Inhibition of protein kinase CK2 by quercetin enhances CD95-mediated apoptosis in a human thymus-derived T cell line

Highlights

• Quercetin is not cytotoxic per se, but sensitizes HPB-ALL cell line to apoptosis.

• Protein kinase CK2 is overexpressed in leukemias and elevated levels of CK2 activity have been associated with an inferior prognosis.

• Quercetin inhibits CK2 activity in an in vitro assay and in HPB-ALL cells.

• CK2 inhibition by quercetin may contribute to sensitize HPB-ALL cells to CD95-induced apoptosis.

Abstract

Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is one of the major dietary flavonoid, found in a broad range of fruits, vegetables and beverages, such as tea and wine, with a daily uptake between 10 and 100 mg depending on the dietary attitude. We previously demonstrated that quercetin sensitizes HPB-ALL cell line to CD95-mediated apoptosis through a mechanism independent of its antioxidant properties and related to its ability to regulate the activity of serine–threonine protein kinases. Here, we report that quercetin is an inhibitor of protein kinase CK2, a kinase involved in apoptotic resistance in several forms of leukemia. Quercetin inhibits CK2 in an in vitro assay and in HPB-ALL cells decreasing its activity of 3.5-fold within 12 h from treatment, in good agreement with the intracellular concentration of the molecule measured by HPLC on the same cells (0.933 ± 0.188 μg/mg of total proteins). Quercetin also inhibits pure CK2 enzyme with an EC50 of about 150 nM in an in vitro assay. A specific CK2 inhibitor, TBB (4,5,6,7-tetra-bromo-benzo-triazole), behaves similarly to quercetin since it abolishes CK2 activity in HPB-ALL cells, but fails to enhance CD95-mediated apoptosis when associated with an anti-CD95 monoclonal antibody mimicking CD95 ligand. The resistance to apoptotic induction in HPB-ALL can be bypassed taking advantage of the pleiotropic activity of quercetin. Combined treatments which include quercetin in association with death receptor inducers may represent a novel therapeutic strategy against leukemia.

Introduction

Many dietary compounds have been identified as potential chemopreventive agents. These include vitamins, minerals, carotenoids, and the large class of phytochemicals (polyphenols, isothiocyanates, organosulfur compounds) (Bilotto et al., 2013). Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is one of the major dietary flavonoid, found in a broad range of fruits, vegetables and beverage such as tea and wine, with a daily uptake between 10 and 100 mg depending on eating habits (Bischoff, 2008, Hertog et al., 1993). This molecule possesses antioxidant, anti-inflammatory, anti-angiogenic, anti-proliferative and pro-apoptotic effects (Lamson and Brignall, 2000, Russo et al., 2012). At molecular level, quercetin effects are multiple, but not fully understood. It modulates cellular neoplastic phenotype by down-regulating the expression of oncogenes (H-ras, c-myc and K-ras) and antioncogenes (p53) (Avila et al., 1996, Ranelletti et al., 2000), or up-regulating p21^WAF1 and p27^KIP1 (Casagrande & Darbon, 2001). Quercetin effects on cell proliferation also depend on its ability to inhibit different tyrosine and serine–threonine kinases, such as PKC, PI₃K, JNK and CK2 (Russo et al., 2014), and also kinases whose activities are linked to survival pathways (MAPK, Akt/PKB) (Nguyen et al., 2004, Spencer, Rice-Evans and Williams, 2003).

There are two main pathways by which apoptosis can be activated: the extrinsic (death receptor-mediated), which includes CD95, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) and other members of TNF receptor gene superfamily, and the intrinsic (mitochondrial dependent) pathway. CD95(Fas/Apo-1) is one of 6 cellular receptor of TNF superfamily having a death domain (DD) in its cytoplasmic portion (Lavrik and Krammer, 2012, Russo, Mupo, Spagnuolo and Russo, 2010). Several tumor cells are resistant to CD95 induced apoptosis, revealing that many molecular aspects of CD95 signaling remain unknown (Russo, Mupo, et al., 2010). We demonstrated that quercetin in association with death receptor (DR) inducers, such as an agonistic antibody (anti-CD95) mimicking CD95 ligand and a recombinant form of TRAIL (rTRAIL), increases synergistically apoptosis in different cell lines (Russo et al., 2007, Russo et al., 1999, Russo et al., 2003). This effect is independent from the antioxidant activity of the molecule. This combination strategy might contribute to kill cancer cells through different targets. More recently, we also demonstrated that in an ex vivo model of chronic lymphocytic leukemia (CLL), quercetin, at low doses (10–20 μM), is able to sensitize B-cells isolated from patients to DR-induced apoptosis (Russo et al., 2010).

Protein kinase CK2 (formerly known as casein kinase 2) is a ubiquitous, highly pleiotropic, and constitutively active dual specificity kinase that phosphorylates serine/threonine and tyrosine residues. CK2 is generally described as a stable tetramer composed of two catalytic (α and/or α′) and two regulatory subunits (β) differently associated to form a α₂β₂, α′₂β₂, or α′αβ₂ complex. Both subunits are regulated by phosphorylation and auto-phosphorylation events. CK2 catalyzes the phosphorylation of a great number of substrates presenting multiple acidic residues surrounding the phosphor-acceptor amino acid (Multi-author review, 2009). CK2 has been associated with the regulation of several biological processes. High levels of CK2 activity have been described in different types of cancer (Multi-author review, 2009). More recently, CK2 has been identified as a player in the pathogenesis of hematopoietic tumors, such as multiple myeloma (MM), CLL, acute myelogenous leukemia (AML), acute lymphoblastic leukemia (T-ALL) and chronic myeloproliferative neoplasms (Piazza et al., 2012). Accordingly, the CK2-inhibitor CX-4945 has been reported to possess pre-clinical activity across a lymphoid malignancies including CLL when combined with first-line drug, fludarabine (Prins et al., 2013). Molecular studies indicate that, in both CLL and T-ALL, CK2 phosphorylates and inhibits PTEN, leading to an over-activation of PI₃K/Akt pathway which results in an increased resistance to apoptosis induced by chemotherapeutic drugs (Barata, 2011, Martins et al., 2010, Shehata et al., 2010). In this context, inhibition of CK2 activity by specific inhibitors may represent a future therapeutic goal against leukemia.

The rationale of the present work is based on the following observations: 1. quercetin sensitizes apoptosis resistant leukemic cells to drug treatment; 2. quercetin is known as potential kinase inhibitor; 3. CK2 is a kinase conferring apoptotic resistance to hematopoietic tumors; therefore, we hypothesized that apoptogenic effect of quercetin in association with CD95 stimulation could be mediated by the inhibition of CK2 activity. This study has been performed on HPB-ALL cells, a cell line strongly resistant to apoptotic stimuli (Russo et al., 1999, Russo et al., 2003), treated with quercetin and the CK2 inhibitor TBB (4,5,6,7-tetrabromo-2-azabenzimidazole) in association with anti-CD95 agonistic antibody.