TY - JOUR
AU - Bhatt, Anant, Narayan
AB - Abstract Interleukin-6 (IL-6)-induced glycolysis and therapeutic resistance is reported in some cell systems; however, the mechanism of IL-6-induced glycolysis in radio-resistance is unexplored. Therefore, to investigate, we treated Raw264.7 cells with IL-6 (1 h prior to irradiation) and examined the glycolytic flux. Increased expression of mRNA and protein levels of key glycolytic enzymes was observed after IL-6 treatment, which conferred glycolysis dependent resistance from radiation-induced cell death. We further established that IL-6-induced glycolysis is activated by Akt signalling and knocking down Akt or inhibition of pan Akt phosphorylation significantly abrogated the IL-6-induced radio-resistance. Moreover, reduction of IL-6-induced pAkt level suppressed the expression of Hexokinase-2 and its translocation to the mitochondria, thereby inhibiting the glycolysis-induced resistance to radiation. IL-6-induced glycolysis also minimized the radiation-induced mitochondrial damage. These results suggest that IL-6-induced glycolysis observed in cells may be responsible for IL-6-mediated therapeutic radio-resistance in cancer cells, partly by activation of Akt signalling. Akt signalling, glycolysis, hexokinase-2, IL-6, radio-resistance Introduction Interleukin-6 (IL-6) is a cytokine synthesized and secreted by various types of human body cells like monocytes, adipocytes, fibroblasts, vascular endothelial cells and tumour cells of different types of cancers (1). It was primarily known as pro-inflammatory cytokine for decades, however, many recent evidences demonstrated its role in tissue remodelling and as anti-inflammatory cytokine (1, 2). IL-6 is also known as myokine as it is secreted by muscle cells during exercise and acts on skeletal muscles to promote myogenesis, regulate energy metabolism and protect them from physical exercise-induced ischaemic–reperfusion injury (3). It also protects cardiomyocytes and lung cells from ischaemic–reperfusion injury and oxidative stress-induced cell death (4, 5). Besides this, IL-6 plays an important role during differentiation and tumour progression in majority of cancers. Increased IL-6 secretion in serum and tumour tissues is one of the key factors that promote rapid growth rate and resulted in aggressive phenotypes of cancer (1, 6). An adaptive consequence of chemotherapy and radiotherapy resulted in elevated levels of IL-6 in tumour microenvironment through NF-kB signalling that impose the major limitation to therapies (7–9). The cytoprotective role of IL-6 is beneficial in normal physiological conditions; however, it brings in an additional challenge in therapeutic management of tumours with high IL-6 levels. The cancer cells and also the normal cells during various types of stress primarily depends on glycolysis for energy production even in the presence of oxygen, due to faster rate of ATP synthesis, this phenomenon is known as Warburg effect (10). Moreover, glycolysis over the oxidative phosphorylation is predominant pathway of energy metabolism in all the cells involved in inflammatory response (11). Hence, it is clear that this phenomenon of aerobic glycolysis, which was originally discovered in cancer cells, is not restricted to cancer cells only (12). It has been recently reported that pro-inflammatory cytokine IL-6 increases the availability of substrates such as glucose and lipids by promoting glycogenolysis and lipolysis in skeletal muscles in an autocrine manner and its deficiency suppresses the key genes of glycolysis pathway (6, 13). These studies suggest that aerobic glycolysis is a common phenomenon among many proliferating cells, which provides advantage during proliferation in terms of faster ATP production to meet the high demand of rapidly dividing cells and cells recovering from injury. It has been demonstrated earlier that IL-6 can induce glycolysis in various types of cell-like mouse embryonic fibroblasts, human skeletal muscles, etc. (14, 15). However, the molecular mechanism of IL-6-induced glycolysis is poorly understood. Recent studies have demonstrated that the IL-6-mediated activation of cellular antioxidant pathway is implicated in the radio-resistance of cancer cells (16–18). Accumulating evidences indicate that induced glycolysis confers radio-resistance in various cancers (6, 7, 19, 20). Although, various mechanisms of IL-6-induced radio-resistance is known but the role of IL-6-induced glycolysis in radio-resistance remains unclear. In the present study, we, therefore, investigated the mechanism of IL-6-induced glycolysis in Raw 264.7 cells and tested the hypothesis, if IL-6 can protect normal cells from ionizing radiation (IR) by inducing glycolysis-mediated radio-resistance. In this study, we have shown that IL-6 treatment induces glycolysis in relatively radio-sensitive Raw 264.7 (murine monocytic) cells (21, 22), which protects it from IR-induced cell death. Results obtained clearly showed that IL-6-induced Akt signalling plays an important role in stimulating glycolysis and protect the cells by conferring glycolysis-mediated radio-resistance. Materials and methods Materials High glucose Dulbecco’s Minimum Essential Medium (HGD), Penicillin G, streptomycin, nystatin, dimethyl sulfoxide (DMSO), Sulforhodamine B (SRB) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemicals Co. (St Louis, MO, USA), whereas mouse IL-6 was purchased from Merck Millipore (Burlington, VT, USA). MK2206 (Akt inhibitor) and Akt1/2 siRNA was purchased from selleckchem (Houston, TX, USA) and Santacruz Biotechnology (Dallas, TX, USA), respectively. Sources of cell line The mouse normal monocyte macrophage (Raw 264.7) was obtained from NCCS, Pune, India and cultured in their respective media containing 10% heat inactivated foetal bovine serum and antibiotics. Stock culture was maintained in the exponential growth phase by passaging them every 3 days with their respective growth medium supplemented with 10% foetal bovine serum and antibiotics in 60 mm tissue culture petri dish (BD Falcon, USA). Radiation treatment All the experiments were carried out using 96, 24 and 6 well plates, 35 and 60 mm tissue culture dishes. Exponentially growing cells were treated with 1 ng/ml IL-6 followed by γ-radiation (2 Gy) and kept for overnight incubation at 37°C in 5% CO2 incubator. All experiments were carried out at a single dose of gamma radiation (2 Gy), cells were irradiated using 60Cobalt-Teletherapy Unit (Bhabhatron-II, Panacea Medical Technologies, Bangalore, India) at a source to sample distance of 80 cm and a field size of 35 × 35 cm2 with dose rate of 1.05 Gy/min. The treatment schedule and concentration of other drug/inhibitors are mention in respective figure legends. Glucose uptake and lactate production assay Raw264.7 cells were incubated in HGD before IL-6 treatment and irradiation. Cells were treated with IL-6, 1 h prior to irradiation. Subsequently cell medium was removed and cells were incubated with 2NBDG (50 μM, 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose) prepared in phosphate-buffered saline (PBS) for 30 min (Fig. 1A). Further the cells were harvested and washed twice with cold PBS to analyse on flow cytometer. Lactate production was estimated in the growth medium using enzymatic assays (Fig. 1B). Lactate was estimated using lactate oxidase method using kit (Randox; Cat. No.