Experiment Study On Breast Cancer Therapy Combining Ad-p16 And Ad-p53 With Tax

Part 1Study on breast cancer p16 and p53 gene protein and mRNA expressionObjectives Breast cancer ranks the top among the malignant tumors of women. About 400,000 people die of breast cancer all over the world every year. Meanwhile, the incidence of breast cancer has been increasing annually and breast cancer patients tend to be younger. A great deal of research shows that abnormality of oncogene and tumor suppressor gene (antioncogene) is closely related to the occurrence and development of breast cancer, such as p16, p53, C-cerbB-2 and p21 genes, p16 antioncogene mutation and /or deficiency is widely available in various tumors, is involved in regulation of tumor cell proliferation and plays an important role in the occurrence and development process of tumor. Through immunohistochemical method, we found out that the positive rate of p16 gene protein expression varied between 24% and 60%. p53 antioncogene mutation is the most popular in the human tumors, and p53 is the gene with the closest relation with the human tumors according to the existing researches. It is known that the p53 gene mutation rate in breast cancer tissue is 15%～60%. Through testing the breast cancer tissue p16 and p53 gene protein and mRNA expression, we analyzed the correlation between the expression and the clinical pathological characteristics of breast cancer and explored the effects of the expression on the molecular biological characteristics of breast cancer to provide a theoretical foundation and basis for treatment and prognosis assessment of breast cancer in clinical and basic researches.Methods We tested p16 and p53 gene protein and mRNA expression within the breast cancer tissue through immunohistochemical SP method and reverse transcription polymerase chain reaction (RT-PCR) technique and analyzed the relationship between the expressions and the clinical pathological characteristics of breast cancer through statistical methods.Results Through immunohistochemical method we found out that the expression rate of p16 gene protein within the tumor-adjacent normal breast tissue was 90%(27/30), including 48.1%(13/27)(+++); 29.6% (8/27)(++); 22.2%(6/27)(+) ; the expression rate of p53 gene protein was 6.67% (2/30) , all as (+) ; while the positive rate of p16 gene protein expression within the breast cancer tissue was 38.3% (23/60) , including 60.9% (14/23) (+++), 26.1% (6/23) (++) and 13.0%(3/23) (+) ; the positive rate of p53 gene protein expression was 48.3% (29/60) , including 79.3% (23/29) (+++) , 13.8% (4/29) (++) and 6.9% (2/29) (+) .The results of RT-PCR test showed that pl6mRNA of tumor-adjacent normal breast tissue was obvious higher than that of breast cancer tissue (P=0.023), while p53mRNA of the breast cancer tissue was significantly higher than that of tumor-adjacent normal breast tissue (P=0.001) . Expression of pl6 and p53 proteins is closely related to histological grade of breast cancer tissue, and lymph node and/or organ metastasis. The higher the cell differentiation of breast cancer is, the higher the positive rate of p16 protein expression. The p53 expression rate was on the contrary. The cases with lymph node and/or organ metastasis showed significantly lower expression rate of p16 protein than that of those without lymph node and / or organ metastasis, while the expression rate of p53 protein in metastasis cases was significantly higher. p16mRNA at histological grade I was obviously higher than that at grade II and III, but no obvious difference was observed between the p16mRNA level at grade II and grade III. No obvious relation was found between p53mRNA and histological classification. The p16mRNA and p53mRNA expression rates for cases with lymph node and /or organ metastasis were obviously higher than those for cases without lymph node and / or organ metastasis.Conclusion (1) Expression of p16 and p53 gene proteins is closely related to histological grade of breast cancer, and lymph node and/or organ metastasis. (2) Expression of pl6mRNA is related to histological grade of breast cancer. Expression of pl6mRNA and p53mRNA is related to lymph node and/or organ metastasis. (3) Deficiency, mutation or methylation, etc of p16 and p53 genes is closely related to the occurrence and development of breast cancer and can be used in prognosis assessment of breast cancer. (4) Exogenous wild-type p16 and p53 genes can be transfected through specific vectors to correct the abnormal changes of p16 and p53 genes and contribute to the treatment of breast cancer.Part 2Construction, identification, purification and determination of recombinant pl6 and p53 adenovirusObjectives