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1.
Cell Death Differ ; 25(2): 340-352, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29099485

RESUMO

The mechanisms of how chemotherapeutic drugs lead to cell cycle checkpoint regulation and DNA damage repair are well understood, but how such signals are transmitted to the cellular apoptosis machinery is less clear. We identified a novel apoptosis-inducing complex, we termed FADDosome, which is driven by ATR-dependent caspase-10 upregulation. During FADDosome-induced apoptosis, cFLIPL is ubiquitinated by TRAF2, leading to its degradation and subsequent FADD-dependent caspase-8 activation. Cancer cells lacking caspase-10, TRAF2 or ATR switch from this cell-autonomous suicide to a more effective, autocrine/paracrine mode of apoptosis initiated by a different complex, the FLIPosome. It leads to processing of cFLIPL to cFLIPp43, TNF-α production and consequently, contrary to the FADDosome, p53-independent apoptosis. Thus, targeting the molecular levers that switch between these mechanisms can increase efficacy of treatment and overcome resistance in cancer cells.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Caspase 10/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/química , Proliferação de Células/efeitos dos fármacos , Feminino , Fluoruracila/farmacologia , Células HCT116 , Células HT29 , Humanos , Ligantes , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Nus
2.
Cancer Biol Ther ; 15(12): 1658-66, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25482930

RESUMO

Current treatment modalities for pancreatic carcinoma afford only modest survival benefits. TRAIL, as a potent and specific inducer of apoptosis in cancer cells, would be a promising new treatment option. However, since not all pancreatic cancer cells respond to TRAIL, further improvements and optimizations are still needed. One strategy to improve the effectiveness of TRAIL-based therapies is to specifically target one of the 2 cell death inducing TRAIL-receptors, TRAIL-R1 or TRAIL-R2 to overcome resistance. To this end, we designed constructs expressing soluble TRAIL (sTRAIL) variants that were rendered specific for either TRAIL-R1 or TRAIL-R2 by amino acid changes in the TRAIL ectodomain. When we expressed these constructs, including wild-type sTRAIL (sTRAIL(wt)), TRAIL-R1 (sTRAIL(DR4)) and TRAIL-R2 (sTRAIL(DR5)) specific variants, in 293 producer cells we found all to be readily expressed and secreted into the supernatant. These supernatants were subsequently transferred onto target cancer cells and apoptosis measured. We found that the TRAIL-R1 specific variant had higher apoptosis-inducing activity in human pancreatic carcinoma Colo357 cells as well as PancTu1 cells that were additionally sensitized by targeting of XIAP. Finally, we tested TRAIL-R1 specific recombinant TRAIL protein (rTRAIL(DR4)) on Colo357 xenografts in nude mice and found them to be more efficacious than rTRAIL(wt). Our results demonstrate the benefits of synthetic biological approaches and show that TRAIL-R1 specific variants can potentially enhance the therapeutic efficacy of TRAIL-based therapies in pancreatic cancer, suggesting that they can possibly become part of individualized and tumor specific combination treatments in the future.


Assuntos
Variação Genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Camundongos , Mutação , Neoplasias Pancreáticas/patologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/administração & dosagem , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Transfecção , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Stem Cell Res ; 7(2): 163-71, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21763624

RESUMO

Cell therapy has the potential to offer novel treatment modalities for a number of diseases including cancer, and stem cells and in particular mesenchymal stem cells (MSCs) have been experimentally used to deliver therapeutic transgenes. However, conflicting reports have on the one side found that human MSCs can promote metastasis, while on the other hand other studies have shown that MSCs can stall the growth of metastatic lesions. In order to clarify the role of MSCs in metastasis development, we tested whether murine MSCs would behave similarly to human cells in mice. We found that the tissue distribution of human and mouse MSCs was nearly identical after intravenous injection. In mice with MDA-MB-231 mammary carcinoma xenografts we found that a fraction of MSCs infiltrated the primary tumor mass, but that the general tissue distribution of MSCs was unaffected by the tumor-burden. About half of the tumor-burdened animals that were treated with murine and human MSCs, respectively, harbored metastatic lesions with only 17% of controls showing metastatic nodules. Hence, both human and mouse MSCs possess metastasis-promoting activity raising concerns about the safe use of MSCs, but at the same time making the use of murine transgenic model systems feasible to study the role of MSCs in metastasis development and possibly finding ways of using them safely as cell therapeutic vehicles.


