Foretinib

Foretinib induces G2/M cell cycle arrest, apoptosis, and invasion in human glioblastoma cells through c‑MET inhibition

Narges K. Gortany1,2 · Ghodratollah Panahi3 · Homanaz Ghafari2 · Maryam Shekari2 · Mahmoud Ghazi‑Khansari1,2

Received: 22 September 2020 / Accepted: 1 February 2021 / Published online: 10 March 2021
© The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature 2021

Abstract

Purpose Glioblastoma multiforme (GBM) is one of the most aggressive human cancers. The c-MET receptor tyrosine kinase (RTK) which is frequently deregulated in GBM is considered as a promising target for GBM treatment. The c-MET plays a key role in cell proliferation, cell cycle progression, invasion, angiogenesis, and metastasis. Here, we investigated the anti- tumour activity of foretinib, a c-MET inhibitor, on three human GBM cells (T98G, U87MG and U251).

Methods Anti-proliferative effect of foretinib was determined using MTT, crystal violet staining, and clonogenic assays. PI
and Annexin V/PI staining flow cytometry were used to evaluate the effects of foretinib on cell cycle and apoptosis, respec- tively. Scratch assay, qRT-PCR, western blot, and zymography analyses were applied to elucidate the molecular mechanisms underlying the anti-tumour activity of foretinib.

Results Foretinib treatment reduced phosphorylation of c-MET on T98G and U251 cells, but not in U87MG cells. The highest inhibitory effect was observed in T98G cells (IC50 = 4.66 ± 0.29 µM) and the lowest one in U87MG cells (IC50 = 29.99 ± 1.31 µM). The results showed that foretinib inhibited the proliferation of GBM cells through a G2/M cell cycle arrest and mitochondrial-mediated apoptosis in association with alternation in expression of the related genes and protein-regulated G2/M phase and apoptosis. Foretinib diminished GBM cell invasion through downregulation of the proteolytic cascade of MMP2, uPA and uPAR and epithelial–mesenchymal transition (EMT)-related genes. A different GBM cell sensitivity pattern was noticeable in all experiments which demonstrated T98G as a sensitive and U87MG as a resistant phenotype to foretinib treatment.

Conclusion The results indicated that foretinib might have the therapeutic potential against human GBM which deserve further investigation.

Keywords : Glioblastoma · Foretinib · c-MET · U251 · T98G · U87MG

Introduction

Glioblastoma multiforme (GBM) is the most common, aggressive, and lethal type of brain tumours known as glio- mas originating from glial cells. It is the highly malignant tumour of central nervous system (CNS) which is resistant to radiation and chemotherapy [1]. GBM accounts for 54% of all gliomas and 16% of all primary brain tumours [2, 3]. Despite the existence of multimodal treatment, GBM still remains an incurable disease with a median survival of 12–15 months post-diagnosis and only 3–5% of patients survive for about 3 years. The standard treatment of GBM is palliative care includes surgery, adjuvant radiotherapy and Temozolomide (TMZ) chemotherapy [4, 5]. However, the severe side effects of TMZ and occurrence of drug resist- ance lead to increase demand to explore effective therapeutic alternatives. GBM affects more than one signalling pathway and causes alternations in several molecular signalling path- ways including receptor tyrosine kinase (RTK)/Ras/phos- phoinositide 3-kinase (PI3K) (87%), retinoblastoma (RB) (78%) and P53 (87%) signalling pathways [5–7]. Mesen- chymal epithelial transition factor (c-MET), as a member of RTKs, is extensively expressed in epithelial and endothe- lial cells and is essential in embryonic development, tissue regeneration, and wound healing [8]. c-MET and its ligand, hepatocyte growth factor (HGF), are known to promote survival, proliferation, invasion and metastasis of human cancers cells. They are overexpressed in carcinomas and other solid tumours such as small cell lung cancer human [9], ovarian tumours [10], esophageal cancer [11], and GBM [12]. Increased level of c-MET expression is correlated with gliomas malignancy grade and its overexpression is associ- ated with poor prognosis and tumour invasiveness in GBM patients [13–16].

Foretinib is an orally small molecule multi-targeted RTK inhibitor of c-MET, vascular endothelial growth factor receptors (VEGFRs), platelet-derived growth factor recep- tors (PDGFR-β), RON/macrophage-stimulating 1 receptor (MST1R), AXL, TIE-2 and ROS1 [8, 17, 18]. Foretinib firmly binds to c-MET and reduces its phosphorylation resulting in inhibition of tumour cell proliferation, inva- sion, and angiogenesis [11, 19]. Several pre-clinical reports have assessed the anti-tumour activity of foretinib against head and neck, gastric, and liver, breast, prostate, and papil- lary renal cell cancer [20, 21]. Foretinib has been studied in different phases of clinical trials of several types of cancer including head and neck [22], papillary renal cancer [23], hepatocellular carcinoma (HCC), gastric cancer [14], non- small cell lung cancer (NSCLC) [24], and others [25, 26]. Foretinib’s ability to target TAM, MerTK receptors and its inhibitory effects against ROS1 fusion kinase in GBM have been reported previously [27–29].

Due to the important role of c-MET in the onset and progression of GBM and the recent therapeutic potential of c-MET inhibitors in clinic, the aim of this study was to evaluate the antitumor activity of foretinib on human GBM cell lines of T98G, U251 and U87MG which are widely used as GBM models. We studied the effects of foretinib on cell survival, cell cycle, and cell apoptosis, and invasion of indicated GBM cell through inhibition of c-MET.

Materials and methods
Reagents and cell lines

Foretinib was purchased from AdooQ Bioscience (Irvine, CA, USA) and all other reagents were purchased from Sigma (St. Louis, MO, USA). Three human glioblastoma cell lines, T98G (ATCC Cat# CRL-1690, RRID: CVCL_0556), U87MG (NCBI_Iran Cat# C531, RRID: CVCL_0022), and
U251 (RRID: CVCL_0021) were purchased from National Cell Bank of Iran (NCBI, Tehran, Iran). All the cell lines were authenticated by STR profiling using Cell ID™ sys- tem (promega) and were routinely checked for mycoplasma infection. Cells were used between passages 8 and 15. All cell lines were maintained in DMEM (Gibco, USA) supple- mented with 10% foetal bovine serum (FBS, Gibco, USA), 100 U/ml penicillin and 100 mg/ml streptomycin (all from Gibco, UK) at 37 °C in humidified incubator containing 5% CO2.