-LC2389). Glucose uptake and lactate production were normalized with number of viable cells in respected wells. Fig. 1 Open in new tabDownload slide IL-6 induced rate of glycolysis was measured by observing glucose uptake and lactate production in cells after IL-6 treatment. Glucose uptake (A) and lactate production (B) was measured in culture media, 2 h after IL-6 treatment. (C) Protein levels of various glycolytic regulatory enzymes (indicated in figure) were measured in untreated and IL-6 (1 ng/ml) treated cells at given time points. β-Actin was used as loading control. Values shown in between the blots are the average fold change value of densitometric analysis of three blots, normalized with respective β-Actin. (D) The mRNA levels of HK-2, PKM2 and PFKFB3 genes at indicated time points after IL-6 (1 ng/ml) treatment. Statistical significance calculated by one-way ANOVA and the Student’s t-test between the groups. Data are expressed as mean ± SD (n = 4). *P < 0.05. Fig. 1 Open in new tabDownload slide IL-6 induced rate of glycolysis was measured by observing glucose uptake and lactate production in cells after IL-6 treatment. Glucose uptake (A) and lactate production (B) was measured in culture media, 2 h after IL-6 treatment. (C) Protein levels of various glycolytic regulatory enzymes (indicated in figure) were measured in untreated and IL-6 (1 ng/ml) treated cells at given time points. β-Actin was used as loading control. Values shown in between the blots are the average fold change value of densitometric analysis of three blots, normalized with respective β-Actin. (D) The mRNA levels of HK-2, PKM2 and PFKFB3 genes at indicated time points after IL-6 (1 ng/ml) treatment. Statistical significance calculated by one-way ANOVA and the Student’s t-test between the groups. Data are expressed as mean ± SD (n = 4). *P < 0.05. Immunoblot for protein levels The protein level of hexokinase 2, phosphofructokinase 1 (PFK-1), PKM2, GLUT4, phospho Akt, Akt and loading control β-Actin were determined in control and irradiated cells (Raw264.7) by immunoblot analysis (Fig. 1C and 4A). Cells were cultured in PD60 incubated in CO2 incubator before treatment. Further cells were harvested post-irradiation at various time points and lysed in ice-cold RIPA lysis buffer (Tris-HCl: 50 mM, pH 7.4, NP-40: 1%, NaCl: 150 mM, EDTA: 1 mM, PMSF: 2 mM, protease inhibitor cocktail, Na3VO4: 1 mM, NaF: 1 mM) containing protease inhibitors. The protein concentration in cell lysates was determined using BCA protein assay kit. Protein (40 μg) was resolved on 10–12% SDS–PAGE (depending on the molecular weight) and electroblotted onto PVDF membrane (MDI). The membrane was then incubated in 4% body surface area for 2 h followed by primary antibody incubation HK-2 (1:1000), PKM2 (1:500), PFK-1 (1:500), GLUT4 (1:500) and β-Actin (1:3000) from Santa Cruz Biotechnology and pAkt (1:1000), Akt (1:1000) and SDH (1:1000) from Cell Signalling Technology. Membrane was washed followed by incubation with the appropriate HRP conjugated secondary antibody (1:5000, Santa Cruz Biotechnology) for 2 h. After washing, the blots were developed using Luminata Forte western HRP substarte (Millipore) The signal was captured by Chemidoc system (Bio-Rad, CA, USA) and band intensities for each individual protein were quantified by densitometry, corrected for background staining, and normalized to the signal for β-Actin. Quantitative PCR for mRNA levels Total RNA was isolated from cells by Qiagen RNA isolation kit according to the manufacturer’s instructions (Qiagen RNeasy mini kit). Further RNA was dissolved in nuclease-free water (Thermo Scientific, USA) and quantified with Nanodrop (Thermo Scientific, USA). About 1 μg of RNA was used for cDNA synthesis via First-strand cDNA synthesis kit (Thermo Scientific, USA) in thermal cycler (Applied Biosystems, CA, USA). Kick start ready to use primers were purchased from Sigma Aldrich (St Louis, MO, USA). For real-time PCR, 25 ng of cDNA was added to 100 nM gene-specific primers and 1× Sybr Green supermix (Bio-Rad, CA, USA). The amplification programme consisted of a hot start at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 15 s. The amount of target gene was normalized by β-Actin (Fig. 1D). Growth kinetics/cell number Cells were seeded in PD35. After IL-6 and radiation treatment cells were kept at 37°C in 5% CO2 incubator for doublings (Fig. 2A). At respective time points cell numbers were counted with a Neubauer-improved counting chamber (Paul Marienfeld GmbH & Co. KG, Germany) under 10 X objective, and 10× eyepiece magnification with compound light microscope (Olympus CH30, Japan). Fig. 2 Open in new tabDownload slide IL-6 confers radio-resistance (A) SRB assay carried out at 48 h post-irradiation using different concentrations of IL-6 (0.1–10 ng/ml). Graph (ΔOD 340 nm) plotted and treatment groups were compared with their respective control. (B) The cell number in control and treatment group was quantified at 0–48 h post-irradiation and graph presented on log scale against time at single concentration of IL-6 (1 ng/ml). Inset graph presents the cell number at 48 h on two different concentrations of IL-6. (C) Dose-response curve of Raw264.7 cells with or without IL-6 treatment. Surviving fraction was plotted against increasing radiation doses (0–8 Gy). Star shows the statistical significance of change between the groups calculated by one-way ANOVA and the Student’s t-test. Data are expressed as mean ± SD (n = 4). *P < 0.05. Fig. 2 Open in new tabDownload slide IL-6 confers radio-resistance (A) SRB assay carried out at 48 h post-irradiation using different concentrations of IL-6 (0.1–10 ng/ml). Graph (ΔOD 340 nm) plotted and treatment groups were compared with their respective control. (B) The cell number in control and treatment group was quantified at 0–48 h post-irradiation and graph presented on log scale against time at single concentration of IL-6 (1 ng/ml). Inset graph presents the cell number at 48 h on two different concentrations of IL-6. (C) Dose-response curve of Raw264.7 cells with or without IL-6 treatment. Surviving fraction was plotted against increasing radiation doses (0–8 Gy). Star shows the statistical significance of change between the groups calculated by one-way ANOVA and the Student’s t-test. Data are expressed as mean ± SD (n = 4). *P < 0.05. Clonogenic cell survival assay Macro-colony assay was performed using pre-plating method of macro-colony assay. Cells were plated at a low density of 100–3200 cells in triplicates in 60 mm Petri dishes. After IL-6 and radiation treatment (0–8 Gy), cells were incubated at 37°C in a humidified CO2 (5%) incubator for 7–10 days. Colonies of at least 50 cells (5–6 generations of proliferation) were scored as survivors. Colonies were washed once with PBS to remove media, fixed with methanol and then stained with 1% crystal violet (dissolved in 7% methanol in PBS). Plating efficiency was calculated as PE = (Number of colonies counted/Number of cells plated) × 100. The surviving fraction (SF) was calculated as SF = PET/PEC, where PET is the plating efficiency of the treated group and PEC is the value of the control. ATP measurement ATP was measured using ATP bioluminescent assay kit (Sigma Aldrich, St Louis, MO, USA) following manufacturer’s protocol. Briefly, cells were treated with IL-6 followed by irradiation at 1 h. At 4 and 24 h, post-irradiation cells were washed and scraped in cold PBS and pelleted at 1000 rpm for 10 min. Cells were lysed in 350 μl of lysis buffer (4 mM EDTA and 0.2% Triton X-100). About 100 μl of this lysate was loaded per well in triplicates with 100 μl