| Objective(s):Usnicoyinamide is a new chemical obtained from the structural transformation of the anticancer active compound Usenamine of our group.In this study,the anticancer activity of the compound and its mechanism of action were preliminarily studied,the difference of the anticancer activity of usnicoyinamide on different human cancer cells in vitro was understood,and the mechanism of its anticancer activity was explored to provide scientific basis for the later research and development of usnicoyinamide.Methods:1.Dose-effect relationship of anticancer in vitro:MTT method was used to detect the OD value of human cancer cells treated with different concentrations of usnicoyinamide for 72h,and calculate the cell viability inhibition rate and half inhibition concentration(IC50).2.Time-effect relationship of anticancer in vitro:MTT method was used to detect the absorbance OD value of human cancer cells treated with different concentrations of usnicoyinamide for 24,48,72 hours,and calculate the cell survival rate and half inhibition concentration(IC50).3.Combined action with clinical anticancer drug DDP:MTT method was used to measure the absorbance of human cancer cells treated with single drug group and combined group after 48h,and the cell viability inhibition rate was calculated,judging the joint action form according to Jin's formula.4.Effects on cell migration:cell scratch test was used to observe the migration of human cancer cells to the injured area at 12,24,36,48 hours after treatment with different concentrations of usnicoyinamide,cell migration status and cell mobility were calculated.5.Effects on cell cycle and apoptosis:48 hours after treatment of human cancer cells with different concentrations of usnicoyinamide,CyStain DNA 1 step and Annexin V/PI double staining methods were used to detect the cycle distribution and apoptosis of cancer cells by flow cytometry,and the cell percentage and apoptosis rate of G1,S and G2 stages were calculated.DAPI and Hoechst fluorescence staining were used to observe the morphological changes of cancer cells after treatment by inverted fluorescence microscope.6.Study on the molecular mechanism of anticancer activity:after treatment of human cancer cells with different concentrations of usnicoyinamide for 48 hours,Western blot analysis measure the expression of Bax,Bcl-2,cleaved-caspase-9,cleaved-caspase-3 protein related to Bax/Bcl-2/caspase-9/caspase-3 apoptosis signal pathway.Western blot analysis measure the expression of Vimentin,N-cadherin,N-cadherin protein related to EMT process.Western blot analysis the expression of PI3k,p-PI3k,AKT,p-AKT protein related to PI3K/AKT pathway.The expression of Bax,Bcl-2,caspase-9 and caspase-3 protein mRNA was detected by RT-qPCR.Results:1.Usnicoyinamide has significant anticancer active on many cancer cell lines in vitro,and its anticancer active has a dose-response relationship.Different cancer cells have different sensitivity to it.0.625,1.25,2.5,5,10 ?mol/L of usnicoyinamide to A-549 cells proliferation inhibition rates were respectively 7.83%,6.89%,0.24%,90.15%,92.76%,and IC50 was 4.12?mol/L.The proliferation inhibition rates of XWLC-05 cells were 17.09%,27.18%,56.11%,82.44%,83.30%,and IC50 was 2.16?mol/L.The proliferation inhibition rate of SPC-A1 cell were 11.90%,25.26%,65.69%,91.75%,95.27%,IC50 is 1.90?mol/L.The proliferation inhibition rate of SGC-7901 cell were 2.95%,27.73%,83.98%,94.92%,95.82%,IC50 is 1.62?mol/L.2.There is a time-effect relationship between the anticancer active of usnicoyinamide in vitro.The 24,48,72h IC50 of SPC-A1 cells were 4.67?mol/L,2.60?mol/L and 1.92?mol/L,and the 24,48,72h IC50 of SGC-7901 cells were 3.45?mol/L,1.43?mol/L and 1.22?mol/L.3.The