Cell proliferation and survival assays

MTT assay

To assess the effect of foretinib on glioblastoma cell growth, cells in logarithmic growth phase were plated in 96-well plates and incubated for 24 h prior to treatment with differ- ent concentrations of foretinib for 48 h. The cell viability assay was carried out using MTT test and the absorbance was measured by ELISA reader (Synergy HT, Biotek). Data are expressed as the percentage of growth relative to that of untreated control cells. Data were normalised and further nonlinear regression curve fit analysis of the normalised data used for determination of IC50 values was performed using GraphPad Prism software.

Crystal violet staining

Crystal violet assay is used to examine the effect of chem- otherapeutics on cell survival and growth inhibition [30]. Briefly, cells were plated at a density of 6 × 104 cells in 6-well plates for 24 h and then treated with foretinib for 48 h. The cultures were fixed in ice-cold methanol and stained with crystal violet (0.5% w/v). The cells were imaged with an inverted microscope at 10 × magnification.

Clonogenic assay

Colony formation or clonogenic assay is a cell survival assay based on the ability of a single cell to grow into a colony to determine cell reproductive death after treatment with cyto- toxic agents. Cells were seeded with a density of 1000–2000 cell in each well in 6-well plates for 24 h. After treatment with foretinib for 48 h, the cell media was changed by fresh media and the cells were incubated at 37 °C in 5% CO2 for 10 days. The cultures stained with crystal violet solution (1% w/v) and the colonies were counted by naked eyes. The surviving fraction is calculated based on following equation [31]: mean colony counts Surviving fraction (SF) = cell plated × plating effiecency where plating efficiency (PE) was determined as below: Plating effieciency (PE) = Cell cycle analysis Cells were collected after centrifugation (2000 rpm, 4 °C for 10 min) of 2 ml of peripheral and then stored at − 80 °C. Total RNA was extracted from a using the Gene All Kit (Korea) according to the manufacturer’s instructions. First-Cell cycle analysis was performed using propidium iodide (PI) staining. The effects of foretinib treatment on cell cycle status were analysed using propidium iodide (PI) flow cytometry. The cells were exposed to 0.1% FBS medium for 24 h prior to treat with foretinib for 48 h. The treated cells were washed with cold PBS, harvested and fixed in 70% (v/v) ethanol. The cells were then incubated with RNase A (100 µg/mL) (Sigma), PI (50 µg/mL) (Sigma) and 0.05% Triton X-100, and analysed on a FACSCalibur (BD Biosci- ence) flow cytometer equipped with CellQuest Pro™ soft- ware (BD Bioscience, USA) to determine G2/M fraction.

Apoptosis assay

Apoptosis assay was performed to determine the percent- age of apoptotic cells in cells treated with foretinib. 48 h post-drug treatment, cells were washed with PBS buffer and centrifuged. Annexin V/propidium iodide (PI) Apoptosis Kit (KeyGENE Biotechnology, The Netherlands) was used to detect the apoptosis cells by flow cytometry.

Quantitative real‑time PCR

Quantitative Real-Time PCR was performed using SYBR®Premix EX Taq II (Takara, biotechnology, LTD, Dalian, Japan). The Primer sequences are shown in Table 1. Quantitative Real-Time PCR reactions were performed in Rotor-Gene Q (Qiagene, Hilden, Germany) in 20 μL of PCR master mix containing 10 μL of SYBR-Green QPCR Master Mix, 1 μL of primer, 1 μL of RT products and 8 μL of RNase free water. The relative expression of genes was calculated using comparative Ct method. All tests were run in triplicate to minimise the experimental error. All assays were inspected for distinct melting curves and the Tm was checked to be within known specifications for each particu- lar assay. Furthermore, the samples must be detected with a Ct < 37 to be included in the analysis. Western blot analysis The GBM cells were lysed for 30 min in ice-cold RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate and 0.1% SDS) con- taining protease inhibitor. Equal amounts of protein (50 µg) were loaded on 12% SDS-PAGE, transferred to PVDF membrane (Membrane Solutions, TX, USA) then probed with primary and horseradish peroxidase (HRP)- conjugated secondary antibodies (Sigma). Primary anti- bodies were used as follows: c-MET, phospho-c-MET (Tyr1234/1235), Bax, Bcl-2, Cdc25c, Foxm1, Cyclin B1, and Plk1 (all from Santa Cruz Biotechnology, USA). β-actin was used as the loading control and proteins were detected using a BM chemiluminescence detection kit (Roche Molecular Biochemicals). All antibody dilutions were 1:500 except for the β-actin antibody, which was used at a dilution of 1:1000. For quantification of protein intensities, western blot bands were visualised on radio- graph film and evaluated by Image J software. Zymography The activity of matrix metalloproteinases (MMPs) enzyme in treated cells with foretinib was detected by zymography method as described previously [32]. Equal amounts of pro- tein from the supernatants were applied to polyacrylamide gels copolymerized with gelatine A (Sigma). After electro- phoresis, the gels were stained with Coomassie Brilliant Blue. The enzymatic activity appeared as clear bands. Scratch assay The GBM cells in logarithmic growth phase were plated in six-well plates and incubated for 24 h prior to treatment with different concentrations of foretinib. After treatment, a thin scratch was made in the central area using a sterile 200-ml pipette tip. Detached and damaged cells were removed with PBS and the plates were incubated at 37 °C, 5% CO2 for 24 h. The wound areas were observed by light microscopy and images were captured at the indicated time points. Blinding and statistical analysis Experimenter conducting the sample analysis for the west- ern blot, qRT-PCR, cell cycle analysis, and apoptosis assay were blinded to the sample treatment details. No randomi- sation and sample size calculation was performed in this study. This study was not pre-registered. All data were evaluated in triplicate against untreated control cells and collected from at least three independent biological repli- cates and at least two technical replicates each. Data are presented as mean ± standard error of the mean (SEM). Data were analysed and graphed by GraphPad Prism Software 6.01 (GraphPad Software Inc., San Diego, CA, USA) using one-way ANOVA and the unpaired two-tailed Student’s t test. P values were determined and P < 0.05 was considered significant. Number of replicates (n) in figure legends indi- cates independent experiments, with averages of technical replicates within each experiment. Results Foretinib inhibits c‑MET activity in human GBM cell lines To confirm c-MET expression and the inhibitory effects of foretinib as a c-MET inhibitor on c-MET phosphorylation, the expression level of c-MET and its phosphorylated form (p-c-MET) were assessed in the studied GBM cell lines by western blotting. As shown in Fig. 1, c-MET and p-c-MET were both expressed in the GBM cells. The results showed the different levels of p-c-MET in the GBM cells. In T98G cell line, the level of p-c-MET was decreased by increasing the concentration of foretinib. There was a slight decrease in p-c-MET in U251 cells and no obvious changes in the level of p-c-MET expression were observed in U87MG cells treated with different concentrations of foretinib. Foretinib inhibits GBM cell proliferation and clonal growth After confirmation of the expression of c-MET in the studied GBM cells, T98G, U251, and U87MG cells, MTT assay was performed to study the cell viability upon treatment with dif- ferent concentrations of foretinib. The result of MTT showed a decrease in cell proliferation of the GBM cells with differ- ent relative sensitivity of cells to foretinib (Fig. 2a1). Upon treatment with 2 µM of foretinib, the cell proliferation was significantly reduced by 34% in T98G cells, while only between 12% and 3% reduction were observed in U251 and U87MG cells, respectively. The calculated IC50 for T98G, U251 and U87MG were 4.66 ± 0.29, 22.35 ± 2.45, and 29.99 ± 1.31 µM, respectively (Fig. 2a2). Based on the IC50 values, T98G was the sensitive and U87MG was the resist- ance cell lines to foretinib. Fig. 1 The expression of c-MET and p-c-MET in T98G, U251, and U87MG GBM cell lines treated with indicated concentrations of foretinib was detected by western blot with β-actin as loading control The inhibitory effect of foretinib on cell viability and pro- liferation was also evaluated by crystal violet staining and clonogenic assays. The microscopic images of GBM cells treated with foretinib and stained in crystal violet are shown in Fig. 2b. The images revealed the different morphologies of the GBM cell lines. U251 cells had an epithelial-like mor- phology, whereas U87MG cells with their long thin protru- sions mostly look like a neuronal morphology. Consistent with results of MTT assay, the microscopic images clearly demonstrated the different inhibitory effects of foretinib in cell survival of T98G, U251 and U87MG cell lines. To con- firm results, a clonogenic assay was performed on GBM cells treated with foretinib for 10 days. The results were in accordance with MTT assay that foretinib treatment dimin- ished colony growth in GBM cell lines in a different sensi- tivity (Fig. 2c). Foretinib significantly reduced the colony formation up to 24% of control with 1 µM foretinib treat- ment (p < 0.01) in T98G cells, while no significant reduc- tion was seen in U87MG at this concentration (p = 0.67). Taken all these results together indicated the sensitivity of the studied GBM cell lines to foretinib as the order of T98MG > U251 > U87MG cells.

