3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), glucose, 2-deoxyglucose (2-DOG), nicotinamide adenine dinucleotide phosphate (NADP⁺), oligomycin (Olig), glucose-6-phosphate dehydrogenase (G6PDH) kit, ATP kit and lactate kit were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Glucox 500 reagent from Doles Reagentes (Goiânia, Goiás, Brazil). All other chemicals were of the highest grade available. Polyvinylidene difluoride (PVDF) membranes were provided by Bio-Rad (Hercules, CA USA) and Millipore (Billerica, MA, USA). Specific antibody against GLUT1 (1:1,000) was from Abcam (Cambridge, MA, USA), and against hexokinase I (HKI) (1:1,000) and HKII (1:1,000) were both purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Cell culture medium Dulbecco's modified Eagle's medium (DMEM) and RPMI-1640 were provided by HiMedia Laboratories (Curitida, Brazil) and fetal bovine serum from Invitrogen Life Technologies (Carlsbad, CA, USA).

Human PTC cells (BCPAP and TPC1) and NTHY-ori cell lineages were generous gifts from Dr Corinne Dupuy, (Institut Gustave Roussy, Paris, France). Cells were cultivated in DMEM (BCPAP and TPC1) and RPMI (NTHY), supplemented with 10% bovine serum medium, and maintained at 37°C in 5% CO₂.

The MTT assay was used to test cell viability in three different experiments. First, we measured the cell viability after 6, 24, 48 and 72 h under a baseline condition. Then, we tested the cytotoxicity after treatment with Olig. In this case, the cells were grown in medium until 80% confluence was achieved. The medium was removed and fresh medium containing 2 µg/ml Olig was added for 2 or 24 h. Also, after the cells achieved confluence, the medium was removed and fresh medium containing 2 mM of 2-DOG was added to the medium for 24 h.

Immediately after treatment, total RNA was extracted using the RNeasy Plus Mini kit (Qiagen Sciences Inc., Germantown, MD, USA) following the manufacturer's instructions. Total RNA was reverse transcribed into cDNA using a high-capacity reverse transcriptase kit (Applied Biosystems Inc., Carlsbad, CA, USA). The specific forward and reverse primers are presented in Table I and were designed to span introns and avoid detection of genomic DNA contamination. Real-time amplifications were carried out using ABI Prism 7500 equipment (Applied Biosystems, Invitrogen Life Technologies, São Paulo, Brazil). The amplification reactions were performed in 96-well plates in 12 µl final volume that contained 3 µl cDNA (diluted 1:100), 7.5 µl of SYBR Master Mix (Applied Biosystems, Invitrogen Life Technologies), and 150 nM of each forward and reverse primers. The amplification program consisted of 55°C for 2 min, 95°C for 10 min followed by 40 cycles of 95°C for 30 sec, and 58°C for 1 min. The RT-PCR efficiency was evaluated using serial dilutions of the cDNA template, and melting curve data were collected to assess RT-PCR specificity. Each cDNA sample was amplified in duplicate, and a corresponding sample without reverse transcriptase (no-RT sample) was included, as a negative control. The content of the target gene transcripts was normalized to that of peptidylprolyl isomerase A (PPIA). The relative quantities of gene expression were determined by the comparative Ct method expressed by the formula 2^−ΔΔCt (19), where Ct refers to the threshold cycle and was determined for each plate by 7500 Real-Time PCR system sequence detection software (Applied Biosystems, Invitrogen Life Technologies).

Glucose uptake was measured as previously described (20). Cells were cultivated in 6-well plates. After confluence, the cells were washed twice, and glucose (1 mM) was added to the PBS saline solution. Aliquots of the incubation medium were collected each 5 min and transferred to 96-well plates. After 60 min, the glucose concentrations in the samples were analyzed by colorimetric methods (20). The specific glucose uptake was corrected by protein concentration that was evaluated using the BCA protein assay kit (Pierce, Thermo Scientific Fisher, Waltham, MA, USA). Lactate production was recorded using the same cell pool. Cells were lysed and the lactate content was measured by procedures according to the instructions provided by the kit (Sigma Chemical Co.).

Cells were cultivated in 12-well plates. After 80% confluence, the medium was removed, and the cells were washed twice, processed using a luciferin-luciferase bioluminescence assay according to the instructions provide by the ADP/ATP ratio assay kit (MAK 135; Sigma Chemical Co.). In the presence of luciferase, ATP immediately reacts with the substrate D-luciferin to produce light. The light intensity is a direct measure of the intracellular ATP concentration.

