Molecular and Cellular Endocrinology

Chien-Liang Liua,b, Yi-Chiung Hsuc, Jie-Jen Leea, Ming-Jen Chena,b, Chi-Hsin Linb,d,
Shih-Yuan Huangb, Shih-Ping Chenga,e,∗

 journal homepage: www.elsevier.com/locate/mce

MolecularandCellularEndocrinology499(2020)110595

 Targeting the pentose phosphate pathway increases reactive oxygen species and induces apoptosis in thyroid cancer cells☆ 

 a Department of Surgery, MacKay Memorial Hospital and Mackay Medical College, Taipei, Taiwan, ROC

b Department of Medical Research, MacKay Memorial Hospital, Taipei, Taiwan, ROC

c Department of Biomedical Sciences and Engineering, National Central University, Taoyuan City,Taiwan, ROC

d Department of Bioscience Technology, Chung Yuan Christian University, Taoyuan City, Taiwan, ROC

e Department of Pharmacology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan, ROC

A R T I C L E I N F O

Keywords:
Pentose phosphate pathway Reactive oxygen species Apoptosis
Thyroid cancer
A B S T R A C T

The pentose phosphate pathway (PPP) plays an important role in the biosynthesis of ribonucleotide precursor and NADPH. Cancer cells frequently increase the flux of glucose into the PPP to support the anabolic demands and regulate oxidative stress. Consistently, metabolomic analyses indicate an upregulation of the PPP in thyroid cancer. In the present study, we found that the combination of glucose-6-phosphate dehydrogenase (G6PD) and transketolase inhibitors (6-aminonicotinamide and oxythiamine) exerted an additive or synergistic effect on cell growth inhibition in thyroid cancer cells. Targeting PPP significantly increased cellular reactive oxygen species (ROS) and induced endoplasmic reticulum (ER) stress and apoptosis. Suppressed cell viability could be partially rescued with treatment with the ROS scavenger or apoptosis inhibitor but not ER-stress inhibitor. Taken to- gether, dual PPP blockade leads to pharmacologic additivity or synergism and causes ROS-mediated apoptosis in thyroid cancer cells.

 Introduction

Cancer cell growth is supported by reprogrammed cellular meta- bolism. To meet both catabolic and anabolic demands, rapidly pro- liferating tumor cells consume glucose at a higher rate compared to normal cells. Enhanced glucose uptake manifested by 18F-fluorodeox- yglucose positron emission tomography (FDG-PET) imaging has diag- nostic, prognostic and predictive importance in thyroid cancer (Santhanam et al., 2017; Manohar et al., 2018). Glucose conjugation has been exploited for the design of new agents for cancer therapy (Calvaresi and Hergenrother, 2013). Along with increased glucose consumption, aerobic glycolysis, also known as the Warburg effect, is one of the metabolic hallmarks of cancer. Aerobic glycolysis is char- acterized by an increased metabolism of glucose to lactate regardless of oxygen availability. We previously demonstrated that old age, a nega- tive prognostic factor in thyroid cancer, is associated with an upregu- lation of glycolysis pathway (Hsu et al., 2016).
Glycolysis is not the only pathway that requires glucose. The pen- tose phosphate pathway provides pentose sugars for ribonucleotidesynthesis and generates reduced nicotinamide adenine dinucleotide phosphate (NADPH) to reduce glutathione and lower reactive oxygen species (ROS) levels (Hay, 2016). The pentose phosphate pathway is composed of two functionally interrelated branches, oxidative and non- oxidative (Stincone et al., 2015). Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme of the oxidative branch to yield ribulose-5-phosphate and NADPH. The non-oxidative branch re- versibly converts pentose sugars into the glycolytic intermediates fructose-6-phosphate and glyceraldehyde-3-phosphate by transketolase and transaldolase. Considerable evidence demonstrates that cancer cells increase the flux of glucose into the pentose phosphate pathway to support anabolic demands and regulate oxidative stress (Patra and Hay, 2014).Although differentiated thyroid cancer is considered clinically in-dolent, nuclear magnetic resonance spectroscopy and gas chromato- graphy-mass spectrometry revealed an elevation in the levels of nucleic acid, nucleosides, and nucleotides (Tian et al., 2015). To facilitate DNA and RNA biosynthesis, an upregulation of the pentose phosphate pathway in cancerous thyroid tissues was confirmed by metabolomic

☆ Part of this study was presented at the 21st European Congress of Endocrinology, Lyon, France, May 2019.

