Combination treatment of cordycepin and radiation induces MA-10 mouse Leydig tumor cell death via ROS accumulation and DNA damage.

Leydig cell tumor is the most frequent non-germ cell tumors of testis. The biggest challenge of using radiotherapy to treat testicular cancer is in effectively killing cancer cells and maintaining reproductive function after treatment. Our recently published article showed that cordycepin could enhance radiosensitivity to induce mouse Leydig tumor cell apoptosis by inducing cell cycle arrest, caspase pathway and endoplasmic reticulum (ER) stress. In the present study, the potency and mechanism of a previous combination treatment protocol on reactive oxygen species (ROS) induction and DNA damage were further investigated. Our results reveal that 25 µM cordycepin plus 4 Gy radiation leads to ROS accumulation accompanied by a decrease in heme oxygenase (HO)-1 protein expression in MA-10 mouse Leydig tumor cells. Subsequently, pronounced DNA damage with phosphorylated H2A histone family member X (γ-H2AX) increase and activation of DNA damage-related signaling pathways including double and single stranded break-induced ataxia telangiectasia mutated (ATM)/checkpoint kinase (Chk)2 and ataxia telangiectasia mutated and Rad3 related (ATR)/Chk1 signaling axes were identified. p53-dependent pathway was then initiated ultimately leading to cell death. Preincubated with free radical scavenger, N-acetylcysteine (NAC), down-regulated γ-H2AX expression in treated cells and partially reduced cell death, indicating that ROS overproduction is involved in combination treatment-induced DNA damage. Furthermore, the combination treatment effectively inhibited tumor growth as reflected in the reduction of tumor volume, size and weight, and high expression level of γ-H2AX in tumor tissue in vivo, suggesting that the combination treatment inhibited tumor growth via causing DNA damage in MA-10 cells. In summary, these results expound that the combination treatment of cordycepin and radiation induces MA-10 mouse Leydig tumor cell death through ROS accumulation and DNA damage. This finding can serve as a reference guideline for future clinical therapy of testicular cancer and provide potential targets for anti-cancer drug design.