Analysis Of Gene Expression Changes Of Two Human Hepatocellular Carcinoma Cell Lines Induced By (-)epigallocatechin-3-gallate

Background:HCC is one of the most common digestive system malignancies and it represents a large proportion in GUANGXI.Combination chemotherapy is the most important strategy for the treatment of HCC.The failure of therapeutic effect is concerned with primitive drug-resistance of 70%HCC.However,it is more critical that the development of resistance to multiple chemotherapeutic drugs is a major obstacle in the treatment of HCC.Nowadays,most reversal agents considered validly in vivo or vitro were not clinically achievable,not only because of signal reversal mechanism,but of manifestly the dosage restricted or toxical required to reverse MDR.Therefore,an urgent need still exists for new MDR reversal modulator agents,which are high activity,low toxicity and effective targes widely in the natural compounds.There may be numerous and diverse mechanisms involved in the development of MDR,such as alterations in expressions and functions of intracellular proteins,enzymes and genes as well as that in the extracellular environment.As we know,the ABC binding cassette transport-based classical MDR mechanisms involve the overexpression of P-glycoprotein.The term non-classical MDR may be related to describe non-transport based mechanisms that affect multiple drug classes.This type of resistance can be caused by altered activity of specific enzyme systems,which can decrease the cytotoxicity of drugs of intracellular drug concentrations.Further research has revealed that anticancer drugs typically induce programmed cell death or apoptosis.which depends upon regulatory interaction of a terms of genes or proteins.Other non-classical MDR based mechanisms include alterations of cellular metabolic transformation,multiple drug classes of pharmacokinetics in vivo,and so on. What to be mentioned is that,furthermore,different cell lines of same tumor may have diversal resistance mechanisms.The mechanisms of MDR are numerous and diverse and that the development of MDR is not a result of single cause but a result of multiple ones.Extensive research in the last few years has revealed that EGCG,catechin monomer of green tea,could suppress the processes of transformation,proliferation and metastasism of tumor.Moreover,EGCG,in coordination with chemotherapeutics,can inhibit HCC MDR activity by suppressing the proliferation as well as by inducing the differentiation and apoptosis,which possess reversal multidrug resistance roles.Respecting multiplicity mechanism of tumor MDR and multipathway-effects of reverse MDR by EGCG,nothing is known about investigating possible molecular mechanism of EGCG as to reversal HCC MDR effects by high-throughput analysis technology cDNA microarray.Objective:To investigate the possible reversal multidrug resistance mechanisms of EGCG,adopting two human hepatocelluar carcinoma cell lines,which have multidrug resistance induced by different chemotherapeutics agents,to study multi-parameters at the same time between occurance mechanisms of HCC MDR and reversal resistance effects of EGCG by cDNA microarrays technology.Research content:1.Cell culture of MDR human hepatocellular carcinoma cell lines of BEL7404/ADMå’ŒBEL7402/5FU:BEL7404 were induced with long-term continuous stepwise exposure and high concentration pulse drug exposure by ADM addition into multidrug-resistant cell line BEL7404/ADM,which inceases resistance index,sustaining with 0.5mg/LADM.BEL7402/5FU was purchased from the Nanjing Kaiji Biotechnology limited company,sustaining with 20mg/L5FU.Both cell lines were maintained in RPMI-1640 medium supplemented streptomycin with 10%calf serum,100U/ml penicillin and 100mg/L streptomycin at 37℃in a humidified atmosphere of 95%air and 5% carbon dioxide.2.MTT drug sensitivity assay2.1 Detected the IC₅₀sensitivity of two parental cell lines and two drug-resistant cell lines to diverse chemotherapeutics respectively and calculated the respective resistance index.2.2 Detected the cytotoxicity of EGCG to two MDR HCC cell lines respectively.The dosage required for 10%inhibition of cell growth(IC₁₀)was calculated by LOGIT software.Concentrations less than IC₁₀were used to test dose-dependent experiment of EGCG reversal effects on HCC MDR cells,in this way to definite EGCG optimization reversal dosage concentration.3.Analysis of the different expressed genes in two MDR hepatocellular carcinoma cell lines optimization reversal dosage concentration EGCG-induced by cDNA microarrays,investigating possible molecule mechanism of EGCG reversal drug resistance effects.3.1 Fabrication of Expression Profile cDNA Microarrays.According to standard references directly,pick up of 8064 genes and other 10 hepatocellular carcinoma and/or resistant related genes from IMAGE cDNA library,probe preparation by RT-PCR amplificaition and purification,printing of probes to the glass slide to make specific cDNA microarray. 