Glutathione S-transferase P1 Expression Research Articles

The protective effect of imatinib against pancreatic β-cell apoptosis induced by dexamethasone via increased GSTP1 expression and reduced oxidative stress

Glucocorticoids (GCs) are known to stimulate pancreatic beta (β)-cell apoptosis via several mechanisms, including oxidative stress. Our previous study suggested an increase in dexamethasone-induced pancreatic β-cell apoptosis via a reduction of glutathione S-transferase P1 (GSTP1), which is an antioxidant enzyme. Imatinib, which is a tyrosine kinase inhibitor, also exerts antioxidant effect. This study aims to test our hypothesis that imatinib would prevent pancreatic β-cell apoptosis induced by dexamethasone via increased GSTP1 expression and reduced oxidative stress. Our results revealed that dexamethasone significantly increased apoptosis in INS-1 cells when compared to the control, and that imatinib significantly decreased INS-1 cell apoptosis induced by dexamethasone. Moreover, dexamethasone significantly increased superoxide production in INS-1 cells when compared to the control; however, imatinib, when combined with dexamethasone, significantly reduced superoxide production in INS-1 cells. Dexamethasone significantly decreased GSTP1, p-ERK1/2, and BCL2 protein expression, but significantly increased p-JNK, p-p38, and BAX protein expression in INS-1 cells—all compared to control. Importantly, imatinib significantly ameliorated the effect of dexamethasone on the expression of GSTP1, p-ERK1/2, p-JNK, p-p38 MAPK, BAX, and BCL2. Furthermore—6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio) hexanol (NBDHEX), which is a GSTP1 inhibitor, neutralized the protective effect of imatinib against pancreatic β-cell apoptosis induced by dexamethasone. In conclusion, imatinib decreases pancreatic β-cell apoptosis induced by dexamethasone via increased GSTP1 expression and reduced oxidative stress.

Irreversible methadone-induced GSTP1 downregulation in SH-SY5Y cells

BackgroundMethadone has been reported to downregulate the expression of glutathione S-transferase P1 (GSTP1) among nine antioxidant genes in SH-SY5Y cells after both short- and long-term treatment. GSTP1 plays a key role in the detoxification of many xenobiotics and is frequently associated with various diseases, especially tumors. The objective of this study is to determine whether this change is reversible.MethodsTwo different treatment protocols were used. The first protocol evaluated the reversibility of the GSTP1 mRNA change, while the second protocol evaluated the methylation status of the GSTP1 promoter site. To investigate the reversibility of the GSTP1 mRNA change, SH-SY5Y cells were treated with methadone. The drug was then removed from the medium and the cells were cultured in methadone-free medium for a period of time. GSTP1 mRNA levels were expressed as cycle threshold (Ct) values using TATA box-binding protein as a calibrator gene. Methylation at the promoter site was detected by bisulfite treatment.ResultsThe analysis of variance revealed no significant change in GSTP1 mRNA levels in the cells after methadone was removed from the medium of methadone-treated cells. The study also examined the methylation status of a CpG island in the promoter of GSTP1 in the treated cells. The results demonstrate that although methadone downregulates the mRNA level of GSTP1 in treated cells, it does not induce methylation in the GSTP1 promoter region.ConclusionsThe expression of the GSTP1 remains downregulated even after methadone removal from SH-SY5Y cell culture medium; however, methylation of the GSTP1 promoter site does not play a role in this process.

Abstract B009: Multi-omics analysis of glutathione S-transferase P1 (GSTP1) knockdown model suggests role in pancreatic ductal adenocarcinoma (PDAC) cell metabolism

