Expression Of GST-pi Research Articles

Arsenic and Protein Expression: It might help to know the mechanism of As toxicity

Arsenic and Protein Expression: It might help to know the mechanism of As toxicity

Regulation of transgelin and GST-pi proteins in the tissues of hamsters exposed to sodium arsenite

Regulation of transgelin and GST-pi proteins in the tissues of hamsters exposed to sodium arsenite

Does and duration of exposure time dependent regulation of proteins in the urinary bladder of hamsters exposed to sodium arsenite

Does and duration of exposure time dependent regulation of proteins in the urinary bladder of hamsters exposed to sodium arsenite

Repeated 28-DAY oral dose study on Boswellia sacra oleo gum resin extract for testicular toxicity in rats

Ethnopharmacological relevanceFrankincense (Boswellia sacra Fluck.,) is a widely used herbal drug and household medicine for treatment of several diseases. Earlier toxicological studies revealed its proulcerogenic effect and no significant hepatotoxic or nephrotoxic effects in rats. However, some other members of Boswellia species such as Boswellia papyrifera (Caill. ex Delile) Hochst and Boswellia carterii have been reported for testicular toxicity in rats. Aim of the studyTesticular toxicity of standardized methanolic extract of B. sacra oleo gum resin was determined through repeated oral dose administration for 28 days. Biochemical, histological and genetic changes in rat testes were evaluated. Materials and methodsB. sacra extract was analyzed for its boswellic acid content by high performance liquid chromatography (HPLC) method. The extract was administered at three different doses to rats. Effect on behavior, weight, food and water consumption along with changes in serum testosterone levels and cytoarchitecture of testis, epididymis and adrenal gland were determined. Gene expression of GSTPi, IGFBP3 and HSP70 in testis was also studied. ResultsBoswellic acids (α and β) were present in highest concentration whereas acetyl-11-keto beta boswellic acid was present in relatively smaller amounts. The extract did not produce any significant change in the behavior of the animals, food/water consumption or weight gain. Serum testosterone levels were significantly decreased only by highest tested dose of Boswellia extract (1000 mg/kg, p.o). Histological examination did not reveal any variation in the structure of testis, adrenal gland and epididymis after administration of the extract while the expression of all three studied genes was significantly decreased. ConclusionThe results indicate that B. sacra extract does not possess any toxic effect on testis. On the contrary, decrease in gene expression of GSTPi, IGFBP3 and HSP70 revealed its antioxidant potential that may protect testes against effect of toxicants. However, a significant reduction in serum testosterone levels point to mechanisms other than direct testicular toxicity.

Erzhu Jiedu decoction ameliorates liver precancerous lesions in a rat model of liver cancer.

Precancerous lesions are the intermediate stage in the development of liver cancer from cirrhosis. Early intervention measures can effectively prevent the occurrence of liver cancer and prolong the lives of patients, resulting in greater economic effects. Erzhu Jiedu decoction (EJD) is a semiempirical formula that is used in the treatment of cirrhosis and liver cancer according to the academic philosophy of “Preventive treatment of disease” and has achieved good curative effects in clinical practice. The purpose of this study was to investigate the effect of EJD on liver precancerous lesions induced by diethylnitrosamine (DEN) in rats. The results showed that EJD improved the general conditions (body weight, ALT, AST, and GGT) and reduced the number of precancerous lesions in the rat model. Notably, the medium dose of EJD (1.05 g/kg) had better treatment effects than the low dose of EJD, and the high dose of EJD did not further improve the liver lesions compared to the medium dose of EJD. Moreover, EJD effectively reduced the DEN-induced GST-Pi, AFP, CK19, c-Myc, and Ki67 protein expression in liver precancerous tissues. Interestingly, EJD significantly reduced YAP and TAZ mRNA expression in the liver precancerous lesions. Collectively, EJD protects against in the initiation of liver cancer and the regulation of c-Myc and Hippo signaling pathways may be the underlying mechanism.