GLUT1, HK1 and HK2 levels were analyzed after lysis of the cells in a buffer [0.05 M Tris-HCl (pH 8.0), 0.15 M NaCl, 0.1% SDS, 0.5% deoxicolic acid, 5 mM MgCl₂, Triton 1%, 1 mM PMSF, 1 mM NaF, 1 mM Na₃VO₄, 1 M leupeptin and 1 M pepstatin]. Subsequently, the samples were centrifuged and then the supernatant was collected. An aliquot was used to determine the concentration of protein by the BCA protein assay kit. Fifty micrograms of protein were then resolved on SDS-PAGE gels, transferred to PVDF membranes, and probed with the indicated primary antibodies. We used a β-actin antibody (1:8,000) from Sigma-Aldrich as the loading control. The detection of proteins was performed by chemiluminescence (ECL; Pierce, Thermo Scientific Fisher) following densitometric analysis.

Initially, we measured cellular viability by trypan blue and corrected the sample to 10⁵ cells. The cells were suspended in lysis buffer [0.05 M Tris-HCl (pH 8.0), 0.15 M NaCl, 0.1% SDS, 0.5% deoxicolic acid, 5 mM MgCl_2, Triton 1%, 1 mM PMSF, 1 mM NaF, 1 mM Na₃VO₄] and HK activity was assayed as previously described (7). Blanks with none of the coupled enzymes were performed to control for non-specific oxidation/reduction. For mitochondrial HK fraction assay, the homogenates were centrifuged at 10,000 × g to separate the soluble fraction of HK (total cell extracted) from the particulate fraction (mitochondrial fraction). The HK activity was measured in each cell fraction.

To measure the glucose-6-phosphate (G6P) amount, cells were corrected to 10⁵ cells and suspended in lysis buffer [0.05 M Tris-HCl (pH 8.0), 0.15 M NaCl, 0.1% SDS, 0.5% deoxycolic acid, 5 mM MgCl₂, Triton 1%, 1 mM PMSF, 1 mM NaF, 1 mM Na₃VO₄]. The reaction medium contained 50 mM Tris-HCl, pH 8.0, 0.5 mM NADP⁺ and 0.2 U/ml G6PDH. Reduction of NADP⁺ was followed by measuring the absorbance at 340 nm in a microplate reader (Victor 4 XR; Perkin-Elmer Inc., Waltham, MA, USA). To measure G6PDH activity, cells were corrected to 1×10⁵ cells and suspended in lysis buffer following the instructions of the G6PDH kit assay (Sigma Chemical Co.) measuring the absorbance at 340 nm in a microplate reader (Victor 4 XR; Perkin-Elmer Inc., São Paulo, Brazil).

Samples (10⁵ cells) were suspended in lysis buffer [0.05 M Tris-HCl (pH 8.0), 0.15 M NaCl, 0.1% SDS, 0.5% deoxicolic acid, 5 mM MgCl₂, Triton 1%, 1 mM PMSF, 1 mM NaF, 1 mM Na₃VO₄] and pyruvate kinase (PK) assay was recorded using the PK kit (Sigma Chemical Co.) measuring the absorbance at 340 nm in a microplate reader (Victor 4 XR; Perkin-Elmer Inc.).

The assay was performed with intact cells (10⁶ cells/ml) in culture medium and oxygen consumption rates were measured polarographically using high-resolution respirometry (Oroboros Oxygraph - O₂K; Oroboros Instruments, Innsbruck, Austria). Assay was started by adding cells in the closed (2 ml) respirometer chamber, and the basal respiration status of the different cell lines was collected. Immediately, we added Olig (1 µg/ml), to acquire the oxygen consumption coupled to ATP synthesis calculated by basal O₂ consumption - Olig O₂ consumption. Finally, we add a selective inhibitor form complex IV (KCN 1 mM) to completely block mitochondrial respiration called the residual oxygen consumption.

The levels of intracellular ROS were measured using a reactive oxygen species assay kit (Applygen Technologies, Inc., Beijing, China). Briefly, the intracellular formation of ROS in the TPC1 and BCPAP cells was determined using 2′7′-dichlorodihydrofluorescein diacetate (DCFH-DA) molecular probes. DCFH-DA is a membrane-permeable indicator of ROS levels and the reaction between these probes and intracellular ROS yields the fluorescent molecule DCF, which may be used as an indicator of intracellular ROS levels. Cells were washed with PBS and loaded with 10 µM DCFH-DA in serum-free DMEM without phenol red in the dark for 30 min at 37̊C. The excess dye was flushed off and the cells were suspended in serum-free DMEM. Fluorescence intensity was quantified using a BD FACSVantage™ SE (BD Biosciences, Franklin Lakes, NJ, USA).

Statistical analyses were performed using GraphPad Software, Inc. (San Diego, CA, USA), and the tests were as follows: Student's t-test or one-way analysis of variance (ANOVA) followed by the Bonferroni multiple comparison test, as described in the figure legends. The level of significance was set at p<0.05.

