| Objective(s):To study the resistance characteristics of drug-resistant lung cancer-Xuanwei lung cancer cells,to explore the synergistic inhibitory effect of Brusatol and NCT503 on the proliferation of Xuanwei lung cancer cells and its specific mechanism,and provide a new basis for the clinical treatment of Xuanwei lung cancer.Methods:1.Detect drug resistance of Xuanwei lung cancer:in four lung adenocarcinoma cell lines PC-9,A549,XLA-07 and XL-jt.Four lung adenocarcinoma cell lines were treated with cisplatin for 24h and three target drugs AG1478(EGFR inhibitor),LY294002(PI3K inhibitor),U0126(MEK1/2 inhibitor)for 72h,then detected absorbance andcalculated cell survival rate and IC50.2.Inhibition effect of Brusatol on lung cancer cells:CCK8 method was used to detect the absorbance of four lung adenocarcinoma cell lines PC-9,A549,XLA-07 and XL-jt after Brusatol treated for 72h,and the half inhibitory concentration(IC50)of cells was calculated.ROS level was detected by ROS detection kit after Brusatol treated for 12h;Proliferation inhibition was detected by EdU-555 cell proliferation assay kit after Brusatol treated for 72 h.3.Inhibition of Nrf2,HMOX1 and NQO1 proteins by Brusatol:the differential expression of Nrf2,HMOX1 and NQO1 in four cells were detected,after treated with different concentrations of Brusatol for 8h,the expression of nuclear Nrf2 was detected,and the total protein expression of HMOX1 and NQO1 was detected after treated with Brusatol for 24h.4.Brusatol induced the up-regulation of serine synthesis pathway and folic acid cycle metabolic enzymes in Xuanwei lung cancer cell lines.The total proteins were extracted from four cell lines treated with different concentrations of Brusatol for 24h,and the expression of serine synthesis pathway and folic acid cycle metabolic enzymes was detected.5.Effects of NCT503 and Pemetrexed on lung cancer cells:CCK8 method was used to detect the absorbance of PC-9,A549,XLA-07 and XL-jt after NCT503 and Pemetrexed treated for 72h,and the half inhibitory concentration(IC50)of cells was calculated.ROS levels were detected by reactive oxygen species detection kit after NCT503 and Pemetrexed treated for 12h;EdU-555 cell proliferation assay kit was used to detect the proliferation inhibition of NCT503 and Pemetrexed.6.The joint action of Brusatol and NCT503 or Brusatol and Pemetrexed treatment:CCK8 method was used to detect the absorbance of the simple administration group,Combinations of Brusatol with NCT503 treatment group,Combinations of Brusatol with Pemetrexed group,and the inhibition rate was calculated.The form of joint action was calculated according to the gold formula.7.After cisplatin,NCT503,Pemetrexed,Brusatol,Brusatol and NCT503,Brusatol and Pemetrexed were treated for 24h,the total proteins were extracted.The the expression of serine synthesis pathway and folic acid circulating metabolic enzymes were detected,and the expression of y-H2AX induced by NCT503,Pemetrexed,Brusatol,Brusatol and NCT503,Brusatol and Pemetrexed was detected.8.NADPH detection:Brusatol was detected using NADP+/NADPH detection kit;NADP+/NADPH levels were measured 24h after Brusatol+NCT503,Brusatol+Pemetrexed treatment.9.Rescue inhibition of Brusatol and NCT503 on tumor cells:After Brusatol and NCT503 were treated in four cell lines,NADPH,NAC and GSH were replenishin four cell lines for 72h,the absorbance was detected and the cell viability curve was plotted.Results:1.Xuanwei lung cancer cell lines XLA-07 and XL-jt were more resistant to cisplatin,EGFR inhibitor AG 1478 and MEK inhibitor U0126 than PC-9 and A549.2.Brusatol could significantly inhibit the survival rates of PC-9,A549,XLA-07 and XL-jt cells at a very low concentration of 20 nmol/L.Compared with PC-9 and A549,Xuanwei lung cancer cell lines XLA-07 and XL-jt also showed stronger resistant to Brusatol.Treatment with4nm/L Brusatolin PC-9,A549,XLA-07and XL-jt cells,increased ROS levels and significantly inhibited the proliferation of lung cancer cells.3.The expression of Nrf2,HMOX1 and NQO1 protein in PC-9 and A549 cells was higher than Nrf2,HMOX1 and NQO1 in XLA-07 and XL-jt cells.Brusatol inhibited the expression of nuclear protein Nrf2 in four cell lines after Brusatol treated for 8h,and inhibited the total protein expression of HMOX1 and NQO1 after treated for 24h.4.After the four cells were treated with Brusatol for 24h,only Xuanwei lung cancer cells XLA-07 and XL-jt successfully induced serine synthesis pathway enzymes PHGDP,PSAT1,PSPH and folic acid circulating metabolic enzymes SHMTs and MTHFDs.5.The sensitivity of XLA-07 and XL-jt to NCT503 was the same,and XLA-07 and XL-jtcells were more tolerant to NCT503 than PC-9 and A549.The tolerance of XLA-07(IC50 74.41μmol/L)and XL-jt(IC50 68.53μmol/L)to Pemetrexed was 100 times higher than that of PC-9(IC50 0.41μmol/L)and A549(IC50 0.57μmol/L).Treatment of PC-9,A549,XLA-07 and XL-jt cells with 10μmol/L LNCT503 and 10μmol/L Lemetrexed for 12h resulted in increased intracellular ROS.PC-9,A549,XLA-07 and XL-jt cells were treated with 10μmol/LNCT503 for 72 h,and PC-9 and A549 cells were treated with 10μmol/L Pemetrexed,XLA-07 and XL-jt,and 0.1μmol/L