2-Deoxyglucose and sorafenib synergistically suppress the proliferation and motility of hepatocellular carcinoma cells

Abstract

Cancer cells consume more glucose than normal cells, mainly due to their increased rate of glycolysis. 2-Deoxy-d-glucose (2DG) is an analogue of glucose, and sorafenib is a kinase inhibitor and molecular agent used to treat hepatocellular carcinoma (HCC). The present study aimed to demonstrate whether combining 2DG and sorafenib suppresses tumor cell proliferation and motility more effectively than either drug alone. HLF and PLC/PRF/5 HCC cells were incubated with sorafenib with or without 1 µM 2DG, and subjected to a proliferation assay. A scratch assay was then performed to analyze cell motility following the addition of 2DG and sorafenib in combination, and each agent alone. RNA was isolated and subjected to reverse transcription-quantitative polymerase chain reaction to analyze the expression of cyclin D1 and matrix metalloproteinase-9 (MMP9) following the addition of 2DG and sorafenib in combination and each agent alone. Proliferation was markedly suppressed in cells cultured with 1 µM 2DG and 30 µM sorafenib compared with cells cultured with either agent alone (P<0.05). In addition, levels of Cyclin D1 expression decreased in cells exposed to 3 µM sorafenib and 1 µM 2DG compared with cells exposed to 2DG or sorafenib alone (P<0.05). Scratch assay demonstrated that the distance between the growing edge of the cell sheet and the scratched line was shorter in cells cultured with sorafenib and 2DG than in cells cultured with 2DG or sorafenib alone (P<0.05). Levels of MMP9 expression decreased more in cells treated with both sorafenib and 2DG than in cells treated with 2DG or sorafenib alone (P<0.05). Therefore, 2DG and sorafenib in combination suppressed the proliferation and motility of HCC cells more effectively than 2DG or sorafenib alone, and a cancer treatment combining both drugs may be more effective than sorafenib alone.

Keywords: 2-Deoxyglucose; HCC; cyclin D1; matrix metalloproteinase; sorafenib.

Figure 1.

Cell proliferation assay. HLF cells (A) and PLC/PRF/5 cells (B) were added to 0, 1, 3, 10, and 30 µM sorafenib either with (white bar), or without (black bar) 1 µM 2DG. The cells were subjected to MTS assay to measure cell proliferation. *P<0.05 against 0 µM 2DG and sorafenib, **P<0.05 against 0 µM 2DG and 30 µM sorafenib. HLF, Human liver fibroblasts; PLC/PRF/5, the human PLC/PRF/5 hepatoma carcinoma cell line; 2DG, 2-deoxy-glucose.

Figure 2.

Levels of cyclin D1 expression. HLF cells (A) and PLC/PRF/5 cells (B) were cultivated with 3 µM sorafenib, 1 µM 2DG, or a combination of the two. Cyclin D1 expression levels were compared with those of a control containing cells without sorafenib or 2DG. RNA was isolated and subjected to RT-qPCR to analyze cyclin D1 expression. *P<0.05 against sorafenib (−) and 2DG (−) **P<0.05 against sorafenib (−) and 2DG (−), sorafenib (−) and 2DG (+), and sorafenib (+) and 2DG (−), n=3. HLF cells, human liver fibroblast cells; PLC/PRF/5, the human hepatoma carcinoma cell line; 2DG, 2-deoxy-glucose; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 3.

Scratch assay. HLF cells (A-D) and PLC/PRF/5 cells (E-H) in 4-well chamber slides were scratched with a sterile razor. Immediately after the scratch (solid line), no sorafenib or 2DG (A, E), 3 µM sorafenib (B, F), 1 µM 2DG (C, G), or both (D, H) were added to the cells. Following 48 h in culture, the cells were stained with hematoxylin and eosin. Distance between the growing edge of the cell sheet and the scratched line was measured at five different points in HLF cells (I) and PLC/PRF/5 cells (J). Original magnification: ×100, scale bar: 200 µM, *P<0.05 against sorafenib (−) and 2DG (−), **P<0.05 against sorafenib (−) and 2DG (−), sorafenib (−) and 2DG (+), and sorafenib (+) and 2DG (−), n=5. HLF, Human liver fibroblast cells; 2DG, 2-deoxyglucose; PLC/PRF/5 cells, human hepatoma carcinoma cell line.

Figure 4.

Expression levels of matrix metalloproteinase. HLF cells (A) and PLC/PRF/5 cells (B) were cultured in 6-well plates, and added to 3 µM sorafenib (+), 1 µM 2DG (+), or a combination of both. Following 48 h in culture, RNA was isolated and subjected to RT-qPCR. Levels of matrix metalloproteinase expression were analyzed. *P<0.05 against sorafenib (−) and 2DG (−), **P<0.05 against sorafenib (−) and 2DG (−), sorafenib (−) and 2DG (+), and sorafenib (+) and 2DG (−), ***P<0.05 against sorafenib (−) and 2DG (−), and sorafenib (−) and 2DG (+), n=3. HLF, Human liver fibroblast cells; 2DG, 2-deoxyglucose; PLC/PRF/5 cells, human hepatoma carcinoma cell line; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.