Foretinib inhibits GBM proliferation via G2/M cell cycle arrest

To further investigate the mechanism by which foretinib inhibits the growth of GBM cells, the cell cycle analysis upon exposed to different concentrations of foretinib for 48 h was performed by flow cytometry (Fig. 3a). The result indicated that foretinib inhibited the proliferation of the GBM cells through a G2/M cell cycle arrest, but at differ- ent level. Foretinib induced a dose-dependent increase in the percentage of cells in G2/M phase in T98G and U251 cells compared to the untreated cells (from 20 to 50% and 11.9–43.9%, respectively). In U87MG, foretinib slightly increased the population of cells in G2/M phase from 13 to 36%. Consistent with the cell proliferation results, T98G cells exhibited the highest sensitivity by the highest cell population in G2/M phase, and U87MG cells showed the least sensitivity to foretinib treatment.

We next determined the effects of foretinib on the expres- sion of proteins and genes that regulate G2/M transition. G2/M checkpoints are controlled by the activation of Cyclin B/CDK1 complex as a key step in mitotic initiation. Cdc25 protein (encoded by the CDC25C gene) permits cell entry into mitosis by activation the Cyclin B/CDK1 complex [33]. In addition, polo-like kinase 1 protein (PLK1) drives entry into mitosis via phosphorylation of CDC25C. AurkA and AurkB are two members of the mammalian Aurora kinases family which play critical roles in the cell division by regu- lating mitosis and the cell cycle through activating PLK1 [34]. Forkhead box protein M1 (FOXM1) is a transcription factor that has important role in controlling the mitotic entry by regulating a group of G2/M-specific genes such as PLK1, CCNB1 (protein coding Cyclin B1), and CCNB2 (protein coding cyclin B2) [35, 36].

In our study, the analysis of qRT-PCR data revealed that foretinib exposure dramatically downregulated the mRNA level of CDK1, CCNB1, CCNB2, CDC25C, AURKA, and AURKB genes in GBM cells (Fig. 3b). Moreover, the western blot analysis showed that foretinib significantly decreased the protein expression of CyclinB1 and Cdc25C, PLK1, and FOXM1 in GBM cells (Fig. 3c). The data indi- cated that foretinib inhibited the proliferation of GBM cells through a G2/M cell cycle arrest. The same pattern of GBM cells sensitivity was also observed in evaluation of foretinib on cell cycle regulation. While 5 µM of foretinib induced a significant reduction in the expression of all studied genes and proteins in T98G and most of them in U251 cells, the reduction was not significant in U87MG cell.