The study shows that the occurrence and development of malignant tumor is closely related to gene mutation, deficiency, phosphorylation, methylation, etc, and gene mutation plays an important role in malignant tumor cell phenotype. It is shown that transfection of exogenous normal target genes can be used to modify the mutant genes and serves as a tumor therapy. The key to designing a gene therapy is to construct an effective target gene vector system for specific transfer of target genes to target cells. The transfection efficiency of non-virus vectors within the body is far lower than that of virus vectors. Adenovirus vectors can effectively infect and transfer the target genes, and are widely applicable to both dividing and static tissues and cells, and the adenovirus DNA does not integrate with DNA of host cells. The disadvantages of adenovirus vectors include severe inflammation and immunoreaction if continuous expression of transgenes is maintained. But such immune characteristics may enhance the effects of target genes on killing tumors. Adenovirus vectors are easily constructed and a large number of adenovirus vectors may be cultured through helper cells. In this study, gene recombinant adenovirus method was adopted to construct recombinant adenovirus type 5 (Ad5) of sub-group C of antioncogene p16 and p53, which were identified, sifted and purified. Human embryonic kidney 293 cells were used for amplification, and the purification rate, virus titer, infection rate, transfection rate, and amplification of recombinant adenovirus p53 (Ad-p53) , p16 (Ad-p16) , as well as expression of p53 and p16 protein after cell infection was determined to provide sound recombinant adenovirus vectors for future experiments, namely Ad-p16 and Ad-p53.Methods HindIII and SpeI enzymes were used for double enzyme digestion of pBluescript-p16 plasmids containing wild type p16cDNA; KpnI and XaI enzymes were used for double enzyme digestion of pGEM-p53 plasmid containing wild type p53cDNA, and then p16cDNA and p53cDNA were recovered through electrophoresis. HindIII/KpnI and SpeI enzymes were used for double enzyme digestion of pAdCMV shuttle plasmid and the recovered pl6cDNA and p53cDNA were then inserted into directional pAdCMV to get pAdCMV-p16 and pAdCMV-p53. Homologous recombination between pAdCMV-p53 recombinant plasmid and adenovirus plasmid pJM17 was made within 293 cells and p53 gene was transferred into the adenovirus gene. The generated El gene-defective recombinant adenovirus multiplied within the 293 cells and proliferated vigorously under transacting effects of El gene products. Adenovirus-, p16-, p53-specific primers were adopted for amplification and PCR amplification products were analyzed through agarose gel electrophoresis. Based on the length of specific fragments of PCR amplification products, the recombinant adenovirus plaques with positive p16cDNA, p53cDNA were selected. The virus in the positive plaques proliferated through subculture 293 cells. The viruses were purified through ultracentrifuge with cesium chloride density gradient. The viruses re-multiplied within 293 cells up to 5 generations to increase titer. 293 cell plaque formation testing was adopted to measure the recombinant adenovirus titer. Reporter gene Lac Z was used to measure the recombinant adenovirus infection rate. Immunohistochemical method (SP method) was used to test the efficiency of recombinant adenovirus-mediated gene transfer, and Western blot method was used to analyze expression of p53 and p16 protein.Results pl6cDNA, p53cDNA were inserted into pAdCMV to get pAdCMV- p16, pAdCMV- p53. Through multiplication within 293 cells, cytopathic effect (CPE) , namely, typical virus plaque occurred after 10-14 days. Then we saw through a microscope that 293 cells rounded up, shrank and fell off, indicating homologous recombination between pAdCMV-pl6/p53 recombinant plasmids and adenovirus plasmid pJM17within 293 cells, p16/p53 gene transferred into adenovirus gene and the upper clean solution of culture media containing the recombinant adenovirus plasmids, with the prepared recombinant adenovirus respectively named Ad-p16, Ad-p53. From PCR amplification, there were 860bp adenovirus DNA segment, 570 bp p16cDNA segment, 642 bp p53cDNA segment, namely, the recombinant p53, pl6 adenovirus. The titer calculated after the plaque formation test was Ad-p53 2.1×10¹⁰; Ad-p16 6.7×10¹⁰; Ad-LacZ1.0×10¹⁰. When MOI of Ad-Lac Z was 25, 50 and 100, the infection rate of 293 cells was 32%, approached 78.9% and reached 98.6% respectively. Therefore, it is feasible to use recombinant adenovirus with MOI≥100 to infect human breast cancer MDA-MB-231 cell strain to observe the gene