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Low-dose 2-deoxyglucose and metformin synergically inhibit proliferation of human polycystic kidney cells by modulating glucose metabolism

Low-dose 2-deoxyglucose and metformin synergically inhibit proliferation of human polycystic... Polycystic kidney disease (PKD) is a common hereditary kidney disease with abnormal proliferation and apoptosis of kidney cystic epithelial cells, eventually leading to chronic renal failure. Currently, there are no effective treatment methods. Similar to tumor cells, cystic epithelial cells have abnormal glycolysis and over-activation of proliferation signaling pathways. In the present study, for the first time, we investigated the effects of low-dose combinational use of 2-deoxyglucose (2-DG) and metformin (MET) on the proliferation and apoptosis in the human cystic kidney epithelial cells. Cystic epithelia cells were divided into control group, 2-DG group, MET group and 2-DG+MET group. Cell Proliferation, apoptosis and glucose metabolism were measured in each group. The results showed that low-dose combinational treatment of 2-DG and MET significantly inhibited the proliferation of renal cystic epithelial cells by suppressing the activities of PKA, mTOR and ERK signaling pathways and upregulating PI3K/Akt pathway. Combination of both drugs increased the apoptosis rates of cystic epithelial cells. Two drugs inhibited glucose metabolic phenotypes, glycolysis and oxidative phosphorylation, and significantly lowered the intracellular ATP level in cystic epithelial cells. 2-DG could also neutralize excessive production of lactate (lactic acidosis) caused by MET and both drugs had complementary effect for cystic epithelial cells. These results reveal that combinational use of low-dose 2-DG and MET can markedly inhibit proliferation via modulating glucose metabolic phenotypes in human polycystic kidney epithelial cells, low-dose combinational use of both drugs can also lower the toxic effects of each drug, and is a novel strategy for future treatment of human polycystic kidney disease. Introduction leading to end-stage kidney disease .The pathogenesis of Polycystic kidney disease (PKD) is a hereditary kidney PKD is still unclear, and there is no effective treatment. disease. Both kidneys in PKD are filled with multiple serous In recent years, the Warburg effect has been found in cysts derived from renal tubules; the cyst epithelial cells show polycystic kidney epithelial cells, similar to tumor cells. abnormal proliferation and gradually increase in volume, Under aerobic conditions, the cystic cells mainly rely on thus compressing normal kidney tissues and eventually glycolytic metabolism for energy supply rather than on 2,3 mitochondrial oxidative phosphorylation . Additionally, the activity of the energy sensor, adenosine monopho- Correspondence: Xiangmei Chen (xmchen301@126.com) or Xue-Yuan Bai sphate activated protein kinase (AMPK), is decreased, (xueyuan_bai@163.com) while the mammalian target of rapamycin (mTOR) Department of Nephrology, Chinese PLA General Hospital, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National signaling pathway is over-activated in cyst epithelial 4,5 Clinical Research Center for Kidney Diseases, Beijing Key Laboratory of Kidney cells . Furthermore, the proliferation-related signaling Diseases, Beijing 100853, China pathways, cyclic adenyl-monophosphate-protein kinase A These authors contributed equally: Jing Zhao, Yuxiang Ma Edited by M.V. Nikilson Chirou © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Zhao et al. Cell Death Discovery (2019) 5:76 Page 2 of 13 (cAMP-PKA) and extracellular-regulated protein kinase treated with different concentrations (0.6, 2.5, 10 and 40 (ERK), are activated, while the activity of phosphoinositide mM) of 2-DG or MET alone for different times (12, 24, 36 3-kinase (PI3K)/Akt signaling pathway that inhibits the and 48 h). When the cells were treated with different over-activation of ERK proliferation signaling pathway is drugs for the same time, the viable cell count gradually significantly inhibited in the cystic cells . declined with increasing concentration of the drugs Numerous anti-proliferative drugs, such as rapamycin compared with that in control group. This finding indi- cates that the inhibitory effects of 2-DG and MET on the (mTOR inhibitor) and octreotide (somatostatin analog), have been used to treat polycystic kidney animal models in proliferation of cystic epithelial cells were dose dependent. recent years. Although these drugs showed good efficacy in When cells were treated with the same drugs for different cells and animal models, the effects were not satisfactory in times, the inhibitory effects of the drugs on cell pro- a number of follow-up clinical trials . Tolvaptan, a vaso- liferation were gradually enhanced as the treatment time pressin V2 receptor antagonist, is also effective; however, increased. These results indicate that the inhibitory clinical studies have shown that patients suffer severe thirst, effects of these drugs were time dependent, and the polyuria, nocturia, polydipsia and liver toxicity, and the US highest inhibition rate was found at 48 h (Fig. 1a, b, Food and Drug Administration (FDA) has not yet approved Tables 1 and 2). this drug for clinical use . Therefore, there is an urgent need The half-maximal inhibitory concentration (IC )for to find new treatment methods. cystic epithelial cells treated with 2-DG or MET alone for 2-Deoxyglucose (2-DG) is a glucose analog that inhibits 48 h was 30.9 and 17.4 mM, respectively (Fig. 1c). Using 9,10 glycolysis . 2-DG can compete with glucose to bind the above IC values, we selected different concentrations hexokinase (the first rate-limiting enzyme of glycolysis) in (1.3+ 0.6, 2.5+ 1.3, 5+ 2.5 and 10+ 5mM) of 2-DG+ cells and inhibit metabolism of tumor cell, thereby inhi- MET to treat cystic epithelial cells for different times (12, biting cell proliferation . Metformin (MET) is a first-line 24, 36 and 48 h). The results showed that the combination drug for the clinical treatment of type 2 diabetes mellitus. of the two drugs significantly inhibited the proliferation of Recent studies have found that MET can specifically cystic epithelial cells. The proliferation inhibition rate inhibit mitochondrial respiratory chain complex I and reached 50% in cystic epithelial cells treated with the decrease oxidative phosphorylation levels in cells, thus combination of 5 mM 2-DG and 2.5 mM MET for 48 h reducing adenosine triphosphate (ATP) synthesis, acti- (Fig. 1d, Table 3). Taking into account the safety of the vating AMPK and inhibiting mTOR proliferation signal- drugs, we selected small dose of 5 mM and 2.5 mM as the 12–16 ing pathway treatment concentrations of 2-DG and MET respectively in . Due to the obvious activation of glycolysis in tumor cells, a large quantity of glucose is the subsequent studies. consumed and high levels of ATP are produced, resulting Subsequently, we evaluated the effects of 5 mM 2-DG, in a decrease in AMP/ATP ratio and significantly inhib- 2.5 mM MET and their combination on the viability of ited AMPK activity . Thus, glycolytic inhibitor 2-DG and HRPTEpiC cells (human renal proximal tubular epithelial AMPK activator MET have been used in the treatment of cells). The results showed that the effects of 2-DG, MET tumors in recent years. The combinational use of MET and their combination on cell viability of HRPTEpiC cells and 2-DG can significantly deplete the ATP supply of were weaker than on cystic epithelial cells, showing a cancer cells and inhibit the over-activation of proliferation therapeutic potential of the combination of 2-DG and signaling pathways in cells, thereby significantly inhibiting MET in PKD (Fig. 1e). the over-proliferation of tumor cells and reducing the side CCK-8 assay evaluated cell proliferation mainly through 18–20 effects caused by high doses of the individual drugs . determining the viable cell counts in the plates; however, In the present study, for the first time, we treated human this method could not fully reflect the proliferation effect of polycystic kidney cyst-lining epithelial cells with a combi- the cells. To further verify whether the combination of 2- nation of low-dose MET and 2-DG. We systematically DG and MET can significantly inhibit the proliferation of analyzed the effects of the combination of these two drugs cystic epithelial cells, we directly observed the inhibitory on the proliferation and apoptosis of cyst epithelial cells and effect of their combination on cell proliferation using 5- explored the possible molecular mechanisms. ethynyl-2'-deoxyuridine (EdU) nucleic acid labeling tech- nique. The results showed that either 5 mM 2-DG alone or Results 2.5 mM MET alone, and their combination significantly Combinational use of low-dose 2-DG and MET significantly inhibited the proliferation of cystic epithelial cells (Fig. 2). inhibits the proliferation of human polycystic kidney epithelial cells Effects of 2-DG and MET on proliferation-related signaling The effects of individual 2-DG and MET on cell pro- pathways in human polycystic kidney epithelial cells liferation were evaluated using a Cell Counting Kit-8 (CCK-8) A previous study showed that the activation levels of B- assay in human polycystic kidney epithelial cells WT9-7 Raf/ERK, mTOR and cAMP-PKA proliferation signaling Official journal of the Cell Death Differentiation Association Zhao et al. Cell Death Discovery (2019) 5:76 Page 3 of 13 Fig. 1 Effects of 2-deoxyglucose (2-DG), metformin (MET) and their combination on the proliferation of WT9-7 cells and human renal proximal tubular epithelial cells (HRPTEC) evaluated using Cell Counting Kit-8 (CCK-8) assay. a WT9-7 cells were treated with 2-DG (0.6, 2.5, 10 and 40 mM) for 12, 24, 36 and 48 h, and the cell viability was determined using CCK-8 assay. b WT9-7 cells were treated with MET (0.6, 2.5, 10 and 40 mM) for 12, 24, 36 and 48 h, and the cell viability was determined using CCK-8 assay. c Cells were treated with various concentrations (0.6, 2.5, 10 and 40 mM) of 2-DG or MET for 48 h, and the half- maximal inhibitory concentration (IC ) was determined using GraphPad Prism software. d The combined treatment with 2-DG and MET enhanced the growth inhibition of WT9-7 cells. WT9-7 cells were treated with 2-DG and MET at different doses (1.3 + 0.6, 2.5+ 1.3, 5 + 2.5 and 10 + 5 mM) for 12, 24, 36 and 48 h, and the cell viability was determined using the CCK-8 assay. e HRPTEC cells were treated with 2-DG (5 mM), MET (2.5 mM) and their combination for 48 h, and the cell viability was determined using CCK-8 assay; *p < 0.05, **p < 0.01, ***p < 0.001 vs Control Table 1 Effect of 2-deoxyglucose (2-DG) on proliferation of polycystic kidney epithelial cells 0 mM 0.6 mM 2.5 mM 10 mM 40 mM 12 h 100.00 ± 2.36 95.86 ± 1.89 91.61 ± 2.31** 86.22 ± 2.75*** 76.92 ± 2.99*** 24 h 100.00 ± 2.41 93.58 ± 0.82* 89.13 ± 1.99*** 82.88 ± 2.60*** 73.43 ± 2.93*** 36 h 100.00 ± 2.34 92.66 ± 1.14** 87.58 ± 0.90*** 70.55 ± 2.06*** 60.48 ± 2.32*** 48 h 100.00 ± 2.28 91.40 ± 1.18*** 82.00 ± 1.49*** 64.75 ± 2.00*** 46.88 ± 1.48*** *P < 0.05, **p < 0.01, ***p < 0.001 vs Control Official journal of the Cell Death Differentiation Association Zhao et al. Cell Death Discovery (2019) 5:76 Page 4 of 13 Table 2 Effect of metformin (MET) on proliferation of polycystic kidney epithelial cells 0 mM 0.6 mM 2.5 mM 10 mM 40 mM 12 h 100.00 ± 2.89 94.22 ± 1.91* 87.09 ± 1.70*** 77.57 ± 2.02*** 64.26 ± 1.17*** 24 h 100.00 ± 2.56 92.28 ± 1.61** 87.23 ± 2.00*** 61.24 ± 1.73*** 51.42 ± 0.57*** 36 h 100.00 ± 2.35 90.68 ± 1.47** 83.14 ± 4.90*** 56.20 ± 1.92*** 43.56 ± 1.31*** 48 h 100.00 ± 3.21 85.26 ± 2.56*** 76.42 ± 3.73*** 55.04 ± 1.59*** 39.93 ± 1.55*** *P < 0.05, **p < 0.01, ***p < 0.001 vs Control Table 3 Effects of combined use of 2-deoxyglucose (2-DG) and metformin (MET) on proliferation of polycystic kidney epithelial cells 0 + 0 mM 1.3 + 0.6 mM 2.5 + 1.3 mM 5 + 2.5 mM 10 + 5mM 12 h 100.00 ± 2.18 85.92 ± 2.15*** 73.47 ± 0.94*** 68.16 ± 1.98*** 58.40 ± 3.25*** 24 h 100.00 ± 3.21 80.02 ± 1.00*** 68.83 ± 1.22*** 56.53 ± 1.20*** 43.21 ± 1.05*** 36 h 100.00 ± 1.97 73.43 ± 2.52*** 63.98 ± 1.44*** 55.11 ± 1.78*** 40.31 ± 1.30*** 48 h 100.00 ± 2.43 67.02 ± 2.00*** 56.41 ± 2.04*** 44.87 ± 1.35*** 35.81 ± 2.12*** *P < 0.05, **p < 0.01, ***p < 0.001 vs Control Fig. 2 The 5-ethynyl-2'-deoxyuridine (EdU)-positive cells represent new dividing cells and are stained (red). The nucleus was counterstained with 4’,6-diamidino-2-phenylindole (DAPI; blue), original magnification ×100. Scale bar = 15 µm; **p < 0.01, ***p < 0.001 vs Control pathways are increased in PKD, while the activation level In MET group, the activity of PKA was significantly of PI3K/Akt pathway is decreased . In the present study, downregulated; the activation level of AMPK was we observed the effects of 2-DG or MET alone and increased; the activation levels of mTOR, p70 S6K and 4E- combination on the phosphorylation level (representing BP1 were decreased; the activation levels of PI3K and Akt the activation level) of proliferation signaling pathway were slightly upregulated; and the activation levels of B- molecules in cystic epithelial cells. Raf, MEK1/2 and Erk1/2 were not significantly decreased The results showed that, in 2-DG group, the activity of compared with those in control group (Fig. 3). PKA was significantly downregulated; the activity of In 2-DG+MET group, the activity of PKA was markedly AMPK was increased; the activating levels of mTOR, p70 downregulated; the activation level of AMPK was sig- S6K and 4E-BP1 were deceased; the activities of PI3K and nificantly increased; the activation levels of mTOR, p70 Akt were markedly upregulated; and the activation levels S6K and 4E-BP1 were significantly decreased; the activa- of B-Raf, MEK1/2, and Erk1/2 were markedly decreased tion levels of PI3K and Akt were markedly upregulated; compared with those in control group (Fig. 3). and the activation levels of B-Raf, MEK1/2 and Erk1/2 Official journal of the Cell Death Differentiation Association Zhao et al. Cell Death Discovery (2019) 5:76 Page 5 of 13 Fig. 3 Effects of 2-deoxyglucose (2-DG), metformin (MET) and 2-DG+MET on the B-Raf/ERK, AMPK-mTOR, AKT and PKA signaling pathway molecules in WT9-7 cells. a, b Protein expression levels of p-PI3K, p-Akt, p-PKA, p-AMPKα, p-mTOR, p-p70 S6K, p-4E-BP1, p-c-Raf, p-MEK1/2 and P-Erk1/2 (p (phospho), represents the protein activation level) were determined using western blotting. c, d Quantification of relative β-actin protein abundance shown in (a, b); *p < 0.05,**p < 0.01, ***p < 0.001 vs Control. ERK: extracellular-regulated protein kinase, AMPK: adenosine monophosphate activated protein kinase, mTOR: mammalian target of rapamycin, PKA: protein kinase A were significantly decreased compared with those in Effects of 2-DG and MET on cell cycle in human polycystic control group. These results indicate that the combina- kidney epithelial cells tional use of these two drugs has synergistic inhibitory Cell proliferation is strongly related to regulation of cell effect for the activation of proliferation signaling pathway cycle. We analyzed the changes of cell cycle in each drug- molecules in cystic epithelial cells (Fig. 3). treated group by flow cytometry. The results showed that Official journal of the Cell Death Differentiation Association Zhao et al. Cell Death Discovery (2019) 5:76 Page 6 of 13 Fig. 4 Effects of 2-deoxyglucose (2-DG), metformin (MET) and 2-DG+MET on proliferating cell nuclear antigen (PCNA), the cell cycle and cell cycle proteins in WT9-7 cells. a–d Results of cell cycle analysis by flow cytometry (FCM) in WT9-7 cells treated with 5 mM 2-DG, 2.5 mM MET and their combination for 48 h. e, f Protein expression levels of PCNA, cyclin D3, cyclin E1, CDK2 and CDK4 in WT9-7 cells; *p < 0.05, **p < 0.01, ***p < 0.001 vs Control 2-DG or MET alone could extend the G0/G1 phase and Table 4 The percentage of WT9-7 cells in G0/G1, S, and G2/M phases reduce the S phase (p < 0.05), but the combination of 2- DG and MET markedly extended the G0/G1 phase and Group G0–G1 (%) S (%) G2-M (%) reduced the S and G2/M phases (Fig. 4a–d, Table 4) compared with those in control group (p < 0.001). These Control 30.71 ± 1.78 35.27 ± 2.05 34.03 ± 2.48 results are indicative of an arrest at the G0/G1 restriction 2-DG 37.64 ± 2.50* 25.76 ± 4.03* 36.60 ± 1.92 point by a reduction in cell cycle progression. The western MET 38.55 ± 2.45* 28.81 ± 1.06** 33.31 ± 1.98 blot results showed that the combination of the two drugs markedly decreased the expression of cell cycle proteins, 2-DG+MET 58.16 ± 2.32*** 21.36 ± 1.52*** 20.48 ± 3.25** cyclin D3 and cyclin E1, as well as the expression of Control normal group, 2-DG 2-deoxyglucose-alone group, MET metformin-alone cyclin-dependent kinases, CDK2 and CDK4 (Fig. 4e, f), group, 2-DG+MET the combination of 2-DG and metformin *P < 0.05, **p < 0.01, ***p < 0.001 vs Control, n = 3 indicating that combinational use of the two drugs can Official journal of the Cell Death Differentiation Association Zhao et al. Cell Death Discovery (2019) 5:76 Page 7 of 13 significantly arrest cell cycle progression by suppressing the expression of cell cycle proteins. Proliferating cell nuclear antigen (PCNA) is present in the nucleus and plays an important role in the initiation of cell proliferation; PCNA is an indicator reflecting the state of cell proliferation. Here, we analyzed the changes of PCNA protein expression in each group by western blot. The results showed that PCNA expression level decreased in all drug-treated groups; however, the effect was more notice- able in 2-DG+MET group than in control group (Fig. 4e, f). Effects of 2-DG and MET on apoptosis of human polycystic kidney epithelial cells The above results show that the combination of 2-DG and MET markedly decreased the viability of cystic epi- thelial cells. We hypothesize that the combination of 2- DG and MET can also induce the apoptosis of cystic epithelial cells while inhibiting the proliferation of cystic epithelial cells. To validate this hypothesis, we also tested the effects of 2-DG, MET and their combination on apoptosis. The results showed that the treatment with MET alone had no effect on apoptosis of cystic epithelial cells. However, the intracellular caspase-3 activity increased at 36 h (p < 0.001) and the early apoptosis rate also slightly increased at 48 h in the cystic epithelial cells treated with 2-DG alone. The combination of 2-DG and MET significantly promoted the apoptosis of the cystic epithelial cells, resulting in significant increases in the intracellular caspase-3 activity (p < 0.001) and early apoptosis rate (Fig. 5, Table 5). Effects of 2-DG or MET alone and combination on glucose Fig. 5 Effects of 2-deoxyglucose (2-DG), metformin (MET) and 2-DG+MET on the apoptosis of WT9-7 cells, as analyzed using metabolic phenotypes in human polycystic kidney annexin V/propidium iodide (PI) staining by flow cytometry epithelial cells (FCM) and a caspase-3 activity assay kit. a Results from analyzing Similar to tumor cells, PKD cyst epithelial cells also the apoptosis rate (D1: necrosis; D2: late apoptosis; D3: living; D4: early show the Warburg effect. In these cells, the glycolytic apoptosis) by FCM in WT9-7 cells treated with 5 mM 2-DG, 2.5 mM activity is significantly enhanced, and the intracellular MET and their combination for 48 h. b Caspase-3 activity in WT9-7 cells treated with 5 mM 2-DG, 2.5 mM MET and their combination for ATP production is increased. Accordingly, the AMPK 12, 24, 36, and 48 h; ***p < 0.001 vs Control activity is significantly decreased, the mTOR activity is increased and the cell proliferation ability is enhanced. To investigate the effects of the combination of 2-DG and MET on glucose metabolism phenotype, we performed Table 5 The activity level of caspase-3 in polycystic kidney epithelial cells extracellular flux analysis on the cystic epithelial cells treated with 2-DG, MET and their combination using a Control 2-DG MET 2-DG+MET Seahorse Extracellular Flux Analyzer. ECAR (extracellular acidification rate) represents the glycolysis level and OCR 12 h 1.00 ± 0.04 1.01 ± 0.04 1.00 ± 0.04 1.14 ± 0.02*** (oxygen consumption rate) represents the mitochondrial 24 h 1.00 ± 0.05 1.02 ± 0.03 1.00 ± 0.04 1.13 ± 0.03*** respiration level. We measured the real-time changes in 36 h 1.00 ± 0.06 1.21 ± 0.07*** 1.00 ± 0.04 1.38 ± 0.09*** ECAR and OCR under baseline (regular measurements, repeated 3 times) and stressed conditions (regular mea- 48 h 1.00 ± 0.03 1.35 ± 0.04*** 1.00 ± 0.06 1.57 ± 0.03*** surements, repeated 5 times) (Fig. 6a, b) to determine the Control: normal group, 2-DG 2-deoxyglucose-alone group, MET metformin alone two key parameters of cell energy metabolism: the base- group, 2-DG+MET the combination of 2-DG and metformin ***p < 0.001 vs Control line phenotype and the stressed phenotype. Official journal of the Cell Death Differentiation Association Zhao et al. Cell Death Discovery (2019) 5:76 Page 8 of 13 Fig. 6 (See legend on next page.) Official journal of the Cell Death Differentiation Association Zhao et al. Cell Death Discovery (2019) 5:76 Page 9 of 13 (see figure on previous page) Fig. 6 Effects of 2-deoxyglucose (2-DG) and metformin (MET) on the metabolism of WT9-7 cells. a, b The real-time mitochondrial bioenergetic profiles and glycolytic profiles were generated using the Seahorse XF96 Analyzer. c, d Quantitative analysis of the total baseline and stressed bioenergetic profiles for the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of the four groups of WT9-7 cells; *p < 0.05,**p # ## ### ▲ ▲▲ ▲▲▲ < 0.01, ***p < 0.001 vs Control, p < 0.05, p < 0.01, p < 0.001 vs 2-DG, p < 0.05, p < 0.01, p < 0.001 vs MET. e Cell energy phenotypes were generated using the Seahorse XF96 Analyzer. f WT9-7 cells were treated with 5 mM 2-DG and 2.5 mM MET for 48 h and their glucose uptake # ## ### ▲ ▲▲ ▲▲▲ was measured; *p < 0.05, **p < 0.01, ***p < 0.001 vs Control, p < 0.05, p < 0.01, p < 0.001 vs 2-DG, p < 0.05, p < 0.01, p < 0.001 vs MET. g WT9-7 cells were treated with 5 mM 2-DG and 2.5 mM MET for 48 h and their lactate production was measured; *p < 0.05, **p < 0.01, ***p < 0.001 vs # ## ### ▲ ▲▲ ▲▲▲ Control, p < 0.05, p < 0.01, p < 0.001 vs 2-DG, p < 0.05, p < 0.01, p < 0.001 vs MET. h WT9-7 cells were treated with 5 mM 2-DG and # ## ### 2.5 mM MET for 48 h and their ATP production was measured; *p < 0.05,**p < 0.01, ***p < 0.001 vs Control, p < 0.05, p < 0.01, p < 0.001 vs 2-DG, ▲ ▲▲ ▲▲▲ p < 0.05, p < 0.01, p < 0.001 vs MET First, we observed the effects of 2-DG, MET and their epithelial cells were treated with 2-DG+MET, both the combination on the ECAR and OCR levels under baseline ECAR and OCR levels declined, suggesting that the conditions in cystic epithelial cells. The results of quan- combination of 2-DG and MET significantly lowered both titative analysis showed that compared with control the glycolysis and oxidative phosphorylation levels in the group, 2-DG group alone had lower ECAR level (reflect- cells (Fig. 6c, d). ing a decrease in the glycolysis level) (p < 0.05), whereas Figure 6e more clearly shows that regardless of whether the OCR level increased. This finding indicates a com- the cells were in the baseline (open marks) or stressed pensatory increase in mitochondrial oxidative phosphor- (solid marks) condition, the ECAR and OCR levels were ylation to supply sufficient energy to the cells. However, very low in the cystic epithelial cells in 2-DG+MET MET alone reduced the OCR level in cystic epithelial cells group. The values were located in the lower left corner of (reflecting a decrease in the oxidative phosphorylation the coordinate system, indicating very low glycolysis and level) (p < 0.001), whereas the ECAR increased. This oxidative phosphorylation levels in these cells. finding indicates a compensatory increase in the cellular glycolytic capacity to supply sufficient energy to the cells Effects of 2-DG or MET alone and combination on glucose (p < 0.001). When cystic epithelial cells were treated with intake, lactate production and ATP concentration in 2-DG+MET, both the ECAR and OCR levels declined, human polycystic kidney epithelial cells suggesting that the combination of 2-DG and MET sig- To further evaluate the effects of 2-DG and MET on nificantly decreased both the glycolysis and oxidative glucose