Amlexanox Enhances Temozolomide-Induced Antitumor Effects in Human Glioblastoma Cells by Inhibiting IKBKE and the Akt-mTOR Signaling Pathway.

Temozolomide (TMZ), as the first-line chemotherapeutic agent for the treatment of glioblastoma multiforme (GBM), often fails to improve the prognosis of GBM patients due to the quick development of resistance. The need for more effective management of GBM is urgent. The aim of this study is to evaluate the efficacy of combined therapy with TMZ and amlexanox, a selective inhibitor of IKBKE, for GBM. We found that the combined treatment resulted in significant induction of cellular apoptosis and the inhibition of cell viability, migration, and invasion in primary glioma cells and in the human glioma cell line, U87 MG. As expected, TMZ enhanced the expression of p-AMPK and amlexanox led to the reduction of IKBKE, with no impact on p-AMPK. Furthermore, we demonstrated that compared to other groups treated with each component alone, TMZ combined with amlexanox effectively reversed the TMZ-induced activation of Akt and inhibited the phosphorylation of mTOR. In addition, the combination treatment also clearly reduced in vivo tumor volume and prolonged median survival time in the xenograft mouse model. These results suggest that amlexanox sensitized the primary glioma cells and U87 MG cells to TMZ at least partially through the suppression of IKBKE activation and the attenuation of TMZ-induced Akt activation. Overall, combined treatment with TMZ and amlexanox may provide a promising possibility for improving the prognosis of glioblastoma patients in clinical practice.

Introduction

Glioblastoma multiforme (GBM), which accounts for more than 60–70% of all gliomas, is the most aggressive and most deadly primary brain tumor in adults.[1,2] Over the past few decades, although there has been progress in human GBM treatment, currently including maximal surgical resection followed by chemotherapy and/or radiotherapy, the prognosis of patients diagnosed with GBM remains extremely grim, with a median survival of approximately 14.6 months and a 5-year survival rate of only 9.8%.[3,4] Thus, there is an overwhelming need for more efficacious therapeutic approaches for this malignancy.

Temozolomide (TMZ), a novel oral alkylating agent, is the drug that is most frequently used against malignant glioma, and it has broad-spectrum antitumor activity.[5] Although TMZ is considered the most promising chemotherapeutic drug against GBM, most patients suffer from tumor recurrence within 7 months due to the development of resistance to TMZ.[6] Accumulating evidence demonstrates that the activation of Akt is responsible for the evolution of resistance in different types of cancers.[7−9] Akt phosphorylates several substrates associated with various cellular processes, such as cell growth, survival, and metabolism.[10,11] Interestingly, Akt activation is enhanced by TMZ treatment, which, in turn, attenuates TMZ-induced apoptosis.[12,13] Recently, Akt is reported to be phosphorylated by IKBKE (known as IKKε and IKKi) in breast cancer,[14] nonsmall-cell cancer (NSCLC),[15] and other cells or tissues.[16]

Amlexanox, a selective IKBKE inhibitor, has been approved for the treatment of aphthous ulcers and asthma without a clear molecular mechanism.[17,18] Amlexanox is also effective for the treatment of obesity and type 2 diabetes.[19,20] Moreover, the potential potency of inhibition in GBM cell lines was recently demonstrated.[21] As far as we know, more and more research studies are being conducted to study the effect of combined therapy on GBM.[4] Cold atmospheric plasma technology, a novel technique investigated as a stand-alone treatment on the tumor in vivo and in vitro, was demonstrated to amply cytotoxicity of TMZ by combining it with TMZ.[22] Moreover, a theranostic nanodrug developed recently, which is a multifunctional nanodrug that contains manganese and TMZ, presents great potential for the treatment of brain glioma.[23] Here, we study if amlexanox could attenuate the chemoresistance of human GBM cells to TMZ by inhibiting the activation of IKBKE and Akt.

In this study, we demonstrated that the combination of TMZ and amlexanox augmented the effects in primary GBM cells and human GBM cells in vitro. Furthermore, the efficacy of the combination was confirmed in vivo in a xenograft mouse model. These results suggest the possibility of TMZ combined with amlexanox for the treatment of GBM.