of ATP mix in a 96-well white luminescence measuring plate. Luminescence of samples along with standards was read at 562 nm and normalized with the cell number (Fig. 3D). ATP concentration is depicted as pg/cell. Fig. 3 Open in new tabDownload slide IL-6-induced radio-resistance is glycolysis dependent. (A) Glucose uptake and lactate production measured 2 h post-irradiation, as described (B) Immuno-blotting of key regulatory glycolytic enzymes was performed at 24 h post-irradiation. β-Actin was used as loading control. Values shown in between the blots are the average fold change value of densitometric analysis of three blots, normalized with respective β-Actin. (C) The mRNA levels of HK-2, PKM2 and PFKFB3 with respect to control were observed after 12 h post-irradiation (D) ATP levels in cells was measured by bioluminescence ATP assay kit at indicated time points and represented as ATP concentration per cell calculated from standard. (E and F) Cell number was quantified at 48 h post-irradiation in various treatment groups and plotted as bar graph with cell number on Y-axis. (G and H) Cells were stained with AO/EtBr 24 h post-irradiation and examined under a fluorescent microscope at 10× magnification. Zoomed images of dying cells are shown in inset for improved view. Dead cells (EtBr stained and marked with arrow) were counted from multiple images and the mean of dead cells per group was plotted as bar graph. Star shows the statistical significance of change between the groups calculated by the Student’s t-test. Data are expressed as mean ± SD (n = 4). *P < 0.05. Fig. 3 Open in new tabDownload slide IL-6-induced radio-resistance is glycolysis dependent. (A) Glucose uptake and lactate production measured 2 h post-irradiation, as described (B) Immuno-blotting of key regulatory glycolytic enzymes was performed at 24 h post-irradiation. β-Actin was used as loading control. Values shown in between the blots are the average fold change value of densitometric analysis of three blots, normalized with respective β-Actin. (C) The mRNA levels of HK-2, PKM2 and PFKFB3 with respect to control were observed after 12 h post-irradiation (D) ATP levels in cells was measured by bioluminescence ATP assay kit at indicated time points and represented as ATP concentration per cell calculated from standard. (E and F) Cell number was quantified at 48 h post-irradiation in various treatment groups and plotted as bar graph with cell number on Y-axis. (G and H) Cells were stained with AO/EtBr 24 h post-irradiation and examined under a fluorescent microscope at 10× magnification. Zoomed images of dying cells are shown in inset for improved view. Dead cells (EtBr stained and marked with arrow) were counted from multiple images and the mean of dead cells per group was plotted as bar graph. Star shows the statistical significance of change between the groups calculated by the Student’s t-test. Data are expressed as mean ± SD (n = 4). *P < 0.05. Acridine orange-ethidium bromide staining Raw 264.7 cells were seeded in 96 well and stained with acridine orange-ethidium bromide according to Deborah Ribble protocol (23) with minimal modifications (Fig. 3G). Images were captured under fluorescence microscope using 10× objective, and 10× eyepiece magnification with fluorescence microscope (Olympus IX51 Fluorescence Microscope, Japan). Formazan quantification The cells were plated in 24 well culture plates (40,000 cells/well) and incubated in CO2 incubator. Next day, treatment was given according to the experimental requirement. Further, at respective time points, 50 μl MTT solutions from the stock (5 mg/ml) was added and cells were incubated in CO2 incubator in the dark for 2 h. The medium was removed and formazan crystals formed by the cells were dissolved using 500 μl of DMSO followed by transfer in 96-well plate. The absorbance was read at 570 nm using 630 nm as reference wavelength on a Multiwell plate reader (Biotech Instruments, USA). Reduced formazan quantification was done with formazan standard. At each respective time points, cell numbers were counted with a Neubauer-improved counting chamber (Paul Marienfeld GmbH & Co. KG, Germany) under 10× objective, and 10× eyepiece magnification with compound light microscope (Olympus CH30, Japan). Measurement of mitochondrial mass and mitochondrial calcium Quantitative analysis of mitochondrial content was carried out using Mitotracker Green (at respective time points, post-irradiation), Cells were incubated with mitotracker green (100 nM; 15 min; 37°C), in PBS, then washed with PBS and resuspended in PBS before analysis. The signals were recorded using BD FACSAria™ III cell sorter. (BD Biosciences, USA). Images of calcium-loaded mitochondria were captured by staining cells with A23187 (6 μM, 20 min). Briefly, Raw264.7 cells were grown in PD-35 having cover slip. At 4 h post-irradiation medium was removed and stained for 20 min in dark. Stain was removed and cells washed with cold PBS. Images were captured under fluorescence microscope with 40× objective. Mitochondrial membrane potential Quantitative and qualitative analysis of mitochondrial membrane potential (MMP) was carried out using TMRM and JC-1 dyes respectively, Cells were incubated with TMRM (50 nM; 30 min; 37°C) in PBS, then washed and resuspended in PBS for analysis. Fluorescence signals were measured by flow cytometer (BD FACSAria™ III cell sorter). For microscopy, cells were stained with JC-1 dye (10 μg/ml; 30 min; 37°C). After staining, cells were washed with PBS and observed at 40× magnification under fluorescence microscope (Olympus IX51 Fluorescence Microscope, Japan). JC-1 accumulates in mitochondria as monomer or J-aggregates depending on the membrane potential. The monomeric form is predominately present in depolarized mitochondria and emits green fluorescence (∼530 nm), whereas the oligomeric (J-aggregate) form in mitochondria with more potentials and emits red fluorescence (∼590 nm). siRNA transfection The control siRNA and Mouse Akt1/2 siRNA pool were purchased from Santacruz Biotechnology (Dallas, TX, USA) to knock down gene expression. siRNA transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions, briefly, 0.1 ×106 cells were seeded in 6-well plate a day before transfection. Next day, transfection was done in Opti-MEM (serum and antibiotic-free medium) for 4 h followed by 24 h recovery in 2× serum-containing medium. Pilot experiments were performed to optimize the amount and time of maximal protein knockdown. Statistical analysis All the experiments were carried out in triplicates or quadruplicates. Means and standard errors were computed. The Student’s t-test and one-way analysis of variance (ANOVA) test were performed for comparisons between two groups and multiple groups, respectively. P-values of <0.05 were considered statistically significant. Results IL-6 induces glycolysis We first tested if IL-6 can induce glycolysis in Raw 264.7 cells. Thus, we measured glucose uptake using non-metabolizing, fluorescent glucose analogue 2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose) in Raw264.7 cells. IL-6 treatment showed significant increase in glucose consumption at both 1 and 10 ng/ml IL-6 with highest 1.5-fold consumption at 1 ng/ml (Fig. 1A). Therefore, we used 1 ng/ml concentration of IL-6 for all the further experiments. Consequently, lactate production, which is the end product of glycolysis was also increased by 1.65-fold after IL-6 treatment (Fig. 1B). Further to understand if the high rate of