The novel NF-κB inhibitor DHMEQ synergizes with celecoxib to exert antitumor effects on human liver cancer cells by a ROS-dependent mechanism
Introduction
Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Several strategies have been suggested for the treatment of HCC patients but, unfortunately, it still has a high lethality. Therefore, novel approaches are required to contrast this tumor.
The nuclear transcription factor-κB (NF-κB) has been implicated in carcinogenesis because it plays a critical role in cell survival, inflammation and cell growth. Recent studies indicate that NF-κB is essential for promoting inflammation-associated cancers and it is therefore a potential target for cancer prevention [1]. Several reports have indicated that NF-κB is constitutively activated in a variety of cancer cells, including hepatocellular carcinoma [2].
Dehydroxymethyl-epoxyquinomicin (DHMEQ) is a novel NF-κB inhibitor which induces apoptosis and cell-cycle arrest in several cancer cell types [3], [4], [5]. We previously demonstrated that DHMEQ promotes ROS generation in human liver cancer cells and that oxidative stress induces ER stress response, DNA damage and release of cytochrome c with activation of the caspase cascade [6].
There are two isoforms of cyclooxygenases (COXs), COX-1 and COX-2, the latter being induced by a variety of stimuli. COX-2 is markedly elevated in many types of tumor, including HCC, as selective COX-2 inhibitors (COXIBs) show anti-proliferative and pro-apoptotic effects in human liver cancer cells [7], [8] suggesting that COXIBs might be effective in HCC treatment. Accumulating evidence suggests that COXIBs inhibit cell proliferation through a COX-2-independent mechanism [9], [10], [11]. The molecular mechanism underlying CLX-mediated apoptosis seems to be associated with the induction of ER stress response through calcium [12] and with the down-regulation of the anti-apoptotic protein survivin [13]. CLX induces the expression of functional death receptors, such as CD95, and a rapid down-regulation of myeloid cell leukemia-1 (Mcl-1) protein, suggesting the activation of intrinsic and extrinsic apoptosis pathways in HCC [14]. CLX has been reported to act synergistically with different drugs in promoting the apoptosis of human liver tumor cells [15]. There are two different apoptotic pathways: death receptor and mitochondrial [16], [17]. Ligation of cell surface death receptors, including CD95 with its specific ligand, triggers a death receptor apoptotic pathway. Fas-associated Death Domain (FADD) is first recruited to the death receptor, followed by the association with pro-caspase 8, which is activated by cleavage. Activation of the mitochondrial apoptotic pathway depends on the release of cytochrome c and consequently the activation of caspase-3/7, -9, which in turn cleaves PARP and inhibits anti-apoptotic proteins, such as survivin. Cytotoxic drugs that cause DNA damage either trigger cell death mediated by mitochondria or induce apoptosis in a CD95-dependent manner. The exact mechanisms by which the two cascades are simultaneously activated by cytotoxic drugs are not known. However, it has been reported that the two death pathways may cross-talk, depending on the cell type or stimuli. Cross-talk may depend on Bid protein cleavage, which transduces an apoptotic signal from the cytoplasmic membrane to mitochondria [18] and also on ROS production in mitochondria, which in turn induces the release of cytochrome c and CD95 aggregation and activation of the FADD-caspase-8 cascade [19].
Therefore, the present study was set up to investigate whether CLX can potentiate the antitumor effects of DHMEQ against human liver cancer cells. We observed synergistic antitumor effects with the DHMEQ–CLX combination. Moreover, we demonstrated that DHMEQ and CLX interact to increase the activity of the intrinsic and extrinsic apoptotic pathways through CD95 activation and down-regulation of Mcl-1. These pro-apoptotic effects are mediated by the production of ROS, resulting in ER stress response.
Section snippets
Reagents and cell culture
DHMEQ was synthesized as previously described [20]. NAC was purchased from Sigma–Aldrich (Milan, Italy). Celecoxib was a gift from the Pfizer Corporation (New York, USA) and was dissolved in dimethyl sulfoxide (DMSO). The human liver cancer cell lines Huh-6, HA22T/VGH and Huh-7 used in this study were a gift from Prof. Massimo Levrero (Department of Internal Medicine, Sapienza University, Rome, Italy). They have different characteristics of differentiation, biological behavior and genetic
DHMEQ–CLX combination synergistically inhibits cell growth, NF-κB p65 DNA-binding capacity, and cell proliferation
DHMEQ has been reported to reduce NF-κB p65 DNA-binding capacity and cell growth in human liver cancer cells [6]. There is increasing evidence that CLX has an anti-cancer activity, associated with its ability to decrease cell survival in HCC [14]. Moreover, it has been reported that the anti-inflammatory activity of CLX depends on the inhibition of NF-κB p65 activation and translocation into the nucleus [10], [23].