metabolism, we measured the glucose intake, phosphorylation levels in the cells (p < 0.001) (Fig. 6c, d). lactate production and ATP concentration in cystic epi- Next, we observed the effects of 2-DG, MET and their thelial cells treated with 2-DG, MET and their combina- combination on the ECAR and OCR levels under stressed tion. The results showed that both glucose intake and conditions in cystic epithelial cells. The stress factors lactate production were decreased, while ATP synthesis include the metabolic modulator oligomycin, which was also significantly decreased in 2-DG group compared inhibits mitochondrial ATP synthase and induces cellular with control group. This finding indicates that 2-DG glycolytic potential, and carbonyl cyanide-p-tri- significantly decreased the glycolysis level in the cystic fluoromethoxyphenylhydrazone (FCCP), which uncouples epithelial cells (Fig. 6f–h). Both glucose intake and lactate mitochondrial oxidative phosphorylation and induces production were markedly increased, whereas ATP maximal mitochondrial respiration . The results of the synthesis was markedly decreased in MET group com- quantitative analysis showed that compared with control pared with control group. This finding indicates that MET group, 2-DG group alone had a lower ECAR level decreased the oxidative phosphorylation level in cystic (reduced glycolytic capacity), whereas the OCR under epithelial cells, resulting in decreased ATP synthesis. stressed conditions increased in the cystic epithelial cells However, a compensatory increase in the glycolysis level (p < 0.05). These findings indicate a compensatory occurred, leading to an increase in lactate metabolites increase in mitochondrial oxidative phosphorylation to (Fig. 6f–h). The glucose intake and lactate production supply sufficient energy to the cells. However, MET alone were decreased, while the ATP synthesis was also sig- reduced the OCR level (reflecting a decrease in the oxi- nificantly reduced in 2-DG+MET group compared with dative phosphorylation level) (p < 0.001), whereas the control group. These findings further confirm that the ECAR level under stressed conditions increased in the combination of 2-DG and MET simultaneously decreased cystic epithelial cells. These results indicate a compensa- the metabolism levels of intracellular glycolysis and oxi- tory increase in the cellular glycolytic capacity to supply dative phosphorylation, thereby depleting the ATP supply sufficient energy to the cells (p < 0.001). When cystic of cells (Fig. 6f–h). Official journal of the Cell Death Differentiation Association Zhao et al. Cell Death Discovery (2019) 5:76 Page 10 of 13 Discussion intracellular ATP production by decreasing the mito- PKD is a hereditary disease caused by the mutations in chondrial oxidative phosphorylation level in polycystic PKD1 or PKD2 gene . The abnormal proliferation of kidney epithelial cells; MET can inhibit the proliferation polycystic kidney epithelial cells is a main cause of the of polycystic kidney epithelial cells by activating AMPKα gradual enlargement of cysts. Except for symptomatic activity (increase in the phosphorylated AMPKα level) treatment, renal transplantation and dialysis for end-stage and inhibiting mTOR pathway activity (decrease in the phosphorylated mTOR, p70 S6K and 4E-BP1). However, renal disease, there have been no clinically effective, tar- geted treatment drugs . when oxidative phosphorylation metabolism was inhib- A study in 2013 accidentally found that polycystic kid- ited, there was a compensatory increase in the glycolysis ney cells mainly rely on aerobic glycolysis to produce a level in the polycystic kidney epithelial cells; the cellular large quantity of ATP; this metabolic phenotype change is intake of glucose increased, and a large quantity of lactate very similar to that in tumor cells. Therefore, inhibition of was produced. Despite the good clinical tolerance of glycolysis may become a method to treat tumor and PKD. MET, lactic acidosis remains the primary side effect. AMPK is a key molecule in the regulation of energy Moreover, because MET is mainly cleared by the kidneys, metabolism. mTOR not only regulates proliferation, but chronic renal disease has been considered a potential also plays an important role in regulating energy meta- predisposing factor for this complication . Thus, treat- bolism. AMPK can specifically inhibit mTOR activity .A ment with MET alone has some limitations. decreased AMPK activity and an increased mTOR activity Given the limitations of 2-DG and MET, at present, were found in polycystic kidney cells . many studies use their combination for the treatment of 19,31 2-DG, the glucose analog, acts as a competitive inhibitor tumors . Therefore, in this study, we investigated of the glycolytic enzyme hexokinase . Upon transport whether the combination of 2-DG and MET at small into the cells, 2-DG is phosphorylated to 2DG-phosphate dose of level can more effectively inhibit the prolifera- (2DG-P) by hexokinase. However, unlike glucose-6- tion of polycystic kidney epithelial cells and lower the phosphate (G-6-P), 2DG-P cannot be further metabo- side effects of the two drugs. The results showed that lized by phosphohexose isomerase, which converts G-6-P their combination simultaneously inhibited glycolysis to fructose-6-phosphate . Thus, 2DG-P is trapped and and oxidative phosphorylation metabolism in polycystic accumulates in the cells, leading to inhibition of glycolysis kidney epithelial cells, significantly decreased glucose mainly at the step of the phosphorylation of glucose by intake, lactate production and ATP yield, and sig- hexokinase. Inhibition of this rate-limiting step by 2-DG nificantly decreased the activation levels of proliferation depletes the cellular ATP, leading to blockage of cell cycle signaling pathway molecules, including mTOR, B-Raf- progression and cell death in vitro . ERK and PKA. Moreover, we found that the combina- Our results showed that 2-DG could decrease the gly- tion of low-dose MET and 2-DG significantly upregu- colysis level in polycystic kidney epithelial cells. The cel- lated the PI3K/Akt activation level, led to cell cycle lular intake of glucose, lactate production and arrest at G1/G0 phase and significantly inhibited cell intracellular ATP production were decreased, whereas the proliferation. Inhibition of apoptosis has been shown to phosphorylated AMPKα level was increased by feedback delay renal cyst growth in some animal models of PKD. in 2-DG group. Further, the activation levels of phos- In present study, either MET or 2-DG alone had little phorylated mTOR, p70 S6K and 4E-BP1 were down- effect on the apoptosis of cystic epithelial cells, whereas regulated in 2-DG group. Additionally, 2-DG increased their combination enhanced early apoptosis and the activation level of PI3K-Akt and indirectly inhibited increased caspase-3 activity in the cells. the over-activation of B-Raf-ERK compared with that in In conclusion, this study presents the first evidence that control group. 2-DG also extended the G1/G0 phase of the combinational use of low-dose 2-DG and MET can polycystic kidney epithelial cells. Although 2-DG alone markedly inhibit cell proliferation via simultaneously inhi- can inhibit the proliferation of polycystic kidney epithelial biting glycolysis and oxidative phosphorylation metabolism, cells, a high dose has side effects. Toxicity due to off- reducing intracellular ATP production, activating AMPK target effects has been attributed to this compound in activity, and thereby inhibiting the activation of the mTOR clinical trials, and 3/10 mice in this study were killed early proliferation signaling pathway in polycystic kidney epi- because of weight loss during 2-DG treatment . There- thelial cells. Furthermore, the combination of these two fore, in this study, in order to decrease side effects, we drugs stimulates the activation level of PI3K/Akt, prevents selected small dose of 2-DG to observe its effect on the the over-activation of B-Raf-ERK signaling pathway mole- proliferation of polycystic kidney epithelial cells. cules and, finally, inhibits over-proliferation by arresting the MET, as first-line hypoglycemic drug, has been used for cells at the G0/G1 phase. More importantly, combinational treatment of type 2 diabetes and clinical trial of cancer use of low-dose 2-DG and MET can lower the side effects of treatment . Our results showed that MET reduced each drug. This study provides a new potential strategy for Official journal of the Cell Death Differentiation Association Zhao et al. Cell Death Discovery (2019) 5:76 Page 11 of 13 the future clinical use of these two drugs for the treatment MET (2.5 mM) and 2-DG+MET (5 and 2.5 mM). Finally, of human PKD. the absorbance of HRPTEpiC cells were measured at 48 h of treatment with different drugs using a microplate Materials and methods reader (Bio-Rad, USA). The percentage cell viability was Cell culture studies calculated based on the absorbance of the drug-treated The human autosomal dominant PKD cyst-lining epithelial cells relative to the absorbance of the control cells. Experiments were performed in triplicate, and sextuplet cell line WT9-7 was purchased from American Type Culture Collection (ATCC, USA). Cells were seeded into Dulbecco’s wells were used. modified Eagle’s medium (ATCC, USA) containing 10% fetal bovine serum (FBS; HyClone, Canada) and cultured in an EdU proliferation assay incubator at 37 °C with 5% CO . Cells were digested using Cell proliferation was assayed using Click-iT® EdU Ima- 0.25% trypsin and subcultured at a ratio of 1:6 when 80% ging kits (Invitrogen™ Thermo Fisher Scientific, USA). The confluence was reached. The experimental groups were as EdU provided in the kit is a nucleoside analog of thymidine follows: (1) control group: normal culture; (2) 2-DG group: and is incorporated into DNA during active DNA synthesis. adherent cells treated with different concentrations of 2-DG Detection is based on a click reaction, a copper-catalyzed (MedChem Express, USA); (3) MET group: adherent cells covalent reaction between an azide and an alkyne. Briefly, treated with different concentrations of MET (Sigma, US); WT9-7 polycystic kidney epithelial cells were seeded in 6- and (4) 2-DG+MET group: adherent cells treated with dif- well plates at 10 /well, synchronized in FBS-free medium ferent concentrations of 2-DG and MET simultaneously. The for 12 h, and then treated with 2-DG (5 mM), MET HRPTEpiC were purchased from ScienCell Research (2.5 mM) or 2-DG+MET (5+ 2.5 mM) for 36 h. Thereafter, Laboratories (ScienCell, USA). Cells were seeded into Epi- 1:1000 EdU probe was added to the medium, and the cells thelial Cell medium (ScienCell, USA) containing 2% FBS were cultured for another 12 h and then fixed with 4% (HyClone, Canada) and cultured in an incubator at 37 °C paraformaldehyde. Following the experimental procedure of with 5% CO . Cells were digested using 0.25% trypsin and the Click-iT® EdU Imaging kits, the nuclei were labeled subculturedataratioof1:2 when 90% confluence was with 4’,6-diamidino-2-phenylindole (DAPI, a fluorescent reached. The experimental groups were as follows: (1) con- dye capable of binding strongly to DNA), and images were trol group: normal culture; (2) 2-DG group: adherent cells acquired by confocal laser microscopy. treated with 5 mM concentration of 2-DG; (3) MET group: adherent cells treated with 2.5 mM concentration of MET; Cell cycle analysis and (4) 2-DG+MET group: adherent cells treated with 5 WT9-7 polycystic kidney epithelial cells were seeded in mM 2-DG and 2.5 mM MET simultaneously. 