Abstract GSTP1 belongs to a large family of detoxification enzymes essential for cellular homeostasis in healthy cells. GSTP1 is overexpressed in many cancers and is associated with poor survival in PDAC patients. Studies in glioblastoma and breast cancer have suggested crucial roles of GST family members in metabolism through increased lactate dehydrogenase A and increased GAPDH activity, respectively. The mechanisms underlying the action of GSTP1 in PDAC metabolism and cell proliferation are not well studied. To begin characterizing the diverse functions of GSTP1 in cancer cells, we knocked down GSTP1 expression in three different PDAC cell lines. We revealed unique changes in the global transcriptomic, proteomic, and metabolomic signatures associated with GSTP1 knockdown compared to control cells through multi-omics techniques, real-time ATP production assays, and comprehensive hydrophilic metabolite and phospholipid panels. Through preliminary transcriptomic and proteomic analyses, we found 461 genes and 57 proteins differentially expressed in our GSTP1 knockdown MIA PaCa-2 cells. Among these, 41 were similarly upregulated or downregulated at the mRNA and protein levels. Pathway analysis of these changes revealed profound effects on energy production and lipid metabolism in GSTP1 knockdown cells. Hydrophilic metabolite and phospholipid panels and real-time ATP production assays supported our findings. Our results indicate that GSTP1 knockdown may directly influence mitochondrial dysfunction and lipid peroxidation pathways through associated reductions in ALDH7A1, GLUT3, and SLC7A11 levels. Continued research to further elucidate precise mechanisms and understand GSTP1’s role in driving cancer cell metabolism will aid in establishing GSTP1 targeting as a therapeutic opportunity for PDAC treatment. Citation Format: Jenna Duttenhefner, Katie M. Reindl. Multi-omics analysis of glutathione S-transferase P1 (GSTP1) knockdown model suggests role in pancreatic ductal adenocarcinoma (PDAC) cell metabolism [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B009.

Prostate Imaging Reporting and Data System score (PI-RADS) and Glutathione S-transferase P1 methylation status (GST-P1) in the diagnosis of prostate cancer patients with borderline PSA values

Objectives. The objective of this study was to evaluate the potential use of Prostate Imaging – Reporting and Data System version 2 (PI-RADS) in combination with Glutathione S-transferase P1 (GST-P1) expression for an improved diagnosis of prostate cancer, in patients with inconclusive values of prostate-specific antigen (PSA). Materials and Methods. The study was conducted on 80 patients for whom PSA values were evaluated and were found to be inconclusive (4-10 ng/ml). These patients underwent imagistic evaluation (PI-RADS), followed by transurethral prostate biopsy, with the evaluation of GST-P1 expression and histopathological examination (for diagnosis confirmation). Results. By combining the results of PI-RADS and GST-P1 the capacity of the tests to correctly identify healthy subjects, with an area under curve of 0,832 (95% CI 0.732–0.907), with a sensitivity of 73,25% and a specificity of 77,78%. Conclusions. PI-RADS lesions and GST-P1 methylation testing when PSA levels are in a “grey zone”, provide a better specificity and sensitivity by comparison through single testing. Testing patients with inconclusive PSA levels allows for a more accurate diagnosis and less over-diagnosis by non-invasive procedures, such as repeated biopsies.

MiR-513a-3p promotes radiation-induced apoptosis of human lung cells by inhibiting glutathione S-transferase P1.

Radiotherapy is a common treatment for lung cancer. However, radiation pneumonitis caused by radiotherapy can affect the quality of life and prognosis of lung cancer patients. miR-513a-3p has been found to sensitize human lung adenocarcinoma cells to chemotherapy by targeting glutathione S-transferase P1 (GSTP1). Here, we found that x-ray induced the apoptosis of BEAS-2B and miR-513a-3p expression in a dose- and time-dependent manner, and miR-513a-3p-mimic significantly increased x-ray induced apoptosis, while miR-513a-3p-inhibitor significantly decreased x-ray induced apoptosis. Dual luciferase gene reporter system showed that miR-513a-3p targeted to inhibit the expression of GSTP1 in BEAS-2B cells. Moreover, knockdown of GSTP1 significantly increased, while overexpression of GSTP1 decreased the apoptosis of BEAS-2B induced by x-ray. Importantly, overexpression of GSTP1 significantly reduced miR-513a-3p-mimic elevated x-ray -induced apoptosis in BEAS-2B cells. In conclusion, x-ray caused increased expression of miR-513a-3p, and miR-513a-3p promoted x-ray-induced apoptosis of human lung cells by inhibiting GSTP1.

Overexpression of GSTP1 promotes colorectal cancer cell proliferation, invasion and metastasis by upregulating STAT3.

