Effects Of Proliferation Inhibition And Apoptosis Induction By Apoptin On Transitional Cellcarcinoma Of Bladder

Transitional cell carcinoma of bladder(TCCB)is the most common malignant tumor in urinary system, and the morbidity rate is very high. Operation, radiotherapy, chemotherapy and immunotherapy are predominant in the clinical treatment of TCCB. Although these means can visibly prolong the survival time of patients, but long-term efficacy and quality of life are not satisfactory owing to its multiple and high recurrence. Furthermore the patients with TBBC in intermediate and advanced stage would always lose the opportunities of operation. As a promising way to cure carcinoma, gene therapy got a late start but have a bright future. In recent years, with the development of molecular biology and cell biology, various oncogenes and tumor suppressor genes were discovered, gene therapy have made great progress and got gratifying achievement. Tumor targeting is the key point of gene therapy, which would kill tumor cells effectively but not kill or interfere normal cell. Tumor targeting insufficient is the bottle neck of gene therapy for clinical application. Genes with the role of anti-tumor selectively would be an ideal choice for tumor gene therapy.Apoptin, which contains 121 amino acids, is encoded by VP3 gene from chicken anemia virus. Its molecular weight is 13.6 KD and it has no homology with any known proteins. Studies show that apoptin could selectively induce apoptosis in tumor cells such as ovarian cancer, liver cancer, malignant lymphoma, while leaving normal cells such as lymph, derma, epithelium, endothelium and other normal human diploid cells intact, especially the diploid cells which are extremely sensitive for chemotherapy and radiotherapy, such as CD34+ hematopoietic stem cells derived from bone marrow, mesenchymal stem cells and lymphocytes were not interfered by Apoptin either, and no toxicity of apoptin had been found with different administration. All of these indicate its prospective anti-tumor value. Therefore, eukaryotic expression plasmid which contains wild type apoptin gene and report gene of EGFP was constructed, and the recombinant plasmid was transfected into transitional cell carcinoma of bladder cell line T24 by mixed with lipofectamin 2000. The therapeutic effects to T24 were detected in vivo and in vitro.Object1. To investigate the role of Apoptin on the proliferation, apoptosis and cell cycle of T24.2. To investigate the role of apoptin in the athymic mouse human bladder tumor transplantation tumor model.3. To investigate the synergistic interaction of apoptin and chemotherapy drug cisplatin on T24 cells.Materials and Methods1. Construction and identification of eukaryotic expression plasmid pApoptin-EGFP: We designed primers according to the wild-type appoptin cDNA sequence from GenBank. Wild type apoptin gene was prepared by PCR from plasmid p3×flag-m-Apoptin-myc which contains a variant-type apoptin gene, then the wild-type apoptin gene was directional cloned to the vector pEGFP-N1. The recombinant plasmid pApoptin-EGFP was identified by double enzyme digestion and sequence.2. The apoptosis effect induced by Apoptin in T24 cells: The identified plasmid pApoptin-EGFP was transfected into T24 cells with Lipofectamine 2000. Apoptin mRNA was detected by RT-PCR. The fusion protein apoptin-EGFP was observed by fluorescent microscope. The proliferation inhibition induced by apoptin in T24 cells were detected by MTT. The morphous and apoptosis of transfected T24 cells was observed by using apoptosis optical kit. FCM was used to detected apoptosis and cell cycle of T24 cells.3. The therapeutic effect of Apoptin on bladder xenograft tumor: Athymic mouse T24 xenograft tumor model was established by inoculating T24 cells subcutaneously. pApoptin-EGFP parceled with liposome was injected into the tumor, when the diameter of xenograft tumor is about 0.5cm, empty plasmid and normal saline were injected as control. Tumor formation, tumor growth, tumor size, athymic mice weight and activity status were observed. The volume of transplant tumor was measured and recorded continually, The mice were sacrificed five weeks later, tumor-inhibition rate was evaluated and the apoptosis rate of cells was detected by in situ TUNEL. 