25 cm culture flasks and synchronized in FBS-free medium for 12 h. The cells were incubated for another CCK-8 assay 48 h after the addition of 2-DG (5 mM), MET (2.5 mM) or After treatment with 2-DG, MET, and 2-DG+MET, the 2-DG+MET (5 + 2.5 mM). The cells were washed twice viability of WT9-7 human polycystic kidney epithelial with phosphate-buffered saline (PBS), suspended in 75% cells were measured using a CCK-8 cell proliferation- ethanol and fixed by incubation in 75% ethanol at 4 °C cytotoxicity assay kit (Beyotime, China), and the optimal overnight. Fixed cells were collected by centrifugation, treatment concentration was selected. The polycystic washed with PBS, treated with RNase (50 μg/mL; Sigma, kidney epithelial cell suspension was adjusted to a con- USA) and stained with propidium iodide (PI; 50 μg/mL; centration of 3 × 10 /L and seeded in 96-well plates at Sigma, USA). A FACS flow cytometer (BD Co., USA) was 3000/well. The cells were synchronized in FBS-free used to determine the cellular DNA contents. The per- medium for 12 h and then treated with different con- centage of cells in the G0/G1, S and G2/M phases was centrations of 2-DG (0.6, 2.5, 10 and 40 mM), MET (0.6, determined using Cell FIT Cell Cycle Analysis software 2.5, 10 and 40 mM) and 2-DG+MET (1.3 + 0.6, 2.5 + 1.3, (version 2.01.2; BD). 5 + 2.5 and 10 + 5 mM). Finally, the absorbance of poly- cystic kidney epithelial cell cultures at 450 nm was mea- Caspase-3 activity assay sured at 12, 24, 36 and 48 h of treatment with different WT9-7 polycystic kidney epithelial cells were seeded in drugs using a microplate reader (Bio-Rad, USA). The 25 cm culture flasks and treated with 2-DG (5 mM), viability of HRPTEpiC was measured using a CCK-8 cell MET (2.5 mM) or 2-DG+MET (5 + 2.5 mM) for 12, 24, proliferation-cytotoxicity assay kit The human renal 36 and 48 h. The proteins were collected, and the protein proximal tubular epithelial cells were adjusted to a con- concentration in each group was measured by the centration of 3 × 10 /L and seeded in 96-well plates at bicinchoninic acid (BCA) assay. The protein concentra- 3000/well. The cells were synchronized in FBS-free tion for each group was adjusted to the same level, and medium for 12 h and then treated with 2-DG (5 mM), 50 μL of protein was taken for analysis. Caspase-3 activity Official journal of the Cell Death Differentiation Association Zhao et al. Cell Death Discovery (2019) 5:76 Page 12 of 13 was evaluated using a Caspase-3/CPP32 Colorimetric Lactate Colorimetric/Fluorometric Assay kit (Biovision, Assay kit (Biovision, USA), according to the manu- USA) and CellTiter-Glo® 2.0 Luminescent Cell Viability facturer’s instructions. Finally, the absorbance of each Assay (Promega, USA), respectively, according to the sample at 405 nm was measured using a microplate manufacturer’s instructions. reader. The percentage of caspase-3 activity was calcu- lated as the absorbance of the drug-treated cells relative to Western blot analysis the absorbance of the control vehicle-treated cells. Proteins were extracted from WT9-7 cells using RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Cell apoptosis evaluated using the annexin V-fluorescein deoxycholate, 1% Nonidet P-40, 0.1% SDS, 1 mM phe- isothiocyanate (FITC) assay nylmethylsulfonyl fluoride and protease cocktail at 1 μg/ Cell apoptosis was evaluated using an Apoptosis Detect- mL). The protein concentrations were determined using a ing kit (BD, USA) and analyzed by flow cytometry (FCM). BCA kit (Pierce, USA). Protein samples (50 μg per lane) Briefly, WT9-7 polycystic kidney epithelial cells were seeded were separated by 6%, 8%, 10% or 12% sodium dodecyl in 25 cm culture flasks and treated with 2-DG (5 mM), sufate–polyacrylamide gel electrophoresis (SDS-PAGE) MET (2.5 mM) or 2-DG+MET (5+ 2.5mM) for 48h. electrophoresis and transferred to nitrocellulose (NC) Following the manufacturer’s instructions, the cells were membranes. After Ponceau S staining, the membranes labeled with annexin V-FITC and PI. Samples were exam- were incubated overnight in 5% non-fat milk at 4 °C and ined using FCM, and the results were analyzed using then incubated with primary antibodies against p- CellQuest software (Becton Dickinson, San Jose, CA). AMPKα, p-mTOR, p-4E-BP1, p-p70 S6 kinase, p-PKA, p- PI3 kinase p85, p-Akt, p-B-Raf, p-MEK1/2, p-p44/42 Assay of cell energy metabolism phenotypes MAPK (Erk1/2), PCNA, cyclin E1, Cdk2, Cdk4, (Cell The Seahorse XF Cell Energy Phenotype Test in con- Signaling Technology, USA), cyclin D3 (Wuhan Sanying junction with a Seahorse Bioscience XF96 Extracellular Biotech Co., Ltd., China) and β-actin (Sigma, USA). Flux Analyzer measures the mitochondrial respiration and Immunoreactive bands were visualized using ECL reagent glycolysis under baseline and stressed conditions and (KeyGen Biotech Co., Ltd., China), according to the provides the two key parameters of cell energy metabo- manufacturer’s instructions, and exposure to X-ray film. lism: the baseline phenotype and the stressed phenotype. The protein band intensities were quantified using ImageJ Briefly, WT9-7 cells were plated at 1000 cells/well (~30% software (NIH, Bethesda, USA). confluence) in a Seahorse XF Cell Culture Microplate (Seahorse Bioscience), and adherent cells were treated Statistical analyses with 2-DG (5 mM), MET (2.5 mM) or 2-DG+MET (5 + Student’s T-test was used to calculate statistical sig- 2.5 mM) for 48 h. Immediately prior to an experiment, the nificance between two experimental groups, analysis of growth medium was removed from each well, the cells variance was used to calculate statistical significance were rinsed with freshly prepared Seahorse assay medium between more than two experimental groups, and multi- (Seahorse Bioscience), and a final volume of 180 μLof variate analysis using factorial analysis. Measurement data assay medium was added to each well. The plate was are expressed as the mean ± SEM. Statistical analyses were incubated in a 37 °C incubator lacking CO for 1 h prior to performed using the SPSS 19.0 software package (SPSS, the assay. Mitochondrial and glycolytic bioenergetic pro- Inc., USA). Values of p < 0.05 were considered statistically files were generated by taking baseline measurements of significant. the OCR and ECAR of live cells in real time for three Acknowledgements measurement cycles and then sequentially injecting oli- This work was supported by grants (No. 81770663, No.81570659) from the gomycin (inhibits ATP synthase, 1 μg/mL final con- National Natural Science Foundation of China, a grant (No. 2016YFA0101002) from the National Key Research and Development Program of China and a centration) and FCCP (uncouples mitochondrial oxidative grant (No. 20158332) from the Natural Science Foundation of Hainan Province. phosphorylation and induces maximal respiration, 1 μg/ The English in this document has been checked by at least two professional mL final concentration) to measure the stressed OCR and editors from AJE, both native speakers of English. ECAR for five measurement cycles. Conflict of interest The authors declare that they have no conflict of interest. Measurement of intracellular glucose intake, extracellular lactate production and intracellular ATP synthesis Publisher’s note Polycystic kidney epithelial cells WT9-7 were seeded in Springer Nature remains neutral with regard to jurisdictional claims in 25 cm culture flasks and treated with 2-DG (5 mM), published maps and institutional affiliations. MET (2.5 mM) or 2-DG+MET (5 + 2.5 mM) for 48 h. The concentrations of glucose, lactate and ATP were Received: 10 January 2019 Revised: 15 February 2019 Accepted: 20 determined using a Glucose Assay kit (Biovision, USA), February 2019 Official journal of the Cell Death Differentiation Association Zhao et al. Cell Death Discovery (2019) 5:76 Page 13 of 13 17. Rehman, G., Shehzad, A., Khan, A. L. & Hamayun, M. Role of AMP- activated protein kinase in cancer therapy. Arch. Pharm. (Weinh.) 347, 457–468 (2014). 18. Ben Sahra, I. et al. Targeting cancer cell metabolism: the combination of References metformin and 2-deoxyglucose induces p53-dependent apoptosis in prostate 1. Chapman, A. B. et al. Autosomal-dominant polycystic kidney disease (ADPKD): cancer cells. Cancer Res. 70, 2465–2475 (2010). executive summary from a Kidney Disease: Improving Global Outcomes 19. Xue, C. et al. Targeting P-glycoprotein expression and cancer cell energy (KDIGO) Controversies Conference. Kidney Int. 88,17–27 (2015). metabolism: combination of metformin and 2-deoxyglucose reverses the 2. Rowe, I. et al. Defective glucose metabolism in polycystic kidney disease multidrug resistance of K562/Dox cells to doxorubicin. Tumour Biol. 37, identifies a new therapeutic strategy. Nat. Med. 19,488–493 (2013). 8587–8597 (2016). 3. Riwanto, M. et al. Inhibition of aerobic glycolysis attenuates disease progres- 20. Bikas, A. et al. Glucose-deprivation increases thyroid cancer cells sensitivity to sion in polycystic kidney disease. PLoS ONE 11, e0146654 (2016). metformin. Endocr. Relat. Cancer 22,919–932 (2015). 4. Wallace,D.P.Cyclic AMP-mediated cystexpansion. Biochim. Biophys. Acta 21. Ferreira, F. M.,Watanabe, E. H. &Onuchic,L.F.in Polycystic Kidney Disease (ed. 1812, 1291–1300 (2011). Li, X) Ch. 7 (Codon Publications, Brisbane, 2015). 5. Shillingford, J. M. et al. The mTOR pathway is regulated by polycystin-1, and its 22. Issaq,S.H., Teicher, B. A. &Monks,A. Bioenergeticpropertiesofhuman inhibition reverses renal cystogenesis in polycystic kidney disease. Proc. Natl. sarcoma cells help define sensitivity to metabolic inhibitors. Cell Cycle 13, Acad. Sci. USA 103,5466–5471 (2006). 1152–1161 (2014). 6. Torres, V. E. & Harris, P. C. Strategies targeting cAMP signaling in the treatment 23. Simms, R. J. Autosomal dominant polycystic kidney disease. BMJ 352,i679 of polycystic kidney disease. J. Am. Soc. Nephrol. 25,18–32 (2014). (2016). 7. Yu,A.S.L., El-Ters, M. &Winklhofer, F. T. in Polycystic Kidney Disease (ed. Li, X) 24. Yuajit, C. & Chatsudthipong, V. Nutraceutical for autosomal dominant Ch. 6 (Codon Publications, Brisbane, 2015). polycystic kidney disease therapy. J. Med. Assoc. Thai. 99(Suppl. 1), S97–S103 8. Torres, V. E. et al. Tolvaptan in patients with autosomal dominant polycystic (2016). kidney disease. N. Eng. J. Med. 367, 2407–2418 (2012). 25. Dong, L. X. et al. Negative regulation of mTOR activity by LKB1-AMPK 9. Xiao, H. et al. Separate and concurrent use of 2-deoxy-D-glucose and 3- signaling in non-small cell lung cancer cells. Acta Pharmacol. Sin. 34,314–318 bromopyruvate in pancreatic cancer cells. Oncol. Rep. 29,329–334 (2013). (2013). 10. Yamasaki, T. et al. Exploring a glycolytic inhibitor for the treatment of an FH- 26. Brown, J. Effects of 2-deoxyglucose on carbohydrate metablism: review of the deficient type-2 papillary RCC. Nat. Rev. Urol. 8,165–171 (2011). literature and studies in the rat. Metabolism 11, 1098–1112 (1962). 11. Zhang, D. et al. 2-Deoxy-D-glucose targeting of glucose metabolism in cancer 27. Weindruch, R. et al. Caloric restriction mimetics: metabolic interventions. J. cells as a potential therapy. Cancer Lett. 355,176–183 (2014). Gerontol.Ser.ABiol.Sci.Med.Sci. 1,20–33 (2001). 12. Zhou, G. et al. Role of AMP-activated protein kinase in mechanism of met- 28. Maher, J. C., Krishan, A. & Lampidis, T. J. Greater cell cycle inhibition and formin action. J. Clin. Invest. 108,1167–1174 (2001). cytotoxicity induced by 2-deoxy-D-glucose in tumor cells treated under 13. Shaw, R. J. et al. The kinase LKB1 mediates glucose homeostasis in liver and hypoxic vs aerobic conditions. Cancer Chemother. Pharmacol. 53,116–122 therapeutic effects of metformin. Science 310, 1642–1646 (2005). (2004). 14. Takiar, V. et al. Activating AMP-activated protein kinase (AMPK) slows renal 29. Csibi, A. & Blenis, J. Appetite for destruction: the inhibition of glycolysis as a cystogenesis. Proc. Natl. Acad. Sci. USA 108, 2462–2467 (2011). therapy for tuberous sclerosis complex-related tumors. BMC Biol. 9, 69 (2011). 15. Morales, D. R. & Morris, A. D. Metformin in cancer treatment and prevention. 30. Chae, Y. K. et al. Repurposing metformin for cancer treatment: current clinical Annu. Rev. Med. 66,17–29 (2015). studies. Oncotarget 7,40767–40780 (2016). 16. Cicero, A. F., Tartagni, E. & Ertek, S. Metformin and its clinical use: new insights 31. Cheng, G. et al. Profiling and targeting of cellular bioenergetics: inhibition of for an old drug in clinical practice. Arch.Med.Sci. 8, 907–917 (2012). pancreatic cancer cell proliferation. Br.J.Cancer 111,85–93 (2014). Official journal of the Cell Death Differentiation Association http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Death Discovery Springer Journals

Low-dose 2-deoxyglucose and metformin synergically inhibit proliferation of human polycystic kidney cells by modulating glucose metabolism

