Therapeutic Potential of Thymoquinone in Glioblastoma Treatment: Targeting Major Gliomagenesis Signaling Pathways

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• Name of Publisher: BioMed Research International
• Published: 31 January 2018
• Cited by: 36
• Field of Research: Pharmacology

Abstract

Glioblastoma multiforme (GBM) is one of the most devastating brain tumors with median survival of one year and presents unique challenges to therapy because of its aggressive behavior. Current treatment strategy involves surgery, radiotherapy, immunotherapy, and adjuvant chemotherapy even though optimal management requires a multidisciplinary approach and knowledge of potential complications from both the disease and its treatment. Thymoquinone (TQ), the main bioactive component of Nigella sativa L., has exhibited anticancer effects in numerous preclinical studies. Due to its multitargeting nature, TQ interferes in a wide range of tumorigenic processes and counteract carcinogenesis, malignant growth, invasion, migration, and angiogenesis. TQ can specifically sensitize tumor cells towards conventional cancer treatments and minimize therapy-associated toxic effects in normal cells. Its potential to enter brain via nasal pathway due to volatile nature of TQ adds another advantage in overcoming blood-brain barrier. In this review, we summarized the potential role of TQ in different signaling pathways in GBM that have undergone treatment with standard therapeutic modalities or with TQ. Altogether, we suggest further comprehensive evaluation of TQ in preclinical and clinical level to delineate its implied utility as novel therapeutics to combat the challenges for the treatment of GBM.