Elemental concentrations in Choroid-RPE and retina of human eyes with age-related macular degeneration

Heavy metals, metallic and toxic elements are reported to play an essential role in the complex multifactorial pathogenesis of age-related macular degeneration (AMD). This study was aimed to measure the concentrations of these elements in choroid-RPE and retina of human donor eyes with and without age-related macular degeneration associated changes. Human cadaver donor eyeballs were obtained from the CU Shah eye bank, Sankara Nethralaya Eye Hospital, India, after removal of the cornea. 39 control and 51 AMD donor eyes were used in this study. Alabama grading was done on the histopathological sections to identify early and late age-related macular degeneration changes. Concentrations of lead, cadmium, chromium, cobalt, nickel, arsenic and selenium were determined in choroid-RPE and retina using Inductively Coupled Plasma Mass Spectrometer (ICP-MS). Further, gene expression of oxidative stress-related genes, Nrf-2, HO-1, GCLC, GCLM, and detoxification related gene GSTpi was performed. The data were analyzed for statistical significance using Graph Pad® Prism 5 software. Donor eyes with early and late AMD had significantly higher levels of lead, cadmium, chromium, arsenic, and nickel in choroid-RPE and retina compared to the control eyes. Selenium was significantly increased in late AMD compared to control. No significant difference was observed in the levels of cobalt between eyes with and without AMD. Decreased transcript levels of oxidative stress-related genes were observed in the choroid-RPE and retinal tissues. Nrf-2 (p < 0.05), HO-1 and GCLC expressions were lowered in the retina of AMD, whereas GCLM and GSTpi expressions were decreased (p < 0.05) with an increase in HO-1 in choroid-RPE of AMD. This study provides evidence that alterations of the heavy metals and toxic elements along with oxidative stress may play a role in the pathogenesis of AMD.

Role of microRNA-21 in uveal melanoma cell invasion and metastasis by regulating p53 and its downstream protein.

To reveal the insight mechanism of liver metastasis in uveal melanoma, we investigated cell functions of microRNA-21 in three different uveal melanoma cell lines and analyze the relationship of target gene p53 and its downstream targets. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect microRNA-21 expression in normal uveal tissue and uveal melanoma cell lines. Lenti-virus expression system was used to construct OCM-1, MuM-2B and M619 cell line with stable overexpression and inhibition of microRNA-21. In vitro cell function tests such as cell proliferation, cell apoptosis, cell circle and abilities of migration and invasion were examined by MTT, BrdU assay, flow cytometry, transwell assay and Matrigel invasion assay respectively. The target gene was predicted by bioinformatics and confirmed by using a dual luciferase reporter assay. The expression of p53 and its suspected downstream targets LIM and SH3 protein 1 (LASP1) and glutathione S transferase pi (GST-Pi) were determined by qRT-PCR in mRNA level and Western blotting analysis in protein level. Finally, the effect of microRNA-21 in a xenograft tumor model was assessed in four-week-old BALB/c nude mice. Compared to normal uveal melanoma, expressions of microRNA-21 were significantly higher in uveal melanoma cell lines. Overexpression of microRNA-21 promoted proliferation, migration, and invasion of OCM-1, M619 and MuM-2B cells, while inhibition of microRNA-21 reveal opposite effects. Wild type p53 was identified as a target gene of microRNA-21-3p, and proved by dual luciferase reporter assay. Up-regulated microRNA-21 inhibited the expression of wild type p53 gene, and the increased expression of LASP1 in mRNA level and protein level, while down-regulated microRNA-21 presented opposite way. However, GST-pi showed the potential pattern as expected, but relative mRNA level showed no statistically significant difference in OCM-1 cells. Furthermore, the mRNA expression of GST-pi was decreased in microRNA-21 overexpressing MuM-2B, and increased in M619 cells with inhibition of microRNA-21. In vivo, inhibition of microRNA-21 reduced tumor growth with statistically significant difference. These findings provide novel insight into molecular etiology of microRNA-21 in uveal melanoma cell lines, and suggest that microRNA-21 might be a potential candidate for the diagnosis and prognostic factor of human uveal melanoma.