combination of usnicoyinamide and DDP had antagonistic and adding effects:at low doses(0.625,1.25?mol/L)the combination showing antagonistic effects,the q values of SPC-A1 cells in the combination group were 0.59 and 0.66,and that of SGC-7901 cells were 0.76 and 0.82.Under the high dose(2.5?mol/L)the combination had an additive effect,the q value of SPC-A1 cells in the combined group was 0.86,and that of SGC-7901 cells was 0.93.4.Usnicoyinamide significantly inhibited the invasion and migration of cancer cells,and the cell migration rate decreased with the increase of drug concentration and the extension of drug treatment time.(1)SPC-A1 cells:The cell migration rates treated by usnicoyinamide 12h of 0,1.25,2.5,5,10?mol/L group were 29.38%,26.27%,24.57%,12.97%and 16.81%;After 24h were 38.05%,29.43%,33.93%,17.21%and 8.60%;After 36h were 59.50%,46.01%,41.53%,25.80%and 14.53%;After 48h were 70.33%,51.15%,47.54%,32.69%and 21.17%.(2)SGC-7901 cells:The cell migration rates treated by usnicoyinamide 12h of 0,1.25,2.5,5,10?mol/L group were 26.62%,27.02%,19.82%,6.32%and 4.51%;After 24h were 39.68%,27.97%,22.32%,5.34%and 9.70%;After 36h were 50.63%,36.18%,22.26%,19.81%and 16.68%;After 48h were 55.69%,39.47%,26.36%,23.86%and 21.84%.5.Usnicoyinamide can block cells in S phase,with the increase of drug concentration,S phase cell percentage increase,G0/G1 phase cell proportion was reduced.SPC-A1 cells:The percentage of S%cells in the 0,1.25,2.5 and 5?mol/L groups were 26.1%,27.62%,42.93%and 52.45%,The percentage of G0/G1%cells were 59.86%,59.49%,49.59%and 38.76%.SGC-7901 cells:The percentage of S%cells in the 0,1.25,2.5 and 5?mol/L groups were 32.27%,39.13%,47.28%and 54.23%;The percentage of G0/G1%cells were 60.34%,45.40%,44.19%and 34.29%.6.Usnicoyinamide can induce apoptosis,and with the increase of drug concentration and the prolongation of drug action time,the overall apoptosis rate of cells is higher.SPC-A1 cells of 0,1.25,2.5,5 microns group total apoptosis rate were 10.03%,18.37%,36.26%,39.58%,SGC-7901 cells to 0,1.25,2.5,5 microns group cell apoptosis rate were 6.09%,7.97%,23.53%and 36.93%respectively.Under the fluorescence microscope,usnicoyinamide can cause the cancer cells to form apoptotic bodies,nuclear concentration and nuclear fragmentation.7.Usnicoyinamide could significantly up-regulate the expression of Bax,cleaved caspase-3 and cleaved caspase-9,and down-regulate the expression of Bcl-2,increasing the Bax/Bcl-2 ratio in SPC-A1 cells and SGC-7901 cells.Usnicoyinamide significantly down-regulated Vimentin and N-cadherin protein expression,and up-regulated E-cadherin protein expression in SPC-A1 cells.Meanwhile,the expression of p-PI3k and p-AKT in s SPC-A1 cells was significantly down-regulated.And with the increase of drug concentration,the trend of change was more obvious.Conclusion:1.Usnicoyinamide has significant anticancer activity in vitro,and SGC-7901 is the most sensitive to usnicoyinamide among the four cancer cell lines detected2.The in vitro anticancer activity of usnicoyinamide has a dose-effect and time-effect relationship.3.High dose usnicoyinamide has additive activity with clinical anticancer drug DDP in vitro,which can improve the sensitivity of chemotherapy drugs.4.Usnicoyinamide significantly inhibited the invasion and migration of cancer cells.5.Usnicoyinamide blocks cancer cells in the S phase.6.Usnicoyinamide induces apoptosis.7.The molecular mechanism of anticancer activity of usnicoyinamide is to regulate the Bax/Bcl-2/caspase-9/caspase-3 signaling pathway to induce apoptosis,affect Bax,Bcl-2,Caspase-9 and Caspase-3 mRNA levels,at the same time regulate PI3K/AKT signaling pathway inhibition of EMT cell transformation. |
PDF Full Text Request