Materials and Methods

Chemical and Reagents

Anti-IKBKE (cat. no. 3416P), anti-Akt (phospho-Ser473, cat. no. 4058S), and anti-AMPK (cat. no. 5831S) antibodies were purchased from Cell Signaling Technology, Inc (Shanghai China). Anti-mTOR (cat. no. 36991), anti-mTOR (phospho-Ser2448, cat. no. 11221), and anti-caspase-3 (cat. no. 27525) antibodies were purchased from Signalway Antibody (College Park, MD). Anti-Akt (cat. no. A18675), anti-Bcl2 (cat. no. A19693), and anti-Bax (cat. no. A7626) antibodies were purchased from ABclonal (Boston, MA). Anti-AMPK (phospho-Ser172, cat. no. ARG51678.100) antibody was purchased from Arigo (Taiwan, China). Goat antimouse IgG-HRP, goat antirabbit IgG-HRP, and GAPDH antibodies were purchased from Utibody (Tianjin, China). Amlexanox was purchased from Selleck.cn (Shanghai, China), and TMZ was purchased from Solarbio Science & Technology (Shanghai, China). Both components were dissolved in dimethyl sulfoxide (DMSO) (Louis, MO) to prepare a stock concentration of 100 mM (TMZ) and 500 mM (amlexanox), respectively, and stored at −20 °C.

Cell Culture

The GBM cell line (U87 MG) was kindly provided by professor Chunsheng Kang and maintained in Dulbecco’s modified Eagle’s medium (DMEM, HyClone, Logan, UT) supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Inc., Waltham, MA), 100 U/mL penicillin/streptomycin (Solarbio Science & Technology, Co., Ltd., Shanghai, China), and cultured in a 95% humidified atmosphere with 5% CO2 at 37 °C.

Primary Human Glioblastoma Cells

After obtaining informed consent, fresh tumor samples, classified as grade IV of astrocytoma based on the World Health Organization (WHO), were obtained from patients undergoing surgical treatment at the Tianjin Medical University General Hospital. Within 1 h of removal, a part of the resected tissues were washed and enzymatically digested at 37 °C for 1 h. The remains were used in the construction of the xenograft model. Then, the undigested tissues were removed through centrifugation, and the rest of the sample was mixed with F12 (Gibco, Thermo Fisher Scientific, Inc.) medium supplemented with 10% FBS and 100 U/mL penicillin/streptomycin and maintained at 37 °C in a 5% CO2 incubator.

Cell Viability Assay

The Cell Counting Kit-8 (CCK-8) assay (Dojindo EU GmbH, Beijing, China) was used to evaluate the cell viability. In brief, U87 MG cells and primary GBM cells (3 × 103 cells/well) were seeded in 96-well plates and incubated overnight. Then, the cells were treated with the designated concentration of TMZ or amlexanox alone or both for 24, 48, and 72 h. After incubation, 10 μL of CCK-8 was added to each well according to the manufacturer’s instructions, and the cells were incubated for 2 h at 37 °C. Finally, the OD value was determined at 450 nm (OD450) by a microplate reader (Synergy2, BioTek, VT). Three experiments were performed independently.

Colony Formation Assay

Five hundred U87 MG or primary GBM cells were seeded in six-well plates. After incubation overnight, both cell types were treated with the desired concentrations of TMZ and amlexanox, alone or in combination, and the medium was changed once every 5 days for 2 weeks. Methanol and crystal violet were used to fix and stain cell colonies, and colonies with more than 50 cells were counted using an inverted microscope (Olympus, Japan). Three experiments were performed independently.