glycolysis is achieved by IL-6-induced enhanced levels of glycolytic enzymes, we checked the levels of key glycolytic enzymes HK-1, HK-2, PFK-1 and PKM2 after IL-6 treatment. We found nearly 1.3–2-fold time-dependent increase in the protein levels of HK-2, PFK-1, and PKM2, while the protein level of HK-1 was found unchanged (Fig. 1C). Enhanced levels of HK-2 and PKM2 protein was further supported by the time-dependent increase in HK-2 and PKM2 mRNA levels (Fig. 1D). We also checked the IL-6-induced gene expression of PFKFB3 gene, which produces Glucose 2,6 bis phosphate for allosteric activation of PFK-1 enzyme (24) We found, IL-6 induced the mRNA levels of PFKFB3 gene also, suggesting it regulates the flow of glucose through glycolysis (Fig. 1D). These findings suggest that IL-6 induces glycolysis by inducing the expression of glycolytic regulatory enzymes in Raw264.7 cells. IL-6 protects Raw264.7 cells from radiation-induced cell death Our previous studies suggest that transient elevation of glycolysis confers radio-resistance (20). Therefore, in order to check the correlation between IL-6-induced glycolysis and radio-resistance, we further carried out SRB assay and growth kinetics in Raw264.7 cells to test the effects of IL-6 on radiation-induced cell death. SRB assay was performed at same concentration range of IL-6 0.1 to 10 ng/ml as used in glucose uptake and we found significant protection of 45–50% at 1 and 10 ng/ml (Fig. 2A). Growth kinetic assay was also performed under similar experimental conditions and nearly 45% reduction in radiation-induced cell death was observed in IL-6 treated cells as compared to irradiated cells (Fig. 2B). The marginal increase in protection, observed at 10 ng/ml with respect to 1 ng/ml IL-6 in SRB assay (Fig. 2A) and marginally low cell count at 10 ng/ml (Fig. 2B inset) was statistically insignificant; Since significant and nearly equal increase in glucose consumption and radioprotection were observed at both 1 and 10 ng/ml doses of IL-6, therefore, we used 1 ng/ml concentration for all further experiments. Further, to validate IL-6 treatment-induced radio-resistance; we irradiated the IL-6 pre-treated cells and then analysed the clonogenicity using macro-colony assay. Indeed, a significant increase in survival was evident in IL-6 pre-treated cells as compared to radiation alone on all radiation doses between 0 and 8 Gy (Fig. 2C). Together, these findings suggest that IL-6-induced glycolysis confers radio-resistance in monocytic Raw264.7 cells. IL-6-induced radio-resistance is glycolysis dependent Further, to validate the correlation between IL-6-induced glycolysis and radio-resistance; we analysed the levels of glucose uptake and lactate production after radiation exposure in IL-6 pre-treated samples. Radiation alone group also showed increase in glucose uptake, however, it was significantly less (∼1.5 fold) as compared to IL-6 alone and combined (IL-6 pre-treatment followed by irradiation) treatment groups (∼2 fold) (Fig. 3A). Similarly, the lactate production was also found significantly higher in IL-6 and radiation combined treatment as compared to radiation-exposed cells (Fig. 3A). This result is substantiated by several fold increased protein levels of glycolytic enzymes (HK-2, PFK-1 and PKM2), glucose transporter GLUT4 (Fig. 3B) and mRNA expression of HK-2, PKM2 and PFKFB3 gene (Fig. 3C) in cells co-treated with IL-6 and radiation as compared to radiation alone. We also found about 1.9-fold increased ATP level at 4 h post-irradiation in IL-6 pre-treated cells with respect to control and radiation alone (Fig. 3D). We further inhibited the glycolysis using non-toxic concentrations of 2-dexoy-D-Glucose (2-DG) and 3-bromo pyruvate (3-BP) in IL-6 pre-treated cells before exposing to radiation and performed growth kinetics. Both the glycolytic inhibitors 2-DG and 3-BP reversed the IL-6-induced protection from radiation-induced cell death (Fig. 3E and F). This observation from growth kinetics assay was correlated with qualitative imaging of ethidium bromide and acridine orange (apoptosis assay) at 24 h. Radiation alone and in combination with glycolytic inhibitors (2-DG and 3-BP) in IL-6 pre-treated group showed nearly similar number of apoptotic and necrotic cell population, which was significantly higher as compared to the IL-6 pre-treated and radiation-exposed treatment group (Fig. 3G and H). Therefore, these findings evidently validate our earlier observation that induced glycolysis confers radio-resistance. It also suggests that IL-6-induced radio-resistance in Raw264.7 cells is glycolysis dependent. IL-6 protects from radiation-induced mitochondrial damage Ionizing radiation is known to damage mitochondria and affect its energy metabolism (25, 26). Therefore, we tested if IL-6 protects from radiation-induced mitochondrial damage also. To test the mitochondrial energy metabolism, we analysed complex II activity of mitochondrial respiratory chain by monitoring formazan formation. We found, radiation induces nearly 2.5-fold increased formazan formation in IL-6 untreated and treated cells at early time point (4 h), which comes down to normal at later time point (24 h, Fig. 4A). Mitochondrial enzymatic activity depends on the mitochondrial mass in cells, and we have shown in our earlier study that radiation-induced mitochondrial damage can induce mitochondrial biogenesis, thereby increases the mitochondrial mass and formazan formation in cells (25). Interestingly, we noted nearly similar formazan formation in IL-6 untreated and treated cells after radiation exposure from 1.8- to 1.25-fold increased mitochondrial mass, respectively (Fig. 4B). This observation shows that radiation-exposed cells have 80% more mitochondrial mass but all may not be contributing to enhanced complex II activity. However, similar formazan formation from only 25% increased mitochondrial mass and nearly similar SDH level (Fig. 4C and D) after radiation exposures in IL-6 pre-treated cells as compared to radiation alone exposed cells suggest reduced mitochondrial damage and higher efficiency of mitochondrial respiration in IL-6 pre-treated cells. Subsequently, we also analysed the MMP under similar experimental conditions to analyse the mitochondrial status in radiation-exposed cells using fluorescent potentiometric dyes JC-1 and TMRM (Fig. 4E and F). We observed radiation induced increased MMP in IL-6 and radiation treated cells (Fig. 4E), however, radiation-induced hyperpolarized mitochondria and increased mitochondrial mass (Fig. 4B) can also show higher dyes uptake in cells giving false information of higher MMP (25). Therefore, we normalized the MMP values with mitochondrial mass of the respective sample to obtain the accurate MMP of the cells. When radiation induced increased MMP was normalized with enhanced mitochondrial mass, it showed significant 30% decrease in MMP, which was restored in IL-6 pre-treated and radiation-exposed samples both at 4 and 24 h (Fig. 4F). This was further validated by microscopic observation of radiation induced damaged mitochondria using A23187 dye (26). We found highly reduced numbers of A23187 puncta positive cells and much smaller intracellular bodies (showing damaged mitochondria) in IL-6 pre-treated cells as compared to radiation alone (Fig. 4G and H). Interestingly, glycolysis inhibition using 2-DG in IL-6 pre-treated cells reverses the protective effects of IL-6 from radiation-induced mitochondrial damage (Fig. 4G and H). These observations suggest that IL-6 