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Fig. 3 | Cancer & Metabolism

Fig. 3

From: A precision therapeutic strategy for hexokinase 1-null, hexokinase 2-positive cancers

Fig. 3

DPI is a synthetically lethal partner in combination with HK2 knockdown or inhibition in HK1HK2+ liver cancer cells. a A high-throughput screen of 3205 drug-like compounds and 119 FDA-approved oncology drugs identified DPI as the best synergistic agent with HK2 knockdown in Hep3B/shHK2DOX cells. Primary screening of all compounds at 10 μM was performed in combination with DOX-induced HK2 knockdown in Hep3B/shHK2DOX cells. Growth inhibition was measured by the CellTiter-Glo assay (left panel). Ninety-two compounds with z scores ≤ − 3 were tested in dose response curves (dose response concentrations 0, 0.0006, 0.0025, 0.01, 0.039, 0.156, 0.625, and 2.5 μM) in Hep3B/shHK2DOX cells cultured with or without DOX. Viable cells were normalized to DMSO-treated cells with or without DOX, respectively (right panel). The dots represent compounds at the tested concentrations. The red line connects the individual DPI concentration points. The chemical structure of DPI is shown. b DPI is synthetically lethal with HK2 knockdown or inhibition in HK1HK2+ liver cancer cells. DOX/DPI 2-day DOX pretreatment prior to 72 h DPI (100 nM) treatment. FDG/DPI 72 h FDG (1 mM) and/or DPI (100 nM) treatments. Viability percentages were determined by trypan blue staining. c FDG/DPI combination irreversibly inhibits Hep3b and Huh7 HK1HK2+ liver cancer cell proliferation. Viable cells were determined by trypan blue staining. d DPI decreases the oxygen consumption rate (OCR) of Hep3B cells. Cells were exposed to 100 nM DPI or vehicle for 2 h before OCR measurements. e DPI increases glycolysis of HK1HK2+ liver cancer cells. Cells were pretreated with vehicle or DOX for 72 h prior to exposure to DPI (100 nM). Twenty-four hours later, culture media were analyzed for glucose and lactate. f, g FDG/DPI treatment activates AMPKα (f) and inactivates the mTOR pathway (g) in Hep3B cells. Cells were treated with 1 mM FDG and/or 100 nM DPI for 4 h. h FDG/DPI treatment triggers apoptosis. Hep3B HK1HK2+ cells were treated with FDG (1 mM) and/or DPI (100 nM) for 24 h prior to apoptosis analyses. i DPI (2 mg/kg) enhances inhibition of tumor progression in response to HK2 silencing in Hep3B/shHK2DOX tumors. Mice-bearing tumor xenografts were treated when tumors reached 200 mm3 (day 0). j Effects of HK2 knockdown and/or DPI (2 mg/kg) treatment on HK2 expression, AMPKα activation, and mTOR pathway inactivation in the xenograft Hep3B/shHK2DOX tumors described in panel i. Each data point in ac and e represents mean ± SD of triplicate samples, and each data point in d and i represents mean ± SEM of five samples. *P < 0.05, ***P < 0.001

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