expression and apoptosis. Immunohistochemical staining results showed the recombinant adenovirus mediated gene transfection rate of pl6 being 93.8±2.5% and of p53 being 96.3±3.8%. No p53 or p16 protein expression was available in Ad-Lac Z infected tumor cells. However, tumor cells infected by both p53 and p16 showed obvious expression of p53 and p16 proteins, indicating that recombinant adenovirus could effectively mediate highly efficient transfer of exogenous genes.Conclusion (1) The recombinant adenovirus can properly carry target genes into target cells and accurately express target gene protein. (2) It is economical to produce recombinant adenovirus vector with high titer. (3) The therapeutic dosage volume of virus is small and will be good for applications within the human body.Part 3 Study on breast cancer therapy combining Ad-p16 and Ad-p53 with TaxObjectives Normal cells rely on checkpoint function to regulate cell cycle progression and maintain balance between cell proliferation and apoptosis. We found that both oncogene and antioncogene act through regulation of cell cycle. Occurrence of tumor often involves mutation, phosphorylation and/or inactivation of a number of antioncogenes. It has become a popular tumor gene therapy to transduce normal tumor suppressor genes into tumor cells to replace dysfunctional genes to reverse malignant phenotype and inhibit tumor growth and even eliminate tumor, p16 and p53 are important tumor suppressor genes in human body and directly or indirectly regulate cell cycle to cause stasis in G1 stage. Deficiency, mutation, rearrangement, phosphorylation or non-expression of p16 and p53 genes may be present in the occurrence and development of various tumors. It has become an important way of anti-tumor gene therapy study to restore the functions of wild type p16 and p53 genes. Tax may induce apoptosis of various tumors, and is especially effective in treatment of metastatic breast cancer. In this study, in-depth research was conducted over the effects of Tax, Ad-p16, Ad-p53 and their combination on growth inhibition and cell apoptosis of human breast cancer cell strain MDA-MB-231. Then Tax, Ad-p16, Ad-p53 and their combination was applied to breast cancer cell MDA-MB-231 in the tumorigenesis model of nude mice for an animal therapy experiment. The therapeutic effects were observed to provide basis for tumor therapy and therapeutic approaches.Methods The effects of Ad-p16, Ad-p53 and their combination on morphology and growth inhibition of MDA-MB-231 cells were observed through a microscope. MTT method was used to monitor and measure the growth inhibition rate of MDA-MB-231 cells applied with Tax, Ad-p16, Ad-p53 or their combination. The flow cytometer Annexin V-FITC/PI method was applied to test and analyze the apoptosis rate of MDA-MB-231 cells after application of Tax, Ad-p16, Ad-p53 and their combination. The flow cytometer was used to analyze the effects of Tax, Ad-p16, Ad-p53 and their combination on cell cycle and apoptosis of MDA-MB-231. MDA-MB-231 cell suspension was injected into the mammary fat pad of nude mice to form a breast cancer model in the nude mice. Ad-Lac Z, Tax, Ad-p16, Ad-p53, Tax+Ad-pl6, Tax+Ad-p53, Ad-pl6+ Ad-p53 and Tax+Ad-p16+Ad-p53 was used as a therapy for tumorigenesis in nude mice, and the changes in tumor size and tumor inhibition rate were observed and then the therapeutic effects were analyzed. In addition, routine pathological examination was made over the tumor specimens after treatment. Terminal deoxynucleotide transferase (TdT) mediated dUTP-biotin nick end labeling (TUNEL) method was used to test and analyze the apoptosis of tumor tissue cells. SPSS11.0 statistical software package was used to analyze the experiment data.Results Ad-p16, .Ad-p53 or their combined infection can inhibit growth of human breast cancer cell MDA-MB-23. Ad-p16 was more effective in inhibiting cell growth than Ad-p53, but with morphologic changes occurring later. Combined Ad-p16 and Ad-p53 infection may almost stop cell growth, with obviously more effective cell growth inhibition and earlier morphologic changes compared with Ad-pl6 or Ad-p53 infection. In case of Ad-Lac Z infection, the cell grew well without obvious morphologic changes. With increasing dose of Tax, the inhibition effects on MDA-MB-231 cells increased with obviously higher inhibition rate and positive correlation between cell inhibition rate and time (r=0.923, P=0.000) . Compared with Ad-Lac Z, Ad-p16, Ad-p53 or their combined infection was obviously effective in inhibiting the tumor cells (P=0.000 in all cases) . The inhibition rate of Ad-p16 ]was higher than that of Ad-p53 in all the time phases (P_24h=0.038, P_48h=0.004, P_72h=0.003, P_96h=0.001, P_120h=0.000) . The inhibition effects of Ad-p16 tended to go up gradually, while the inhibition effects of Ad-p53 tended to go down after reaching the peak value at 72h, but without statistical significance in both cases. In case of combined Ad-p16 and Ad-p53 infection, the inhibition rate of MDA-MB-231 cells was higher than that of Ad-p16 or Ad-p53 infection in all the time phases (vs Ad-p16, P = 0.001-0.004; vs Ad-p53,P=0.000); and obviously increased with time( 120h vs72h, P=0.006) , indicating obvious positive correlation with infection time (r=0.89, P=0.001) . After application of combined Tax and Ad-p16 into MDA-MB-231 cells, the cell growth inhibition rate was notably higher than that of Tax or Ad-p16application in all the time phases and reached the peak value at 96h up to 70.96% (70.96±3.30) (vsAd-p16,P=0.001;vsTax,P=0.000) ; and notably increased with time (120h vs 72h,P=0.028) . Same effects were observed in case of combined application of Tax and Ad-p53 but without obvious difference of inhibition rate in various time phases (P = 0.069-0.86) compared with the inhibition rate in case of application of Tax and Ad-p16. Inhibition of MDA-MB-231 cells was more obvious in case of combined application of Tax, Ad-pl6 and Ad-p53, and increased in all the time phases (P = 0.000 in all phases) with the highest inhibition rate up to over 90%. Application of Tax, Ad-p16 or Ad-p53 to MDA-MB-231 cells may induce cell apoptosis and the apoptosis rate obviously increased with time and reached 15.31%, 19.34% and 29.05% respectively at 72h. However, Ad-p16-infected MDA-MB-231 cell apoptosis occurred later than apoptosis of Tax- or Ad-p53-infected cells with apoptosis rate for Ad-p16 obviously lower than that for Ad-p53. Application of combined Tax and Ad-p16 or Ad-p53 can obviously increase the apoptosis rate of MDA-MB-231 cells, and combined application of Ad-p16 and Ad-p53 can also increase apoptosis of MDA-MB-231 cells. Application of combined Tax, Ad-p16 and Ad-p53 obviously improved the ability to induce MDA-MB-231 cell apoptosis with more notable effects. Tax may cause MDA-MB-231 cell stasis in G2/M stage, while application of Ad-p16, Ad-p53 or their combined infection may cause MDA-MB-231 cell stasis in G0/G1 stage. Application of combined Tax, Ad-p16 and Ad-p53 caused stasis of most cells in G0/G1 and G2/M stage. In case of application of Tax, Ad-p16, Ad-p53 or their combination into MDA-MB-231 cells, 72h agarose gel electrophoresis showed typical apoptosis trapezoidal belt, while the control group only showed gene group DNA. The tumors in the nude mice were obviously inhibited for Tax, Ad-p16 and Ad-p53 group, and P = 0.000 for all cases at the 20^th day compared with Ad-Lac Z group. Although the tumor size decreased for all groups compared with pre-treatment but without statistical significance (P = 0.216, 0.895, 0.138 respectively) . Application of combined Ad-p16 and Ad-p53 can both inhibit tumor growth and obviously reduce tumor size (0d vs 20d,P=0.034; 0d vs 20d,P=0.007). Application of combined Tax, Ad-pl6 and Ad-p53 more obviously reduced tumor size (0d vs 12d, P=0.041; 0d vs 16d, P=0.003; 0d vs 20d, P=0.000) . For the therapy group, tumor tissue stroma increased obviously and the tumor cells and nuclear division decreased notably with light blue nucleus, decreasing ratio between nucleus and cell plasma and scattered crescent chromatin and nucleus fragments visible. The tumor tissue cell apoptosis rate was 1.4±0.9%, 14.7±2.6%, 19.5±2.5%, 21.3±3.1%, 30.5±3.2% and 42.3±4.3 respectively in case of application of Ad-Lac Z, Tax, Ad-p16, Ad-p53, Ad-p16+Ad-p53 and Tax+Ad-p16+Ad-p53.Conclusion (1) Ad-p16, Ad-p53 or combined application of both can contribute to morphologic change and growth inhibition of oncogenic MDA-MB-231 cells, while this is due to the functions of exogenous p16 and p53 genes, instead of adenovirus virus. (2) There is obvious synergetic effect of Tax, Ad-p16 and Ad-p53 in inducing apoptosis of MDA-MB-231 cells, expression of p16 and p53 genes can enhance sensitivity of Tax to MDA-MB-231 cells, and p16 genes can contribute to apoptosis induced by p53 genes. (3) Tax can cause MDA-MB-231 cell stasis in G2/M stage, while Ad-p16 infection, Ad-p53 infection or combined Ad-pl6 and Ad-p53 infection can cause MDA-MB-231 cell stasis in G0/G1 stage. Action of combined Tax, Ad-p16 and Ad-p53 causes stasis in G0/G1 stage and G2/M stage of most cells. (4) Tax, Ad-p16 and Ad-p53 have synergetic effects of inhibiting tumor growth in Balb/c nu/nu, and the synergetic effects of combined Tax, Ad-p16 and Ad-p53 are more obvious. Besides inhibiting the tumor growth, they can also cause apoptosis of tumor cells.