Foretinib‑induced GBM cell apoptosis

We next inquire the effects of foretinib on cell death in GBM cells. T98G, U251 and U87MG cells were treated with dif- ferent concentrations of foretinib and the degree of apoptosis was analysed by Annexin V-PI staining and flow cytom- etry to assess early apoptotic, late apoptotic, and necrotic cell populations (Fig. 4a). The results showed that the early apoptotic cell population was increased from 0.98% to 89.2% in T98G, 1.68–14.6% in U251, and 0.84–18.4% in U87MG cells by increasing the concentration of foretinib. The results obviously demonstrated that foretinib induces the highest rate of cell death via apoptosis in T98G cell line compared to the other GBM cells which was consistent with our previ- ous results regarding cell sensitivity to foretinib treatment. To investigate the mechanisms underlying foretinib- induced apoptosis, the effect of drug on expression of apoptosis-related regulator genes and proteins including BAX, BCL2 and Survivin was evaluated by qRT-PCR and western blots, respectively (Fig. 4b, c). The BCL-2 family of proteins include pro-apoptotic (BAX) and anti-apoptotic proteins (BCL2) play a crucial role to induce apoptosis via controlling mitochondrial outer membrane permeability [37]. Survivin is a member of the inhibitor of apoptosis (IAP) protein family involved in inhibition of apoptosis and regulation of cell cycle [38]. The results showed a sig- nificant upregulation of BAX and concomitant downregu- lation of BCL2 genes and proteins in all three GBM cell lines by increasing the concentration of foretinib (Fig. 4b,c) which were more significant in T98G compared to U251 and U87MG cells. The lowest concentration foretinib (5 µM) enhanced the expression of BAX and diminished BCL2 in T98G and U251 cells (P < 0.05), but had no effect in U87MG cells. In addition, foretinib induced significant downregulation of Survivin gene in all three cell lines. Our data suggested that foretinib impeded the proliferation of GBM cells via apoptosis induction. ◂Fig. 2 Foretinib inhibited the proliferation and clonal growth of T98G, U251 and U87MG. a1 MTT assay was applied to evaluate the cell viability after 48 h of treatment with foretinib. a2 Determi- nation of foretinib IC50 values by non-liner regression. b The GBM cells were treated with foretinib, stained with crystal violet and imaged by an inverted microscope. c, d GBM cells were cultured for 10 days after treatment with indicated concentration of foretinib, and were then stained with 0.1% crystal violet to conduct the clonogenic assay to evaluate the effects of foretinib on clonal proliferation. Data presented were obtained from three independent experiments. Data are given as mean ± SEM. Data were analysed by GraphPad Prism using one-way ANOVA. Statistically significant values of *p < 0.05, **p < 0.01 and ***p < 0.001 were determined compared with the control Foretinib inhibited invasion‑related genes in GBM cells Cancer cell migration and invasion are primary steps in metastasis. GBM invasion is activated by proteolytic deg- radation of brain extracellular matrix (ECM) components via several upregulated proteases including MMPs (zinc- dependent proteases), uPA (a serine protease) and its recep- tor uPAR [39]. The transformation from epithelial to mesen- chymal status, known as epithelial–mesenchymal transition (EMT) is required to progress invasion. EMT is a complex process which is mediated by a set of EMT-activating tran- scription factors (EMT-TFs) mainly including the families of Twist, Snail, zinc-finger E-box-binding homeobox (ZEB) [40]. To evaluate the effects of foretinib on migration and inva- sion of GBM cells, the GBM cells were subjected to the different concentrations of foretinib for 24 h and their abil- ity to migrate was studied by scratch assay. As shown in Fig. 5a, foretinib inhibited cellular migration in T98G cells compared to the control. The anti-migratory of foretinib could also be observed in U251 cells, but did not observe in U87MG cells. Then, the expression level of genes involved in ECM degradation of GBM cells (MMP2, uPA and uPAR) was evaluated by real-time RT-PCR. In addition, the gelati- nolytic activity of MMP2 was measured by zymography assay. Next, the effect of foretinib on the mRNA expression level of the EMT markers of TWIST, SNAIL1 (SNAIL) and SNAIL2 (SLUG), ZEB1, ZEB2 and CDH2 (which encodes N-cadherin) was assessed by real-time PCR. The results indicated that foretinib inhibited the expression of MMP2, uPA and uPAR genes in all studied GBM cells (Fig. 5b). As represented in Fig. 5c, the intensities of clear bands demon- strated the decreasing gelatinolytic activities of MMP2 by increasing concentrations of foretinib suggesting an obvious inhibition of MMP2 enzyme activity on T98G cells with- out inhibitory effect on U87MG cells. Regarding EMT- related genes, the results showed that the mRNA levels of all indicated EMT transcription factors were significantly decreased in a dose-dependent manner in T98G and U251 cells (P < 0.05), but had relatively weak effect on U87MG cells (Fig. 5d). Foretinib had no effect on the expression level of SNAIL and ZEB2 and the expression of TWIST and SLUG were reduced only at the high concentration of foretinib (P < 0.05). Discussion Glioblastoma multiforme as the most lethal malignant brain tumour still remained incurable and new therapeutic targets are highly demanding. Among the main signalling pathways of GBM, RTK/Ras/PI3K happened more frequently (87%) identified by mutation in EGFR, ERBB2 (epidermal growth factor receptor), PDGFRα (platelet-derived growth factor receptor α), and c-MET [6, 41]. c-MET mediates a range of complex biological activities including cell migration, cell proliferation, apoptosis, angiogenesis, tumour propagation, and treatment resistance [6, 41, 42]. c-MET overexpres- sion has been reported in a high proportion of GBM cases [12, 43, 44]. Targeted inhibition of c-MET is particularly effective in GBM therapy of the cells in which display high expression of c-MET [6]. Small molecule c-MET kinase inhibitors such as foretinib, target activation sites of c-MET resulting in blocking the downstream signalling pathways [8, 41]. In this study, the expression of c-MET in three human GBM cells (T98G, U87MG, and U251) and the inhibitory effect of foretinib through inhibition of phosphorylation of c-MET expression were investigated. The results showed that c-MET was expressed in all studied GBM cells and foretinib treatment inhibited the p-c-MET in T98G and U251 cells, but with no obvious effect on U87MG cells (Fig. 1). As shown in MTT, crystal violet, and colony growth assays, foretinib treatment inhibited the proliferation of GBM cells on both setting of short time and long term (Fig. 2a–c). The results of MTT assay were consistent with the crystal violet and colony growth assays that foretinib suppressed the GBM cells growth in a different cell sensitivity pattern. T98G cells presented the most sensitivity to foretinib with a low IC50 value of 4.66 µM compared to U87MG cells that were more resistant to low concentration of foretinib with the IC50 value of 29.99 µM. U251 cells with IC50 value of 22.35 showed a moderate sensitivity between these two cell lines. Notably, the T98G cell proliferation was significantly inhibited in a dose-dependent manner. However, foretinib treatment had less effect on U87MG cells proliferation. The effect of foretinib on cell proliferation demonstrated a pattern of sensitivity in GBM cells in the following order T98MG > U251 > U87MG. We found that foretinib-treated T98G cells which shown the lowest expression of phos- phorylation of c-Met, expressed the highest sensitivity in response to foretinib treatment.

