Investigation Of The Target Genes Involved In Tumor Cell Apoptosis Induced By HDAC Inhibitor

ObjectiveTo construct a noval functional-screening-based method--- SMART (Suppression ofMortality by Antisense Rescue Technique) for the screening of target genes responsible forHDAC inhibitor induced apoptosis and explore the mechanism underlying the action of thegenes.MethodsTo confirm the time course of apoptosis in MCF-7 induced by different concentrationsof Trichostatin A.To induce apoptosis in MCF-7 by treated with 250 nM Trichostatin A.mRNA were extracted from cells collected at multiple time-points after Trichostatin Atreatment.cDNA were synthesized and inserted reversely into plasmid pCEP4 to constrctan anti-sense cDNA library.Library DNA was transfected randomly into Hela,positivetransfected cell clones were screened by Trichostatin A and Hygromycin B.Screening wasstoped when all control cells were died.Surviving cell clones then were amplified andplasmids DNA in cells were acquired by the method of Hirt DNA Extraction.Insertedsequences were then sequenced and EST fragments were obtained.The data were analyzedby the method ofbioinformatics and functional identification.ResultsApoptosis of MCF-7 induced by Trichostatin A started at 36 hours later after drugtreatment and climaxed at about 72 hours.Antisense cDNA library was constructed formcells treated with Trichostatin A within 48 hours at multiple time-points.Insert efficiencywas about 90%,average inserted fragment was about 15 kb and the volum of library wasabout 4×10⁵.Library plasmid DNA were transfected into Hela,after two rounds ofscreening,several cell clones that showed an anti-apoptosis activity were formed.Theseclones were amplified and Hirt DNA was extracted.76 EST fragments were obtained bysequencing.After bioinformatics analysis and functional identification,we acquiredLIV-lgene.Further study showed that these genes knockdown in tumor cell lines couldresist HDACI induced apoptosis,which is caused,at least in part,by disruption ofintracellular zinc homeostasis. ConclusionsSMART cDNA library with high quantity and quality based on HDAC inhibitorinducing apoptosis was successfully constructed;LIV1 gene may be one of the essentialgenes with anti-tumor effects on apoptosis induced by HDAC inhibitors;down-regulationof LIV1 is capable of conferring a significant resistance to HDACi-induced cell death intumor cells,which is caused,at least in part,by disruption of intracellular zinc homeostasis. Evidence for a protective role of CD146 in SAHA-inducederadication of tumorIntroductionHistone deacetylase inhibitors (HDACi) show promise as a novel class of anticanceragents with potent activity to inhibit proliferation,induce apoptosis and differentiation in awide spectrum of tumors.At least 14 HDACi are being tested in over 100 clinical trials anddisplay encouraging therapeutic response with surprisingly good safety profiles^1,2.Theclinical potential of HDACi has been well exemplified by the successful development ofVorinostat (suberoylanilide hydroxamic acid,SAHA),which has recently been approved bythe US Food and Drug Administration for treating cutaneous T-cell lymphoma³.However,despite the rapid clinical progress achieved,the mechanisms of action of HDACi are not yetwell understood.Early studies using gene profiling techniques have revealed that up to 17%of all known genes are affected by HDACi at the transcriptional level⁴.A large number ofpro- or anti-apoptotic genes and cell cycle regulatory genes have been independentlyidentified by different groups as the downstream targets to HDACi.In addition to HDACs,many non-histone proteins are also regulated by HDACi including transcription factors,nuclear import proteins,signal transduction molecules,cytoskeletal proteins and DNArepair enzymes⁴.One of the central problems with respect to the action of HDACi is thatmodulation of such extensive substrates by these agents might result in the initiation of boththerapeutic responses and unanticipated protective procedures,which significantly limitsthe optimal application of this class of drugs.Identification of the potential protectivesubstrates in HDACi-induced eradication of tumors would set critical biomarkers andfacilitates development of novel HDACi.Recently,a set of clinical data show that there is limited efficacy for HDACi as asingle agent.For example,patients with MDS and AML have had biologic responsesfollowing HDACi therapy;however,true PRs or CRs are infrequent².The majority of thenew trials are combination studies looking at HDACi in combination with other agents^5,6Two Phaseâ… studies demonstrated that the combination of SAHA with conventionalchemotherapeutic agents such as carboplatin/paclitaxel and FOLFOX regimen (fluorouracil, leucovorin and oxaliplatin) displayed synergistic effect in the treatment of advanced solidmalignancies^7,8.Three Phaseâ… /â…¡trials combining HDACi (PB or VPA) with thedemethylating agents of 5-azacytidine or decitabine in patients with AML and MDSshowed both tolerability and promising efficacy^9-11.Similarly,synergism has also beenobserved between HDACi and other agents such as all-trans-retinoic acid,proteasomeinhibitor,HSP90 antagonists,tyrosine kinase inhibitors and so on^12-15.All of thesecombination trials seek to increase the tumoricidal impact.However,although thesecombination strategies follow a rational molecular approach in some cases;in most cases,they are relatively empirical.Furthermore,unfortunately,synergism in efficacy might beaccompanied by adverse effects that are rarely or never seen with HDACi alone such asmyelosuppression⁶.And high-dose HDACi with substantially longer half-lives show ahigher toxicity that precludes daily treatment¹⁶.Therefore,revealing the molecularmechanisms underlying the low potency of HDACi is pivotal to develop potentially lesstoxic and more effective treatments and promote the optimal application of this class oftherapeutic drugs.In this study we analyzed the HDACi-induced expression pattern using cDNAmicroarrays and confirmed that many of adhesion molecules could be significantlyup-regulated.Interestingly,among these inducible adhesion molecules,CD146 expressionlevel was the highest.To further elucidate whether the increased expression levels ofadhesion molecules influenced HDACi activity we took CD146 for example investigatingthe therapeutic effects of combined treatment with CD146 mAb and HDACi onangiogenesis,tumor growth and metastasis.