Experimental Study Of The Effect On Prostate Cancer Cell Line DU145 Via ShRNA Expression Vector-mediated Glutathione S-transferase P1 Gene Silencing

Background Hormone-refractory prostate cancer (HRPC) has no effective treatment at present, and the new therapy for HRPC is a focus of research around the world. After endocrine treatment of androgen deprivation the prostate carcinoma will usually get the progression of androgen-independent prostate cancer (AIPC) or even hormone-refractory prostate cancer. The AIPC is responsive to some secondary endocrine treatment, but the HRPC is irresponsive to any endocrine treatment and the disease gets development during the endocrine treatment. About 28,900 people died of HRPC annually in America. The incidence of prostate cancer increases is increasing rapidly in China as the improvement of living quality and prolongation in life span. In our country the prostate cancer took the third place in common malignant tumor in the genitourinary system. Two thirds of the prostate cancer patients lose the chance of radical cure, for whom the maximum androgen blockade (MAB) is the only choice. But after a median period of 12-18 months,70%-80% converted to androgen independent prostate cancer and recurrence or metastasis occurs. The mean survival time is about 1 year. As lack of efficient treatment for HRPC, it is the focus in carcinoma research.Glutathione S-transferases (GSTs) is a kind of dimeride isoenzyme, which has powerful catalytic action. The GSTs could catalyse electron binding centre of the substrate molecule to conjugate with reduced glutathione to promote the metabolism and inactivation of the substrate. According to the location in chromosome and the sequence homology, the superfamily of GSTs consists ofα,μ,π,θfour subfamily. The GST-πprotein produced by GSTP1 gene has close relation with the malignant tumors, which became the focus of research. The reports of research in gastrointestinal tumor show that inhibition of GSTP1 could reduce the drug resistance of tumors. The main inhibitive methods include the signal path block drug and RNA interference to lower the expression level. During the early stage of prostate cancer, the promoter sequence of GSTP1 got hypermethylation, which caused the deletion of GSTP1 expression. As a result the prostate cancer took place and progressed. The detection of GSTP1 promoter methylation condition could be an early diagnostic method for prostate cancer. The effect of androgen deprivation on the expression of GSTP1 has been reported that after androgen deprivation therapy, the tissue got stress reaction and regained the expression of GSTP1. Research reports shew that GSTP1 expressed in HRPC primary lesion and the metastatic lesion. The recovery expression of GSTP1 may result in the resistance of chemotherapy of HRPC.RNA interference (RNAi) is the technique that invented in 1998 and the development of RNAi is rapid. RNAi is a kind of gene silencing technique, which has high degree of specificity, efficiency and the interfere effect is diffusive. As a result the inhibitive effect on target gene is powerful. RNA interference is an effective gene silencing process, and the mechanism is post transcription inhibition. The process could divide into initiation and effector stage. In initiation stage the RNAaseⅢfabricated the lipofectamine or virus imported dsRNA in to siRNA of 21-23nt. In the effector phase the double strand of the siRNA unrolled and the anti-sense siRNA combined the related protein to form the active RNA induced silencing complex (RISC). RISC then recognize the mRNA with the completely complementary sequences and make the mRNA degrade. The process has cascade amplification. At present the RNAi via lipofectamine or virus to treat the prostate cancer is in the experiment stage.In this experiment GSTP1 is taken as the target of gene therapy, and the application of short hairpin RNA is taken to silence the GSTP1 gene. The GSTP1 shRNA is constructed and transfected to androgen independent prostate cancer cell line DU145. Then RT-PCR, Western Blotting are used to detect the mRNA and protein level of GSTP1 to evaluate the effect of RNA interference. After