The Function And Molecular Mechanism Of 4HPR Combined With Parthenolide On Hepatoma Cell Apoptosis

Fenretinide(N-4-hydroxyphenyl retinamide,4HPR),a synthetic anticancer retinoid,is a well-known apoptosis-inducing agent.Recently,we observed that the apoptosis induced by fenretinide could be effectively enhanced in hepatoma cells by concomitant treatment with parthenolide,a known inhibitor of nuclear factor NF-kB Furthermore,treatment with fenretinide triggered the activation of NF-kB.during apoptosis,an effect that could be substantially inhibited by parthenolide,which suggests that NF-κB activation during fenretinide-induced apoptosis might have an anti-apoptotic effect.In this study,we investigated the molecular mechanism of this apoptotic potentiation by NF-κB inhibition.Using the differential display-PCR (DD-PCR) method,and subsequent Northern blot or semiquantitative reverse transcriptase PCR(RT-PCR) analysis we identified genes which are involved in enhanced fenretinide-induced apoptosis by parthenolide.We identified 35 apoptosis-related genes including 12 unknown genes that are up- or down-regulated by parthenolide.Intriguingly,one up-regulated gene(HA1A2) was isolated and cloned from liver cDNA,and turned out to be identical to ANKRD1,which is also referred to as the CARP gene.Ectopic expression of ANKRD1 led to reduced colony formation and to enhanced apoptotic cell death in hepatoma cells,compared with controls treated with an empty vector or with antisense cDNA.These results imply that ANKRD1 and other genes whose expressions were substantially modulated by parthenolide-mediated inhibition of NF-κB activation may play roles in the enhanced drug-induced apoptosis and further,that those identified genes may be useful in anticancer strategies against hepatoma.The results are as below:1.Parthenolide enhances fenretinide-induced apoptotic cell death. apoptotic cell death was determined by TUNEL staining in Hep 3B cells and in SK-HEP-1 cells treated for 72 h with 10μM or 15μM fenretinide,respectively,in the presence or absence of 4μM parthenolide(P),or treated with vehicle alone.Vertical bars represent the means±SE of quadruplicate experiments.^**P＜0.01,compared with cells treated with fenretinide alone,quantitation of the apoptotic fraction by FCM analysis in Hep3B cells(upper panels) and SK-HEP-1 cells(lower panels) treated with 10μM fenretinide in the presence or absence of 4μM parthenolide(P) for 72 h,or were treated with vehicle alone.The Sub-G₁ fraction was estimates by gating hypodiploid cells in the histogram using the LYSISâ…¡program.DNA contents are plotted on the linear abscissa(M₁,apoptotic fraction).Each value represents the mean±SE of triplicate experiments.^** P＜0.01,compared with cells treated with fenretinide alone.2.Parthenolide effectively inhibits NF-κB activation during fenretinide-induced apoptosis.Hep 3B cells and SK-HEP cells(1×10⁶) were treated with 10μM or 15μM fenretinide,respectively,for the indicated time periods,or were treated with vehicle alone.DNA-binding activity for NF-kB was detected by autoradiography. Experiments were performed at least three times in duplicate,and the result of one representative experiment is shown.3.Differential display analysis of genes expressed in apoptotic Hep 3B cells. Differential display using three one-base anchored oligo-dT primers(H-T₁₁G,H-T₁₁A and H-T₁₁C) in combination with 80 arbitrary 13-mers.Representative patterns of differentially expressed genes were obtained from DD-PCR analysis using duplicate RIgA samples from control Hep 3B cells(C) and from apoptotic Hep 3B cells(A) induced by treatment with 10μM fenretinide for 72 h using H-T₁₁C in combination with 67-79th-arbitrary 13-mers.The HC70A1,HC71A1,HC75A1 and HC75C2 cDNA fragments are marked by arrows.4.ANKRD1 overexpression during drug-induced apoptosisIsolated ankyrin repeat domain 1/cardia ankyrin repeat protein gene(ANKRD1/CARP) cDNA and its up-regulation by the NF-kB inhibitor parthenolide.,Northern