Apoptosis Assay

Both U87 MG and primary GBM cells (1–1.5 × 105 cells/well) were plated in six-well plates and treated with the desired concentration of TMZ and amlexanox, alone or in combination, for 48 h. Then, the treated cells were harvested through trypsinization without ethylenediaminetetraacetato (EDTA) and washed with ice-cold phosphate-buffered saline (PBS). Next, the cells were resuspended in 500 μL of the binding solution containing 5 μL of annexin V and 5 μL of propidium iodide (PI) (annexin V, FITC Apoptosis Detection Kit, Dojindo EU GmbH, Beijing, China) according to the manufacturer’s instruction. After incubating for 15 min at room temperature, cell apoptosis was immediately analyzed by a FACScan flow cytometer (BD Biosciences), and further statistical analysis of apoptotic cells was conducted using FlowJo software. Three experiments were performed independently.

Western Blotting

U87 MG and primary GBM treated with the desired concentrations of TMZ and/or amlexanox were harvested and then lysed in RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.) supplemented with a protease inhibitor mixture (APExBIO). The concentrations of the proteins were determined using a BCA protein assay kit (Thermo Fisher Scientific, Inc.), based on the manufacturer’s instructions, and the protein was denatured in 100 °C for 5 min. Equivalent amounts of protein were separated by 6, 10, or 12% SDS-PAGE gels and then transferred to poly(vinylidene fluoride) (PVDF) membranes (Billerica, MA). After blocking in 5% skim milk for 1 h at 37 °C, the membranes were incubated with specific primary antibodies overnight at 4 °C, followed by incubation with the corresponding horseradish peroxidase (HRP)-conjugated second antibodies for 1 h at room temperature. GAPDH was considered as an internal reference for loading. Antigen-bound antibodies were detected using the supersignal west pico plus chemiluminescent substrate (Thermo Fisher Scientific).

Migration Assay

To assess the migration ability of U87 MG and primary GBM cells after treatment with TMZ and amlexanox, alone or in combination, for 48 h, a scratch wound healing assay was conducted. In brief, the treated cells (3 × 105 cells/well) were seeded into six-well plates; when the cells reached 80–90% confluence in a monolayer, scratch wounds were made using a 200 μL pipette tip. Then, cell debris was removed, and a microscope was used to confirm the uniform scratch width of every group. After incubation at 0, 12, and 24 h, five different fields of each well were measured and photographed using a phase-contrast microscope. In addition, transwell filters with 8 μm pores (Corning Costar, NY) (without Matrigel) were also used to evaluate the migration ability of treated cells. The assay was conducted as described in a previous report.[21]

Invasion Assay

The invasion capacity of the treated U87 MG cell and primary GBM cell with TMZ and/or amlexanox was evaluated by the Transwell assay with inserts of 8 μm pore size. The treated cells were resuspended in 200 μL of serum-free DMEM, seeded into the upper chamber covered with Matrigel (BD Bioscience), diluted with serum-free DMEM, and incubated for 24 h at 37 °C. After removing noninvading cells from the top well, the bottom cells were fixed in 5% methanol, stained with 0.1% crystal violet, and then three independent 10× regions were photographed for each insert. Three experiments were independently conducted.

Immunohistochemistry (IHC)

For histological analysis, the tissues were fixed in 4% formaldehyde for IHC analysis. In brief, the slides (5 μm) were dewaxed using xylene and rehydrated using graded alcohols. Antigen retrieval was performed with sodium citrate (pH = 6) buffer at 92–99 °C for 15 min, and then the slides were cooled at room temperature. The slides were washed three times for 5 min in PBS and incubated with 3% H2O2 for 30 min to block endogenous peroxidases. The slides were blocked using 1% bovine serum albumin (BSA) for 30 min at room temperature. Next, the slides were incubated with specific primary antibodies at 4 °C overnight before being incubated using biotin-labeled secondary antibody for 1 h at 37 °C and incubated again with diaminobenzidine (DAB) (Solarbio Science & Technology, Beijing, China). Finally, the slides were counterstained using hematoxylin and mounted. All experiments were repeated independently at least three times.

