| Background:The inhibitor of apoptosis proteins (IAPs) are a family of highly conserved cell apoptosis inhibitors that have been found in yeast, invertebrates and vertebrates. The basal level of apoptosis is tightly controlled by endogenous IAPs in mammalian cells. The dysregulation of IAPs expression results in Tumorigenesis. Up to now, eight IAP-family members have been identified in human's cells: NAIP, c-IAP1 (MIHB, HIAP-2), c-IAP2 (HIAP-1, MIHC, API2) .XIAP (hILP, MIHA, ILP-1), Survivin, BRUCE (apollon), ILP-2 and Livin (ML-IAP, KIAP). Livin is one of the novel human IAPs family members, which encodes negative regulatory proteins that prevent cell apoptosis. It was expressed during the development of embryonal. Interestingly, it was not detected in human adult tissues but expressed in the transformed cell lines and a variety of human tumors, including melanoma, bladder cancer, renal cancer, lung cancer, neuroblastomas and leukemia. It could be involved in carcinogenesis and it was important for cell survival in carcinomas. Clinical experiments also demonstrated that livin may be involved in the progression of superficial bladder cancer and used as a marker of early recurrence. In 30 patients with bladder cancer, Livin expression was measured by reverse transcription-PCR. In this study, patients with increased Livin had a shorter time to relapse compared with patients without increased Livin expression (3.5 versus 25.8 months).Our former study was to detect of Livin expression in urine of bladder cancer patients by using RT-PCR. We found that 42 of 52(86.5%)patients with bladder cancer showed positive Livinαsignals in urine,while none showed positive Livinβsignals in urine;none showed positive Livinαand Livinβsignals in urine from non-cancerous patients or healthy volunteers.It shows that Livinαis applicable to detect cancer cells in urine of patients with bladder cancer and may be helpful in screening bladder cancer.Objective:To investigate the effect of antisense o1igonuc1eotides targeting livin on the inhibition of livin mRNA and protein expression and apoptosis in human bladder cancer cells in vitro and in vivo. To discuss the mechanism of inhibition effect to bladder cancer cells apoptosis by Livin gene. To investigate the potency of Livin gene as a new target for bladder cancer's treatment.Methods:1. Specific phosphorothioate antisense oligodeoxynucleotide targeting Livin was synthesized, and phosphorothioate missense oligodeoxy- nucleotide was synthesized as control nucleotide. And were transfected into bladder cancer cells.2. The inhibiton effect of Livin antisense oligodeoxynucleotide (ASODN) on the biological effect of bladder cancer cell line (5637).(1)Transfected Livin ASODN into bladder cancer cells in different concentrations. The inhibition of the proliferating of 5637 cells was assayed by MTT method.(2) Investigate the effect of antisense o1igonuc1eotides targeting livin on the inhibition of livin mRNA and protein expression by RT-PCR, Western blot and immunofluorescence in 5637 cells.(3)Biological effect of Livin ASODN on 5637 cells①Morphology of 5637 cells transfected with Livin ASODN was observed by electromicroscope and confocal laser scanning microscope (CLSM).And the expression and location of livin were observed by CLSM.②Apoptosis rate of 5637 cells transfected with Livin ASODN was assayed by AO/EB stain and flow cytometry.3. Investigate the inhibitory effects of Livin ASODN on development and growth ability of Human bladder cancer cells xenograft in nude mice and it's mechanism.(1) A human bladder cancer animal model was set up by hypodermic injection of 5637 cells into nude mice.(2) The tumor-bearing mice were randomized into 2 groups: ASODN group , and MSODN group. The o1igonuc1eotides were separately injected into the lumor bodies of mice.(3) Investigate the effect of livin ASODN on the inhibition of livin mRNA and protein expression by RT-PCR, Western blot and immuno- histochemistry of human bladder cancer cells xenograft in nude mice.