Cancer Letters

Cancer Letters

Volume 322, Issue 1, 1 September 2012, Pages 35-44
Cancer Letters

The novel NF-κB inhibitor DHMEQ synergizes with celecoxib to exert antitumor effects on human liver cancer cells by a ROS-dependent mechanism

https://doi.org/10.1016/j.canlet.2012.02.008Get rights and content

Abstract

In a previous work of ours dehydroxymethyl-epoxyquinomicin (DHMEQ), an inhibitor of NF-κB, was shown to induce apoptosis through Reactive Oxygen Species (ROS) production in hepatoma cells. The present study demonstrated that DHMEQ cooperates with Celecoxib (CLX) to decrease NF-κB DNA binding and to inhibit cell growth and proliferation more effectively than treatment with these single agents alone in the hepatoma cell lines HA22T/VGH and Huh-6. ROS production induced by the DHMEQ–CLX combination in turn generated the expression of genes involved in endoplasmic reticulum (ER) stress and silencing TRB3 mRNA significantly decreased DHMEQ–CLX-induced cell growth inhibition. Moreover, the DHMEQ–CLX combination was associated with induction of PARP cleavage and down-regulation of the anti-apoptotic proteins Bcl-2, Mcl-1 and survivin, as well as activated Akt. CD95 and CD95 ligand expression increased synergistically in the combination treatment, which was reversed in the presence of NAC. Knockdown of CD95 mRNA expression significantly decreased DHMEQ–CLX-induced cell growth inhibition in both cell lines. These data suggest that the DHMEQ–CLX combination kills hepatoma cells via ROS production, ER stress response and the activation of intrinsic and extrinsic apoptotic pathways.

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.

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