protects from radiation-induced mitochondrial damage and this protective effect is also linked with IL-6-induced glycolysis. Fig. 4 Open in new tabDownload slide Open in new tabDownload slide IL-6 prevents mitochondrial damage from radiation. (A) The formazan formed per cell was quantified spectro-photometrically and presented as bar graph at indicated time points. (B) The mitochondrial mass was analysed by staining cells with MitoTracker Green FM (100 nM; 20 min) at indicated time points. Graph showing mean fluorescence intensity, presented as fold change with respect to control. (C and D) Immunoblot showing protein expression of mitochondrial complex-II subunit SDH-A presented in Raw264.7 cells. The bar graph represents the fold increase in SDH levels quantified by densitometry and normalized with β-Actin. (E) Microscopic evaluation of mitochondrial membrane potential shown by distribution of JC-1-loaded mitochondria in response to radiation, the retention of monomer and aggregates (marked with arrow) represents the shift in mitochondrial membrane potential. Images were captured at 40× (objective) and 10× (eyepiece) magnification. (F) Quantitative estimation of MMP by TMRM and normalized with respective mitochondrial content (data obtained from mitotracker green, Fig. 4B) at 4 and 24 h post-irradiation. (G) Photomicrograph shows the radiation induced changes (marked with arrow) in mitochondrial calcium by staining the cells with A23187 (6 μM, 30 min). Images were captured under fluorescence microscope with 40× objective. (H) Graph representing quantification of I-bodies formation per cell in different treatment groups. Data were obtained from n = 10 fields per group. Star shows the statistical significance of change between the groups calculated by the Student’s t-test. Data are expressed as mean ± SD (n = 4). *P < 0.05, # represents the significance with respect to the MMP/mitochondrial mass of control group. Fig. 4 Open in new tabDownload slide Open in new tabDownload slide IL-6 prevents mitochondrial damage from radiation. (A) The formazan formed per cell was quantified spectro-photometrically and presented as bar graph at indicated time points. (B) The mitochondrial mass was analysed by staining cells with MitoTracker Green FM (100 nM; 20 min) at indicated time points. Graph showing mean fluorescence intensity, presented as fold change with respect to control. (C and D) Immunoblot showing protein expression of mitochondrial complex-II subunit SDH-A presented in Raw264.7 cells. The bar graph represents the fold increase in SDH levels quantified by densitometry and normalized with β-Actin. (E) Microscopic evaluation of mitochondrial membrane potential shown by distribution of JC-1-loaded mitochondria in response to radiation, the retention of monomer and aggregates (marked with arrow) represents the shift in mitochondrial membrane potential. Images were captured at 40× (objective) and 10× (eyepiece) magnification. (F) Quantitative estimation of MMP by TMRM and normalized with respective mitochondrial content (data obtained from mitotracker green, Fig. 4B) at 4 and 24 h post-irradiation. (G) Photomicrograph shows the radiation induced changes (marked with arrow) in mitochondrial calcium by staining the cells with A23187 (6 μM, 30 min). Images were captured under fluorescence microscope with 40× objective. (H) Graph representing quantification of I-bodies formation per cell in different treatment groups. Data were obtained from n = 10 fields per group. Star shows the statistical significance of change between the groups calculated by the Student’s t-test. Data are expressed as mean ± SD (n = 4). *P < 0.05, # represents the significance with respect to the MMP/mitochondrial mass of control group. IL-6-induced Akt signalling promotes glycolysis and confers radio-resistance Enhanced glycolytic metabolism is known to be regulated by two main signalling pathways in normal cells namely, HIF1α and Akt. IL-6 activates both HIF1α and Akt signalling pathway (1, 27). Whereas, we found that IL-6 at 1 ng/ml concentration does not induce detectable levels HIF1α in Raw264.7 cells. Therefore, we tested Akt pathway by estimating the time-dependent protein levels of pAkt (active form) in IL-6 treated cells. We found more than 2-fold increase in pAkt levels as early as 1 h of IL-6 treatment (Fig. 5A). Further, to authenticate if IL-6-induced radio-resistance is dependent on Akt signalling and enhanced glycolysis mediated by it; we inhibited Akt signalling using pan Akt inhibitor MK2206 (28) and also knock down the Akt expression (53% depletion in Akt1/2 level) using Akt1/2 siRNA. Down-regulation of Akt signalling not only reduced the IL-6-induced pAkt levels to un-induced basal level (Fig. 5B) but also significantly brought down the level of IL-6-induced glucose uptake, and lactate production to the basal level (Fig. 5C). Further, low pAkt level reduced the IL-6 induced total and mitochondrial bound fraction of HK-2 (Fig. 5D and F) and reversed the IL-6-induced radio-resistance, indicated by significant reduction in cell number of IL-6 pre-treatment combined with Akt1/2 siRNA and MK2206 in irradiated cells nearly to the level of radiation control (Fig. 5E). These results suggest that IL-6-induced Akt signalling up-regulate the levels of glycolytic enzymes, which leads to enhanced glycolysis and radio-resistance in Raw264.7 cells. Fig. 5 Open in new tabDownload slide IL-6-induced glycolysis is Akt dependent. (A) Phosphorylation of Akt at ser473 was detected at indicated time points after IL-6 treatment. (B) Cells treated with MK2206 (2.5 μM) 15 min prior to IL-6 treatment followed by irradiation were harvested 1 h post-irradiation for western blotting. Akt1/2 siRNA (100 nM) and Control (scramble) siRNA transfected cells were also treated with IL-6 followed by irradiation and harvested for western blotting of pAkt and Akt levels. Total Akt levels were normalized with the values of beta-actin (loading control). (C) Graph represents fold change in glucose uptake and lactate production per cell respectively in various treatment groups at 2 h post-irradiation. (D) Immunoblot of total HK-2 protein with MK2206 and Akt siRNA. β-Actin used as loading control and (E) Cell number quantified at 48 h post-irradiation in various treatment groups is presented as bar diagram. (F) Showing immunoblot of mitochondrial bound fraction of HK-2 where VDAC used as loading control. Values shown in between the blots are the average fold change value of densitometric analysis of three blots, normalized with respective β-Actin. Star shows the statistical significance of change between the groups calculated by one-way ANOVA and the Student’s t-test. # represents the statistical significance between siRNA or MK2206 group with respect to IL-6 pre-treated irradiated group. Data are expressed as mean ± SD (n = 4). *P < 0.05. Fig. 5 Open in new tabDownload slide IL-6-induced glycolysis is Akt dependent. (A) Phosphorylation of Akt at ser473 was detected at indicated time points after IL-6 treatment. (B) Cells treated with MK2206 (2.5 μM) 15 min prior to IL-6 treatment followed by irradiation were harvested 1 h post-irradiation for western blotting. Akt1/2 siRNA (100 nM) and Control (scramble) siRNA transfected cells were also treated with IL-6 followed by irradiation and harvested for western blotting of pAkt and Akt levels. Total Akt levels were normalized with the values of beta-actin (loading control). (C) Graph represents fold change in glucose uptake