Study On Detecting The Differential Genes Expression As To Anti-Hcc And Reversing MDR Of Traditional Chinese Medicine By Q-PCR Microarray

Objective To establish a high throughput and multi-target method for screening traditional Chinese medicine as to anti-hepatocellular carcinoma and reversing MDR in the course of genetic transcription .Methods Human hepatocellular carcinoma cell line Bel-7402 and 5-FU resistant cell line Bel-FU were cultured in vitro. The multidrug resistance(MDR) of Bel-FU ,drug sensitivity of 5 kinds of traditional Chinese medicine(quercetin,EGCG,IA , ACO-IA,loureirin B)to Bel-FU and their dose-dependen reversal effects were determined by Methyl thiazolyl tetrazolium (MTT) assay.To confine the scope of the hepatocarcinogenesis, 42 gene expression profiles in Bel-FU were detected by q-PCR microarray before and after treatment of traditional Chinese medicine. Then the gene expression of H-ras,MDR1 in Bel-FU treated with quercetin,EGCG, ACO-IA respectively were detected by real-time quantitative PCR to confirm the results of q-PCR microarray.Results In Bel-FU the resistance index (IR)of 5-fluorouracil ,cytarabine, vincristin, doxorubicin, daunorubicin were 89.6,3.4,37.1, 15.0, 3.7respectively. The 50% inhibitory concentration(IC50) of quercetin ,EGCG ,IA ,ACO-IA and loureirin B were 193.9μM,1230.8μM,507.8μM,244.9μΜ,163.7μΜrespectively. The 10% inhibitory concentration(IC10) were 58.2μM,159.5μM,75.3μM, 64.7μM, 25.8μM respectively. 43.6,87.2,130.8μM EGCG reversed the MDR by 1.04,1.36,2.10 folds respectively .16.5,33,49.5μM quercetin reversed the MDR by 1.14,1.68,2.38 folds respectively . 13.2,33,66μM IA reversed the MDR by 1.13,1.54,1.33 folds respectively . 21,42,63μM ACO-IA reversed the MDR by 1.44, 2.87, 1.76 folds respectively . 6, 12, 24μM loureirin B reversed the MDR by 2.25,3.80,5.76 folds respectively . Positive control VRP(5μg·mL-1)was 1.11 folds .Compared with those in Bel-7402, the gene expression of H-ras,Bcl-2,PDGFA,MDR1,BCRP were up-regulated (P<0.05)and cyclinB1 was down-regulated in Bel-FU(P<0.05). H-ras,EGFR,FGF,VEGFA,PDGFA,MDR1,Erk1 were down-regulated in Bel-FU treated with quercetin(P<0.05). The gene expression of H-ras,EGFR,PDGFA,MDR1,VEGFA were down-regulated in Bel-FU treated with EGCG(P<0.05). The gene expression of H-ras, VEGFA,PDGFA,C-myc,NF-κB were down-regulated in Bel-FU treated with ACO-IA ( P < 0.05 ) . The gene expression of Wnt1,VEGFA were down-regulated in Bel-FU treated with both IA and loureirin B(P<0.05). Real-time quantitative PCR showed that quercetin and EGCG could down-regulate the gene expression of H-ras,MDR1 in Bel-FU(P<0.05),then ACO-IA just could down-regulate the gene expression of H-ras in Bel-FU(P<0.05).Conclusion qPCR microarray is a high throughput and multi-target method for screening traditional Chinese medicine as to anti-hepatocellular carcinoma and reversing MDR in the course of genetic transcription . Objective To screen differentially expressed proteins in the human HCC MDR cell line before and after treatment of 3 kinds of traditional Chinese medicine respectively,to found a method for screening traditional Chinese medicine as to anti-hepatocellular carcinoma and reversing MDR by detecting target protein.Methods Detected the differentially expressed proteins between human hepatocellular carcinoma sensitive cell line Bel-7402 and drug -resistant cell line Bel-FU,and that in Bel-FU treated by 3 kinds of traditional Chinese medicine respectively.Differentially expressed proteins were defined as whose absolute ratio values were greater than 2.5. Western blot was used to detect the expression of P-gp ,FAK and CRT in Bel-FU treated with 3 kinds of traditional Chinese medicine.Results Compared with that in Bel-7402,24 proteins expression were up-regulated , including FAK,TM3,ORP150,CRT,NSE ,80K-H protein,EF-1-D,phosphoprotein and so on ,then 12 were down-regulated in Bel-FU.EGCG could down-regulate the proteins expression of FAK,CRT,TM3,80K-H protein,EF-1-D. quercetin could down-regulate the proteins expression of phosphoprotein,CRT,TM3,EF-1-D. COA-IA could down-regulate the proteins expression of FAK,80K-H protein,CRT,TM3 in Bel-FU. The results of Western blot showed that both EGCG and quercetin could down-regulate the proteins expression of P-gp(P < 0.05), EGCG and COA-IA could down-regulate the proteins expression of FAK(P < 0.05), 3 kinds of traditional Chinese medicine could down-regulate the proteins expression of CRT(P < 0.05).Conclusion EGCG , quercetin and COA-IA can reverse the MDR of HCC .Detecting protein differential expression as to anti-HCC and reversing MDR by proteomics technique provides a platform for high throughput drug screen.