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.
Androgens ; metabolism ; Antineoplastic Agents ; pharmacology ; Apoptosis ; genetics ; Cell Line, Tumor ; Cell Proliferation ; Gene Silencing ; Glutathione S-Transferase pi ; genetics ; Humans ; Male ; Prostatic Neoplasms ; genetics ; pathology ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection

Aneuploidy ; Gene Rearrangement ; Glutathione S-Transferase pi ; metabolism ; Humans ; Male ; Methylation ; Neoplasm Grading ; Neoplasm Staging ; Neoplasms, Multiple Primary ; genetics ; metabolism ; pathology ; surgery ; Oncogene Proteins, Fusion ; genetics ; Prostate-Specific Antigen ; metabolism ; Prostatic Intraepithelial Neoplasia ; genetics ; pathology ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; surgery

Carcinoma, Hepatocellular ; genetics ; metabolism ; DNA Methylation ; DNA Restriction Enzymes ; metabolism ; DNA, Neoplasm ; genetics ; Glutathione S-Transferase pi ; genetics ; metabolism ; Humans ; Liver Neoplasms ; genetics ; metabolism ; Polymerase Chain Reaction ; methods

OBJECTIVETo investigate the methylation frequencies of multiple tumor suppressor genes (TSGs) in hepatocellular carcinoma (HCC) and the clinical implication of aberrant DNA methylation in molecular carcinogenesis of HCC.

METHODSSixty samples of HCC and the paired adjacent liver tissue, 16 samples from post-hepatitis cirrhotic livers, 5 from livers with chronic hepatitis and 5 from normal livers were collected. Eight TSGs frequently silenced by hypermethylation of their promoters in various types of digestive tumors were selected, including APC, RASSF1A, p16, GSTP1, MGMT, DAPK, SOCS-1 and RIZ1. The status of promoter methylation in these 8 genes was investigated using methylation-specific polymerase chain reaction. The clinicopathological data of HCC were also analyzed in order to evaluate the clinical implication of aberrant methylation in HCC.

RESULTSMethylation of the 8 TSGs was quite frequent in HCC, with a methylation rate of 95.0% in RASSF1A, 90.0% in APC, 73.3% in GSTP1, 65.0% in p16, 61.6% in RIZ1 and 60.0% in MGMT. Methylation of the 6 genes was more frequent in HCC than that in adjacent tissues (P < 0.05). The methylation rate of MGMT, GSTP1 and RIZ1 in the adjacent tissues was 41.6%, 40.0% and 25.0%, respectively, significantly higher than that in cirrhotic liver (P < 0.05). p16 methylation was more frequently observed in HCC in elderly patients. The frequency of MGMT methylation was tended to be higher in giant HCC than that in the other types of HCC. Patients with MGMT methylation in the tumor were found to have a shorter disease free survival.

CONCLUSIONDifferent frequency of methylation in hepatocellular carcinomas, adjacent liver tissues and cirrhotic livers implies that epigenetic alteration in the hepatocellular carcinogenesis may be a gradually progressive process. Methylation status of MGMT, GSTP1 and RIZ1 may be promising in risk assessment of hepatocellular carcinoma and in early diagnosis. Furthermore, MGMT methylation might be also used as a potential prognostic biomarker for hepatocellular carcinoma patients.

Adult ; Aged ; Base Sequence ; Carcinoma, Hepatocellular ; genetics ; metabolism ; DNA Methylation ; DNA Modification Methylases ; genetics ; metabolism ; DNA Repair Enzymes ; genetics ; metabolism ; DNA, Neoplasm ; genetics ; DNA-Binding Proteins ; genetics ; metabolism ; Disease-Free Survival ; Female ; Follow-Up Studies ; Gene Expression Regulation, Neoplastic ; Genes, Tumor Suppressor ; Glutathione S-Transferase pi ; genetics ; metabolism ; Hepatitis B, Chronic ; genetics ; metabolism ; Histone-Lysine N-Methyltransferase ; Humans ; Liver ; metabolism ; Liver Cirrhosis ; genetics ; metabolism ; Liver Neoplasms ; genetics ; metabolism ; Male ; Middle Aged ; Molecular Sequence Data ; Neoplasm Recurrence, Local ; Nuclear Proteins ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism ; Tumor Suppressor Proteins ; genetics ; metabolism ; Young Adult