transfection the MTT, flow cytometry are used to assess the RNAi effect on the biological behavior of DU145. The sensitivity to chemotherapeutics of DU145 before and after the transfecion are compared. TUNEL was used to detect the effect of ERK inhibitor PD98059 on the apoptosis of prostate cancer cell line.Part IStudy on the expression of GSTP1 in the prostate cancer tissue and plasmaObjective Analyze and compare the expression level of GSTP1 in the prostate cancer tissue and plasma of the hormone-refractory prostate cancer and prophase localized prostate cancer.Methods Immunohistochemistry analysis is used to detect the expression of GSTP1 in 10 cases of prophase localized prostate cancer(PCa) tissue, and 10 cases of hormone-refractory prostate cancer(HRPC) tissue. In control 10 cases of benign prostate hyperplasia(BPH) tissue were analyzed.Enzyme-linked immunosorbent assay (ELISA) is used to detect the expression of GSTP1 in plasma of 10 prophase localized prostate cancer patients, and 10 cases of HRPC patients. In control 10 cases of BPH patients'plasma were analyzed.Results The expression result of GSTP1 in 10 cases of prophase localized prostate cancer tissue:Negative 8 cases, weakly positive 1 case, middling positive 1 case, the positive ratio was 20%. The expression result of GSTP1 in 10 cases of HRPC tissue:Negative 2 cases, weakly positive 1 case, middling positive 4 cases, obviously positive 3 cases, the positive ratio was 80%. In control 10 cases of BPH tissue:no negative case,1 case weakly positive,2 cases middling positive and 7 cases obviously positive, the positive ratio was 100%. Wilcoxon rank test shew that the GSTP1 positive ratio of HRPC is higher than that of PCa, P <0.01, but lower than that of BPH, P<0.01. The concentration of GSTP1 in plasma was 2033.39±232.75pg/mL in 10 cases of PCa patients; 3879.89±210.32pg/mL in 10 cases of HRPC patients; 4019.07±170.33pg/mL in 10 cases of BPH patients. The HRPC group had higher GSTP1 concentration than the PCa group, P<0.01; the HRPC group had lower GSTP1 concentration than the BPH group, P<0.01.Conclusion1. The prostate cancer tissue of hormone-refractory prostate cancer has higher GSTP1 expression than that of prophase localized prostate cancer.2. The concentration of GSTP1 in plasma of patients with hormone-refractory prostate cancer is higher than that of patients with prophase localized prostate cancer.PartⅡDetection of the mRNA and protein expression level of gene GSTP1 in the prostate cancer cells line DU145, PC3 and LNCaPObjective Compare the mRNA and protein expression level of gene GSTP1 among the prostate cell line DU145, PC3 and LNCaP.Methods RPMI 1640 nutrient medium is used to culture the prostate cancer cell line DU145, PC3 and LNCaP. RT-PCR is used to detect the mRNA level of gene GSTP1 of the three cell lines, and western blot is used to detect the GSTP1 protein level.Results Semi-quantitive RT-PCR was used to detect the level of the GSTP1 mRNA and the absorbance of light indicate the expression ratio. Of the androgen independent prostate cancer cell line DU145 the GSTP1 mRNA was 346.7±7.3; in PC3 229.0±1.4; of the androgen dependent prostate cancer cell line LNCaP the GSTP1 mRNA was 73.5±2.2. Compared with the androgen dependent prostate cancer cell line LNCaP, the androgen independent prostate cancer cell line DU145 and PC3 had higher level of GSTP1 mRNA, P<0.01. DU145 had higher level of GSTP1 mRNA than that of PC3, P<0.01. Western blot was used to detect the level of GSTP1 protein level in the three cell lines. The absorbance of light indicated the GSTP1 protein level. Of the androgen independent prostate cancer cell line DU145 the GSTP1 protein was 103.1±1.1; PC3 78.5±0.5; of the androgen dependent prostate cancer cell line LNCaP the GSTP1 protein was 24.4±1.2. Compared with the androgen dependent prostate cancer cell line LNCaP, the androgen independent prostate cancer cell line DU145 and PC3 had higher level of GSTP1 protein, P<0.01. DU145 had higher level of GSTP1 protein than that of PC3,P<0.01.Conclusion1. The androgen independent prostate cancer cell lines DU145 and PC3 have higher level of GSTP1 mRNA and GSTP1 protein, compared with that