal toxicity in mice. Taken together, these results support the conclusion that MT477 acts as a direct PKC-α inhibitor in non-Ras mutated cancer, with maximum effectiveness when given in a continuous treatment schedule.
Assuntos
Apoptose/efeitos dos fármacos , Mutação/genética , Neoplasias/patologia , Proteína Quinase C-alfa/antagonistas & inibidores , Quinolinas/efeitos adversos , Quinolinas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Citoesqueleto de Actina/metabolismo , Animais , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Adesões Focais/efeitos dos fármacos , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Camundongos , Modelos Biológicos , Neoplasias/enzimologia , Neoplasias/genética , Fosforilação/efeitos dos fármacos , Proteína Quinase C-alfa/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas ras/genéticaRESUMO
Molecular topology (MT) was used to develop quantitative structure-activity relationship (QSAR) models to screen databases for new anticancer compounds. One of the selected compounds was MT103, an isoborneol derivative, with a promising profile predicted to slow tumor growth through pro-apoptotic signaling and protein kinase C inhibition. We found that MT103 inhibited the growth of a wide variety of cancer cell types as verified by the NCI-60 cancer cell line panel. MTT cell viability assay showed that MT103 inhibited 50% of the growth of HOP-92, ACHN, NCI-H226, MCF-7, and A549 cancer cell lines at much lower concentrations than that required for HUVECs and human fibroblasts. MT103 stimulated apoptosis in NCI-H226 lung carcinoma cells as measured by oligonucleosomal DNA fragmentation. However, protein kinase C was not targeted by MT103, as predicted by in silico modeling. MT103 slowed in vivo tumor growth and metastatic spread of NCI-H226 cells injected subcutaneously into NOD/SCID mice, without eliciting any severe adverse events as monitored by animal survival, blood serum analysis, and histological analysis of organs. Oral administration of MT103 nanoparticles (200 nm in diameter), which were generated with ElectroNanospray™ technology, inhibited in vivo growth of HOP-92 lung carcinoma cells almost as effectively as intraperitoneal injections of cisplatin. Taken together, our study of a novel anti-cancer drug identified using a molecular topology-based approach to drug discovery demonstrates that MT103 has anti-tumor activity in vitro and in vivo, although additional studies are needed to elucidate its mechanism of action.
Assuntos
Apoptose/efeitos dos fármacos , Canfanos/farmacologia , Canfanos/toxicidade , Neoplasias Pulmonares/patologia , Sulfonamidas/farmacologia , Sulfonamidas/toxicidade , Administração Oral , Animais , Canfanos/administração & dosagem , Canfanos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Neoplasias Pulmonares/ultraestrutura , Metástase Linfática/patologia , Camundongos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Sulfonamidas/administração & dosagem , Sulfonamidas/química , Análise de Sobrevida , Fatores de Tempo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Emerging evidence continues to demonstrate that disrupted insulin signaling and altered energy metabolism may play a key role underpinning pathology in neurodegenerative conditions. Intranasally administered insulin has already shown promise as a memory-enhancing therapy in patients with Alzheimer's and animal models of the disease. Intranasal drug delivery allows for direct targeting of insulin to the brain, bypassing the blood brain barrier and minimizing systemic adverse effects. In this study, we sought to expand upon previous results that show intranasal insulin may also have promise as a Parkinson's therapy. We treated 6-OHDA parkinsonian rats with a low dose (3 IU/day) of insulin and assessed apomorphine induced rotational turns, motor deficits via a horizontal ladder test, and dopaminergic cell survival via stereological counting. We found that insulin therapy substantially reduced motor dysfunction and dopaminergic cell death induced by unilateral injection of 6-OHDA. These results confirm insulin's efficacy within this model, and do so over a longer period after model induction which more closely resembles Parkinson's disease. This study also employed a lower dose than previous studies and utilizes a delivery device, which could lead to an easier transition into human clinical trials as a therapeutic for Parkinson's disease.
Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Atividade Motora/efeitos dos fármacos , Transtornos Parkinsonianos/fisiopatologia , Parte Compacta da Substância Negra/efeitos dos fármacos , Administração Intranasal , Adrenérgicos/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Neurônios Dopaminérgicos/patologia , Movimento/efeitos dos fármacos , Oxidopamina/toxicidade , Doença de Parkinson , Transtornos Parkinsonianos/patologia , Parte Compacta da Substância Negra/patologia , Ratos , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
NAD(P)H:quinone oxidoreductase (NQO1) has been reported to play an important role in cell death caused by beta-lapachone (beta-lap), 3,4-dihydro-22,2-dimethyl-2H-naphthol[1,22b]pyran-5,6-dione. This study investigated whether cisplatin (cis-diamminedichloroplatinum) sensitizes cancer cells to beta-lap by upregulating NQO1. The cytotoxicity of cisplatin and beta-lap alone or in combination against FSaII fibrosarcoma cells of C3H mice in vitro was determined with a clonogenic survival assay and assessment of gamma-H2AX foci formation, a hallmark of DNA double-strand breaks. The cellular sensitivity to beta-lap progressively increased during the 24 h after cisplatin treatment. The expression and enzymatic activity of NQO1 also increased during the 24 h after cisplatin treatment, and dicoumarol, an inhibitor of NQO1, was found to nullify the cisplatin-induced increase in beta-lap sensitivity. The role of NQO1 in the cell death caused by beta-lap alone or in combination with cisplatin was further elucidated using NQO1-positive and NQO1-negative MDA-MB-231 human breast cancer cells. Cisplatin increased the sensitivity of the NQO1-positive but not the NQO1-negative MDA-MB-231 cells to beta-lap treatment. Combined treatment with cisplatin and beta-lap suppressed the growth of FSaII tumors in the legs of C3H mice in a manner greater than additive. It is concluded that cisplatin markedly increases the sensitivity of cancer to beta-lap in vitro and in vivo by upregulating NQO1.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Cisplatino/farmacologia , Naftoquinonas/farmacologia , Regulação para Cima/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Feminino , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos C3H , NAD(P)H Desidrogenase (Quinona) , NADPH Desidrogenase/metabolismoRESUMO
Enzastaurin (LY317615.HCI), a protein kinase C (PKC)-beta inhibitor, has a radiosensitising effect on 4T1 murine breast cancer and human glioma cells; however, the exact mechanism of this action has not been evaluated. The present study investigated the effects of enzastaurin and gamma irradiation on PKC activity in MCF-7 human breast cancer cells in vitro and in vivo. Enzastaurin (5 microM) in combination with irradiation (2-8 Gy) produced a synergistic decline in MCF-7 clonogenic cell survival. Analysis of MCF-7 cells stained with Annexin V and 7-aminoactinomycin D showed a dose-dependent increase in apoptosis in response to enzastaurin (3, 5 and 7 microM) and irradiation (10 Gy) compared to irradiation alone. This pro-apoptotic effect was confirmed by increases in caspase-3 and -9 activity. In a MCF-7 xenograft model, irradiation with 25 Gy increased PKC-alpha activity by 2.5-fold compared to untreated controls, whereas PKC-epsilon and -betaII activity was increased by 1.8-fold. Radiation-induced activation of all three anti-apoptotic isoforms of PKC was reversed by pre-treatment with enzastaurin (75 mg/kg, twice daily for 3 days). We conclude that enzastaurin has a radiosensitising effect on MCF-7 human xenograft tumours through the reversal of anti-apoptotic activation of PKC isoforms.