and lactate production per cell respectively in various treatment groups at 2 h post-irradiation. (D) Immunoblot of total HK-2 protein with MK2206 and Akt siRNA. β-Actin used as loading control and (E) Cell number quantified at 48 h post-irradiation in various treatment groups is presented as bar diagram. (F) Showing immunoblot of mitochondrial bound fraction of HK-2 where VDAC used as loading control. Values shown in between the blots are the average fold change value of densitometric analysis of three blots, normalized with respective β-Actin. Star shows the statistical significance of change between the groups calculated by one-way ANOVA and the Student’s t-test. # represents the statistical significance between siRNA or MK2206 group with respect to IL-6 pre-treated irradiated group. Data are expressed as mean ± SD (n = 4). *P < 0.05. Discussion IL-6, which is known to be a cytoprotective cytokine in normal physiological conditions, also protects tumour cells from radiotherapy and chemotherapeutic agents posing major limitation in therapeutic gain in cancer treatment (1). IL-6 protects the cells from therapeutic stress-induced cell death by inducing various pro-survival signalling namely inhibition of apoptosis, induce survival and proliferation (1, 6, 14). Therefore, IL-6-induced cellular defense to therapeutic stress causes therapeutic resistance (1, 6, 16–19). It is known that IL-6 can induce aerobic glycolysis in cells (12, 15), and we have demonstrated earlier that induced glycolysis caused radio-resistance in cells (20). IL-6-mediated radio-resistance in cells has been attributed to IL-6-induced anti-oxidant defense system and STAT-3-mediated pro-survival signalling (14, 18); however, the role of IL-6-induced energy metabolism mainly glycolysis in radio-resistance is not known. Therefore, we tested the hypothesis, if IL-6-induced glycolysis plays any role in cellular radio-resistance, which may be diminishing the therapeutic gain in cancer treatment. IL-6-induced radio-resistance can also be exploited in protecting the normal tissues from radiation hazards. We selected murine monocytic cell line Raw264.7 to test the IL-6-induced radio-resistance because haematopoietic cells are relatively more sensitive to radiation (21, 22). The data presented above demonstrate the potential of IL-6 to induce glycolysis in haematopoietic Raw264.7 cells. It induced the glycolysis by increasing the levels of many regulatory glycolytic enzymes viz. HK-2, PFK-1 and PKM2 (Fig. 1C and D). Enhanced expression of HK-2 and its association with mitochondria ensures rapid phosphorylation of glucose using mitochondrial ATP (29). The PFK-1 is the first critical and irreversible step of glycolysis which diverts the glucose through glycolysis (catabolic pathway) and ensures that glucose should not enter to pentose phosphate pathway or gluconeogenesis, both anabolic pathways. At the time of stress or cellular injury, ATP generated from catabolic pathways is required for macromolecular repair and cell survival, therefore, diverting glucose towards glycolysis for more ATP production is vital and decisive for cell survival (24). We found higher ATP levels in IL-6 treated cells (Fig. 3D), which acts as inhibitor of PFK-1, however, increased expression of PFKFB3 (Fig. 1D) produces fructose 2,6bisphosphate, which acts as allosteric activator of PFK-1 and ensures the continuous activation of PFK-1, even in the presence of high ATP (15, 24). Further, IL-6 induced higher protein levels of PKM2 (Fig. 1C and D) maintains the smooth running of glucose through glycolysis by reducing the level of its substrate phosphoenolpyruvate, which can inhibit the PFK-1 and stop the flow of glucose towards glycolysis. These results suggest that IL-6 induces the glycolysis by elevating the levels of all the crucial regulatory enzymes of this pathway. Interestingly, IL-6 pre-treatment to induce glycolysis could protect the radio-sensitive Raw264.7 cells from radiation-induced cell death (Fig. 2). The protein levels of glucose transporter and glycolytic enzymes, which was decreased after radiation exposure was found high in IL-6 alone and combined treatment group, suggesting that IL-6 induced the glycolysis in radiation-exposed cells also. Radiation is also known to induce the glycolysis (30), which can be observed by enhanced glucose uptake, lactate production and higher levels of glycolytic enzymes, HK-2, PFK-1 and PKM2 in radiation-exposed sample as compared to control (Fig. 3A–C). However, these levels were found further increased in IL-6 pre-treated and radiation-exposed (combined treatment) sample. These findings suggest that radiation-induced glycolysis, which is marginally higher than the control cannot meet the requirement of energy to rescue the cells from radiation-induced cell death, however, IL-6 can induce the glycolysis at sufficiently higher level to accomplish the requirement of energy for repair and survival of cells battling with radiation-induced damage. This proposition is further confirmed by significantly higher levels of ATP in radiation-exposed cells pre-treated with IL-6 as compared to control and radiation alone (Fig. 3D). The IL-6 induced high levels of ATP in IL-6 alone and combined treatment was noted at early time point (4 h), when it was obligatory to rescue the cells, as ATP is essentially required for energy-consuming processes like macromolecular repair, mainly DNA (20). The reversal of IL-6-induced radio-resistance by glycolytic inhibitors 3-BP and 2-DG (Fig. 3E–H) authenticated the role of glycolysis in IL-6-induced radio-resistance. Radiation-induced mitochondrial damage also contributes in radiation-induced cell death; we observed that IL-6 reduced the mitochondrial damage in IL-6 pre-treated cells. Interestingly, inhibition of IL-6-induced glycolysis also reverses the protective effect of IL-6 from radiation-induced mitochondrial damage (Fig. 4). IL-6 after binding to its receptor on the cell surface induces the phosphorylation of STAT-3 and PI3K (Phosphoinositol-3-kinase), which further phosphorylates the Akt (1). Increased Akt phosphorylation is found to be associated with increased rates of glucose metabolism in cells (31). Akt signalling influence the glycolysis directly by regulating the localization of the GLUT to the plasma membrane (32), HK-2 expression and mitochondrial interaction (33), and expression of PFK-1 and PFKFB3 (34). Since we found the expression levels of all these genes were increased by IL-6 treatment in Raw264.7 cells; we envisaged that IL-6-induced glycolysis in Raw264.7 cells could be mediated by Akt pathway. IL-6-induced Akt phosphorylation suggested the involvement of Akt signalling in IL-6 induced enhanced glycolysis, which was further verified by knocking down the Akt expression and inhibition of Akt signalling under similar experimental settings (Fig. 5). Down-regulation of Akt signalling in IL-6 pre-treated samples not only reduced the glycolysis (glucose uptake and lactate production) but also the expression level of key glycolytic enzyme HK-2 and its association with mitochondria, which resulted in reversal of IL-6-induced radio-resistance (Fig. 5). HK-2 association with mitochondrial outer membrane not only facilitates the quick phosphorylation of glucose using mitochondrial ATP but also prevents the cytochrome C release from mitochondria thereby inhibiting the apoptosis (35). Therefore, IL-6-induced and Akt-mediated translocation of HK-2 to mitochondrial membrane may cause radio-resistance