∗ Corresponding author. Department of Surgery, MacKay Memorial Hospital, 92, Section 2, Chung-Shan North Road, Taipei, 10449, Taiwan, ROC.
E-mail address: [email protected] (S.-P. Cheng).

https://doi.org/10.1016/j.mce.2019.110595

Received 28 April 2019; Received in revised form 24 September 2019; Accepted 24 September 2019
Availableonline26September2019
0303-7207/©2019ElsevierB.V.Allrightsreserved.analyses (Chen et al., 2015; Wojakowska et al., 2015). The upregulation might have a prognostic impact on oncological outcomes. For instance, overexpression of transketolase like 1 (TKTL1) was associated with extrathyroidal extension, vascular invasion, and lymph node metastases in papillary thyroid cancer (Zerilli et al., 2008). In the current study, we aimed to explore the therapeutic potential of dual inhibition of the oxidative and the non-oxidative branches of the pentose phosphate pathway in thyroid cancer cells.

2. Materials and methods
2.1. Cell culture and reagents
Human thyroid cancer cell lines BCPAP, K1, and 8505C were pur- chased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) or the European Collection of Authenticated Cell Cultures (ECACC; Salisbury, UK). SW1736 thyroid cancer cell line was obtained from CLS Cell Lines Service GmbH, Eppelheim, Germany. All are authenticated cell lines recommended for biomedical research (Marlow et al., 2018). An immortalized human
2.3. Cellular metabolism
Oxygen consumption rate (OCR) was determined using the XF24 Extracellular Flux Analyzer (Seahorse Bioscience, Agilent Technologies, North Billerica, MA, USA) as reported previously (Liu et al., 2019). For assessing respiratory capacity, cells were subjected to a mitochondrial stress test. After three basal OCR measurements, the OCR was mon- itored upon serial injections of an ATP synthase inhibitor oligomycin (1 μM), a protonophore uncoupler carbonyl cyanide 4-(tri- fluoromethoxy) phenylhydrazone (FCCP, 1 μM), and a mitochondrial respiratory chain complex I inhibitor rotenone (2 μM). Spare reserve capacity was calculated as FCCP-induced maximum OCR relative to baseline OCR. Non-mitochondrial respiration was calculated based on residual respiration in response to rotenone. Extracellular acidification rate (ECAR) was obtained for assessing glycolytic capacity. Both OCR and ECAR were normalized to the CyQUANT assay readout.
2.4. Apoptotic assay
Apoptosis is characterized by cell shrinkage, blebbing of the plasma membrane, nuclear fragmentation, and apoptotic body formation. Cellsdifferentiated thyrocyte cell line Nthy-ori 3-1 (abbreviated as Nthywere treated with vehicle control (DMSO), 6AN, oxythiamine, orthereafter) was obtained from ECACC. Cells were cultured in RPMI- 1640 medium (containing 2 g/L glucose) supplemented with 10% fetal bovine serum. Cell identity has been regularly confirmed by short tandem repeat profiling (performed by Mission Biotech Ltd., Taipei, combined therapy with 6AN and oxythiamine for 24 h. Histone/DNA complexes released from the nucleus to the cytosol were measured by the Cell Death Detection ELISA kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions.

Taiwan using Applied Biosystems 3730xl DNA Analyzer; reference,
In addition, cells were fixed with 2% formaldehyde followingLanda et al., 2019) and tested for being mycoplasma free (using EZ-PCR Mycoplasma Detection Kit purchased from Biological Industries Israel Beit-Haemek, Israel).

6-Aminonicotinamide (6AN), dehydroepiandrosterone (DHEA), oxythiamine, genistein, tunicamycin, and N-acetylcysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Z-VAD-FMK and GSK2606414 were obtained from BD Biosciences (San Jose, CA, USA) and Selleck Chemicals (Houston, TX, USA), respectively.

