Expression of adenine nucleotide translocase2 regulates 18F-FDG accumulation in thyroid cancer cells

Objectives 18F-FDG (FDG) provides valuable functional information based on the glycolysis of cancer cells and depicts metabolic abnormalities. Thyroid cancers increase FDG uptake during dedifferentiation. Though the role of glucose transporter-1 (GLUT-1) and hexokinase II (HKII) for FDG uptake have been emphasis, this is unknown that correlation between increased FDG uptake and thyroid cancer stage. Therefore, better understanding of FDG accumulation is required. Adenine nucleotide translocase 2 (ANT2) imports glycolytic ATP into mitochondria and was shown to be directly associated with glucose metabolisms. However, the correlation between ANT2 expression and FDG uptake was not reported.

Methods For immunohistochemistry (IHC), 37 human thyroid tissue-array cores separated from TNM stage (7 benign, 15 stageI, 4 stageII, 7 stageIII, 4 stageIV) were investigated for ANT2 expression. And human thyroid cell lines such as N-thy-ori (normal), WRO (follicular cancer), BHP10-3 and TPC-1 (papillary cancer), FRO (anaplastic cancer) were used for this research. ANT2 expression was measured by RT-PCR and western blot. 2’-methoxy (2’-OMe) modified siRNAs were used for down-regulation of ANT2, and pcDNA3.1-ANT2 vector was used for up-regulating ANT2 expression. A gamma counter was used for measuring FDG uptake. Luciferase-expressing FRO cells were subcutaneously grafted in a BALB/c nude mouse, and siRNA was directly injected into the tumor. 18F-FDG PET and in vivo bioluminescent imaging were obtained using animal PET and IVIS 100. Tumor tissue isolated for IHC.

Results In immunohistochemistry, ANT2 expression in stageIV was 1.8-fold higher than benign and other stages (P<0.05). In vitro FDG uptake in FRO was 3.5-fold higher than N-thy-ori and 2.0-fold higher than WRO, 1.8-fold higher than BHP10-3, and 1.5-fold higher than TPC-1, respectively (P<0.001). FRO and TPC-1 were chosen to investigate the role of ANT2 in FDG uptake because ANT2 mRNA and protein expressions were higher in FRO and lower in TPC-1, relatively. FRO cells treated with ANT2 siRNA showed that reduced ANT2 expression and FDG uptake as concentration dependently. Especially, FDG uptake in 200 nM ANT2 siRNA treatment was significantly decreased in 0.55-fold (P<0.001) compared to the scramble siRNA treatment. TPC-1 cells treated with 12 ug of pcDNA3.1-ANT2 expression vector showed increased ANT2 expression, and FDG uptake in TPC-1 overexpressed ANT2 was significantly higher (1.7-fold) than control. In the xenograft mouse model, 100 uM ANT2 siRNA reduced 18F-FDG uptake in FRO at 0.75-fold (P<0.01) of the scramble treatment.

Conclusions We showed that ANT2 expression is related with FDG uptake in the thyroid carcinoma cells by modulating ANT2 expression. Also we showed correlation between ANT2 expression and thyroid cancer stage. Our result suggests that the evaluation of ANT2 expression can be used as a possible prognostic marker for FDG PET positive tumor.