Ecto-5′-nucleotidase/CD73 contributes to the radiosensitivity of T24 human bladder cancer cell line

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.

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