Induction of apoptosis through extrinsic/intrinsic pathways and suppression of ERK/NF‐κB signalling participate in anti‐glioblastoma of imipramine

Abstract Glioblastomas are the most aggressive type of brain tumour, with poor prognosis even after standard treatment such as surgical resection, temozolomide and radiation therapy. The overexpression of the nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) in glioblastomas is recognized as an important treatment target. Thus, an urgent need regarding glioblastomas is the development of a new, suitable agent that may show potential for the inhibition of extracellular signal‐regulated kinase (ERK)/NF‐κB–mediated glioblastoma progression. Imipramine, a tricyclic antidepressant, has anti‐inflammatory actions against inflamed glial cells; additionally, imipramine can induce glioblastoma toxicity via the activation of autophagy. However, whether imipramine can suppress glioblastoma progression via the induction of apoptosis and blockage of ERK/NF‐κB signalling remains unclear. The main purpose of this study was to investigate the effects of imipramine on apoptotic signalling and ERK/NF‐κB–mediated glioblastoma progression by using cell proliferation (3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide [MTT] assay), flow cytometry, Western blotting, and cell invasion/migration assay analysis in vitro. The ERK and NF‐κB inhibitory capacity of imipramine is detected by NF‐κB reporter gene assay and Western blotting. Additionally, a glioblastoma‐bearing animal model was used to validate the therapeutic efficacy and general toxicity of imipramine. Our results demonstrated that imipramine successfully triggered apoptosis through extrinsic/intrinsic pathways and suppressed the invasion/migration ability of glioblastoma cells. Furthermore, imipramine effectively suppressed glioblastoma progression in vivo via the inhibition of the ERK/NF‐κB pathway. In summary, imipramine is a potential anti‐glioblastoma drug which induces apoptosis and has the capacity to inhibit ERK/NF‐κB signalling.


| INTRODUC TI ON
Glioblastomas, also known as grade IV gliomas, are the most common and aggressive type of tumour of the central nervous system (CNS) in adults. 1 Rapid tumour progression, as an unfavourable prognostic factor, is related to poor outcomes in patients with glioblastomas. 2,3 Genetic and epigenetic alterations modulate the hyperactivation of oncogenic signalling transduction and are associated with the pathogenesis of glioblastomas. The upstream oncogenic kinases trigger downstream transcription factor-regulated oncogene expression, promoting the progression of glioblastomas. 1,4,5 The blockage of oncogenic signalling transduction contributes to the inhibition of tumour progression.
Apoptosis is programmed cell death which triggers cell death by extrinsic and intrinsic (mitochondrial) apoptotic pathways. 6 Chemotherapeutic agents modulate the reduction in tumour progression through the induction of apoptosis by deoxyribonucleic acid (DNA) damage. The high expressions of DNA repair and anti-apoptotic proteins diminish the anticancer efficacy of chemotherapy through the repair of DNA lesions and blockage of apoptotic signalling transduction. 7,8 High expressions of DNA repair and anti-apoptotic proteins were associated with poor survival in patients with glioblastoma. 9,10 The nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) p50/p65 heterodimer is the oncogenic transcription factor driving the expression of oncogenes and plays the critical mediator role in tumour progression. Constitutive NF-κB activation controls the expression of tumour progression-associated proteins which participate in tumour cell growth, anti-apoptosis, angiogenesis and metastasis. [11][12][13] In addition, the expression of several DNA repair proteins is also linked to NF-κB activation. 14,15 Puliyappadamba et al demonstrated that glioblastoma had higher NF-κB activation compared to normal brain and lower grade glioma. 1 NF-κB activation cannot be inhibited with chemotherapy or radiotherapy. 16,17 Therefore, the development of complementary agents which repress NF-κB signalling may offer therapeutic benefits for patients with glioblastomas. Anticancer effects and antidepressant mechanisms for glioma have been demonstrated. 18,19 Fluoxetine, the antidepressant of the selective serotonin reuptake inhibitor (SSRI), was demonstrated to reduce NF-κB activation and sensitize glioblastomas to the chemotherapeutic agent temozolomide (TMZ). 20 Fluoxetine also induced apoptosis through the calcium-mediated intrinsic apoptotic pathway in glioblastoma. 19 Long-term use of tricyclic antidepressants (TCAs) has been indicated to decrease glioma risk. 21 Imipramine, the TCA, has been shown to induce an accumulation of reactive oxygen species and inhibits NF-κB p65 gene expression in glioblastoma. 22 Jeon et al 23 presented imipramine-inhibited tumour cell growth through autophagy in glioblastoma in vitro. Furthermore, to overcome glioblastoma, whether imipramine can cross blood-brain barrier (BBB) is the major issue of drug selection. As compared to various antidepressant drug, such as duloxetine, fluoxetine and mirtazapine, the BBB penetration ability of imipramine was relatively better. 24 However, whether the induction of apoptosis and suppression of NF-κB signalling are associated with the imipramine-inhibited progression of glioblastomas is ambiguous. The major purpose of this study was to verify the efficacy and mechanism of imipramine on tumour progression in glioblastoma in vitro and in vivo.

