The Effect Of Histone De-acetylase Inhibitor On RECK Gene Expression In Breast Cancer

Breast cancer is the most common cancer in women worldwide.Its invasion and metastasis contributes mainly to the breast cancer death.Degradation of the extra-cellular matrix is an essential component of the invasive metastatic process.The major proteases responsible for turnover of the ECM belong to matrix metalloproteinases(MMPs)family.A novel MMP inhibitor,reversion-inducing cysteine-rich protein with Kazal motifs(RECK),found by Takahashi C in 1998,was identified by screening a fibroblast expression library for cDNAs that changed the round morphology in v-Ki-ras-transformed NIH 3T3 cells into the non-transformed flat morphology.It inhibits MMP-9,MMP-2,and membrane Type 1(MT1)-MMP(MMP-14)secretion and activity by an as yet unknown mechanism.We assessed RECK expression and its prognostic value in tumor tissue specimens from 39 breast cancer patients.RECK mRNA levels were measured by RT-PCR,RECK protein was measured by western-blot,as a tumor suppressor gene.It was down-regulated in breast cancer.Tumor suppressor genes(TSGs)silenced in virtually every cancer type.silencing of these genes was shown to involve dense hyper-methylation of 5,CpG islands and hypo-acetylation of lysine 9 and 14 on histone H3.Histone acetylation has been associated with transcriptional activation,whereas conversely,de-acetylatin of histones is associated with gene silencing and transcriptional repress.The balance of Histone acetylases and de-acetylases in cell can determine transcription of many tumor suppressor genes.De-acetylases function as a transcription repressor.So we select Histone deacetylases-1 and assess its relation with RECK gene in breast cancer tissue and cell lines.And using histone de-actylase(HDAC)inhibitors (HDACIs):MS-275 to treat tumor cells,meaning to make TSGs(including RECK) reactivated.Lastly we explore the suppressive effect of MS-275 on transplanted human breast cancer in nude mice and its effect on the expression of tumor suppressor genes —RECK in breast cancer tissues.The PartⅠObjective:To investigate the effects of the RECK gene in the progression and invasion on the breast cancer by evaluating the expression level of the RECK -mRNA,RECK protein and MMP-9-mRNA,MMP-9 protein in 39 breast cancer tissues and normal breast tissues.Methods:The mRNA level of breast cancer and normal breast tissues was measured by RT-PCR and western-blot technique.Results:The mRNA and protein level of RECK in the breast cancer tissues was lower than that in the normal breast tissues(0.247±0.092VS0.914±0.126) while the MMP-9 level were higher in cancer than normal tissues(1.642±0.217VS0.892±0.184).P＜0.01.Conclusion:RECK gene was supposed to inhibit the expression of the MMP-9 and metastasis in breast cancer.The PartⅡObjective:To investigate the relation between RECK and Histone Deacetylases-1 in breast cancer tissue and normal breast tissues.Methods:The expression of HDAC1-mRNA in 39 cases of surgically resected breast cancer and 39 cases of normal breast tissues were detected by RT-PCR.and HDAC1 protein was tested by immuno-histochemical assays.Results:HDAC1-mRNA in breast cancer,normal breast tissues was 1.061±0.406 and 0.820±0.119 respectively(P＜0.05).The level of RECK expression was inversely correlated with HDAC1(at mRNA level,r=-0.39).immunohistochemically manifestation:expression of HDAC1 protein increased in breast cancer tissues than normal breast tissues.Conclusion:De-acetylatin of histones is associated with gene silencing and transcriptional repress.HDAC1 maybe an inhibitor of RECK in breast cancer tissue. PartⅢObjective:Test the possibility that HDAC inhibitor may increase RECK expression to inhibit MMP activation in vitro and their effect on proliferation, apoptosis of breast cancer cell.Methods:Breast cancer cell was treated with MS-275 at 0.25,0.5,1.0,2.5umol/L for 48h respectively,,at the same time we set control group to compare the RECK expression using RT-PCR,Western-blot analysis;MMP-9 activity of the cell was detected by zymography.Flow cytometry used to examine the distribution of cell cycle and apoptosis of the cells.Results:MS-275 at 1uM can up-regulated RECK in MDA-MB-231 breast cancer cells.RT-PCR and Western-blot analysis demonstrated that RECK mRNA and protein were increased after treatment of MS-275.Moreover,up-regulation of RECK expression by MS-275 attenuated MMP-9 activity in gelatin zymography analysis. The proliferation of the breast cancer cell was inhibited by MS-275 at 1 to 2.5 uM time and dose-dependently,cells were retarded at the stage of G2/M,G1.Conclusion:In breast cancer cell,some oncogenes represses RECK expression via a histone deacetylation mechanism.HDAC inhibitor MS-275 can potently antagonize the inhibitory action of oncogenes on RECK,also can induce apoptosis and cell cycle arrest.PartⅣObjective:To investigate the effects of MS-275 on the RECK,MMP-9 expression and invation ability of MDA-MB-231 breast cancer cell.Methods:Different concentration of MS-275(the same as above)added into cell culture fluid.Cancer cell's Invasion ability was tested with Transwell chambers.Results:The invasion ability of cancer cells decreased in MS-275 treated group(1.0uM or more).In matrigel chambers,the number of cells transfer through the gel is 186±16.4(MS-275 treated group)VS93±9.5(control group).Conclusion:MS-275 is another novel class of drugs which can restore RECK expression in human cancer cells to inhibit MMP activation and tumor metastasis.PartⅤObjective:To explore the suppressive effect of MS-275 on transplanted human breast cancer in nude mice and its effect on the expression of tumor suppressor genes—RECK in breast cancer tissues.Methods:18 nude mice were transplanted with human breast cancer MDA-MB-231 cells to construct tumor models,MS-275 were injected into the tumor or around the tumor,A total of 4 injections were given,once every 3 days,the control group nude mice were injected DMSO or without treatment.Observations were made on tumor suppression rate,tumor volume and changes in weight of the animals. western blot was used to detemine the expression of RECK and MMP-9 protein in the 3 groups,their mRNA level were measured by RT-PCR.HDAC1 protein was measured by immuno-histochemical assays.Results:The subcutaneous tumor model in nude mice was successfully established.The tumor volume in the group of MS-275 treated decreased during treatment;while the tumor volume of the other two groups increased.(250±15mm~3 VS 680±26m~3)(P＜0.05)The expression of RECK mRNA and Protein in MS-275 group increased significantly(P＜0.05).HDAC1 protein was down-regulated in transplanted human breast cancer in nude mice of MS-275 treated group than control group.Conclusions:MS-275 can effectively suppress human breast cancer cells growth rate in subcutaneous tumor of nude mice and up-regulate the RECK expression in transplanted neuplasma.Histone deacetylase(HDAC)inhibitors can exert anti-cancer activity in vivo.