Fabrícia Dietrich1, Fabrício Figueiró1,2, Eduardo Cremonese Filippi-Chiela3, Angélica Regina Cappellari4, Liliana Rockenbach1,5, Alain Tremblay5, Patrícia Boni de Paula2, Rafael Roesler6,7, Aroldo Braga Filho8, Jean Sévigny5,9, Fernanda Bueno Morrone4, Ana Maria Oliveira Battastini10,11. 1. Programa de Pós-Graduação em Ciências Biológicas: Bioquímica, Instituto de Ciências Básicas da Saúde, UFRGS, Porto Alegre, RS, CEP 90035-003, Brazil. 2. Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, UFRGS, Porto Alegre, RS, CEP 90035-003, Brazil. 3. Programa de Pós-Graduação Ciências em Gastroenterologia e Hepatologia, Faculdade de Medicina, UFRGS, Porto Alegre, RS, CEP 90035-003, Brazil. 4. Laboratório de Farmacologia Aplicada, Escola de Ciências da Saúde, PUCRS, Porto Alegre, RS, CEP 90619-900, Brazil. 5. Centre de Recherche du CHU de Québec, Université Laval, Quebec, QC, G1V 4G2, Canada. 6. Laboratório de Câncer e Neurobiologia, Centro de Pesquisa Experimental, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, CEP 90035-003, Brazil. 7. Departamento de Farmacologia, Instituto de Ciências Básicas da Saúde, UFRGS, Porto Alegre, RS, CEP 90050-170, Brazil. 8. Serviço de Radioterapia, Hospital São Lucas da PUCRS, Porto Alegre, RS, CEP 90619-900, Brazil. 9. Département de Microbiologie-infectiologie et d'immunologie, Faculté de Médecine, Université Laval, Quebec, QC, G1V 0A6, Canada. 10. Programa de Pós-Graduação em Ciências Biológicas: Bioquímica, Instituto de Ciências Básicas da Saúde, UFRGS, Porto Alegre, RS, CEP 90035-003, Brazil. abattastini@gmail.com. 11. Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, UFRGS, Porto Alegre, RS, CEP 90035-003, Brazil. abattastini@gmail.com.
Abstract
PURPOSE: Trimodal therapy is a reasonable bladder-preserving option to radical cystectomy. However, many tumors are radioresistive. In this sense, the identification of new prognostic and predictive biomarkers that allow the selection of patients with better responses to radiation therapy would improve outcomes. With the aim of using ecto-5'-nucleotidase/CD73 as a predictive biomarker, the role of this enzyme in the context of radiotherapy in T24 human bladder cancer cell line was investigated. METHODS: T24 cell line was exposure to a single dose of radiation (4 Gray) and trypan blue assay (pharmacological assays of viability/cumulative population doubling), flow cytometry (cell cycle/cell death/active caspase-3/ecto-5'-nucleotidase/CD73 protein staining), DAPI staining (nuclear morphometric assay), RT-PCR and real-time PCR, malachite green method (ectonucleotidase enzymatic assay), and HPLC (analysis of AMP metabolism) were carried out. T24 cell line in which ecto-5'-nucleotidase/CD73 has been completely silenced (5'KO) was also used. RESULTS: The exposure of T24 cell line to a single dose (4 Gray) of radiation-induced cell death and triggered a transitory increase in ecto-5'-nucleotidase/CD73 expression, increased ectonucleotidase activity, and led to adenosine and inosine accumulation in the extracellular medium. Pharmacological inhibition or knocking out ecto-5'-nucleotidase/CD73 rescued cells' proliferative capacity, reducing their sensitivity to radiation. CONCLUSION: Our findings show that the induction of ecto-5'-nucleotidase/CD73 by radiation contributes to the radiosensitivity of T24 cell line.
PURPOSE: Trimodal therapy is a reasonable bladder-preserving option to radical cystectomy. However, many tumors are radioresistive. In this sense, the identification of new prognostic and predictive biomarkers that allow the selection of patients with better responses to radiation therapy would improve outcomes. With the aim of using ecto-5'-nucleotidase/CD73 as a predictive biomarker, the role of this enzyme in the context of radiotherapy in T24 humanbladder cancer cell line was investigated. METHODS: T24 cell line was exposure to a single dose of radiation (4 Gray) and trypan blue assay (pharmacological assays of viability/cumulative population doubling), flow cytometry (cell cycle/cell death/active caspase-3/ecto-5'-nucleotidase/CD73 protein staining), DAPI staining (nuclear morphometric assay), RT-PCR and real-time PCR, malachite green method (ectonucleotidase enzymatic assay), and HPLC (analysis of AMP metabolism) were carried out. T24 cell line in which ecto-5'-nucleotidase/CD73 has been completely silenced (5'KO) was also used. RESULTS: The exposure of T24 cell line to a single dose (4 Gray) of radiation-induced cell death and triggered a transitory increase in ecto-5'-nucleotidase/CD73 expression, increased ectonucleotidase activity, and led to adenosine and inosine accumulation in the extracellular medium. Pharmacological inhibition or knocking out ecto-5'-nucleotidase/CD73 rescued cells' proliferative capacity, reducing their sensitivity to radiation. CONCLUSION: Our findings show that the induction of ecto-5'-nucleotidase/CD73 by radiation contributes to the radiosensitivity of T24 cell line.
Entities:
Keywords:
Bladder carcinoma; Ectonucleotidase; Purinergic signaling; Radiotherapy; T24 cell line
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