| Plasmid transfection of U-87 MG cells
The vector containing NF-κB-luciferrase 2 vector (pNF-κB/luc2) was prepared in advance (Promega). U-87 MG cells were grown on a 6 cm plate and maintained at 70% confluency before the transfection process. Cationic polymer transfection reagent (Polyplus transfection) was used to transport these NF-κB/luc2 plasmids into the intrathecal region. First, 10 µL jetPEI™ reagent in 250 µL of NaCl buffer was added into plasmid solution (5 µg plasmids with 250 µL of NaCl buffer) followed by incubation for 25 minutes at room temperature. Second, the mixture was added onto a 6-cm plate and incubated for one day. Third, cells were then selected by hygromycin B 200 µg/mL for 2 weeks as a U87/NF-κB/luc2 stable clone for further investigation. [28][29][30] F I G U R E 1 Cell viability was decreased, and cell morphology was changed by imipramine in glioblastoma cells. Cells were treated with imipramine concentrations of 0, 20, 40, 60 or 80 µmol/L for 24 or 48 h. A, Cell viability was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. B, Cell morphology was examined by contrast-phase microscopy. Significant differences from the control and imipramine-treated groups were recorded at *P < .05 and **P < .01

| Sub-G1 phase (apoptosis) assays
Briefly, U-87 MG and GBM8401 cells were placed at a concentration F I G U R E 2 Cell mobility, migration and invasion ability of glioblastoma cells were suppressed by imipramine. U-87 MG and GBM8401 were grown in 6-well plates until 80% confluence, and cell monolayers were scraped and incubated in medium containing 0, 40 and 80 µmol/L of imipramine for 48 h. After incubation, the closure of the gap generated by the scraping was assayed as described in the Section 2. A, B, The representative figures for the closures of scraped areas. C, D, The percentage of area reduction was calculated by Image J. Transwell assays were performed to detect the (E) migration and (F) invasion activity. The quantification of (G) migrated and (H) invaded cells was assayed by Image J. *P < .05 and **P < .01: significant differences between imipramine-treated groups and the control as analysed by Student's t test. # P < .05 and ## P < .01: significant differences between 40 µmol/L imipramine-treated groups and 80 µmol/L imipraminetreated groups Animal NF-κB activation signals were also acquired by IVIS after 100 µL d-luciferin (150 mg/kg) injection as previously described. 34

| Invasion and migration assay
The assessment of in vitro invasion and migration activities were carried out using matrigel-coated or uncoated transwell cell culture chambers (8 μm pore size) as described previously. 35

| Wound healing assay
U-87 MG and GBM8401 cells (5 × 10 5 cells/well) maintained in 6-well plates were grown to complete confluency. Cell monolayers were scraped using a sterile yellow micropipette tip and washed with PBS three times. Cells were then cultured in DMEM medium con-

| Animal
Twenty male athymic BALB/c nu/nu mice at 6-8 weeks of age were

| Haematoxylin and eosin stain
Liver tissues were fixed in 10% formalin and embedded in paraffin. Paraffin-embedded specimens were then sliced, deparaffinized, rehydrated and stained with haematoxylin and eosin (H&E) by Bio-Check Laboratories Ltd as previously described. 39

| Statistical analysis
Data were expressed as mean ± SEM. Student's t test was used to compare the means of vehicle and imipramine groups by Excel 2017 (Microsoft). A P value of <.05 was considered statistically significant. 40

| Imipramine decreased cell viability and induced cell morphological changes of U-87 MG and GBM8401 glioblastoma cells
U-87 MG and GBM8401 glioblastoma cells were treated with various concentrations of imipramine for 24 or 48 hours; then, the total viable cells were determined and the cell morphology examined.