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Copyright © 2019 by The Author(s)
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Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Stem Cells; Apoptosis; Cell Cycle Analysis
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2058-7716
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Abstract

Polycystic kidney disease (PKD) is a common hereditary kidney disease with abnormal proliferation and apoptosis of kidney cystic epithelial cells, eventually leading to chronic renal failure. Currently, there are no effective treatment methods. Similar to tumor cells, cystic epithelial cells have abnormal glycolysis and over-activation of proliferation signaling pathways. In the present study, for the first time, we investigated the effects of low-dose combinational use of 2-deoxyglucose (2-DG) and metformin (MET) on the proliferation and apoptosis in the human cystic kidney epithelial cells. Cystic epithelia cells were divided into control group, 2-DG group, MET group and 2-DG+MET group. Cell Proliferation, apoptosis and glucose metabolism were measured in each group. The results showed that low-dose combinational treatment of 2-DG and MET significantly inhibited the proliferation of renal cystic epithelial cells by suppressing the activities of PKA, mTOR and ERK signaling pathways and upregulating PI3K/Akt pathway. Combination of both drugs increased the apoptosis rates of cystic epithelial cells. Two drugs inhibited glucose metabolic phenotypes, glycolysis and oxidative phosphorylation, and significantly lowered the intracellular ATP level in cystic epithelial cells. 2-DG could also neutralize excessive production of lactate (lactic acidosis) caused by MET and both drugs had complementary effect for cystic epithelial cells. These results reveal that combinational use of low-dose 2-DG and MET can markedly inhibit proliferation via modulating glucose metabolic phenotypes in human polycystic kidney epithelial cells, low-dose combinational use of both drugs can also lower the toxic effects of each drug, and is a novel strategy for future treatment of human polycystic kidney disease. Introduction leading to end-stage kidney disease .The pathogenesis of Polycystic kidney disease (PKD) is a hereditary kidney PKD is still unclear, and there is no effective treatment. disease. Both kidneys in PKD are filled with multiple serous In recent years, the Warburg effect has been found in cysts derived from renal tubules; the cyst epithelial cells show polycystic kidney epithelial cells, similar to tumor cells. abnormal proliferation and gradually increase in volume, Under aerobic conditions, the cystic cells mainly rely on thus compressing normal kidney tissues and eventually glycolytic metabolism for energy supply rather than on 2,3 mitochondrial oxidative phosphorylation . Additionally, the activity of the energy sensor, adenosine monopho- Correspondence: Xiangmei Chen (xmchen301@126.com) or Xue-Yuan Bai sphate activated protein kinase (AMPK), is decreased, (xueyuan_bai@163.com) while the mammalian target of rapamycin (mTOR) Department of Nephrology, Chinese PLA General Hospital, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National signaling pathway is over-activated in cyst epithelial 4,5 Clinical Research Center for Kidney Diseases, Beijing Key Laboratory of Kidney cells . Furthermore, the proliferation-related signaling Diseases, Beijing 100853, China pathways, cyclic adenyl-monophosphate-protein kinase A These authors contributed equally: Jing Zhao, Yuxiang Ma Edited by M.V. Nikilson Chirou © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Zhao et al. Cell Death Discovery (2019) 5:76 Page 2 of 13 (cAMP-PKA) and extracellular-regulated protein kinase treated with different concentrations (0.6, 2.5, 10 and 40 (ERK), are activated, while the activity of phosphoinositide mM) of 2-DG or MET alone for different times (12, 24, 36 3-kinase (PI3K)/Akt signaling pathway that inhibits the and 48 h). When the cells were treated with different over-activation of ERK proliferation signaling pathway is drugs for the same time, the viable cell count gradually significantly inhibited in the cystic cells . declined with increasing concentration of the drugs Numerous anti-proliferative drugs, such as rapamycin compared with that in control group. This finding indi- cates that the inhibitory effects of 2-DG and MET on the (mTOR inhibitor) and octreotide (somatostatin analog), have been used to treat polycystic kidney animal models in proliferation of cystic epithelial cells were dose dependent. recent years. Although these drugs showed good efficacy in When cells were treated with the same drugs for different cells and animal models, the effects were not satisfactory in times, the inhibitory effects of the drugs on cell pro- a number of follow-up clinical trials . Tolvaptan, a vaso- liferation were gradually enhanced as the treatment time pressin V2 receptor antagonist, is also effective; however, increased. These results indicate that the inhibitory clinical studies have shown that patients suffer severe thirst, effects of these drugs were time dependent, and the polyuria, nocturia, polydipsia and liver toxicity, and the US highest inhibition rate was found at 48 h (Fig. 1a, b, Food and Drug Administration (FDA) has not yet approved Tables 1 and 2). this drug for clinical use . Therefore, there is an urgent need The half-maximal inhibitory concentration (IC )for to find new treatment methods. cystic epithelial cells treated with 2-DG or MET alone for 2-Deoxyglucose (2-DG) is a glucose analog that inhibits 48 h was 30.9 and 17.4 mM, respectively (Fig. 1c). Using 9,10 glycolysis . 2-DG can compete with glucose to bind the above IC values, we selected different concentrations hexokinase (the first rate-limiting enzyme of glycolysis) in (1.3+ 0.6, 2.5+ 1.3, 5+ 2.5 and 10+ 5mM) of 2-DG+ cells and inhibit metabolism of tumor cell, thereby inhi- MET to treat cystic epithelial cells for different times (12, biting cell proliferation . Metformin (MET) is a first-line 24, 36 and 48 h). The results showed that the combination drug for the clinical treatment of type 2 diabetes mellitus. of the two drugs significantly inhibited the proliferation of Recent studies have found that MET can specifically cystic epithelial cells. The proliferation inhibition rate inhibit mitochondrial respiratory chain complex I and reached 50% in cystic epithelial cells treated with the decrease oxidative phosphorylation levels in cells, thus combination of 5 mM 2-DG and 2.5 mM MET for 48 h reducing adenosine triphosphate (ATP) synthesis, acti- (Fig. 1d, Table 3). Taking into account the safety of the vating AMPK and inhibiting mTOR proliferation signal- drugs, we selected small dose of 5 mM and 2.5 mM as the 12–16 ing pathway treatment concentrations of 2-DG and MET respectively in . Due to the obvious activation of glycolysis in tumor cells, a large quantity of glucose is the subsequent studies. consumed and high levels of ATP are produced, resulting Subsequently, we evaluated the effects of 5 mM 2-DG, in a decrease in AMP/ATP ratio and significantly inhib- 2.5 mM MET and their combination on the viability of ited AMPK activity . Thus, glycolytic inhibitor 2-DG and HRPTEpiC cells (human renal proximal tubular epithelial AMPK activator MET have been used in the treatment of cells). The results showed that the effects of 2-DG, MET tumors in recent years. The combinational use of MET and their combination on cell viability of HRPTEpiC cells and 2-DG can significantly deplete the ATP supply of were weaker than on cystic epithelial cells, showing a cancer cells and inhibit the over-activation of proliferation therapeutic potential of the combination of 2-DG and signaling pathways in cells, thereby significantly inhibiting MET in PKD (Fig. 1e). the over-proliferation of tumor cells and reducing the side CCK-8 assay evaluated cell proliferation mainly through 18–20 effects caused by high doses of the individual drugs . determining the viable cell counts in the plates; however, In the present study, for the first time, we treated human this method could not fully reflect the proliferation effect of polycystic kidney cyst-lining epithelial cells with a combi- the cells. To further verify whether the combination of 2- nation of low-dose MET and 2-DG. We systematically DG and MET can significantly inhibit the proliferation of analyzed the effects of the combination of these two drugs cystic epithelial cells, we directly observed the inhibitory on the proliferation and apoptosis of cyst epithelial cells and effect of their combination on cell proliferation using 5- explored the possible molecular mechanisms. ethynyl-2'-deoxyuridine (EdU) nucleic acid labeling tech- nique. The results showed that either 5 mM 2-DG alone or Results 2.5 mM MET alone, and their combination significantly Combinational use of low-dose 2-DG and MET significantly inhibited the proliferation of cystic epithelial cells (Fig. 2). inhibits the proliferation of human polycystic kidney epithelial cells Effects of 2-DG and MET on proliferation-related signaling The effects of individual 2-DG and MET on cell pro- pathways in human polycystic kidney epithelial cells liferation were evaluated using a Cell Counting Kit-8 (CCK-8) A previous study showed that the activation levels of B- assay in human polycystic kidney epithelial cells WT9-7 Raf/ERK, mTOR and cAMP-PKA proliferation signaling Official journal of the Cell Death Differentiation Association Zhao et al. Cell Death Discovery (2019) 5:76 Page 3 of 13 Fig. 1 Effects of 2-deoxyglucose (2-DG), metformin (MET) and their combination on the proliferation of WT9-7 cells and human renal proximal tubular epithelial cells (HRPTEC) evaluated using Cell Counting Kit-8 (CCK-8) assay. a WT9-7 cells were treated with 2-DG (0.6, 2.5, 10 and 40 mM) for 12, 24, 36 and 48 h, and the cell viability was determined using CCK-8 assay. b WT9-7 cells were treated with MET (0.6, 2.5, 10 and 40 mM) for 12, 24, 36 and 48 h, and the cell viability was determined using CCK-8 assay. c Cells were treated with various concentrations (0.6, 2.5, 10 and 40 mM) of 2-DG or MET for 48 h, and the half- maximal inhibitory concentration (IC ) was determined using GraphPad Prism software. d The combined treatment with 2-DG and MET enhanced the growth inhibition of WT9-7 cells. WT9-7 cells were treated with 2-DG and MET at different doses (1.3 + 0.6, 2.5+ 1.3, 5 + 2.5 and 10 + 5 mM) for 12, 24, 36 and 48 h, and the cell viability was determined using the CCK-8 assay. e HRPTEC cells were treated with 2-DG (5 mM), MET (2.5 mM) and their combination for 48 h, and the cell viability was determined using CCK-8 assay; *p < 0.05, **p < 0.01, ***p < 0.001 vs Control Table 1 Effect of 2-deoxyglucose (2-DG) on proliferation of polycystic kidney epithelial cells 0 mM 0.6 mM 2.5 mM 10 mM 40 mM 12 h 100.00 ± 2.36 95.86 ± 1.89 91.61 ± 2.31** 86.22 ± 2.75*** 76.92 ± 2.99*** 24 h 100.00 ± 2.41 93.58 ± 0.82* 89.13 ± 1.99*** 82.88 ± 2.60*** 73.43 ± 2.93*** 36 h 100.00 ± 2.34 92.66 ± 1.14** 87.58 ± 0.90*** 70.55 ± 2.06*** 60.48 ± 2.32*** 48 h 100.00 ± 2.28 91.40 ± 1.18*** 82.00 ± 1.49*** 64.75 ± 2.00*** 46.88 ± 1.48*** *P < 0.05, **p < 0.01, ***p < 0.001 vs Control Official journal of the Cell Death Differentiation Association Zhao et al. Cell Death Discovery (2019) 5:76 Page 4 of 13 Table 2 Effect of metformin (MET) on proliferation of polycystic kidney epithelial cells 0 mM 0.6 mM 2.5 mM 10 mM 40 mM 12 h 100.00 ± 2.89 94.22 ± 1.91* 87.09 ± 1.70*** 77.57 ± 2.02*** 64.26 ± 1.17*** 24 h 100.00 ± 2.56 92.28 ± 1.61** 87.23 ± 2.00*** 61.24 ± 1.73*** 51.42 ± 0.57*** 36 h 100.00 ± 2.35 90.68 ± 1.47** 83.14 ± 4.90*** 56.20 ± 1.92*** 43.56 ± 1.31*** 48 h 100.00 ± 3.21 85.26 ± 2.56*** 76.42 ± 3.73*** 55.04 ± 1.59*** 39.93 ± 1.55*** *P < 0.05, **p < 0.01, ***p < 0.001 vs Control Table 3 Effects of combined use of 2-deoxyglucose (2-DG) and metformin (MET) on proliferation of polycystic kidney epithelial cells 0 + 0 mM 1.3 + 0.6 mM 2.5 + 1.3 mM 5 + 2.5 mM 10 + 5mM 12 h 100.00 ± 2.18 85.92 ± 2.15*** 73.47 ± 0.94*** 68.16 ± 1.98*** 58.40 ± 3.25*** 24 h 100.00 ± 