Modulation of G6PD affects bladder cancer via ROS accumulation and the AKT pathway in vitro

  • Authors:
    • Xiaoyi Chen
    • Zhijie Xu
    • Zhijian Zhu
    • Anqi Chen
    • Guanghou Fu
    • Yimin Wang
    • Hao Pan
    • Baiye Jin
  • View Affiliations

  • Published online on: July 25, 2018     https://doi.org/10.3892/ijo.2018.4501
  • Pages: 1703-1712
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Glucose-6-phosphate dehydrogenase (G6PD) is a rate-limiting enzyme of the pentose phosphate pathway. Multiple studies have previously revealed that elevated G6PD levels promote cancer progression in numerous tumor types; however, the underlying mechanism remains unclear. In the present study, it was demonstrated that high G6PD expression is a poor prognostic factor in bladder cancer, and the levels of G6PD expression increase with increasing tumor stage. Patients with bladder cancer with high G6PD expression had worse survival rates compared with those with lower G6PD expression in resected tumors. In vitro experiments revealed that knockdown of G6PD suppressed cell viability and growth in Cell Counting Kit-8 and colony formation assays, and increased apoptosis in bladder cancer cell lines compared with normal cells. Further experiments indicated that the weakening of the survival ability in G6PD-knockdown bladder cancer cells may be explained by intracellular reactive oxygen species accumulation and protein kinase B pathway suppression. Furthermore, it was additionally revealed that 6-aminonicotinamide (6-AN), a competitive G6PD inhibitor, may be a potential therapy for bladder cancer, particularly in cases with high G6PD expression, and that the combination of cisplatin and 6-AN may optimize the clinical dose or minimize the side effects of cisplatin.

Introduction

Bladder cancer is the most common urinary malignancy in China, with an annual incidence rate and estimated mortality rate of 80.5/100,000 and 32.9/100,000, respectively (1). Despite recent improvements in treatment strategies, the overall survival rates of patients with an advanced stage of the disease remains poor (2). Tumor progression and metastasis are the main causes of bladder cancer-associated mortality, yet the exact mechanisms underlying these processes have not been fully elucidated. Hence, it is imperative to determine the key mechanisms implicated in bladder cancer development and progression, in order to identify potential therapeutic targets to improve patient prognosis.

The pentose phosphate pathway (PPP), one of the alternative routes for glucose metabolism, provides products for biosynthesis and antioxidant defense in cells (3). PPP has been attracting increased attention due to its ability to facilitate tumor progression or chemotherapy resistance by satisfying the considerable biosynthetic demands of rapidly growing cancer cells, in addition to their resistance and survival under stress conditions (3). Glucose-6-phosphate dehydrogenase (G6PD) is a rate-limiting enzyme of the PPP, and it is well known to promote intracellular anabolic reactions and redox homeostasis (4). Recently, multiple studies have demonstrated that elevated G6PD levels promote cancer progression in numerous tumor types, including melanoma, leukemia and colon cancer (5-10). Considering the function of G6PD in the critical processes of cancer cells, it is imperative to identify the mechanisms underlying the function of G6PD in bladder cancer in order to develop potent and selective G6PD inhibitors (10).

The aim of the present study was to investigate whether the high expression of G6PD in bladder cancer is associated with tumor aggressiveness and poor clinical prognosis and to determine whether targeting G6PD may be of value as a therapeutic option for bladder cancer, particularly in advanced cases.

Materials and methods

Online database

Information on G6PD mRNA expression in bladder cancer and normal tissues were acquired from the Oncomine database (https://www.oncomine.org) using the following searching terms: G6PD and bladder cancer (11). Dyrskjøt et al (12) bladder and Lee et al (13) bladder were 2 independent studies with bladder cancer samples recorded in the Oncomine database. The information between G6PD expression and clinical significance were downloaded from The Cancer Genome Atlas (TCGA) database (https://cancergenome.nih.gov/) using the following searching terms: Project, TCGA-Bladder Urothelial Carcinoma; Primary site, bladder; expiration date, January 2018.

Cell culture

Human bladder cancer cell lines 5637 (thought to have the same molecular features as high-risk superficial bladder cancer) (14), T24 (thought to have the same molecular features as muscle invasive bladder cancer with grade III pathological grading) (15), TCCSUP (poorly differentiated and high-risk muscle invasive bladder cancer with grade IV pathological grading) (14), normal uroepithelial cell line SV-HUC-1 (16) and the engineered 293T cell line were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and short tandem repeat DNA profiling analysis were performed by the supplier to validate all cell lines. 5637, T24 and TCCSUP cells were cultured in RPMI-1640 medium, SV-HUC-1 cells were cultured in Minimum Essential medium and 293T cells were cultured in DMEM. All the aforementioned mediums without any antibiotics (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) were pre-supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), and all cell lines were incubated under standard conditions (37°C and 5% CO2).