Introduction

Glioblastoma multiforme (GBM) is a primary neuroepithelial tumor of the brain, characterized by an aggressive clinical phenotype derived from inter- and intrapatient genomic and histopathological diversity [1]. In the latest reclassification of the World Health Organization (WHO), the GBMs are listed in the group of diffuse astrocytic and oligodendroglial tumors reflecting their highly malignant behavior [2]. It constitutes more than 40% of all malignant brain tumors and approximately 54.4% of all malignant gliomas with mean age at diagnosis being 64 years and 1.5 times more common in men than women [3]. Even after the treatments by multimodal therapy that involved surgery, radiotherapy, and combined chemotherapy, GBM is nearly incurable with approximate survival rate of around 8 to 15 months after diagnosis [4]. Genomic analysis for prognostic markers of GBM has been conducted with large-scale genomic characterization. These investigations found mutations or amplifications of different signaling pathways [5–8]. The most commonly disrupted signaling cascades in GBM are pathways related to receptor tyrosine kinase (RTK), including epidermal growth factor receptor (EGFR), platelet derived growth factor receptor alpha (PDGFRA), basic fibroblast growth factor receptor 1 (FGFR-1), and insulin-like growth factor receptor (IGFR-1) [9], and nuclear factor-κB (NF-κB) [10]. GBM has also been associated with aberration in signaling through the mitogen-activated protein kinase (RAS/MAPK), phosphatase inosine 3 kinase/protein kinase B (also known as AKT)/mammalian target of rapamycin (PI3K/AKT/mTOR), cell cycle-regulating retinoblastoma (RB) tumor suppressor related pathways, tumor protein p53 (TP53) [11], promoter methylation of O-6-methylguanine-DNA methyltransferase (MGMT), isocitrate dehydrogenase (IDH) mutation, and altered expression of cyclin dependent kinase (CDK) genes [12]. The logical leap from such investigation is that targeting disrupted pathways may be an effective means of treating GBM as is the case for other type of cancers [13]. The changes in RTK, PI3K, TP53, cell cycle, neoangiogenesis, cellular metabolism, NF-κB [10], signal transducer, and activator of transcription 3 (STAT3) [14, 15] signaling pathways have already paved the way for considering them as feasible targets in GBM [10, 16, 17]. Among the RTKs, the instance of increased gene copy number of EFGR is prevalent in GBM, which is frequently responsible for increased proliferation, transformation, adhesion, migration, and escape from apoptosis [18]. Though extensive preclinical studies with GBM have shown promising results in EGFR targeting, several clinical trials designed for therapeutic targeting of EGFR in GBM patients have failed so far [19]. The lipid kinases PI3K family are situated downstream of RTKs and with the interaction of numerous intermediary signal transduction kinases (e.g., Akt, PTEN, and mTOR), control protein translation, ribosome biosynthesis, and cell growth [20, 21]. Targeting PI3K, PTEN, and mTOR pathways have shown moderate success in combination with conventional therapy at clinical level [17, 22, 23]. Alterations in cell cycle regulatory signaling pathways, for example, CDK signaling (especially mutation of CDK4, CDK6, and CDKN2A followed by E2F1 transcription factor dysregulation), and inactivation of TP53 (either dependent or independent of MDM2 mutation), have also been extensively targeted in GBM [8, 24]. Moreover, some studies with cell cycle inhibitors in GBM therapy have shown promise in radiosensitization as well as in the promotion of senescence and apoptosis of tumor cells [25, 26]. Neoangiogenesis, a characteristic histopathologic feature of GBM, is in part secondary to the hypoxic tumor microenvironment that induces hypoxia-inducible factor-1α (HIF-1α) followed by subsequent VEGF accumulation, RTK activation, fibroblast growth factor (FGF), PDGF, hepatocyte growth factor (HGF, also known as scatter factor), integrins, angiopoietins, and STAT3 upregulation [14, 27]. Depriving cell of oxygen and nutrients to halt further growth is the initial justification for targeting angiogenesis. The Food and Drug Administration (FDA) of the United States of America has already approved a monoclonal antibody targeting VEGF (Bevacizumab) in GBM therapy [4] but other interventions targeting angiogenesis have not shown improvement in overall survival in GBM [28, 29]. Clinical trials targeting multiple players in the angiogenesis pathways in GBM are underway but the outcomes are yet to be published [17]. The mutation of IDH (a component of the tricarboxylic acid cycle) occurs early in the gliomagenesis, leading towards neoenzymatic activity that converts α-ketoglutarate to 2-hydroxyglutarate and disrupts cellular metabolism in GBM [30, 31] providing rationale for considering IDH as a therapeutic target and it has already prompted several clinical trials whose outcome is yet to be available [30, 32, 33]. Glioma stem cells (GSCs) represent another viable target in GBM treatment due to their important role in mediating therapeutic resistance [34]. A number of other novel targets, such as poly-ADP ribose polymerase (PARP) (DNA repair protein), BRAF (a protein kinase that mediates MAP kinase signaling), bone-marrow X-linked kinase (BMX), Bruton’s tyrosine kinase, and gamma-secretase, are now under investigation for GBM treatment at preclinical level[17, 35–37]. However, chemotherapeutic drugs still remains the mainstay in glioblastoma treatment. At present, the chemotherapeutic drugs for GBM approved by FDA act as alkylating agents (Temozolomide (TMZ) and Nitrosourea) [4, 38] which are not sufficient to combat GBM. Based on this situation, there have always been a need to find new therapeutics for GBM. Thymoquinone (2-methyl-5-isopropyl-1, 4-benzoquinone; TQ) is the principle active ingredient of the volatile oil of black cumin or black seed (Nigella sativa L. (NS)) (family Ranunculaceae) [39]. People in different societies used NS as condiment and different traditional medicinal system such as Ayurvedic and Unani systems consider NS for the treatment of various maladies [40–43]. The pharmacological investigations of TQ [44] are almost as old as its isolation from NS in 1963 by El-Dakhakhny [45]. Since then, numerous preclinical studies have been performed including those to determine the anticancer effects of TQ. The molecular mechanism of through what TQ shows selective cytotoxicity for human cancer cells is widely reported [46]. Studies have shown that TQ causes selective cancer cell death and possess tumor growth inhibitory activities in addition to its role in interference with other tumorigenic processes such as angiogenesis, invasion, and metastasis [47, 48]. TQ is involved in tumorigenesis or development of drug resistance [49] as well as in the sensitization of cancer cells to chemotherapeutic agents and radiation therapy through the resistance mechanisms [31, 50]. The result of a registered investigation for studying the role of NS in a precancerous disease, actinic keratosis (AK), is yet to be reported (ClinicalTrials.gov Identifier: NCT01735097; website: https://clinicaltrials.gov/ct2/show/record/NCT01735097 accessed on 26 June, 2017). Hence, the usefulness of TQ in cancers including GBM is now more than a speculation and it can target different hallmarks [51] of GBM. There are several reviews on GBM [52], its pathology [53, 54], possible therapeutic targets [17, 55, 56], and current challenges in its therapies [54]. The treatment with TQ alone has shown antitumor efficacy in several in vitro and in vivo studies [57, 58] and also as in adjuvant therapy either to prevent carcinogenesis [59] or to potentiate the efficiency of conventional therapeutic modalities [60]. However, there is no systemic compilation of the potential role of TQ as a therapeutic agent or as an adjuvant agent for the treatment or the prevention of GBM or an agent for slowing the progress of GBM. In this review, we have compiled the potential role of TQ in GBM therapeutics focusing on the major gliomagenesis signaling pathways.