| Imipramine decreased the cell mobility, migration and invasion ability of U-87 MG and GBM8401 glioblastoma cells
In Figure 2A

| Imipramine-induced apoptotic cell death in U-87 MG and GBM8401 glioblastoma cells
We further investigated whether imipramine may trigger an apoptosis effect of U-87 MG and GBM8401 glioblastoma cells. As shown in Figure 3A,B, the apoptosis marker cleaved caspase-3 was noticeably activated by a dose elevation of imipramine. In annexin V staining, apoptosis effect was both induced in U-87 MG and GBM8401 cells by imipramine after 48-hour treatment ( Figure 3C-E). In addition, the sub-G1 population, which was recognized as apoptotic cell death, was also increased by imipramine ( Figure 3F,G). These results proved that an apoptosis effect was effectively triggered by imipramine on both types of glioblastoma cells.

| Imipramine triggered the death receptordependent extrinsic apoptosis effect of U-87 MG and GBM8401 glioblastoma cells
To identify whether the apoptosis effect of imipramine was modulated by extrinsic death receptor-dependent apoptosis signalling, we investigated several extrinsic apoptosis markers. First of all, Fas activation was increased by imipramine on U-87 MG and GBM8401 cells ( Figure 4A,B). The Fas corresponded ligand Fas-L was also markedly activated by imipramine, as shown in Figure 4C,D in both types of glioblastoma cells. Moreover, we found that the downstream molecular of Fas and Fas-L, cleaved caspase-8, was also increased by imipramine on two types of glioblastoma cells ( Figure 4E,F).

| Imipramine-induced mitochondrial-dependent intrinsic apoptosis effect of U-87 MG and GBM8401 glioblastoma cells
To further validate whether imipramine-induced apoptosis is mediated by mitochondria-dependent intrinsic apoptosis, we performed MMP (ΔΨ m ) and Ca 2+ concentration analysis. As shown in Figure 5A,B, the loss of ΔΨ m was increased 15%-45% in imipraminetreated cells as compared to controls. Results from Figure 5C,D indicate that Ca 2+ production increased in imipramine treatments, which may indicate that imipramine-induced cell apoptosis is associated with Ca 2+ production.

| Imipramine inhibited tumour progression via targeting ERK/NF-κB and its associated protein levels in U-87 MG and GBM8401 glioblastoma cells
In order to examine the mechanism by which imipramine affects the DNA damage/repair, proliferation, migration and invasion of F I G U R E 6 (Continued) glioblastoma cells, its effects on the levels of NF-κB activation and certain proteins were examined. As shown in the reporter gene assay, imipramine markedly suppressed NF-κB activation in U87/ NF-κB/luc2 cells ( Figure 6A). Imipramine was found to significantly reduce the NF-κB upstream mediator protein phosphorylation level of p-ERK in both types of glioblastoma cells ( Figure 6C,D). In addition, the DNA damage repair-related marker MGMT was also effectively suppressed by imipramine ( Figure 6C,D). Moreover, tumour progression-related proteins such as MMP-2, MMP-9, uPA, VEGF, Cyclin D1 and XIAP were all decreased by imipramine in U-87 MG and GBM8401 cells ( Figure 6E,F). Based on these observations, it is concluded that imipramine inhibits tumour progression through the inhibition of the ERK//NF-κB pathway.

| Imipramine suppressed the growth of U-87 MG tumour xenograft in vivo
An animal experimental flowchart is displayed in Figure 7A, and all experiments were repeated twice. A total of 10 mice was divided into 2 groups: a 0.1% DMSO-treated vehicle group and a 10 mg/ kg imipramine-treated group. BLI scan was performed on days 0, F I G U R E 7 No general toxicity, but tumour growth inhibition, was found in imipramine-treated glioblastoma-bearing mice. A, Animal experimental procedure. B, Mice body weight was recorded every 3 days and calculated. C, Mice liver tissue was investigated with H&E staining. D, Tumour tissues extracted from mice on day 21 are displayed. E, Tumour volume was also recorded every 3 days. F, Tumour weight was measured after extraction from mice. **P < .01: significant difference between imipramine-treated groups and vehicle as analysed by Student's t test 10 and 20 after treatment. Tumour volume (TV) and body weight (BW) were measured every three days. Mice were killed on day 21 for IHC, H&E staining and ex vivo Western blotting. In Figure 7B, the mice's body weight remained unchanged during 21 days of treatment. The lack of an obvious liver pathology difference between vehicle and imipramine group mice indicated that no general toxicity was found ( Figure 7C). The tumours extracted on day 21 showed that sizes in the imipramine-treated group were markedly smaller than the vehicle group ( Figure 7D). In Figure 7E, effective tumour growth inhibition was found in imipramine group after 3-day administration until the end of the experiment. Moreover, the tumour weight from both groups was measured after being isolated from killed mice. As can be seen in Figure 7F, the tumour weight was effectively reduced by imipramine, and this result corresponded with Figure 7D,E.

