Curcumin inhibits adverse psychological stress‐induced proliferation and invasion of glioma cells via down‐regulating the ERK/MAPK pathway

Abstract Curcumin is a natural polyphenol extracted from the rhizome of Curcuma that has an important antitumour effect, but its effect on adverse psychological stress‐induced tumour proliferation and invasion has not been reported to date. Here, we found that curcumin not only inhibited the growth of xenografts in chronically stressed nude mice, but also decreased the expression of matrix metalloproteinase (MMP)‐2/9 and CD147 in tumour tissues. Exogenous norepinephrine (NE) was used to stimulate glioma cells to simulate the stress environment in vitro, and it was found that curcumin inhibited the NE‐induced proliferation and invasion of glioma cells in a dose‐dependent manner. Further research found that the effects of NE on glioma cells could lead to the activation of the mitogen‐activated protein kinase (MAPK) signalling pathway through β‐adrenergic receptor, while curcumin suppressed the level of extracellular signal–regulated kinase (ERK)1/2 phosphorylation. In addition, blocking ERK1/2 expression with U0126 resulted in the down‐regulated expression of CD147, which further led to the decreased expression of MMP‐2 and MMP‐9. Curcumin could also inhibit the expression of cyclin D1/CDK4/6 and anti‐apoptotic protein Bcl‐2/Bcl‐XL induced by NE, and induced cell cycle changes and increased apoptosis. Therefore, curcumin may be a potential candidate drug for preventing and treating the progression of glioma induced by adverse psychological stress.

stress could promote the proliferation and invasion of glioma. 7 Glioblastoma multiforme (GBM) is the most common malignant brain tumour in adults, with a survival rate of <5% in 5 years. 8 At present, the standard treatments for glioma include surgical resection and radiotherapy. However, the invasive growth of GBM often leads to the inability to completely eradicate GBM, and adverse psychological stress promotes further deterioration. 9 Therefore, further comprehensive and effective interventions are urgently needed to improve the treatment of patients with glioma.
Curcumin is a polyphenolic compound found in the rhizome of turmeric plants, which has various biological effects, including antiinflammatory, anti-oxidation and anti-infection effects. 10,11 In recent years, curcumin has been found to play a key role in inhibiting the initiation, progression and metastasis of several tumours, 12-14 but its effect on stress-induced tumour proliferation and invasion has not been reported to date. More importantly, curcumin is also the main ingredient of the Traditional Chinese Medicine formula Jieyu Pill, which is typically used for its mood stabilizing/enhancing properties, and therefore is mainly employed for the treatment of depression. 15,16 A recent study found that curcumin could enhance resistance to adverse stress and relieve stress-induced anxiety-like behaviour in mice. 17 Thus, it was speculated that curcumin may play an important role in alleviating glioma progression, which is exacerbated by adverse psychological stress.
Several studies have shown that NE could induce the production of matrix metalloproteinases (MMPs) in tumour cells by acting on the adrenergic receptors on the surface of target cells. The gelatindegraded MMPs, including MMP-2 and MMP-9, are known to play an important role in the invasion and metastasis of cancer cells by degrading type IV collagen, which is the main component of the basement membrane. 18 The activity of MMPs is regulated by a variety of mechanisms, including growth factors, cytokines and CD147.
Accumulating evidence has revealed the role of CD147 in the development and progression of various cancer types, including glioma, ovarian cancer, renal cell carcinoma, laryngeal squamous cell carcinoma and skin cancer. [19][20][21] As an MMP inducer, CD147 can also stimulate surrounding fibroblasts to secrete abundant MMPs to further enhance tumour cell invasion. Therefore, inhibiting the expression of MMPs and/or their upstream regulatory pathways may be critical in the treatment of malignant tumours such as glioma. Curcumin inhibits the expression of MMPs by targeting a variety of intracellular signalling pathways, including MAPK, nuclear factor-κB (NF-κB) and PI3K/Akt. [12][13][14] MAPK is a crucial kinase that promotes proliferation and transmits stress signals. Its family members include ERK1/2, c-Jun NH-2 terminal kinase (JNK) and p38 MAPK. The MAPK signalling pathway is activated by the stress hormone NE in various tumours. 22,23 Our previous study found that the stress hormone NE could stimulate the progression of glioma cells by enhancing the activity of ERK and the expression of phosphorylated (p)-ERK1/2, 7 but it is unclear whether curcumin has an inhibitory effect on this process.
The present study established an adverse stress model in mice and cells to evaluate whether curcumin had an inhibitory effect on the proliferation and invasion of glioma induced by adverse psychological stress. In addition, the effect of curcumin on the NE-induced MAPK signalling pathway and MMP-2/9 activation was investigated to reveal the antitumour mechanism of curcumin, with the aim of relieving stress.