Role of MRP-1 and GST-Pi in MDR and their inhibition by indomethacin in AML

BackgroundMDR continues to be a major challenge to effective chemotherapeutic interventions against cancer. Defining major factor contributing to MDR and inhibiting their action may thus be used for reversing MDR. AimThis work aimed to evaluate the role played by MRP-1 and GST-Pi in MDR, and to explore the possible role of indomethacin as an inhibitor of chemotherapy resistance in patients with AML. Subjects and methodsThe study included 2 groups, one included 20 healthy volunteers and the second included 50 AML patients. All patients received one cycle of standard induction chemotherapy, then regrouped according to their response to either CR group or unremitted group. Unremitted patients received a second cycle of chemotherapy combined with indomethacin. From each subject a blood sample was drawn before and after the 1st cycle of chemotherapy and after the 2nd cycle. From blood, mononuclear cells were separated, mRNA was extracted, and RT-PCR was carried out to detect GST-Pi and MRP-1 gene expression. ResultsGST-Pi expression in CR group was 60% before therapy that significantly decreased to 30% after therapy. While in unremitted group, its expression significantly increased from 30% before to 80% after therapy. GST-Pi positive patients had a significantly lower overall and disease free survival time than GST-Pi negative patients (P=0.000 and 0.039, respectively). While MRP-1 expression was so low (20%) and remained unchanged after therapy in both groups. MRP-1 expression did not affect overall or disease free survival. Taking indomethacin with 2nd cycle of chemotherapy in unremitted patients resulted in a significant inhibition of GST-Pi expression and a significantly longer overall survival time than those taking 2nd cycle chemotherapy alone (P=0.034). ConclusionMRP-1 is not likely to contribute to MDR, while GST-Pi might have a role in MDR phenotype in AML patients. Furthermore, GST-Pi inhibition significantly reduced MDR in AML patients.

Phosphodiesterase-5 inhibition promotes remyelination by MCP-1/CCR-2 and MMP-9 regulation in a cuprizone-induced demyelination model

While it has recently been shown that sildenafil (Viagra®) has a protective effect on myelination/remyelination, the mechanism of this protection is still unknown. In general, cytokines, chemokines and metalloproteinases have a pro-inflammatory action, but can also exert a role in modulating glial cell activation, contributing to the balance of cell response. Investigating these molecules can contribute to clarifying the mechanisms of sildenafil neuroprotection. In addition, it is not known whether sildenafil is able to restore an already installed neurodegenerative process or if the treatment period is critical for its action. The aim of the present study was to evaluate, in a cuprizone (CPZ)-induced demyelination model, the effects and mechanisms of time-dependent treatment with sildenafil (beginning 15days after neurodegeneration and continuing for 15days, or starting concomitantly with neurodegeneration and continuing for 30days) on neuroinflammation and remyelination. Neuroinflammation and demyelination induced by CPZ in rodents has been widely used as a model of multiple sclerosis (MS). In the present study, five male C57BL/6 mice aged 7–10weeks were used per group. For four weeks, the groups received either cuprizone (CPZ) 0.2% mixed in feed or CPZ combined with the administration of sildenafil (Viagra®, Pfizer, 25mg/kg) orally in drinking water, starting concurrently with (sild-T0) or 15days (sild-T15) after the start of CPZ treatment. Control animals received pure food and water. The cerebella were dissected and processed for immunohistochemistry, immunofluorescence (frozen), Western blotting, Luxol fast blue staining and transmission electron microscopy. Magnetic resonance was performed for live animals, after the same treatment, using CPZ 0.3%. CPZ induced an increase in the expression of IL-1β and a decrease in MCP-1, CCR-2, MBP and GST-pi, as well as promoting damage in the structure and ultra-structure of the myelin sheath. Interestingly, the administering of sild-T0 promoted a further increase of MMP-9, MCP-1, and CCR-2, possibly contributing to changes in the microglia phenotype, which becomes more phagocytic, cleaning myelin debris. It was also observed that, after sild-T0 treatment, the expression of GST-pi and MBP increased and the myelin structure was improved. However, sild-T15 was not efficient in all aspects, probably due to the short treatment period and to starting after the installation of the degenerative process. Therefore, the present study shows that sildenafil modulates inflammation, with the involvement of MMP-9, MCP-1, and CCR-2, and also contributes to myelin repair. These protective effects were dependent on the therapeutic strategy used. This clarification can strengthen research proposals into the mechanism of action of sildenafil and contribute to the control of neurodegenerative diseases such as MS.