Assuntos
Neoplasias da Mama/enzimologia , Inibidores Enzimáticos/farmacologia , Raios gama , Indóis/farmacologia , Proteína Quinase C/antagonistas & inibidores , Radiossensibilizantes/farmacologia , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Caspase 3/metabolismo , Caspase 9/metabolismo , Sobrevivência Celular , Citosol/química , Dactinomicina/análogos & derivados , Dactinomicina/metabolismo , Relação Dose-Resposta a Droga , Feminino , Humanos , Fosforilação/efeitos dos fármacos , Proteína Quinase C/efeitos da radiação , Proteínas Repressoras/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
BACKGROUND: Systemic Capillary Leak Syndrome (SCLS) is an extremely rare and life-threatening vascular disorder of unknown etiology. SCLS is characterized by abrupt and transient episodes of hypotensive shock and edema due to plasma leakage into peripheral tissues. The disorder has garnered attention recently because its initial presentation resembles more common vascular disorders including systemic anaphylaxis, sepsis, and acute infections with the Ebola/Marburg family of filoviruses. Although approximately 70-85% of patients with SCLS have a concurrent monoclonal gammopathy of unknown significance (MGUS), any contribution of the paraprotein to acute flares is unknown. PROCEDURE: To identify circulating factors that might trigger acute SCLS crises, we profiled transcriptomes of paired peripheral blood mononuclear cell fractions obtained from patients during acute attacks and convalescent intervals by microarray. RESULTS: This study uncovered 61 genes that were significantly up- or downregulated more than 2.5-fold in acute samples relative to respective baselines. One of the most upregulated genes was ADM, which encodes the vasoactive peptide adrenomedullin. A stable ADM protein surrogate (pro-ADM) was markedly elevated in SCLS acute sera compared to remission samples or sera from healthy controls. Monocytes and endothelial cells (ECs) from SCLS subjects expressed significantly more ADM in response to proinflammatory stimuli compared to healthy control cells. Application of ADM to ECs elicited protective effects on vascular barrier function, suggesting a feedback protective mechanism in SCLS. CONCLUSIONS: Since ADM has established hypotensive effects, differentiating between these dual actions of ADM is crucial for therapeutic applications aimed at more common diseases associated with increased ADM levels.
Assuntos
Adrenomedulina/metabolismo , Biomarcadores/metabolismo , Síndrome de Vazamento Capilar/patologia , Endotélio Vascular/patologia , Leucócitos Mononucleares/patologia , Monócitos/patologia , Doença Aguda , Idoso , Síndrome de Vazamento Capilar/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Endotélio Vascular/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismoRESUMO
Arylsulfatase B (ARSB; N-acetylgalactosamine 4-sulfatase) is reduced in several malignancies, but levels in melanoma have not been investigated previously. Experiments were performed in melanoma cell lines to determine ARSB activity and impact on melanoma invasiveness. ARSB activity was reduced ~50% in melanoma cells compared to normal melanocytes. Silencing ARSB significantly increased the mRNA expression of chondroitin sulfate proteoglycan(CSPG)4 and pro-matrix metalloproteinase(MMP)-2, known mediators of melanoma progression. Also, invasiveness and MMP activity increased when ARSB was reduced, and recombinant ARSB inhibited invasiveness and MMP activity. Since the only known function of ARSB is to remove 4-sulfate groups from the N-acetylgalactosamine 4-sulfate residue at the non-reducing end of chondroitin 4-sulfate (C4S) or dermatan sulfate, experiments were performed to determine the transcriptional mechanisms by which expression of CSPG4 and MMP2 increased. Promoter activation of CSPG4 was mediated by reduced binding of galectin-3 to C4S when ARSB activity declined. In contrast, increased pro-MMP2 expression was mediated by increased binding of the non-receptor tyrosine phosphatase SHP2 to C4S. Increased phospho-ERK1,2 resulted from SHP2 inhibition. Combined effects of increased C4S, CSPG4, and MMP2 increased the invasiveness of the melanoma cells, and therapy with recombinant ARSB may inhibit melanoma progression.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/genética , Metaloproteinase 2 da Matriz/genética , Melanoma/genética , Proteínas de Membrana/genética , N-Acetilgalactosamina-4-Sulfatase/genética , Proteínas Sanguíneas , Linhagem Celular Tumoral , Sulfatos de Condroitina/metabolismo , Galectina 3/metabolismo , Galectinas , Humanos , N-Acetilgalactosamina-4-Sulfatase/metabolismo , Invasividade Neoplásica , Regiões Promotoras Genéticas , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismoRESUMO
