Secreted APE1/Ref-1 inhibits doxorubicin-induced cardiotoxicity via suppressing ROS generation and p53 pathway
Authors: An S Y , Jin S A , Seo H J , Lee Y R , Choi E J , Kim S M , Jeon B H , Jeong J O 
Published: January 2023

Abstract

Funding Acknowledgements
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This research is supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare and Basic Science Research Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Republic of Korea.

Background
Doxorubicin is an anthracycline anticancer drug with a potent therapeutic effect against various malignancies. However, its use is limited due to cardiotoxicity, which can lead to heart failure. Oxidative stress is a key player in the development of anthracycline cardiotoxicity. Apurinic/apyrimidinic endonuclease 1/redox factor-1(APE1/Ref-1) is a multifunctional protein that regulates oxidative stress and anti-inflammatory function. Secreted APE1/Ref-1 has shown anti-oxidant and anti-inflammatory properties, but its therapeutic potential in various diseases is largely unknown.

Purpose
This study aims to test the protective effect of secreted APE1/Ref-1 in the doxorubicin-induced cardiomyopathy model in vivo and in vitro.

Methods
H9C2 cells were treated with doxorubicin(2uM) to induce cardiotoxicity. Adenovirus preprotrypsin leading sequence APE1/Ref-1(AdPPT-LS-APE1/Ref-1), a secretory APE1/Ref-1 adenovirus encoding human APE1/Ref-1 gene, was amplified in HEK293T cells. H9C2 cells were then treated with the supernatant of the amplified AdPPT-LS-APE1/Ref-1. In vivo model of doxorubicin-induced cardiotoxicity was established by injecting doxorubicin(15mg/kg) into C57BL/6 mice via the peritoneum. The conditioned media of HEK293T cells with AdPPT-LS-APE1/Ref-1 transfection was injected i.p. after 24h. The severity of cardiotoxicity was evaluated by echocardiography and plasma NT-proBNP. Cell survival, morphology, and ROS level were examined. The expression of cellular APE1/Ref-1, p53, Bax/Bcl-2, and Caspase-3 were analyzed by Western blot and ELISA. Redox activity was measured using the DTNB reduction assay.

Results
Doxorubicin caused apoptosis and oxidative stress in H9C2 cells and cardiomyopathy in mice. NT-proBNP level increased both in vitro(p&lt;0.001) and in vivo(p&lt;0.05) upon administering doxorubicin. Interestingly, doxorubicin increased p53 level in a dose-dependent manner in 48 hr, but the cellular APE1/Ref-1 level did not change. Secreted APE1/Ref-1 in the conditional media showed redox activity and decreased NT-proBNP in doxorubicin-treated H9C2 cells and mice(p&lt;0.001 and p&lt;0.05, respectively). Doxorubicin aggravated cell death(p&lt;0.001) and oxidative tissue damage(p&lt;0.001) in H9C2 cells, while pre-treatment of secreted APE1/Ref-1 inhibited cell death in H9C2 cells(p&lt;0.05). Secreted APE1/Ref-1 significantly reduced the expression of p53(p&lt;0.001) and its effectors, Bax/Bcl-2(p&lt;0.05) and Caspase-3(p&lt;0.05) in doxorubicin-treated H9C2 cells. It also reduced p53(p&lt;0.05) along with Bax/Bcl-2(p&lt;0.05) and Caspase-3(p&lt;0.05) in doxorubicin-treated mice(n=4 in each group). Finally, secreted APE1/Ref-1 significantly decreased intracellular ROS production(p&lt;0.001).

Conclusion
Secreted APE1/Ref-1 is protective against doxorubicin-induced cardiomyocyte apoptosis and oxidative stress by suppressing ROS generation and p53 pathway. APE1/Ref-1 showed therapeutic potential for the prevention and treatment of doxorubicin-induced cardiotoxicity.