of androgen dependent prostate cancer cell line LNCaP.2. DU145 has higher level of GSTP1 mRNA and GSTP1 protein than that of PC3.PartⅢThe effect of gene GSTP1 silencing via shRNA transfection on the androgen independent prostate cancer cell line DU145Objective To design special shRNA interference sequence for transfection to the androgen independent prostate cancer cell line DU145 to silence the gene GSTP1, and then to investigate the effect on proliferate activity and sensitivity to chemotherapeutics.Methods Through software design and review of related references, the target sequence was picked up to form the shRNA, and the DNA template was cloned to the shRNA expression vector pGPU6/GFP/Neo to construct the plasmid with the targeting inhibitive shRNA of gene GSTP1. The shRNA was identified by gene sequencing. The screening experiment was taken to pick up the shRNA expression vector with highest transfection ratio and best gene silencing result. Lipofectamine was used to mediate transfection of plasmid to DU145, and RT-PCR, western blot was used to detect the level of mRNA and protein of GSTP1 which indicated the effect of transfection. MTT analysis and flow cytometry were used to evaluate the effectiveness of shRNA transfection. TO compare the sensitivity to chemotherapeutics before and after transfecion, MTT analysis and flow cytometry were used to detect the proliferate activity of DU145 after the 5-FU or PA of different concentration added.Results The constructed vectors including three shRNA expression vector and the negative vector. They were pGPU6/GFP/Neo-shRNA255, pGPU6/GFP/Neo-shRNA554, pGPU6/GFP/Neo-shRNA593 and Negative-shRNA. In preliminary experiment, the transfection efficiency of shRNA255,shRNA554 and shRNA593 was 63.3±1.04%,76.2±0.68% and 72.7±0.33% respectively. In which the shRNA554 had highest transfection efficiency, P< 0.01. In preliminary experiment, after transfection of shRNA255, shRNA554 and shRNA593 the gene GSTP1 mRNA level detected by RT-PCR of DU145 was 128.31±2.5,43.24±4.3 and 85.62±6.3 respectively, and the GSTP1 protein level detected by western blot of DU145 was 163.92±12.4,65.38±9.3 and 114.25±16.7 respectively. Statistical analysis indicated that shRNA554 of highest transfection ratio and best gene silencing result, P< 0.01. After transfection of shRNA554 to DU145, on the 2 day,4 day and 6 day the level of GSTP1 protein was 125.44±10.85,106.52±11.2 and 56.43±8.76 respectively. While the level after the transfection of blank plasmid was 174.35±7.2,168.09±6.54 and 171.72±8.25 respectively. The data indicated that after transfection of shRNA554 to DU145 the GSTP1 protein level decreased(P<0.01), and decreased progressively as the extension of transfection time, which indicated the time dependence(P< 0.01). Similarly MTT analysis shew that after 2 days,4 days and 6 days after transfection the survival ratio of DU145 decreased progressively as the extension of transfection time, which indicated the time dependence(P < 0.01). Flow cytometry indicated that the ratio of subGl phase transfected with shRNA554 was higher than that of blank plasmid(P< 0.01), and the proliferate ratio decreased(P< 0.01). MTT analysis indicated that before transfection, the survival ratio of DU145 added different concentration of 5-FU (μg/ml) were:95.6±2.11%(30), 90.2±0.86%(60),83.1±3.12%(120) and 74.6±1.32%(240); while after transfection the survival ratio of DU145 added different concentration of 5-FU (μg/ml) were:91.3±1.43%(30),84.6±2.13%(60),73.2±1.52%(120) and 65.5±0.94%(240). Flow cytometry indicated that before transfection, the subGl phase(apoptosis cell) ratio of DU145 with different concentration of 5-FU (μg/ml) added were:5.37±0.43%(30), 6.49±2.06%(60),9.02±0.34%(120) and 12.33±2.46%(240); the proliferate index were:47.54±2.33%(30),46.69±2.12%(60), 46.01±1.87%(120) and 44.29±1.78%(240); while after transfection the subGl phase(apoptosis cell) ratio of DU145 with different concentration of 5-FU (μg/ml) added were:7.69±1.04%(30),11.44±0.98%(60), 13.57±3.44%(120) and 22.42±0.84%(240); the proliferate index were: 40.97±0.92%(30),38.51±2.03%(60),36.39±2.67%(120) and 34.04±3.21%(240). Compared with the data before