Plasmid construction, lentiviral packaging and transfection

The G6PD-overexpression plasmid was acquired from Vigene Biosciences, Inc. (Rockville, MD, USA). Validated sequences of short hairpin RNA (shRNA) against G6PD (shG6PD) were screened from Sigma-Aldrich online (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Scramble sequences were designed with the corresponding shG6PD sequences using the siRNA Wizard v3.1 online software (https://www.invivogen. com/sirnawizard/) declaring the absence of preclusive mRNA and miRNA seed sequence matches. Oligonucleotides of G6PD shRNA and control shRNA were then synthesized (Tsingke, Hangzhou, China) and inserted into the GIPZ lenti-viral vector. The sequences used were as follows: shG6PD forward, 5′-CCGGCAACAGATACAAGAACGTGAACTCGAGTTCACGTTCTTGTATCTGTTGTTTTTG-3′ and reverse, 5′-AATTCAAAAACAACAGATACAAGAACGTGAACTCGAGTTCACGTTCTTGTATCTGTTG-3′; Scramble forward, 5′-CCGGGCAAAGCAAACGTGACATAAACTCGAGTTTATGTCACGTTTGCTTTGCTTTTTG-3′ and reverse, 5′-AATTCAAAAAGCAAAGCAAACGTGACATAAACTCGAGTTATGTCACGTTTGCTTTGC-3′. Then the vectors were co-transfected with pSPAX2 and pMD2G (purchased from Addgene, Inc., Cambridge, MA, USA) plasmids (4:3:1 for vectors, pSPAX2 and pMD2G, respectively) into the 293T cell line cultured in 60 mm plates (at 60% cell density) using a calcium phosphate precipitation method (17). The supernatant, which contained lentivirus, was harvested 48 or 72 h post-transfection. Subsequent to virus packaging, T24 and TCCSUP cells cultured in 60 mm plates (at 30% cell density) were infected for 48 or 72 h with polybrene (5 µl/ml; Sigma-Aldrich; Merck KGaA) and the medium was changed 6–12 h later. Successfully transfected G6PD-shRNA cell lines were screened with 0.5 mg/ml puromycin (Sigma-Aldrich; Merck KGaA) and the transfection efficiency was validated using green fluorescent protein (488 nm) expression and western blotting.

Cell counting kit-8 (CCK-8) assay and colony formation assay

The proliferation of different groups of cells were compared using a CCK-8 kit (Dojindo Molecular Technologies, Inc., Kumamoto Japan) according to the manufacturer's protocol. 5637, T24 and TCCSUP cell lines with or without transfection were firstly prepared at a density of 1,000 cells/plate in 96-well plates under standard conditions (37°C and 5% CO2). Then, premixed medium (as aforementioned) with a 10% concentration of CCK-8 reagent was added into each well and placed in standard conditions (37°C and 5% CO2) for 1 h prior to measurement at an optical density of 450 nm. The preparations of cells exposed to 10 µM 6-aminonicotinamide (6-AN; Sigma-Aldrich; Merck KGaA), 0, 5, 10, 20, 40 or 80 µg/ml cisplatin (Sigma-Aldrich; Merck KGaA), 5 or 10 µM SC79 (MedChemExpress, Monmouth Junction, NJ, USA) or controlled DMSO for 24 or 48 h following the same procedure. As for colony formation assay, cells were seeded at a density of 500 cells/plate in 6-well plates and cultured for 8–10 days under standard conditions followed by 15 min fixation at room temperature and 15 min staining (0.5% crystal violet) at room temperature of the colonies which were performed prior to comparison.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) was used for cells total RNA extraction according to manufacturer's protocol. Takara PrimeScript™ RT and SYBR EX Taq™ kits (Takara Bio, Inc., Otsu, Japan) were used according to manufacturer's protocol. The specify thermocycling conditions used were as follows: Step 1, 95.0°C for 30 sec; step 2, 40 cycles of 95°C for 5 sec and 60°C for 30 sec; and step 3, melt curve analysis at 65°C to 95°C, increasing in 0.5°C increments for 5 sec. The primers were designed as follows: G6PD forward, 5′-ACCGCATCGACCACTACCT-3′ and reverse 5′-TGGGGCCGAAGATCCTGTT-3′; β-actin forward, 5′-GCAAGCAGGAGTATGACGAG-3′ and reverse, 5′-CAAATAAAGCCATGCCAATC-3′. Control groups were used to confirm the absence of the pollution of agents or primer dimers, and melt-curve analysis was used to identify the specificity of amplification. All genes were normalized to β-actin expression, and the gene mRNA relative expressions were calculated using the ΔΔCq method (18) using SPSS version 22.0 software (IBM Corp., Armonk, NY, USA).

Western blotting

5637, T24 and TCCSUP cell lines were firstly washed using PBS twice prior to being lysed using RIPA Lysis buffer with 1% cocktail protease inhibitor (Thermo Fisher Scientific, Inc.) for 4 h at 4°C and purified by centrifugation (4°C, 15,000 × g, 15 min). Subsequent to concentration measurement using a BCA protein assay (Pierce; Thermo Fisher Scientific, Inc.), 15 µg/10 µl protein samples were loaded in 12% Tris-acetate gels (Invitrogen; Thermo Fisher Scientific, Inc.) and then separated by electrophoresis. Next, the proteins were transferred onto a polyvinylidene fluoride membrane. Then, the membrane was blocked with 5% non-fat milk in Tris-buffered saline containing 1% Tween-20 (TBST) for 1 h at room temperature and further incubated with primary antibodies for 12 h at 4°C. Subsequent to washing with TBST three times, the membrane was incubated with secondary antibodies for 1 h at room temperature. The primary antibodies used in this experiment were: Rabbit polyclonal antibody G6PD (1:10,000; cat no. ab993; Abcam, Cambridge, UK); Rabbit monoclonal antibodies phosphorylated-protein kinase B (AKT; Ser473; 1:1,000; cat no. 4060; CST Biological Reagents Co., Ltd., Shanghai, China), AKT (1:1,000; cat no. 4685; CST Biological Reagents Co., Ltd.), P21 (1:2,000; cat no. ab109520; Abcam), P27 (1:2,000; cat no. ab32034; Abcam), cleaved caspase-3 (1:1,000; cat no. 9664; CST Biological Reagents Co., Ltd.), cleaved caspase-7 (1:1,000; cat no. 8438; CST Biological Reagents Co., Ltd.), cleaved caspase-9 (1:1,000; cat no. 7237; CST Biological Reagents Co., Ltd.) and Mouse monoclonal antibody β-actin (1:2,000; cat no. ab6276; Abcam). The secondary antibodies used in this experiment were: Goat anti Rabbit-horseradish peroxidase (HRP; 1:5,000; cat no. PDR007; Fdbio Science, Hangzhou, China) and Goat anti Mouse-HRP (1:5,000; cat no. PDM007; Fdbio Science). Immunodetection was performed by EZ-ECL chemiluminescence detection kit (Biological Industries, Kibbutz Beit Haemek, Israel). Protein bands were analyzed using Image-Pro Plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA), and β-actin was selected as an internal reference.

