Inhibitions Of PTEN Gene To Bladder Transitional Cell Carcinoma And Their Mechanisms

Part 1 PTEN Expression in Bladder Cancer and Its Relationship to BloodVessel Productions1, PTEN Expression in Bladder Cancer and the Relationship between PTEN and Microvessel DensityObjective PTEN (phosphatase and tensin homologue deleted from chromosome 10) is the first anti-oncogene being a phosphatase, and its expression correlates with tumor development and prognosis. Our task is to study the expression of anti-oncogene PTEN in bladder cancer and the relationship between PTEN expression and microvessel density(MVD) in tumor tissues. Methods Using immunohistochemical S-P Methods, the expression of PTEN and FVIIIRAg(a specific mark of vascular endothelial cell used to measure MVD) were examined in 62 specimens of bladder cancer tissues and 18 specimens of bladder benign lesions. Gender and age of patients, tumor grades and staging were taken into account. The influences of the factors above on PTEN expression and the relation between PTEN and MVD were analyzed. Results In the 62 bladder cancers, 53. 23% (33/62) was positive for PTEN expression. All bladder benign lesions were positive for PTEN expression. The expression of PTEN was well correlated with tumor grades(P<0. 05). The MVD in bladder cancer was higher than that in benign lesions (P<0. 01) . MVD significantly correlated with tumor grades, but other factors not. There was a negative correlation between PTEN expression and MVD in bladder cancer (r=-0. 532, P<0. 01). Conclusion PTEN gene could inhibit microvessel production. So PETN mutations or deletions might play an important role in tumor progression and angiogenesis of bladder cancer. 2, Relationship between Expression of PTEN and VEGF and Angiogenesis in Bladder CancerObjective To study the relationship between expression of PTEN, vascular endothelial growth fact or (VEGF) and angiogenesis in bladder cancer, and to evaluate the role of PTEN and VEGF in bladder cancer progression. Methods Using immunohistochemical S-P Method, the expression of PTEN and VEGF were examined in 62 bladder cancer and 18 benigh bladder lesion samples, and microvessei density (MVD) was compared according to different expression of PTEN and /EGF in bladder cancer. According to expression statue of PTEN and VEGF, bladder cancer specimen were divided into 4 groups:group 1(PTEN-VEGF+, n=19), group 2(PTEN-VEGF-, n=10), group 3(PTEN+VEGF+, n=20) and group 4 (PTEN+VEGF-, n=13). Results In the 62 bladder cancers, 53.23% (33/62) was positive for PTEN expression,and 62.90% (39/62) was positive for VEGF. In the 18 control specimen, 100.0% (18/18) was positive for PTEN and 27. 8% (5/18) was positive for VEGF. There was a negative correlation between PTEN and VEGF expression (r = - 0.832, P<0. 01) .MVDs of the 4 groups and control group were 41.53 ± 10. 09, 31. 40 ± 8. 28, 26. 55 ± 8. 57, 25. 15 ± 8. 73 and 19. 44 ± 7. 32, respectively. MVD in bladder cancer was significantly higher when PTEN was negative and VEGF positive compared with other groups(P< 0.05). MVDs of other 3 groups were of no differernce. Conclusion PTEN expression in bladder cancer was significantly lower than that in control group, and VEGF was opposite. There was a negative correlation between PTEN and VEGF expression. When PTEN-VEGF+, MVD was highest. Inactivation of PTEN in bladder cancer might promote angiogenesis through pathway of VEGF, and thus induce tumor progressing.Part 2 Effects of PTEN Gene Transfection on Biological Activities of Human Bladder Cancer Cell Lines BIU-871, Effects of PTEN Gene Transfection on Proliferation and Invasion of Human Bladder Cancer Cell Lines BIU-87Objective To study the effects of anti-oncogene PTEN transfection on proliferation and invasiong of human bladder cancer cell lines, BIU-87, and to bring a new method of gene therapy for bladder cancer. Methods A eukaryotic expression plasmid containing PTEN, pBp-PTEN, was introduced into E. coli DH5. α and amplified. Plasmid was prepared and purified, and then identified by restriction enzyme. pBp-PTEN was transfected into BIU-87 (pBp-PTEN-BIU87) , and positive cell clones were selected and amplified. With empty plasmid transfected BIU-87 (pBp-BIU87) and normal BIU-87 as control groups, expression of PTEN was detected by RT-PCR and cell immunohistochemistry. Proliferation and invasion ability were measured before and after transfection by MTT and cell invasion assay. Three cells were inoculated on nude mice, and tumor growth were observed to see whether PTEN has influence on BIU-87 proliferation in vivo. Results Identification with restriction enzyme confirmed that pBp-PTEN contained PTEN;RT-PCR found that pBp-PTEN-BIU87 had positive strip, while cells of control groups not, and immunohistochemistry also confirmed PTEN successfully transfected;MTT showed, absorbance of the second., third and fourth day of pBp-PTEN-BIU87 were significantly lower than those of control groups ( P < 0.05 ) , and using normal BIU-87 as control groups, inhibition rates of cell growth of the first, second, third and fourth day were 4-27%, 18.92%, 19.54% and 17.69%, respectively;Cell invasion assay showod, number of pBp-PTEN-BIU87 cell penetrating ECM was 39. 3±7. 7, obviously lower than those of control groups, 48.1 ± 13.2 and 48.9±11.0 . P<0.05) .nude mice test found that pBp-PTEN-BIU87 proliferated faster than control. Conclusion Transfection of PTEN might suppress, proliferation and invasion ability of bladder cancer cells so as to inhibit the occurrence and development of tumors. 