The abnormal expression of glutathione S-transferase P1 (GSTP1) is associated with the progression of several tumor types. However, its role and molecular mechanism in the progression of colorectal cancer (CRC) are largely unknown. To examine the effect of GSTP1 in CRC and determine its possible mechanisms. In the present study, immunohistochemistry (IHC) and the quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis were used to detect the expression of GSTP1 and signal transducer and activator of transcription 3 (STAT3) in CRC tissues. Western blotting was applied to detect the expression of GSTP1 and proteins of the Janus kinase (JAK)-STAT3 pathway in different CRC cell lines. The interaction and co-localization of GSTP1 and STAT3 were detected using co-immunoprecipitation (co-IP) and immunofluorescence (IF) in the SW620 cell line. A positive correlation was identified between the expression of GSTP1 and STAT3 in human CRC tissues. The overexpression of GSTP1 promoted the proliferation, invasion and metastasis of CRC cells by upregulating STAT3. The GSTP1 and STAT3 can directly bind to and regulate each other. The interaction between them is regulated by the upstream gene called F-box only protein 8 (FBX8). The present study demonstrated that GSTP1 could enhance the expression of STAT3 to promote the proliferation, invasion and metastasis of CRC cells, which provides a potential therapeutic target for the clinical treatment of CRC.

Chemoprevention of pancreatic cancer by inhibition of glutathione-S transferase P1.

Pancreatic cancer is among the most refractory malignancies with poor prognosis. Thus, preventive approaches, in addition to the development of novel therapeutic strategies are essential for this type of cancer. KRAS mutations occur very early in the development of pancreatic cancers and could be targeted for its prevention, yet specific inhibitors for mutated KRAS are lacking. Accordingly, Glutathione-S Transferase p1 (GSTP1), which we recently found to be an autocrine stimulator of mutated KRAS signaling, is predicted to be an alternative target for chemoprevention of pancreatic cancer. In this study, chemopreventive effects of O-Hexadecyl-γ-glutamyl-S-benzyl-cysteinyl-D-phenyl glycine-Ethylester (HGBPE), which we previously synthesized to inhibit GSTP1 activity, was analyzed for its effect on the prevention of a rat pancreatic carcinogenesis model induced by 7,12-dimethyl-benzanthracene (DMBA). Rats administered with DMBA were grouped into five cohorts. In the treated group I, which was treated neither with HGBPE nor vehicle, sequential appearance of precancerous lesions, ductal complexes, and adenocarcinoma was confirmed as previously reported. We also confirmed in this group that mutations of KRAS and expression of GSTP1 simultaneously occurred in the ductal complex. To rats of groups II and IV, HGBPE was administered, and vehicle to those of group III and V. In groups of II and IV, the incidence of both ductal complex and adenocarcinoma were significantly lower than those in groups III and V. These data clearly suggest the efficacy of HGBP as a potential chemopreventive agent for pancreatic cancer.

Glutathione S-transferases P1-mediated interleukin-6 in tumor-associated macrophages augments drug-resistance in MCF-7 breast cancer

Glutathione S-transferase P1 (GSTP1), a phase II detoxifying enzyme, is overexpressed and plays an important role during breast cancer drug resistance. Tumor-associated macrophages (TAMs), representing most of the leukocyte population in solid tumors, are involved in cancer cell resistance to chemotherapy. Although GSTP1 exists in TAMs, whether GSTP1 in TAMs promotes drug resistance is still unclear. In the current study, we found a novel mechanism that GSTP1 in TAMs contributes breast cancer cell drug resistance. GSTP1 is aberrantly expressed in TAMs from breast cancer tissues of patients after chemotherapy than that without chemotherapy. Adriamycin (ADR) time-dependently induced the expression of GSTP1 in TAMs in vitro. Conditional medium of TAMs significantly inhibited ADR-induced cell death of MCF-7 breast cancer cells. Meanwhile, overexpression of GSTP1 in TAMs promoted the expression and release of interleukin-6 (IL-6) associated with reduced ADR-induced breast cell death, which was reversed by IL-6 antibody. Mechanistically, GSTP1 interacted with inhibitor of nuclear factor κB kinase β (IKKβ) to activate nuclear factor-κB (NF-κB) to induced the expression and release of IL-6 in TAMs. Moreover, IL-6 further upregulated GSTP1 through c-Jun, and ultimately mediated drug resistance in MCF-7 cells. Taken together, our data demonstrated for the first time that GSTP1 in TAMs promoted ADR-resistance in breast cancer by regulating interleukin-6 release.

