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OBJECTIVE@#To design short hairpin RNA (shRNA) interference sequence to silence glutathione S-transferase P1 (GSTP1) gene of androgen independent prostate cancer cell line DU145, and to explore its effect on proliferation and sensitivity to chemotherapeutics.@*METHODS@#The target sequence was picked up to form the shRNA, and the 3 shRNA expression vectors were shRNA255, shRNA554 and shRNA593. The DNA template was cloned to plasmid pGPU6/GFP/Neo. The shRNA was identified by enzyme digesting and gene sequencing. The screening experiment was done to pick up the shRNA expression vector with the highest transfection ratio and best gene silencing results. DU145 cells were divided into a blank plasmid group and a shRNA transfected group. According to the chemotherapeutics the DU145 cells were divided into a fluorouracil (FU) group and a paclitaxel (PA) group, and the 2 groups were subdivided into 4 subsets according to the chemotherapeutic concentrations (FU: 30, 60, 120, and 240 μg/mL; PA: 0.2, 2, 10, and 20 μg/mL), meanwhile a blank control group was included respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate the proliferation after the transfection. MTT and terminal de-oxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were used to detect the inhibition effect of different concentrations of 5-FU or PA on the proliferation and induction of apoptosis of DU145.@*RESULTS@#The transfection ratio of the 3 shRNA expression vectors (shRNA255, shRNA554, and shRNA593) was (63.30±1.04)%, (76.20±0.68)%, and (72.70±0.33)%, and the transfection ratio of shRNA554 was the highest. there was significant difference among the above 3 shRNA expression vectors (P<0.01). After the transfection, the mRNA was 128.31±2.50, 43.24±4.30 and 85.62±6.30, the GSTP1 protein was 163.92±12.40, 65.38±9.30 and 114.25±16.70. After the transfection of shRNA554, the mRNA and protein of GSTP1 were the lowest level. there was significant difference among the above 3 shRNA expression vector (P<0.01). MTT analysis showed that before the transfection, the survival ratio of cells under different concentrations of FU (30, 60, 120, and 240 μg/mL) was (95.60±2.11)%, (90.20±0.86)%, (83.10±3.12)% and (74.60±1.32)%; however after the transfection, the survival ratio of cells was (91.30±1.43)%, (84.60±2.13)%, (73.20±1.52)%, and (65.5±0.942)%. TUNEL assay showed that before the transfection, the apoptosis ratio of cells under different concentrations of FU (30, 60, 120, and 240 μg/mL) was (5.50±0.88)%, (10.20±1.64)%, (15.20±2.39)%, and (25.10±2.59)%; however after the transfection, the apoptosis ratio of cells was (10.8±0.62)%, (15.7±1.32)%, (20.4±1.89)%, and (34.9±2.54)%. After the transfection, the cell survival ratio decreased under the same concentration of FU, and the apoptosis ratio increased, with statistical significance (both P<0.01). MTT analysis showed that before the transfection, the survival ratio of cells under different concentrations of PA (0.2, 2, 10, and 20 μg/mL) was (98.50±2.34)%, (95.20±1.32)%, (89.40±0.68)%, and (82.70±1.73)%; after the transfection the survival ratio of cells was (94.20±0.78)%, (86.50±2.13)%, (78.70±1.34)%, and (70.10±0.76)%. TUNEL assay showed that before the transfection, the apoptosis ratio of cells under different concentrations of PA (0.2, 2, 10, and 20 μg/mL) were (2.40±1.07)%, (5.20±1.33)%, (10.50±2.41)%, (20.70±1.92)%; after the transfection the apoptosis ratio of cells was (5.46±2.13)%, (13.80±1.24)%, (21.20±2.39)%, and (29.20±2.21)%. After the transfection, the cell survival ratio decreased under the same PA concentration, and the apoptosis ratio increased, with statistical significance (both P<0.01).@*CONCLUSION@#gene GSTP1 silence via shRNA transfection to androgen independent prostate cancer cell line DU145 can inhibit its proliferation in time dependent manner, and induce apoptosis and raise its sensitivity to chemotherapeutics.