2.2. Cell proliferation assay
Cells were treated with increasing doses of 6AN (0, 16, 40, and 100 μM), DHEA (0, 25, 100, and 400 μM), oxythiamine (0, 4, 10, and
25 mM), and genistein (0, 1, 4, and 16 mM) individually or in combi- nation. In some experiments, cells were simultaneously treated with Z- VAD-FMK (10 μM), GSK2606414 (1 μM), or NAC (3 mM).
Cell proliferation was determined by CyQUANT Cell Proliferation Assay (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s recommendations. The assay is based on the measure- ment of cellular DNA content via the binding of a fluorescent dye. Briefly, cells were seeded on 96-well plates and allowed to attach overnight. The cells were incubated under the experimental conditions for an additional 24–72 h. The medium was removed, and plates were frozen at −80 °C. Plates were then thawed and incubated with the CyQUANT GR dye/cell-lysis buffer. RNA fluorescence was eliminated by pretreating cell lysates with DNase-free RNase. Fluorescence in- tensity was measured with an excitation of 480 nm and an emission of 520 nm.
For combination therapy studies, cells were treated with 6AN and
oxythiamine at a fixed-dose ratio to determine therapeutic synergy, which was evaluated using the Chou-Talalay combination index (CI) (Chang et al., 2014). The CI for each two drug interactions was calcu- lated using the following equation: CI = Ac/Ai + Bc/Bi, where Ac and Bc are drug concentrations used in combination needed to achieve a specific effect, and Ai and Bi are the individual drug concentrations needed to achieve that effect. CI < 1 indicates synergism, CI = 1 indicates additive effect, and CI > 1 indicates antagonism.
Treatment and incubated with fluorescein isothiocyanate (FITC)-an- nexin V and propidium iodide (PI) using the Annexin V-FITC Apoptosis Kit (BioVision, Milpitas, CA, USA) (Zhou et al., 2018). After washing, cells were mounted and observed with a phase-contrast and fluores- cence microscope (Olympus IX71; Olympus, Tokyo, Japan).
2.5. Western blot analysis
Eight paired samples of classic papillary thyroid cancer were col- lected during surgery and immediately snap-frozen in liquid nitrogen (Cheng et al., 2015). For cellular experiments, cells were treated with 6AN and oxythiamine in combination and harvested at 0, 4, 6, and 12 h. Tissue or cellular proteins were extracted and quantified, and Western blotting assays were performed according to the standard procedure (Lee et al., 2017). Quantitative densitometric analysis of Western blots was performed using ImageJ software (version 1.52d; National In- stitutes of Health, Bethesda, MD, USA). The primary antibodies were G6PD (sc-373887; Santa Cruz Biotechnology, Dallas, TX, USA), trans- ketolase (sc-390179; Santa Cruz), TKTL1 (sc-271296; Santa Cruz), PERK (#5683; Cell Signaling Technology, Danvers, MA, USA), phospho-IRE1 (Ser724) (ab48187; Abcam, Cambridge, UK), and β-actin (A5441; Sigma-Aldrich). TKTL1-overexpression HEK293T cell lysate(catalog number NBL1-16941) as the positive control was obtainedfrom Novus Biologicals, Centennial, CO, USA.
2.6. Endoplasmic reticulum (ER) staining
Cells were treated with vehicle control, 6AN, oxythiamine, com- bined therapy with 6AN and oxythiamine, a combination of 6AN/ oxythiamine and GSK2606414 (1 μM), or tunicamycin (5 μg/ml) for 24 h. To selectively label the ER, we stained the cells with ER-Tracker Blue-White DPX (Thermo Fisher Scientific) for 30 min. After that, cells were fixed and observed under a fluorescence microscope.

2.7. Analysis of the NADP/NADPH ratio
The cytoplasmic NADP/NADPH ratio was determined using the NADP/NADPH Assay Kit (ab65349; Abcam) according to the protocol provided by the manufacturer. Cells were treated with vehicle controlor combined therapy with 6AN and oxythiamine for 24 h and collected with the recommended extraction buffer. The measured NADP and NADPH levels were calculated by comparison with a standard curve. Protein concentration was determined and normalized for each sample.