transfection, statistical analysis indicated that after transfection, under the same concentration of 5-FU, the survival ratio decreased, apoptosis increased and the proliferate index decreased of statistical significance, P<0.01. MTT analysis indicated that before transfection, the survival ratio of DU145 added different concentration of PA (μg/ml) were:98.5±2.34%(0.2), 95.2±1.32%(2),89.4±0.68%(10) and 82.7±1.73%(20); while after transfection the survival ratio of DU145 added different concentration of PA (μg/ml) were:94.2±0.78%(0.2),86.5±2.13%(2),78.7±1.34%(10) and 70.1±0.76%(20). Flow cytometry indicated that before transfection, the subG1 phase(apoptosis cell) ratio of DU145 with different concentration of PA (μg/ml) added were:3.51±0.35%(0.2),5.41±1.03%(2), 9.48±1.09%(10) and 10.91±1.03%(20); the proliferate index were: 46.25±1.27%(0.2),46.05±1.98%(2),41.75±2.44%(10) and 41.04±1.71%(20); while after transfection the subGl phase(apoptosis cell) ratio of DU145 with different concentration of PA(μg/ml) added were: 5.66±1.32%(0.2),11.02±0.87%(2),19.53±1.23%(10) and 26.31±2.01%(20); the proliferate index were:44.20±2.52%(0.2), 41.37±2.51%(2),35.04±2.41%(10) and 32.97±2.89%(20). Compared with the data before transfection, statistical analysis indicated that after transfection, under the same concentration of PA, the survival ratio decreased, apoptosis increased and the proliferate index decreased of statistical significance, P<0.01.Conclusion1. The shRNA expression vector-transfected to androgen independent prostate cancer cell line DU145 could reduce the level of GSTP1 mRNA and GSTP1 protein, in vitro inhibition of the proliferate activity of DU145 in a manor of time dependent.2. The shRNA expression vector-transfected to androgen independent prostate cancer cell line DU145 could induce apoptosis and decrease the proliferate index. 3. The shRNA expression vector-transfected to androgen independent prostate cancer cell line DU145 could raise the sensitivity to chemotherapeutics, under the same concentration of chemotherapeutics, the survival ratio of DU145 decreased, apoptosis increased and the proliferate index decreased.Part IVThe effect of ERK inhibitor PD98059 on the expression of GSTP1 protein and apoptosis of DU145Objective To detect the effect of the extracellular regulated kinase (ERK) inhibitor PD98059 on the expression of GSTP1 of DU145, and the effect on the survival ratio and apoptosis level of DU145.Methods ERK inhibitor PD98059 was added to DU145, then western blot was used to detect the effect of PD98059 with different concentration on the level of P-ERK and GSTP1. MTT analysis was used to detect the cell survival ratio and TUNEL was used to detect the apoptosis level of DU145 when treated with PD98059.Results After treated with different concentration of PD98059, the protein level of P-ERK and GSTP1 protein were detected by western blot and the absorbance of light indicated the protein level. The DU145 was treated with different concentration of PD98059 0μmol/L,5μmol/L, 10μmol/L,20μmol/L,40μmol/L; the P-ERK level was 485.93±12.1, 397.02±7.8,351.11±7.2,230.54±6.5,142.97±13.2 respectively; the GSTP1 protein level was 380.74±14.4,285.57±9.87,231.45±6.43, 190.87±11.2,102.08±14.4 respectively. The level of P-ERK and GSTP1 protein decreased progressively as the concentration of PD98059 incresed, and the difference between every two different concentration group is statistically significant, P<0.01. The DU145 was treated with different concentration of PD98059 0μmol/L,5μmol/L,10μmol/L,20μmol/L, 40μmol/L; the apoptosis ratio detected by TUNEL was 5.54±0.39%, 10.46±0.88%,19.8±1.64%,36.2±2.39%,49.2±2.59% respectively. The apoptosis ratio increased progressively as the concentration of PD98059 incresed, and the difference between every two different concentration group is statistically significant, P<0.01.Conclusion1. The ERK inhibitor PD98059 has inhibitive effect on the expression of P-ERK, GSTP1 of DU145 in the manner of obvious dose-effect relationship.2. ERK inhibitor PD98059 induced the DU145 apoptosis in the manner of significant dose-dependent.3. The ERK pathway may participate in regulation of GSTP1 protein expression.