Subject(s)
Humans , Male , Androgens , Metabolism , Antineoplastic Agents , Pharmacology , Apoptosis , Genetics , Cell Line, Tumor , Cell Proliferation , Gene Silencing , Glutathione S-Transferase pi , Genetics , Prostatic Neoplasms , Genetics , Pathology , RNA Interference , RNA, Small Interfering , Genetics , TransfectionABSTRACT
Objective To study the apoptosis-inducing effect of Oridonin on PC-3 cells line and the role of Survivin in the process.Methods After PC-3 cells were incubated with different concentrations of Oridonin,cell viability was analyzed with MTT assay.The percentage of earlier apoptosis cell was analyzed by flow cytometry.The protein expression of Survivin in PC-3 cells were detected by Western blot and fluorescent quantitative PCR.Results Oridonin effectively inhibited the proliferation of PC-3 cells in a concentration-time dependent way.After PC-3 cells were treated with Oridonin ( 2.5,5,10,20,40 μmol/L)for 48 hours,the cytotoxicity index were 9.2％,25.3％,39.3％,77.2％,92.5％ and the IC50 of PC-3 cells was 10.29 μmol/L,respectively.Flow cytometry was used to detect the effect of different concentration of Oridonin (0,10,20,40 μmol/L) for 48 hours,the apoptotic rates of PC-3 cells were 4.8％,15.4％,19.5％,27.4％ ( P ＜ 0.05).Oridonin down-regulated Survivin protein in a concentration-dependent way in PC-3 cells.Conclusions Oridonin can induce the apoptosis of PC-3 cells by a concentration-dependent manner.Oridonin can induce the apoptosis of PC-3 cells by down-regulated Survivin protein.
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Objective To observe the proliferation inhibition and apoptosis promotion effect of oridonin on PC-3 cells.Methods PC-3 cells were treated with different concentrations of oridonin.MTT assay and drug concentration-time survival curve were used to test the effect of oridonin on the PC-3 cells.The percentage of earlier apoptosis cells was analyzed by flow cytometry.The protein expression of caspase-3,Bcl-2,and Bax in the PC-3 cells was detected by Western blot.Results Oridonin effectively inhibited the proliferation of PC-3 cells in both concentration- and time-dependent manner,and the IC50 of PC-3 cells was 10.29 μmol/L.Hochest33258 staining and flow eytometry deteced that oridonin induced the apoptosis of PC-3 cells in a concentration-dependent manner (P ＜ 0.05 ).Oridonin down-regulated Bcl-2,up-regulated Bax protein,and activated caspase-3 in a concentration-dependent manner in the PC-3 cells.Conclusion The apoptosis of PC-3 cells induced by oridonin might be associated with the mitochondrial pathway.
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Objective To observe the effect of extraeorporeal shock wave lithotripsy (ESWL) on residual stones after different methods of surgery. Methods Clinical resources of 100 patients with residual stones after different methods of surgery treated with extracorporeal shock wave lithotripsy from May 2006 to May 2008 were retrospeetively studied. Of the 100 patients, ureteroscopic laser lithotripsy was used for 15 patients (Group Ⅰ) , ureteroscopic pneumatic lithotripsy was used for 25 (Group Ⅱ), mini-percutaneous nephrolithotomy (MPCNL) with holmium laser for 11 (Group Ⅲ), mini-percutaneous nephrolithotomy (MPCNL) with airpressure path lithotripterfor 12 (Group Ⅳ) , open surgery for the other 37 (Group Ⅴ). Results About 94% of the residual stones were shattered, and 86 % of the residual stones were cleared successfully. The clearance rate of residual stones from Group Ⅰ to Ⅴ was 100% , 100% , 81.8% , 83.3% , and 73.0% , respectively. The clearance rate of residual stones in Group Ⅰ + Ⅱ was higher than that of Group Ⅲ + Ⅳ and Group V (P<0.05). Conclusion Extracorporeal shock wave lithotripsy is good for the treatmentof residual stones after different methods of surgery, especially the management of residual stones after trans-urethral ureteroscope technique.