| Cell culture and reagents
The human glioma LN229 cell line was obtained from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH. The human glioma U87 MG cell line was obtained from American Type Culture Collection. The cells were cultured in DMEM containing 10% foetal bovine serum (FBS, Gibco BRL), 100 U/ml penicillin and 100 mg/ml streptomycin in a humidified atmosphere of 5% CO 2 at 37°C. Curcumin, propranolol, NE and U0126 were purchased from Sigma-Aldrich (Merck KGaA).

| Nude mouse model of adverse stress restraint
In total, 20 female 4-6 weeks-old BALB/c-nu/nu nude mice were free DMEM and injected subcutaneously into the right axilla of nude mice. One week later, tumour-bearing mice were randomly divided into four groups: control (DMEM injected), stress (DMEM injected and stressed), curcumin (curcumin injected) and curcumin and stress (curcumin injected and stressed). Five mice were assigned to each group. The nude mice were placed in a 50 ml multi-well centrifuge tube without squeezing the animal's body during the process in order to establish an adverse stress model through restraint. 4,24 The restraint stress lasted for 8 hours per day. During rest, the mice were provided free access to food and water. Consistent with previously reported literature and our preliminary results, a single dose of curcumin (60 mg/kg) was administered via an intraperitoneal injection once every other day. 25 In the control group, curcumin was replaced

| Analysis of serum NE and EPI levels
Whole blood was extracted from the posterior venous plexus of the eye socket of the mice and was placed at 4℃ for 12 hours and then centrifuged at 356 ×g for 20 minutes to obtain serum. EPI and NE concentrations in the serum were measured with an ELISA kit (Rapid Bio Lab, DRE-H80507C) according to the manufacturer's instructions. Each sample was analysed in triplicate.

| Cell proliferation assay
Cell growth was detected using Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Inc) according to the manufacturer's instructions. A total of 5 × 10 3 cells per well were inoculated into 96-well plates and cultured for 24 hours and then cultured for 24-72 hours in the presence or absence of curcumin (8, 16, 24 and 32 μmol/L). Next, 10 μl CCK-8 reagent per well was added and incubated for 2 hours, and then, the absorbance value was measured at 450 nm. The control group was exposed only to DMEM and CCK-8 reagent. All experiments were repeated three times. The proliferation inhibition rate was calculated as 1-(OD sample /OD control ) × 100%.

| Colony formation assay
A total of 100 LN229 and U87 MG cells per well were inoculated into a 6-well plate and cultured for 24 hours. Next, the cells were treated with or without curcumin (8, 16 and 24 μmol/L) or 10 μmol/L NE. After continuous culture for 12 days, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 20 minutes and stained with 0.1% crystal violet for 10 minutes. Colonies that contained ≥50 cells were counted under the microscope (magnification, × 100).

| Wound healing assay
LN229 and U87 MG cells were seeded in 6-well plates at 5 × 10 5 cells per well, and cells were incubated for 24 hours until reaching ~90% confluence. A linear wound was created by scraping with a 10 µl pipette tip on a monolayer of cells. Cells were pre-treated with or without curcumin (8, 16 and 24 μmol/L) for 4 hours and then treated with or without NE (10 μmol/L) for 24 hours. Wound healing was observed using a microscope (magnification, × 100) and photographed.

