Rationale and Objectives
In spite of various therapies developed, hepatocellular carcinoma still shows poor prognosis. In this study, we introduced ethylbromopyruvate (EBrP), a hydrophobic derivative of 3-bromopyruvate, as an agent for intraarterial therapy of hepatocellular carcinoma.
Materials and Methods
In in vitro study, we evaluated whether EBrP induced apoptotic cell death in Huh-BAT cells. Chemical degradation products of EBrP were identified by performing proton nuclear magnetic resonance spectroscopy and thin layer chromatography. VX2 carcinoma was implanted and grown in the liver of 25 rabbits for in vivo study. By transfemoral intraarterial approach, 0.4 mL of 10 mM and 40 mM EBrP dissolved in an iodized oil (Lipiodol) was infused into the proper hepatic artery in 8 and 10 rabbits, respectively. In the remaining seven rabbits, 0.4 mL of Lipiodol alone was intraarterially injected as a control. One week later, tumor necrosis rate was calculated with histopathologic examination and hepatotoxicity was evaluated with biochemical analysis.
Results
EBrP induced apoptosis in human HCC cells via mitochondrial apoptotic signaling cascades. EBrP dissociated into 3-bromopyruvate and ethanol in the aqueous environment. In VX2 liver tumor models, the group of intraarterial delivery of 40 mM EBrP/Lipiodol solution showed higher tumor necrosis rates (96.1% ± 3.8) than the other groups (38.9% ± 15.9 of a control, 90.5% ± 2.9 in 10 mM) ( P < .05). There was transient elevation of AST and ALT enzyme levels without any mortality.
Conclusions
Intraarterial infusion of EBrP/Lipiodol solution is a feasible intraarterial therapy for liver tumors with potent antitumor effects and transient hepatotoxicity.
Hepatocellular carcinoma (HCC) is an aggressive cancer showing a poor prognosis even though various treatments have been developed. It is one of the most common, lethal malignancies around the world, and about 10% of patients survive 5 years after a diagnosis and the median survival period in patients with unresectable tumors is 4–6 months . Surgical resection is still the treatment of choice in patients with a good residual liver function, but only 15%–25% of HCC in patients with liver cirrhosis are resectable as most patients have a poor hepatic function and advanced stage of disease. Therefore, in advanced stage of HCC, various palliative or investigational therapies have been performed. They include intraarterial chemotherapy such as transcatheter arterial chemoembolization or infusion chemotherapy, internal or external radiotherapy, and newly developed systemic anticancer drugs targeting tumor angiogenesis. Hepatic intraarterial chemotherapy, which is based on the dominant blood supply of HCC from the hepatic artery and the direct delivery of high-dose anticancer agents, is widely used, especially in Asian countries, to treat HCC using various chemotherapeutic drugs such as mitomycin, doxorubicin, cisplatin, 5-fluorouracil, and epirubicin. However, its therapeutic efficacy is still limited and its overall results remain unsatisfactory .
Recently, 3-bromopyruvate (3-BrP), a hexokinase II inhibitor, was introduced as a novel intraarterial therapy for liver cancer and its potential anticancer effects have been studied . Highly malignant cells such as HCC spend more energy in keeping their functions and growth than normal cells, so they are always short of energy called ATP no matter how much energy they produce effectively through an oxygen-dependent mitochondrial ATP-generating system. The shortage spurs them to conduct a salvage pathway called a glycolytic system converting glucose into lactate in a cell, which is then taken out of the cell by specialized transporters . Hexokinase is the first-step enzyme in the pathway and it is essential in maintaining the high glycolytic phenotype. Hexokinase II among the four mammalian types (I-IV) is the predominantly overexpressed form in HCC . 3-BrP selectively blocks hexokinase II to inhibit the pathway and induces depletion of ATP and loss of cell viability . 3-BrP is known also to inhibit glyceraldehyde-3-phosphate dehydrogenase and to overcome protective autophagy of tumor cells, a cellular response to stress resisting to chemotherapy, and induce apoptosis in the end . In vitro and in vivo studies presented that 3-BrP has anticancer effects and that it can be an effective anticancer agent in advanced malignant tumors . Nonetheless, 3-BrP was rapidly neutralized in vivo and had a single-pass effect during rather short intraarterial infusion . Therefore, it will be meaningful to find out chemical agents analogous to 3-BrP will take a role as a hexokinase II inhibitor and stay in the tumor neovasculature with prolonged release and action.
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Materials and methods
Preparation of EBrP/Lipiodol Solutions
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Stability of EBrP Solutions
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In Vitro Study
Cell line and culture
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Quantitation of apoptosis
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Preparation of mitochondrial and cytosolic extracts
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Immunoblot analysis
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In Vivo Study
Rabbit VX2 liver tumor model and experimental groups
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Transcatheter arterial embolization
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Evaluation of antitumor effects
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Measurement of plasma biochemicals
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Statistical Analysis
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Results
Degradation of Ethylbromopyruvate in Aqueous Solutions
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EBrP Induces Apoptosis in Human HCC Cells via Mitochondrial Apoptotic Signaling Cascades
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Antitumor Effects and Hepatotoxicity of EBrP
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Discussion
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