4. Synergistic killing effects of Apoptin and cisplatin (DDP) on T24 cells: Concentration gradient cisplatin was administrated to T24 cells which transfected with pApoptin-EGFP. 24 hours later, the proliferation of T24 cells was detected by MTT and the apoptosis of T24 cells was assayed by flow cytometry.Conclusion1. Wild-type apoptin gene was amplified by PCR successfully and the product was correctly cloned to vector pEGFP-N1 between the enzyme site of EcoRⅠand BamHⅠ. An integrated reading frame was constructed consist of apoptin gene and EGFP gene. With no frame shift mutation, the recombinant plasmid pApoptin-EGFP would express apoptin-EGFP fusion protein in theory.2. Wild-type Apoptin mRNA could be amplified from the transfected T24 cells by RT-PCR method and the fluorescence of EGFP could be observed by using fluorescence microscopy. Both indicated fusion protein could be express in T24 cell. The results of MTT showed that Apoptin could inhibit the proliferation of T24 cells. The inhibition rates were 19.4%, 37.5%, 49.5% respectively at the time-point of 24h, 48h, 72h after transient transfection. Analyzed with student's t test, the mean of proliferation rate on T24 cells showed significant difference between pApopotin-EGFP group and the control. The morphology of T24 cells showed typical apoptosis changes detected by staining with apoptosis detected kit. The apoptosis rate of T24 was 21.25%, much higher than that of the control at the time-point of 48 hours post transient transfection. Transfected cells tagged with APC and PI were analyzed by FCM, the results indicated that the early apoptotic rate of pApoptin-EGFP group could reach 18.3%, while empty plasmid group only 4.7%, significant difference was found between the two groups. Furthermore, the advanced apoptosis or necrosis of Apoptin group was 17.8% compared to 1.6% in the control group, significant difference could be found either. All these suggested that Apoptin play an important role which could obviously induce apoptosis on bladder tumor cells. FCM was performed to study the cell life cycle at different time-point post transfection. We found that the percentage of S phase is reduced and the G1/G2, G2/M phase arrested 24 hours post the transfection, as the time go on, these changes become weaken and the cell cycle is nearly to the cells without transfection.72 hour later. In contrast, pApoptin-EGFP group got S phase reduced at 24h post transfection and as the time go on, these changes become strength, especially at the 72 hours time-point, the percentage of S phase have been reduced from 46.8% to 28.5%, both G1/G0 and G2/M phase were arrested.3. T24 xenograft tumor model was established successfully. Compared with the control group, the Athymic mice that injected with pApoptin-EGFP got better status and weight gain, without any cachexia syndrome. The tumor weight of pApoptin-EGFP, pEGFP-N1 and NS groups are 0.26±0.08g, 0.83±0.20g, 1.02±0.23g respectively. There are significant difference between pApoptin-EGFP group and the control, No statistical difference have been found between the pEGFP-N1 group and the NS group. The tumor inhibition ratio of pApoptin-EGFP group was 74.5% and pEGFP-N1 group was 18.6% as contrast. xenograft tumor tissue slice stained by H.E, the tissue was identified to be TCCB. The apoptosis rate of the three group (pApoptin-EGFP, pEGFP-N1, NS) were (23.24±6.12)%, (3.52±1.20)%, (1.76±0.44)% respectively detected by in situ TUNEL. There are significant difference between pApoptin- EGFP group and the control.(P<0.01). It suggested that Apoptin as exogenous gene product could inhibit TCCB in vitro and no side effects had been found in our experiment.4. The proliferation inhibition ratio of the three groups(DDP,pEGFP-N1+DDP,pApoptin-EGFP+DDP) on T24 cells increased as the concentration of DDP raise. Inhibition ratio of each dose point between pApoptin-EGFP+DDP and the other two groups had been found to be significant different by using statistical test, except 64ug/ml dose point because of the curve of inhibition ratio had been reach platform. The IC50 of each group(DDP,pEGFP-N1+DDP, pApoptin-EGFP+DDP) were 10.61ug/ml, 7.9ug/ml and 2.4ug/ml respectively. Calculated by using Jin-formula, we found pApoptin-EGFP and DDP had synergistic effect on inhibiting the proliferation of T24 cells. In contrast inhibition effects of pEGFP-N1and DDP on T24 cells were only simple addition. Same result was found for the apoptosis induction on T24 cells between the pApoptin-EGFP and DDP.Conclusions1. The recombinant plasmid pApoptin-EGFP was constructed successfully which could express the apoptin-EGFP fusion protein in transitional cell carcinoma of bladder cell lines T24.2. Compared to the empty group, the Apoptin gene could significantly inhibit the T24 cells'proliferation , induce apoptosis and make cell life cycle arrested in G1/G0 and G2/M stage.3. Apoptin can inhibit the growth of xenograft transitional cell carcinoma of bladder by inhibiting proliferation and inducing apoptosis, no side effects were observed during the treatment.4. Apoptin recombinant plasmid transfection combined with cisplatin could enhance proliferation inhibition and apoptosis induction effects on T24 cells, and the two therapies have synergies.