Erk-Creb pathway suppresses glutathione-S-transferase pi expression under basal and oxidative stress conditions in zebrafish embryos.

Transcriptional activation of phase II enzymes including glutathione-S-transferase pi class (Gst Pi) is important for redox regulation and defense from xenobiotics. The role of extracellular signal-regulated kinase (Erk) and protein kinase B (Akt) in regulation of Gst Pi expression has been described using adult mammalian cells. Whether these signaling pathways contribute to Gst Pi expression during embryogenesis is unknown. Using zebrafish embryo model, we provide novel evidence that Erk signaling acts as a specific suppressor of gstp1-2 mRNA during early embryogenesis. Addition of Erk inhibitor U0126 enhanced gstp1-2 mRNA expression during transition from blastula to the segmentation stage and from pharyngula until the hatching stage. Basal Erk activity did not affect gstp1-2 expression in tert-butylhydroquinone-exposed embryos. Addition of phorbol 12-myristate 13-acetate increased Erk activity leading to suppression of gstp1-2 mRNA. Activation of cAMP/Creb pathway by forskolin prevented gstp1-2 expression, whereas U0126 suppressed Creb phosphorylation, thus setting up Creb as a proximal transmitter of Erk inhibitory effect. Collectively, these findings suggest that Erk-Creb pathway exerts suppressive effect on gstp1-2 mRNA in a narrow developmental window. This study also provides a novel link between Erk and gstp1-2 expression, setting apart a possible differential regulation of gstp1-2 in adult and embryonic cells.

Zebularine partially reverses GST methylation in prostate cancer cells and restores sensitivity to the DNA minor groove binder brostallicin

Brostallicin is a DNA minor groove binder that shows enhanced antitumor activity in cells with high glutathione S-transferase (GST)/glutathione content. Prostate cancer cells present, almost invariably, methylation of the GSTP1 gene promoter and, as a consequence, low levels of GST-pi expression and activity. In these cells, brostallicin shows very little activity. We tested whether pretreatment of heavily GST-methylated prostate cancer cells with demethylating agents could enhance the activity of brostallicin. Human prostate cancer cells LNCaP and DU145 were used for these studies both in vitro and in vivo. The demethylating agent zebularine was used in combination with brostallicin. Methylation specific PCR and pyrosequencing were used to determine the level of GST methylation. Pretreatment with demethylating agents enhanced the in vitro activity of brostallicin in LNCaP cells. Zebularine, in particular, induced an enhancement of activity in vivo comparable to that obtained by transfecting the human GSTP1 gene in LNCaP cells in vitro. Molecular analysis performed on tumor xenografts in mice pretreated with zebularine failed to detect re-expression of GST-pi and demethylation of GSTP1. However, we found demethylation in the GSTM1 gene, with consequent re-expression of GST-mu at the mRNA level. These results indicate that zebularine, both in vitro and in vivo, enhances the activity of brostallicin and that this enhancement correlates with re-expression of GST-pi and GST-mu. These findings highlight the potential therapeutic value of combining demethylating agents and brostallicin in tumors with GST methylation that poorly respond to brostallicin.

Glutathione S-transferase P1 single nucleotide polymorphism predicts permanent ototoxicity in children with medulloblastoma.