3.2 Monitoring the Expression Profiles of EGCG-induced two MDR hepatocellular carcinoma cells by cDNA microarray.EGCG(optimization reversal dosage concentration)induced MDR HCC for 48h.Compare to MDR HCC non-exposed,total RNA prepared from these cells was used to synthesize Cy5/Cy3-labeled cDNAs by reverse transcription,followed by hybridization to the human cDNA microarray,and scanning to analyze.4.Expression of Cyclin G1 protein in same exposing condition to two MDR HCC cells by Western blot,as to confirm cDNA microarray.Results:1.BEL7404/ADM cultured by ADM-induced was resistant to ADM,with its resistance index 39.6 folds higher than BEL7404,and cross-resistance to VCR,CDDP,but not resistance to 5FU.BEL7402/5FU was resistant to 5FU significantly with its resistance index 127.6 folds higher than BEL7402,but not resistance to CDDP and VCR.2.The IC₁₀of EGCG on BEL7404/ADM and BEL7402/5FU were 24.76 mg/L and 20.60 mg/L respectively.So,7 mg/L,15 mg/L,20 mg/L EGCG were used to perform the dose-dependent reversal experiments respectively, compared to 5mg/L VRP,which indicated sensitivity of MDR cell lines to anticancer agents increased somewhat in the presence of three dosages EGCG combinations with corresponding chemotherapeutics.The reversal folds BEL7404/ADM were 2.69-,5.37-and 9.66-fold,respectively,as compared with 5mg/L VRP 6.48-fold,whereas which had inapparently effect of BEL7402/5FU with EGCG combination with 5FU,same to combination with VRP.3.The experiment of investigating mechanisms after 48h induced with 20mg/L dosage concentration EGCG for different samples.The biochemical functions of the differential genes in expression profile are diverse,which include cell proliferation and apoptotic genes,DNA duplication and transcription and damage repair factors,oncogenes and tumor suppressor genes,cell metabolism genes,as well as signal transduction cell cycle regulators.Compared to non-induction,in dipl-fluorescence signal pathways,210 genes were differentially expressed significantly deviation in Be17404/ADM with EGCG,among which 38 genes were up-regulated and 172 genes were down-regulated.These genetic alterations participated in potential multidrug resistance mechanism that probably involve ABCB 10(MDR/TAP),TOP2A, TOP2B,CCNG1 up-regulated,ABCB1,MVP,ARHD,HDAC5,GSS,GSTPI,HSPA1B,HSPB7,CDKN1A,RAB11B,RAB9P40 down-regulated. Equally above-mentioned process,179 genes expressing deviation in Be17402/5FU with EGCG,among which 31 up-regulating and 148 genes down-regulating.Probalblly participated in mechanisms related gene: ABCG(BCRP),CCNG2,GADD34,RB1,RBBP4 up-regulated,DTYMK,GPX1,USP5,BAX,BAK1,HSPA1L down-regulated,and so on.4.The results of Cyclin G1 protein expression in Western Blot were increased respectively in same exposing condition to two MDR HCC cells,compased to non-induction of EGCG,with one accord of CCNG1 gene expression.Conclusion:1.Reversing effects of HCC MDR induced by ADM,which exposured to EGCG,were multigenes,multifactorial and multipathways influencing various biochemical pathways.Referring to the altered expression of genes encoded classical transmembrane transport protein,for instance,down-regulating MDR1 gene expression due to ABCB1,ARHD,HDAC5,and so on,accordingly to degrade P-gp protein expression to reverse MDR.Others of ABC membrane transport protein familys gene expression altering,MVP,ABCD10 (MDR/TAP).Relation to multitargets roles,reverse MDR mechanisms of EGCG were concerned with non-classical MDR biochemical pathways,which include genes encoded glutathione detoxication enzyme systems,specific topoisomerase enzyme,mainly with other more genes encoded MDR related proteins as to HSP70,PKC,MMP.2.Reversing effects of HCC MDR induced by 5FU,which exposured to EGCG,was inapparent,on account of resistance mechanisms by 5FU-induced other than classical transmembrane transport protein,which probably concerned with regulating cell cycle checkpoint targets or exerting complex interplay of regulatory roles in out of tune apoptosis genes.3.Effects exposured to EGCG with non-cytotoxico dosage concentration were in coordination with chemotherapeutic agents,to influence regulation of cell cycle and apoptosis.4.Difference type of same category tumor involved in diversal drug resistance mechanisms,in which reversal effects exposured to EGCG presented complex distinction.5.Results of western blot were in accord with gene chips,which is feasible for cDNA microarrays to obtain molecule identification marker of mechanism of drug action that had characteristics of high speed,high throughput and sensitivity,so it was predominant ascendancy for analyzing mechanisms and relations between MDR and reversal agents.