Pemetrexed for 72 h.The proliferation of lung cancer cells was inhibited.6.According to the gold formula,Brusatol combined with NCT503 was synergistic effect in four cells.Brusatol combined with Pemetrexed showed synergistic effect on PC-9,synergistic and additive effect on A549,additive effect on XLA-07 and synergistic and antagonistic effect on XL-jt.7.Combinations of Brusatol with NCT503 treatment and Combinations of Brusatol with Pemetrexed treatment inhibited Brusatol-induced up-regulation of serine synthesis pathway and folate cycle metabolic enzyme in Xuanwei lung cancer cells,while cisplatin did not induce up-regulation of serine synthesis pathway and folate cycle metabolic enzyme in Xuanwei lung cancer cells.The DNA damage levels of Combinations of Brusatol with NCT503 treatment group and Combinations of Brusatol with Pemetrexed treatment group were higher than that of NCT503,Pemetrexed and Brusatol alone.In XLA-07 and XL-jt cells,Brusatol did not upregulate the expression of serine synthesis pathway and folate cycle metabolic enzymes in PC-9 and A549 cells,so Co-treatment with Brusatol and NCT503 or co-treatment with Brusatol and Pemetrexed did not induce more significant DNA damage in PC-9 and A549 cells.8.Brusatol combined with NCT503 or Pemetrexate treatment further reduced NADPH levels in four cell lines compared with Brusatol alone,the group with lowest level of NADPH in four cell lines was the one co-treatment with Brusatol and NCT503.9.The rescue experiments of tumor inhibition of co-treatment with Brusatol and NCT503 showed that NADPH,NAC and GSH can compensate the cell inhibition of co-treatment with Brusatol and NCT503 in lung cancer cells,and the survival rates of the four cell lines were the highest after NADPH replenishment.Conclusion(s):1.Drug resistance results showed that Xuanwei lung cancer was generally resistant to chemotherapy drugs and had poor prognosis.2.Brusatol can significantly inhibit the survival rates of PC-9,A549,XLA-07 and XL-jt cells,inhibit the proliferation of lung cancer cells and improve the ROS level in lung cancer cells,indicating that Brusatol may be a potential targeted drug for Xuanwei lung cancer.However,Xuanwei lung cancer cell lines XLA-07 and XL-jt also showed stronger resistance to Brusatol.3.The expression of Nrf2,HMOX1 and NQO1 protein in PC-9 and A549 cells was higher than that in XLA-07 and XL-jt cells;indicated that PC-9 and A549 are more vulnerable to Brusatol.Brusatol inhibited the protein expression of Nrf2,HMOX1 and NQO1.4.After Brusatol treatment,only Xuanwei lung cancer cells XLA-07 and XL-jt successfully inducedserine synthesis pathway enzymes PHGDP,PSAT1,PSPH and folic acid circulating metabolic enzymes SHMTs and MTHFDs,so as to adapt to the oxidative stress caused by Nrf2 inhibition.These results suggest that serine synthesis pathway and folate metabolism pathway are up-regulated only in Xuanwei lung cancer cells to compensate for Nrf2 inhibition.5.XLA-07 and XL-jt are more resistant to NCT503 than PC-9 and A549.The tolerance of XLA-07 and XL-jt to Pemetrexed was 100 times higher than that of PC-9 and A549 cells.NCT503 and Pemetrexed could induce the increase of ROS and inhibit the proliferation of lung cancer cells.6.Co-treatment with Brusatol and NCT503 showed synergistic effects on the four cells;co-treatment with Brusatol and Pemetrexed showed synergistic effect on PC-9,synergistic and additive effect on A549,additive effect on XLA-07 and synergistic and antagonistic effect on XL-jt.7.Brusatol-induced upregulation of serine synthesis pathway and folate metabolism enzymes in Xuanwei lung cancer cells is Brusatol-specific.co-treatment with Brusatol and NCT503 and co-treatment with Brusatol and Pemetrexed inhibited Brusatol-induced upregulation of serine synthesis pathway and folate cycle metabolic enzymes in Xuanwei lung cancer cells,thus promoted cell inhibition of Xuanwei lung cancer cells.The DNA damage levels of Xuanwei lung cancer cells treated with Combinations of Brusatol with NCT503 treatment and Combinations of Brusatol with Pemetrexed treatment were higher than that of NCT503,Pemetrexed and Brusatol alone,which explained the synergistic effect of Brusatol and NCT503.Co-treatment with Brusatol and NCT503 or co-treatment with Brusatol and Pemetrexed inhibited Xuanwei lung cancer cell proliferation by inducing more significant DNA damage.Brusatol did not upregulate the expression of serine synthesis pathway and folate cycle metabolic enzymes in PC-9 and A549 cells,so Co-treatment with Brusatol and NCT503 or co-treatment with Brusatol and Pemetrexed did not induce more significant DNA damage in PC-9 and A549 cells.8.Co-treatment with Brusatol and NCT503 further reduced NADPH levels in four cell lines,especially in PC-9 and A549 cells,compared with co-treatment with Brusatol and Pemetrexed.Co-treatment with Brusatol and NCT503 inhibited lung cancer cell proliferation by significantly reducing NADPH levels.9.In cell inhibition of Co-treatment with Brusatol and NCT503 treatment,NADPH is a key metabolite for maintaining redox balance to maintain the survival of lung cancer cells. |
PDF Full Text Request