Blockade of calcium-activated chloride channel ANO1 ameliorates ionizing radiation-induced intestinal injury.

INTRODUCTION

Radiation enteritis is one of the most frequent clinical complications of radiotherapy (RT), yet few effective strategies currently exist to protect against that. Anoctamin 1 (ANO1) functions both as a chloride channel and a signal transduction protein, influencing numerous pathophysiological processes.

OBJECTIVES

This study aimed to investigate whether targeting ANO1 could mitigate radiation-induced enteritis while enhancing tumor radiosensitivity.

METHODS

Quantitative PCR (qPCR) and Western blot (WB) were used to assess ANO1 expression and its changes after irradiation. Survival rates were recorded to evaluate the effects of ANO1 agonist and inhibitors. A cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor was administered to irradiated mice to investigate the role of chloride channel in radiation protection. qPCR and WB were executed to analyze the expression of relevant ion channels in intestinal epithelium. Functional validation was conducted using inhibitors in mice and 3D organoids. Fluorescent probe kits detected intracellular ion levels and membrane potential, and WB was performed to elucidate the underlying mechanisms. Finally, the radiosensitizing effect of CaCCinh-A01 was assessed in colorectal cancer (CRC) cells and validated in in vivo models.

RESULTS

Blocking the calcium-activated chloride channel (CaCC) protein ANO1, which is highly expressed in the colon, protects the intestine from radiation-induced damage. The ANO1 inhibitor CaCCinh-A01, suppresses CaCC currents, downregulates ANO1 protein expression, alleviates radiation-induced intestine injury, and enhances the radiosensitivity of CRC. Mechanistically, CaCCinh-A01 upregulates Na-K-Cl Cotransporter 1 (NKCC1) protein expression, leading to an increase in intracellular Cl- concentration and the inhibition of membrane depolarization in MODE-K cells. This subsequently inhibits p53-mediate DNA damage signaling, ultimately alleviating ionizing radiation-induced intestinal injury.

CONCLUSION

These findings suggest that targeting ANO1 not only alleviates radiation-induced intestinal injury in mice but also enhances CRC radiosensitivity. Thus, ANO1 represents a promising therapeutic target for mitigating the side effects of RT in CRC patients.