Summary and Future Perspective

GBM is one of the least understood diseases. Highly heterogeneous cell populations and complex pathogenesis add further complexities for effective therapeutic agent developments. The presence of BBB adds another layer of complexity in combating this disease. Though considerable advancements have been accomplished in GBM molecular pathogenesis and thereby in treatment strategies, the overall survival rates remain poor. Targeting a particular molecule or signaling pathway, involved in one of the singular aspects of the multistep complex tumorigenesis processes, has recently been deemed as extravagant attempt to curtail malignant progression. Due to the inherent heterogeneous nature, GBM can always evade a particular therapeutic modality and continue to survive on alternative pathways followed by recurrence of tumor at a far more aggressive form. Therefore, the paradigm in cancer treatment strategy is now shifting from targeted therapy to combination or multitargeted approaches or targeting cancer with modalities that affect multiple signaling pathways. As a multitargeting therapeutic substance, TQ has been investigated in numerous disease models along with different types of cancer in vivo and in vitro models including GBM. Studies have focused on various signaling pathways providing evidence for its potential use in the GBM therapeutics. The prominent GBM signaling pathways includes the role of TQ in interfering in the phosphorylation and subsequent activation of several upstream tyrosine kinases (e.g., MAPK, Akt, mTOR, and PIP3) that are involved in tumor cell proliferation signaling pathways [49, 180]. Transcriptional factors (e.g., Nrf2, NF-κB, and STAT-3) that are considered as key players in various oncogenesis process are other crucial molecular targets of TQ [49, 57, 84]. It has been suggested by multiple studies that, by regulating the activation of these transcription factors, TQ might counteract different tumorigenic processes including inflammation, cell proliferation, cell survival, angiogenesis, cell invasions, and metastasis. Furthermore, TQ shows chemopreventive properties by downregulating carcinogen metabolizing enzymes (e.g., CYP 1A2 and CYP 3A4), upregulating cytoprotective enzymes (e.g., glutathione S-transferase, superoxide dismutase, and oxidoreductase), attenuated production of proinflammatory mediators (e.g., cytokines, chemokines, and prostaglandins) [49]. Among different signaling pathways several are significant in the context of GBM therapy with TQ; the JAK/STAT and NF-κB are getting increasing attention in the context of GBM. The JAK/STAT signaling in GBM consists of four JAKs (JAKs1–3 and TYK2) and seven STATs (STATs1–4, 5a, 5b, and 6) [194] but STAT3 is generally considered as the most eminent among cancers [195]. In GBM, protein kinase Cε has been shown to drive serine phosphorylation of STAT3 in a RAK/MEK/ERK-dependent fashion, and this modification of STAT3 enhances the invasive capacity and apoptosis resistance of GBM [196, 197]. STAT3 upregulation, hyperactivation, and nuclear accumulation is a well-known feature of GBM [198]. Studies have shown that TQ inhibits proliferation in gastric cancer via STAT3 pathway in vivo and in vitro [171] alone and also in combination with other drugs in breast cancer [199]. We propose further investigation for the role of TQ in GBM in JAK/STAT3 pathways. Further investigations are also required whether TQ affect specific parts of NF-κB such as IκK complex that is involved in the regulating NF-κB activation or regulate NF-κB signaling in a more selective manner by specifically interacting with NF-κB dimers or whether TQ blocks NF-κB by directly targeting the subunits (p65 and p50) themselves. This is of particular interest because of the fact that one available drug, TMZ, has opposite effects in the subunits [200]. Previous study in GBM cells has shown that Ikk inhibitors decrease proliferation and increase apoptosis directly [201] or via inhibiting nuclear p65 translocation [202]. Study regarding the effects of TQ on proteasomes is also suggested since inhibitors of proteasomes has shown to have beneficiary effects on GBM [203] but whether such effects are mediated by TQ has not been investigated. TQ induces selective and time-dependent proteasome inhibition, both in isolated enzymes and in GBM cells, suggesting that this inhibition leads to intracellular increases in the levels of apoptotic proteins such as p53 and Bax, and may be linked to the onset of apoptotic events [204]. Therefore, we propose further investigations on TQ as its potential application as an adjuvant in the treatment of cancer and other diseases. In the clinical settings, no such study has been conducted with TQ for the treatment of GBM but one general conclusion is that improved understanding of the molecular mechanism by which GBM is regulated is a strategy that can make a significant impact in the successful management of GBM. Interestingly, even the lower efficacy [205] and poor bioavailability [206, 207] of TQ are the primary bottleneck of TQ, its volatile nature [208] provides opportunity to be exploited for use in novel drug delivery strategy via intranasal pathway to brain due to unique connection provided by the olfactory and/or trigeminal nerve system present between the olfactory epithelium and the central nervous system. Such delivery system provides opportunity to bypass both the BBB and hepatic first-pass metabolism [209]. It is evident that TQ is multitargeting in its nature but majority of the signaling pathways in the GBM pathogenic context is yet to be explored. Due to its lower efficacy and systemic bioavailability, we propose further investigation on its role as adjuvant therapy with other chemotherapeutic courses. Further investigation could also be conducted for its more efficacious analogues and formulating those into different delivery systems to cross BBB in GBM treatment along with determination of their pharmacokinetic behavior, efficacy, and toxicity. To better understand these differential cellular effects of TQ, more in vitro, in vivo, and in silico studies could be conducted at both proteomic and genomic level. Findings from such studies will enable us to devise clinically effective combination therapeutics where TQ or its derivatives can potentiate the antitumorigenic potential of various conventional and established GBM therapeutic courses.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

Muhammad Shahdaat Bin Sayeed and A. G. M. Mostofa conceived the idea, Fabliha Ahmed Chowdhury and Muhammad Shahdaat Bin Sayeed wrote the major sections of the manuscript, Md Kamal Hossain wrote Section 5, and Maruf Mohammad Akbor provided critical insights.