Targeting G6PD reverses paclitaxel resistance in ovarian cancer by suppressing GSTP1

Ovarian cancer is one of the leading causes of mortality in women worldwide. Currently, paclitaxel is one of the most effective chemotherapies. However, resistance to paclitaxel is a major cause of therapy failure and the precise mechanism of paclitaxel resistance remains unclear. In this study, we demonstrated that the oxidative pentose phosphate pathway (PPP) enzyme glucose-6-phosphate dehydrogenase (G6PD) promotes paclitaxel resistance. We showed that G6PD expression was higher in paclitaxel-resistant cancer cells than in their paclitaxel-sensitive counterparts. Furthermore, we demonstrated that suppressing G6PD using shRNA, or an inhibitor, either as single agents or in combination, sensitized paclitaxel-resistant cancer cells to paclitaxel treatment and thereby improving the therapeutic efficacy of paclitaxel. Interestingly, we found that the upregulation of G6PD in paclitaxel-resistant cells was due to the decreased expression of protein arginine methyltransferase 6 (PRMT6), which targets the promoter of G6PD. We further identified that G6PD promotes paclitaxel resistance by regulating the expression of glutathione S-transferase P1 (GSTP1), which confers resistance to chemotherapy by detoxifying several anticancer drugs. Taken together, our results suggest that G6PD is a novel potential target to overcome paclitaxel resistance.

Long intergenic noncoding RNA 00844 promotes apoptosis and represses proliferation of prostate cancer cells through upregulating GSTP1 by recruiting EBF1.

Accumulating evidence have suggested the function of long noncoding RNAs as crucial players in the pathogenesis of prostate cancer (PC), a urologic tumor in male with poor prognosis. This study was designed to explore the functions of long intergenic noncoding RNA 00844 (LINC00844) in PC progression. The expression of LINC00844 and glutathione S-transferase P1-1 (GSTP1) was detected by reverse transcription quantitative polymerase chain reaction, followed by the identification of the relationship among LINC00844, GSTP1, and early B cell factor 1 (EBF1) by dual luciferase reporter gene assay, RNA immunoprecipitation assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay. Using loss- and gain-of-function assays, the effects of LINC00844, GSTP1, and EBF1 on the biological characteristics of PC cells were assessed by cell counting kit-8 assay, 5-ethynyl-2'-deoxyuridine assay, and flow cytometry. Lastly, the results from in vitro experiments were verified in vivo by establishing a xenograft tumor model in nude mice. LINC00844 and GSTP1 both displayed low expression in PC tissues and cells. LINC00844 positively regulated the expression of GSTP1 via recruiting EBF1. Overexpression of LINC00844 reduced proliferation and elevated apoptosis of PC cells through recruiting EBF1, which subsequently upregulated GSTP1. In vivo experiments confirmed that LINC00844 or GSTP1 upregulation attenuated tumor growth. LINC00844 elevated GSTP1 expression by recruiting EBF1 to the promoter region of GSTP1, thereby suppressing PC progression. Hence, LINC00844 is a novel therapeutic target for the development of new treatment protocols for PC.

Multidrug resistance affects the prognosis of primary epithelial ovarian cancer.

