Synergistic Cytotoxicity Between Alkannin And PARP Inhibition In Human Osteosarcoma Cells

Osteosarcoma is a common primary malignancy,mainly occurring in adolescents,and has a high mortality rate.Chromosomes of osteosarcoma cel s are prone to mutatio n,and the location and form of mutation are uncertain,which brings great difficulty to the treatment.At present,surgical resection is the main treatment for osteosarcoma,and chemotherapy is not a routine treatment for osteosarcoma.Therefore,it is urgent to develop new therapeutic strategies.Alkannin is the main pharmacological component of the traditional Chinese herbal medicine shikonin,and its extensive activities have recently been demonstrated,for example,to have antimicrobial and anti-inflammatory effects,especially inhibiting the proliferation of human tumor cell lines.Experimental studies have found that alkannin can form oxidation-redox cycling to lead to the production of ROS,and can directly bind and inhibit the thioredoxin reductase-1(Trx R1)to cause the accumulation of ROS,resulting in DNA oxidative damage in cancer cell,such as DNA base oxidation and DNA single strand breakage(SSB),etc,to cause apoptosis.However,the DNA damage response signaling pathway promotes the survival of cancer cel s by regulating cell cycle checkpoints and promoting DNA repair.Therefore,the combination of alkannin with inhibition of DNA repair by PARP inhibitors,is expected to produce specific synergistic lethal effects in cancer cel s.PARP1 and PARP2(PARP1/2)are key proteins in the repair of DNA base damage and single strand breaks.It has long been known that the loss of PARP1/2 activity makes cel s more sensitive to ionizing radiation and DNA damaging agents.As a result,many clinical studies have tested PARP inhibitors combined with radiotherapy or chemotherapy,but it has not being successful due to their severe toxic and side effects in normal tissues.Studies have shown that one of the common features of cancer cells is the rise in reactive oxygen species(ROS)that accompanies maligna nt transformation.High intrinsic oxidative stress makes cancer cel s more sensitive to exogenous oxidative shock than normal cel s.Therefore,the purpose of this study was to investigate whether alkannin combined with PARP inhibitor can induce specific synergistic lethal effect in human osteosarcoma cancer cel s and the mechanism of action.In this study,two osteosarcoma cell lines,MG-63 and U2 OS,were used as the research objects to explore the synergistic anti-cancer effect of alkannin combined with PARP inhibitor.First,we detected the IC50 of alkannin in MG-63 and U2 OS cells by MTT,and found that alkannin had a good inhibitory effect on osteosarcoma cel s.Next,colony formation assay showed that the combination of PARP inhibitor and alkannin had stronger cytotoxicity on U2 OS cells and MG-63 cells than alkannin alone,while the cytotoxicity to normal intestinal epithelial NCM460 cells was very weak.Calcu Syn software was used to calculate the Combination Index(CI)of PARP inhibitors and alkannin,and the data showed that the CI values were far less than 1,indicating that PARP inhibitors and alkannin had a strong synergistic effect.Next,the specific mechanism of the synergistic anti-cancer activity was explored.Flow cytometry was used to analyze different stages of the cell cycle.The results showed that treatment by both PARP inhibitor and alkannin for 24 h caused G2 phase arrest.The cell cycle arrest was more obvious after treatment by the combination of the two drugs,while the cell cycle of the normal intestinal epithelial NCM460 cell line was not affected.Staining with DCFH-DA showed that alkannin combined with PARP inhibitor caused increase in oxidative stress,which was blocked by the antioxida nt N-acetyl cysteine(NAC).53BP1 staining showed that the number of double-stranded DNA breaks(DSBs)in the nucleus was significantly increased with alkannin combined with PARP inhibitor.The alkaline comet experiment demonstrated that the combinat io n of akannin and PARP inhibitors triggerred stronger DNA breakage.In addition,cell apoptosis was detected by different methods.Westerning Blot analysis showed that the expression of pro-apoptotic protein Bax was significantly increased,while the expression of anti-apoptotic protein Bcl-2 was decreased,and the endogenous apoptotic pathway was induced.Results of annexin V-FITC and PI double-staining were consistent with the above results,again indicating a significant increase in apoptosis after treatment by the combination.Finally,addition of NAC reversed the strong DNA damage and apoptosis induced by the combination treatment of alkannin and PARP inhibitor.To sum up,DNA oxidative damage caused by alkannin in the MG-63 or U2 OS osteosarcoma cel s significantly increased the efficacy of PARP inhibitors,the synergistic activity induced more DNA damage in the nucleus,caused serious G2 cell cycle arrest,and intracellular increase in ROS levels to lead to apoptosis.These studies provide new ideas and valuable information for the design of novel therapeutic strategies for osteosarcoma based on the high intrinsic oxidative pressure of cancer cells.