This study was purposed to investigate the relationship of expressions of gluthatione-S-transferase-pi (GST-pi), multidrug resistance protein-1 (MRP-1), lung resistance protein (LRP) with multidrug resistance of acute leukemia (AL), the correlation between 3 kinds protein expressions and the correlation of their protein expression with clinical features of AL patients. The S-P immunohistochemical staining method was used to determine the expressions of GST-pi, MRP1 and LRP proteins in 80 AL patients and 30 normal subjects. The results showed that there was the correlation between GST-pi, MRP1, LRP protein expression and chemotherapy resistance, meanwhile CR rates of patients with positive expression of those proteins were lower than that of patients with negative expression (P<0.05), so those protein expressions may be accounted for poor prognosis. There was the positive relationship between expression of GST-pi and MRP1 in refractory group (r=0.851, P<0.01). It is concluded that co-examination of GST-pi and MRP1 has greater significance than examination of one kind of protein in evaluating poor prognosis of leukemia patients. LRP protein expression increase obviously when WBC counts >or= 10 x 10(9)/L (63.6%, P<0.05), therefore LRP protein has great judging value for evaluating drug resistance and prognosis of acute leukemia patients whose peripheral blood WBC counts were high.
ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; genetics ; Adolescent ; Adult ; Aged ; Drug Resistance, Multiple ; genetics ; Drug Resistance, Neoplasm ; genetics ; Female ; Glutathione S-Transferase pi ; biosynthesis ; genetics ; Humans ; Leukemia, Myeloid, Acute ; drug therapy ; metabolism ; Male ; Middle Aged ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; metabolism ; Vault Ribonucleoprotein Particles ; biosynthesis ; genetics

BACKGROUND: The multidrug resistance (mdr1), multidrug resistance associated protein (mrp1), and glutathione-s-transferase (gst) pi genes have been associated with treatment failure in acute myeloid leukemia (AML). c-jun N-terminal kinase (JNK) activity is increased in response to chemotherapeutic agent. METHODS: To investigate the significance of multidrug resistance (mdr) parameters and JNK activity, bone marrow or peripheral blood cells from 52 patients with AML were analyzed. RT-PCR was performed for mdr1, mrp1, and gst pi gene expression. JNK expression and activity were measured using an immunoe- nzymatic kinase assay and a western blot method. RESULTS: High level expression of mdr1, mrp1, and gst pi mRNA was observed in 38.5%, 48.1% and 54.3% of AML cases, respectively. The remission rate was significantly low in cases with an older age (>55 yr), a high WBC count, poor chromosomal abnormalities, a high level expression of mdr1 and mrp1. The WBC count and mdr1 mRNA expression were independent predictors for the outcome to induction chemotherapy. There was a shorter duration of overall survival in the patients with an older age, a high WBC count, chromosome aberrations, high level expressions of mdr1 and mrp1 mRNA, and JNK activation. The patient's age, WBC count and chromosomal abnormalities were independent predictors for overall survivals. The majority (28/30) of AML cases did not show any levels of JNK activation except for two cases, which were associated with an extremely high WBC count, chromosomal aberration, high level expressions of mdr1, mrp1 and gst pi mRNA, and treatment resistance. CONCLUSIONS: These data indicate the influences of mdr1 and mrp1 mRNA expression on the clinical outcome of AML to induction chemotherapy. But it will be necessary to investigate further whether blast cells of AML resistant to chemotherapy retain the capacity to activate JNK, and relate to MDR parameters.
Adolescent ; Adult ; Aged ; *Drug Resistance, Multiple/genetics ; *Drug Resistance, Neoplasm/genetics ; Female ; Glutathione S-Transferase pi/genetics ; Humans ; JNK Mitogen-Activated Protein Kinases/*metabolism ; Leukemia, Myeloid, Acute/*drug therapy/genetics/metabolism ; Male ; Middle Aged ; Multidrug Resistance-Associated Proteins/genetics ; P-Glycoprotein/genetics ; RNA, Messenger/*metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Survival Analysis ; Treatment Outcome

OBJECTIVETo elucidate gene expressions of phase II enzymes in the mouse embryonic stem (ES) cell-derived hepatocytes.

METHODSEmbryoid body (EB) formation cultures were applied in directed differentiation of ES to hepatic-like cells. The expressions of hepatic-specific genes, including AFP, ALB, Cyp7a1, were detected by RT-PCR during the differentiation course. Albumin was detected by immunocyto- chemistry. The gene expressions of mGST1 and UGTs family, including UGT1a1, UGT1a6, UGT1a9 and UGT2b5, were investigated using RT-PCR.

RESULTSA notable gene expression of AFP and ALB was observed on d 8. On d 18, AFP gene failed to express, while ALB and Cyp7a1 genes were detected.Albumin-positive staining was detected in hepatic-like cells. Phase II enzyme genes expressed in variance during the differentiation course, UGT1a1 and UGT1a9 were expressed stably, UGT1a6 expression increased gradually, and UGT2b5 failed to express. Little mGST1 gene expression could been detected in the early course until d 18. In addition, all the enzymes gene expressions in the derived hepatocytes on d 18 were similar to those from mature mouse hepatocytes.

CONCLUSIONSMouse ES cell-derived mature hepatocytes express phase II enzyme UGTs and mGST1 genes similar to those in mature hepatocytes. The system may offer an alternative animal testing model related to phase enzymes in further research.