Multidrug-resistant tumor cells can tolerate different structures, functions and antidrug action mechanisms, therefore, allowing these cells to respond to various structurally unrelated mechanisms of different chemotherapy drugs and to exhibit cross-resistance. The present study aimed to investigate the role of Multi-drug resistance gene (MDR1), Placental glutathione S-transferase-P1 (GSTP1), Lung resistance protein (LRP) and Ras association domain family member 1 (RASSF1A) in primary epithelial ovarian cancer (PEOC). The mRNA (protein) expression levels of MDR1, product P glycoprotein, LRP and GSTP1 were evaluated with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis in all tissue samples, ovarian cancer cell line A2780 and A2780/DDP. Methylation-specific PCR (MSP) was used to detect RASSF1A gene methylation in all tissue samples. The resistance genes/proteins were either poorly or not expressed in A2780, however were highly expressed in A2780/DDP cell line. The expression of resistance genes/proteins decreased following different concentrations of zebularine-stimulated A2780/DDP. Hypermethylation and low expression of RASSF1A gene were detected in PEOC and A2780/DDP. Subsequent to being exposed to different concentrations of zebularine-stimulated A2780/DDP, the RASSF1A methylation level was decreased, while the unmethylation level was increased. The expression of RASSF1A gene/protein was gradually restored, and the gene/protein expression was enhanced with the increase in drug concentration. Multivariate logistic regression indicated that the expression level of gene LRP and GSTP1 was a risk factor for PEOC prognosis. Furthermore, the expression of LRP and GSTP1 in the negative-group survival curves was higher compared with the positive group. High expression of resistance genes may serve an important role in cancer primary resistance. Low expression caused by hyper-methylation of RASSF1A gene may serve an important role in cancer-acquired resistance in PEOC. The present study suggested that resistant gene expression may be a potential prognostic biomarker.

Sulodexide up-regulates glutathione S-transferase P1 by enhancing Nrf2 expression and translocation in human umbilical vein endothelial cells injured by oxygen glucose deprivation.

IntroductionSulodexide (SDX) is used for the treatment of many vascular disorders due to its anticoagulant, anti-inflammatory and anti-atherosclerotic properties. However, the detailed molecular mechanism of its endothelioprotective action is still not completely understood. There is increasing evidence suggesting that antioxidant enzymes play an important role in anti-ischemic properties of SDX. We postulate that up-regulation of glutathione-S-transferase P1 (GSTP1) mediated by the transcription factor Nrf2 could be associated with the antioxidant effect of SDX on vascular endothelial cells.Material and methodsIn the present study, we investigated whether SDX affects GSTP1 and Nrf2 in oxygen glucose deprivation (OGD) treated human umbilical vein endothelial cells (HUVECs). The cells treated with/without SDX (0.5 LRU/ml) were subjected to OGD for 1–6 h. To study the influence of SDX on the Nrf2 nucleus accumulation, the cells were incubated with 0.5 LRU/ml SDX in OGD for 1 h.ResultsWe found that after short-term OGD (1–3 h), the drug increased the expression of both GSTP1 and Nrf2 mRNA/protein in HUVECs (p < 0.05), as determined by real-time PCR and enzyme-linked immunosorbent assay (ELISA). SDX treatment also enhanced the nuclear accumulation of Nrf2 in HUVECs after 1 h of OGD (p < 0.05).ConclusionsSDX induces a rapid onset of the antioxidant response by up-regulating the expression of GSTP1 and Nrf2 in endothelial cells subjected to in vitro simulated ischemia.

Accumulation of hypoxia imaging probe "18F-FMISO" in macrophages depends on macrophage polarization in addition to hypoxic state.

Macrophages play an essential role in immune response, and are closely related to the progression of diseases such as cancer and atherosclerosis. Macrophages polarize to M1 or M2 type, which is related to the environmental hypoxic state. Previously, we found that 18F-FMISO uptake varied according to expression levels of biomolecules such as glutathione S-transferase P1 (GST-P1), which catalyzes the conjugation of glutathione to 18F-FMISO metabolites, and multidrug resistance-associated protein 1 (MRP1), which exports glutathione-18F-FMISO metabolite conjugates out of cells. However, the relationship between macrophage polarization and 18F-FMISO accumulation remains unclear. Mouse peritoneal macrophages were polarized to either the M1 or M2 type, and were treated with 18F-FMISO. Then, their radioactivity after a 4h incubation period under normoxic (21% O2) or hypoxic (1% O2) condition was measured. GST-P1 and MRP1 expression levels were measured by qRT-PCR. M2 macrophages exhibited a significantly higher uptake of 18F-FMISO than non-polarized (M0) macrophages, whereas M1 macrophages had a significantly lower uptake than M0 macrophages (M0: 1.05 ± 0.22, M1: 0.34 ± 0.02, M2: 4.17 ± 0.36 %dose/mg protein). The GST-P1 expression level in M1 macrophages was higher than that in M2 and M0 macrophages [GST-P1/β-actin normalized by M0: 9.0 ± 3.7 (M1), 1.2 ± 0.2 (M2)]. The MRP1 expression level in M1 macrophages was significantly higher than that in M2 and M0 macrophages [MRP1/β-actin normalized by M0 macrophages: 5.1 ± 2.1 (M1), 2.8 ± 1.0 (M2)]. 18F-FMISO accumulation in macrophages may depend on the polarization state in addition to hypoxic condition.

