Experimental Research On Antisense-rescue Treatment Of Breast Cancer Targeting P53 Gene In Vitro And In Vivo

Objectives:There are mutation points on p53 gene exon 8 in MDA-MB-231 cells.Antisense-rescue is a double gene therapy which not only specifically block themutation point with the corresponding antisense RNA, but also add wt-p53. Based onthe results of previous experiments, this study was to investigate the arrest of cellcycles and the expression of mt-p53 protein when the MDA-MB-231 cells weretransferred antisense RNA targeting p53 exon 8(ASp53exon8' RNA) and wild typep53 (wt-p53) gene. Following that, we researched the inhibitory effect ofASp53exon8' RNA and wt-p53 for the cancer of nude mice which were inoculated byMDA-MB-231 cells and the curative effect of double gene therapy andcis-diarnminedichloroplatinum(C-DDP) treatment for the cancer of these mice.Methods:1 Antisense RNA targetting the mt-p53 exon8 was synthesized in vitro(ASp53exon8'RNA) through the single strand antisense transcription systemcontaining mt-p53 exon8 sequence constructed in our previous research.2 The breast cancer cell line MDA-MB-231 (containing mt-p53 exon 8) weretransfected with ASp53exon8' RNA and pCDNA3.1-p53 plasmid containing wt-p53gene mediated by cationic liposome; Cell cycle distribution was determined by flowcytometry (FCM) and the expression of mt-p53 protein was examined by westernblot;3 The MDA-MB-231 tumor cells were transplanted into nude mice to establish thenude mice model; 4 Nude mice models were used to explore the administer way and the dose ofantisense RNA and of C-DDP;5 At last, according to the results, we went on the experimental study on theantisense-rescue therapy and C-DDP treatment, including therapeutic efficacy andtoxicity analysis. The long and short diameter were measured regularly by verniercaliper and the volumes were calculated, at the same time the curves of the growthof cancer were drew. 21 days after therapy, when these mice were executed weobserved the change of cancer dyed by H&E and scaled the inhibitory instance ofmt-p53 protein by immunohistochemical staining.Results:1. 48h after transfection, the antisense-rescue treatment (Both the pCDNA 3.1-p53and Asp53exon8' RNA were transfected into MDA-MB-231 cells) induced: (1)The cell cycle was arrested on G₂/M phase which was clearly than the singleASp53exon8' RNA or wt-p53 transfection group, furthermore which has statisticsdifference contrast the blank group. (2) Westernblot results indicated thatthe expression of mr-p53 protein was suppressed at 84.29% on contrastgroup which also has statistical significance.2. The optimal administer way and dose of antisense RNA and C-DDP weredetermined: the dose of sense RNA, antisense RNA and pCDNA 3.1-p53 plasmid were all10 ug per nude mouse, intratumorally injection; C-DDP 3mg/kg weight, qd*3d, 3weeks as one course of treatment, intraperitoneal injection.3. The antisense-rescue treatment (The Asp53exonS' RNA and pCDNA 3.1-p53plasmid were both injected to the mice bearing cancer mediated by lipid)obtainedprospective effects:â‘ the growth of cancer was inhibited and the speed of growthslowed down;â‘¡histopathologic examination displayed completely necrosis in largearea of the tumor tissue after the antisense-rescue treatment.â‘¢the slice of cancer tissue dyed by immunohistochemistry showed the expression of mt-p53 proteinmostly turned to negative after double gene antisense-rescue therapy and showeddistinctive curative effect compared with control group;4. The antisense-rescue therapy combined with C-DDP improved the treatmenteffects: the inhibition of the growth of cancer, the tumor necrosis and mr-p53expression inhibition was more prominent after double gene therapy and C-DDPtreatment, compared with the control, especially with the C-DDP alone group.5. No toxicity was found after the antisense-rescue therapy: the general conditionsof the nude mice after antisense-rescue therapy were normal and no abnormalitywas found in their tissue of heart, brain, liver, spleen, lung, kidney, adrenal gland,stomach, intestine, skin.Conclusion:1. The antisense-rescue therapy can effectively arrest the cell cycle and inhibit theexpression of mr-p53 protein.2. The study showed that the curative effect of double gene therapy whichcontained wt-p53 gene in nude mice made by MDA-MB-231 cells andAsp53exon8'RNA antisense-rescue therapy was better than a single gene therapyor single C-DDP therapy for breast cancer.3. The antisense-rescue therapy is specific, only targeting to the tumor cells withp53 mutation.4. Double gene therapy combined with C-DDP treatment had cooperative andpromotive effect for the treatment of breast cancer.