Glutathione S-transferase (GST) enzymes are involved in detoxifying chemotherapy and clearing reactive oxygen species formed by radiation. We explored the relationship between the host GSTP1 105 A > G polymorphism (rs1695), tumor GSTpi protein expression, and clinical outcomes in pediatric medulloblastoma. We hypothesized that the GSTP1 105 G-allele and increased tumor GSTpi expression would be associated with lower progression-free survival and fewer adverse events. The study included 106 medulloblastoma/primitive neuroectodermal tumor (PNET) patients seen at Texas Children's Cancer Center. Genotyping was performed using an Illumina HumanOmni1-Quad BeadChip and GSTpi expression was assessed using immunohistochemistry. We used the Kaplan-Meier method for survival analyses and logistic regression for toxicity comparisons. Patients with a GSTP1 105 AG/GG genotype (vs. AA) or who had received high dose craniospinal radiation (≥34 Gy vs. <26 Gy) had a greater risk of requiring hearing aids than their counterparts (OR 4.0, 95% CI 1.2-13.6, and OR 3.1, 95% CI 1.1-8.8, respectively, n = 69). Additionally, there was a statistically significant interaction between these variables. Compared with the lowest risk group (GSTP1 105 AA-low dose radiation), patients with a GSTP1 105 AG/GG genotype who received high dose radiation were 8.4 times more likely to require hearing aids (95% CI 1.4-49.9, p-trend = 0.005, n = 69). When adjusted for age, cumulative cisplatin dose, and amifostine use, the association remained. The GSTP1 105 G-allele is associated with permanent ototoxicity in pediatric medulloblastoma/PNET and strongly interacts with radiation dose. Patients with this allele should be considered for clinical trials employing radiation dose modifications and cytoprotectant strategies.

Identification and Functional Studies of a New Nrf2 Partner IQGAP1: A Critical Role in the Stability and Transactivation of Nrf2

Nuclear factor-erythroid-related factor 2 (Nrf2) is a critical transcriptional factor that is used in regulating cellular defense against oxidative stress. This study is aimed at investigating new interacting protein partners of Nrf2 using One-strep tag pull-down coupled with LTQ Orbitrap LC/MS/MS, and at examining the impact on Nr2 signaling by the newly identified IQ motif containing GTPase activating protein 1 (IQGAP1). Using the One-strep tag pull-down and LTQ Orbitrap LC/MS/MS, we identified IQGAP1 as a new Nrf2 interacting partner. Direct interactions between IQGAP1 and Nrf2 proteins were verified using in vitro glutathione S-transferase (GST) pull-down, transcription/translation assays, and in vivo utilizing Nrf2 overexpressing cells. Coexpression of Dsredmono-IQGAP1 and eGFP-Nrf2 increased the stability of eGFP-Nrf2 and enhanced the expression of Nrf2-target gene heme oxygenase-1 (HO-1). To confirm the functional role of IQGAP1 on Nrf2, knock-downed IQGAP1 using siIQGAP1 attenuated the expression of endogenous Nrf2, HO-1 proteins, and Nrf2-target genes GSTpi, GCLC, and quinone oxidoreductase 1 (NQO-1). Furthermore, the stability of Nrf2 was dramatically decreased in IQGAP1-deficient mouse embryonic fibroblast (MEF) cells. Since IQGAP1 signaling could be mediated by calcium, treating the cells with calcium showed the translocation of IQGAP1/Nrf2 complex into the nucleus, suggesting that IQGAP1 may play a critical role in Nrf2 stability. Interestingly, consistent with calcium signaling for IQGAP1, treating the cells with calcium functionally enhanced Nrf2-mediated antioxidant responsive element-transcription activity and enhanced the expression of the endogenous Nrf2-target gene HO-1. In the aggregate, our current study identifies and functionally characterizes a new Nrf2 partner protein IQGAP1, which may contribute to Nrf2's regulation of antioxidant enzymes such as HO-1. IQGAP1 may play a critical role in the stability and transactivation of Nrf2.

