Synergistic Lethality Between Alkannin-induced Oxidative DNA Damage And PARP Inhibition In Colorectal Cancer Cells

With the growing recognition that most malignantly transformed cells exhibit rewired metabolic pathways,targeting cancer-specific biochemical and metabolic vulnerabilities to selectively attack tumor cells is considered as a promising anti-cancer strategy.One of the transformation-associated metabolic feature shared by most cancer cells is the increase in reactive oxygen species(ROS)levels.A large number of basic and clinical studies have shown that cancer cells are more sensitive to further oxidative stress than normal cells due to inherent high oxidative pressure.Therefore,drugs that increase ROS or inhibit cellular antioxidant capacity can selectively cause oxidative DNA damage,such as DNA base damage and single-strand DNA break(SSB),in cancer cells.Rapid generation of large number of oxidative DNA damage will collide with DNA replication,repair or transcription,resulting in increased DNA replication stress and formation of fatal double-strand DNA breaks(DSBs),which will lead to cancer cell death.However,activation of DNA damage response(DDR)signaling can enhance the survival of cancer cells by regulating cell cycle checkpoints and promoting DNA repair and genome maintenance mechanisms.Therefore,combining exogenous oxidative attack to selectively induce oxidative DNA damage in cancer cells with inhibition of DNA damage response or DNA repair,may produce cancer-specific synergistic lethal effect.PARP1 and PARP2(PARP1/2)initiate repair of damaged DNA base and SSB and play a regulatory role in DSB repair.In addition,PARP1 also participates in the stabilization and restart of stalled DNA replication forks.Consequently,PARP1/2play important roles in the repair of oxidative DNA damage and DDR signaling pathways.It has long been known that the absence of PARP1/2 activity is not lethal in adult cells with normal homology-directed repair(HDR)competency but can sensitize cells to ionizing radiation and therapeutic drugs that cause DNA damage.In the past decades,many clinical trials have tested the combination of PARP inhibitors with radiotherapy or chemotherapy,but so far,no successful protocol has been developed for clinical application.The main obstacle is the serious side-effects caused to normal tissues.As mentioned above,exogenous oxidative stress can selectively induce oxidative DNA damage in cancer cells,which inspires us to explore whether the combination of oxidative-promoting or antioxidant capacity-inhibiting drugs with PARP inhibitors can form synergistic lethal effect specifically in cancer cells.Radix Arnebiae is a perennial plant of Boraginaceae.Its crude preparations have a variety of pharmacological activities and the dried powder of its roots has been commonly used to treat burn injuries in traditional Chinese medicine.Radix Arnebiae is native to Inner Mongolia and Northeast China and its main active pharmaceutical components have been identified to be alkannin,shikonin and their derivatives.A large number of studies have shown that both alkannin and shikonin have robust and broad-spectrum antitumor activities.Alkannin and shikonin can generate ROS through redox cycling and can also directly bind to and inhibit thioredoxin Reductase-1(Trx R1)to induce ROS accumulation,resulting in increase in intracellular oxidative pressure.Inhibition of ROS by N-acetylcysteine(NAC)completely blocks the antitumor activities of alkannin and shikonin,indicating that increased oxidative stress is the main source of their cancer cell toxicity.However,given the non-specificity of ROS toxicity,it is difficult to achieve therapeutic effects without significant side-effects using alkannin or shikonin as monotherapy.In this study,we investigated the effects and underlying mechanisms of combining alkannin and PARP inhibitors in colorectal cancer cells.First,we found that non-cytotoxic doses of alkannin induced ROS elevation and oxidative DNA damage,including 8-oxo G accumulation and DNA-strand breaks,in colorectal cancer but not in normal colon epithelial cells.The increases in ROS levels and DNA damage were inhibited by the ROS inhibitor NAC,indicating that low-dose,non-cytotoxic alkannin caused oxidative DNA damage specifically in colorectal cancer cells.Second,MTT and colony formation assay showed that the combination of non-toxic doses of alkannin and the PARP inhibitor olaparib produced a prominent synergistic lethal effect in colorectal cancer but not in normal colon epithelial cells.Rad51 immunofluorescent staining showed that drug treatment had no effect on the homologous recombination function of the cancer cells,indicating that the synergistic lethal effect of the drug combination was not related to the state of homologous recombination-directed DNA repair.The ROS inhibitor NAC significantly reduced the oxidative DNA damage induced by alkannin and the synergistic lethal effect of the alkannin and olaparib combination in colorectal cancer cells,suggesting that increased oxidative DNA damage were the basis of the synergistic effect of alkannin and olaparib.OGG1 inhibitor also significantly reduced the synergistic cancer cell toxicity of the alkannin and olaparib combination,indicating that the DNA single-strand breaks produced in the process of OGG1-mediated base-excision repair was the source of drug synergistic toxicity.In addition,the PARP inhibitor veliparib that has no PARP-trapping ability did not synergize with alkannin,which indicated that the synergistic effect of alkannin and olaparib was due to the trapped PARP-DNA complex caused by the PARP-trapping inhibitor olaparib.Further experiments showed that the combination of non-cytotoxic alkannin and olaparib caused intense DNA replication stress and extensive DNA double-strand breaks in colorectal cancer cells,leading to activation of the G₂/M cell cycle checkpoint and G₂arrest,which was followed by massive apoptosis.In nude mice,the combination of alkannin and olaparib significantly induced regress of tumor xenografts,while each drug alone had no effect on tumor growth.In summary,our studies showed that non-toxic doses of alkannin produced oxidative DNA damage specifically in colorectal cancer cells,which sensitized the cancer cells to the PARP inhibitor olaparib,resulting in synergistic lethality between alkannin and olaparib specifically in colorectal cancer cells.The results showed that the main cause of alkannin and olaparib synergism was the oxidative DNA damage and PARP trapping induced by alkannin and olaparib,respectively.These studies support further exploration of the synergistic cytotoxicity between PARP inhibitors and alkannin to exploit a cancer vulnerability common to most oncogenically transformed cells.