| Imipramine suppressed tumour growth via inactivation of ERK/NF-κB signalling transduction
To identify whether the NF-κB inhibition effect of imipramine also worked effectively on in vivo animal models, we performed in vivo NF-κB activation analysis by IVIS. The result in Figure 8A showed the dramatic suppression of NF-κB activation found in imipraminetreated mice as compared to vehicle mice. The signal intensity of NF-κB was markedly reduced in imipramine tumours. Then, we further validated whether NF-κB upstream signalling was also suppressed by imipramine. In IHC staining results, the phosphorylation of ERK and phosphorylation of P-65 NF-κB were both inhibited by imipramine ( Figure 8B). NF-κB-associated proteins involved in tumour progression, such as MMP-2, MMP-9, uPA, VEGF, XIAP and Cyclin D1, were all decreased by imipramine ( Figure 8C). Importantly, MGMT, which is involved in the treatment-induced resistance of glioblastomas, was also decreased by imipramine ( Figure 8D). Furthermore, ex vivo Western blotting was also performed to doubly confirm the protein expression alteration of imipramine treatment. As shown in Figure 8E,F, P-ERK(Thr202/Tyr204), P-P65 NF-κB(Ser276), MGMT and NF-κB-related protein expression was decreased 70%-80% by imipramine as compared with vehicle treatment. The proposed mechanism of imipramine was displayed as Figure 8G.

| D ISCUSS I ON
Pro-apoptotic proteins, the critical components of extrinsic and intrinsic apoptotic pathways, are decreased in glioblastomas. 41,42 Caspase family members act as activators, executioners or mediators modulating the induction, transduction and amplification of apoptotic signalling cascades. Caspase-8, the apoptotic activator in the extrinsic pathway, can be activated with death receptor/ death receptor-ligand interaction. Caspase-3, the apoptotic executioner, participates in apoptotic deoxyribonucleic acid (DNA) fragmentation. 43 Decreased expression of active caspase-8 and caspase-3 were recognized as poor prognostic biomarkers related to worse survival in patients with glioblastomas. 42,44 Our data showed that imipramine significantly increased the activation of both caspase-3 and caspase-8 in glioblastoma U87 MG and GBM-8401 cells (Figures 3 and 4). In addition, the down-regulation of apoptotic pathways and up-regulation of anti-apoptotic proteins were associated with apoptosis evasion, contributing to tumour progression. 45 The overexpression of anti-apoptotic proteins abrogates chemotherapeutic agent-induced cell death through the disruption of apoptotic signalling transduction. 46 X-linked inhibitor of apoptosis (XIAP), the anti-apoptotic protein, hinders chemotherapy-induced apoptosis by the reduction of caspase-3, -7 and -9 activation. 47 Glioblastoma patients with high XIAP expression had worse survival rates than those with low XIAP expression. 48 As shown in Figures 6 and 8, XIAP expression was significantly decreased by imipramine in glioblastoma U87 MG and GBM-8401 cells.
Loss of mitochondrial membrane potential (ΔΨ m ) activates caspase-mediated apoptosis through the release of cytochrome C and apoptotic protease activating factor 1 (APAF1) from mitochondria. 49  Here, we summarized our proposed mechanism of imipramine in glioblastoma ( Figure 8G). First, imipramine may inhibit ERK/NF-κB signalling transduction and thus reduced tumour progression-related proteins expression, including MGMT, MMP-2, MMP-9, uPA, VEGF, Cyclin D1 and XIAP. Thus, tumour progression of glioblastoma was effectively blocked by imipramine treatment. In addition, imipramine may also trigger death receptor-dependent extrinsic and mitochondria-dependent intrinsic apoptosis signalling. In sum, the inhibition of ERK/NF-κB signalling transduction and the induction of apoptosis are associated with imipramine-inhibited tumour progression. In conclusion, we suggest that imipramine may be used as a potential complementary agent, affording additional therapeutic efficacy to patients with glioblastomas. Thus, validated the progression-free survival data from patients who taking this antidepressant may be our further step.

ACK N OWLED G EM ENT
Experiments and data analysis were performed in part through the use of the Medical Research Core Facilities Center, Office of Research & Development at China Medical University, Taichung, Taiwan, ROC.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflicts of interest.