Expression of resistance gene and prognosis of chemotherapy in primary epithelial ovarian cancer

The sensitivity of tumor cells to chemotherapy drugs may become attenuated accounts for various reasons. Reduced drug sensitivity may cause the failure of chemotherapy and affect the prognosis of patients with cancer. This study investigates the relationship between the expression levels of lung resistance protein (LRP) and placental glutathione S-transferase-P1 (GSTP1), the resistance of primary epithelial ovarian cancer (PEOC) to chemotherapy, and the prognosis of patients with platinum drug-resistant PEOC.Quantitative PCR (QT-PCR) was used to detect the mRNA level of the resistance genes LRP, GSTP1 in all tissue and cell lines.The expression levels of resistance gene (LRP, GSTP1) in PEOC were the highest, followed by borderline adenoma tissues, and the lowest levels found in benign tumor tissues, the difference of genes expression between different tissues was statistically significant; the difference between the expression rates and relative expression level of drug resistance genes was statistically significant in platinum sensitive group compare with the platinum resistant group. The difference between resistant gene negative-expression and positive-expression of chemotherapy efficiency, disease free survival time, and recurrence time were statistically significant. The resistant genes expression in the PEOC patients of the negative-group survival curves was higher than that in the positive group. With ascites non-cellular component (ANCC) stimulated SKOV3 cells, the cell proliferation inhibition rate (CPIR) increased, and with ANCC stimulated SKOV3/DDP, the expression of LRP and GSTP1 also increased.ANCC may promote the expression of drug resistance genes, and the expression of genes may predict the poorly prognosis of epithelial ovarian cancer.

Small hepatitis delta antigen selectively binds to target mRNA in hepatic cells: a potential mechanism by which hepatitis D virus downregulates glutathione S-transferase P1 and induces liver injury and hepatocarcinogenesis.

Liver coinfection by hepatitis B virus (HBV) and hepatitis D virus (HDV) can result in a severe form of hepatocellular carcinoma with poor prognosis. Coinfection with HDV and HBV causes more deleterious effects than infection with HBV alone. Clinical research has shown that glutathione S-transferase P1 (GSTP1), a tumor suppressor gene, is typically downregulated in liver samples from hepatitis-infected patients. In the present study, our data indicated that small HDV antigen (s-HDAg) could specifically bind to GSTP1 mRNA and significantly downregulate GSTP1 protein expression. For the human fetal hepatocyte cell line L-02, cells transfected with s-HDAg, along with decreased GSTP1 expression, there was a significant accumulation of reactive oxygen species (ROS) and increased apoptotic ratios. Restoring GSTP1 expression through silencing s-HDAg via RNAi or overexpressing exogenous GSTP1 could largely recover the abnormal cell status. Our results revealed a novel potential mechanism of HDV-induced liver injury and hepatocarcinogenesis: s-HDAg can inhibit GSTP1 expression by directly binding to GSTP1 mRNA, which leads to accumulation of cellular ROS, resulting in high cellular apoptotic ratios and increased selective pressure for malignant transformation. To our knowledge, this is the first study to examine s-HDAg-specific pathogenic mechanisms through potential protein-RNA interactions.

