Secreted APE1/Ref-1 inhibits doxorubicin-induced cardiotoxicity via suppressing ROS generation and p53 pathway

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.

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.

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

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.

Doxorubicin caused apoptosis and oxidative stress in H9C2 cells and cardiomyopathy in mice. NT-proBNP level increased both in vitro(p<0.001) and in vivo(p<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<0.001 and p<0.05, respectively). Doxorubicin aggravated cell death(p<0.001) and oxidative tissue damage(p<0.001) in H9C2 cells, while pre-treatment of secreted APE1/Ref-1 inhibited cell death in H9C2 cells(p<0.05). Secreted APE1/Ref-1 significantly reduced the expression of p53(p<0.001) and its effectors, Bax/Bcl-2(p<0.05) and Caspase-3(p<0.05) in doxorubicin-treated H9C2 cells. It also reduced p53(p<0.05) along with Bax/Bcl-2(p<0.05) and Caspase-3(p<0.05) in doxorubicin-treated mice(n=4 in each group). Finally, secreted APE1/Ref-1 significantly decreased intracellular ROS production(p<0.001).

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.