Xenograft Models

All mouse experiments were conducted according to the protocols approved by the Tianjin Medical University Animal Care and Use Committee and followed guidelines for animal welfare. Female BALB/c nude mice (4 weeks old, approximately 12 g) were purchased from Beijing HFK Bioscience Co., Ltd. To establish an intracranial tumor model, primary GBM cells (5 × 104 cells) infected with luciferase-encoding lentivirus were stereotactically injected into the right hemisphere. A burr hole was located at a point situated 2 mm lateral from bregma and between bregma and fonticuli minor with a syringe under stereotactic guidance. Seven days after injection, the mice were divided randomly into four groups with 15 mice in each group: control, amlexanox alone, TMZ alone, and combination (TMZ and amlexanox). The negative control group was treated with DMSO, and the treated groups were intraperitoneally given amlexanox alone (100 mg/kg), TMZ alone (5 mg/kg), or TMZ (5 mg/kg) and amlexanox (100 mg/kg), respectively, for 5 days. After 2 days without injections, the same dosing regimen was continuously repeated. Tumor growth was measured once every week using bioluminescence (BLI) at a designated time with the IVIS Spectrum Live Imaging System (PerkinElmer). After 4 weeks postinjection, three mice in each group were sacrificed, and the brains were extracted and fixed in 10% formalin then embedded in paraffin for HE and IHC. The remaining mice were used for survival analysis.

Before constructing the xenograft model of patient-derived tissues, consent was received. The samples resected from the same GBM patient were minced in a sterile dish by a sterile scalpel blade. The blood was cleared and equal amounts of tissues were injected into the right flanks of anesthetized nude mice. The nude mice were divided randomly into three groups with 5 mice in each group when the tumors reaching a volume of 100 mm3. The administration for each group was the same as described above. The volume (mm3) of the tumor was calculated as follows: length × width2/2 and measured every 3 days. Four weeks after implantation, all mice were sacrificed and the tumors were removed for IHC analysis.

Statistical Analysis

All experimental data are represented as the mean ± standard error of mean (SEM). Statistical analysis was performed using GraphPad Prism 6 software. One-way analysis of variance (ANOVA) was carried out to assess differences between multiple groups. The Kaplan–Meier method was used to evaluate the difference in survival among the groups. The value of p < 0.05 was regarded as statistically significant.

Results

Amlexanox Enhanced the TMZ-Induced Suppression of GBM Cell Proliferation

To evaluate the effect of the association with TMZ and amlexanox on cell viability, the CCK-8 assay was conducted. U87 MG and primary GBM cells were treated with different doses of TMZ, amlexanox, or combination for 24, 48, and 72 h. As expected, the inhibition of proliferation in both cell types was gradually amplified with an increased concentration of either agent alone (Figure A), which was consistent with the previous studies.[4,21] Moreover, the proliferation of cells treated with the combination, whether U87 MG or primary GBM cells, was efficaciously attenuated (Figure A). In addition, the 48 h IC50 values for TMZ and amlexanox in U87 MG cells were 400 and 300 μM, respectively, which were reduced to 150 and 200 μM with the combination treatment. Similarly, the IC50 value of either component alone was higher than that of the combination treatment in primary GBM cells. Based on the IC50 values in our study, the concentrations of 100 and 50 μM for TMZ and amlexanox, respectively, were used in the subsequent experiments. Next, the colony-forming ability of U87 MG and primary GBM cells was determined. The colony formation analysis showed that the colony formation rates were significantly decreased in cells with the combination treatment compared to those in the cells treated with either agent alone (Figure B,C).

Figure 1

TMZ combined with amlexanox effectively inhibited the proliferation of U87 MG and primary GBM cells. (A) Results of CCK-8 assay. (B, C) Representative images of the colony-forming assay (left) and statistical analysis in the form of histogram (right) after exposure to TMZ (100 μM), amlexanox (50 μM), or both for two weeks. The data presented are shown as mean ± SED (*p < 0.05, **p < 0.01, and ***p < 0.005).