3.21 80.02 ± 1.00*** 68.83 ± 1.22*** 56.53 ± 1.20*** 43.21 ± 1.05*** 36 h 100.00 ± 1.97 73.43 ± 2.52*** 63.98 ± 1.44*** 55.11 ± 1.78*** 40.31 ± 1.30*** 48 h 100.00 ± 2.43 67.02 ± 2.00*** 56.41 ± 2.04*** 44.87 ± 1.35*** 35.81 ± 2.12*** *P < 0.05, **p < 0.01, ***p < 0.001 vs Control Fig. 2 The 5-ethynyl-2'-deoxyuridine (EdU)-positive cells represent new dividing cells and are stained (red). The nucleus was counterstained with 4’,6-diamidino-2-phenylindole (DAPI; blue), original magnification ×100. Scale bar = 15 µm; **p < 0.01, ***p < 0.001 vs Control pathways are increased in PKD, while the activation level In MET group, the activity of PKA was significantly of PI3K/Akt pathway is decreased . In the present study, downregulated; the activation level of AMPK was we observed the effects of 2-DG or MET alone and increased; the activation levels of mTOR, p70 S6K and 4E- combination on the phosphorylation level (representing BP1 were decreased; the activation levels of PI3K and Akt the activation level) of proliferation signaling pathway were slightly upregulated; and the activation levels of B- molecules in cystic epithelial cells. Raf, MEK1/2 and Erk1/2 were not significantly decreased The results showed that, in 2-DG group, the activity of compared with those in control group (Fig. 3). PKA was significantly downregulated; the activity of In 2-DG+MET group, the activity of PKA was markedly AMPK was increased; the activating levels of mTOR, p70 downregulated; the activation level of AMPK was sig- S6K and 4E-BP1 were deceased; the activities of PI3K and nificantly increased; the activation levels of mTOR, p70 Akt were markedly upregulated; and the activation levels S6K and 4E-BP1 were significantly decreased; the activa- of B-Raf, MEK1/2, and Erk1/2 were markedly decreased tion levels of PI3K and Akt were markedly upregulated; compared with those in control group (Fig. 3). and the activation levels of B-Raf, MEK1/2 and Erk1/2 Official journal of the Cell Death Differentiation Association Zhao et al. Cell Death Discovery (2019) 5:76 Page 5 of 13 Fig. 3 Effects of 2-deoxyglucose (2-DG), metformin (MET) and 2-DG+MET on the B-Raf/ERK, AMPK-mTOR, AKT and PKA signaling pathway molecules in WT9-7 cells. a, b Protein expression levels of p-PI3K, p-Akt, p-PKA, p-AMPKα, p-mTOR, p-p70 S6K, p-4E-BP1, p-c-Raf, p-MEK1/2 and P-Erk1/2 (p (phospho), represents the protein activation level) were determined using western blotting. c, d Quantification of relative β-actin protein abundance shown in (a, b); *p < 0.05,**p < 0.01, ***p < 0.001 vs Control. ERK: extracellular-regulated protein kinase, AMPK: adenosine monophosphate activated protein kinase, mTOR: mammalian target of rapamycin, PKA: protein kinase A were significantly decreased compared with those in Effects of 2-DG and MET on cell cycle in human polycystic control group. These results indicate that the combina- kidney epithelial cells tional use of these two drugs has synergistic inhibitory Cell proliferation is strongly related to regulation of cell effect for the activation of proliferation signaling pathway cycle. We analyzed the changes of cell cycle in each drug- molecules in cystic epithelial cells (Fig. 3). treated group by flow cytometry. The results showed that Official journal of the Cell Death Differentiation Association Zhao et al. Cell Death Discovery (2019) 5:76 Page 6 of 13 Fig. 4 Effects of 2-deoxyglucose (2-DG), metformin (MET) and 2-DG+MET on proliferating cell nuclear antigen (PCNA), the cell cycle and cell cycle proteins in WT9-7 cells. a–d Results of cell cycle analysis by flow cytometry (FCM) in WT9-7 cells treated with 5 mM 2-DG, 2.5 mM MET and their combination for 48 h. e, f Protein expression levels of PCNA, cyclin D3, cyclin E1, CDK2 and CDK4 in WT9-7 cells; *p < 0.05, **p < 0.01, ***p < 0.001 vs Control 2-DG or MET alone could extend the G0/G1 phase and Table 4 The percentage of WT9-7 cells in G0/G1, S, and G2/M phases reduce the S phase (p < 0.05), but the combination of 2- DG and MET markedly extended the G0/G1 phase and Group G0–G1 (%) S (%) G2-M (%) reduced the S and G2/M phases (Fig. 4a–d, Table 4) compared with those in control group (p < 0.001). These Control 30.71 ± 1.78 35.27 ± 2.05 34.03 ± 2.48 results are indicative of an arrest at the G0/G1 restriction 2-DG 37.64 ± 2.50* 25.76 ± 4.03* 36.60 ± 1.92 point by a reduction in cell cycle progression. The western MET 38.55 ± 2.45* 28.81 ± 1.06** 33.31 ± 1.98 blot results showed that the combination of the two drugs markedly decreased the expression of cell cycle proteins, 2-DG+MET 58.16 ± 2.32*** 21.36 ± 1.52*** 20.48 ± 3.25** cyclin D3 and cyclin E1, as well as the expression of Control normal group, 2-DG 2-deoxyglucose-alone group, MET metformin-alone cyclin-dependent kinases, CDK2 and CDK4 (Fig. 4e, f), group, 2-DG+MET the combination of 2-DG and metformin *P < 0.05, **p < 0.01, ***p < 0.001 vs Control, n = 3 indicating that combinational use of the two drugs can Official journal of the Cell Death Differentiation Association Zhao et al. Cell Death Discovery (2019) 5:76 Page 7 of 13 significantly arrest cell cycle progression by suppressing the expression of cell cycle proteins. Proliferating cell nuclear antigen (PCNA) is present in the nucleus and plays an important role in the initiation of cell proliferation; PCNA is an indicator reflecting the state of cell proliferation. Here, we analyzed the changes of PCNA protein expression in each group by western blot. The results showed that PCNA expression level decreased in all drug-treated groups; however, the effect was more notice- able in 2-DG+MET group than in control group (Fig. 4e, f). Effects of 2-DG and MET on apoptosis of human polycystic kidney epithelial cells The above results show that the combination of 2-DG and MET markedly decreased the viability of cystic epi- thelial cells. We hypothesize that the combination of 2- DG and MET can also induce the apoptosis of cystic epithelial cells while inhibiting the proliferation of cystic epithelial cells. To validate this hypothesis, we also tested the effects of 2-DG, MET and their combination on apoptosis. The results showed that the treatment with MET alone had no effect on apoptosis of cystic epithelial cells. However, the intracellular caspase-3 activity increased at 36 h (p < 0.001) and the early apoptosis rate also slightly increased at 48 h in the cystic epithelial cells treated with 2-DG alone. The combination of 2-DG and MET significantly promoted the apoptosis of the cystic epithelial cells, resulting in significant increases in the intracellular caspase-3 activity (p < 0.001) and early apoptosis rate (Fig. 5, Table 5). Effects of 2-DG or MET alone and combination on glucose Fig. 5 Effects of 2-deoxyglucose (2-DG), metformin (MET) and 2-DG+MET on the apoptosis of WT9-7 cells, as analyzed using metabolic phenotypes in human polycystic kidney annexin V/propidium iodide (PI) staining by flow cytometry epithelial cells (FCM) and a caspase-3 activity assay kit. a Results from analyzing Similar to tumor cells, PKD cyst epithelial cells also the apoptosis rate (D1: necrosis; D2: late apoptosis; D3: living; D4: early show the Warburg effect. In these cells, the glycolytic apoptosis) by FCM in WT9-7 cells treated with 5 mM 2-DG, 2.5 mM activity is significantly enhanced, and the intracellular MET and their combination for 48 h. b Caspase-3 activity in WT9-7 cells treated with 5 mM 2-DG, 2.5 mM MET and their combination for ATP production is increased. Accordingly, the AMPK 12, 24, 36, and 48 h; ***p < 0.001 vs Control activity is significantly decreased, the mTOR activity is increased and the cell proliferation ability is enhanced. To investigate the effects of the combination of 2-DG and MET on glucose metabolism phenotype, we performed Table 5 The activity level of caspase-3 in polycystic kidney epithelial cells extracellular flux analysis on the cystic epithelial cells treated with 2-DG, MET and their combination using a Control 2-DG MET 2-DG+MET Seahorse Extracellular Flux Analyzer. ECAR (extracellular acidification rate) represents the glycolysis level and OCR 12 h 1.00 ± 0.04 1.01 ± 0.04 1.00 ± 0.04 1.14 ± 0.02*** (oxygen consumption rate) represents the mitochondrial 24 h 1.00 ± 0.05 1.02 ± 0.03 1.00 ± 0.04 1.13 ± 0.03*** respiration level. We measured the real-time changes in 36 h 1.00 ± 0.06 1.21 ± 0.07*** 1.00 ± 0.04 1.38 ± 0.09*** ECAR and OCR under baseline (regular measurements, repeated 3 times) and stressed conditions (regular mea- 48 h 1.00 ± 0.03 1.35 ± 0.04*** 1.00 ± 0.06 1.57 ± 0.03*** surements, repeated 5 times) (Fig. 6a, b) to determine the Control: normal group, 2-DG 2-deoxyglucose-alone group, MET metformin alone two key parameters of cell energy metabolism: the base- group, 2-DG+MET the combination of 2-DG and metformin ***p < 0.001 vs Control line phenotype and the stressed phenotype. Official journal of the Cell Death Differentiation Association Zhao et al. Cell Death Discovery (2019) 5:76 Page 8 of 13 Fig. 6 (See legend on next page.) Official journal of the Cell Death Differentiation Association Zhao et al. Cell Death Discovery (2019) 5:76 Page 9 of 13 (see figure on previous page) Fig. 6 Effects of 2-deoxyglucose (2-DG) and metformin (MET) on the metabolism of WT9-7 cells. a, b The real-time mitochondrial bioenergetic profiles and glycolytic profiles were generated using the Seahorse XF96 Analyzer. c, d Quantitative analysis of the total baseline and stressed bioenergetic profiles for the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of the four groups of WT9-7 cells; *p < 0.05,**p # ## ### ▲ ▲▲ ▲▲▲ < 0.01, ***p < 0.001 vs Control, p < 0.05, p < 0.01, p < 0.001 vs 2-DG, p < 0.05, p < 0.01, p < 0.001 vs MET. e Cell energy phenotypes were generated using the Seahorse XF96 Analyzer. f WT9-7 cells were treated with 5 mM 2-DG and 2.5 mM MET for 48 h and their glucose uptake # ## ### ▲ ▲▲ ▲▲▲ was measured; *p < 0.05, **p < 0.01, ***p < 0.001 vs Control, p < 0.05, p < 0.01, p < 0.001 vs 2-DG, p < 0.05, p < 0.01, p < 0.001 vs MET. g WT9-7 cells were treated with 5 mM 2-DG and 2.5 mM MET for 48 h and their lactate production was measured; *p < 0.05, **p < 0.01, ***p < 0.001 vs # ## ### ▲ ▲▲ ▲▲▲ Control, p < 0.05, p < 0.01, p < 0.001 vs 2-DG, p < 0.05, p < 0.01, p < 0.001 vs MET. h WT9-7 cells were treated with 5 mM 2-DG and # ## ### 2.5 mM MET for 48 h and their ATP production was measured; *p < 0.05,**p < 0.01, ***p < 0.001 vs Control, p < 0.05, p < 0.01, p < 0.001 vs 2-DG, ▲ ▲▲ ▲▲▲ p < 0.05, p < 0.01, p < 0.001 vs MET First, we observed the effects of 2-DG, MET and their epithelial cells were treated with 2-DG+MET, both the combination on the ECAR and OCR levels under baseline ECAR and OCR levels declined, suggesting that the conditions in cystic epithelial cells. The results of quan- combination of 2-DG and MET significantly lowered both titative analysis showed that compared with control the glycolysis and oxidative phosphorylation levels in the group, 2-DG group alone had lower ECAR level (reflect- cells (Fig. 6c, d). ing a decrease in the glycolysis level) (p < 0.05), whereas Figure 6e more clearly shows that regardless of whether the OCR level increased. This finding indicates a com- the cells were in the baseline (open marks) or stressed pensatory increase in mitochondrial oxidative phosphor- (solid marks) condition, the ECAR and OCR levels were ylation to supply sufficient energy to the cells. However, very low in the cystic epithelial cells in 2-DG+MET MET alone reduced the OCR level in cystic epithelial cells group. The values were located in the lower left corner of (reflecting a decrease in the oxidative phosphorylation the coordinate system, indicating very low glycolysis and level) (p < 0.001), whereas the ECAR increased. This oxidative phosphorylation levels in these cells. finding indicates a compensatory increase in the cellular glycolytic capacity to supply sufficient energy to the cells Effects of 2-DG or MET alone and combination on glucose (p < 0.001). When cystic epithelial cells were treated with intake, lactate production and ATP concentration in 2-DG+MET, both the ECAR and OCR levels declined, human polycystic kidney epithelial cells suggesting that the combination of 2-DG and MET