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1.
Gut ; 63(2): 310-6, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23408349

RESUMO

BACKGROUND: Hormone replacement therapy increases the risk of developing ulcerative colitis in postmenopausal women. Chronic intestinal inflammation predisposes to colon cancer development, but effects of female hormones on colitis-associated cancer development have not been examined. AIM: To investigate the role of female hormones in the dextran sodium sulfate (DSS)-azoxymethane (AOM) mouse model for colitis-associated cancer. DESIGN: We performed ovariectomies, or sham operations, on mice, and supplemented these animals with indicated hormones. Additionally, we used oestrogen receptor α or ß (Erα or Erß) mutant mice. To study colitis or colitis-associated cancer, we used DSS only, or DSS and AOM, respectively. RESULTS: Ovariectomy protects female mice against colitis-associated tumour development. Hormone replacement in ovariectomised mice with either oestradiol (E2), medroxyprogesterone acetate or a combination of both suggests that oestrogens are the ovary-derived factor that promotes tumour development in the context of inflammatory damage. E2-treated animals showed increased clinical symptoms and Il-6 production upon DSS-induced colitis and enhanced epithelial proliferation. Treatment with E2 markedly increased the numbers of polyps in ovariectomised mice and also strongly promoted tumour progression with all E2-treated animals developing at least one invasive adenocarcinoma, whereas, placebo-treated animals developed adenomas only. Using Er mutant mice, we find that the protumorigenic effect of oestrogen depends on both Erα and Erß. CONCLUSIONS: Our results suggest that oestrogens promote inflammation-associated cancer development by impairing the mucosal response to inflammatory damage.


Assuntos
Carcinogênese/induzido quimicamente , Colite/induzido quimicamente , Neoplasias do Colo/induzido quimicamente , Modelos Animais de Doenças , Estradiol/efeitos adversos , Estrogênios/efeitos adversos , Medroxiprogesterona/efeitos adversos , Animais , Azoximetano/toxicidade , Citocinas/metabolismo , Sulfato de Dextrana/toxicidade , Feminino , Terapia de Reposição Hormonal/efeitos adversos , Imuno-Histoquímica , Camundongos , Ovariectomia
2.
Cancer Lett ; 587: 216678, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38360143

RESUMO

Endoplasmic reticulum (ER) stress and the adaptive response that follows, termed the unfolded protein response (UPR), are crucial molecular mechanisms to maintain cellular integrity by safeguarding proper protein synthesis. Next to being important in protein homeostasis, the UPR is intricate in cell fate decisions such as proliferation, differentiation, and stemness. In the intestine, stem cells are critical in governing epithelial homeostasis and they are the cell of origin of gastrointestinal malignancies. In this review, we will discuss the role of ER stress and the UPR in the gastrointestinal tract, focusing on stem cells and carcinogenesis. Insights in mechanisms that connect ER stress and UPR with stemness and carcinogenesis may broaden our understanding in the development of cancer throughout the gastrointestinal tract and how we can exploit these mechanisms to target these malignancies.


Assuntos
Neoplasias , Resposta a Proteínas não Dobradas , Humanos , Estresse do Retículo Endoplasmático/fisiologia , Carcinogênese , Neoplasias/patologia , Células-Tronco/patologia , Trato Gastrointestinal
3.
Cell Death Dis ; 10(7): 490, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227689