The purpose of the present study was to investigate the effects of Pentoxifylline (PTX), Vinpocetine (VPT) and Ticlopidine Hydrochloride (TCD), used commonly for vascular disorders in humans, on the pO2 in SCCVII tumors of C3H/HeJ mice and on the radioresponse of SCCVII tumors. The pO2 in the SCCVII tumors, which were measured 30 min after intraperitoneal (i.p.) injection of PTX (5 mg/kg), VPT (5 mg/kg), or TCD (10 mg/kg) using polarography, was compared to that in saline-treated control tumors. All the three drugs, PTX, VPT and TCD, yielded significant increase of the pO2 in the SCCVII tumors from 25.6 to 26.9 mmHg, from 18.6 to 22.9 mmHg, and from 22.6 to 25.9 mmHg, respectively. Frequency histogram of the pO2 distribution in the saline-treated SCCVII tumors did not show hypoxic fraction of less than 10 mmHg. The radioresponses of the drugs were investigated by tumor growth delay assay. In the drug-treated groups, the SCCVII tumors were irradiated with a single dose of 15 Gy 30 min after injection of the drugs at the same doses as those used in the experiments for intratumoral pO2 measurement. Compared with the irradiation alone group, significant tumor growth delays were observed in all the drug-treated groups. The time required to reach a four-fold increase in the initial tumor volume were 4 days in the saline-treated control group, 22 days in the irradiation (IR) alone group, 28 days in the PTX + IR group, 29 days in the VPT + IR group, and 32 days in TCD + IR group. In conclusion, VPT and TCD are potentially promising drugs for increasing the intratumoral pO2 although the mechanism for radiopotentiation observed in the present study is unknown due to small hypoxic fraction in the SCCVII tumors. Further studies on other mechanisms for radiopotentiation of PTX, VPT or TCD, besides of increasing the pO2 in the tumor, are needed.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/radioterapia , Oxigênio/metabolismo , Pentoxifilina/administração & dosagem , Ticlopidina/administração & dosagem , Alcaloides de Vinca/administração & dosagem , Animais , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Camundongos , Camundongos Endogâmicos C3H , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/administração & dosagem , Resultado do TratamentoRESUMO
BACKGROUND: Chondroitin sulfate proteoglycan-4 (CSPG4) is commonly expressed in melanoma cells and induces melanoma cell proliferation and migration by enhancement of activation of the extracellular signal-regulated kinase 1, 2 (ERK1,2) pathway. The phosphoinositide 3-kinase (PI3K) -protein kinase B (AKT) and mammalian target of rapamycin (mTOR) pathways are also frequently de-regulated in melanoma. We hypothesized that CSPG4, by sustained activation of PI3K, may reduce the effect of the dual inhibition of PI3K-AKT and mTOR pathways. MATERIALS AND METHODS: CSPG4-negative melanoma cell line WM1552C was transfected with CSPG4 and CSPG4 lacking cytoplasmic domain (melanoma-associated chondroitin sulfate proteoglycan (MCSP)ΔCD). To assess the effect of CSPG4 on the mTOR pathway, PF-5212384, a dual PI3K/mTOR inhibitor was used. Cell proliferation and downstream signaling from mTOR was assayed in the presence of CSPG4. RESULTS: Forced CSPG4 expression did not provide any protection to melanoma cells from the pharmacological inhibition of mTOR pathway in vitro. In addition, we demonstrated that inhibition of signaling molecules downstream of AKT and mTOR was not diminished in the presence of CSPG4 when the cells were treated with the PI3K/mTOR inhibitor. CONCLUSION: CSPG4 expression does not have any impact on survival and signaling activity of melanoma cells during PI3K/mTOR inhibition.