Fig. 3 Foretinib inhibited human GBM cells (T98, U87MG and U251) proliferation by G2/M arrest. a After treatment with differ- ent concentrations of foretinib for 48 h, cells were fixed and stained with PI to analyse the cell cycle distributions by a flow cytometer. The graphs are representative of three independent experiments with similar results. Quantitative analysis of distribution of GBM cells in each phase of G0/G1, S and G2/M was performed and plotted. b The GBM cells were treated with different concentrations of foretinib for 48 h. the mRNA expression of CCNB1, CCNB2, CDK1, CDC25C, AURKA and AURKB were detected by qRT-PCR, and results represent the mean ± SD of three experiments done triplicate. c Effect of foretinib on the expression of CDC25c, CyclinB1, FoxM1 and Plk1 protein determined by western blot analysis. Blots are representative of one of three separate experiments. Densitometry values were nor- malised to β-actin used as loading and shown as the mean ± SEM of three separate experiments. Data presented were obtained from three independent experiments. Data are given as mean ± SEM. Data were analysed by GraphPad Prism using one-way ANOVA. Statistically significant values of *p < 0.05, **p < 0.01 and ***p < 0.001 were determined compared with the control. Foretinib diminished the proliferation of the indicated GBM cells by induction of G2/M arrest and apoptosis. Foretinib significantly suppressed CyclinB1, Cdc25C, FOXM1, and PLK1 proteins as well as CCNB1, CCNB2, CDK1, CDC25C, AURKA and AURKB genes which conformed the induction of G2/M arrest and inhibition of mitosis in GBM cells that are in agreement with previ- ous reports [11, 17, 45–47]. The same pattern of sensi- tivity of GBM cells to foretinib treatment was observed too. Foretinib significantly downregulated the indicated genes and proteins in T98G cells, but not in U87MG cells. FOXM1 plays an important role in cell cycle progres- sion and stimulates the expression of a number of genes that are critical for the G2/M phase progression, such as Plk1, Aurora B, Cyclin B1, CDC25B, and Survivin [48]. There is a complex inter-regulatory relationship between FOXM1 and PLK1 which contributes to the aberrant cell proliferation leads to malignancy. Indeed, the overexpres- sion of them was found in several aggressive human car- cinomas including GBM [49]. The PLK1/FOXM1 axis has been presented as an important regulator in carcino- genesis and also a potential therapeutic target [36, 50]. In this study, FOXM1 and PLK1 proteins were expressed in GBM cells and foretinib treatment inhibited both of them which may provide a new approach insight into the anti-tumour activity of foretinib through inhibition of the PLK1–FOXM1 axis. The cell apoptosis assay revealed a dose-dependent increase in early apoptosis in GBM cells treated with foretinib (Fig. 4). Foretinib treatment upregulated Bax and concomitant downregulation of Bcl-2 genes and proteins in all three GBMs cell lines which were observed significantly in T98G and U251 cells compared to U87MG cells. Moreover, foretinib induced significant downregulation of survivin in all three cell lines which is mostly observed in T98G and U251 cells. Survivin is an important prognostic marker in many cancers, including GBM. It is only expressed in the G2/M phase in tumour cells, with dual function as an apoptosis suppressor and mitosis regulator [51–53]. Fig. 4 Foretinib-induced apoptosis in T98, U87MG and U251 cells. a GBM cells were incubated with indicated concentrations of foretinib for 48 h and apoptotic cell death was measured by flow cytometry after staining with Annexin V-PI staining. Annexin V-positive cells are considered early apoptotic, whereas PI uptake indicates necrosis. Cells positive for both stains are considered late apoptotic. b Foretinib increased the mRNA expression levels of BAX and decreased the anti-apoptotic genes of BCL2 and Survivin as measured by qRT-PCR after 48 h treatment. c Effects of foretinib on the expression of BAX and BCL2 proteins measured by west- ern blot analysis. Blots are representative of one of three separate experiments. Densitometry values were normalised to β-actin used as loading and shown as the mean ± SEM of three separate experi- ments. Data presented were obtained from three independent experi- ments. Data are given as mean ± SEM. Data were analysed by Graph- Pad Prism using one-way ANOVA. Statistically significant values of *p < 0.05, **p < 0.01 and ***p < 0.001 were determined compared with the control. In addition to the effect on cell proliferation, the inhibi- tory effect of foretinib on migration and invasion of GBM cells was evaluated by scratch assay and qRT-PCR. The initial step of migration/invasion involves the detachment of tumour cells from the main site, which is proceeded by degradation of ECM upon activation of various proteases such as MMPs, uPA and uPAR. In GBM, there is a correla- tion between the increased MMP expression and increased invasiveness. MMP overexpressions, in particular MMP2 and MMP9, have been correlated to brain tumour malig- nancy and metastasis formation in GBM and their inhibition reduces the GBM invasion [54, 55]. Several studies have shown that uPA and its receptor uPAR promotes tumour progression and have a critical role in invasion of GBM cells [56]. Our results demonstrated that foretinib not only reduced the enzymatic activity of MMP2, but also down- regulated gene expressions of MMP2, uPA, and uPAR. Con- sistently, a recent study reported the reduction of MMP2 enzyme and gene by foretinib treatment [57]. EMT process has been found to have a critical role in invasive nature of GBM. EMT transcription factors including SNAI1, SLUG, Twist1, and ZEB1/2 enhanced the proliferation, invasion, and migration of GBM cells in animal model and human specimens [58–60]. Our results showed that foretinib sig- nificantly suppressed the migratory and invasive ability of GBM cells