Flow cytometry analysis of cell apoptosis

Prepared T24 and TCCSUP cell lines (60% cell density in 6 mm dishes) with or without the shRNA-mediated G6PD knockdown were harvested with non-EDTA trypsin (Biological Industries), washed twice with cold PBS and then incubated using BD Annexin V-APC/7-aminoactinomycin D Apoptosis Detection Kit (BD Biosciences, San Jose CA, USA) according to the manufacturer's protocol. All aforementioned cells were harvested and stained with Annexin V (10 µl/200 µl) and 7-aminoactinomycin D (10 µl/200 µl) for 15 min in the dark at room temperature, and then apoptosis was analyzed using BD FACSCanto™ II (BD Biosciences), and data was then analyzed by FlowJo 7.6 software (FlowJo LLC, Ashland, OR, USA).

Intracellular reactive oxygen species (ROS) detection

T24, TCCSUP and 5637 cell lines with or without shRNA-mediated G6PD knockdown or G6PD overexpression or in the presence or absence of H2O2 were prepared in 9 wells in 96-well plates. Then, each group of cells was stimulated with or without 50 µM H2O2 for 30 min at 37°C prior to being co-incubated with 20 µM diochloro-dihydro-fluorescein diacetate (Invitrogen; Thermo Fisher Scientific, Inc.) for 30–60 min at 37°C. Then, 3 wells of each group of cells (selected as the cell counting group) were used for cell counting. The remaining wells (selected as the measuring group) were used to detect the ROS levels. Fluorescence was read at 485 nm/520 nm by a fluorescent enzyme meter, Varioskan™ Flash (Thermo Fisher Scientific, Inc.). Levels of cellular ROS were normalized to the total number of cells.

Isobolographic analysis

In order to determine the combination effects of cisplatin and 6-AN, isobolographic analysis was performed. T24 and TCCSUP cell lines were firstly prepared at a density of 5,000 cells/plate in 96-well plates under standard conditions (37°C and 5% CO2). Then cells were incubated with combination of different concentrations of 6-AN (C1 = 0, 0.5, 1, 4, 8 or 16 µM) and cisplatin (C2 = 0, 5, 10, 20, 40 or 80 µg/ml). A total of 24 h later, premixed medium with a 10% concentration of CCK-8 reagent was added into each well and placed in standard conditions for 1 h prior to measurement at an optical density of 450 nm. Once the background density was excluded, the 450 nm density (D) of each combination was used for cytotoxicity calculation using the following formula: Cytotoxic effect (CE) =1-D (C1, C2)/D (0, 0), and D(0, 0) was selected as the 0% cytotoxic effect. Isoboles are defined as isoeffect curves that reveal the concentration of the combination of two drugs which results in a similar CE to a previous study (19,20). The curves were obtained by ligaturing plots which represent the concentration combination of two drugs resulting in a 50% CE. The straight lines refer to the theoretical additivity line resulting in a 50% CE, and all data were analyzed using SPSS 22.0 (IBM Corp., Armonk, NY, USA) software.

Statistical analysis

SPSS 22.0 (IBM Corp.) was used to analyze the data. The normality of the data was initially determined using a Kolmogorov-Smirnov test. Data was presented as the mean ± standard deviation. The correlations between G6PD expression and clinicopathological characteristics were analyzed using a Pearson's χ2 test or a continuity correction χ2 test. Overall survival and disease-free survival rates curves were plotted using the Kaplan-Meier method, and data was analyzed by a log-rank test. One-way analysis of variance test was used to examine the differences between different groups. Student-Newman-Keuls test was used as a post hoc test. P<0.05 was considered to indicate a statistically significant difference. Data was derived from at least 3 repeated experiments.

Results

High G6PD expression is a poor prognostic factor in bladder cancer

To investigate whether G6PD expression is associated with bladder cancer progression and prognosis, the present study compared the G6PD expression levels in bladder cancer using the Oncomine database. G6PD mRNA expression levels were significantly higher in bladder cancer tissues compared with that in adjacent normal tissues (P<0.05; Fig. 1A and B). Furthermore, the levels of G6PD expression notably increased with increasing T stage (Fig. 1A and B). In addition, the results of qPCR analysis and western blotting revealed that G6PD mRNA levels were significantly upregulated in three tumor cell lines compared with a normal urothelial cell line (P<0.05) and that the protein levels were notably upregulated. Furthermore, T24 and TCCSUP, which have a higher malignant potential compared with 5637, exhibited higher G6PD expression levels compared with 5637 (Fig. 1C and D). Next, the clinical significance of G6PD in 408 patients with muscle-invasive bladder cancer (MIBC) from the TCGA database was investigated. Analysis revealed a significant association of G6PD expression with stage (P<0.05) and sex (P<0.001) (Table I). Kaplan-Meier survival analysis of 402 patients with MIBC (as the data for 6 patients was inaccessible) revealed that patients with high G6PD expression levels had a worse overall survival rates (P=0.057, close to 0.05) and a significantly worse disease-free survival rates (P=0.0013) compared with those with lower G6PD expression levels (Fig. 1E and F).