In attempt to described differences in energy behavior between thyroid carcinoma cell lineages, we analyzed the cell growth rate and glycolic capacity of all cells (Fig. 1). BCPAP showed a higher cell growth rate different from TPC1 after 48 h (Fig. 1A). Both TPC1 and the non-tumor cell line NTHY-ori exhibited the same growth rate (Fig. 1B). The glucose uptake of tumor cell lines was 2- to 3.5-fold higher than the uptake found in non-tumor cells. The lactate production was also much higher in the BCPAP and TPC1 cell lines when compared to the NTHY-ori cells (Fig. 1C). These data suggest that thyroid carcinoma cell lines show a high ability to convert glucose into lactate (glycolytic efficiency), an important phenomenon possibly involved in tumor survival.

To investigate the mechanisms involved in the differences found in the metabolism of these cell lines, we analyzed the mRNA expression and levels of proteins involved in the glycolytic flux. As shown in Fig. 2A, we observed that Glut1 mRNA and protein levels were more highly expressed in both carcinoma cell lines than these levels in the NTHY-ori cells. Moreover, the protein levels of HK1 and HK2 isoforms were more highly expressed in the cancer cell lines (Fig. 2B and C) in the same way as gene expression of other glycolytic targets such as PKM1 and PKM2.

According to previous results, we analyzed the activity of specific regulatory enzymes of glycolysis and the pentose phosphate pathway (PPP). As shown in Fig. 3A, we observed a higher HK activity pattern in the BCPAP and TPC1 cell lines compared to this pattern in the NTHY-ori cells. However, the amount of G6P in the BCPAP cell line was lower than that in the TPC1 cells, indicating a difference in the use of this substrate (Fig. 3B). As G6P is consumed by both glycolysis and PPP, we evaluated the G6PDH activity, the limiting step enzyme of PPP. Although, the mRNA levels of G6PDH did not differ among all thyroid cell lines (data not shown), we observed a higher G6PDH activity in the TPC1 cells but not in the BCPAP cells (Fig. 3C). Adding to these, we evaluated the PK activity and our results showed that BCPAP and TPC1 differ from each other in PK activity (Fig. 3D).

In Fig. 3A, the HK activity was the same in both thyroid carcinoma cell lines. Nevertheless, their activity was different when we analyzed the specific cell fraction (cytosolic and mitochondrial fractions). In an attempt to evaluate the degree of HK bound to mitochondria, we observed that BCPAP cells had >90% of total HK activity present in the mitochondrial fraction, while NTHY-ori and TPC1 cells had equally distributed HK activity in both cytosolic and mitochondrial fractions (Fig. 3E).

Since our results indicated a high glycolytic potential in these tumor cell lineages, we then evaluated the contribution of the different metabolic pathways for ATP generation in these cells. First, we showed that high levels of ATP and ADP were found in the BCPAP and TPC1 cells in comparison to the NTHY cells (Fig. 4A). Second, we incubated all cells with Olig C, an inhibitor of ATP-synthase complex V of mitochondria electron transport chain, for 2 h. The ATP and ADP levels were reduced following treatment with Olig (Fig. 4A). Moreover, only the NTHY and TPC1 cells showed a modulation of the ADP/ATP ratio in the presence of Olig, suggesting a dependency of mitochondrial oxidative metabolism for ATP generation in TPC1 and non-tumoral NTHY-ori cells (Fig. 4B). On the other hand, Olig treatment caused no modulation in the ADP/ATP content and lactate production in the BCPAP cell line. Yet, the TPC1 cell line showed an increase in lactate production after 24 h in the presence of Olig (Fig. 4C and D). To assess the importance of mitochondrial ATP production to cell viability, we treated the cells with Olig for 24 h or 2-DOG, an inhibitor of glycolytic flux. No differences in cell viability were observed after 24 h of Olig treatment (Fig. 4E) although we found an increase in lactate production in the TPC1 cells at 24 h (Fig. 4D). However, when we inhibited the glycolysis pathway using 2 mM of 2-DOG, we observed a decreased in cell viability after 24 h of incubation in all cell lineages but the effect was more severe in the BCPAP cells when compared with this effect in the TPC1 cells (Fig. 4E).

The above results indicated that the PTC cells are very different. Because of this finding, we evaluated the oxidative profile in all thyroid cell lines. Baseline oxygen consumption in the BCPAP cells was lower than that in the TPC1 cells (Fig. 5A). However, both cell lines showed lower oxygen consumption coupled to ATP production acquired after treatment with Olig (2 µg/ml) (Fig. 5B). These results suggest that these cells could redirect the oxygen to another metabolic pathway. Analysis of residual oxygen, after inhibition of oxidative phosphorylation in tumor cells by KCN (2 mM) showed high residual oxygen consumption by both PTC cells (Fig. 5C). Using a fluorescence probe, we observed that BCPAP and TPC1 cells produced high ROS levels. Moreover, the ROS production by the BCPAP cell line was higher than that noted in the TPC1 cells (Fig. 5D).