Metformin—an Adjunct Antineoplastic Therapy—Divergently Modulates Tumor Metabolism and Proliferation, Interfering with Early Response Prediction by 18F-FDG PET Imaging

Abstract

Over the last several years, epidemiologic data have suggested that the antidiabetes drug metformin (MET), an adenosine monophosphate-activated protein kinase (AMPK) activator, improves progression-free survival of patients with multiple cancers; more than 30 clinical trials are under way to confirm this finding. We postulated that the role of AMPK as a central cellular energy sensor would result in opposite effects on glucose uptake and proliferation, suggesting different roles for (18)F-FDG and 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) in assessing its effectiveness as an antineoplastic agent. Colon cancer cell lines HT29 (human) and MC26 (murine) were treated for 24 or 72 h with a range of MET concentrations (0-10 mM). Western blotting was used to study the activation of AMPK after MET treatment. Glucose uptake and cell proliferation were measured by cell retention studies with either (18)F-FDG or (18)F-FLT. EdU (ethynyl deoxyuridine, a thymidine analog) and annexin-propidium iodide flow cytometry was performed to determine cell cycle S-phase and apoptotic changes. In vivo (18)F-FDG and (18)F-FLT PET images were acquired before and 24 h after MET treatment of HT29 tumor-bearing mice. After 24 h of MET incubation, phosphorylated AMPK levels increased severalfold in both cell lines, whereas total AMPK levels remained unchanged. In cell retention studies, (18)F-FDG uptake increased but (18)F-FLT retention decreased significantly in both cell lines. The numbers of HT29 and MC26 cells in the S phase decreased 36% and 33%, respectively, after MET therapy. Apoptosis increased 10.5-fold and 5.8-fold in HT29 and MC26 cells, respectively, after 72 h of incubation with MET. PET imaging revealed increased (18)F-FDG uptake (mean ± SEM standardized uptake values were 0.71 ± 0.03 before and 1.29 ± 0.11 after MET therapy) (P < 0.05) and decreased (18)F-FLT uptake (mean ± SEM standardized uptake values were 1.18 ± 0.05 before and 0.89 ± 0.01 after MET therapy) (P < 0.05) in HT29 tumor-bearing mice. MET, through activation of the AMPK pathway, produces a dose-dependent increase in tumor glucose uptake while decreasing cell proliferation in human and murine colon cancer cells. Thus, changes in (18)F-FDG uptake after MET treatment may be misleading. (18)F-FLT imaging is a promising alternative that correlates with the tumor response.