Abstract LB-389: miR133a-3p sensitizes breast cancer cells by targeting GSTP1

Abstract Breast cancer is a main threat for women's health and mammary carcinogenesis is attributed to estrogen receptor α (ERα)-mediated signaling pathways. Tamoxifen is one of the most effective ERα-targeting antiestrogen therapies to treat breast cancer. However, tamoxifen resistance remains a major hurdle in the long-term management of breast cancer. Glutathione S-transferase P1-1 (GSTP1) is one of the major non-hepatic GST family and plays important role in detoxification. Our previous study has shown that GSTP1 is a modulator for ERα signaling in tamoxifen-resistant MCF7 breast cancer cells (LCC3) and other studies have proposed that GSTP1 is one of biomarkers for the diagnosis of breast cancer risk and drug resistance. In the present study, a microRNA (miR) 133a-3p is identified as a functional modulator for GSTP1 expression and cell proliferation in LCC3 cells. We have found that level of miR133a-3p was much lower and the GSTP1 expression at both mRNA and protein was higher in LCC3 cells compared to MCF7 cells. Treatment of miR133a-3p mimics decreased the levels of GSTP1 in LCC3 cells. Finally, the miR133a-3p mimics resensitized the LCC3 cells against tamoxifen-induced cell death. Our data demonstrated that GSTP1 is the target of miR133a-3p in MCF7-derived breast cancer cells and miR133a-3p may play a critical role to overcome tamoxifen resistance in breast cancer chemotherapy. Citation Format: Minsun Chang, Xiyuan Liu, Eun Young Park. miR133a-3p sensitizes breast cancer cells by targeting GSTP1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-389.

OP-35 - Glutathione S-transferase P influences the Nrf2-dependent control of cellular glutathione metabolism

Glutathione-S-transferase P-1 (GSTP) was recently proposed to interact with Nrf2 thus regulating its transcriptional activity. The possible consequences of such functional interaction on the metabolism and redox of cellular GSH remain unexplored and were investigated for the first time in this study using both genetic and pharmacological manipulation models of GSTP in murine embryonic fibroblasts (MEFs). The GSTP-/- genotype was associated with a higher nuclear translocation of Nrf2 and apoptotic signaling of MEFs, and then with a marked increase of Cys uptake that resulted in a higher de novo biosynthesis and secretion of cellular GSH. Se-organic compounds (SOC) further stimulated these responses with a potency that correlated with the thiol peroxidase activity of the compounds. A defective expression of GSTP and SOC treatments also influenced the oxidation of GSH to GSSG and protein glutathionylation (PSSG) that was confirmed to be a GSTP-dependent process. The transient manipulation of Nrf2 gene interfered with the SOC-stimulated synthesis and secretion of GSH in the GSTP-/- aplotype, but not in GSTP+/+ cells. In conclusion, the GSTP-Nrf2 interaction strongly influences the metabolism and redox-signaling function of cellular GSH.

MiR-133b reverses cisplatin resistance by targeting GSTP1 in cisplatin-resistant lung cancer cells.

MicroRNAs play a critical role in chemoresistance and are implicated in various biological and pathological processes of cells. The objective of the present study was to explore the role of miR-133b and its mechanism in the regulation of cisplatin resistance and tumor progression in cisplatin-resistant non-small cell lung cancer (NSCLC) cells. Reverse transcription-quantitative polymerase chain reaction and western blot assays of the cisplatin-resistant cell lines A549/DPP and H1299/DDP displayed the reduced expression of miR-133b and increased expression of glutathione-S-transferase P1 (GSTP1) in the resistant cells compared with the respective parental cell lines A549 and H1299. Cell Counting kit-8, flow cytometry, colony formation and Transwell migration assays indicated that the overexpression of miR-133b increased the chemosensitivity to cisplatin and attenuated the proliferation and migration capacities of the cisplatin-resistant NSCLC cell lines in vitro. A dual-luciferase reporter assay demonstrated that miR-133b negatively regulated the expression of GSTP1 by targeting its 3′-untranslated region. In addition, the knockdown of GSTP1 by transfection with small interfering RNA exerted similar effects on cell chemosensitivity, proliferation and migration as did ectopic miR-133b expression, in addition to the upregulation of Bax and down-regulation of Bcl-2, survivin and matrix metalloproteinase expression. In conclusion, the present study findings provide the insights that miR-133b reduces cisplatin resistance and its overexpression contributes to the suppression of the malignant growth and aggressiveness of cisplatin-resistant NSCLC cells by targeting GSTP1. This could potentially be exploited as a novel therapeutic strategy for the reversal of cisplatin resistance.