QUALITY OF LIFE/AFTERCARE

BACKGROUND: Glutathione S-transferase (GST) enzymes are involved in detoxifying chemotherapy agents and clearing reactive oxygen species formed by radiation. In this study, we explored the relationship between the host GSTP1-105 polymorphism (rs1695), tumor GSTpi protein expression, and clinical outcomes in pediatric medulloblastoma. We hypothesized that the GSTP1-105 G-allele and increased tumor GSTpi expression would be associated with lower progression-free survival and fewer adverse events. METHODS: The study included 106 medulloblastoma/primitive neuroectodermal tumor (PNET) patients seen at Texas Children’s Cancer Center. Genotyping was performed using an Illumina HumanOmni1-Quad BeadChip and tumor GSTpi expression was assessed using immunohistochemistry. We used the Kaplan-Meier method for survival analyses and multivariable logistic regression for toxicity comparisons. RESULTS: Patients with a GSTP1-105 AG/GG genotype or who had received a higher dose of craniospinal radiation (median 36 Gy) had a greater risk of requiring hearing aids than their respective counterparts (OR 4.0, 95%CI 1.2 - 13.6, and OR 3.1, 95%CI 1.1 - 8.8, respectively). Additionally, there was a statistically significant interaction between the two variables. Compared with the lowest risk group (GSTP1-105 AA-lower dose radiation) patients with a GSTP1-105 AG/GG genotype who received a higher dose radiation were 8.4 times more likely to require hearing aids (95%CI 1.4 - 49.9, p-trend ¼ 0.005). When adjusted for age, gender, and amifostine use, the association remained. CONCLUSIONS: The GSTP1-105 G-allele is associated with permanent ototoxicity in pediatric medulloblastoma/PNET and strongly interacts with radiation dose. A possible mechanism for this finding is that the GSTP1-105 G-allele leads to reduced GSTpi free radical detoxification in the setting of multimodality therapy including cisplatin and radiation. Patients with this allele should be considered for clinical trials employing radiation dose modifications and more targeted cytoprotectant strategies than are currently being used with amifostine.

Glutathione S-transferase Pi expression predicts response to adjuvant chemotherapy for stage C colon cancer: a matched historical control study

BackgroundThis study examined the association between overall survival and Glutathione S-transferase Pi (GST Pi) expression and genetic polymorphism in stage C colon cancer patients after resection alone versus resection plus 5-fluourouracil-based adjuvant chemotherapy.MethodsPatients were drawn from a hospital registry of colorectal cancer resections. Those receiving chemotherapy after it was introduced in 1992 were compared with an age and sex matched control group from the preceding period. GST Pi expression was assessed by immunohistochemistry. Overall survival was analysed by the Kaplan-Meier method and Cox regression.ResultsFrom an initial 104 patients treated with chemotherapy and 104 matched controls, 26 were excluded because of non-informative immunohistochemistry, leaving 95 in the treated group and 87 controls. Survival did not differ significantly among patients with low GST Pi who did or did not receive chemotherapy and those with high GST Pi who received chemotherapy (lowest pair-wise p = 0.11) whereas patients with high GST Pi who did not receive chemotherapy experienced markedly poorer survival than any of the other three groups (all pair-wise p <0.01). This result was unaffected by GST Pi genotype.ConclusionStage C colon cancer patients with low GST Pi did not benefit from 5-fluourouracil-based adjuvant chemotherapy whereas those with high GST Pi did.

Abstract 3284: Organ-specific Gst-pi expression in new metastatic androgen-independent prostate cancer animal model

Abstract Introduction: Prostate cancer is major leading cause of death in western world. And elucidating the mechanisms of metastasis in prostate cancer, particularly to the bone, is a major issue for treatment of this malignancy. Reactive oxygen species (ROS) have been identified as important chemical mediators in cell growth and differentiation, and glutathione oxidation-reduction (redox) system is the main mechanism against ROS in human body. We established a new metastatic model of prostate cancer, using new cell line, and we investigated the role of glutathione redox system in the androgen-independent prostate cancer. Methods: A new cell line, PCai1, was established from the androgen-independent cancer derived from transgenic rat model for adenocarcinoma of the prostate (TRAP), and investigated its metastatic potential in nude mice. By cDNA microarray analysis in our model, glutathione S-transferase Pi (Gst-pi), which was the major component protein of glutathione redox system, had higher expression in androgen independent prostate cancer. Therefore, effects of Gst-pi knocked down in vitro with iRNA strategy were analyzed in PCai1 cells, and the quantification of ROS was performed by DCFH assay. In addition, to evaluate roles of Gst-pi in castration-resistant cell proliferation, Gst-pi siRNA transfected PCai1 cells were transplanted subcutaneously in castrated or normal nude mice. Results: Gst-pi-siRNA in vitro significantly suppressed cell proliferation rate of PCai1. In addition, high levels of intracellular ROS were recognized in the Gst-pi knockout. Strong expression of Gst-pi was realized castration-resistant prostate cancer specimen, while no tumor was stained in hormone naïve prostate cancer. PCai1 frequently formed metastatic lesions in the lung and lymph nodes after orthotopic implantation in the prostate. Intravenous injections of PCai1, metastasis to lung and bone were obvious. Tumors metastasized in the bone marrow after intravenous injection expressed high levels of Gst-pi, while metastatic lesions in the lung and lymph nodes failed to express Gst-pi as seen in cases of the orthotopic implantation. Knock down of Gst-pi expression of PCai1 significantly inhibited tumor growth of PCai1 compared to negative controls in castrated mice. On the other hand, Gst-pi expression did not affect tumor growth in non-castrated mice Conclusion: Establishment of a new metastatic prostate cancer model allows for the systematic analysis of genetic and molecular characteristics of human tumors. And these results suggest that glutathione redox system can regulate cancer cell growth in androgen free environment. Gst-pi expression of the prostate cancers are dependent on metastatic site, and that Gst-pi has an important role in adapting prostate cancer for growth and metastasis involving an alteration of ROS signals. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3284. doi:1538-7445.AM2012-3284