Table I

Association between G6PD expression level and clinicopathological features of 408 patients with muscle-invasive bladder cancer in The Cancer Genome Atlas database.

Table I

Association between G6PD expression level and clinicopathological features of 408 patients with muscle-invasive bladder cancer in The Cancer Genome Atlas database.

CharacteristicG6PD expression levels
P-value
LowHigh
Sex<0.001b
 Male104151
 Female10053
Age (years)0.265
 <657586
 ≥65129118
Tumor grade
0.207
 Low159
 High189195
Stage0.034a
 I or II7656
 III or IV128148
Lymph node metastasis0.070
 Absent14856
 Present13173
Distant metastasis0.221c
 Absent2013
 Present1968

a P<0.05;

b P<0.01;

c continuity correction. G6PD, glucose-6-phosphate dehydrogenase.

Knockdown of G6PD suppresses cell proliferation and growth, while increasing intracellular ROS levels

In order to determine the effect of upregulated G6PD expression in bladder cancer cell lines highly expressing G6PD, T24 and TCCSUP cells were transfected with sh-G6PD lentivirus. The infection efficiency was validated by western blotting and fluorescence microscopy, and G6PD was demonstrated to be significantly lower in transfected cell lines compared with their respective scramble controls (P<0.05; Fig. 2A). The CCK8 cell proliferation assay revealed that the knockdown of G6PD in the two cell lines significantly reduced cell proliferation (P<0.05; Fig. 2B). Furthermore, the colony-forming ability of the two cell lines was substantially suppressed in the G6PD knockdown groups compared with the control groups (Fig. 2C). No significant difference was observed between G6PD-overexpressing and control T24 or TCCSUP cell lines, however the overexpression of G6PD was observed in the 5637 cell line, which exhibited lower G6PD expression prior to transfection, in addition to enhanced cell proliferation and colony-forming abilities compared with the control group (Fig. 2B and C). Additionally, the knockdown of G6PD in the two cell lines resulted in significantly higher ROS accumulation compared with the corresponding control groups, which indicated a weaker ability to survive oxidative stress (P<0.05; Fig. 2D) (12,13).

Knockdown of G6PD induces intracellular apoptosis and suppresses the phosphorylated-AKT/AKT pathway

Multiple studies have reported that toxic ROS levels tend to induce apoptosis or other adverse reactions in cells (21-23). The present study compared the apoptosis between G6PD-knockdown and control groups. Interestingly, significantly increased apoptosis was demonstrated by flow cytometry analysis in the G6PD-knockdown groups compared with the control (P<0.05; Fig. 2E), in addition to the significant upregulation of cleaved caspase-3, -7 and -9 levels in G6PD-knockdown cells compared with scramble control cells (P<0.05; Fig. 3A). As reported in previous studies, the AKT pathway, which serves a vital function in the proliferation and apoptosis of tumor cells, was also revealed to sensitize cells to oxidative apoptosis (24,25). In addition, the AKT signaling pathway was also reported to promote the progression of bladder cancer (26). Therefore the present study investigated whether the knockdown of G6PD additionally suppressed AKT signaling. The western blotting results revealed a significant decrease of phosphorylated AKT (P<0.05), but no significant change was observed of the total AKT in two G6PD-knockdown cell lines (T24 and TCCSUP) compared with the corresponding controls (Fig. 3B). Furthermore, P27, a cell cycle regulator known to be inhibited by AKT, was also revealed to be upregulated in G6PD-knockdown cell lines (Fig. 3B). Interestingly, a rescue assay with SC79, a specific AKT activator, successfully restored the partial effects of G6PD knockdown (P<0.05; Fig. 4A). All this evidence suggests that the suppression of AKT signaling in G6PD-knockdown bladder cancer cells may exert a substantial tumor inhibitory effect.

Inhibition of G6PD activity with 6-AN exerts antineoplastic effects and functions synergistically with cisplatin