2.8. Detection of intracellular ROS
Cells were treated with vehicle control, 6AN, oxythiamine, com- bined therapy with 6AN and oxythiamine, or a combination of 6AN/ oxythiamine and NAC (3 mM) for 24 h. Intracellular free radicals were measured using the Cellular Reactive Oxygen Species Detection Assay (Abcam) per the manufacturer’s protocols (Yang et al., 2018). In- tracellular ROS content was further visualized by a fluorescent probe dihydroethidium (DHE; Sigma-Aldrich). DHE interacts with ROS and gets oxidized to ethidium cation which in turn binds to DNA and gives a red fluorescence (Zielonka and Kalyanaraman, 2010). Following the above treatment for 24 h, cells were stained with DHE for 30 min. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich).
2.9. Analysis of The Cancer Genome Atlas (TCGA) database
Transcriptome sequencing data of TCGA papillary thyroid cancer (THCA) were obtained from the Genomic Data Commons of the National Cancer Institute (https://portal.gdc.cancer.gov/). Gene ex- pression levels were expressed as RNA-Seq by expectation maximiza- tion (RSEM) values. For paired comparisons, 59 cases which had data available for both tumor and matched normal samples were included in the analysis. The Wilcoxon signed-rank test was used to compare ex- pression levels between matched normal and tumor samples. A total of 387 cases had confident mutational information of telomerase reverse transcriptase (TERT) promoter of tumor samples (Chien et al., 2018). Among them, 39 (10%) harbored TERT promoter mutations. The dif- ference between TERT promoter wild-type and mutant groups was determined using the Mann-Whitney U test.
2.10. Statistical analysis
Experimental data were reported as the mean ± standard error of the mean from the results of at least three independent experiments. Comparisons were made by using the two-tailed Student’s t-test or one- way analysis of variance, followed by Dunnett’s multiple comparison tests as appropriate. A p value less than 0.05 was considered statisti- cally significant.
3. Results

3.1. Expression of key enzymes of the pentose phosphate pathway

Protein expression of glucose-6-phosphate dehydrogenase (G6PD), transketolase (TKT), and transketolase like 1 (TKTL1) determined by im- munoblot analysis in eight paired papillary thyroid cancer samples (N, normal thyroid tissue; T, tumor). β-Actin served as a loading control. **, p < 0.01. PC, positive control.

3.2. Cytotoxicity of inhibitors for the pentose phosphate pathway
BCPAP, K1, 8505C, SW1736, and Nthy cells had high expression of G6PD and transketolase (data not shown). The cells were treated with each of G6PD inhibitors (6AN and DHEA) and transketolase inhibitors(oxythiamine and genistein) at serially diluted concentrations for

We first investigated the expression of key enzymes of the pentose phosphate pathway in eight paired samples of papillary thyroid cancer. As shown in Fig. 1, the expression of G6PD and transketolase was sig- nificantly higher in the tumor part than the matched normal thyroid tissue. The expression of TKTL1 was not detectable in our normal or tumorous thyroid tissues.
To substantiate our findings, an analysis of TCGA data was per- formed. The mRNA expression of transketolase was more abundant than G6PD, and the transketolase expression in tumor tissues was higher than that in the normal counterpart (Supplementary Fig. S1). The mRNA expression of TKTL1 in papillary thyroid cancer was rela- tively low.

In glioma cells, a positive correlation between TERT and G6PD/ transketolase has been reported (Ahmad et al., 2016). Thus, we com- pared the expression levels between TERT promoter wild-type and mutant groups. The expression of G6PD, transketolase, and TKTL1 was not different between the two groups (Supplementary Fig. S2).
24–72 h. As shown in Fig. 2A and B and Supplementary Fig. S3, in- hibitors of the pentose phosphate pathway suppressed the proliferation of all tested cell lines in a dose- and time-dependent manner.
Drug synergy was determined using CI analyses according to the median effect methods. In Nthy cells, a synergistic effect with combi- nation therapy of 6AN and oxythiamine was observed only when the fractional effect was less than 15%. Otherwise, combination therapy had an antagonistic effect. In other thyroid cancer cells, the synergism was observed with a wider range of fractional effect (Fig. 2C). At the median 50% effective dose (ED50), the CI for BCPAP, K1, 8505C, SW1736, and Nthy cells was 0.99 ± 0.27, 0.75 ± 0.24, 0.94 ± 0.42,0.86 ± 0.28, and 3.47 ± 1.50, respectively. The CI of Nthy cells was significantly higher than that of four thyroid cancer cell lines (all p < 0.01). In summary, the drug combination at the median effective dose showed additivity or slight synergism in thyroid cancer cells but antagonism in thyroid follicular epithelial cells.