besides glycolysis also. Moreover, besides the direct role of PKM2 in glucose catabolism, it was demonstrated to facilitate the homologous recombination of DNA repair in nucleus (36). Hence, we can assume that IL-6 induced high levels of PKM2 and HK-2 may be causing radio-resistance through glycolysis as well as other moonlighting functions. It will be pertinent to see the role of IL-6-induced glycolytic enzymes in DNA repair and other moonlighting functions. Pending this insight to be unravelled, results of the present studies lend support to our hypothesis that IL-6-induced glycolysis is a favourable metabolic change partly responsible for radio-resistance. This study adds induced glycolysis also as one of the factors among the list of various causes responsible for IL-6-induced radio-resistance. Conclusion In conclusion, the results of this study suggest that IL-6 induced the glycolysis in Raw264.7 cells by activating Akt signalling, which further induced the expression of key regulatory glycolytic enzymes and glucose transporters. The IL-6-induced aerobic glycolysis and reduced mitochondrial damage at the time of radiation exposure ensures continuous and sufficient supply of energy for repair of radiation-induced macro-molecular and cellular damages, thereby causing radio-resistance (Fig. 6). The moonlighting functions of IL-6-induced glycolytic enzymes also need to be understood to know their role in IL-6-induced radio-resistance. Further, understanding the mechanisms underlying IL-6 induced Glycolysis may help in unravelling critical molecular targets responsible for therapeutic resistance and facilitate the design and/or identification of molecules/agents that specifically overcome resistance linked to enhanced glycolysis, thereby enhancing the efficacy of radio- and chemotherapies. Clinical use of costly anti-IL-6 or anti IL-6 receptor antibodies in combination with therapies in IL-6 over-expressing tumours is challenging. Instead, use of metabolic inhibitors will be economical in achieving a similar level of therapeutic gain; however, more research is required to validate this proposition. This study also throws the light that IL-6 can protect the normal cells from radiation-induced cell death and it has potential to be developed as radio-protector. Fig. 6 Open in new tabDownload slide The picture illustrates that IL-6 treatment before irradiation activates PI3K-Akt pathway which resulted in the up-regulation of important regulatory genes of glycolysis. Akt also phosphorylates HK-2, which allows its binding to outer membrane of mitochondria where it facilitates efficient glucose phoshphorylation using mitochondrial ATP and ensures rapid rate of glycolysis. Efficient supply of energy (ATP) for repair of radiation-induced macro-molecular and cellular damages and moonlighting functions of glycolytic enzymes like prevention of cytochrome C release from mitochondria by HK-2 resulted in IL-6-induced radio-resistance. Fig. 6 Open in new tabDownload slide The picture illustrates that IL-6 treatment before irradiation activates PI3K-Akt pathway which resulted in the up-regulation of important regulatory genes of glycolysis. Akt also phosphorylates HK-2, which allows its binding to outer membrane of mitochondria where it facilitates efficient glucose phoshphorylation using mitochondrial ATP and ensures rapid rate of glycolysis. Efficient supply of energy (ATP) for repair of radiation-induced macro-molecular and cellular damages and moonlighting functions of glycolytic enzymes like prevention of cytochrome C release from mitochondria by HK-2 resulted in IL-6-induced radio-resistance. Acknowledgements N.K. thanks the Defence Research and Development Organisation, Government of India, for fellowship support. The authors acknowledge Director INMAS for constant support and encouragement. They also acknowledge Yogesh Rai, Dhananjay Sah and Ankit Chauhan for support during experiments. They extend sincere thanks to Dr Ravi Soni and the staff of the institutional Central Instrumentation Facility. Author contributions A.N.B.: Conceptualization, writing-reviewing and editing, supervision. N.K.: Data curation, investigation and writing-original draft. A.D.: Writing-reviewing and editing. All authors reviewed and approved the final version of the manuscript for submission. Funding This work was supported by grant (INM 311/1.4) funded by Defence Research and Development Organisation, Government of India. Conflict of Interest None declared. References 1 Kumari N. , Dwarakanath B.S. , Das A. , Bhatt A.N. ( 2016 ) Role of interleukin-6 in cancer progression and therapeutic resistance . Tumor Biol. 37 , 11553 – 11572 Google Scholar Crossref Search ADS WorldCat 2 Scheller J. , Chalaris A. , Schmidt-Arras D. , Rose-John S. ( 2011 ) The pro- and anti-inflammatory properties of the cytokine interleukin-6 . Biochim. Biophys. Acta 1813 , 878 – 888 Google Scholar Crossref Search ADS PubMed WorldCat 3 Muñoz-Cánoves P. , Scheele C. , Pedersen B.K. , Serrano A.L. ( 2013 ) Interleukin-6 myokine signaling in skeletal muscle: a double-edged sword? FEBS J. 280 , 4131 – 4148 Google Scholar Crossref Search ADS PubMed WorldCat 4 Smart N. , Mojet M.H. , Latchman D.S. , Marber M.S. , Duchen M.R. , Heads R.J. ( 2006 ) IL-6 induces PI 3-kinase and nitric oxide-dependent protection and preserves mitochondrial function in cardiomyocytes . Cardiovasc. Res . 69 , 164 – 177 Google Scholar Crossref Search ADS PubMed WorldCat 5 Kolliputi N. , Waxman A.B. ( 2009 ) IL-6 cytoprotection in hyperoxic acute lung injury occurs via suppressor of cytokine signaling-1-induced apoptosis signal-regulating kinase-1 degradation . Am. J. Respir. Cell Mol. Biol. 40 , 314 – 324 Google Scholar Crossref Search ADS PubMed WorldCat 6 Mauer J. , Denson J.L. , Brüning J.C. ( 2015 ) Versatile functions for IL-6 in metabolism and cancer . Trends Immunol . 36 , 92 – 101 Google Scholar Crossref Search ADS PubMed WorldCat 7 Ishibashi K. , Koguchi T. , Matsuoka K. , Onagi A. , Tanji R. , Takinami-Honda R. , Hoshi S. , Onoda M. , Kurimura Y. , Hata J. , Sato Y. , Kataoka M. , Ogawsa S. , Haga N. , Kojima Y. ( 2018 ) Interleukin-6 induces drug resistance in renal cell carcinoma . Fukushima J. Med. Sci . 64 , 103 – 110 Google Scholar Crossref Search ADS PubMed WorldCat 8 Ahmed K.M. , Li J.J. ( 2008 ) NF-kappa B-mediated adaptive resistance to ionizing radiation . Free Radic. Biol. Med . 44 , 1 – 13 Google Scholar Crossref Search ADS PubMed WorldCat 9 Yoon S. , Woo S.U. , Kang J.H. , Kim K. , Shin H.-J. , Gwak H.-S. , Park S. , Chwae Y.-J. ( 2012 ) NF-κB and STAT3 cooperatively induce IL6 in starved cancer cells . Oncogene 31 , 3467 – 3481 Google Scholar Crossref Search ADS PubMed WorldCat 10 Warburg O. ( 1956 ) On the origin of cancer cells . Science 123 , 309 – 314 Google Scholar Crossref Search ADS PubMed WorldCat 11 Wang T. , Liu H. , Lian G. , Zhang S.-Y. , Wang X. , Jiang C. ( 2017 ) HIF1α-induced glycolysis metabolism is essential to the activation of inflammatory macrophages . Mediators Inflamm. 2017 , 9029327 Google Scholar PubMed WorldCat 12 Wang T. , Marquardt C. , Foker J. ( 1976 ) Aerobic glycolysis during lymphocyte proliferation . Nature 261 , 702 – 705 Google Scholar Crossref Search ADS PubMed WorldCat 13 Inoue H. , Ogawa W. , Asakawa A. , Okamoto Y. , Nishizawa A. , Matsumoto M. , Teshigawara K. , Matsuki Y. , Watanabe E. , Hiramatsu R. , Notohara K. , Katayose K. , Okamura H. , Kahn C.R. , Noda T. , Takeda K. , Akira S. , Inui A. , Kasuga M. ( 2006 ) Role of hepatic STAT3 in brain-insulin action on hepatic glucose production . Cell Metab . 