The Therapeutic Significance Of Ku-70Gene Silencing On Drug-resistant Breast Cancer

Breast cancer is the major malignant tumour that endangers the health of women,in worldwide, more than100million women are diagnosed with breast cancer,accounting for10%of the patients with cancer, accounting for23%of the femalecancer cases every year[1]. In the United States,40,000people died of breast cancerone year[2]. Breast cancer is one of the most sensitive tumor to chemotherapy in solidtumors, but tumor multidrug resistance (MDR) often leads to the failure ofchemotherapy, which has become a major factor for the lower survival rate ofpatients. Solve the MDR problem of the tumor is important for the treatment of avariety of tumors, therefore, scholars hope to understand more of cancer causes andmechanisms of multidrug resistance in tumor therapy, to provide a new target forcancer treatment.Ku protein is a heterodimeric protein, composed by Ku70and Ku80two-subunit,and was found and identified earliest in the serum of patients with autoimmunediseases, later proved to be a versatile regulatory protein involved in DNA repair,telomere maintenance, apoptosis. Thus, Ku protein is considered to play an importantrole in the chromosomal integrity and cell survival.Reports in recent years suggested that the Ku protein expression was associatedwith tumor development[3], the more expression of Ku protein promoted tumorphenotype occurred, induced cell proliferation and apoptosis resistance, on the otherhand, less expression of Ku protein leaded to genomic instability and the occurrenceof tumors, a variety of experiments concluded that Ku protein also plays dual role-atumor suppressor and tumor factors. Ku becomes an important anticancer drugcandidate targets, causing attentions of many cancer treatment researchers. rapysensitivity of cervical carcinoma cell line, Hela[4]; Pim-1gene knockout enhancedDown-regulating Ku70protein expression could promote chemothepaclitaxelinduced hormone-refractory prostate cancer apoptosis by down-regulating the expression and nuclear localization of Ku protein, inhibiting of Ku70, Ku80pairof non-homologous end-joining DNA repair[5]. Amphiregulin promoted non-smallcell lung cancer resistante to gemcitabine through inhibitiing gemcitabine-inducedacetylation of Ku70protein[6]. The materials above prompts, Ku70play an importantrole in a variety of tumor resistance to chemotherapy treatment. Ku70proteinexpression in breast cancer and correlations with breast cancer resistance has not beenreported.The purpose of this experiment is to reveal the Ku70protein expression in breastcancer and its role in the treatment of breast cancer resistance, details are as follows:1The experimental route1.1Drug resistance test of MCF7-ADR–a breast cancer cell line whichresistant epirubicin.1.1.1MTS assay detected MCF7, MCF7-ADR proliferative activity.1.1.2Observed MCF7, MCF7-ADR cell morphological differences via invertedphase contrast microscope.1.1.3Stimulated MCF7, MCF7-ADR cells with different concentrations ofepirubicin and calculated the drugs half fatality rate (IC50) and drug resistance indexof MCF7-ADR to pharmorubicin.1.2RT-PCR detected Ku-70mRNA levels in breast cancer cell line MCF7andMCF7-ADR cellsCultured human breast cancer cell line MCF7and breast cancer cell lineMCF7-ADR which resistance to epirubicin to logarithmic phase, extracted total RNA,then detected Ku-70mRNA of the two tumor cells by RT-PCR.1.3Western blot detected Ku-70protein levels of MCF7and MCF7-ADR cells.Cultured human breast cancer cell line MCF7and breast cancer cell lineMCF7-ADR which resistance to epirubicin to logarithmic phase, extracted totalprotein, transferred to a membrane after vertical electrophoresis, hybridized withanti-Ku70antibody, observed Ku70protein expression levels of MCF7ofMCF7-ADR cells via chemiluminescence imaging system after ECL.1.4Transfected Ku70SiRNA fragment into MCF7-ADR cells, RT-PCR, Westernblot analysis Ku70gene silencing results.1.5Stimulated MCF7, MCF7-ADR, MCF7-ADR/Ku70-SiRNA with differentconcentration epirubicin, MTS assay tested cell proliferative activity, then calculated the median lethal dose of epirubicin to different cell lines, compared resistant indexchanges of MCF7-ADR after Ku70silenced.1.6Observed apoptosis morphological differences of MCF7-ADR cells andMCF7-ADR cells knoched out