blot analysis of ANKRD1 in Hep 3B cells(upper panels) and in SK-HEP-1 cells(middle panels) treated either with fenretinide alone or in combination with parthenolide (4HPR + P) at the indicated time intervals,or treated with vehicle alone.Total RNAs were extracted and fractionated by electrophoresis on 1%agarose gels containing formaldehyde,and were then transferred to membranes.Each blot was stripped and subsequently rehybridized with a probe for 18S cDNA as a loading control. Experiments were performed at least three times,and the result of one representative experiment is shown.Relative densitometric plots on the quantification of ANKRD1 mRNA induction(%of control)(lower panel).5.Decreased clonogenicity and apoptotic morphology by ectopic ANKRD1 overexpression.6.Increased drug-susceptibility by ectopic ANKRD1 overexpression. Isolated ankyrin repeat domain 1/cardia ankyrin repeat protein gene(ANKRD1/CARP) cDNA and its up-regulation by the NF-kB inhibitor parthenolide C,immunoblot analysis of ANKRD1 expression in Hep 3B cells(upper panels) and in SK-HEP-1 cells(lower panels) treated either with fenretinide alone or in combination with parthenolide(4HPR + P) at the indicated time intervals,or treated with vehicle alone (C).Thirtyμg of extracted proteins were resolved by 12%SDS-PAGE and were transferred to the membrane.The blots were probed with a polyclonal antibody against ANKRD1 and were then stripped and reprobed with a monoclonal antibody to actin as a loading control.Experiments were performed at least three times,and the result of one representative experiment is shown.7.We transiently transfected the cells with the ANKRD1 tagged at its amino terminus with the enhanced green fluorescent protein(GFP) and simultaneously performed immunohistochemistry with a mouse monoclonal antibody for ANKRD1. GFP fused to ANKRD1 protein expression is confined to mainly cytoplasm and less nuclei of the Hep 3B,SK-Hep1 and Hep G2,Huh7,which is perfectively merged with immunoreactivity for ANKRD1 expression.8 The ectopic overexpression of ANKRD1 sensitizes the Hep 3B cells to drug-induced apoptosis.Ectopic over-expression of ANKRD1 sensitizes Hep 3B cells to drug-induced apoptosis.Expression of ANKRD1 protein in stably transfected Hep 3B cells with a sense GFP-ANKRD1(S1 and S6) were compared with antisense GFP-ANKRD1(AS8 and AS13) or with GFP vector control cells(VC2 and VC3) (upper).Ectopic,ectopic expression of ANKRD1 protein.Endogenous,endogenous expression of ANKRD1 protein.The stable transfectants and vector control cells were treated with 10 M fenretinide or 4 M parthenolide(P) for 48 h as indicated. Quantification of apoptotic fractions was performed using a FACScan.The sub-G₁ fraction was estimated by gating hypodiploid cells in the histogram using a C-30 program.DNA contents are plotted on the linear abscissa(M₁,apoptotic fraction). Each value represents the mean±SE of two independent experiments performed in triplicate±SE(^** P＜0.01 compared with control).Our conclusions:1.Parthenolide enhances fenretinide-induced apoptosis in hepatoma cells,The Sub-G₁ fraction was estimates by gating hypodiploid cells in the histogram using the LYSISâ…¡program.2.Parthenolide inhibit NF-kB activation induced by fenretinide and enhance fenretinide-induced apoptosis.3.Identified 35 apoptosis-related genes including 12 tmknown genes that are up- or down-regulated by parthenolide,one up-regulated gene(HA1A2) was isolated and cloned from liver cDNA,and turned out to be identical to ANKRD1,which is also referred to as the CARP gene.4.Ectopic expression of ANKRD1 led to reduced colony formation and to enhanced apoptotic cell death in hepatoma cells.5.These results imply that ANKRD1 and other genes whose expressions were substantially modulated by parthenolide-mediated inhibition of NF-kB activation may play roles in the enhanced drug-induced apoptosis and further,that those identified genes may be useful in anticancer strategies against hepatoma.