Subsequently, the effect of G6PD inhibition on T24 and TCCSUP cell lines using 6-AN, a competitive G6PD inhibitor, was examined. The specific effect of 6-AN on G6PD was confirmed by the fact that 6-AN treatment in the two cell lines resulted in similar results with G6PD knockdown (Figs. 4B–D and 5A). The 6-AN treatment in the two cell lines significantly reduced cell proliferation determined using a CCK8 cell proliferation assay (P<0.05; Fig. 4B) and resulted in a significantly higher ROS accumulation compared with the corresponding control groups (P<0.05; Fig. 2C). A rescue assay with SC79, a specific AKT activator, successfully restored the partial effects of the treatment of 6-AN (P<0.05; Fig. 4D). Additionally, the significant upregulation of cleaved caspase-3, -7 and -9 levels in cells with 6-AN treatment compared with cells with DMSO treatment was observed (P<0.05; Fig. 3A). Of note, it was revealed that the T24 and TCCSUP cell lines (high G6PD expression) displayed a higher sensitivity to 6-AN compared with the SVHUC and 5637 cell lines (lower G6PD expression), suggesting the specificity and rationale of targeting higher G6PD activity in bladder cancer (*P<0.05; Fig. 5B). Interestingly, it was revealed that the level of G6PD protein increased subsequent to 6-AN treatment. 6-AN may be metabolized to 6-amino-NAD(P+), a competitive inhibitor of NAD(P+)-requiring processes, particularly G6PD. Additionally, it does not directly affect the expression of G6PD protein itself (27). Thus, the increase of G6PD may be explained as a compensatory increasing. Finally, the present study investigated whether 6-AN has the ability to enhance the antitumor effects of cisplatin, a classical drug mostly used in bladder cancer chemotherapy. In the T24 and TCCSUP cell lines, 6-AN and cisplatin functioned synergistically to enhance cytotoxicity in the CCK8 assay, and a substantial dose reduction with the combination of the two drugs by using isoeffective drug concentrations resulting in 50% of the cytotoxic effect was observed (Fig. 5C). All these cumulative results indicate that inhibition of G6PD activity by 6-AN may be a potential therapeutic method for bladder cancer, particularly in cases with high G6PD expression, and that the combination of cisplatin with 6-AN may optimize the clinical dose of cisplatin or minimize the cisplatin-associated side effects.

Discussion

It has become apparent that cancer cells require sugars to drive oncogenic processes (28,29). For example, rapidly dividing cells require a constant supply of building blocks to maintain their elevated biosynthetic activity. In line with this increased metabolism are increased ROS levels (23). Considered to be by-products of oxygen consumption and cellular metabolism, ROS are formed by the partial reduction of molecular oxygen (30,31). ROS homeostasis is crucial for cell survival and normal cell signaling, in addition to protecting cells from damage. To some extent, cancer cells must maintain cellular ROS at levels that favor growth (32). Glutathione (GSH), an important antioxidant for ROS detoxification, serves a key role in maintaining ROS homeostasis in cells (32). The content of activated GSH were revealed to be closely associated with the content of NADPH, the product of G6PD (23). G6PD is the rate-limiting enzyme in PPP; the oncogenic properties of G6PD have been attracting increasing attention and its increased activity in cancer cells was also recently demonstrated (33-35). In the present study, it was revealed that high G6PD expression was associated with a higher stage and poorer prognosis in patients with bladder cancer. G6PD expression was upregulated in tumor tissues compared with that in adjacent normal tissues, and the level of G6PD expression increased with increasing T stage. Analysis from the TCGA database revealed a significant association of G6PD expression with stage and sex in patients with MIBC. Patients with bladder cancer exhibited worse overall and disease-free survival rates compared with those with lower G6PD expression in the resected tumors.

Knockdown of G6PD in bladder cancer cell lines resulted in an increase of intracellular ROS levels. Inhibition of G6PD restricts the function of PPP, resulting in lower NADPH production, an unstable GSH/oxidized GSH ratio and subsequent intracellular ROS accumulation, ultimately resulting in the disruption of the intracellular redox equilibrium (21-23). Consistent with the results of these previous studies, the toxic ROS levels induced apoptosis in bladder cancer cell lines, which may explain why the inhibition of G6PD suppressed the cell proliferation and colony formation ability of bladder cancer cell lines in vitro.

The AKT signaling pathway is known to serve a key role in the proliferation and apoptosis of tumor cells (24). An increasing number of studies demonstrated that activated AKT suppresses apoptosis by the phosphorylation of certain sites in Bcl2 associated agonist of cell death, caspase-9, protease-activated receptor 4 or other pro-apoptotic proteins (36,37). AKT signaling was also reported to promote the progression of bladder cancer (25) and activated AKT signaling has been demonstrated to be positively correlated with tumor progression and poor clinical prognosis in patients with bladder cancer (38). The present results demonstrated that the knockdown of G6PD suppressed AKT signaling and increased the apoptosis of bladder cancer cells. This may explain the weakening of survival ability in G6PD-knockdown cancer cells. However, the detailed association between G6PD and the AKT pathway and the mechanism underlying the mode of action of AKT in bladder cancer requires further study.

6-AN, a competitive G6PD inhibitor (39), mostly used for radiosensitization combined with 2-deoxy-D-glucose (40-42), was also demonstrated to exert antineoplastic effects on cancer cells, alone or combined with other agents (43,44). Similar to other studies, the present results demonstrated that G6PD inhibition with a fixed concentration of 6-AN significantly reduced the proliferation of bladder cancer cells, particularly those highly expressing G6PD, whereas it did not induce a substantial decrease of cell survival in normal cells (P<0.05; Fig. 5B) (45). Furthermore, the results demonstrated that 6-AN functioned synergistically with cisplatin in bladder cancer treatment. Cisplatin-based chemotherapy remains the first-line chemotherapeutic treatment in patients with MIBC pre- and postoperatively (46). However, despite great success in tumor suppression, the application of cisplatin is restricted by drug resistance and side effects, the underlying mechanisms of which remain unclear (47). However, the hypothesis that the dysregulation of cell metabolism sustains drug resistance has gained the support of an increasing number of researchers (9,10,48). Several agents are known to sensitize cancer cells to cisplatin, one of which is 6-AN (49). This effect is primarily due to the action of 6-amino-NAD(P+), which results in intracellular cisplatin enrichment and the accumulation of plasma tumor DNA adducts (50). Pretreatment with 6-AN has been demonstrated to sensitize different tumor cells to cisplatin cytotoxicity, even cisplatin-resistant cells; this may explain the synergistic effects of 6-AN and cisplatin on bladder cancer cells (49,50). However, it has also been reported that 6-AN may cause neurotoxicity or hematological toxicity under certain conditions (43-46). Thus, further in vivo studies are required to optimize the therapeutic window and dose of 6-AN in cancer treatment in clinical practice.