Amlexanox Promoted TMZ-Induced Apoptosis of GBM Cells

Apoptosis is one of the main mechanisms by which TMZ acts.[24,25] To evaluate whether amlexanox can enhance TMZ-induced apoptosis, an annexin V-FITC assay was conducted. As shown in Figure A, the percentage of apoptotic cells was significantly increased in U87 MG cells with the combination treatment compared to the cells treated with either drug alone, which was consistent with the findings in primary GBM cells. Furthermore, the effect of the combination on apoptosis was validated through a Western blot assay in which changes in apoptosis-related proteins were induced. The activation of proapoptotic proteins, Bax, and caspase-3 were higher in both cells treated with the combination than that in the cells treated with either drug alone (Figure B). The expression of antiapoptotic protein Bcl2 was decreased in the combination group, in both U87 MG and primary GBM cells, compared to that in the cells treated with each drug alone. These results suggest the possibility that amlexanox enhanced the TMZ-induced antiproliferative activity by promoting apoptosis.

Figure 2

Amlexanox prompted TMZ-induced apoptosis in U87 MG and primary GBM cells. (A) Representative images of apoptosis for U87 MG and primary GBM cells after treatment with TMZ (100 μM), amlexanox (50 μM), or combination for 48 h. (B) Quantified results in histograms for apoptosis of U87 MG and GBM cells. (C) Levels of expression of Bcl2, Bax and active caspase-3 were measured by Western blotting. The data presented here are shown as mean ± SED (*p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001).

Amlexanox Augmented the TMZ-Induced Inhibition of Migration and Invasion in GBM Cells

Migration and invasion are the primary characteristics of GBM cells. To evaluate the effect of TMZ combined with amlexanox on the migration and invasion of U87 MG and primary GBM cells, wound healing and transwell assays were conducted. The results of the wound healing assay showed that the inhibition of cellular migration in the combination group was more apparent compared to that in other groups treated with a single drug (Figure A,B), which was in accordance with the results of the Transwell migration assay (without Matrigel) (Figure C,D). For the Transwell assay (with Matrigel), the results indicated that after different treatments for 24 h, amlexanox efficaciously augmented TMZ-induced inhibition of invasion in U87 MG and primary GBM cells (Figure C,D).

Figure 3

Combined treatment of TMZ with amlexanox efficaciously inhibited the migration and invasion of U87 MG and primary GBM cells. (A) Representative images of the wound healing assay for each cell type after 0, 12, and 24 h seeding. (B) Data of the wound healing assay were quantified and shown as a histogram. (C) Representative images of both cell types were captured after 24 seedings in the Transwell assay (without Matrigel). (D) Results of both cell types from the Transwell assay (without Matrigel) were quantified and are shown in a histogram. (E) Representative images of both cell types were captured after 24 seedings in the Transwell assay (with Matrigel). (F) Results of both cell types from the Transwell assay (with Matrigel) were quantified and are shown in a histogram (**p < 0.01, ***p < 0.005, and ****p < 0.001).

TMZ Combined with Amlexanox Effectively Decreased the Activation of Akt

TMZ treatment has been suggested to increase the activation of Akt, which, in turn, results in resistance to TMZ.[12] Moreover, the IκB kinase, IKBKE, is affirmed to be responsible for activating Akt.[14,16] To investigate whether the amlexanox-mediated inhibition of IKBKE can attenuate the TMZ-induced activation of Akt, the alteration of relevant proteins was measured following treatment with TMZ and amlexanox, alone or combined, in U87 MG and primary GBM cell for 48 h. The effect of TMZ or amlexanox alone on the IKBKE and Akt signaling pathways was first evaluated. The results after treatment with the designated dose of either agent for 48 h in U87 MG and primary GBM cells are shown in Figure . The Western blot assay indicated that TMZ induced the activation of Akt and AMPK and decreased the phosphorylation of mTOR; the activation of IKBKE was decreased after amlexanox treatment, which was consistent with previous studies.[14,21,25] After treating U87 MG and primary GBM cells with both agents, the results showed that TMZ combined with amlexanox resulted in an enhanced reduction of p-Akt and p-mTOR. Taken together, these results show that after treatment with TMZ alone, the slightly decreased level of p-mTOR may be due to the TMZ-induced activation of AMPK, but Akt was activated at the same time, which may have resulted in the resistance of GBM cells to TMZ. However, amlexanox reversed the TMZ-mediated expression of p-Akt by inhibiting IKBKE activation, which may be part of the mechanism by which amlexanox can sensitize GBM cells to TMZ treatment.

