Studies On Anti-mdr Tumor Cells Activities Of P100 And Its Mechanism Of Action

Objective: In this paper,we selected several human tumor cells and mouse H22 transplant tumor as the experimental object to study the anti-tumor effect of novel podophyllotoxin derivative P100.In addition,the K562 cells and multidrug resistant K562/A02 cells were used to study the mechanism of multidrug resistance and anti-tumor multidrug resistance of P100.Methods:1 The drug resistance of K562A/02 cells and suppressive effect on the several human tumor cells of P100 were detected by MTT assay.2 Kun ming mouse was used to make model of H22 transplant tumor in order to detect anti-tumor effect of P100(5 mg/kg'weight?10 mg/kg'weight?20 mg/kg'weight)by tail vein injection in vivo.3 The apoptosis morphological changes of K562 cell and K562/A02 cell induced by P100 were observed by Hoechst33342/PI staining,In addition,the apoptosis morphological changes of K562/A02 cell induced by P100 was observed by DAPI staining.4 Effects of P100 on apoptosis rates of K562 cell and K562/A02 cell were detected by Flow cytometry.5 The mRNA expressions of PI3 K,Akt,NF-?B,MDR-1,MRP,Caspase3,Caspase9 in K562 cell and K562A/02 cell treated by P100 were detected by Real-time fluorescent quantitative PCR.6 The protein expressions of PI3 K p110?,Akt1,Phospho-Akt1,I?B?,Phospho-I?B?,NF-?B p65,Phospho-NF-?B p65,P-gp,Caspase3,Cleaved Caspase3,Caspase9,Cleaved Caspase9,Caspase8,Cleaved Caspase8 in K562 cell and K562A/02 cell treated by P100 were detected by Western Blot.7 The expression of MDR-1,NF-?B P65 and Akt1 gene in K562A/02 cell were interfere by RNAi.The ADM resistance of K562A/02 cell as well as expression changes of related protein were detected.Results:1 Results of MTT assay showed that K562A/02 cells had significant resistance to various antitumor drugs with different mechanisms of action;The P100 had better inhibitory activity on H4?Hela?A549?MCF-7?K562?K562/A02 cell(IC50: 0.06 ?mol/L~9.6 ?mol/L),and its inhibitory activity was generally higher than positive drug VP-16.In particular,the inhibitory effect of VP-16 on multidrug resistant K562/A02 cell line was poor(21.83±4.78 ?mol/L),but P100 can effectively inhibit the growth of K562A/02(IC50: 4.90±0.86 ?mol/L).In addition,there was no obvious toxicity of P100 on 926 cell and H9C2 cell.2 Mouse transplanted tumor experiment showed that the inhibition rate of P100(5,10,20 mg/kg'weight)on transplanted tumor were 31.3%,50.6%,70.0% respectively.The inhibition rate of positive control drug VP-16(20 mg/kg'weight)was 51.3%.3 Hoechst 33342/PI staining indicated that the morphology of K562 cell and K562A/02 cell were intacted,after treated with different concentration of P100,the cells began to appear irregular morphology,shrinkage,fragmentation and apoptosis body.With the increase of drug concentration,the apoptosis was more obvious.DAPI staining showed that different concentration of P100 evoked K562A/02 cell shrinkage and some nucleus pycnosis.4 The results of flow cytometry manifested that the apoptosis rate of K562 cell induced by different concentrations of P100(1 ?mol/L,2 ?mol/L,4 ?mol/L)were 23.92±2.15%?54.37±4.97%?65.09±5.63%,the apoptosis rate of VP-16 group was 43.46±2.30%.Different concentrations of P100(2.5 ?mol/L,5 ?mol/L,10 ?mol/L)could induce K562A/02 cell apoptosis,the apoptosis rates were 16.06±1.23%,27.29±3.36%,55.56±2.69% respectively,the apoptosis rate of VP-16 group was 21.08±3.76%.It can clearly be seen that the ability to induce apoptosis of P100 was stronger than the VP-16.5 The results of qPCR showed that the mRNA expression of PI3 K,Akt,NF-?B,MDR-1 and MRP in K562A/02 cells were increased,compared with K562 cells.The mRNA expression of PI3 K,Akt,NF-?B,MDR-1 and MRP were decreased with different concentration of P100(2.5 ?mol/L,5 ?mol/L,10 ?mol/L).In addition,the mRNA expression of Caspase9 and Caspase3 were raised with P100 treating.6 The results of Western Blot showed that the protein expression of PI3 K p110?,Phospho-Akt1,Phospho-I?B,NF-?B p65,Phospho-NF-?B p65,P-gp in K562A/02 cells were increased compared with K562 cells.The protein expression of PI3 K p110?,Phospho-Akt1,Phospho-I?B?,NF-?B p65,Phospho-NF-?B p65,P-gp,Caspase9,Caspase8 and Caspase3 were decreased with different concentration of P100(2.5 ?mol/L,5 ?mol/L,10 ?mol/L).In addition,the protein expression of Fas,Cleaved Caspase9,Cleaved Caspase8 and Cleaved Caspase3 were raised with P100 treating.7 The expression of P-gp was inhibited and the sensitivity of K562A/02 cell to ADM was increased by interfering MDR-1 shRNA.Compared with NC group,IC50 value decreased from 17.13±3.05 ?mol/L to 9.28±0.53 ?mol/L.The expression of Phospho-NF-?B p65 was inhibited and the sensitivity of K562A/02 cell to ADM was increased by tansfecting NF-?B p65 shRNA.In addition,the expression of P-gp was inhibited by interfering NF-?B p65 shRNA.Compared with NC group,IC50 value decreased from 17.13±3.05 ?mol/L to 11.53±1.52 ?mol/L.The expression of Phospho-Akt1,Akt1 mRNA and NF-?B mRNA was inhibited but the expression of P-gp and sensitivity of K562A/02 to ADM were not changed by transfecting Akt1 si RNA.Conclusion: P100 treatment could play a significant inhibitory effect on the proliferation of series tumor cells and multi-drug resistant cells in vitro and the growth of H22 transplant tumor in vivo.Compared with the positive control drug VP-16 has the following advantages:(1)P100 has good physical and chemical properties,the water solubility of P100 is 100 times higher than VP-16;(2)The cytotoxicity of P100 to normal cells was significantly lower than VP-16.The antitumor activity and antitumor spectrum of P100 are better than VP-16;(3)The activity of P100 on multidrug resistant cell line K562/A02 was nearly 5 times higher than VP-16.P100 can not only induce the apoptosis of K562 cells,but also induce the apoptosis of multidrug resistant cells K562/A02.The same dose of VP-16 can induce the apoptosis of K562 cells,but can not obviously induce the apoptosis of K562/A02 cells.The study found between multidrug resistant cells K562/A02 and parental cells K562 not only in the P glycoprotein expression have great differences,but there are great differences in the PI3K/Akt pathway and NF-?B inflammation pathway.The apoptosis mechanism of K562/A02 cell induced by P100 may be related with inhibition of PI3K/Akt pathway and down-regulation of P-gp resulting in the inhibition of NF-?B pathway,and the initiation of Fas apoptosis pathway and Caspase cascade.These results suggest that P100 can overcome the multidrug resistance through multiple targets in the process of anti-tumor multidrug resistance.Therefore,as the study of anti-tumor multidrug resistance,P100 is a valuable novel podophyllotoxin compound.What is more,the novel compound of P100 is very valuable to drug research of anti-tumor multidrug resistance.