2, Effects of PTEN Gene Transfection on Apoptosis of Human Bladder Cancer Cell Lines BIU-87Objective To study the effects of anti-oncogene PTEN transfection on apoptosis of human bladder cancer cell lines, BIU-87, and to bring a new method of gene therapy for bladder cancer. Methods A eukaryotic expression plasmid containing PTEN, pBp-PTEN, was introduced into E. coli DH5α and amplified. Plasmid was prepared and purified, and then identified by restriction enzyme. pBp-PTEN was transfected into BIU-87( pBp-PTEN-BIU87 ) , and positive cell clones were selected and amplified. With empty plasmid transfected BIU-87 (pBp-BIU87) and normal BIU-87 as control groups, expression of PTEN was detected by RT-PCR. Apoptosis of BIU-87 were measured before and after transfection by in situ cell apoptosis detection kit and flow cytometry. Results PTEN transfected BIU-87 could steadily express PTENmRNA. In situ cell apoptosis detection found that apoptosis indexes of pBp-PTEN-BIU87, pBp-BIU87 and BIU-87 were(14.28 ± 2.49)%, (9. 18 ± 2. 85) % and (10. 75 ± 3. 15) %, respectively. Apoptosis index of pBp-PTEN-BIU87 significantly increased compared with control groups (P<0. 05) .Flow cytometry found that apoptosis rates of the 3 cells were (14. 23±4. 17) %, (3. 49±1.26) % and (2.79±1.22) %, respectively, and apoptosis rate of pBp-PTEN-BIU87 significantly increased (P<0. 05) .In addition, pBp-PTEN-BIU87 cell in G0-G1 of cell cycle was more than those of control groups (P<0. 01) , and cell in S , less (P<0.01) .It was clear that PTEN gene block cell cycle at G1-S. Conclusion Transfection of PTEN might induce apoptosis of bladder cancer cells so as to inhibit the occurrence and development of tumor. So PTEN has potential of being target of gene therapy. Part 3 Effect of PTEN gene on ehemosensitivity of Bladder Cancer CellLines BIU-87 and on Cancer Xenografts in Nude MiceObjective To study the effects of antioncogene PTEN transfection on chemosensitivity in human bladder cancer cell lines, BIU-87 and the anti-tumor effect of PTEN gene on bladder cancer xenografts in nude mice. Methods A eukaryotic expression plasmid containing PTEN, pBp-PTEN, was introduced into E. coli DH5 a and amplified. Plasmid was prepared and purified, and then identified by restriction enzyme. pBp-PTEN was transfected into BIU-87 (pBp-PTEN-BIU87) , and positive cell clones were selected and amplified. With empty plasmid transfected BIU-87 (pBp-BIU87) and normal BIU-87 as control groups, expression of PTEN was detected by RT-PCR, ehemosensitivity of BIU-87 to mitomycin C(MMC, 1 μ g/mL 10 μg/ml and 100 μ g/ml), hydroxycamptothecin for injection (0. 25 μ g/ml, 2. 5μ g/ml and 25 μ g/ml) and pirarubicin (2 μ g/mK 20 μ g/ml and 200 μ g/ml) before and after transfection were measured by MTT. Drug concentrations were calculated according to 0.1,1.0, and 10 times of clinical plasm peak concentration (PPC). Drug inhibition rate(IR) = (Absorbency of control-Absorbency of test)/ Absorbency of control X 100%, IR<25%, not sensitive, 25%≤IR≤50%, sensitive, 50%≤IR≤75%, median sensitive, IR> 75%, highly sensitive Multiple direct intratumoral injection of PTEN expressing plasmid was given to nude mice, an tumor volume was observed regularly. Results After transfection, and chemosensitivity to mitomycin C of BIU-87 proved to be significantly increased by MTT, but chemosensitivity to hydroxycamptothecin and pirarubicin proved to be no change. Tumor growth showed no difference after 2 weeks of plasmid injection compared with control group. Conclusion Transfection of PTEN might increase chemosensitivity to some chemotherapy drugs, and the effects depend on mechanism of drugs, intratumoral injection of plasmid as a means of gene therapy showed no anti-tumor effect on bladder cancer xenografts in nude mice.Part 4 Effects of Gene PTEN Transfection on PI3K-Akt Signal Path ofBladder Cancer Cell BIU-87Objective PI3K-Akt is an important signal transduction pathway that can promote cell proliferation and inhibit apoptosis , and PIP₃ is an important signal molecule in the pathway. Our task is to study the effects of anti-oncogene PTEN transfection on signal transduction path of PI3K-Akt of human bladder cancer cell BIU-87. Methods A eukaryotic expression plasmid containing PTEN, pBp-PTEN, was introduced into E. coli DH5α and amplified. Plasmid was prepared and purified, and then identified by restriction enzyme. pBp-PTEN was transfected into BIU-87( pBp-PTEN-BIU87 ) , and positive cell clones were selected and amplified. With empty plasmid transfected BIU-87 (pBp-BIU87) and normal BIU-87 as control groups, expression of PTEN was detected by RT-PCR. After transfecting PTEN eukaryotic expressing plasmid into bladder cancer cell lines BIU-87 expressions of P110(catalyzing subunit of PI3K), phosphorylated P110, Akt and phosphorylated Akt were examined by western blot with normal BILJ-87 cell and empty plamid transfected cell as control groups. Results All cells of three group expressed the same level of total P110 and Akt, but expressions of phosphorylated P110 and phosphorylated Akt were significantly weaker in PTEN transfected cells than those in other two groups. Conclusion PTEN might downregulate PI3K-Akt signal path as a phosphatase, block phosphorylating of PI3K, suppress activating of oncogene Akt, and finally inhibit tumor development;the signal transduction path, PTEN-PI3K-Akt, might be an important mechanism of PTEN functons.