by downregulation of CDH2, SLUG, SNAIL, TWIST, ZEB1 and ZEB2. There are several challenges in developing suc- cessful therapeutic agents for GBM due to the high cellular heterogeneity between GBM patients, even within one patient’s tumour, and the resistance to the current ther- apy in clinics [61–64]. The most important obstacles of TMZ in treating GBM patients are its variable sensitivity to different GBM cell lines and the drug resistance. Studies on TMZ resistance identified the U87MG and U251 cell lines as sensitive, and T98G as resistance cell lines to TMZ treatment [28, 65, 66]. There are different approaches to enhance the therapeutic efficacy of TMZ by concomitant and adjuvant therapy [65, 67–69]. Based on the result of this study which indicated the high sensitivity of T98G cells to foretinib treatment, it might be beneficial to add foretinib to the standard treatment of GBM to observe more inhibi- tory effects on GBM cell proliferation over the TMZ alone treatment. It has been demonstrated that GBM combination therapy of TMZ and foretinib on both TMZ sensitive (U251 and A172) and resistance cells (SF188) was effectively worked out [28]. Future in vivo and in vitro studies need to be done to evaluate and determine the best sequence and concentration for the combination therapy. Fig. 5 The effects of foretinib on migration and invasion of GBM cells. a Cells were treated with different concentrations of foretinib for 24 h and subjected to scratch assay and were photographed (b) T98, U87MG and U251 cells were treated with different concentra- tions of foretinib for 48 h, then total RNA was extracted and qRT- PCR was performed. The effects of foretinib on ECM-degradation genes. Foretinib decreased the expression levels of MMP2, uPA, and uPAR. c Effect of foretinib on the enzymatic activity of MMP2 in GBM cells. MMP2 enzyme was collected and separated on a non-reducing polyacrylamide gel containing gelatine A. Gelatino- lytic activities are visualised as clear bands against the background of stained gelatine. d The effects of foretinib on EMT-related genes. Foretinib altered the expression levels of CDH2, SLUG, SNAIL, TWIST, ZEB1, and ZEB2. Data presented were obtained from three independent experiments. Data are given as mean ± SEM. Data were analysed by GraphPad Prism using one-way ANOVA. Statistically significant values of *p < 0.05, **p < 0.01 and ***p < 0.001 were determined compared with the control. In conclusion, we demonstrated that foretinib as a c-MET inhibitor, inhibited GBM cell proliferation via G2/M arrest and mitochondria-mediated apoptotic, and also downregu- lated of several transcription factors and mediator genes and proteins which are involved in migration and invasion of GBM cells. These findings suggest that foretinib has the potential to be considered as a therapeutic agent in GBM treatment in future. Further studies are necessary to evaluate the effect of foretinib on GBM patients with tumours char- acterised by c-MET amplification in clinical trial settings. Supplementary Information The online version contains supplemen- tary material available at https://doi.org/10.1007/s00280-021-04242-0. Acknowledgements This work was supported by Cancer Biology Research Center in Cancer Institute of I.R. Iran at Tehran University of Medical Sciences and also partially supported by National Institute for Medical Research Development (NIMAD) Project No. 963331. Special thanks goes to Dr. Majid Momeny and Farima Moghaddaskho for the preliminary work on this project, Dr. Mohammad Hossein Ghahrem- ani and Dr. Amir Shadboorestan for the technical support in western blot experiments, and MS Raziyeh khoshsohbat for the administrative support. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Conflict of interest The authors disclose no potential conficts of interest. References 1. Haas-Kogan D, Shalev N, Wong M, Mills G, Yount G, Stokoe D (1998) Protein kinase B (PKB/Akt) activity is elevated in glio- blastoma cells due to mutation of the tumor suppressor PTEN/ MMAC. Curr Biol 8(21):1195-S1191 2. Thakkar JP, Dolecek TA, Horbinski C, Ostrom QT, Lightner DD, Barnholtz-Sloan JS, Villano JL (2014) Epidemiologic and molecular prognostic review of glioblastoma. Cancer Epidemiol Prev Biomarkers 23(8):2199 3. Tamimi AF, Juweid M (2017) Epidemiology and outcome of glio- blastoma. In: Glioblastoma. Codon Publications 4. Omuro A, DeAngelis LM (2013) Glioblastoma and other malig- nant gliomas: a clinical review. JAMA 310(17):1842–1850 5. Pearson JR, Regad T (2017) Targeting cellular pathways in glio- blastoma multiforme. Signal Transduct Target Ther 2:17040 6. Cruickshanks N, Zhang Y, Yuan F, Pahuski M, Gibert M, Abou- nader R (2017) Role and therapeutic targeting of the HGF/MET pathway in glioblastoma. Cancers 9(7):87 7. Hanif F, Muzaffar K, Perveen K, Malhi SM, Simjee SU (2017) Glioblastoma multiforme: a review of its epidemiology and patho- genesis through clinical presentation and treatment. Asian Pac J Cancer Prev 18(1):3 8. Puccini A, Marín-Ramos NI, Bergamo F, Schirripa M, Lonardi S, Lenz H-J, Loupakis F, Battaglin F (2019) Safety and tolerability of c-MET inhibitors in cancer. Drug Saf 42(2):1–23 9. Maulik G, Kijima T, Ma PC, Ghosh SK, Lin J, Shapiro GI, Schaefer E, Tibaldi E, Johnson BE, Salgia R (2002) Modulation of the c-Met/hepatocyte growth factor pathway in small cell lung cancer. Clin Cancer Res 8(2):620–627 10. Sawada K, Radjabi AR, Shinomiya N, Kistner E, Kenny H, Becker AR, Turkyilmaz MA, Salgia R, Yamada SD, Vande Woude GF, Tretiakova MS, Lengyel E (2007) c-Met overexpression is a prog- nostic factor in ovarian cancer and an effective target for inhibition of peritoneal dissemination and invasion. Can Res 67(4):1670– 1679. https://doi.org/10.1158/0008-5472.can-06-1147 11. Chen G-Z, Dai W-S, Zhu H-C, Song H-M, Yang X, Wang Y-D, Min H, Lu Q, Liu S, Sun X-C (2017) Foretinib enhances the radio- sensitivity in esophageal squamous cell carcinoma