145 - GSTP1-catalyzed PKM2 S-Glutathionylation Regulates Glycolysis in Non-small Cell Lung Cancer and is Attenuated with a Clinically Relevant inhibitor of Glutathione-S-transferase P

Rationale Lung cancer is the most lethal form of any of the common cancers, resulting in over 160,000 deaths in the US every year. Unfortunately, the 5-year survival rate has remained almost unchanged over the past 3 decades. Glutathione S-transferase P1 (GSTP1) is expressed significantly higher in lung tumor tissue from NSCLC compared to matched non-tumorous lung tissue. Originally this was attributed to its role in phase II drug metabolism. However, GSTP1 was recently described as a catalyst of protein S-glutathionylation (PSSG), a reversible cysteine oxidation and post-translational modification pointing towards a different role of GSTP in lung cancer. The goal of this study is to determine whether GSTP1 expression and subsequent enhanced PSSG are important in tumorigenesis. Methods In a tissue micro array of 106 human NSCLC specimens together with adjacent healthy tissue GSTP1 expression and overall PSSG was evaluated. The most common “driver” mutation in NSCLC occurs in the KRAS gene. To study NSCLC in mice we established an inducible KRASG12D-driven lung adenocarcinoma model that enables lung-epithelial specific expression of mutant KRAS. Alternatively, primary Mouse Tracheal Epithelial (MTE) Cells derived from KRASG12D mice were exposed to adeno-Cre to activate mutant KRAS. Results Assessment of 106 NSCLC TMAs revealed that 47% were completely negative and 41% were highly positive for GSTP1, and the lung cancers expressing GSTP1 had high PSSG reactivity. GSTP1 expression and PSSG were associated with a worse prognosis. In a mouse model of NSCLC, activation of mutant KRAS resulted in an increase of GSTP1 protein and PSSG in the lung. Further, lactate content and glycolysis regulators were significantly upregulated in lungs following KRASG12D activation. Following pharmaceutical inhibition of GSTP with TLK117, a phase II tested compound, KRAS-induced increases in PSSG and lactate were strongly decreased in lungs compared to vehicle- treated animals. KRASG12D activation in primary lung epithelial cells by adeno-cre resulted in a strong increase of GSTP1 protein and a striking increase in overall protein S-glutathionylation. More specifically Pyruvate kinase M2 (PKM2), an important enzyme in cancer metabolism, was highly S-glutathionylated in KRASG12D activated mouse lung and in MTE cells and increase in lactate production and expression of glycolysis enzymes was observed. Pharmacological GSTP1 inhibition with TLK117 decreased PKM2-and overall PSSG, glycolysis and tumor migration. Conclusion Together, these data reveal a potential pivotal new role of GSTP1 in regulating lung adenocarcinoma metabolism by promoting glycolysis in part through oxidative inactivation (S-glutathionylation) of PKM2.

P 140 - GSTP expression influences the metabolism and redox of cellular glutathione

Recently we described the role of glutathione-S-transferase P-1 (GSTP) expression in regulating Nrf2 transcriptional activity by protein-protein interaction. A functional interplay between Nrf2 and GSTP to control glutathione (GSH) biosynthesis, detoxification function and signal transduction through protein Cys glutathionylation can be hypothesized. To explore such interplay the effect of GSTP gene manipulation on the cellular levels and redox of main thiols in murine embryonic fibroblasts (MEFs) was investigated. GSTP knockout MEFs showed increased levels of all the identified forms of thiols that included GSH, GSSG, GSSP, Cys, Cyss, CyssP, HCys, HCyss, HCyssP, γ-GluCys and CysGly. The exposure to the Se-organic thiol-peroxidase compound Ebselen, further and massively up-regulated GSH, Cys and HCys and their corresponding disulfides in GSTP-KO, but not in wild-type (WT) cells. GSSP (protein glutathionylation), and to a lower extent HCyssP, increased after treatment with Se-organic compounds only in WT cells (confirmed by both HPLC and immunoblot analysis). These findings confirms the role of GSTP as a feedback component of Nrf2 pathway (control of GSH levels) and identify with an unbiased approach the role of this gene in mediating protein glutathionylation signalling during the exposure to Se-organic thiol-peroxidases.