Assuntos
Antígenos/fisiologia , Melanoma/patologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteoglicanas/fisiologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Transdução de Sinais/fisiologiaRESUMO
Idiopathic systemic capillary leak syndrome (SCLS) is a rare and potentially fatal vascular disorder characterized by reversible bouts of hypotension and edema resulting from fluid and solute escape into soft tissues. Although spikes in permeability-inducing factors have been linked to acute SCLS flares, whether or not they act on an inherently dysfunctional endothelium is unknown. To assess the contribution of endothelial-intrinsic mechanisms in SCLS, we derived blood-outgrowth endothelial cells (BOEC) from patients and healthy controls and examined gene expression patterns. Ednra, encoding Endothelin receptor A (ETA)-the target of Endothelin 1 (ET-1)-was significantly increased in SCLS BOEC compared to healthy controls. Although vasoconstriction mediated by ET-1 through ETA activation on vascular smooth muscle cells has been well characterized, the expression and function of ETA receptors in endothelial cells (ECs) has not been described. To determine the role of ETA and its ligand ET-1 in SCLS, if any, we examined ET-1 levels in SCLS sera and functional effects of endothelial ETA expression. ETA overexpression in EAhy926 endothelioma cells led to ET-1-induced hyper-permeability through canonical mechanisms. Serum ET-1 levels were elevated in acute SCLS sera compared to remission and healthy control sera, suggesting a possible role for ET-1 and ETA in SCLS pathogenesis. However, although ET-1 alone did not induce hyper-permeability of patient-derived BOEC, an SCLS-related mediator (CXCL10) increased Edrna quantities in BOEC, suggesting a link between SCLS and endothelial ETA expression. These results demonstrate that ET-1 triggers classical mechanisms of vascular barrier dysfunction in ECs through ETA. Further studies of the ET-1-ETA axis in SCLS and in more common plasma leakage syndromes including sepsis and filovirus infection would advance our understanding of vascular integrity mechanisms and potentially uncover new treatment strategies.
Assuntos
Síndrome de Vazamento Capilar/metabolismo , Endotélio Vascular/metabolismo , Receptor de Endotelina A/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Síndrome de Vazamento Capilar/genética , Síndrome de Vazamento Capilar/patologia , Estudos de Casos e Controles , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Quimiocina CXCL10/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotelina-1/metabolismo , Endotélio Vascular/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Masculino , Pessoa de Meia-Idade , Receptor de Endotelina A/genéticaRESUMO
BACKGROUND: The novel compound thiopyrano [2,3-c]quinoline (MT477) has been shown to exhibit antitumor activity in both in vitro and in vivo studies. The present study examined the expression levels of 10,000 genes and how they changed after MT477 treatment in three cancer cell lines: H226, MDA231 and MiaPaCa-2. Materials and Methods/ RESULTS: Molecular function analysis revealed changes in genes involved in cell death, cell-cycle progression and cellular growth and proliferation in all three cancer cell lines. Canonical pathway analysis showed the involvement of the NRF2-mediated oxidative stress response, glucocorticoid, p53, RXR-VDR, G(1)/S checkpoint regulation, ERK, SAPK/JNK and JAS/Stat signaling. Analysis of 234 kinases and phosphatases using a kinase inhibition assay demonstrated a strong inhibitory effect for MAPK14 (104 ± 2%), AMPK A2/B1/G1 (89%) and FGR (83 ± 2%). AURKA was inhibited at 77 ± 1%. MiaPaCa-2 tumor xenograft studies showed a 49.5 ±1 4.8% inhibitory effect in mice treated with 100 µg/kg MT477 compared to untreated mice (p=0.0021). CONCLUSION: MT477 induces molecular mechanisms related to cell death, survival, and inhibition of cellular growth in vitro and in vivo.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Quinolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Aurora Quinase A , Aurora Quinases , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Pancreáticas/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The insulin-like growth factor receptor is overexpressed on many types of cancer cells and has been implicated in metastasis and resistance to apoptosis. We report here the development of a novel covalent conjugate that contains the antifolate drug methotrexate coupled to an engineered variant of insulin-like growth factor-1 (IGF-1), long-R3-IGF-1, which was designed to target methotrexate to tumor cells that overexpress the membrane IGF-1 receptor. The IGF-methotrexate conjugate was found to contain at least 4 methotrexate molecules per IGF-1 protein. The IGF-methotrexate conjugate bound to MCF7 breast cancer cells with greater than 3.3-fold higher affinity than unconjugated long-R3-IGF-1 in a competition binding assay against radiolabeled wild-type IGF-1. Compared with free methotrexate, the IGF-methotrexate conjugate required slightly higher concentrations to inhibit the in vitro growth of the human prostate cancer cell line LNCaP. In vivo, however, in a mouse xenograft model using LNCaP cells, the IGF-methotrexate conjugate was more effective than free methotrexate even at a 6.25-fold lower molar dosage. Similarly, MCF7 xenografts were inhibited more effectively by the IGF-methotrexate conjugate than free methotrexate, even at a 4-fold lower molar dosage. Our results suggest that the targeting of the IGF receptor on tumor cells and tumor-related tissues with IGF-chemotherapy conjugates may substantially increase the specific drug localization and therapeutic effect in the tumor.