sig- To further evaluate the effects of 2-DG and MET on nificantly decreased both the glycolysis and oxidative glucose metabolism, we measured the glucose intake, phosphorylation levels in the cells (p < 0.001) (Fig. 6c, d). lactate production and ATP concentration in cystic epi- Next, we observed the effects of 2-DG, MET and their thelial cells treated with 2-DG, MET and their combina- combination on the ECAR and OCR levels under stressed tion. The results showed that both glucose intake and conditions in cystic epithelial cells. The stress factors lactate production were decreased, while ATP synthesis include the metabolic modulator oligomycin, which was also significantly decreased in 2-DG group compared inhibits mitochondrial ATP synthase and induces cellular with control group. This finding indicates that 2-DG glycolytic potential, and carbonyl cyanide-p-tri- significantly decreased the glycolysis level in the cystic fluoromethoxyphenylhydrazone (FCCP), which uncouples epithelial cells (Fig. 6f–h). Both glucose intake and lactate mitochondrial oxidative phosphorylation and induces production were markedly increased, whereas ATP maximal mitochondrial respiration . The results of the synthesis was markedly decreased in MET group com- quantitative analysis showed that compared with control pared with control group. This finding indicates that MET group, 2-DG group alone had a lower ECAR level decreased the oxidative phosphorylation level in cystic (reduced glycolytic capacity), whereas the OCR under epithelial cells, resulting in decreased ATP synthesis. stressed conditions increased in the cystic epithelial cells However, a compensatory increase in the glycolysis level (p < 0.05). These findings indicate a compensatory occurred, leading to an increase in lactate metabolites increase in mitochondrial oxidative phosphorylation to (Fig. 6f–h). The glucose intake and lactate production supply sufficient energy to the cells. However, MET alone were decreased, while the ATP synthesis was also sig- reduced the OCR level (reflecting a decrease in the oxi- nificantly reduced in 2-DG+MET group compared with dative phosphorylation level) (p < 0.001), whereas the control group. These findings further confirm that the ECAR level under stressed conditions increased in the combination of 2-DG and MET simultaneously decreased cystic epithelial cells. These results indicate a compensa- the metabolism levels of intracellular glycolysis and oxi- tory increase in the cellular glycolytic capacity to supply dative phosphorylation, thereby depleting the ATP supply sufficient energy to the cells (p < 0.001). When cystic of cells (Fig. 6f–h). Official journal of the Cell Death Differentiation Association Zhao et al. Cell Death Discovery (2019) 5:76 Page 10 of 13 Discussion intracellular ATP production by decreasing the mito- PKD is a hereditary disease caused by the mutations in chondrial oxidative phosphorylation level in polycystic PKD1 or PKD2 gene . The abnormal proliferation of kidney epithelial cells; MET can inhibit the proliferation polycystic kidney epithelial cells is a main cause of the of polycystic kidney epithelial cells by activating AMPKα gradual enlargement of cysts. Except for symptomatic activity (increase in the phosphorylated AMPKα level) treatment, renal transplantation and dialysis for end-stage and inhibiting mTOR pathway activity (decrease in the phosphorylated mTOR, p70 S6K and 4E-BP1). However, renal disease, there have been no clinically effective, tar- geted treatment drugs . when oxidative phosphorylation metabolism was inhib- A study in 2013 accidentally found that polycystic kid- ited, there was a compensatory increase in the glycolysis ney cells mainly rely on aerobic glycolysis to produce a level in the polycystic kidney epithelial cells; the cellular large quantity of ATP; this metabolic phenotype change is intake of glucose increased, and a large quantity of lactate very similar to that in tumor cells. Therefore, inhibition of was produced. Despite the good clinical tolerance of glycolysis may become a method to treat tumor and PKD. MET, lactic acidosis remains the primary side effect. AMPK is a key molecule in the regulation of energy Moreover, because MET is mainly cleared by the kidneys, metabolism. mTOR not only regulates proliferation, but chronic renal disease has been considered a potential also plays an important role in regulating energy meta- predisposing factor for this complication . Thus, treat- bolism. AMPK can specifically inhibit mTOR activity .A ment with MET alone has some limitations. decreased AMPK activity and an increased mTOR activity Given the limitations of 2-DG and MET, at present, were found in polycystic kidney cells . many studies use their combination for the treatment of 19,31 2-DG, the glucose analog, acts as a competitive inhibitor tumors . Therefore, in this study, we investigated of the glycolytic enzyme hexokinase . Upon transport whether the combination of 2-DG and MET at small into the cells, 2-DG is phosphorylated to 2DG-phosphate dose of level can more effectively inhibit the prolifera- (2DG-P) by hexokinase. However, unlike glucose-6- tion of polycystic kidney epithelial cells and lower the phosphate (G-6-P), 2DG-P cannot be further metabo- side effects of the two drugs. The results showed that lized by phosphohexose isomerase, which converts G-6-P their combination simultaneously inhibited glycolysis to fructose-6-phosphate . Thus, 2DG-P is trapped and and oxidative phosphorylation metabolism in polycystic accumulates in the cells, leading to inhibition of glycolysis kidney epithelial cells, significantly decreased glucose mainly at the step of the phosphorylation of glucose by intake, lactate production and ATP yield, and sig- hexokinase. Inhibition of this rate-limiting step by 2-DG nificantly decreased the activation levels of proliferation depletes the cellular ATP, leading to blockage of cell cycle signaling pathway molecules, including mTOR, B-Raf- progression and cell death in vitro . ERK and PKA. Moreover, we found that the combina- Our results showed that 2-DG could decrease the gly- tion of low-dose MET and 2-DG significantly upregu- colysis level in polycystic kidney epithelial cells. The cel- lated the PI3K/Akt activation level, led to cell cycle lular intake of glucose, lactate production and arrest at G1/G0 phase and significantly inhibited cell intracellular ATP production were decreased, whereas the proliferation. Inhibition of apoptosis has been shown to phosphorylated AMPKα level was increased by feedback delay renal cyst growth in some animal models of PKD. in 2-DG group. Further, the activation levels of phos- In present study, either MET or 2-DG alone had little phorylated mTOR, p70 S6K and 4E-BP1 were down- effect on the apoptosis of cystic epithelial cells, whereas regulated in 2-DG group. Additionally, 2-DG increased their combination enhanced early apoptosis and the activation level of PI3K-Akt and indirectly inhibited increased caspase-3 activity in the cells. the over-activation of B-Raf-ERK compared with that in In conclusion, this study presents the first evidence that control group. 2-DG also extended the G1/G0 phase of the combinational use of low-dose 2-DG and MET can polycystic kidney epithelial cells. Although 2-DG alone markedly inhibit cell proliferation via simultaneously inhi- can inhibit the proliferation of polycystic kidney epithelial biting glycolysis and oxidative phosphorylation metabolism, cells, a high dose has side effects. Toxicity due to off- reducing intracellular ATP production, activating AMPK target effects has been attributed to this compound in activity, and thereby inhibiting the activation of the mTOR clinical trials, and 3/10 mice in this study were killed early proliferation signaling pathway in polycystic kidney epi- because of weight loss during 2-DG treatment . There- thelial cells. Furthermore, the combination of these two fore, in this study, in order to decrease side effects, we drugs stimulates the activation level of PI3K/Akt, prevents selected small dose of 2-DG to observe its effect on the the over-activation of B-Raf-ERK signaling pathway mole- proliferation of polycystic kidney epithelial cells. cules and, finally, inhibits over-proliferation by arresting the MET, as first-line hypoglycemic drug, has been used for cells at the G0/G1 phase. More importantly, combinational treatment of type 2 diabetes and clinical trial of cancer use of low-dose 2-DG and MET can lower the side effects of treatment . Our results showed that MET reduced each drug. This study provides a new potential strategy for Official journal of the Cell Death Differentiation Association Zhao et al. Cell Death Discovery (2019) 5:76 Page 11 of 13 the future clinical use of these two drugs for the treatment MET (2.5 mM) and 2-DG+MET (5 and 2.5 mM). Finally, of human PKD. the absorbance of HRPTEpiC cells were measured at 48 h of treatment with different drugs using a microplate Materials and methods reader (Bio-Rad, USA). The percentage cell viability was Cell culture studies calculated based on the absorbance of the drug-treated The human autosomal dominant PKD cyst-lining epithelial cells relative to the absorbance of the control cells. Experiments were performed in triplicate, and sextuplet cell line WT9-7 was purchased from American Type Culture Collection (ATCC, USA). Cells were seeded into Dulbecco’s wells were used. modified Eagle’s medium (ATCC, USA) containing 10% fetal bovine serum (FBS; HyClone, Canada) and cultured in an EdU proliferation assay incubator at 37 °C with 5% CO . Cells were digested using Cell proliferation was assayed using Click-iT® EdU Ima- 0.25% trypsin and subcultured at a ratio of 1:6 when 80% ging kits (Invitrogen™ Thermo Fisher Scientific, USA). The confluence was reached. The experimental groups were as EdU provided in the kit is a nucleoside analog of thymidine follows: (1) control group: normal culture; (2) 2-DG group: and is incorporated into DNA during active DNA synthesis. adherent cells treated with different concentrations of 2-DG Detection is based on a click reaction, a copper-catalyzed (MedChem Express, USA); (3) MET group: adherent cells covalent reaction between an azide and an alkyne. Briefly, treated with different concentrations of MET (Sigma, US); WT9-7 polycystic kidney epithelial cells were seeded in 6- and (4) 2-DG+MET group: adherent cells treated with dif- well plates at 10 /well, synchronized in FBS-free medium ferent concentrations of 2-DG and MET simultaneously. The for 12 h, and then treated with 2-DG (5 mM), MET HRPTEpiC were purchased from ScienCell Research (2.5 mM) or 2-DG+MET (5+ 2.5 mM) for 36 h. Thereafter, Laboratories (ScienCell, USA). Cells were seeded into Epi- 1:1000 EdU probe was added to the medium, and the cells thelial Cell medium (ScienCell, USA) containing 2% FBS were cultured for another 12 h and then fixed with 4% (HyClone, Canada) and cultured in an incubator at 37 °C paraformaldehyde. Following the experimental procedure of with 5% CO . Cells were digested using 0.25% trypsin and the Click-iT® EdU Imaging kits, the nuclei were labeled subculturedataratioof1:2 when 90% confluence was with 4’,6-diamidino-2-phenylindole (DAPI, a fluorescent reached. The experimental groups were as follows: (1) con- dye capable of binding strongly to DNA), and images were trol group: normal culture; (2) 2-DG group: adherent cells acquired by confocal laser microscopy. treated with 5 mM concentration of 2-DG; (3) MET group: adherent cells treated with 2.5 mM concentration of MET; Cell cycle analysis and (4) 2-DG+MET group: adherent cells treated with 5 WT9-7 polycystic kidney epithelial cells were seeded in mM 2-DG and 2.5 mM MET simultaneously. 