RESUMO
BACKGROUND & AIMS: In the gut, tumorigenesis is thought to arise from the stem cell population located near the base of intestinal and colonic crypts. The RNA binding protein musashi-1 (Msi-1) is a putative intestinal and progenitor/stem cell marker. Msi-1 expression is increased during rat brain development and in APC(min/+) mice tumors. This study examined a potential role of Msi-1 in tumorigenesis. METHODS: Msi-1 small interfering RNA (siRNA) was administered as a liposomal preparation to HCT116 colon adenocarcinoma xenografts in athymic nude mice and tumor volume was measured. Cell proliferation was assessed by hexosaminidase and 3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyltetrazolium bromide MTT assays. siRNA-transfected cells were subjected to 12 Gy gamma-irradiation. Apoptosis was assessed by immunoreactive activated caspase-3 and mitosis was assessed by phosphorylated histone H3 staining. The tumor xenografts were stained similarly for phosphorylated histone H3, activated caspase-3, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, Notch-1, and p21(WAF1). Furthermore, siRNA-transfected cells were subjected to cell-cycle analysis and Western blot analyses for Notch-1 and p21(WAF1). RESULTS: Knockdown of Msi-1 resulted in tumor growth arrest in xenografts, reduced cancer cell proliferation, and increased apoptosis alone and in combination with radiation injury. siRNA-mediated reduction of Msi-1 lead to mitotic catastrophe in tumor cells. Moreover, there was inhibition of Notch-1 and up-regulation of p21(WAF1) after knockdown of Msi-1. CONCLUSIONS: Our results show the involvement of Msi-1 in cancer cell proliferation, inhibition of apoptosis, and mitotic catastrophe, suggesting an important potential mechanism for its role in tumorigenesis.
Assuntos
Adenocarcinoma/patologia , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Proteínas do Tecido Nervoso/genética , RNA Neoplásico/genética , Proteínas de Ligação a RNA/genética , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animais , Apoptose , Biomarcadores Tumorais , Western Blotting , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Progressão da Doença , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias Experimentais , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante HeterólogoRESUMO
The anti-apoptotic effect of PGE(2) was examined in Jurkat cells (human T-cell leukemia) by incubation with PGE(2) (5 nM) prior to treatment with the cancer chemotherapeutic agent camptothecin. Apoptosis was evaluated by caspase-3 activity in cell extracts and flow cytometry of propidium iodide-labeled cells. Pre-incubation with PGE(2) reduced camptothecin-induced caspase activity by 30% and apoptosis by 35%, respectively. Pharmacological data demonstrate that the EP4 receptor is responsible for mediating the protection from camptothecin-induced apoptosis. Pre-treatment of the cells with the EP4 antagonist (EP4A) prior to PGE(2) and camptothecin abolished the increased survival effect of PGE(2). Specific inhibition of the downstream of PI3 kinase or AKT/protein kinase but not protein kinase A prevents the observed increase in cell survival elicited by PGE(2). These findings have critical implications regarding the mechanism and potential application of PGE(2) receptor specific inhibition in cancer therapy.
Assuntos
Dinoprostona/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Prostaglandina E/metabolismo , Transdução de Sinais/efeitos dos fármacos , Camptotecina/farmacologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células Jurkat , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Prostaglandina E/agonistas , Receptores de Prostaglandina E/antagonistas & inibidores , Receptores de Prostaglandina E/genética , Receptores de Prostaglandina E Subtipo EP4RESUMO
Alkali burns are known to possess high pathological potential because of their inherent ability to lyse cell membranes and penetrate intraocular structures with devastating results. The authors aimed to evaluate the most common cause of this presentation, the current treatment approaches to injury, and eventual outcome as related to severity. The authors performed a retrospective review of all patients who sustained chemical-related ocular injuries seen at the Concord Hospital Burns Unit, Australia between January 2005 and March 2012. Management was based on cooperation between ophthalmic staff and the burns unit, with emphasis on early aggressive intervention and rigorous follow-up. The records of 39 patients who presented with chemical-related injury were assessed, 12 of whom had confirmed alkali burns involving the cornea. The most commonly implicated agent was sodium hydroxide, usually in the context of otherwise trivial domestic accidents. Acute medical management included copious irrigation and the use of analgesics, cycloplegics, and topical antibiotics. In half the cases, steroid drops and oral vitamin C were also used. Ten of the 12 patients (83%) had return to premorbid visual acuity. Complications included cicatrical ectropion (n = 1), pseudoexfoliative syndrome (n = 1), and symblepharon (n = 1). Surgical correction was needed in the one patient with cicatrical ectropion. This case series shows that appropriate acute management minimizes the potentially devastating sequelae of ocular alkali burns. Emphasis should be placed on prevention of domestic and workplace injuries when using alkaline products.