Figure 4

Amlexanox sensitized U87 MG and primary GBM cells to TMZ through the inhibition of AKT activation. After treatment with TMZ and/or amlexanox for 48 h, the cells were harvested, and Western blotting was conducted to detect the expression of relevant proteins.

Combination of TMZ and Amlexanox Inhibited the Growth of Tumors in Xenograft Models

To evaluate the efficacy of the combination of TMZ and amlexanox in vivo, nude mice were subcutaneously injected with patient-derived tissues to validate the effect of the combination of TMZ and amlexanox on tumor growth. The results showed that the tumors in the group treated with the combination grew slower than those in the TMZ group and the control group at every time point (Figure A–C). Furthermore, the weight of the tumors was significantly decreased in the combination group compared with that in the other groups (Figure D). The IHC staining assay of subcutaneous tumors showed that there was an apparent decrease in p-Akt and p-mTOR staining in the mice treated with the combination compared to that in other groups (Figure E). In addition, intracranial tumor models were constructed with primary GBM cells expressing a luciferase reporter to further validate the above finding. The results are shown in Figure A–C. The volume of intracranial tumors in the group treated with amlexanox alone or TMZ alone was modestly reduced, and the survival (24 days or 25.5 days) was slightly improved compared with that in the control group (21 days). However, there was a significant reduction of tumor burden and improvement of survival (31 days) in the combination group compared with that in the amlexanox alone group or the TMZ alone group. The median survival in the TMZ and amlexanox co-treatment group was longer than that in the control, amlexanox alone, and TMZ alone groups. Moreover, the molecular mechanism was assessed by IHC staining assay (Figure D). The results showed that compared to the treatment with control, amlexanox alone or TMZ alone, treatment of TMZ with amlexanox obviously reduced the levels of p-Akt and p-mTOR. Overall, these results suggested that amlexanox was able to penetrate the blood–brain barrier in vivo and enhance the TMZ-induced inhibition of tumor growth but decreased the expression of p-Akt and p-mTOR.

Figure 5

Combination of TMZ and amlexanox effectively reduced the growth of the tumor of the patient tissue-derived models. (A) Representative images of subcutaneous tumors 4 weeks postinjection. (B) Dissected tumors and (C) tumor growth and (D) tumor weight curves. (E) Representative images from the IHC assay were captured for IKBKE, p-AKT, p-mTOR, and p-AMPK (***p < 0.005 and ****p < 0.001).

Figure 6

Amlexanox treatment sensitized orthotopic intracranial tumors of primary GBM cells to TMZ. (A) Representative images of the bioluminescence (BLI) of intracranial tumor models were captured on days 7, 14, 21, and 28. (B) Quantified analysis of these bioluminescence images for each group. (C) Survival analysis of the mice in each group. (D) Representative images of HE staining of full-brain sections and representative images of the IHC staining assay for IKBKE, p-AKT, p-mTOR, and p-AMPK (×200 magnification) (**p < 0.01, ***p < 0.005, and ****p < 0.001).

Discussion

In the present study, we demonstrated that amlexanox enhanced the sensitization of GBM cells to TMZ in vitro and in vivo. As far as we know, this study was the first to use primary GBM cell and patient-derived GBM tissue models, which maintained important histopathological and molecular characteristics of primary GBM tumor, to evaluate the effect of the combination of TMZ with amlexanox. Moreover, we found that amlexanox could indirectly inhibit the expression of Akt through reducing activity of IKBKE, which suggested a connection between Akt and IKBKE in the development of GBM and might be a potential inhibitory target of GBM.