RESUMO

The unfolded protein response (UPR) acts through its downstream branches, PERK-eIF2α signaling, IRE1α-XBP1 signaling and ATF6 signaling. In the intestine, activation of the UPR through the kinase PERK results in differentiation of intestinal epithelial stem cells and colon cancer stem cells, whereas deletion of XBP1 results in increased stemness and adenomagenesis. How downstream activation of XBP1 and ATF6 influences intestinal stemness and proliferation remains largely unknown. We generated colorectal cancer cells (LS174T) that harbor doxycycline inducible expression of the active forms of either XBP1(s) or ATF61-373. Activation of either XBP1 or ATF6 resulted in reduced cellular proliferation and reduced expression of markers of intestinal epithelial stemness. Moreover, XBP1 and ATF6 activation reduced global protein synthesis and lowered the threshold for UPR activation. XBP1-mediated loss of stemness and proliferation resulted from crossactivation of PERK-eIF2α signaling and could be rescued by constitutive expression of eIF2α phosphatase GADD34. We thus find that enforced activation of XBP1 and ATF6 results in reduction of stemness and proliferation. We expose a novel interaction between XBP1 and PERK-eIF2α signaling.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Neoplasias do Colo/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Proteína 1 de Ligação a X-Box/metabolismo , Fator 6 Ativador da Transcrição/genética , Western Blotting , Pontos de Checagem do Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/fisiologia , Neoplasias do Colo/genética , Humanos , Células-Tronco Neoplásicas/citologia , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Resposta a Proteínas não Dobradas/genética , Proteína 1 de Ligação a X-Box/genética
4.
Cancer Res ; 78(21): 6098-6106, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30232220

RESUMO

Deletion of endoplasmic reticulum resident chaperone Grp78 results in activation of the unfolded protein response and causes rapid depletion of the entire intestinal epithelium. Whether modest reduction of Grp78 may affect stem cell fate without compromising intestinal integrity remains unknown. Here, we employ a model of epithelial-specific, heterozygous Grp78 deletion by use of VillinCreERT2-Rosa26ZsGreen/LacZ-Grp78+/fl mice and organoids. We examine models of irradiation and tumorigenesis, both in vitro and in vivo Although we observed no phenotypic changes in Grp78 heterozygous mice, Grp78 heterozygous organoid growth was markedly reduced. Irradiation of Grp78 heterozygous mice resulted in less frequent regeneration of crypts compared with nonrecombined (wild-type) mice, exposing reduced capacity for self-renewal upon genotoxic insult. We crossed mice to Apc-mutant animals for adenoma studies and found that adenomagenesis in Apc heterozygous-Grp78 heterozygous mice was reduced compared with Apc heterozygous controls (1.43 vs. 3.33; P < 0.01). In conclusion, epithelium-specific Grp78 heterozygosity compromises epithelial fitness under conditions requiring expansive growth such as adenomagenesis or regeneration after γ-irradiation. These results suggest that Grp78 may be a therapeutic target in prevention of intestinal neoplasms without affecting normal tissue.Significance: Heterozygous disruption of chaperone protein Grp78 reduces tissue regeneration and expansive growth and protects from tumor formation without affecting intestinal homeostasis. Cancer Res; 78(21); 6098-106. ©2018 AACR.


Assuntos
Adenoma/metabolismo , Proteínas de Choque Térmico/metabolismo , Neoplasias Intestinais/metabolismo , Intestinos/citologia , Células-Tronco/citologia , Adenoma/genética , Alelos , Animais , Diferenciação Celular , Proliferação de Células , Transformação Celular Neoplásica/genética , Chaperona BiP do Retículo Endoplasmático , Feminino , Deleção de Genes , Genótipo , Proteínas de Choque Térmico/genética , Heterozigoto , Imuno-Histoquímica , Mucosa Intestinal/metabolismo , Neoplasias Intestinais/genética , Masculino , Camundongos , Chaperonas Moleculares , Organoides , Fenótipo , Regeneração , Resposta a Proteínas não Dobradas
5.
J Vis Exp ; (98)2015 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-25938265

RESUMO

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.


Assuntos
Mucosa Intestinal/fisiologia , Intestino Delgado/fisiologia , Lentivirus/genética , Transdução Genética/métodos , Animais , Diferenciação Celular/fisiologia , Técnicas de Silenciamento de Genes/métodos , Vetores Genéticos/genética , Mucosa Intestinal/citologia , Mucosa Intestinal/virologia , Intestino Delgado/citologia , Intestino Delgado/virologia , Camundongos , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Organoides/fisiologia , Organoides/virologia , Interferência de RNA , Células-Tronco/citologia , Células-Tronco/virologia , Transgenes
6.
Cell Rep ; 13(3): 489-494, 2015 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-26456824

