NFκB inhibitors induce cell death in glioblastomas

Abstract

Identification of novel target pathways in glioblastoma (GBM) remains critical due to poor prognosis, inefficient therapies and recurrence associated with these tumors. In this work, we evaluated the role of nuclear-factor-kappa-B (NFκB) in the growth of GBM cells, and the potential of NFκB inhibitors as antiglioma agents. NFκB pathway was found overstimulated in GBM cell lines and in tumor specimens compared to normal astrocytes and healthy brain tissues, respectively. Treatment of a panel of established GBM cell lines (U138MG, U87, U373 and C6) with pharmacological NFκB inhibitors (BAY117082, parthenolide, MG132, curcumin and arsenic trioxide) and NFκB-p65 siRNA markedly decreased the viability of GBMs as compared to inhibitors of other signaling pathways such as MAPKs (ERK, JNK and p38), PKC, EGFR and PI3K/Akt. In addition, NFκB inhibitors presented a low toxicity to normal astrocytes, indicating selectivity to cancerous cells. In GBMs, mitochondrial dysfunction (membrane depolarization, bcl-xL downregulation and cytochrome c release) and arrest in the G2/M phase were observed at the early steps of NFκB inhibitors treatment. These events preceded sub-G1 detection, apoptotic body formation and caspase-3 activation. Also, NFκB was found overstimulated in cisplatin-resistant C6 cells, and treatment of GBMs with NFκB inhibitors overcame cisplatin resistance besides potentiating the effects of the chemotherapeutics, cisplatin and doxorubicin. These findings support NFκB as a potential target to cell death induction in GBMs, and that the NFκB inhibitors may be considered for in vivo testing on animal models and possibly on GBM therapy.

Introduction

Glioblastoma (GBM) is an aggressive, invasive, and difficult to treat primary brain tumor. Standard therapy includes surgical resection, external beam radiation and chemotherapy, with no known curative therapy [1]. A number of dysregulated signaling cascades have been described in GBMs, including the MEK/ERK pathway, PLC/PKC pathway, and the PI3K/Akt pathway. Dysregulation of these pathways is driven by mutation, amplification, or overexpression of multiple genes such as PTEN, EGFR, PDGFR-a, p53, and mTOR [2], [3], [4]. Understanding these dysregulated pathways has provideded the basis for designing molecular targeted therapies as monoclonal antibodies against EGF, VEGF and PDGF receptors, as well as new combination therapies and drug delivery systems [4], [5], [6]. Despite these new treatment strategies, median survival has remained approximately 1 year for decades [1]. Thus, it is urgent to determine novel molecular targets in GBMs in order to develop more effective therapies and new therapeutic opportunities to patients.

Studies have compelling evidence that the transcription factor NFκB (Nuclear factor κB) plays a role in the control of oncogenesis, tumor progression and chemotherapy resistance of diverse types of malignances as lymphoma, leukemia, breast and ovarian cancers [7], [8], [9], [10], [11]. NFκB is formed by homo or heterodimers comprising members of the Rel family of proteins (p50/p105, p52/p100, p65, c-Rel and RelB) which form, upon non-stimulated conditions, a ternary and inactive cytoplasmic complex by interacting with inhibitory proteins of the IκB family. Upon stimulation by cytokines (TNF-alpha and IL1-beta), growth factors (EGF and PDGF), anticancer drugs (cisplatin, doxorubicin and vincristine) and other stressor stimuli, IκBs are phosphorylated by IKK (IκB kinase) proteins thus releasing the active NFκB, which translocates into nucleus and binds to DNA sequences in gene promoters. NFκB binding to DNA modulates the expression of a wide range of genes as that involved in inflammation (IL1-beta, IL-6, COX2, and TNF), apoptosis resistance (bcl-xL, cIAP1/2, XIAP, FLICE and survivin), cell invasion (ICAM-1, VCAM-1, MMP2, MMP9), angiogenesis (VEGF), proliferation (cyclin D1, MYC) and metastasis (CXCR4 and TWIST) [7], [8], [9], [10], [11], [12]. These groups of genes are directly related to tumor-associated phenomena suggesting NFκB as a potential target to cell death induction and chemosensitization in cancer [9], [13], [14], [15].

Aberrant NFκB activity has been described in some types of cancer cells upon basal and following anticancer drugs treatments. Besides, constitutive NFκB up-regulation has been found in chemotherapy-resistant cell lines, which correlates with therapy failure [9], [13], [15], [16], [17]. In this context, studies have suggested that molecules with NFκB inhibitory properties are potential anticancer agents [9], [15], [17], [18]. In this intent, researchers have blocking NFκB activity via IKK/NFκB inhibitors (as BAY117082, BAY117085, parthenolide, curcumin, arsenic trioxide and PDTC) or proteasome/NFκB inhibitors (PS-341 and MG132) in order to inhibit the growth of myeloma [10], leukemia [17], [19], esophageal [14] and breast cancer cells [20]. Recently, analysis of brain tumor biopsies identified that NFκB and its target genes are overexpressed in GBM and astrocytoma tumors compared to normal brain tissues [21], [22]. In addition, a positive correlation between NFκB activation and poor GBM prognosis was reported, suggesting that the known role of NFκB as a survival factor in other cancers could also be considered in GBMs [23]. Taken into account the aforementioned, this study was undertaken in order to determine the role of NFκB in GBM growth, and the selectivity, apoptotic potential and chemosensitizing activity of the NFκB inhibitors BAY117082, parthenolide, curcumin, arsenic trioxide, MG132, and p65 small-interfering RNA in GBM cell lines.