TMZ is a common chemotherapeutic drug, and the induction of DNA adducts is the primary mechanism of exerting cellular toxicity.[26] Although the administration of TMZ has improved the prognosis of GBM patients, resistance against TMZ was quickly developed.[9] The overexpression of O6-methylguanine-DNA-methyltransferase (MGMT) is one of the main resistance mechanisms for repairing O6-methylguanine, which is an important TMZ-induced lesion, resulting in the breakage of DNA double-strand and subsequent apoptosis.[5,6] In addition, immune escape after TMZ treatment,[27] dysfunction of the DNA mismatch repair (MMR) system,[1] abnormal expression of nuclear factor erythroid 2-related factor 2 (Nrf2),[28] and high expression of ATP-binding cassette (ABC) membrane transporters[2] were also demonstrated to be involved in resistance development. However, what greatly interested us is the activation of Akt, which involved various cellular processes, such as proliferation, cell growth, and survival.[14] A growing number of studies have proved that the activation of Akt is associated with resistance to TMZ treatment.[29] Moreover, administration of TMZ for GBM was proved to induce Akt activation.[4,12,13,30] Given that the above factors resulted in decreased TMZ-induced cellular toxicity, it is impractical to treat GBM patients with a single agent. Therefore, in this study, we conducted a series of assays to evaluate the effect of a combination of TMZ and an inhibitor, amlexanox, in the treatment of human GBM cell line and primary GBM cell.

The results of our study showed that TMZ combined with amlexanox not only effectively inhibited proliferation, invasion, and migration, which were the main challenges in the treatment of GBM, but also greatly promoted cellular apoptosis in human GBM cell line and primary GBM cell. The upregulated expression of Bax and caspase-3 validated the occurrence of apoptosis. Moreover, Western blot assays suggested that TMZ treatment induced AMPK phosphorylation, which contributed to apoptosis via mTOR inhibition.[25] To better understand the potential mechanism of amlexanox enhancing the TMZ-induced cellular toxicity, the relevant proteins were examined following treatment of U87 MG and primary GBM cells with either agent alone or in combination. It was revealed that amlexanox treatment induced the reduction of IKBKE activation, subsequently reversed TMZ-mediated expression of p-Akt, and enhanced the suppression of p-mTOR. These results indicated that amlexanox attenuated the chemoresistance of GBM cells to TMZ partially through the amlexanox-induced inhibition of IKBKE, which resulted in the repression of TMZ-induced Akt activation. In addition, TMZ combined with amlexanox exhibited notable antitumor efficacy in PDX models, which is consistent with their antiproliferation in vitro. Orthotopic intracranial mouse models were constructed to demonstrate the ability of amlexanox to cross the blood–brain barrier (BBB). Our results verify that the combined treatment with TMZ and amlexanox not only be able to significantly inhibit brain tumor growth but also prolong the survival time of the intracranial mouse models, which suggest a good permeability of BBB for amlexanox. To date, a growing number of studies have focused on the inhibition of the Akt signaling pathway with the combined administration in GBM and other tumors.[7,31−36] Yu and his colleagues suggested that TMZ combined with NVP-BEZ235 synergistically inhibited GBM cell proliferation by downregulating Akt/mTOR signaling pathway,[4] which was similar to our findings. However, in our study, the primary GBM cell and patient-derived tissue models, which are more similar in histopathological and molecular characteristics to primary GBM tumors, were used to evaluate the effect of combination treatment. Nevertheless, it is undeniable that there are some limitations to our study. Only one kind of GBM cell line or primary GBM cell was used. If more cell lines and primary GBM cells were used, the results would be more convincing. Additionally, more assays should be conducted to further validate that the inhibition of the Akt signaling pathway was induced by amlexanox, which enhanced TMZ-induced cellular toxicity. The side effect was always the issue that should be considered when it comes to treatment with a new combination in GBM patients. More assays, such as blood biochemistry analyses for hematologic, hepatic, and nephric functions, should be conducted to evaluate the side effect when nude mice were managed with a combination of TMZ and amlexanox in the present study.

In conclusion, we have demonstrated that amlexanox can enhance the TMZ-induced cellular toxicity first using primary GBM cell and patient-derived GBM tissue models; the mechanism occurs partially through that the downregulated activation of IKBKE induced by amlexanox reverses TMZ-induced activation of Akt, suggesting that the combination of TMZ and amlexanox is a possible treatment for GBM patients.