Abstract B61: Picropodophyllin (PPP) increases glucose metabolism and lactate production in paediatric glioblastoma cells

Abstract

Abstract Introduction: Picropodophyllin (PPP) is of interest as an anti-cancer agent, as it has been shown to induce growth inhibition and apoptosis in a variety of human cancer cells and xenografts. Recently, PPP was found to have dramatic antitumor effects in human glioblastoma cells and in subcutaneous and intracerebral xenografts and reported to be an insulin-like growth factor-1 receptor inhibitor; however, its full mechanisms of action remain unclear. Aims: To examine the effect of PPP on glucose metabolism in cancer cells and to use hyperpolarized [1-13C]pyruvate 13C-magnetic resonance spectroscopy (MRS) and conventional 1H-MRS to measure lactate formation/production in real-time and in steady state, respectively, in order to develop a non-invasive biomarker of response for PPP treatment in a pediatric glioblastoma cell line. Methods: Pediatric glioblastoma KNS42 cells were treated for 24 hours with 2.4μM (5×IC50) of PPP. Real-time 13C exchange from hyperpolarized [1-13C] pyruvate to lactate was performed by DNP-13C MRS. Culture media and extracts from the PPP-treated and control KNS42 cells were analyzed by 1H-MRS to examine the effect of PPP on glucose metabolism and lactate production. Lactate dehydrogenase (LDH) expression was examined by western blot and LDH activity was also measured enzymatically. Cellular protein content and size were also evaluated. Results and Discussion: The rate of real-time 13C exchange from pyruvate to lactate measured by DNP-13C MRS showed a significant increase in the PPP-treated group (P = 0.027 versus control group). These DNP changes in PPP-treated KNS42 cells are consistent with the significant increase in intracellular lactate (P = 0.003) and lactate excretion to the culture media (P < 0.001). This increase in lactate production is also consistent with significantly elevated LDH activity (P = 0.003) in the PPP-treated group. However, no change in LDH expression was observed in the PPP-treated group. Significant increases in intracellular glucose (P = 0.006) and glucose uptake (P = 0.008) were also found in PPP-treated KNS42 cells. These changes indicate an up-regulation of glucose metabolism following PPP treatment and a subsequent increase in lactate production. Twenty-four hours PPP treatment caused a significant decrease in cell number when compared to controls (∼40%, P = 0.001). No significant difference in cellular protein concentration and size were found in PPP-treated cells when compared to controls, indicating that the observed increases in glucose metabolism, lactate production and LDH activity are due to changes in cellular metabolism following PPP treatment and not from changes in cellular size or protein concentration. The increase in lactate production following PPP treatment found in this study is an unusual observation, as most cancer targeting-treatments cause a decrease in lactate production. Hence, the use of DNP and 13C MRS to measure lactate production could provide a more specific non-invasive biomarker of response for PPP treatment. Conclusions: PPP treatment resulted in increased glucose metabolism and lactate production in KNS42 cells, as shown by both real time and steady-state measurements. These changes have potential as specific biomarkers of response for PPP treatment. We acknowledge the support received from the CRUK and EPSRC Cancer Imaging Centre with the MRC and Department of Health (England) grant C1060/A10334, NHS funding to the NIHR Biomedical Research Centre.