Assuntos
Neoplasias da Mama/patologia , Células-Tronco Mesenquimais/patologia , Animais , Neoplasias da Mama/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Terapia Baseada em Transplante de Células e Tecidos/efeitos adversos , Modelos Animais de Doenças , Feminino , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica
4.
Stem Cells ; 28(11): 2109-20, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20882532

RESUMO

Disseminating tumors are one of the gravest medical problems. Here, we combine the tumor-specific apoptosis-inducing activity of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with the ability of mesenchymal stem cells (MSCs) to infiltrate both tumor and lymphatic tissues to target primary tumors as well as disseminated cancer cells in a human pancreatic cancer mouse model. Furthermore, we targeted X-linked inhibitor of apoptosis protein (XIAP) by RNA interference (RNAi) inside the cancer cells to make use of the apoptosis sensitization as well the antimetastatic effect that is afforded by XIAP silencing. We generated MSCs, termed MSC.sTRAIL, that express and secrete a trimeric form of soluble TRAIL (sTRAIL). MSC.sTRAIL triggered limited apoptosis in human pancreatic carcinoma cells that were resistant to soluble recombinant TRAIL, which is most likely due to the enhanced effect of the direct, cell-mediated delivery of trimeric TRAIL. MSC.sTRAIL-mediated cell death was markedly increased by concomitant knockdown of XIAP by RNAi in the cancer cells. These findings were confirmed in xenograft models, in which tumors from the parental pancreatic carcinoma cells showed only growth retardation on treatment with MSC.sTRAIL, whereas tumors with silenced XIAP that were treated with MSC.sTRAIL went into remission. Moreover, animals with XIAP-negative xenografts treated with MSC.sTRAIL were almost free of lung metastasis, whereas animals treated with control MSCs showed substantial metastatic growth in the lungs. In summary, this is the first demonstration that a combined approach using systemic MSC-mediated delivery of sTRAIL together with XIAP inhibition suppresses metastatic growth of pancreatic carcinoma.


Assuntos
Células-Tronco Mesenquimais/metabolismo , Neoplasias Pancreáticas/terapia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Animais , Apoptose/fisiologia , Western Blotting , Diferenciação Celular , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Camundongos Nus , Ligante Indutor de Apoptose Relacionado a TNF/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética
5.
Exp Mol Pathol ; 78(2): 144-9, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15713441

RESUMO

T-2 toxin, a kind of trichothecene mycotoxins produced by the genus Fusarium, induces apoptosis in basal keratinocytes when topically applied to the dorsal skin of rats. In the present study, direct effects of T-2 toxin on keratinocyte primary cultures obtained from newborn rats were examined after the third passage. Keratinocyte medium containing 0.25 microg/ml of T-2 toxin dissolved in dimethyl sulfoxide or solvent alone was added to 4-day cultures and incubated at 37 degrees C. At 0.5, 1, 3, 5, 7, and 9 h after treatment (h), feeder layer was separated from flasks, and cells were trypsinized. Cell viability was estimated by trypan blue exclusion method. In addition, RNA was obtained and RT-PCR was performed. Samples obtained from slide cultures at 3, 6, 9, and 12 h were fixed in 4% paraformaldehyde or 2.5% glutaraldehyde for morphological examination. After T-2 toxin application, cell viability decreased to 40% at 3 h. At 6 h, small-sized keratinocytes showed pyknosis or karyorrhexis, resulting in detachment from slides. The number of such cells increased until 12 h. These small-sized keratinocytes showed ultrastructural changes characteristic for apoptosis. At the same time, large squamous keratinocytes showed intracytoplasmic edema. The expression of apoptosis-related genes (c-fos and c-jun) and cytokines (TNF-alpha and IL-1beta) mRNAs markedly increased before the development of apoptosis. These findings indicate that c-fos and c-jun oncogenes and TNF-alpha and IL-1beta play an important role in the development of T-2 toxin-induced apoptosis in keratinocytes.


Assuntos
Apoptose/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Toxina T-2/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Queratinócitos/patologia , Queratinócitos/ultraestrutura , Microscopia Eletrônica de Transmissão , Proteínas Proto-Oncogênicas c-fos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/análise , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa
6.
Cell Biol Int ; 26(1): 123-5, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-11779229

RESUMO

The presence of a tumor generally changes the mitotic activity of the normal cell population in mice. In the present work, the mitotic activity of the duodenal crypt enterocytes in EA21a mammary carcinoma-bearing mice was determined. The results show that there is a patent circadian variation in normal mice and, in the presence of the EA21a mammary tumor, cell proliferation is stimulated. Stimulation was evident in enterocytes from the intermediate as well as the superficial regions of the crypt. Some humoral factors produced by the transplanted tumor could interfere with the regulatory mechanism of the mitotic activity of duodenal crypt enterocytes.


Assuntos
Carcinoma/metabolismo , Duodeno/metabolismo , Neoplasias Mamárias Animais/metabolismo , Mitose , Animais , Divisão Celular , Ritmo Circadiano , Enterócitos/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C3H , Transplante de Neoplasias , Fatores de Tempo
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