Altogether, the results of the present study demonstrate that high G6PD expression is associated with a higher stage and poorer prognosis in patients with bladder cancer. Inhibition of G6PD may suppress the growth of bladder cancer cells via ROS accumulation and AKT pathway suppression. Thus, targeting G6PD may be a potential therapeutic method for bladder cancer, particularly in cases with high G6PD expression. Furthermore, 6-AN used as a supplementary component in cisplatin-based chemotherapy may optimize the therapeutic doses of cisplatin, thereby minimizing the side effects (51).

Acknowledgments

The authors would like to thank The Key Laboratory of Combined Multi-Organ Transplantation, Ministry of Public Health (Hangzhou, China) for the use of the facilities and assistance from the technicians.

Funding

The present study was funded by the Key Project of the Science and Technology Program of Zhejiang Province (grant no. 2014C03028) and Science and Technology Program of Zhejiang Province (grant no. LY15H050002).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

XC and BJ designed the experiments, performed the experiments, analyzed the data and wrote the paper. ZX, AC and GF performed the experiments. ZZ, YW and HP provided the reagents and helped with the experiments and the writing of the paper. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References

1 

Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, Yu XQ and He J: Cancer statistics in China, 2015. CA Cancer J Clin. 66:115–132. 2016. View Article : Google Scholar : PubMed/NCBI

2 

Bellmunt J, Orsola A, Leow JJ, Wiegel T, De Santis M and Horwich A; Group EGW; ESMO Guidelines Working Group: Bladder cancer: ESMO Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 25(Suppl 3): iii40–iii48. 2014. View Article : Google Scholar : PubMed/NCBI

3 

Riganti C, Gazzano E, Polimeni M, Aldieri E and Ghigo D: The pentose phosphate pathway: An antioxidant defense and a crossroad in tumor cell fate. Free Radic Biol Med. 53:421–436. 2012. View Article : Google Scholar : PubMed/NCBI

4 

Tian WN, Braunstein LD, Pang J, Stuhlmeier KM, Xi QC, Tian X and Stanton RC: Importance of glucose-6-phosphate dehydrogenase activity for cell growth. J Biol Chem. 273:10609–10617. 1998. View Article : Google Scholar : PubMed/NCBI

5 

Hu T, Zhang C, Tang Q, Su Y, Li B, Chen L, Zhang Z, Cai T and Zhu Y: Variant G6PD levels promote tumor cell proliferation or apoptosis via the STAT3/5 pathway in the human melanoma xenograft mouse model. BMC Cancer. 13:2512013. View Article : Google Scholar : PubMed/NCBI

6 

Batetta B, Pulisci D, Bonatesta RR, Sanna F, Piras S, Mulas MF, Spano O, Putzolu M, Broccia G and Dessì S: G6PD activity and gene expression in leukemic cells from G6PD-deficient subjects. Cancer Lett. 140:53–58. 1999. View Article : Google Scholar : PubMed/NCBI

7 

Van Driel BE, Valet GK, Lyon H, Hansen U, Song JY and Van Noorden CJ: Prognostic estimation of survival of colorectal cancer patients with the quantitative histochemical assay of G6PDH activity and the multiparameter classification program CLASSIF1. Cytometry. 38:176–183. 1999. View Article : Google Scholar : PubMed/NCBI

8 

Polat MF, Taysi S, Gul M, Cikman O, Yilmaz I, Bakan E and Erdogan F: Oxidant/antioxidant status in blood of patients with malignant breast tumour and benign breast disease. Cell Biochem Funct. 20:327–331. 2002. View Article : Google Scholar : PubMed/NCBI

9 

Philipson KA, Elder MG and White JO: The effects of medroxyprogesterone acetate on enzyme activities in human endometrial carcinoma. J Steroid Biochem. 23A:1059–1064. 1985. View Article : Google Scholar

10 

Zhang C, Zhang Z, Zhu Y and Qin S: Glucose-6-phosphate dehydrogenase: A biomarker and potential therapeutic target for cancer. Anticancer Agents Med Chem. 14:280–289. 2014. View Article : Google Scholar

11 

Rhodes DR, Yu J, Shanker K, Deshpande N, Varambally R, Ghosh D, Barrette T, Pandey A and Chinnaiyan AM: ONCOMINE: A cancer microarray database and integrated data-mining platform. Neoplasia. 6:1–6. 2004. View Article : Google Scholar : PubMed/NCBI

12 

Dyrskjøt L, Kruhøffer M, Thykjaer T, Marcussen N, Jensen JL, Møller K and Ørntoft TF: Gene expression in the urinary bladder: A common carcinoma in situ gene expression signature exists disregarding histopathological classification. Cancer Res. 64:4040–4048. 2004. View Article : Google Scholar : PubMed/NCBI

13 

Lee JS, Leem SH, Lee SY, Kim SC, Park ES, Kim SB, Kim SK, Kim YJ, Kim WJ and Chu IS: Expression signature of E2F1 and its associated genes predict superficial to invasive progression of bladder tumors. J Clin Oncol. 28:2660–2667. 2010. View Article : Google Scholar : PubMed/NCBI

14 

Fogh J: Cultivation, characterization, and identification of human tumor cells with emphasis on kidney, testis, and bladder tumors. Natl Cancer Inst Monogr. 49:5–9. 1978.

