Evaluation of the Role of Hexokinase Type II in Cellular Proliferation and Apoptosis Using Human Hepatocellular Carcinoma Cell Lines

The F-18-FDG uptake pattern on PET could be an indicator of the prognosis and aggressiveness of various tumors, including hepatocellular carcinoma (HCC). Hexokinase, especially hexokinase type II (HKII), plays a critical role in F-18-FDG uptake in rapidly growing tumors. We established a stable cell line overexpressing HKII by the transfection of full DNA of HKII to HCC cells (SNU449) that express low levels of HKII and investigated how F-18-FDG uptake mechanisms, especially overexpression of HKII, are linked to tumor proliferation mechanisms. Methods: The HKII gene was stably transfected to SNU449 cells with an expression vector. HKII expression in the cells was verified by reverse-transcriptase polymerase chain reaction, Western blot analysis, adenosine triphosphate (ATP) and lactate production, F-18-FDG uptake measurement, and confocal microscopy. Cellular proliferation activity and response to the anticancer drug cisplatin were evaluated by cell counting using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. For the evaluation of molecular pathways involved in tumor proliferation, the phosphatidylinositol 3- kinase (PI3K)/Akt pathway was investigated. Results: The stable cell line produced HKII effectively, but expression of other enzymes or transporters for glycolysis, such as glucose-6-phosphatase (G6Pase), HKI and III, and glucose transporter type 1 and 2 (Glut-1 and Glut- 2), did not show any changes. F-18-FDG uptake was significantly increased after transfection. ATP and lactate production was also increased after transfection. Overexpressed HKII was associated with mitochondria on confocal microscopy. Cells with overexpression of HKII, compared with the nontransfected cell line, showed 1.5- to 2-fold higher cell survival and resistance to the anticancer agent cisplatin (2- to 8-fold). In the molecular study, the activated form of Akt was increased after transfection, and PI3K inhibitor dissociated the mitochondrial HKII to the cytoplasm. In addition, the adenosine monophosphate-activated protein kinase (AMPK) pathway is also involved in Akt signaling. Conclusion: HKII plays an important role in F-18-FDG uptake and tumor proliferation by both the PI3K-dependent and the PI3K-independent Akt signal pathways; therefore, the F-18-FDG uptake pattern on a PET scan can be a surrogate marker of prognosis in HCC.