(4) Biological effect of Livin ASODN on xenograft in nude mice.①The tumor weight and volume were measured.②Morphology of xenograft in nude mice was observed by microscope.③Apoptosis index of xenograft in nude mice was assayed by TUNEL stain.④The expression of caspase-3 of xenograft in nude mice was detected by immunohistochemistry.Results:1. Livin ASODN was transfected into bladder cancer cells, the transfection rate was about 55%.2. The effect of Livin ASODN on the inhibition of livin mRNA and protein expression.The expression of livinαand livinβin 5637 cells was detected by RT-PCR and Western blot.After the transfection of Livin ASODN, the expression of livin mRNA and protein was decreased (P<0.01), which was detected by RT-PCR and Western blot.In RT-PCR procedure and gel electrophoresis, the luminosity ratio of livin strip and GAPDH strip were represented the intensity of the expression of livin. The luminosity ratio of Livin ASODN group was 0.46±0.05, and the control group was 0.88±0.05 (MSODN group), 0.92±0.04 (Lipo group), 0.91±0.06 (PBS group) respectively.In Western blot procedure, the luminosity ratio of livin strip was smaller than the control group too.The fluorescence signal of livin observed by CLSM was significantly decreased.3. Biological effect of Livin ASODN on 5637 cells①Livin ASODN could inhibit the proliferating activity of bladder cancer cells, and the inhibiting effection depended on the concentration of Livin ASODN.②Under electromicroscope and confocal laser scanning microscope, the morphology changes of 5637 cells transfected with Livin ASODN was: intumesce, degeneration, cellular organ swelling, some of cells necrosis and to form apoptotic body.③The apoptosis rate of 5637 cells transfected with Livin ASODN was increased assayed by AO/EB stain and flow cytometry. The apoptosis rate was (46.39±9.23)%( ASODN group), (5.10±1.56)% (MSODN group), (5.70±1.61)% (Lipo group), (4.54±1.84)% (PBS group), P﹤0.01.4. Experimentation of xenograft in nude mice(1) The effect of livin ASODN on the inhibition of livin mRNA and protein expression.The expression of livin mRNA and protein detected by RT-PCR, Western blot and immunohistochemistry of human bladder cancer cells xenograft in nude mice was decreased.In RT-PCR and Western blot procedure, the luminosity of livin strip was smaller than the control group.The expression of livin detected by immunohistochemistry of human bladder cancer cells xenograft in nude mice was decreased compare to control group(AIOD: 0.1661±0.1028 VS 0.2490±0.1483), P﹤0.05.(2) Biological effect of Livin ASODN on xenograft in nude mice①Tumor sizes were significantly smaller in Livin ASODN group than MSODN group from 18 to 30 days after inoculation, P < 0.05. Tumors weight were significantly lighter in Livin ASODN group(1.31±0.88g) than those in MSODN group(2.41±0.41g) at 30 days after inoculation,P<0.05②Necrosis foci and inflammatory cell infiltration were observed in xenograft in nude mice of Livin ASODN group by microscopy.③Apoptotic index detected by TUNEL stain in xenograft in nude mice of Livin ASODN group(19.60±5.91) was significantly higher than MSODN group(3.48±2.35), P﹤0.05.④The expression of Caspase-3 detected by immunohistochemistry in xenograft in nude mice of Livin ASODN group (AIOD: 0.4501±0.1618)was increased compare to MSODN group(AIOD: 0.3601±0.1065), P﹤0.05.Conclusions:1. Human cancer cell line 5637 could express Livinαand Livinβwhich was detected by RT-PCR and Western blot.2. Livin plays an important role in the inhibition of apoptosis and proliferation with bladder cancer cells.3. One of the possible mechanical of the inhibition of apoptosis by Livin was the inhibition of the Caspase-3.4. Livin ASODN could remarkably down-regulate the expression of livin gene in 5637cells, induce apoptosis and inhibit tumor growth.5. Livin ASODN might be a potential target and strategies for the treatment of bladder cancer. |
PDF Full Text Request