RESUMO

Colon cancer stem cells (colon-CSCs) are more resistant to conventional chemotherapy than differentiated cancer cells. This subset of therapy refractory cells is therefore believed to play an important role in post-therapeutic tumor relapse. In order to improve the rate of sustained response to conventional chemotherapy, development of approaches is warranted that specifically sensitize colon-CSCs to treatment. Here, we report that ER-stress-induced activation of the unfolded protein response (UPR) forces colon-CSCs to differentiate, resulting in their enhanced sensitivity to chemotherapy in vitro and in vivo. Our data suggest that agents that induce activation of the UPR may be used to specifically increase sensitivity of colon-CSCs to the effects of conventional chemotherapy.


Assuntos
Antineoplásicos/farmacologia , Diferenciação Celular , Neoplasias do Colo/metabolismo , Estresse do Retículo Endoplasmático , Células-Tronco Neoplásicas/metabolismo , Compostos Organoplatínicos/farmacologia , Animais , Antineoplásicos/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Humanos , Camundongos , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Compostos Organoplatínicos/uso terapêutico , Oxaliplatina , Esferoides Celulares , Resposta a Proteínas não Dobradas
7.
World J Gastroenterol ; 20(44): 16683-9, 2014 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-25469037

RESUMO

AIM: To investigate if azathioprine could reduce adenoma formation in Apc(Min/+) , a mouse model of sporadic intestinal tumorigenesis. METHODS: Azathioprine was administered via drinking water (estimated 6-20 mg/kg body weight per day) to Apc(Min/+) and wildtype mice. Control animals received vehicle only (DMSO) dissolved in drinking water. At 15 wk of age all mice were sacrificed and intestines of Apc(Min/+) were harvested for evaluation of polyp number. Azathioprine induced toxicity was investigated by immunohistochemical analysis on spleens. RESULTS: All azathioprine treated mice showed signs of drug-associated toxicity such as weight loss and development of splenic T-cell lymphomas. Although this suggests that the thiopurine concentration was clearly in the therapeutic range, it did not reduce tumor formation (48 ± 3.1 adenomas vs 59 ± 5.7 adenomas, P = 0.148). CONCLUSION: We conclude that in the absence of inflammation, azathioprine does not affect intestinal tumorigenesis.


Assuntos
Polipose Adenomatosa do Colo/prevenção & controle , Anticarcinógenos/farmacologia , Azatioprina/farmacologia , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/metabolismo , Polipose Adenomatosa do Colo/patologia , Animais , Anticarcinógenos/toxicidade , Azatioprina/toxicidade , Feminino , Genes APC , Linfoma de Células T/induzido quimicamente , Linfoma de Células T/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Esplênicas/induzido quimicamente , Neoplasias Esplênicas/patologia , Fatores de Tempo
8.
Cell Rep ; 3(4): 1128-39, 2013 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-23545496

RESUMO

Stem cells generate rapidly dividing transit-amplifying cells that have lost the capacity for self-renewal but cycle for a number of times until they exit the cell cycle and undergo terminal differentiation. We know very little of the type of signals that trigger the earliest steps of stem cell differentiation and mediate a stem cell to transit-amplifying cell transition. We show that in normal intestinal epithelium, endoplasmic reticulum (ER) stress and activity of the unfolded protein response (UPR) are induced at the transition from stem cell to transit-amplifying cell. Induction of ER stress causes loss of stemness in a Perk-eIF2α-dependent manner. Inhibition of Perk-eIF2α signaling results in stem cell accumulation in organoid culture of primary intestinal epithelium. Our findings show that the UPR plays an important role in the regulation of intestinal epithelial stem cell differentiation.


Assuntos
Estresse do Retículo Endoplasmático , Mucosa Intestinal/metabolismo , Células-Tronco/citologia , Resposta a Proteínas não Dobradas , Animais , Diferenciação Celular , Células Cultivadas , Chaperona BiP do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Mucosa Intestinal/citologia , Camundongos , Mutação , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , eIF-2 Quinase/antagonistas & inibidores , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
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