Synthetic Lethality And Mechanism Of Action Between ATR Inhibitor VE821 And Alantolactone In SW480 Colorectal Cancer Cells

Physiological amount of reactive oxygen species?ROS?are generated in cells during normal cellular metabolism,and increased levels of ROS may cause severe damage to cellular macromolecules such as nucleic acids,proteins and lipids,potentially leading to cellular malfunctions or even cell death.Fast proliferation and robust anabolic metabolism in rapidly growing cancer cells usually lead to higher than normal ROS levels.Although cancer cells can survive such toxic levels of ROS through rewiring of biochemical,metabolic and signaling pathways,as well as enhancement of antioxidative defense,both clinical practice and experimental studies have shown that most cancer cells are highly sensitive to additional oxidative stress.Alantolactone?ATL?is a class of compounds that can rapidly and dramatically increase ROS generation in cancer cells to cause extensive oxidative DNA damage and subsequently lead to apoptotic cancer cell death.Thus,through increasing ROS to cause selective cytotoxicity to cancer cells,ATL is considered to possess good anticancer activity.However,the drug dose that is needed to achieve optimal therapeutic effects may be too high such that some noncancer normal cells may also be affected.Therefore,identifying drugs that can synergize with ATL to produce synthetic lethality in cancer cells would be of high significance.ATR is one of the key protein kinases mediating the DNA damage response?DDR?.It is activated by single-stranded DNA?ssDNA?associated with replication stress or repair of double-strand DNA breaks.Through Chk1,activated ATR causes the cell cycle to slow down to resolve the tension between DNA replication and replicative stress while promoting repair,or induces apoptosis if the damage is too severe or irreparable,which removes problematic cells to prevent carcinogenesis.However,due to inactivating mutations in downstream apoptosis initiators such as p53 or RB proteins,or block of apoptotic pathways,ATR does not induce apoptosis in cancer cells but instead plays only important protective roles to maintain their proliferation and survival.Thus,targeting ATR is considered as a promising anticancer strategy and several small molecule ATR inhibitors have entered clinical trials.However,ATR inhibitor alone is ineffective in most types of cancer,and so all ATR inhibitors are used together with a classical chemotherapeutic drug such as a topoisomerase inhibitor.Considering the side-effects and drug resistance associated with most chemotherapy drugs,it is important to identify novel and more effective agents that can synergize with ATR inhibitors to achieve synthetic lethal anticancer effects.In this study,we tested the effects of combining ATL with the ATR inhibitor VE821 in the SW480 colorectal cancer cells.At first,the result of MTT assay showed that the IC₅₀0 of 48-hour treatment of SW480 cancer cells by ATL was 15.20?m,therefore,a sublethal 10?m dose of ATL was chosen for all subsequent experiments.MTT and flow cytometry analyses showed that treatment of SW480 cells with 10?m ATL did not cause any cytotoxicity but the proportion of cells in S and G2/M phases was increased,indicating cell cycle arrest in S and G2/M phases.Moreover,immunofluorescent staining showed that a dramatic increase in ROS levels was obvious 3 hours after 10?m ATL treatment,which was followed by significant increases in the number of?H2AX positive cells and phosphorylated Chk1 levels?Chk1-pT14?,suggesting existence of extensive DNA damage and ATR activation.Blocking ROS by NAC suppressed the S-and G2/M-phase arrest as well as the?H2AX signal,indicating that,although sublethal dose of ATL did not induce cancer cell death,the ROS-triggered DNA damage and replicative stress induced activation of ATR and S phase checkpoint.On the other side,because ATR inhibitors do not have significant cytotoxicity,25,12.5,6.25 and 3.125?m VE821 were chosen according to reports in the literature to study its effects in SW480 cells.The results showed that VE821 alone did not induce apoptosis or changes in ROS and?H2AX,but it did suppress Chk1-pT14 levels,indicating inhibition of ATR.Interestingly,when combined with 10?m ATL,all the VE821 doses above showed potent anticancer activity in SW480 cells,and the combination indexes?CI?derived from calculation using the CompuSyn software showed synergistic activity.In addition,VE821+ATL also significantly suppressed SW620 colorectal cancer,HT1080 fibrosarcoma,HepG2 hepatocarcinoma,SiHa cervical squamous cell carcinoma and A549 lung adenocarcinoma,supporting its broad-spectrum anticancer activity.Immunofluorescent staining and Western blot analyses showed that the number of?H2AX positive cells and the intensity of RPA-pS4/8 signal was much higher in the cells treated with VE81+ATL than those treated with each drug alone,suggesting that when ATR and S phase checkpoint were suppressed by VE821,the ATL-induced DNA damage could not be repaired normally and ran into conflict with ongoing DNA replication,resulting in increase in?H2AX and RPA-pT4/8.Comet assay showed that the number of DNA breaks were much higher in VE821+ATL-treated cells,supporting that without the protection of ATR,the conflict between DNA damage and replication induced replication fork collapse and DNA strand breaks.Flow cytometry analyses confirmed the disappearance of S phase arrest and aggravation of G2/M phase arrest and apoptosis,indicating that DNA breaks induced activation of the G2/M checkpoint and apoptosis most likely through the ATM-Chk2-p53 pathway.Finally,blocking ROS with NAC blocked the synergistic lethality of VE821+ATL,confirming that ATL-induced ROS were the basis of the synergy between VE821 and ATL.Taken together,these studies showed that although a sublethal dose of ATL did not induce cell death,it did trigger ROS increase and replication stress in cancer cells;when combined with VE821,ATR and S phase cell cycle checkpoint were suppressed,the ATL-related replication stress was converted into DNA strand breaks,which subsequently promoted G2/M arrest and apoptosis through ATM-Chk2-p53 pathway.The results above showed that there is a good synergistic lethality between VE821and ATL,thus providing valuable information for further development of ATL as well as screening of anticancer drugs sharing synergy with ATR inhibitors.