Clinicopathological correlates and prognostic significance of glutathione S-transferase Pi expression in 468 patients after potentially curative resection of node-positive colonic cancer

This study investigated the association between glutathione S-transferase Pi (GST Pi) expression, histopathology and overall survival in 468 patients after resection of stage C colonic adenocarcinoma. Data were drawn from a prospective hospital registry of consecutive bowel cancer resections with a minimum follow-up of 5 years. Nuclear and cytoplasmic GST Pi expression, assessed by both intensity of staining and percentage of stained cells at both the central part of the tumour and the invasive tumour front, were evaluated retrospectively by tissue microarray immunohistochemistry on archival specimens. The most effective measure of GST Pi expression was the percentage of immunostained nuclei in central tumour tissue, where >40% stained was associated significantly with high grade, invasion beyond the muscularis propria, involvement of a free serosal surface or apical node, and invasion into an adjacent organ or structure. After adjustment of other predictors, GST Pi expression remained independently prognostic for reduced overall survival (hazard ratio 1.4, P = 0.002). In patients with clinicopathological stage C colonic cancer, GST Pi expression is associated with features of tumour aggressiveness and with reduced overall survival. Further appropriately designed studies should aim to discover whether GST Pi can predict response to adjuvant chemotherapy.

Differences in glutathione S-transferase pi expression in transgenic mice with symptoms of neurodegeneration.

Glutathione S-transferase pi (GST pi) is an enzyme involved in cell protection against toxic electrophiles and products of oxidative stress. GST pi expression was studied in transgenic mice hybrids (B6-C3H) with symptoms of neurodegeneration harboring SOD1G93A (SOD1/+), Dync1h1 (Cra1/+) and double (Cra1/SOD1) mutations, at presymptomatic and symptomatic stages (age 70, 140, 365 days) using RT-PCR and Western blotting. The main changes in GST pi expression were observed in mice with the SODG93A mutation. In SOD1/+ and Cra1/SOD1 transgenics, with the exception of cerebellum, the changes in GST pi-mRNA accompanied those in GST pi protein. In brain cortex of both groups the expression was unchanged at the presymptomatic (age 70 days) but was lower at the symptomatic stage (age 140 days) and at both stages in hippocampus and spinal cord of SOD1/+ but not of Cra1/SOD1 mice compared to age-matched wild-type controls. In cerebellum of the presymptomatic and the symptomatic SOD1/+ mice and presymptomatic Cra1/SOD1 mice, the GST pi-mRNA was drastically elevated but the protein level remained unchanged. In Cra1/+ transgenics there were no changes in GST pi expression in any CNS region both on the mRNA and on the protein level. It can be concluded that the SOD1G93A but not the Dync1h1 mutation significantly decreases detoxification efficiency of GST pi in CNS, however the Dync1h1 mutation reduces the effects caused by the SOD1G93A mutation. Despite similarities in neurological symptoms, the differences in GST pi expression between SOD1/+ and Cra1/+ transgenics indicate a distinct pathogenic entity of these two conditions.

