Abstract
Activation of the nuclear transcription factor-κB (NF-κB) has been implicated in liver tumorigenesis. We evaluated the effects of a novel NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), in two human liver cancer cell lines HA22T/VGH and HuH-6. DHMEQ treatment dose dependently decreased the DNA-binding capacity of the NF-κB p65 subunit, inhibited cell growth and proliferation, and increased apoptosis as shown by caspase activation, release of cytochrome c, poly(ADP-ribose) polymerase cleavage, and down-regulation of survivin. DHMEQ also induced a dose-dependent activation of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling, and inhibition of this pathway significantly reduced cell growth. It is noteworthy that we observed that DHMEQ stimulated reactive oxygen species (ROS) production in a dose-dependent manner and that pretreatment of the cells with the antioxidant N-acetyl-l-cysteine (NAC) significantly reduced DHMEQ-induced ROS generation. Accordingly, NAC completely reversed the DHMEQ-induced growth inhibition, caspase activation, and cell death. DHMEQ-treated cells exhibited DNA damage, as evaluated by accumulation in nuclear foci of phospho-H2AX, which was completely reversed by NAC. Moreover, DHMEQ induced the expression of genes involved in the endoplasmic reticulum stress response (GRP78, CHOP, TRB3) and promoted the splicing of XBP1 mRNA in a dose-dependent fashion in both cell lines, which was reversed in the presence of NAC. Knockdown of TRB3 mRNA expression by small interference RNA significantly decreased DHMEQ-induced cell growth inhibition. These data suggest that DHMEQ antitumor effects are primarily mediated through ROS generation. Thereby, considering that cancer cells are under increased ER stress and oxidative stress conditions, DHMEQ may greatly improve various anticancer strategies.
Footnotes
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This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC) and the Italian National Research Council (CNR) [Istituto di Biomedicina e Immunologia Molecolare]
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ABBREVIATIONS: HCC, hepatocellular carcinoma; ROS, reactive oxygen species; ERK, extracellular signal-regulated kinase; ER, endoplasmic reticulum; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase; GRP78, glucose-regulated protein 78; CHOP, CAAT/enhancer-binding protein homologous transcription factor; TRB3, tribbles homolog 3; NF-κB, nuclear factor-κB; DHMEQ, dehydroxymethylepoxyquinomicin; NAC, N-acetyl-l-cysteine; FBS, fetal bovine serum; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; AM, acetoxymethyl ester; TTFA, thenoyltrifluoroacetone; DPI, diphenyleneiodonium; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene; BrdU, 5-bromo-2′-deoxyuridine; PARP, poly(ADP-ribose) polymerase; RT-PCR, reverse transcription-polymerase chain reaction; H2DCFDA, 2′,7′-difluorodihydrofluorescein diacetate; PBS, phosphate-buffered saline; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; MRC, mitochondrial respiratory chain; XBP1, X-box binding protein 1.
- Accepted May 20, 2009.
- Received February 16, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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