by inhibiting phosphorylation of c-Met. J Cancer 8(6):983 12. Petterson SA, Dahlrot RH, Hermansen SK, Munthe SK, Gundesen MT, Wohlleben H, Rasmussen T, Beier CP, Hansen S, Kristensen BW (2015) High levels of c-Met is associated with poor prognosis in glioblastoma. J Neurooncol 122(3):517–527 13. Nabeshima K, Shimao Y, Sato S, Kataoka H, Moriyama T, Kawano H, Wakisaka S, Koono M (1997) Expression of c-Met correlates with grade of malignancy in human astro- cytic tumours: an immunohistochemical study. Histopathology 31(5):436–443 14. Shah MA, Wainberg ZA, Catenacci DV, Hochster HS, Ford J, Kunz P, Lee F-C, Kallender H, Cecchi F, Rabe DC (2013) Phase II study evaluating 2 dosing schedules of oral foretinib (GSK1363089), cMET/VEGFR2 inhibitor, in patients with meta- static gastric cancer. PLoS ONE 8(3):e54014 15. Qian F, Engst S, Yamaguchi K, Yu P, Won K-A, Mock L, Lou T, Tan J, Li C, Tam D (2009) Inhibition of tumor cell growth, invasion, and metastasis by EXEL-2880 (XL880, GSK1363089), a novel inhibitor of HGF and VEGF receptor tyrosine kinases. Cancer Res 69(20):8009–8016 16. Joo KM, Jin J, Kim E, Kim KH, Kim Y, Kang BG, Kang Y-J, Lathia JD, Cheong KH, Song PH (2012) MET signaling regulates glioblastoma stem cells. Cancer Res 72(15):3828–3838 17. Zillhardt M, Park S-M, Romero IL, Sawada K, Montag AG, Krausz T, Yamada SD, Peter ME, Lengyel E (2011) Foretinib (GSK1363089), an orally available multi-kinase inhibitor of c-Met and VEGFR, blocks proliferation, induces anoikis, and impairs ovarian cancer metastasis. Clin Cancer Res 3387:2010 18. Chen H-M, Tsai C-H, Hung W-C (2015) Foretinib inhibits angi- ogenesis, lymphangiogenesis and tumor growth of pancreatic cancer in vivo by decreasing VEGFR-2/3 and TIE-2 signaling. Oncotarget 6(17):14940 19. Naing A, Kurzrock R, Adams LM, Kleha JF, Laubscher KH, Bonate PL, Weller S, Fitzgerald C, Xu Y, LoRusso PM (2012) A comparison of the pharmacokinetics of the anticancer MET inhibitor foretinib free base tablet formulation to bisphosphate salt capsule formulation in patients with solid tumors. Invest New Drugs 30(1):327–334 20. Zhang Y, Xia M, Jin K, Wang S, Wei H, Fan C, Wu Y, Li X, Li X, Li G (2018) Function of the c-Met receptor tyrosine kinase in carcinogenesis and associated therapeutic opportunities. Mol Cancer 17(1):45 21. Logan TF (2013) Foretinib (XL880): c-MET inhibitor with activ- ity in papillary renal cell cancer. Curr Oncol Rep 15(2):83–90 22. Seiwert T, Sarantopoulos J, Kallender H, McCallum S, Keer HN, Blumenschein G (2013) Phase II trial of single-agent foretinib (GSK1363089) in patients with recurrent or metastatic squa- mous cell carcinoma of the head and neck. Invest New Drugs 31(2):417–424 23. Choueiri TK, Vaishampayan U, Rosenberg JE, Logan TF, Har- zstark AL, Bukowski RM, Rini BI, Srinivas S, Stein MN, Adams LM (2013) Phase II and biomarker study of the dual MET/ VEGFR2 inhibitor foretinib in patients with papillary renal cell carcinoma. J Clin Oncol 31(2):181 24. Leighl NB, Tsao M-S, Liu G, Tu D, Ho C, Shepherd FA, Murray N, Goffin JR, Nicholas G, Sakashita S (2017) A phase I study of foretinib plus erlotinib in patients with previously treated advanced non-small cell lung cancer: Canadian cancer trials group IND 196. Oncotarget 8(41):69651 25. Eder JP, Shapiro GI, Appleman LJ, Zhu AX, Miles D, Keer H, Cancilla B, Chu F, Hitchcock-Bryan S, Sherman L (2010) A phase I study of foretinib, a multi-targeted inhibitor of c-Met and vascular endothelial growth factor receptor 2. Clin Cancer Res 16(13):3507–3516 26. Shapiro GI, McCallum S, Adams LM, Sherman L, Weller S, Swann S, Keer H, Miles D, Müller T, LoRusso P (2013) A Phase 1 dose-escalation study of the safety and pharmacokinetics of once- daily oral foretinib, a multi-kinase inhibitor, in patients with solid tumors. Invest New Drugs 31(3):742–750 27. Bender S, Gronych J, Warnatz H-J, Hutter B, Gröbner S, Ryzhova M, Pfaff E, Hovestadt V, Weinberg F, Halbach S (2016) Recurrent MET fusion genes represent a drug target in pediatric glioblas- toma. Nat Med 22(11):1314 28. Knubel KH, Pernu BM, Sufit A, Nelson S, Pierce AM, Keating AK (2014) MerTK inhibition is a novel therapeutic approach for glioblastoma multiforme. Oncotarget 5(5):1338 29. Davare MA, Saborowski A, Eide CA, Tognon C, Smith RL, Elferich J, Agarwal A, Tyner JW, Shinde UP, Lowe SW (2013) Foretinib is a potent inhibitor of oncogenic ROS1 fusion proteins. Proc Natl Acad Sci USA 110(48):19519–19524 30. Feoktistova M, Geserick P (2016) Leverkus M (2016) Crystal vio- let assay for determining viability of cultured cells. Cold Spring Harb Protoc 4:prot087379 31. Franken NA, Rodermond HM, Stap J, Haveman J, Van Bree C (2006) Clonogenic assay of cells in vitro. Nat Protoc 1(5):2315 32. Toth M, Sohail A, Fridman R (2012) Assessment of gelatinases (MMP-2 and MMP-9) by gelatin zymography. In: Metastasis research protocols. Springer. pp 121–135 33. Stark GR, Taylor WR (2006) Control of the G 2/M transition. Mol Biotechnol 32(3):227–248 34. Archambault V, Carmena M (2012) Polo-like kinase-activat- ing kinases: aurora A, aurora B and what else? Cell Cycle 11(8):1490–1495 35. Laoukili J, Kooistra MR, Brás A, Kauw J, Kerkhoven RM, Mor- rison A, Clevers H, Medema RH (2005) FoxM1 is required for execution of the mitotic programme and chromosome stability. Nat Cell Biol 7(2):126–136 36. Dibb M, Han N, Choudhury J, Hayes S, Valentine H, West C, Ang Y, Sharrocks A (2012) The FOXM1-PLK1 axis is com- monly upregulated in oesophageal adenocarcinoma. Br J Cancer 107(10):1766–1775 37. Gross A, McDonnell JM, Korsmeyer SJ (1999) BCL-2 fam- ily members and the mitochondria in apoptosis. Genes Dev 13(15):1899–1911 38. Garg H, Suri P, Gupta JC, Talwar G, Dubey S (2016) Survivin: a unique target for tumor therapy. Cancer Cell Int 16(1):49 39. Hatoum A, Mohammed R, Zakieh O (2019) The unique invasive- ness of glioblastoma and possible drug targets on extracellular matrix. Cancer Manag Res 11:1843 40. Stemmler MP, Eccles