P-0076 18F-FDG and Gossypol: Studies in Hepatocellular Carcinoma

Abstract

IntroductionMalignant cells require high energy via glycolytic generation of ATP for cell proliferation and survival, a process that is thought to be mediated by glucose transporters (GLUTs). Notably, GLUT1 is overexpressed and confers poor prognosis in a wide range of solid tumors, including liver tumors. A recent study has shown that expression of GLUT1 is increased in Hepatocellular Carcinoma (HCC) and promotes tumorigenesis. Conversely, suppression of GLUT1 expression by small interfering RNA significantly impaired tumorigenicity of HCC cells in in vitro models, suggesting that GLUT1 as an innovative therapeutic target for this highly aggressive tumor. Positron Emission Tomography (PET) using the radiolabeled glucose analogue 18F-FDG enables the detection of increased glycolysis events in cancer cells. Glucose is transported into the cell mainly through the GLUT-1 and -3. Gossypol is a natural compound that has demonstrated anticancer effect in several tumor types. It is known that gossypol is a competitive inhibitor of the GLUT1. The aim of this study is to test the anticancer effect of gossypol in HCC cell lines, as well as check its effect on 18F-FDG uptake. MethodsThe cell lines used are HepG2 (wp53), HuH7 (mp53) and Hep3b2.1-7 (p53 null). These cell lines were propagated in the presence of gossypol in several concentrations. Cell proliferation was evaluated by the MTT test, in order to obtain dose-response curves. After that the type of cell death (necrosis or apoptosis) and the percentage of live cells were assessed by flow cytometry for the concentration of drug able to induce half maximal inhibitory concentration (IC50).For uptake studies, 18F-FDG was incubated in a cell suspension with 2×106 cells/ml (25µCi/ml) in cells pre-incubated with gossypol and control cells. Samples were collected to Eppendorf tubes for tracer uptake calculation. Eppendorfs were then centrifuged and radioactivity of cell pellets and supernatants was measured with a well-type gamma counter. ResultsThis study demonstrates that the concentrations of gossypol necessary to achieve the IC50 is higher for HuH7 cell line (IC50(24h)=7.5µM). Interestingly, the cell line more sensitive to this compound is Hep3B2.1-7 (IC50(24h)=2.6µM), which may be related to the fact that these cells do not express p53. The cellular response is dependent on time, for each of the cell lines studied. Flow cytometry results show that gossypol induces high apoptosis in HepG2 and HuH7 cell lines with lower induction of necrosis. In Hep3B2.1-7 cell line there is a balance between apoptosis and necrosis. In addition, for the three cell lines studied, gossypol was able to decrease the percentage of 18F-FDG uptake in the similar way. ConclusionThese results shown that gossypol has anti-proliferative effect on HCC. Gossypol also has shown ability to decrease the uptake of 18F-FDG in the three cell lines. More studies must be done to clarify the role of p53 in the anti-proliferative effect of this compound in HCC as well as the effect in the uptake of the 18F-FDG. These preliminary results are quite useful in clinical practice.