. Effects of inhibitors of the pentose phosphate pathway on cell proliferation. BCPAP, K1, 8505C, SW1736, and Nthy-ori 3-1 (abbreviated as Nthy) cells were treated with (A) 6-aminonicotinamide (6AN, 0–100 μM), (B) oxythiamine (Oxy, 0–25 mM), or a fixed-ratio combination for 24–72 h. Cell proliferation was de- termined by CyQUANT assay. *p < 0.05 versus control; **p < 0.01; ***p < 0.001. (C) The in vitro interaction between 6AN and Oxy was demonstrated by the combination index (CI) plots. Trend lines represent CI values at any given effect (fraction affected, % inhibition), and dashed lines indicate 95% confidence limits. CI < 1 corresponds to synergistic cytotoxicity.

Bioenergetic state after treatment with inhibitors of the pentose phosphate pathway. (A) Oxygen consumption rate (OCR) and (B) extracellular acidification rate (ECAR) were determined in BCPAP, K1, 8505C, SW1736, and Nthy-ori 3-1 (abbreviated as Nthy) cells following treatment with vehicle control (DMSO) or combined therapy with 100 μM 6-aminonicotinamide plus 25 mM oxythiamine (6AN/Oxy) for 24 h *p < 0.05 versus control; **p < 0.01.

Apoptosis after treatment with inhibitors of the pentose phosphate pathway. BCPAP, 8505C, and Nthy-ori 3-1 (abbreviated as Nthy) cells were treated with vehicle control (DMSO), 100 μM 6-aminonicotinamide (6AN), 25 mM oxythiamine (Oxy), or combined therapy with 6AN and Oxy (6AN/Oxy) for 24 h. (A) Apoptosis was determined by the Cell Death Detection ELISA kit. *p < 0.05 versus vehicle control; **p < 0.01; ***p < 0.001. (B) Cells were stained with fluorescein isothiocyanate (FITC)-annexin V and propidium iodide (Annexin V/PI). Phase contrast and corresponding fluorescence images were captured. Scale bars, 200 μm. (C) Cells were treated with vehicle control, 6AN, Oxy, 6AN/Oxy, or a combination of 6AN/Oxy and 10 μM Z-VAD-FMK for 24 h. Cell proliferation was determined by CyQUANT assay. *p < 0.05 versus vehicle control; **p < 0.01; ***p < 0.001. #p < 0.05 and ##p < 0.01 for 6AN/Oxy group versus combined therapy with 6AN/Oxy and Z-VAD-FMK.
3.3. Metabolic effects of inhibitors for the pentose phosphate pathway
To determine the effects on metabolic reprogramming following dual inhibition of the oxidative and the non-oxidative branches of the pentose phosphate pathway, we measured the OCR, an indicator of oxidative phosphorylation, and ECAR, an indicator of glycolytic activity mainly attributable to lactate production, using a flux analyzer. For consistency, fixed doses of 6AN (100 μM) and oxythiamine (25 mM) were used in the subsequent experiments.
As illustrated in Fig. 3, combination therapy of 6AN and oxythia- mine decreased the basal OCR and ECAR in BCPAP, K1, SW1736, and Nthy cells but not in 8505C cells. Spare reserve capacity and non-mi- tochondrial respiration were not affected by combination therapy. Furthermore, the ratio of OCR to ECAR, an indicator of cellular pre- ference for oxidative phosphorylation versus glycolysis, was not sig- nificantly changed following combination therapy in tested cell lines. Overall, acute inhibition of the pentose phosphate pathway does not influence the metabolic state of thyroid cancer cells.
3.4. Apoptosis induced by inhibitors of the pentose phosphate pathway
To identify the mechanisms which might account for the decrease in
cell proliferation following the treatment with inhibitors of the pentose phosphate pathway, the Cell Death Detection ELISA was used to quantify DNA fragmentation. As shown in Fig. 4A, combined therapy with 6AN and oxythiamine significantly increased DNA fragmentation in BCPAP and 8505C thyroid cancer cells.
To further discriminate apoptotic and necrotic cell death, cells treated with the combination of 6AN and oxythiamine were stained with FITC-annexin V and PI. Annexin V is a phospholipid-binding protein with a high affinity for phosphatidylserine. One of the early membrane alterations in apoptosis is the translocation of phosphati- dylserine from the inner side of the plasma membrane to the outer layer. Therefore, annexin V is frequently used as a probe for detecting apoptosis. PI is a well-established viability stain which is impermeable to cells with an intact plasma membrane. When cell integrity becomes compromised, PI gains access to the nucleus and yields red fluorescent chromatin. Following the combination therapy, the majority of cells exhibited both annexin V and PI staining (Fig. 4B and Supplementary Fig. S4). Furthermore, phase images showed characteristic cellular morphology including cell rounding and shrinkage, chromatin con-densation, and the formation of apoptotic bodies. These results indicatethat apoptosis is the major pathway contributing to the synergistic cytotoxic effect of 6AN and oxythiamine.