Assuntos
Álcalis/efeitos adversos , Queimaduras Químicas/terapia , Lesões da Córnea/induzido quimicamente , Lesões da Córnea/terapia , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/terapia , Acidentes Domésticos/estatística & dados numéricos , Acidentes de Trabalho/estatística & dados numéricos , Idoso , Queimaduras Químicas/etiologia , Queimaduras Químicas/fisiopatologia , Terapia Combinada , Lesões da Córnea/fisiopatologia , Queimaduras Oculares/fisiopatologia , Feminino , Humanos , Escala de Gravidade do Ferimento , Masculino , Pessoa de Meia-Idade , New South Wales , Medição de Risco , Estudos de Amostragem , Resultado do Tratamento , Acuidade Visual , Adulto JovemAssuntos
Cartilagem Costal/lesões , Fixação Interna de Fraturas/métodos , Fraturas das Costelas/cirurgia , Placas Ósseas , Humanos , Imageamento Tridimensional , Masculino , Pessoa de Meia-Idade , Fraturas das Costelas/diagnóstico por imagem , Fraturas das Costelas/patologia , Tomografia Computadorizada por Raios XRESUMO
The microcolony assay following gamma irradiation (IR) is a functional assay of intestinal stem cell fate. The cyclin-dependent kinase (CDK) inhibitor p21(Waf1/Cip1/Sdi1) (p21) regulates cell cycle arrest following DNA damage. To explore the role of p21 on stem cell fate, we examined the effects of p21 deletion on intestinal crypt survival following IR and expression of the stem/progenitor cell marker Musashi-1 (Msi-1) and the antiapoptotic gene survivin. Intestinal stem cell survival in adult wild-type (WT) and p21(-/-) mice was measured using the microcolony assay. Msi-1, p21, and survivin mRNA were measured using real-time PCR and immunohistochemical analysis. Laser capture microdissection (LCM) was used to isolate mRNA from the crypt stem cell zone. No differences in radiation-induced apoptosis were observed between WT and p21(-/-) mice. However, increased crypt survival (3.0-fold) was observed in p21(-/-) compared with WT mice 3.5 days after 13 Gy. Msi-1 and survivin mRNA were elevated 12- and 7.5-fold, respectively, in LCM-dissected crypts of p21(-/-) compared with WT mice. In conclusion, deletion of p21 results in protection of crypt stem/progenitor cells from IR-induced cell death. Furthermore, the increase in crypt survival is associated with increased numbers of Msi-1- and survivin-expressing cells in regenerative crypts.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Intestino Delgado/efeitos da radiação , Células-Tronco/efeitos da radiação , Irradiação Corporal Total , Animais , Apoptose/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Inibidor de Quinase Dependente de Ciclina p21/deficiência , Inibidor de Quinase Dependente de Ciclina p21/genética , Feminino , Proteínas Inibidoras de Apoptose , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/efeitos da radiação , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras , Células-Tronco/metabolismo , Células-Tronco/patologia , Survivina , Fatores de TempoRESUMO
Azoxymethane (AOM) is a potent DNA-damaging agent and carcinogen that induces intestinal and colonic tumors in rodents. Evaluation of the stem cell population by colony formation assay reveals that, within 8 h after treatment, AOM (10 mg/kg) elicited a prosurvival response. In wild-type (WT) mice, AOM treatment induced a 2.5-fold increase in intestinal crypt stem cell survival. AOM treatment increased stem cell survival in cyclooxygenase (COX)-2(-/-) but not COX-1(-/-) mice, confirming a role of COX-1 in the AOM-induced increase in stem cell survival. COX-1 mRNA and protein expression as well as COX-1-derived PGE(2) synthesis were increased 8 h after AOM treatment. Immunohistochemical staining of COX-1 demonstrated expression of the enzyme in the crypt epithelial cells, especially in the columnar epithelial cells between the Paneth cells adjacent to the stem cell zone. WT mice receiving AOM exhibited increased intestinal apoptosis and a simultaneous reduction in crypt mitotic figures within 8 h of injection. There were no significant differences in baseline or AOM-induced intestinal epithelial apoptosis between WT and COX-1(-/-) mice, but there was a complete reversal of the AOM-mediated reduction in mitosis in COX-1(-/-) mice. This suggests that COX-1-derived PGE(2) may play a key role in the early phase of intestinal tumorigenesis in response to DNA damage and suggests that COX-1 may be a potential therapeutic target in this model of colon cancer.