RL, Brabletz S, Brabletz T (2019) Non- redundant functions of EMT transcription factors. Nat Cell Biol 21(1):102–112 41. Awad AJ, Burns TC, Zhang Y, Abounader R (2014) Targeting MET for glioma therapy. Neurosurg Focus 37(6):E10 42. Organ SL, Tsao M-S (2011) An overview of the c-MET signaling pathway. Ther Adv Med Oncol 3(1 suppl):S7–S19 43. Kwak Y, Kim S-I, Park C-K, Paek SH, Lee S-T, Park S-H (2015) c-MET overexpression and amplification in gliomas. Int J Clin Exp Pathol 8(11):14932 44. Liu W, Fu Y, Xu S, Ding F, Zhao G, Zhang K, Du C, Pang B, Pang Q (2011) c-Met expression is associated with time to recur- rence in patients with glioblastoma multiforme. J Clin Neurosci 18(1):119–121 45. Simiczyjew A, Dratkiewicz E, Van Troys M, Ampe C, Styczeń I, Nowak D (2018) Combination of EGFR inhibitor lapatinib and MET inhibitor foretinib inhibits migration of triple negative breast cancer cell lines. Cancers 10(9):335 46. Dratkiewicz E, Pietraszek-Gremplewicz K, Simiczyjew A, Mazur AJ, Nowak D (2018) Gefitinib or lapatinib with foretinib synergis- tically induce a cytotoxic effect in melanoma cell lines. Oncotarget 9(26):18254 47. Moghimi M, Sadeghi Tafti H, Namazi F, Salehi M (2019) Investi- gation of the impact of foretinib on AURKA and AURKB expres- sion in T98 glioblastoma cell line. Res Mol Med 7(1):1–7 48. Maachani UB, Shankavaram U, Kramp T, Tofilon PJ, Camphausen K, Tandle AT (2016) FOXM1 and STAT3 interaction confers radi- oresistance in glioblastoma cells. Oncotarget 7(47):77365 49. Zhang X, Qiao-Li L, Huang Y-T, Zhang L-H, Zhou H-H (2017) Akt/FoxM1 signaling pathway-mediated upregulation of MYBL2 promotes progression of human glioma. J Exp Clin Cancer Res 36(1):105 50. Mancini M, De Santis S, Monaldi C, Bavaro L, Martelli M, Castagnetti F, Gugliotta G, Rosti G, Santucci M, Martinelli G (2019) Hyper-activation of aurora kinase a-polo-like kinase 1-FOXM1 axis promotes chronic myeloid leukemia resistance to tyrosine kinase inhibitors. J Exp Clin Cancer Res 38(1):1–11 51. Chakravarti A, Zhai GG, Zhang M, Malhotra R, Latham DE, Delaney MA, Robe P, Nestler U, Song Q, Loeffler J (2004) Survivin enhances radiation resistance in primary human glio- blastoma cells via caspase-independent mechanisms. Oncogene 23(45):7494 52. Li F, Ambrosini G, Chu EY, Plescia J, Tognin S, Marchisio PC, Altieri DC (1998) Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 396(6711):580–584. https://doi. org/10.1038/25141 53. Zhang S, Zhang C, Song Y, Zhang J, Xu J (2018) Prognostic role of survivin in patients with glioma. Medicine 97(17):e0571 54. Kesanakurti D, Chetty C, Maddirela DR, Gujrati M, Rao J (2012) Functional cooperativity by direct interaction between PAK4 and MMP-2 in the regulation of anoikis resistance, migration and invasion in glioma. Cell Death Dis 3(12):e445 55. Badiga AV, Chetty C, Kesanakurti D, Are D, Gujrati M, Klop- fenstein JD, Dinh DH, Rao JS (2011) MMP-2 siRNA inhibits radiation-enhanced invasiveness in glioma cells. PLoS ONE 6(6):e20614 56. Gondi CS, Kandhukuri N, Dinh DH, Gujrati M, Rao JS (2007) Down-regulation of uPAR and uPA activates caspase-mediated apoptosis and inhibits the PI3K/AKT pathway. Int J Oncol 31(1):19–27 57. Fotoohi M, Hadi N, Namazi F (2019) Effect of foretinib on matrix metalloproteinase-2 (MMP2) expression in glioblastoma. Res Mol Med 6(4):20–27
58. Mikheeva SA, Mikheev AM, Petit A, Beyer R, Oxford RG, Kho- rasani L, Maxwell J-P, Glackin CA, Wakimoto H, González- Herrero I (2010) TWIST1 promotes invasion through mesen- chymal change in human glioblastoma. Mol Cancer 9(1):194
59. Han S-P, Kim J-H, Han M-E, Sim H-E, Kim K-S, Yoon S, Baek S-Y, Kim B-S, Oh S-O (2011) SNAI1 is involved in the prolif- eration and migration of glioblastoma cells. Cell Mol Neurobiol 31(3):489–496
60. Oh S-J, Ahn E-J, Kim O, Kim D, Jung T-Y, Jung S, Lee J-H, Kim K-K, Kim H, Kim EH (2019) The role played by SLUG, an epithelial-mesenchymal transition factor, in invasion and therapeutic resistance of malignant glioma. Cell Mol Neurobiol 56:1–14
61. Sottoriva A, Spiteri I, Piccirillo SG, Touloumis A, Collins VP, Marioni JC, Curtis C, Watts C, Tavaré S (2013) Intratumor het- erogeneity in human glioblastoma reflects cancer evolutionary dynamics. Proc Natl Acad Sci 110(10):4009–4014
62. Patel AP, Tirosh I, Trombetta JJ, Shalek AK, Gillespie SM, Waki- moto H, Cahill DP, Nahed BV, Curry WT, Martuza RL (2014) Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science 344(6190):1396–1401
63. Parker NR, Khong P, Parkinson JF, Howell VM, Wheeler HR (2015) Molecular heterogeneity in glioblastoma: potential clinical implications. Front Oncol 5:55
64. Noch EK, Ramakrishna R, Magge R (2018) Challenges in the treatment of glioblastoma: multisystem mechanisms of therapeutic resistance. World Neurosurg 116:505–517
65. Akbarnejad Z, Eskandary H, Dini L, Vergallo C, Nematollahi- Mahani SN, Farsinejad A, Abadi MFS, Ahmadi M (2017) Cytotoxicity of temozolomide on human glioblastoma cells is enhanced by the concomitant exposure to an extremely low-fre- quency electromagnetic field (100 Hz, 100 G). Biomed Pharma- cother 92:254–264
66. Lee C-Y, Lai H-Y, Chiu A, Chan S-H, Hsiao L-P, Lee S-T (2016) The effects of antiepileptic drugs on the growth of glioblastoma cell lines. J Neurooncol 127(3):445–453
67. Ryu CH, Yoon WS, Park KY, Kim SM, Lim JY, Woo JS, Jeong CH, Hou Y, Jeun S-S (2012) Valproic acid downregulates the expression of MGMT and sensitizes temozolomide-resistant glioma cells. BioMed Res Int
68. Messaoudi K, Clavreul A, Lagarce F (2015) Toward an effective strategy in glioblastoma treatment. Part I: resistance mechanisms
and strategies to overcome resistance of glioblastoma to temozo- lomide. Drug Discov Today 20(7):899–905
69. Towner RA, Ihnat M, Saunders D, Bastian A, Smith N, Pavana RK, Gangjee A (2015) A new anti-glioma therapy, AG119: pre- clinical assessment in a mouse GL261 glioma model. BMC Can- cer 15(1):522
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