The Study On The Synergistic Anti-cancer Effects Of ROS Elevating Drugs And PARP Inhibitors

Studies have shown that moderate amounts of reactive oxygen species?ROS?are essential for normal activities of diverse types of cell;however,the ROS content in cancer cells is far higher than that necessary for normal cellular activity.Excess ROS can damage macro-molecules if not antagonized in time.Oxidative DNA damage caused by ROS can result in DNA mutations to suppress the survival or growth of cancer cells.However,various types of cancer cells still survive and grow rigorously even under intense oxidative pressure,suggesting that cancer cells have evolved special mechanisms to escape the lethal impact of oxidative damage.Nevertheless,many studies do show that cancer cells are highly vulnerable to further ROS insult.Alantolactone?Ala?is a type of sesquiterpene lactone,which is mainly extracted from the root of the plant of compositae.As a traditional Chinese medicine,it has anti-inflammatory and antibacterial,as well as other biological activities.Some studies have shown that it has anti-tumor effect,mainly by promoting production of excessive ROS in cancer cells,which generates oxidative DNA damage and initiates apoptotic cancer cell death.But as a traditional Chinese medicine,by elevating ROS levels to kill cancer cells is associated with many side effects.Previous studies have shown that the IC₅₀ of Ala on cancer cells is as high as 20?M.Thus,there is a need to find feasible synergistic interactions with other drugs to achieve combination therapeutic effects in the safe concentration range of Ala.Poly-ADP-ribose polymerase?PARP?is a family of key enzymes involved in various types of DNA damage repair.PARP is not essential to normal cells but inhibition of PARP is lethal to BRCA mutated cancer cells and may also make other types of cancer cells more sensitive to DNA damage.Olaparib?Ola?is one of thePARP inhibitors approved by the US Food and Drug Administration?FDA?for the treatment of BRCA mutated breast and ovary cancers.Although highly effective in indicated patients,its therapeutic effect in other types of cancer is limited,and drug resistance is becoming an increasingly problematic issue.Therefore,Ola is usually used in combination with conventional chemotherapy drugs in the clinic.In this study,we explored synergistic interactions between the ROS-elevating Ala and the PARP inhibitor Ola,hoping to find combined anticancer effects.First,we used the MTT assay to detect the effects of Ala and Ola mono-and combination-therapy on the cellular activity of four different cancer and one noncancer cell lines.The results showed that both the mono-and combination treatments can influence all four cancer cell lines to different degrees,however,theimpact on the noncancer cells was obviously weaker,and most significant synergistic cytotoxic effects were found in the prostate cancer cell line PC-3.Further assays,including calculation of Combination Index?CI?and colony formation,verified the results of the MTT experiment and confirmed that combined use of Ala and Ola showed significantly higher cytotoxicity than monotherapy on PC-3 cancer cells,thus the follow experiments were conducted on the PC-3 cells.Second,we examined the dynamic changes of cell numbers in each phase of the cell cycle by flow cytometry.Treatment by Ala alone did not result in obvious changes,except slight increase in the number of cells in G₂/M phase.On the contrary,treatment by Ola alone caused significant increase in the number of cells in G₂/M phase,indicating that Ola arrested PC-3 cells in G₂/M phase.Surprisingly,combining Ala and Ola significantly increased the number of cells in S phase,suggesting that the combination of Ala and Ola caused cell cycle arrest in S phase.At the same time,AnnexinV-FITC and PI double staining and JC-1 mitochondrial membrane potential detection experiments found that the combination of Ala and Ola increased the number of apoptotic PC-3 cells.Furthermore,53BP1 immunofluorescence staining found that the combination of the two drugs significantly increased the number of positive cells with 53BP1,and alkaline comet assay showed that the combination of the two drugs significantly increased the number of cells with DNA breaks and thelevel of DNA breaks?tail moment?in a single cell.Finally,through detection of DCFH-DA fluorescence by flow cytometry,we found that Ala alone or in combination with Ola significantly increased the level of ROS in PC-3 cells,which was blocked by the ROS scavenging agent-N-acetylene cysteine?NAC?.NAC treatment also significantly reduced the DNA damage,apoptosis,and cell cycle changes.To sum up,Ala increased ROS levels in PC-3 cells,and Ala in combination with Ola caused significantly higher number of DNA strand breaks than each drug alone.Consequently,the combined drugs blocked cells in S phase which was earlier than the G₂/M phase blockage caused by Ola alone.The apoptosis rate of PC-3 cells was increased and the cell proliferation was inhibited by the combined drugs,all of which were blocked by NAC.These studies validated our hypothesis that further increase in ROS levels caused by Ala resulted in more oxidative DNA damage,and in the presence of the DNA repair inhibitor Ola,the DNA damage was converted into double strand DNA breaks to induce cell cycle arrest and apoptosis and achieve the synergistic therapeutic anticancer effects at lower drug concentration.