. Endoplasmic reticulum (ER) stress after treatment with inhibitors of the pentose phosphate pathway. (A) BCPAP and 8505C thyroid cancer cells were treated with combined therapy with 100 μM 6-aminonicotinamide plus 25 mM oxythiamine (6AN/Oxy) for 0–12 h. The expression of PERK and phosphorylated IRE1 was determined by immunoblot. (B) BCPAP, 8505C, and Nthy-ori 3-1 (abbreviated as Nthy) cells were treated with vehicle control (DMSO), 6AN, Oxy, 6AN/Oxy, or a combination of 6AN/Oxy and 1 μM GSK2606414 for 24 h and stained with the ER marker (ER-Tracker Blue-White DPX). Treatment with tunicamycin (5 μg/ml) served as a positive control. Scale bars, 30 μm. (C) Cell proliferation was determined by CyQUANT assay. **p < 0.01 and ***p < 0.001 versus vehicle control.

We further examined the rescue effect of an apoptosis inhibitor Z- VAD-FMK. Co-treatment with Z-VAD-FMK significantly dampened the inhibitory effects of combined therapy with 6AN and oxythiamine in BCPAP, K1, 8505C, and SW1736 thyroid cancer cells but not in Nthy cells (Fig. 4C and Supplementary Fig. S4). Taken together, the cytotoxic effects of inhibitors for the pentose phosphate pathway are mediated, at least in part, by the induction of apoptotic cell death.

ER stress induced by inhibitors of the pentose phosphate pathway
Although the enzymes of the pentose phosphate pathway are mainly localized in the cytosol, the microsomal pentose phosphate pathway may also be a target for the inhibitors. ER stress-induced apoptosis can result from the inactivation of the anti-apoptotic protein BCL-2 by CHOP, a downstream effector of the protein kinase R-like endoplasmic reticulum kinase (PERK), or by c-Jun N-terminal kinase activated by inositol-requiring enzyme 1 (IRE1) (Iurlaro and Munoz-Pinedo, 2016). We examined the protein expression of PERK and phosphorylated IRE1 in BCPAP and 8505C thyroid cancer cells following combined therapy with 6AN and oxythiamine. Both ER stress markers began to increase at 4 h following treatment (Fig. 5A).
To corroborate these results, we performed an immunofluorescence staining using a cell-permeable dye that selectively labels the ER. Previous observations have shown that the process of ER stress is ac- companied by massive ER membrane expansion (Schuck et al., 2009). As such, the fluorescence intensity of ER-Tracker reflects the extent of ER stress. Cells were treated with the combination of 6AN and oxy- thiamine and stained with ER-Tracker Blue-White DPX. Treatment with tunicamycin, a known inducer of ER stress, was used as a positive control. As shown in Fig. 5B and Supplementary Fig. S5, combined therapy with 6AN and oxythiamine profoundly increased peripheral and perinuclear ER fluorescence.
GSK2606414 is a selective inhibitor for PERK. As expected, co- treatment with GSK2606414 attenuated the ER stress induced by the combination therapy (Fig. 5B and Supplementary Fig. S5). Nonetheless, co-treatment with GSK2606414 failed to revert the cytotoxicity elicited by the combination of 6AN and oxythiamine except in SW1736 cells (Fig. 5C and Supplementary Fig. S5). Collectively, although inhibitors of the pentose phosphate pathway induce ER stress in thyroid cancer cells, ER stress is not the prime pathway leading to apoptotic cell death.