15 

Bubeník J, Baresová M, Viklický V, Jakoubková J, Sainerová H and Donner J: Established cell line of urinary bladder carcinoma (T24) containing tumour-specific antigen. Int J Cancer. 11:765–773. 1973. View Article : Google Scholar : PubMed/NCBI

16 

Christian BJ, Loretz LJ, Oberley TD and Reznikoff CA: Characterization of human uroepithelial cells immortalized in vitro by simian virus 40. Cancer Res. 47:6066–6073. 1987.PubMed/NCBI

17 

Graham FL and van der Eb AJ: A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 52:456–467. 1973. View Article : Google Scholar : PubMed/NCBI

18 

Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)). Method. 25:402–408. 2001. View Article : Google Scholar

19 

Loewe S: The problem of synergism and antagonism of combined drugs. Arzneimittelforschung. 3:285–290. 1953.PubMed/NCBI

20 

Tallarida RJ: An overview of drug combination analysis with isobolograms. J Pharmacol Exp Ther. 319:1–7. 2006. View Article : Google Scholar : PubMed/NCBI

21 

Ichijo H, Nishida E, Irie K, ten Dijke P, Saitoh M, Moriguchi T, Takagi M, Matsumoto K, Miyazono K and Gotoh Y: Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science. 275:90–94. 1997. View Article : Google Scholar : PubMed/NCBI

22 

Moon DO, Kim MO, Choi YH, Hyun JW, Chang WY and Kim GY: Butein induces G(2)/M phase arrest and apoptosis in human hepatoma cancer cells through ROS generation. Cancer Lett. 288:204–213. 2010. View Article : Google Scholar

23 

Moloney JN and Cotter TG: ROS signalling in the biology of cancer. Semin Cell Dev Biol. 80:50–64. 2018. View Article : Google Scholar

24 

Vivanco I and Sawyers CL: The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer. 2:489–501. 2002. View Article : Google Scholar : PubMed/NCBI

25 

Nogueira V, Park Y, Chen CC, Xu PZ, Chen ML, Tonic I, Unterman T and Hay N: Akt determines replicative senescence and oxidative or oncogenic premature senescence and sensitizes cells to oxidative apoptosis. Cancer Cell. 14:458–470. 2008. View Article : Google Scholar : PubMed/NCBI

26 

Calderaro J, Rebouissou S, de Koning L, Masmoudi A, Hérault A, Dubois T, Maille P, Soyeux P, Sibony M, de la Taille A, et al: PI3K/AKT pathway activation in bladder carcinogenesis. Int J Cancer. 134:1776–1784. 2014. View Article : Google Scholar

27 

Street JC, Alfieri AA and Koutcher JA: Quantitation of metabolic and radiobiological effects of 6-aminonicotinamide in RIF-1 tumor cells in vitro. Cancer Res. 57:3956–3962. 1997.PubMed/NCBI

28 

Ward PS and Thompson CB: Metabolic reprogramming: A cancer hallmark even warburg did not anticipate. Cancer Cell. 21:297–308. 2012. View Article : Google Scholar : PubMed/NCBI

29 

Wittig R and Coy JF: The role of glucose metabolism and glucose-associated signalling in cancer. Perspect Medicin Chem. 1:64–82. 2008.PubMed/NCBI

30 

Giorgio M, Trinei M, Migliaccio E and Pelicci PG: Hydrogen peroxide: A metabolic by-product or a common mediator of ageing signals? Nat Rev Mol Cell Biol. 8:722–728. 2007. View Article : Google Scholar : PubMed/NCBI

31 

Zorov DB, Juhaszova M and Sollott SJ: Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release. Physiol Rev. 94:909–950. 2014. View Article : Google Scholar : PubMed/NCBI

32 

Vander Heiden MG, Cantley LC and Thompson CB: Understanding the Warburg effect: The metabolic requirements of cell proliferation. Science. 324:1029–1033. 2009. View Article : Google Scholar : PubMed/NCBI

33 

Kuo W, Lin J and Tang TK: Human glucose-6-phosphate dehydrogenase (G6PD) gene transforms NIH 3T3 cells and induces tumors in nude mice. Int J Cancer. 85:857–864. 2000. View Article : Google Scholar : PubMed/NCBI

34 

Jiang P, Du W and Yang X: A critical role of glucose-6-phosphate dehydrogenase in TAp73-mediated cell proliferation. Cell Cycle. 12:3720–3726. 2013. View Article : Google Scholar : PubMed/NCBI

35 

Rao X, Duan X, Mao W, Li X, Li Z, Li Q, Zheng Z, Xu H, Chen M, Wang PG, et al: O-GlcNAcylation of G6PD promotes the pentose phosphate pathway and tumor growth. Nat Commun. 6:84682015. View Article : Google Scholar : PubMed/NCBI

36 

Song G, Ouyang G and Bao S: The activation of Akt/PKB signaling pathway and cell survival. J Cell Mol Med. 9:59–71. 2005. View Article : Google Scholar : PubMed/NCBI

37 

Goswami A, Burikhanov R, de Thonel A, Fujita N, Goswami M, Zhao Y, Eriksson JE, Tsuruo T and Rangnekar VM: Binding and phosphorylation of par-4 by akt is essential for cancer cell survival. Mol Cell. 20:33–44. 2005. View Article : Google Scholar : PubMed/NCBI