Assuntos
Antimetabólitos Antineoplásicos/farmacocinética , Neoplasias da Mama/tratamento farmacológico , Fator de Crescimento Insulin-Like I/análogos & derivados , Fator de Crescimento Insulin-Like I/farmacocinética , Metotrexato/farmacocinética , Animais , Antimetabólitos Antineoplásicos/química , Antimetabólitos Antineoplásicos/toxicidade , Ligação Competitiva , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Desenho de Fármacos , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Fator de Crescimento Insulin-Like I/química , Metotrexato/química , Metotrexato/toxicidade , Camundongos , Camundongos Nus , Receptor IGF Tipo 1/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
MT477 is a novel quinoline with potential activity in Ras-mutated cancers. In this study, MT477 preferentially inhibited the proliferation of K-ras-mutated human pulmonary (A549) and pancreatic (MiaPaCa-2) adenocarcinoma cell lines, compared with a non-Ras-mutated human lung squamous carcinoma cell line (H226) and normal human lung fibroblasts. MT477 treatment induced apoptosis in A549 cells and was associated with caspase-3 activation. MT477 also induced sub-G1 cell-cycle arrest in A549 cells. Although we found that MT477 partially inhibited protein kinase C (PKC), it inhibited Ras directly followed in time by inhibition of 2 Ras downstream molecules, Erk1/2 and Ral. MT477 also caused a reorganization of the actin cytoskeleton and formation of filopodias in A549 cells; this event may lead to decreased migration and invasion of tumor cells. In a xenograft mouse model, A549 tumor growth was inhibited significantly by MT477 at a dose of 1 mg/kg (P < 0.05 vs vehicle control). Taken together, these results support the conclusion that MT477 acts as a direct Ras inhibitor. This quinoline, therefore, could potentially be active in Ras-mutated cancers and could be developed extensively as an anticancer molecule with this in mind.
Assuntos
Adenocarcinoma/tratamento farmacológico , Apoptose/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Proteína Oncogênica p21(ras)/antagonistas & inibidores , Quinolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Citoesqueleto de Actina/efeitos dos fármacos , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fibroblastos/citologia , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Nus , Mutação , Proteína Oncogênica p21(ras)/genética , Proteína Oncogênica p21(ras)/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
MT477 is a novel thiopyrano[2,3-c]quinoline that has been identified using molecular topology screening as a potential anticancer drug with a high activity against protein kinase C (PKC) isoforms. The objective of the present study was to determine the mechanism of action of MT477 and its activity against human cancer cell lines. MT477 interfered with PKC activity as well as phosphorylation of Ras and ERK1/2 in H226 human lung carcinoma cells. It also induced poly-caspase-dependent apoptosis. MT477 had a dose-dependent (0.006 to 0.2 mM) inhibitory effect on cellular proliferation of H226, MCF-7, U87, LNCaP, A431 and A549 cancer cell lines as determined by in vitro proliferation assays. Two murine xenograft models of human A431 and H226 lung carcinoma were used to evaluate tumor response to intraperitoneal administration of MT477 (33 microg/kg, 100 microg/kg, and 1 mg/kg). Tumor growth was inhibited by 24.5% in A431 and 43.67% in H226 xenografts following MT477 treatment, compared to vehicle controls (p < 0.05). In conclusion, our empirical findings are consistent with molecular modeling of MT477's activity against PKC. We also found, however, that its mechanism of action occurs through suppressing Ras signaling, indicating that its effects on apoptosis and tumor growth in vivo may be mediated by Ras as well as PKC. We propose, therefore, that MT477 warrants further development as an anticancer drug.
Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Proteína Quinase C/efeitos dos fármacos , Quinolinas/farmacologia , Animais , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Masculino , Camundongos , Camundongos Nus , Quinases de Proteína Quinase Ativadas por Mitógeno/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Quinolinas/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/efeitos dos fármacos , Proteínas ras/metabolismoRESUMO
Radiation therapy is a widely used treatment for metastatic bone cancer, but the rapid onset of tumor radioresistance is a major problem. We investigated the radiosensitizing effect of enzastaurin, a protein kinase Cbeta (PKCbeta) inhibitor, on bone tumor growth and tumor-related pain. We found that enzastaurin enhanced the effect of ionizing radiation on cultured murine 4T1 breast cancer and murine endothelial cells, suppressing their proliferation and colony formation. Enzastaurin and ionizing radiation also induced caspase-mediated apoptosis of 4T1 cells to a greater degree than radiation alone. Enzastaurin treatment of 4T1 cells blocked the phosphorylation of PKCbeta, as well as Ras and two of its downstream effectors ERK1/2 and RAL-GTP. Using an orthotopic model of bone metastasis, we observed that a combination of enzastaurin and localized radiation treatment reduced tumor blood vessel density, bone destruction and pain compared to single modality treatment. In conclusion, we demonstrate that inhibition of PKCbeta in combination with localized radiation treatment suppresses tumor growth and alleviates pain as compared to radiation-only treatment. We also show that the radiosensitizing effect of enzastaurin is associated with suppression of tumor cell proliferation and tumor-induced angiogenesis possibly through inhibition of the Ras pathway.
Assuntos
Indóis/farmacologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/radioterapia , Proteína Quinase C/antagonistas & inibidores , Radiossensibilizantes/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/radioterapia , Neoplasias Ósseas/secundário , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Terapia Combinada , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/efeitos da radiação , Feminino , Indóis/uso terapêutico , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos SCID , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/radioterapia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoclastos/efeitos da radiação , Dor/tratamento farmacológico , Dor/radioterapia , Proteína Quinase C beta , Radiossensibilizantes/uso terapêutico , Radioterapia Adjuvante/métodos , Transdução de Sinais/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/efeitos da radiação , Proteínas ras/metabolismoRESUMO
The receptor tyrosine kinase inhibitor, SU11248, was added to localised radiation to evaluate the response of bone metastases and to define the basic mechanism of radiosensitisation. Treatment with SU11248 and radiation was assessed in vitro using cultured 4T1 breast cancer cells and in vivo using an orthotopic 4T1 murine mammary tumour model of breast cancer bone metastasis. Cultured 4T1 cells treated with SU11248 (1 microM) and radiation (10 Gy) showed an almost 7.5-fold increase in caspase-mediated apoptosis after 24 h of incubation, compared to either treatment alone. Mice treated with SU11248 (40 mg/kg/daily) and radiation (15 Gy/single-dose) had a relatively greater reduction in tumour growth, bone osteolysis, osteoclast maturation and microvessel density. Combined modality treatment resulted in improvements in behavioural pain assessment scores and normalisation of neurochemical changes in the spinal cord receiving primary afferent innervation from tumour-bearing femora. Our study demonstrates that SU11248 enhances the radiation control of metastatic breast tumours in bone and tumour-induced pain.