25 cm culture flasks and synchronized in FBS-free medium for 12 h. The cells were incubated for another CCK-8 assay 48 h after the addition of 2-DG (5 mM), MET (2.5 mM) or After treatment with 2-DG, MET, and 2-DG+MET, the 2-DG+MET (5 + 2.5 mM). The cells were washed twice viability of WT9-7 human polycystic kidney epithelial with phosphate-buffered saline (PBS), suspended in 75% cells were measured using a CCK-8 cell proliferation- ethanol and fixed by incubation in 75% ethanol at 4 °C cytotoxicity assay kit (Beyotime, China), and the optimal overnight. Fixed cells were collected by centrifugation, treatment concentration was selected. The polycystic washed with PBS, treated with RNase (50 μg/mL; Sigma, kidney epithelial cell suspension was adjusted to a con- USA) and stained with propidium iodide (PI; 50 μg/mL; centration of 3 × 10 /L and seeded in 96-well plates at Sigma, USA). A FACS flow cytometer (BD Co., USA) was 3000/well. The cells were synchronized in FBS-free used to determine the cellular DNA contents. The per- medium for 12 h and then treated with different con- centage of cells in the G0/G1, S and G2/M phases was centrations of 2-DG (0.6, 2.5, 10 and 40 mM), MET (0.6, determined using Cell FIT Cell Cycle Analysis software 2.5, 10 and 40 mM) and 2-DG+MET (1.3 + 0.6, 2.5 + 1.3, (version 2.01.2; BD). 5 + 2.5 and 10 + 5 mM). Finally, the absorbance of poly- cystic kidney epithelial cell cultures at 450 nm was mea- Caspase-3 activity assay sured at 12, 24, 36 and 48 h of treatment with different WT9-7 polycystic kidney epithelial cells were seeded in drugs using a microplate reader (Bio-Rad, USA). The 25 cm culture flasks and treated with 2-DG (5 mM), viability of HRPTEpiC was measured using a CCK-8 cell MET (2.5 mM) or 2-DG+MET (5 + 2.5 mM) for 12, 24, proliferation-cytotoxicity assay kit The human renal 36 and 48 h. The proteins were collected, and the protein proximal tubular epithelial cells were adjusted to a con- concentration in each group was measured by the centration of 3 × 10 /L and seeded in 96-well plates at bicinchoninic acid (BCA) assay. The protein concentra- 3000/well. The cells were synchronized in FBS-free tion for each group was adjusted to the same level, and medium for 12 h and then treated with 2-DG (5 mM), 50 μL of protein was taken for analysis. Caspase-3 activity Official journal of the Cell Death Differentiation Association Zhao et al. Cell Death Discovery (2019) 5:76 Page 12 of 13 was evaluated using a Caspase-3/CPP32 Colorimetric Lactate Colorimetric/Fluorometric Assay kit (Biovision, Assay kit (Biovision, USA), according to the manu- USA) and CellTiter-Glo® 2.0 Luminescent Cell Viability facturer’s instructions. Finally, the absorbance of each Assay (Promega, USA), respectively, according to the sample at 405 nm was measured using a microplate manufacturer’s instructions. reader. The percentage of caspase-3 activity was calcu- lated as the absorbance of the drug-treated cells relative to Western blot analysis the absorbance of the control vehicle-treated cells. Proteins were extracted from WT9-7 cells using RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Cell apoptosis evaluated using the annexin V-fluorescein deoxycholate, 1% Nonidet P-40, 0.1% SDS, 1 mM phe- isothiocyanate (FITC) assay nylmethylsulfonyl fluoride and protease cocktail at 1 μg/ Cell apoptosis was evaluated using an Apoptosis Detect- mL). The protein concentrations were determined using a ing kit (BD, USA) and analyzed by flow cytometry (FCM). BCA kit (Pierce, USA). Protein samples (50 μg per lane) Briefly, WT9-7 polycystic kidney epithelial cells were seeded were separated by 6%, 8%, 10% or 12% sodium dodecyl in 25 cm culture flasks and treated with 2-DG (5 mM), sufate–polyacrylamide gel electrophoresis (SDS-PAGE) MET (2.5 mM) or 2-DG+MET (5+ 2.5mM) for 48h. electrophoresis and transferred to nitrocellulose (NC) Following the manufacturer’s instructions, the cells were membranes. After Ponceau S staining, the membranes labeled with annexin V-FITC and PI. Samples were exam- were incubated overnight in 5% non-fat milk at 4 °C and ined using FCM, and the results were analyzed using then incubated with primary antibodies against p- CellQuest software (Becton Dickinson, San Jose, CA). AMPKα, p-mTOR, p-4E-BP1, p-p70 S6 kinase, p-PKA, p- PI3 kinase p85, p-Akt, p-B-Raf, p-MEK1/2, p-p44/42 Assay of cell energy metabolism phenotypes MAPK (Erk1/2), PCNA, cyclin E1, Cdk2, Cdk4, (Cell The Seahorse XF Cell Energy Phenotype Test in con- Signaling Technology, USA), cyclin D3 (Wuhan Sanying junction with a Seahorse Bioscience XF96 Extracellular Biotech Co., Ltd., China) and β-actin (Sigma, USA). Flux Analyzer measures the mitochondrial respiration and Immunoreactive bands were visualized using ECL reagent glycolysis under baseline and stressed conditions and (KeyGen Biotech Co., Ltd., China), according to the provides the two key parameters of cell energy metabo- manufacturer’s instructions, and exposure to X-ray film. lism: the baseline phenotype and the stressed phenotype. The protein band intensities were quantified using ImageJ Briefly, WT9-7 cells were plated at 1000 cells/well (~30% software (NIH, Bethesda, USA). confluence) in a Seahorse XF Cell Culture Microplate (Seahorse Bioscience), and adherent cells were treated Statistical analyses with 2-DG (5 mM), MET (2.5 mM) or 2-DG+MET (5 + Student’s T-test was used to calculate statistical sig- 2.5 mM) for 48 h. Immediately prior to an experiment, the nificance between two experimental groups, analysis of growth medium was removed from each well, the cells variance was used to calculate statistical significance were rinsed with freshly prepared Seahorse assay medium between more than two experimental groups, and multi- (Seahorse Bioscience), and a final volume of 180 μLof variate analysis using factorial analysis. Measurement data assay medium was added to each well. The plate was are expressed as the mean ± SEM. Statistical analyses were incubated in a 37 °C incubator lacking CO for 1 h prior to performed using the SPSS 19.0 software package (SPSS, the assay. Mitochondrial and glycolytic bioenergetic pro- Inc., USA). Values of p < 0.05 were considered statistically files were generated by taking baseline measurements of significant. the OCR and ECAR of live cells in real time for three Acknowledgements measurement cycles and then sequentially injecting oli- This work was supported by grants (No. 81770663, No.81570659) from the gomycin (inhibits ATP synthase, 1 μg/mL final con- National Natural Science Foundation of China, a grant (No. 2016YFA0101002) from the National Key Research and Development Program of China and a centration) and FCCP (uncouples mitochondrial oxidative grant (No. 20158332) from the Natural Science Foundation of Hainan Province. phosphorylation and induces maximal respiration, 1 μg/ The English in this document has been checked by at least two professional mL final concentration) to measure the stressed OCR and editors from AJE, both native speakers of English. ECAR for five measurement cycles. Conflict of interest The authors declare that they have no conflict of interest. Measurement of intracellular glucose intake, extracellular lactate production and intracellular ATP synthesis Publisher’s note Polycystic kidney epithelial cells WT9-7 were seeded in Springer Nature remains neutral with regard to jurisdictional claims in 25 cm culture flasks and treated with 2-DG (5 mM), published maps and institutional affiliations. MET (2.5 mM) or 2-DG+MET (5 + 2.5 mM) for 48 h. The concentrations of glucose, lactate and ATP were Received: 10 January 2019 Revised: 15 February 2019 Accepted: 20 determined using a Glucose Assay kit (Biovision, USA), February 2019 Official journal of the Cell Death Differentiation Association Zhao et al. Cell Death Discovery (2019) 5:76 Page 13 of 13 17. Rehman, G., Shehzad, A., Khan, A. L. & Hamayun, M. Role of AMP- activated protein kinase in cancer therapy. Arch. Pharm. (Weinh.) 347, 457–468 (2014). 18. Ben Sahra, I. et al. Targeting cancer cell metabolism: the combination of References metformin and 2-deoxyglucose induces p53-dependent apoptosis in prostate 1. Chapman, A. B. et al. Autosomal-dominant polycystic kidney disease (ADPKD): cancer cells. Cancer Res. 70, 2465–2475 (2010). executive summary from a Kidney Disease: Improving Global Outcomes 19. Xue, C. et al. Targeting P-glycoprotein expression and cancer cell energy (KDIGO) Controversies Conference. Kidney Int. 88,17–27 (2015). metabolism: combination of metformin and 2-deoxyglucose reverses the 2. Rowe, I. et al. Defective glucose metabolism in polycystic kidney disease multidrug resistance of K562/Dox cells to doxorubicin. Tumour Biol. 37, identifies a new therapeutic strategy. Nat. Med. 19,488–493 (2013). 8587–8597 (2016). 3. Riwanto, M. et al. Inhibition of aerobic glycolysis attenuates disease progres- 20. Bikas, A. et al. Glucose-deprivation increases thyroid cancer cells sensitivity to sion in polycystic kidney disease. PLoS ONE 11, e0146654 (2016). metformin. Endocr. Relat. Cancer 22,919–932 (2015). 4. Wallace,D.P.Cyclic AMP-mediated cystexpansion. Biochim. Biophys. Acta 21. Ferreira, F. M.,Watanabe, E. H. &Onuchic,L.F.in Polycystic Kidney Disease (ed. 1812, 1291–1300 (2011). Li, X) Ch. 7 (Codon Publications, Brisbane, 2015). 5. Shillingford, J. M. et al. The mTOR pathway is regulated by polycystin-1, and its 22. Issaq,S.H., Teicher, B. A. &Monks,A. Bioenergeticpropertiesofhuman inhibition reverses renal cystogenesis in polycystic kidney disease. Proc. Natl. sarcoma cells help define sensitivity to metabolic inhibitors. Cell Cycle 13, Acad. Sci. USA 103,5466–5471 (2006). 1152–1161 (2014). 6. Torres, V. E. & Harris, P. C. Strategies targeting cAMP signaling in the treatment 23. Simms, R. J. Autosomal dominant polycystic kidney disease. BMJ 352,i679 of polycystic kidney disease. J. Am. Soc. Nephrol. 25,18–32 (2014). (2016). 7. Yu,A.S.L., El-Ters, M. &Winklhofer, F. T. in Polycystic Kidney Disease (ed. Li, X) 24. Yuajit, C. & Chatsudthipong, V. Nutraceutical for autosomal dominant Ch. 6 (Codon Publications, Brisbane, 2015). polycystic kidney disease therapy. J. Med. Assoc. Thai. 99(Suppl. 1), S97–S103 8. Torres, V. E. et al. Tolvaptan in patients with autosomal dominant polycystic (2016). kidney disease. N. Eng. J. Med. 367, 2407–2418 (2012). 25. Dong, L. X. et al. Negative regulation of mTOR activity by LKB1-AMPK 9. Xiao, H. et al. Separate and concurrent use of 2-deoxy-D-glucose and 3- signaling in non-small cell lung cancer cells. Acta Pharmacol. Sin. 34,314–318 bromopyruvate in pancreatic cancer cells. Oncol. Rep. 29,329–334 (2013). (2013). 10. Yamasaki, T. et al. Exploring a glycolytic inhibitor for the treatment of an FH- 26. Brown, J. Effects of 2-deoxyglucose on carbohydrate metablism: review of the deficient type-2 papillary RCC. Nat. Rev. Urol. 8,165–171 (2011). literature and studies in the rat. Metabolism 11, 1098–1112 (1962). 11. Zhang, D. et al. 2-Deoxy-D-glucose targeting of glucose metabolism in cancer 27. Weindruch, R. et al. Caloric restriction mimetics: metabolic interventions. J. cells as a potential therapy. Cancer Lett. 355,176–183 (2014). Gerontol.Ser.ABiol.Sci.Med.Sci. 1,20–33 (2001). 12. Zhou, G. et al. Role of AMP-activated protein kinase in mechanism of met- 28. Maher, J. C., Krishan, A. & Lampidis, T. J. Greater cell cycle inhibition and formin action. J. Clin. Invest. 108,1167–1174 (2001). cytotoxicity induced by 2-deoxy-D-glucose in tumor cells treated under 13. Shaw, R. J. et al. The kinase LKB1 mediates glucose homeostasis in liver and hypoxic vs aerobic conditions. Cancer Chemother. Pharmacol. 53,116–122 therapeutic effects of metformin. Science 310, 1642–1646 (2005). (2004). 14. Takiar, V. et al. Activating AMP-activated protein kinase (AMPK) slows renal 29. Csibi, A. & Blenis, J. Appetite for destruction: the inhibition of glycolysis as a cystogenesis. Proc. Natl. Acad. Sci. USA 108, 2462–2467 (2011). therapy for tuberous sclerosis complex-related tumors. BMC Biol. 9, 69 (2011). 15. Morales, D. R. & Morris, A. D. Metformin in cancer treatment and prevention. 30. Chae, Y. K. et al. Repurposing metformin for cancer treatment: current clinical Annu. Rev. Med. 66,17–29 (2015). studies. Oncotarget 7,40767–40780 (2016). 16. Cicero, A. F., Tartagni, E. & Ertek, S. Metformin and its clinical use: new insights 31. Cheng, G. et al. Profiling and targeting of cellular bioenergetics: inhibition of for an old drug in clinical practice. Arch.Med.Sci. 8, 907–917 (2012). pancreatic cancer cell proliferation. Br.J.Cancer 111,85–93 (2014). Official journal of the Cell Death Differentiation Association

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