Ku70gene cultured with doxorubicin via opticalmicroscopy and Hochest-33258staining.1.7Compared the apoptosic cells number of MCF7-ADR cells andMCF7-ADR cells knoched out Ku70gene treated with doxorubicin via flowcytometry after Annexin V-PI staining.1.8PCR-array detected apoptosis-related gene changes in MCF7-ADR cellsafter knoched out Ku70gene to investigate the possible mechanisms of Ku70genesilence reversed drug resistance of MCF7-ADR to doxorubicin.2Results2.1Drug resistance test of MCF7-ADR–a breast cancer cell line whichresistant epirubicin.MTS assay tested MCF7, MCF7-ADR cells proliferative differences, the resultsshowed no significant proliferative difference between MCF7and MCF7-ADR. Therewere no significant different between MCF7and MCF7-ADR cells as far as cell sizeand shape concernd. The two kinds of cells were nearly round, nuclei were large, andhad abundant cytoplasm. Using different concentrate epirubicin to stimulate MCF7and MCF7-ADR cells, the median inhibitory concentration (IC50) were4.16+0.72ug/ml and34.38+6.75ug/ml individely. The resistance index of MCF7-ADR cellsto epirubicin was8.26.2.2Ku-70mRNA levels in breast cancer cell line MCF7and MCF7-ADR cells.RT-PCR detected Ku-70mRNA levels of MCF7and MCF7-ADR cells, theresults showed no significant difference between two cell lines.2.3Expression difference of Ku-70protein in MCF7and MCF7-ADR cellsAnalysis Ku-70protein expression of MCF7and MCF7-ADR cells via Westernblot, the results showed there was no significant difference between two cell lines.2.4Analysised Ku70gene silencing results after transfected with Ku-70SiRNAAnalysis Ku70gene silencing results via RT-PCR and Western blot assay aftertransfected Ku70SiRNA fragment into MCF7-ADR cells24h, the results showedKu-70expression was significately less than the cells that transfected control SiRNA fragment. The results indicated that Ku-70silencing result was good and followed testcould be carried out.2.5The effect of Ku70silencing on drug resistance of MCF7-ADRMTS assay tested cell proliferative activity of MCF7, MCF7-ADR,MCF7-ADR/Ku70-SiRNA stimulated with different concentration epirubicin, theresults showed that MCF7-ADR drug resistance to epirubicin was reversed aftertransfected Ku70-SiRNA, the IC50of the three cell lines was4.16+0.72ug/ml、34.38+6.75ug/ml、13.06+2.62ug/ml individely. Epirubicin resistance index of MCF7-ADRcells decreased from8.26to3.14Ku70after transfected Ku70SiRNA.2.6Analysis epirubicin-induced apoptosis sensitivity change of MCF7-ADRafter Ku70silence through morphology observe.Observed morphology changs of MCF7-ADR, MCF7-ADR/Ku70-siRNA treatedwith10ug/ml epirubicin via Hochest-33258staining, the results showed that thenumber of MCF7-ADR transfected Ku70SiRNA decreased than the control group,MCF7-ADR transfected Ku70SiRNA was smaller and cells edge shrinked, chromatincondensated or pyknosis, obvious apoptotic bodies could be found in some cells.2.7Analysis epirubicin-induced apoptosis sensitivity change of MCF7-ADRafter Ku70silence through flow cytometry. Epirubicin-induced apoptosis sensitivityof MCF7-ADR increaced significately after transfected Ku70SiRNA, AnnexinⅤpositvie stained rate of MCF7-ADR, MCF7-ADR transfected contol SiRNA,MCF7-ADR transfected Ku70SiRNA was4.72+0.56%,4.63+0.78%and16.76+2.93%.2.8In MCF7-ADR/Ku70SiRNA cells apoptosis-related genes APAF1, BBC3,BCL2, BCL2, BIRC6, CASP3, DAPK1, TNFRSF10A expression increased andapoptosis-related genes NAIP, HUK expression decreased.3Conclusions3.1Ku70mRNA and protein levels had no significant diference betweenMCF7-ADR cells and MCF7cells, that indicated Ku70can not be a marker of breastcancer drug resistance.3.2Ku70gene silencing was able to reverse epirubicin resistance ofMCF7-ADR cells.3.3Ku70gene silencing enhanced epirubicin induced MCF7-ADR apoptosis, suggesting that Ku70-SiRNA might work as breast cancer chemotherapy sensitizer.3.4In MCF7-ADR/Ku70SiRNA cells apoptosis-related genes APAF1, BBC3,BCL2, BCL2, BIRC6, CASP3, DAPK1, TNFRSF10A expression increased andapoptosis-related genes NAIP, HUK expression decreased. Ku70gene silencingprompted expression of most apoptosis signaling molecules, indicating that Ku70gene silencing activated apoptotic signaling in breast cancer cells which resistedepirubicin, thereby increased the sensitivity ofMCF7-ADR to epirubicin and reversedthe resistance to epirubicin.