Centipeda Minima Sensitizes Lung Cancer To Chemotherapy Via Inhibiting DNA Damage Repair Pathways

ObjectiveChemoresistance remains a critical challenge in lung cancer chemotherapy and a major cause of chemotherapy failure.To date,there is no effective method to control chemoresistance in clinical practice.It is urgent to find novel strategies to overcome the chemotherapeutic resistance of lung cancer.Fanconi anemia(FA)pathway plays a key role in antagonizing the cytotoxic effects of chemotherapeutics by repairing DNA breaks.We recently demonstrated that the traditional Chinese medicinal herb,Centipeda minima(C.minima),possessed anti-inflammatory and antioxidant properties.The crude extract and several components of C.minima have been identified to exert promising anticancer activities in various human malignancies.However,the chemosensitizing effects of C.minima have not been reported.We aimed to investigate the anticancer effects of the ethanol extract of C.minima(ECM)and Arnicolide C(ArnC)in combination with DNA cross-linking agents respectively on non-small cell lung cancer(NSCLC),and elucidate the underlying mechanisms.MethodsThe study is divided into three parts:In part one,ECM was extracted by reflux extraction and analyzed by high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS).For in vitro experiments,A549 and H1299 NSCLC cells were used to test the synergistic cytotoxicity of ECM and DNA-crosslinking agents such as cisplatin(CDDP)or mitomycin C(MMC).MTT assay was used to detect cell viability,Flow cytometry to detect cell apoptosis,and Western blotting to determine the expression of apoptosis-associated proteins,respectively.The combined effects of the two drugs were evaluated using online synergy software.Online human gene expression dataset analysis was used to assess the correlation between FANCD2 expression levels and lung cancer patients’prognosis.Western blotting and immunofluorescence assays were used to detect the effects of ECM on DNA damage repair pathways,comet assay was applied to evaluate the level of DNA damage,and flow cytometry was used to detect the cell cycle distribution.In part two,for in vivo experiments,4-week-old male BALB/c nude mice were selected for subcutaneous inoculation with A549 cells to establish an NSCLC xenograft model.After tumors were palpable,tumor sizes were measured every second days.After 14 days subcutaneous injection,they were randomly divided into 6 groups(six mice per group):(1)vehicle control(Control,gavage with saline containing 10%ethanol);(2)ECM low-dose group(ECM-L,gavage,100 mg/kg,daily);(3)ECM high-dose group(ECM-H,gavage,200 mg/kg,daily);(4)CDDP group(CDDP,i.p.,2 mg/kg,once every 3 days);(5)CDDP+ECM low-dose group(CDDP+ECM-L);(6)CDDP+ECM high-dose group(CDDP+ECM-H).The body weight of each animal was recorded every 3 days and the treatment lasted for a total of 27 days.To evaluate the combined anticancer effects of ECM and CDDP,Western blotting and immunohistochemistry were used to detect the protein expression related to DNA damage,proliferation and apoptosis in the tumor tissues.The levels of creatinine(Cr),urea nitrogen(BUN),alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in the serum of mice were measured to observe the effects of ECM on the liver and kidney functions.In the last part,to determine the effects of ArnC and DNA cross-linking agents on NSCLC cells and the mechanism of synergistic cytotoxicity,cell viability was measured by MTT assay.The combined effects of the two drugs were evaluated by synergistic software.Western blotting was used to determine the expression of apoptosis-related proteins as well as FA pathway proteins.Further,A549-CDDP resistant cells(A549-Re)and H1299-CDDP resistant cells(H1299-Re)were established to examine the effects of ArnC on reversing chemotherapeutic resistance.ResultsHPLC-MS/MS analysis confirmed that ECM contains chlorogenic acid,caffeic acid,rutin,isochlorogenic acid B,isochlorogenic acid A,isochlorogenic acid C and brevilin A,which were consistent with the literature reports.For in vivo experiments,ECM treatment alone showed moderate cytotoxicity against NSCLC cells,while combined treatment showed that ECM significantly enhanced the cytotoxicity of CDDP and that the combination of ECM and CDDP had a synergistic effect.The combination of ECM and MMC also produced a synergistic cytotoxic effect,suggesting that ECM can enhance the cytotoxicity of DNA crosslinkers.Flow cytometry showed that CDDP treatment alone induced low levels of apoptosis in H1299 cells;however,ECM significantly increased the level of CDDP-induced apoptosis in a concentration-dependent manner.The combination of ECM and CDDP significantly induced the cleavage of caspase-3 and its substrate PARP,compared to CDDP treatment alone.Similar results were observed in NSCLC cells treated with ECM and MMC,suggesting that ECM can promote DNA