To determine the potential of DHMEQ to inhibit cell growth, we examined its
Discussion
NF-κB is a major stress-inducible anti-apoptotic transcription factor [24] which is frequently activated in many types of cancer, including HCC [2]. The NF-κB inhibitor DHMEQ has been reported to induce apoptosis in liver cancer cells through the induction of oxidative stress, mainly via the mitochondrial pathway [6].
The tumorigenic potential of COX-2 over-expression has frequently been associated with resistance to apoptosis in certain types of tumors, including HCC. Celecoxib, a selective
Acknowledgments
CM and GM have been supported in part by Grants PRIN 2008 and FIRB-MERIT n. RBNE08YYBM from the Italian Ministry for Education, the University and Research MIUR. This work was supported in part by a Grant to the CNR from the Italian Ministry of Economy and Finance for the Project FaReBio di Qualità.
References (46)
- et al.
Inflammation and cancer: how hot is the link?
Biochem. Pharmacol.
(2006) - et al.
Activation of NF-κB, AP-1 and STAT transcription factors is a frequent and early event in human hepatocellular carcinomas
J. Hepatol.
(2002) - et al.
Proapoptotic and antiproliferative potential of selective cyclooxygenase-2 inhibitors in human liver tumor cells
Hepatology
(2002) - et al.
Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis
Cell
(1998) - et al.
Preparation and biological activities of optically active dehydroxymethylepoxyquinomicin, a novel nuclear factor-κB inhibitor
Tetrahedran
(2004) - et al.
Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors
Adv. Enzyme Reg.
(1984) - et al.
Celecoxib potently inhibits TNF-α-induced nuclear translocation and activation of NF-κB
Biochem. Pharmacol.
(2008) - et al.
Oxidative stress and nuclear factor-κB activation: a reassessment of the evidence in the light of recent discoveries
Biochem. Pharmacol.
(2000) - et al.
Interrelated roles for Mcl-1 and BIM in regulation of TRAIL-mediated mitochondrial apoptosis
J. Biol. Chem.
(2006) - et al.
Cyclooxygenase-2 inducing Mcl-1-dependent survival mechanism in human lung adenocarcinoma CL1.0 cells. Involvement of phosphatidylinositol 3-kinase/Akt pathway
Biol. Chem.
(2001)
Enhanced killing of chemo-resistant breast cancer cells via controlled aggravation of ER stress
Cancer Lett.
Induction of apoptosis by cigarettes smoke via ROS-dependent endoplasmic reticulum stress and CAAT/enhancer-binding protein-homologous protein (CHOP)
Free Rad. Biol. Med.
Interaction between caspase-8 activation and endoplasmic reticulum stress in glycochenodeoxycholic acid-induced apoptotic HepG2 cells
Toxicology
HM. Shen, CN. Ong, Role of intracellular thiol depletion, mitochondrial dysfunction and reactive oxygen species in Salvia Miltiorrhiza-induced apoptosis in human hepatoma HepG2 cells
Life Sci.
Caspase-dependent apoptosis and independent- poly(ADP-ribose) polymerase cleavage induced by transforming growth factor B1
Int. J. Biochem. Cell Biol.
Role of reactive oxygen intermediates in activation-induced CD95 (APO-1/Fas) ligand expression
Biol. Chem.
Induction of thyroid cancer cell apoptosis by a novel nuclear factor κB inhibitor, dehydroxymethyl-epoxyquinomicin
Clin. Cancer Res.
DHMEQ, a novel NF-kB inhibitor, induces apoptosis and cell-cycle arrest in human hepatoma cells
Int. J. Oncol.
Antitumor effects of the novel NF-κB inhibitor dehydroxymethyl-epoxyquinomicin on human hepatic cancer cells analysis of synergy with cisplatin and of possible correlation with inhibition of pro-survival genes and IL-6 production
Int. J. Oncol.
Antitumor effects of dehydroxymethylepoxyquinomicin, a novel nuclear factor-κB inhibitor, in human liver cancer cells are mediated through a reactive oxygen species-dependent mechanism
Mol. Pharmacol.
Induction of apoptosis and inhibition of cell growth in human hepatocellular carcinoma cells by COX-2 inhibitors
Ann. N. Y. Acad. Sci.
Targeting apoptosis pathway by celecoxib in cancer
Cancer Lett.
COX-2-Dependent and COX-2-Independent mode of action of celecoxib in human liver cancer cells
Omics
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