3.6. ROS increment induced by inhibitors of the pentose phosphate pathway
The oxidative branch of the pentose phosphate pathway is a major source of NADPH, which maintains the pool of reduced glutathione to balance redox state. As expected, combined therapy with 6AN and oxythiamine significantly increased the NADP/NADPH ratio in BCPAP and 8505C thyroid cancer cells (Supplementary Fig. S6).
The NADPH produced in the pentose phosphate pathway can be utilized for lipid biosynthesis or ROS detoxification. We further in- vestigated the ROS content in thyroid cancer cells following combined therapy with 6AN and oxythiamine. The combination therapy sig- nificantly increased the intracellular ROS levels, while the ROS sca- venger NAC significantly reduced the ROS content (Fig. 6A). The results were confirmed with DHE staining. Strong DHE staining was observed in thyroid cancer cells treated with combined therapy with 6AN and oxythiamine, and the staining was attenuated with co-treatment with NAC (Fig. 6B and Supplementary Fig. S7).
Notably, co-treatment with NAC provided a robust rescue effect on cytotoxicity induced by inhibitors of the pentose phosphate pathway in BCPAP, K1, 8505C, and SW1736 thyroid cancer cells but not in Nthy cells (Fig. 6C and Supplementary Fig. S7). Taken together, apoptotic cytotoxicity elicited by inhibition of the pentose phosphate pathway is mediated, at least partly, by ROS.

Reactive oxygen species (ROS) content after treatment with inhibitors of the pentose phosphate pathway. BCPAP, 8505C, and Nthy-ori 3-1 (abbreviated as Nthy) cells were treated with vehicle control (DMSO), 100 μM 6-aminonicotinamide (6AN), 25 mM oxythiamine (Oxy), combined therapy with 6AN and Oxy (6AN/ Oxy), or a combination of 6AN/Oxy and 3 mM N-acetylcysteine (NAC) for 24 h. Intracellular ROS was quantified by the Cellular Reactive Oxygen Species Detection Assay Kit (A) or dihydroethidium (DHE) staining (B). Scale bars, 100 μm. (C) Cell proliferation was determined by CyQUANT assay. *p < 0.05 versus vehicle control; **p < 0.01; ***p < 0.001. ##p < 0.01 for 6AN/Oxy group versus combined therapy with 6AN/Oxy and NAC.

4. Discussion
Metabolic reprogramming is tuned for the proliferation and survival of cancer cells. An analysis of TCGA data revealed that metabolic dys- regulation is associated with recurrence in papillary thyroid cancer (Chien et al., 2017). In this regard, the FDG-PET metabolic parameters can be used for dynamic risk stratification (Manohar et al., 2018). Therapies that target tumor metabolism are anticipated to suppress tumor growth or re-sensitize tumors to chemotherapy and radio- therapy. Currently, more and more antimetabolites and inhibitors tar- geting metabolic enzymes are being evaluated in preclinical models or clinical trials (Luengo et al., 2017).
Cancer cells predominantly employ the non-oxidative branch of the
pentose phosphate pathway for de novo nucleotides biosynthesis (Boros et al., 1997). Interestingly, retinoic acid-mediated re-differentiation led to reduced transketolase levels in thyroid cancer cells (Trojanowicz et al., 2010). Our findings are in agreement with previous studies that demonstrated that the expression of transketolase is upregulated in papillary thyroid cancer. The seeming downregulation of TKTL1 in TCGA data probably reflects variation caused by the low-level expres- sion.
In the present study, we examined the effects of two transketolase inhibitors (oxythiamine and genistein). Given that transketolase is a thiamine-dependent enzyme, oxythiamine is a thiamine antagonist and an analog of antimetabolite. A recent study showed that oxythiamine inhibited cell proliferation of ARO cells (Wang et al., 2018), although

