3-Bromopyruvate Regulates the Status of Glycolysis and BCNU Sensitivity in Human Hepatocellular Carcinoma Cells.

Chloroethylnitrosoureas (CENUs) are bifunctional antitumor alkylating agents, which exert their antitumor activity through inducing the formation of dG-dC interstrand crosslinks (ICLs) within DNA double strand. However, the complex process of tumor biology enables tumor cells to escape the killing triggered by CENUs, as for instance with the detoxifying activity of O6-methylguanine DNA methyltransferase (MGMT) to accomplish DNA damage repair. Considering the fact that most tumor cells highly depend on aerobic glycolysis to provide energy for survival even in the presence of oxygen (Warburg effect), inhibition of aerobic glycolysis may be an attractive strategy to overcome the resistance and improve the chemotherapeutic effects of CENUs. Especially, 3-bromopyruvate (3-BrPA), a small molecule alkylating agent, has been emerged as an effective glycolytic inhibitor (energy blocker) in cancer treatment. In view of its tumor specificity and inhibition on cellular multiple targets, it is likely to reduce the chemoresistance when chemotherapeutic drugs are combined with 3-BrPA. In this study, we investigated the effects of 3-BrPA on the chemosensitivity of two human hepatocellular carcinoma (HCC) cell lines to the cytotoxic effects of l,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and the underlying molecular mechanism. The sensitivity of SMMC-7721 and HepG2 cells to BCNU was significantly increased by 2 h pretreatment with micromolar dosage of 3-BrPA. Moreover, 3-BrPA decreased the cellular ATP and GSH levels, and extracellular lactate excreted by tumor cells, and the effects were more effective when 3-BrPA was combined with BCNU. Cellular hexokinase-II (HK-II) activity was also reduced after exposure to the treatment of 3-BrPA plus BCNU. Based on the above results, the effects of 3-BrPA on the formation of dG-dC ICLs induced by BCNU was investigated by stable isotope dilution high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The results indicated that BCNU produced higher levels of dG-dC ICLs in SMMC-7721 and HepG2 cells pretreated with 3-BrPA compared to that without 3-BrPA pretreatment. Notably, in MGMT-deficient HepG2 cells, the levels of dG-dC ICLs were significantly higher than MGMT-proficient SMMC-7721 cells. In general, these findings revealed that 3-BrPA, as an effective glycolytic inhibitor, may be considered as a potential clinical chemosensitizer to optimize the therapeutic index of CENUs.