Synergistic Lethality Between Cinobufagin-Induced Oxidative DNA Damage And ATM Inhibition In Cancer Cells

Cancer is a disease caused by genetic changes in somatic cells.The current treatment methods are mainly chemotherapy,radiotherapy and surgery.However,despite the significant benefits of chemotherapy and radiotherapy in cancer treatment,the side effects they have on patients are extremely severe and most patients can't be cured.In addition to radiotherapy,chemotherapy,and surgery,targeted therapy for specific genes or signaling pathways also has a good therapeutic effect on a small number of specific patients.However,due to the diversity and plasticity of cancer cell genotypes,and the heterogeneity of tumors,it is difficult for the treatment to have a fundamental impact on the improvement of the overall treatment effect of cancer.Therefore,finding a broad-spectrum,high-efficiency,low-side effect anti-cancer strategy is an important goal of cancer research.Modern biomedical studies have shown that a common metabolic feature of malignantly transformed cells is the increase in reactive oxygen species(ROS)levels.On the one hand,ROS can cause abnormal cell proliferation by activating intracellular signaling pathways and causing genetic mutations;on the other hand,ROS at toxic levels can damage cellular components and endanger cell survival.Studies have found that compared to normal cells,cancer cells have increased oxidative stress and limited reserves of antioxidant capacity,so cancer cells are more sensitive to additional oxidative insult than normal cells.Studies have shown that increasing ROS or inhibiting antioxidant capacity can selectively cause the oxidative pressure in cancer cells to reach a toxic level and cause cell death,while normal cells have low basal ROS outputs and sufficient antioxidant capacity reserves to survive.Therefore,the difference in oxidative pressure between cancer and normal cells is expected to be useful in designing new anti-cancer strategies that selectively target cancer cells.Some chemicals secreted by the skin and special exocrine glands of amphibians can resist predators and microbial infections.Therefore,various forms of toad extracts have been made into various forms of traditional Chinese medicine for thousands of years.In China and many Asian countries,a dried toad skin extract,Chansu,is widely used to treat pain,infection,various inflammations,and cancer.Both experimental and clinical studies have shown that Huachansu,a soluble form of Chansu,has obvious anticancer effects.Recent studies have found that bufadienolide glycosides are the main pharmacological compounds for the anti-tumor activity of Huachansu,while Cinobufagin(CBG)and Bufalin are the major pharmaceutical components of Huachansu.Both of the two main bufadienolides have shown significant anti-tumor effects in vitro and in vivo,but the exact mechanism of action is still unclear.Some studies have shown that CBG can increase ROS in cancer cells,but the effect of the increased ROS on cancer cells is also unclear.In this study,we first analyzed the anti-cancer activity of CBG and the role of ROS caused by CBG.The IC₅₀ values of 24-h CBG treatment in SW480 and SW1116 colon cancer cell lines were 103.60 n M and 267.50 n M,respectively,indicating that CBG has strong cytotoxicity to these two colon cancer cells.Treatment with a concentration close to IC₅₀(100 n M)for 24 hours also significantly inhibited the survival of more than 10other cancer cell lines,but the survival of three non-cancer cell lines,including human colonic epithelial cell line NCM460,lung epithelial cell line BEAS-2B and liver cell line L-O2,was not affected.These results indicate that CBG not only has significant and broad-spectrum anti-cancer activity,but also its cytotoxicity has certain selectivity to cancerous cells.The experimental results also showed that treatment with a sublethal dose of 100 n M CBG specifically caused a rapid increase in ROS levels in cancer cells,while the ROS levels of the three non-cancer cells did not change significantly.Next,we examined the effects of ROS on cancer cells.Experimental data showed that treatment with 100 n M CBG for 24 hours significantly increased the content of oxidized guanine(8-oxo G)in cancer cells,resulting in DNA strand breaks and strong replication stress.The ROS inhibitor N-acetyl cysteine(NAC)completely blocked the above changes caused by CBG in cancer cells,indicating that the increase in ROS caused by CBG resulted in serious oxidative DNA damage in cancer cells.Consistent with this,100 n M CBG treatment resulted in up-regulation of p-Chk1,p-Chk2,and p-CDC25C in cancer cells,indicating DNA damage response(DDR)and activation of G₂/M cell cycle checkpoint.Flow cytometry analysis revealed significant G₂ phase cell cycle arrest and time-dependent apoptosis.NAC treatment blocked the DDR and G₂cell cycle arrest and apoptosis caused by CBG,which proves that the toxicity in cancer cells comes from the oxidative DNA damage caused by CBG.Experiments on transplanted tumors in nude mice proved that CBG has a good anti-cancer effect in vivo.A large number of studies have reported that the combination of DNA damaging drugs and DDR inhibitors can significantly increase the anti-cancer effects of both.To this end,we further explored the combined effects of low-dose CBG and DDR pathway inhibitors.The results of the study showed that the combination of low-dose,non-cytotoxic CBG and similarly non-cytotoxic ATM inhibitor KU-55933 produced significant synergistic cancer cell toxicity.Low-dose CBG caused the cancer cell-specific ROS to increase,causing a certain degree of DNA damage and activating the DDR pathway and G₂/M cell cycle checkpoint.The same non-cytotoxic ATM inhibitor KU-55933 relieved CBG-induced cell cycle arrest and blocked DNA repair,thereby increasing CBG-induced DNA damage,and causing cancer cells with severe DNA damage to enter mitosis,leading to mitotic disaster and cell death.Inhibition of OGG1-mediated base excision repair-related DNA breaks significantly reduced the combined effect of CBG and KU-55933,which proves that the synergistic toxicity of cancer cells is related to oxidative DNA damage caused by CBG;in addition,CBG and Wee1inhibitors also showned great synergistic effect,proving that the combined interactions is related to DDR pathway.Nude mouse experiments fully showed that CBG and ATM inhibitor KU-55933 have a significant synergistic anti-tumor effect in vivo.In summary,the studies in this paper prove that low-dose CBG can specifically cause oxidative DNA damage and DNA damage response in cancer cells.It also proves that the combined application of CBG and KU-55933 has a good synergistic anti-cancer effect.The research results of this paper provide experimental and theoretical information for the clinical application of CBG single drug and the combination of CBG and KU-55933,and provide new ideas for anti-tumor therapy.