Organ specific Gst‐pi expression of the metastatic androgen independent prostate cancer cells in nude mice

Elucidating the mechanisms of metastasis in prostate cancer, particularly to the bone, is a major issue for treatment of this malignancy. We previously reported that an androgen-independent variant had higher expression of glutathione S-transferase pi (Gst-pi) compared with a parent androgen-dependent transplantable rat prostate carcinoma which was established from the transgenic rat for adenocarcinoma of the prostate (TRAP). A new cell line, PCai1, was established from the androgen-independent tumor and investigated its metastatic potential in nude mice. The tumorigenesis of PCai1 cells in vivo was studied by subcutaneous transplantations into nude mice. The growth in the microenvironment of the prostate was studied by orthotopic transplantation of PCai1 cells into nude mice. The metastatic potential of PCai1 cells was studied by tail vein injections. Effects of Gst-pi knocked down were analysis in PCai1 cells. PCai1 frequently formed metastatic lesions in the lung and lymph nodes after orthotopic implantation in the prostate. Intravenous injections of PCai1, metastasis to lung and bone were obvious. PCai1 had strong expression for Gst-pi, therefore we tried knocked down Gst-pi. Gst-pi-siRNA in vitro significantly suppressed cell proliferation rate. In addition, high levels of intracellular reactive oxygen species (ROS) were recognized in the Gst-pi knockout. Gst-pi expression of the prostate cancers are dependent on metastatic site, and that Gst-pi has an important role in adapting prostate cancer for growth and metastasis involving an alteration of ROS signals.

Abstract 218: GSTP1 expression in human cancers decreases p53 protein levels and markedly attenuates p53-dependent responses to therapy-induced DNA damage

Abstract The antioxidant enzyme glutathione transferase-pi (GSTP1) is expressed at high levels in most human cancers and this has been well linked to increased malignancy, enhanced anticancer drug resistance, and poor prognosis. While the enzymic inactivation anticancer drugs by GSTP1 can explain the tumor drug resistance, at least in part, it cannot fully account for the impact of this protein on aggressive tumor growth and patient's poor response to therapy. Relevant to this notion and analogous to the known negative regulation of JNK and other stress-activated kinases by GST-pi, here, we report for the first time a physical association between p53 and GSTP1 proteins both in vitro and in tumor cells, decreased levels of basal p53, and a significant attenuation in the biological responses of p53 to DNA damage in GSTP1 expressing cancer cell lines. Enforced expression of GSTP1 in GSTP1-deficient MCF-7 breast cancer and UW228 medulloblastoma cell lines induced a reproducible decrease (approx. 40%) in the basal and DNA-damage inducible p53 protein levels. In nuclear extracts from camptothecin-treated MCF-7 and UW228 cells, p53 binding to DNA was 35-45% lower after enforced expression of GST-pi. In contrast, inhibiting GSTP1 by ethacrynic acid or its silencing by specific siRNA in the pi-proficient A549 and H460 lung cancer cells resulted in the increase of basal p53 levels. In all these experiments, the greater levels of basal p53 found in the GSTP1 knock-out cells translated into enhanced binding of the tumor suppressor to DNA in EMSA or DNA-affinity immunoblotting procedures. These changes were also reflected in the activity of p53 as a transcriptional regulator. Semi-quantitative RT-PCR showed that several targets of p53 transcriptional activation viz., p21cip1, MDM2, IGFBP3 and Bax were upregulated in H460 cells transfected with GSTP1 siRNA, while their mRNA levels were significantly lower in MCF-7 cells stably expressing GSTP1 as compared to the untransfected parent cells. Contrary to our expectation, the presence of GSTP1 in MCF-7 cells actually increased the half-life and stability of p53 protein, albeit slightly, indicating that other mechanisms, possibly involving the JNK, may mediate the decrease of basal p53 protein. Significant differences in the cell cycle progression were also evident in this setting; treatment of GSTP1-silenced H460 cells with vincristine or camptothecin resulted in a marked increase of sub G1 DNA content and apoptotic cells. The findings highlight a novel role for GSTP1 in diminishing the DNA damage response of p53 in human cancers. Such observations have been made in chemically-induced hepatic preneoplastic foci that express GSTP1 de novo. Our results bear huge significance for increasing the efficacy of anticancer drugs, not only the alkylating agents, but also of other classes by restoring p53 responsiveness through GSTP1 inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 218. doi:10.1158/1538-7445.AM2011-218