Sensitization of hepatocellular carcinoma cells towards doxorubicin and sorafenib is facilitated by glucosedependent alterations in reactive oxygen species, P-glycoprotein and DKK4

Altered glucose uptake and metabolism is the key characteristic of cancer cells including hepatocellularcarcinoma (HCC). However, role of glucose availability in chemotherapeutic outcome of HCC is unclear. Thepresent study investigates the effect of glucose facilitated sensitization of HCC cells towards doxorubicin(DOX) and sorafenib (SORA). In HCC cells, we observed that hyperglycemic culture condition (HG) isassociated with increased sensitivity towards DOX and SORA. P-glycoprotein (P-gp), a transporter involved indrug efflux, was elevated in HCC cells in NG, rendering them less susceptible to DOX and SORA. Further, thisstudy demonstrated that knockdown of dickkopf protein 4 (DKK4), a Wnt antagonist protein, causes enhancedglucose uptake and reduction in P-gp level rendering HCC cells in NG sensitive to DOX and SORA.Moreover, HG elevates the level of intracellular reactive oxygen species (ROS), which regulates P-gp.Alteration in intracellular ROS did not directly affect regulation of DKK4 in HCC cells. Functional assayssuggest that alterations in DKK4 and P-gp level in HCC cells are dependent on glucose availability andchanges in ROS level because of enhanced glucose utilization, respectively. Collectively, the present studyhighlights direct involvement of glucose-induced ROS, DKK4 and P-gp in altering the sensitivity of HCC cellstowards DOX and SORA.

Demonstration of a visual cell-based assay for screening glucose transporter 4 translocation modulators in real time.

Insulin-stimulated translocation of glucose transporter 4 (GLUT4) to cell membrane leading to glucose uptake is the rate-limiting step in diabetes. It is also a defi ned target of antidiabetic drug research. Existing GLUT4 translocation assays are based on time-consuming immunoassays and are hampered by assay variability and low sensitivity. We describe a real-time, visual, cell-based qualitative GLUT4 translocation assay using CHO-HIRc-myc-GLUT4eGFP cells that stably express myc- and eGFP-tagged GLUT4 in addition to human insulin receptor (HIRc). GLUT4 translocation is visualized by live cell imaging based on GFP fl uorescence by employing a cooled charge-coupled device camera attached to a fl uorescent microscope. This video imaging method and further quantitative analysis of GLUT4 on the cell membrane provide rapid and foolproof visual evidence that this method is suitable for screening GLUT4 translocation modulators.