38 

Sun CH, Chang YH and Pan CC: Activation of the PI3K/Akt/mTOR pathway correlates with tumour progression and reduced survival in patients with urothelial carcinoma of the urinary bladder. Histopathology. 58:1054–1063. 2011. View Article : Google Scholar : PubMed/NCBI

39 

Hothersall JS, Gordge M and Noronha-Dutra AA: Inhibition of NADPH supply by 6-aminonicotinamide: Effect on glutathione, nitric oxide and superoxide in J774 cells. FEBS Lett. 434:97–100. 1998. View Article : Google Scholar : PubMed/NCBI

40 

Sharma PK and Varshney R: 2-Deoxy-D-glucose and 6-aminonicotinamide-mediated Nrf2 down regulation leads to radiosensitization of malignant cells via abrogation of GSH-mediated defense. Free Radic Res. 46:1446–1457. 2012. View Article : Google Scholar : PubMed/NCBI

41 

Sharma PK, Bhardwaj R, Dwarakanath BS and Varshney R: Metabolic oxidative stress induced by a combination of 2-DG and 6-AN enhances radiation damage selectively in malignant cells via non-coordinated expression of antioxidant enzymes. Cancer Lett. 295:154–166. 2010. View Article : Google Scholar : PubMed/NCBI

42 

Varshney R, Gupta S and Dwarakanath BS: Radiosensitization of murine Ehrlich ascites tumor by a combination of 2-deoxy-D-glucose and 6-aminonicotinamide. Technol Cancer Res Treat. 3:659–663. 2004. View Article : Google Scholar : PubMed/NCBI

43 

Stolfi RL, Colofiore JR, Nord LD, Koutcher JA and Martin DS: Biochemical modulation of tumor cell energy: Regression of advanced spontaneous murine breast tumors with a 5-fluoro-uracil-containing drug combination. Cancer Res. 52:4074–4081. 1992.PubMed/NCBI

44 

Koutcher JA, Alfieri AA, Stolfi RL, Devitt ML, Colofiore JR, Nord LD and Martin DS: Potentiation of a three drug chemotherapy regimen by radiation. Cancer Res. 53:3518–3523. 1993.PubMed/NCBI

45 

Poulain L, Sujobert P, Zylbersztejn F, Barreau S, Stuani L, Lambert M, Palama TL, Chesnais V, Birsen R, Vergez F, et al: High mTORC1 activity drives glycolysis addiction and sensitivity to G6PD inhibition in acute myeloid leukemia cells. Leukemia. 31:2326–2335. 2017. View Article : Google Scholar : PubMed/NCBI

46 

Alfred Witjes J, Lebret T, Compérat EM, Cowan NC, De Santis M, Bruins HM, Hernández V, Espinós EL, Dunn J, Rouanne M, et al: Updated 2016 EAU Guidelines on muscle-invasive and metastatic Bladder Cancer. Eur Urol. 71:462–475. 2017. View Article : Google Scholar

47 

Köberle B, Tomicic MT, Usanova S and Kaina B: Cisplatin resistance: Preclinical findings and clinical implications. Biochim Biophys Acta. 1806:172–182. 2010.PubMed/NCBI

48 

Liu H, Liu Y and Zhang JT: A new mechanism of drug resistance in breast cancer cells: Fatty acid synthase overexpression-mediated palmitate overproduction. Mol Cancer Ther. 7:263–270. 2008. View Article : Google Scholar : PubMed/NCBI

49 

Budihardjo II, Walker DL, Svingen PA, Buckwalter CA, Desnoyers S, Eckdahl S, Shah GM, Poirier GG, Reid JM, Ames MM, et al: 6-Aminonicotinamide sensitizes human tumor cell lines to cisplatin. Clin Cancer Res. 4:117–130. 1998.PubMed/NCBI

50 

Catanzaro D, Gaude E, Orso G, Giordano C, Guzzo G, Rasola A, Ragazzi E, Caparrotta L, Frezza C and Montopoli M: Inhibition of glucose-6-phosphate dehydrogenase sensitizes cisplatin-resistant cells to death. Oncotarget. 6:30102–30114. 2015. View Article : Google Scholar : PubMed/NCBI

51 

Zhelev Z, Ivanova D, Bakalova R, Aoki I and Higashi T: Inhibition of the pentose-phosphate pathway selectively sensitizes leukemia lymphocytes to chemotherapeutics by ROS-independent mechanism. Anticancer Res. 36:6011–6020. 2016. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

October 2018
Volume 53 Issue 4

Print ISSN: 1019-6439
Online ISSN:1791-2423

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
APA
Chen, X., Xu, Z., Zhu, Z., Chen, A., Fu, G., Wang, Y. ... Jin, B. (2018). Modulation of G6PD affects bladder cancer via ROS accumulation and the AKT pathway in vitro. International Journal of Oncology, 53, 1703-1712. https://doi.org/10.3892/ijo.2018.4501
MLA
Chen, X., Xu, Z., Zhu, Z., Chen, A., Fu, G., Wang, Y., Pan, H., Jin, B."Modulation of G6PD affects bladder cancer via ROS accumulation and the AKT pathway in vitro". International Journal of Oncology 53.4 (2018): 1703-1712.
Chicago
Chen, X., Xu, Z., Zhu, Z., Chen, A., Fu, G., Wang, Y., Pan, H., Jin, B."Modulation of G6PD affects bladder cancer via ROS accumulation and the AKT pathway in vitro". International Journal of Oncology 53, no. 4 (2018): 1703-1712. https://doi.org/10.3892/ijo.2018.4501