crosslinking agent-induced apoptosis of NSCLC cells.Mechanistically,ECM significantly reduced both total protein levels and monoubiquitination levels of FANCD2 in NSCLC cells.Database analysis showed that FANCD2 geneexpression levels were significantly higher in NSCLC tissues than that in normal tissues,and the high expression level of FANCD2 correlated with the poor prognosis of NSCLC patients.In line with this,exposure to low concentration of CDDP for 2 weeks led to increased expression level of FANCD2 protein in A549 cells.DNA cross-linking agents such as CDDP,MMC and HU significantly increased the monoubiquitinated level of FANCD2 in NSCLC cells,whereas ECM significantly attenuated this effect of DNA cross-linking agents.Furthermore,ECM dose-dependently inhibited CDDP-induced FANCD2 nuclear foci formation and increased the expression of y H2AX.In H1299 cells,the combined treatment with ECM and CDDP significantly increased tail moment levels,compared to ECM treatment alone.These results suggest that ECM can enhance DNA crosslinking agent induced DNA damage by inhibiting monubiquitination of FANCD2.Further research revealed that CDDP alone significantly induced S-phase arrest,whereas ECM significantly reduced CDDP-induced S-phase delay and induced G2/M-phase accumulation in H1299 cells,while ECM treatment alone also induced G2/M-phase arrest.The combined treatment significantly inhibited CDDP-induced Chkl phosphorylation.For In vivo experiment,ECM administration alone appropriately reduced tumor volume and weight.Tumor volume in the CDDP+ECM-H group was significantly reduced since day 21 of administration,compared to the CDDP group;on day 27 of administration,the tumor volume in the CDDP+ECM-L group was significantly smaller than that in the CDDP group.Tumor weight was significantly reduced in the combination groups,compared to the CDDP group.Compared with the control group,the expression of Ki67 was slightly lower and cleaved-caspase 3 was slightly higher in the tumor tissues of the CDDP group;whereas the expression of Ki67 was significantly lower while cleaved-caspase 3 was significantly higher in the tumor tissues of the combination groups compared with the CDDP group.These results demonstrated ECM exerted chemosensitizing effects in vivo,and the combination of ECM and CDDP significantly inhibited the proliferation of NSCLC cells,promoted cell apoptosis and inhibited tumor growth.Mechanistically,the expression level of FANCD2 in tumor tissues in the CDDP group was significantly higher than that in the control group,however,ECM significantly reversed this effect,and the expression level of FANCD2 in the combination groups was significantly lower than that in the CDDP group.The level of γ H2AX in tumor tissue of the combination group was significantly higher than that of the CDDP group.These results suggests that the chemosensitizing effects of ECM relied on DNA damage repair pathways.In terms of drug safety,the administration of ECM alone did not cause either weight loss or significant hepatotoxicity and nephrotoxicity in mice.The mice in the CDDP and combination groups showed moderate weight loss,but the difference between the combined group and the CDDP group were not statistically significant.ALT,AST,Cr and BUN levels in serum of the mice showed no statistically significant differences between the groups.These results show the safety for the combination application of ECM and CDDP.In the last part,ArnC treatment alone did not have significant cytotoxic effects on NSCLC cells;however,ArnC significantly enhanced the cytotoxic effects of CDDP and they obtained a synergistic effect on NSCLC cells.A similar phenomenon was observed for the combination of ArnC and MMC.These results indicate that ArnC can sensitise NSCLC cells to DNA crosslinking agents.In terms of cell apoptosis,CDDP treatment alone slightly increased PARP cleavage levels;however,the combination of ArnC and CDDP significantly increased PARP cleavage levels,indicating that ArnC significantly enhanced CDDP-induced apoptosis.Mechanistically,CDDP dose-dependently increased the monoubiquitination of FANCD2,however,ArnC significantly reversed the effect,suggesting that ArnC could exert anti-cancer activity against NSCLC by targeting the FA pathway.Further studies revealed that the resistance of H1299-Re and A549-Re cells was significantly enhanced compared to sensitive strains and that ArnC reversed the resistance of NSCLC CDDP-resistant strain cells.ConclusionIn this study,we investigated the chemosensitizing effects of ECM and its active component ArnC,and elucidated the underlying mechanisms.We found that ECM and ArnC as novel FA pathway inhibitors,can inhibit DNA crosslinking agent induced FA pathway activation.ECM and ArnC in combination with CDDP respectively exerted synergistic anti-cancer effects,providing a new strategy for the clinical treatment of NSCLC.