Chemosensitization Of Solid Tumors By Inhibition Of Bcl-xL Expression Using DNAzyme

As an important therapeutic modulity, chemotherapy benefits many cancer patients. However side effects and cancer recoverence remain a significant clinical problem. Chemotherapy failure mainly results from the intrinsic and acquired drug resistance of tumor cells. Previous studies have shown that the expression level of Bcl-xL strongly correlated with chemo-resistance in the NCI60cells. DNAzyme is a new molecular tool for gene therapy. It represses the expression of target gene by cleaving mRNA at specific phosphodiester bond, which is located between an unpaired purine and paired pyrimidine. In this present study, we designed a10-23type DNAzyme with specific cleavage of Bcl-xL mRNA. This DNAzyme effectively down-regulated the Bcl-xL expression in types of cancer cell lines, thus overcame the anti-apoptotic barrier. Furthermore, it sensitized the cancer cells to taxol in xenograft animal models. In all, our data indicated that the Bcl-xL DNAzyme could be an effective chemo-adjuvant agent for potential clinical applicationsPart ⅠPurpose:To design, screen and analyze anti-Bcl-xL DNAzymes.Method:By bioinformatics analysis,81potential cleavable sites were identified and thermodynamic stability of the enzyme-substrate heteroduplex is predicted by the hybridization free energy.26potential sites were chosen for further multiplex cleavage assay. According to these targeting sites, the DNAzymes were designed based on "10-23" model, and were modified by1+1,3+3,5+5phosphorothioate (PS) linkages. Then the DNAzyme stability of three PS modification schemes were compared in human serum and an indicator for cleavage activity Kobs were measured-under a signal turnover condition.Results:The DNAzyme stability improved with the increase of number of PS linkages, but inversely the cleavage efficiency decreased. To strike a balance between the stability and catalytic activity, we selected the3+3PS modification scheme for the further study. By the multiplex selection, we got10active DNAzymes that could efficiently cleave Bcl-xL mRNA.Conclusion:In vitro we successfully tested10active DNAzyme with cleavage ability to Bcl-xL mRNA, which provided an experiment basis for the further studies in vitro and in vivo.Part ⅡPurpose:To validate the effect of Bcl-xL DNAzymes on Bcl-xL expression.Method:We transfected these10active DNAzymes into PC3cells by TMP, then tested the inhibitory effect on Bcl-xL expression. The screened DNAzyme’s activity was validated in a panel of cancer cell lines (PC3, prostate cancer; T24, bladder cancer; A549, lung carcinoma; CNE1, nasopharyngeal carcinoma; HCT116, colon cancer).Result:Three DNAzymes (DT882, DT883, and DT884) significantly declined the level of Bcl-xL protein in PC3cells (P<0.05). Together with biochemical analysis, we selected DNAzyme DT882for the further studies. The effect of DNAzyme DT882was validated in different cancer cell lines.Conclusion:DT882could be effectively transfected into the cells, find its target mRNA and inhibit Bcl-xL expression in cells.Part IIIPurpose:To test the Bcl-xL specific DNAzyme ability of apoptosis inducing and explore the mechanism. Method:DT882was transfected into PC3and CNE2R by TMP. The cells were stained by using Annexin V/PI kit, the apoptotic cells were detected by flow cytometry48h after transfection and the expression of Bcl-xL and Cytochrome C were detected by westwen blot.Result:The apoptosis rate was remarkably increased in the DNAzyme-treated cells, compared with the control-treated cells (P<0.05) in the both cell lines, the expression of bcl-xL protein was significantly reduced but the release of CytoC was increased in the DNAzyme-treated PC3cells (P<0.05).Conclusion:The DNAzyme targeting Bcl-xL induced cell apoptosis via the mitochondria pathway in PC3cells.Part ⅣPurpose:To investigate the chemosensitization effect of anti-Bcl-xL DNAzyme. Method:(1) DT882was transfected into seven cell lines driving from different tissues by TMP. The treatment was divided into seven groups: antisense control (AS), DNAzyme group (DT882), negative control (Scr Ctr), Taxol group and Taxol plus AS (AS+Taxol), Taxol plus DT882(DT882+Taxol), Taxol plus ScrCtr (ScrCtr+Taxol). The cell survival rate was measured by MTS assay. Cell death (%) was calculated as (untreated-treated)/untreated. Sensitization index was expressed as [(DT882+Taxol)-Taxol]/Taxol x100.(2) CNE2R. cells was seeded into96well plate at a density of3*103.The next day the cells was transfected with DNAzyme by lip2000, then treated with Taoxl at concentration of0nM、10nM、20nM and40nM, the survival of cells was tested by MTS assay48h after transfection. The survival rate of cells was calculated as treated/untreated, and plotted the survival curve.(3) Animal experiment:The PC3cells were transplanted into female Balb/c nude mice. When tumours reached an average volume of20-30mm3, an Alzet osmotic pump, was surgically implanted in the peritoneum of the mouse via the abdominal route. The mice were randomed seven groups (8mice per group) and treated respectively by Saline, DNAzyine, AS, Control, Taxol, Taxol+DNAzyme, and Taxol+AS. DNAzyme was delivered at a rate of12.5mg/kg/d over a period of14days. Then25mg/kg taxol was injected into abdominal cavity once a week post-surgery during the study period.(4) To analyze the in vivo stability of DNAzyme and the pump delivery efficiency, the DNAzyme oligonucleotide was extracted from tumor tissues, plasma and the pumps in the mice treated with DT882plus Taxol, and lablled with γ-32P-ATP by T4polynucleotide kinase and assayed on PAGE.Result:(1) The sensitization index was increased for over140%in the cancer cells treated with DT882, and for266%in MDA-MB-231cells.(2) The Taxol treatment in a range of concentrations did not cause detectable cell death of the resistant cells or the control-treated resistant cells, but DT882treated resistant cells shown a significant reduction in cell survival.(3) The combination of DT882and Taxol remarkedly inhibited PC3tumour growth compare with DT882, Taxol alone or control DNAzyme plus Taxol.(4) PAGE analysis showed that the remaining DNAzyme was the highest in plasma, followed by tumour tissues and Alzet osmotic pump. Conclusion:DNAzyme targeting Bcl-xL DT882sensitized the Taxol-resistant cells to chemotherapeutics in vitro, promoted the antitumor effect of taxol in vivo. Furthermore, DT882was stable in the animal body. These result generalized that DNAzyme is a useful tool for chemosensitization in the clinic.