3 , 267 – 275 Google Scholar Crossref Search ADS PubMed WorldCat 14 Chen X. , Wei J. , Li C. , Pierson C.R. , Finlay J.L. , Lin J. ( 2018 ) Blocking interleukin-6 signaling inhibits cell viability/proliferation, glycolysis, and colony forming activity of human medulloblastoma cells . Int. J. Oncol . 52 , 571 – 578 Google Scholar PubMed WorldCat 15 Ando M. , Uehara I. , Kogure K. , Asano Y. , Nakajima W. , Abe Y. , Kawauchi K. , Tanaka N. ( 2010 ) Interleukin 6 enhances glycolysis through expression of the glycolytic enzymes hexokinase 2 and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 . J. Nippon Med. Sch. 77 , 97 – 105 Google Scholar Crossref Search ADS PubMed WorldCat 16 Matsuoka Y. , Nakayama H. , Yoshida R. , Hirosue A. , Nagata M. , Tanaka T. , Kawahara K. , Sakata J. , Arita H. , Nakashima H. , Shinriki S. , Fukuma D. , Ogi H. , Hiraki A. , Shinohara M. , Toya R. , Murakami R. ( 2016 ) IL-6 controls resistance to radiation by suppressing oxidative stress via the Nrf2-antioxidant pathway in oral squamous cell carcinoma . Br. J. Cancer 115 , 1234 – 1244 Google Scholar Crossref Search ADS PubMed WorldCat 17 Tamari Y. , Kashino G. , Mori H. ( 2017 ) Acquisition of radioresistance by IL-6 treatment is caused by suppression of oxidative stress derived from mitochondria after γ-irradiation . J. Radiat. Res . 58 , 412 – 420 Google Scholar Crossref Search ADS PubMed WorldCat 18 Brown C.O. , Salem K. , Wagner B.A. , Bera S. , Singh N. , Tiwari A. , Choudhury A. , Buettner G.R. , Goel A. ( 2012 ) Interleukin-6 counteracts therapy-induced cellular oxidative stress in multiple myeloma by up-regulating manganese superoxide dismutase . Biochem. J. 444 , 515 – 527 Google Scholar Crossref Search ADS PubMed WorldCat 19 Wang T. , Ning K. , Sun X. , Zhang C. , Jin L.F. , Hua D. ( 2018 ) Glycolysis is essential for chemoresistance induced by transient receptor potential channel C5 in colorectal cancer . BMC Cancer 18 , 207 Google Scholar Crossref Search ADS PubMed WorldCat 20 Bhatt A.N. , Chauhan A. , Khanna S. , Rai Y. , Singh S. , Soni R. , Kalra N. , Dwarakanath B.S. ( 2015 ) Transient elevation of glycolysis confers radio-resistance by facilitating DNA repair in cells . BMC Cancer 15 , 335 Google Scholar Crossref Search ADS PubMed WorldCat 21 Huang B. , Zhang Q. , Yuan Y. , Xin N. , He K. , Huang Y. , Tang H. , Gong P. ( 2018 ) Sema3a inhibits the differentiation of Raw264.7 cells to osteoclasts under 2Gy radiation by reducing inflammation . PLoS One 13 , e0200000 Google Scholar Crossref Search ADS PubMed WorldCat 22 Kato K. , Omori A. , Kashiwakura I. ( 2013 ) Radiosensitivity of human haematopoietic stem/progenitor cells . J. Radiol. Prot. 33 , 71 – 80 Google Scholar Crossref Search ADS PubMed WorldCat 23 Ribble D. , Nathaniel B Goldstein D.A.N. , Shellman Y.G. ( 2005 ) A simple technique for quantifying apoptosis in 96-well plates . BMC Biotechnol . 5 , 12 Google Scholar Crossref Search ADS PubMed WorldCat 24 Berg J.M. , Tymoczko J.L. , Stryer L. ( 2002 ) The glycolytic pathway is tightly controlled in Biochemistry, 5th edn. W H Freeman , New York . https://www.ncbi.nlm.nih.gov/books/NBK22395/ COPAC 25 Rai Y. , Pathak R. , Kumari N. , Sah D.K. , Pandey S. , Kalra N. , Soni R. , Dwarakanath B.S. , Bhatt A.N. ( 2018 ) Mitochondrial biogenesis and metabolic hyperactivation limits the application of MTT assay in the estimation of radiation induced growth inhibition . Sci. Rep . 8 , 1531 Google Scholar Crossref Search ADS PubMed WorldCat 26 Verma A. , Bhatt A.N. , Farooque A. , Khanna S. , Singh S. , Dwarakanath B.S. ( 2011 ) Calcium ionophore A23187 reveals calcium related cellular stress as “I-Bodies”: an old actor in a new role . Cell Calcium 50 , 510 – 522 Google Scholar Crossref Search ADS PubMed WorldCat 27 Zhang F. , Duan S. , Tsai Y. , Keng P.C. , Chen Y. , Lee S.O. , Chen Y. ( 2016 ) Cisplatin treatment increases stemness through upregulation of hypoxia-inducible factors by interleukin-6 in non-small cell lung cancer . Cancer Sci. 107 , 746 – 754 Google Scholar Crossref Search ADS PubMed WorldCat 28 Oki Y. , Fanale M. , Romaguera J. , Fayad L. , Fowler N. , Copeland A. , Samaniego F. , Kwak L.W. , Neelapu S. , Wang M. , Feng L. , Younes A. ( 2015 ) Phase II study of an AKT inhibitor MK2206 in patients with relapsed or refractory lymphoma . Br. J. Haematol . 171 , 463 – 470 Google Scholar Crossref Search ADS PubMed WorldCat 29 Chen Z. , Zhang H. , Lu W. , Huang P. ( 2009 ) Role of mitochondria-associated hexokinase II in cancer cell death induced by 3-bromopyruvate . Biochim. Biophys. Acta 1787 , 553 – 560 Google Scholar Crossref Search ADS PubMed WorldCat 30 Zhong J. , Rajaram N. , Brizel D.M. , Frees A.E. , Ramanujam N. , Batinic-Haberle I. , Dewhirst M.W. ( 2013 ) Radiation induces aerobic glycolysis through reactive oxygen species . Radiother. Oncol . 106 , 390 – 396 Google Scholar Crossref Search ADS PubMed WorldCat 31 Elstrom R.L. , Bauer D.E. , Buzzai M. , Karnauskas R. , Harris M.H. , Plas D.R. , Zhuang H. , Cinalli R.M. , Alavi A. , Rudin C.M. , Thompson C.B. ( 2004 ) Akt stimulates aerobic glycolysis in cancer cells . Cancer Res. 64 , 3892 – 3899 Google Scholar Crossref Search ADS PubMed WorldCat 32 Barthel A. , Okino S.T. , Liao J. , Nakatani K. , Li J. , Whitlock J.P. , Roth R.A. ( 1999 ) Regulation of GLUT1 gene transcription by the serine/threonine kinase Akt1 . J. Biol. Chem. 274 , 20281 – 20286 Google Scholar Crossref Search ADS PubMed WorldCat 33 Miyamoto S. , Murphy A.N. , Brown J.H. ( 2008 ) Akt mediates mitochondrial protection in cardiomyocytes through phosphorylation of mitochondrial hexokinase-II . Cell Death Differ. 15 , 521 – 529 Google Scholar Crossref Search ADS PubMed WorldCat 34 Deprez J. , Vertommen D. , Alessi D.R. , Hue L. , Rider M.H. ( 1997 ) Phosphorylation and activation of heart 6-phosphofructo-2-kinase by protein kinase B and other protein kinases of the insulin signaling cascades . J. Biol. Chem. 272 , 17269 – 17275 Google Scholar Crossref Search ADS PubMed WorldCat 35 Pastorino J.G. , Shulga N. , Hoek J.B. ( 2002 ) Mitochondrial binding of hexokinase II inhibits Bax-induced cytochrome c release and apoptosis . J. Biol. Chem. 277 , 7610 – 7618 Google Scholar Crossref Search ADS PubMed WorldCat 36 Sizemore S.T. , Zhang M. , Cho J.H. , Sizemore G.M. , Hurwitz B. , Kaur B. , Lehman N.L. , Ostrowski M.C. , Robe P.A. , Miao W. , Wang Y. , Chakravarti A. , Xia F. ( 2018 ) Pyruvate kinase M2 regulates homologous recombination-mediated DNA double-strand break repair . Cell Res. 28 , 1090 – 1102 Google Scholar Crossref Search ADS PubMed WorldCat Abbreviations Abbreviations 2-DG 2-dexoy-D-glucose 3-BP 3-bromo pyruvate ANOVA analysis of variance DMSO dimethyl sulfoxide HGD high glucose Dulbecco’s HK-2 hexokinase-2 IL-6 interleukin-6 IR ionizing radiation MMP mitochondrial membrane potential PBS phosphate-buffered saline PFK-1 phosphofructokinase 1 PFKFB3 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 PKM2 pyruvate kinase M2 SF surviving fraction SRB sulforhodamine B © The Author(s) 2019. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Interleukin-6 confers radio-resistance by inducing Akt-mediated glycolysis and reducing mitochondrial damage in cells
JF - The Journal of Biochemistry
DO - 10.1093/jb/mvz091
DA - 2020-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/interleukin-6-confers-radio-resistance-by-inducing-akt-mediated-akUmXO6SJa
SP - 303
VL - 167
IS - 3
DP - DeepDyve
ER -