Animals ; Cell Differentiation ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; enzymology ; Fluorescent Antibody Technique ; Glucuronosyltransferase ; genetics ; metabolism ; Glutathione S-Transferase pi ; genetics ; metabolism ; Hepatocytes ; cytology ; enzymology ; Isoenzymes ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Microsomes ; enzymology ; Reverse Transcriptase Polymerase Chain Reaction

We have investigated the susceptibility of rat lung's GSTP1 gene to hypobaric hypoxia and explored its role in the body's possible adaptation mechanism at the moleuclar lever. Thirty male SD rats were randomly divided into five groups(0,1,3,5 and 7 d) and were exposed for 12 h per day at a simulating altitude of 7000 +/- 50 m in a hypobaric hypoxia chamber with 1 h's rest after 6 h's exposure. Then the expression of GSTP1 mRNA in the lung tissue of SD rats was examined using fluorescence quantitative RT-PCR. Meanwhile the activity of glutathione S-transferases (GSTs) enzyme and the change of maleic dialdehyde (MDA) in the lung tissue of SD rats were determined using spectrophotometer. In comparison with the non-exposure group,the expression of GSTP1 gene showed statistically significant differnce from the first to the seventh day (P<0.05). The level of GSTs decreased and MDA increased from the first to the seventh day (P<0.05). In conclusion, GSTP1 gene is susceptible to hypobaric hypoxia and may be a new marker of gene screening for the body's adaptation to special environment.
Animals ; Biomarkers ; analysis ; Glutathione S-Transferase pi ; analysis ; biosynthesis ; genetics ; Hypoxia ; genetics ; metabolism ; Lung ; metabolism ; Male ; RNA, Messenger ; analysis ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo explore the expression of multidrug resistance gene 1 ( MDR1), glutathione-S-transferases-pi (GST-pi) in osteosarcoma and soft tissue sarcoma tissues from 34 patients and their correlation with chemotherapy resistance.

METHODSMDR1 and GST-pi expressions were analyzed by real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) and flow cytometry (FCM) at mRNA and protein levels, respectively. Chemotherapy sensitivity on adriamycin, cisplatinum, fluorouracil, mitomycin C, dacarbazine, vincristine, methotrexate in tumor tissues were detected by MTT assay.

RESULTSThe nonsensitive rates on adriamycin, cisplatinum, fluorouracil, mitomycin C, dacarbazine, vincristine, methotrexate in tumor tissues were 41.18%, 17.7%, 47.1%, 50.0%, 76.5%, 61.8% and 52.9%, respectively. The expression of P-glycoprotein (P-gp) and GST-pi in tumor tissues was 1.54 and 2.58 (relative fluorescence intensity). Chi2 analysis showed that there was a positive correlation between P-gp expression and drug resistance on ADM, GST-pi expression and resistance on ADM, DDP and MMC (P < 0.05). There was not seen obvious correlation between expression of MDR1, GST-pi and age, gender, pathological type, tumor size in osteosarcoma and soft tissue sarcoma patients (P > 0.05). The expression of GST-pi was increased in patients receiving preoperative chemotherapy. The rate of postoperative recurrence was higher in patients with higher GST-pi expression level than those with lower GST-pi expression level before operation (P < 0.05).

CONCLUSIONIndividual differences exist in chemotherapy sensitivity and expression of MDR1 and GST-pi in osteosarcoma and soft tissue sarcomas patients. Chemotherapy can induce up-regulation of GST-pi protein expression. Primary high expression of GST-pi is the main mechanism of resistance of osteosarcoma and soft tissue sarcomas to chemotherapy and is related to poor prognosis.

ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; genetics ; Adolescent ; Adult ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Bone Neoplasms ; drug therapy ; genetics ; metabolism ; Child ; Cisplatin ; therapeutic use ; Doxorubicin ; therapeutic use ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Female ; Flow Cytometry ; Follow-Up Studies ; Glutathione S-Transferase pi ; biosynthesis ; genetics ; Humans ; Male ; Middle Aged ; Mitolactol ; therapeutic use ; Mitomycins ; therapeutic use ; Osteosarcoma ; drug therapy ; genetics ; metabolism ; Prognosis ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sarcoma ; drug therapy ; genetics ; metabolism

AIMTo investigate the relationship between glutathione S-transferases gene polymorphism and susceptibility response to hypoxia.

METHODSIn the case-control study, the gene polymorphisms of glutathione S-transferases were tested in Tibetan mountaineers and sea-level Han Chinese by multiple-PCR and PCR-RELP.

RESULTSThe frequency of GSTT1 null genotype was significant different between Tibetan mountaineers and sea-level Han Chinese (P < 0.05), OR = 1.86 (95% CI = 1.01-3.39), and also for GSTP(1-105) mutant genotype in two groups (P < 0.01), OR = 2.19 (95% CI = 1.16-4.13). There was significant difference between A allele and G allele of GSTP(1-105) groups (P < 0.01). There was no difference for GSTM1 null genotype between two groups (P > 0.05), OR = 0.78 (95% CI = 0.43 - 1.42).

CONCLUSIONGSTT1 and GSTP(1-105) genotype may be associated with susceptibility response to altitude hypoxia.

Adult ; Alleles ; China ; Genotype ; Glutathione S-Transferase pi ; genetics ; Glutathione Transferase ; genetics ; Humans ; Hypoxia ; genetics ; Male ; Mountaineering ; Polymorphism, Genetic ; Reactive Oxygen Species ; metabolism ; Young Adult