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Objective To evaluate the treatment of bilateral hydronephrosis caused by prostate cancer. Methods Twenty-four eases with mean age of 71 years old (ranging from 64--81 years old) were diagnosed with bilateral hydronephrosis caused by prostate cancer and treated with complete androgen deprivation. Surgical castration plus Bicalutamide 50 mg/d was offered to 18 eases and medical castration (Goserelin, 3. 6 mg/month) plus Bicalutamide 50 mg/d was offered to 6 cases. There were 19 cases developed severe lower urinary tract symptoms. Among these 19 cases, 13 cases had accepted Foley catheter and 6 cases accepted suprapubic tube drainage. Results Before and after the treatment, the prostate volume decreased from (70. 3±11.2)ml to (42.6±15.8)ml(P=0. 001). Total PSA decreased from (40. 3±27.2)ng/ml to (9.5±8.3)ng/ml(P=0.02). Of the 24 cases, hydrone phrosis improved in 18 cases, remained unchanged in 3 cases and deteriorated in 3 cases. There were 14 patients developed renal insufficiency. After the treatment, Serum urea nitrogen decreased from (12. 8±6. 5) mmol/L to (6. 3 ± 4. 2) mmol/L (P = 0. 004) and serum ereatinine decreased from (206.8±152.3)μmol/L to (85.3±43.6)μmol/L(P=0.03), respectively. For those 6 cases with hy dronephrosis unchanged or deteriorated during the treatment, 4 cases accepted percutaneous nephros tomy and 2 cases accepted chtaneous ureterostomy. Conclusion The combination of complete androgen deprivation and bladder drainage through Foley catheter or suprapubic tube is an effective option in the treatment of bilateral hydronephrosis caused by prostate cancer.
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23 cases of the patients with supraorbital neuralgia were treated by puncturing Yangbai (GB 14) toward Yuyao (Extra), Zanzhu (BL 2), Taiyang (Extra), Touwei (ST 8),Zhongzhu (TE 3) and Neiting (ST 44) on the sick side, plus laser radiation on a site about 1 cm apart from the midpoint of the eyebrow of the sick side. After 10 treatments, the results showed cure in 19 cases and remarkable effect in 4 cases.
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Objective To determine the expression of hepatocyte growth factor and its receptor c-met in bladder transitional cell carcinoma, and to investigate correlations of the expression and clinic features of the tumor. Methods Samples of 45 human bladder carcinoma and 8 normal bladder tissues were detected with immunohistochemical method for HGF and c-met ,then relationships between the expressions and clinic features of the tumor were analyzed. Results There was no HGF expression and only 1 weak positive c-met expression in the normal urinary bladder tissues. While in bladde cancer specimens, positive expression rates of HGF and c-met were as high as 64.4%,62.2%, respectively.Coexpression rate of HGF and c-met was 42.2%. The positive rates were significantly higher in BTCC specimens than in normal tissues (P
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Objective To explore the relationship between the expression of caveolin-1 and pathological grade, clinical stage in bladder carcinoma. Methods Caveolin-1 expression was detected in 63 cases of bladder carcinoma and 10 cases of normal bladder epithelial tissue using SABC immunohistochemistry. The clinical and pathological data of the patients were collected and used to make statistical analysis. Results The normal bladder mucosal epithelia had not caveolin-1 expression, while the positive rate of caveolin-1 expression was 34.92% in bladder carcinoma. The positive rates of caveolin-1 expression in the grades I, II and III of bladder carcinoma were 0%, 2.86%, 54.28%, respectively, which had a significant difference(P