ARO cells are likely cross-contaminated by HT-29 colon cancer cells (Schweppe et al., 2008). Genistein is a plant isoflavonoid which could decrease ribose synthesis primarily through the non-oxidative reactions of the pentose phosphate pathway (Boros et al., 2001). Genistein could significantly inhibit the proliferation and invasion of papillary thyroid cancer cells (Zhang et al., 2019). Overall, these findings suggest that targeting transketolase is probably beneficial in the management of thyroid cancer.
G6PD is the key enzyme of the oxidative branch of the pentose phosphate pathway. Ectopic G6PD overexpression is sufficient to transform NIH 3T3 cells (Kuo et al., 2000). Increased G6PD enzymatic activity or expression has been reported in multiple types of cancer (Kowalik et al., 2017). In this study, two G6PD inhibitors (6AN and DHEA) were investigated. In the past, 6AN, a competitive G6PD in- hibitor, was used in chemotherapy for various malignancies (Herter et al., 1961). However, the clinical use of 6AN is hampered by its neurologic toxicity at high concentrations. DHEA is a 17-ketosteroid that uncompetitively inhibits G6PD. An animal study demonstrated that administration of DHEA inhibited the development of thyroid tumors (Moore et al., 1986). Nonetheless, DHEA may exert endocrine and in- tracrine effects following conversion to androgens and estrogens.
In this context, the combined treatment of 6AN and oxythiamine is a
viable option. Potential pharmacologic synergism shown in this study may reduce the dosage of both agents and thus decrease the possible adverse effects, including 6AN-associated nerve damage and oxythia- mine-associated thiamine deficiency. Of note, synergistic inhibitory effects of DHEA combined with oxythiamine has been reported in pancreatic and colon cancer cells (Boros et al., 1997; Ramos-Montoya et al., 2006). Likewise, we observed an additive or slightly synergistic effect in thyroid cancer cells. A possible explanation is that compared to normal thyroid follicular cells, thyroid cancer cells generally exhibit higher glycolysis efficiency and a higher rate of glucose consumption (Coelho et al., 2016).
In the present study, we demonstrated that inhibitors of the pentose phosphate pathway cause ROS-induced apoptosis in thyroid cancer cells. In the thyroid gland, ROS are actively generated during the pro- cess of iodide metabolism and thyroid hormone synthesis. Oxidative DNA damage may promote chromosomal instability, tumorigenesis, and dedifferentiation (Ameziane et al., 2019). Interestingly, some ROS- related genes such as FMO2, AOX1, and SOD3 were downregulated during tumor progression (Cheng et al., 2017). By contrast, other scavenging mechanisms such as antioxidant transcription factor NFE2- related factor 2 (Nrf2) and heme oxygenase-1 were upregulated in thyroid cancer (Ziros et al., 2013; Wang et al., 2015). As expected, dual inhibition of the pentose phosphate pathway decreased NADPH avail- ability and increased ROS content. Accumulation of ROS may increase ER stress and exacerbate ER stress-driven cell death (Mele et al., 2018).
Nonetheless, our results suggest that ER stress does not account for
apoptotic cell death in thyroid cancer cells. In conclusion, combined inhibition of the oxidative and the non- oxidative branches of the pentose phosphate pathway results in phar- macologic additivity or synergism that leads to apoptosis of thyroid cancer cells. Furthermore, targeting the pentose phosphate pathway significantly increased cellular ROS and induced ER stress. The cyto- toxicity could be partially rescued with the ROS scavenger or apoptosis inhibitor but not ER-stress inhibitor. These results are summarized in Table 1. Our findings suggest that combined inhibition of the pentose phosphate pathway can serve as a potential targeted therapeutic option in thyroid cancer.

Funding support
This work was supported by research grants from the Ministry of Science and Technology of Taiwan (MOST-107-2314-B-195-001-MY3) and Mackay Memorial Hospital (MMH-10813 and MMH-E−108-10).
Declaration of competing interest
The authors declare no conflict of interest.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.mce.2019.110595.
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