| Transwell Matrigel invasion assay
After pre-treatment of LN229 and U87 MG cells without or with

| Gelatin zymography analysis
The activity of MMP-2 and MMP-9 secreted by LN229 cells was determined by gelatin zymography. LN229 cells were seeded in 6well plates, allowed to grow to ~90% confluence for 24 hours and then maintained in serum-free medium. The cells were pre-treated with or without curcumin (24 μmol/L) for 4 hours and then treated with or without NE (10 μmol/L) for 24 hours. Subsequently, the conditioned medium was collected and subjected to 10% SDS-PAGE in the presence of 0.1% gelatin. Samples were not boiled prior to electrophoresis. After electrophoresis, the gels were washed three times for 15 minutes each at room temperature in an eluate with or without 2.5% Triton X-100 to remove SDS. Next, the gels were incubated in a solution containing 50 mmol/l Tris-HCL, 5 mmol/l CaCl 2 , 1 μmol/l ZnCl 2 and 0.02% Brij-35 for 42 hours at 37°C. The gels were subsequently stained with 0.25% Coomassie Brilliant Blue (G250) in 10% acetic acid (v/v) and 30% methanol (v/v) and then de-stained with the same solution but without Coomassie Brilliant Blue. Gelatinolytic activity was detected as unstained bands on a blue background.

| Reverse transcription-quantitative PCR (RT-qPCR) analysis
Total RNA was extracted using TRIzol ® (Invitrogen; Thermo Fisher Scientific, Inc) according to the manufacturer's instructions, and then, 1 μg total RNA was used to synthesize cDNA using PrimeScript™ RT Master Mix (Takara Biotechnology Co., Ltd.). Using cDNA as a template, amplification was performed for 35 cycles using the SYBR ® Premix Ex Taq TM II kit (Takara Biotechnology Co., Ltd.), and gene-specific primer sequences were designed based on the literature. 26 The primer se-

| Statistical analysis
Statistical analysis was performed using GraphPad Prism 5 software (GraphPad Software, Inc), and all data are expressed as the mean ± SD. Differences between two groups were evaluated by Student's t test, and differences among groups were assessed by one-way analysis of variance (ANOVA). P < .05 was considered to indicate a statistically significant difference.

| Curcumin inhibits the psychological stressinduced growth of transplanted tumours in nude mice
Previous reports have demonstrated that curcumin ( Figure 1A) inhibited the growth of xenografted tumours in nude mice. 25 To evaluate the effect of curcumin on tumour proliferation induced by adverse psychological stress, the progression of postimplantation tumour growth was examined. The results showed that the tumour volume of the control group was smaller than that of the stress group from the third week to the end of the experiment.
Moreover, curcumin was used to treat stressed nude mice. It was found that the tumour volume of the stress and curcumin group was significantly smaller than that of the stress only group. These results suggested that curcumin was able to inhibit tumour growth induced by psychological stress (Figure 1B-C). To evaluate whether each group of nude mice was in a state of stress, blood samples were collected to determine the levels of serum stress hormones in each group. The results showed that the levels of EPI and NE in the stress group were significantly higher than those in the non-stress group. Remarkably, serum NE was elevated by 2.5-fold in nude mice with psychological stress ( Figure 1D). However, there was no impact on NE or EPI levels with curcumin treatment. In addition, Western blot analysis showed that psychological stress could induce the expression of MMP-2, MMP-9 and CD147, which was inhibited by curcumin ( Figure 1E).

| Curcumin inhibited NE-induced proliferation of glioma cells
Previous studies have shown that curcumin elicits an antiproliferative activity against numerous tumour types. 19,20 To investigate the killing effect and cytotoxicity of curcumin on glioma cells, Exogenous NE was used to stimulate glioma LN229 and U87 MG cells to simulate the stress environment in vitro, 29 and it was found that NE enhanced the proliferation of LN229 and U87 MG cells in a dose-dependent manner, and the maximal stimulatory effect was observed at a dose of 10 μmol/L ( Figure 2B). In order to further study the effect of curcumin on NE-induced proliferation of LN229 and U87 MG cells, the cells were exposed to 10 μmol/L NE without or with curcumin (8, 16 or 24 μmol/L). These results showed that the colony formation ability of LN229 and U87 MG cells was increased after NE treatment, while curcumin could inhibit the NE-induced colony formation of LN229 and U87 MG cells in a dose-dependent manner ( Figure 2C).

| Curcumin inhibited NE-triggered G1 to S phase transition and apoptosis in glioma cells
To investigate the mechanism of curcumin inhibiting the NE-

| Curcumin inhibits the NE-induced migration and invasion of glioma cells
Glioma LN229 and U87 MG cells were exposed to NE without or

| Curcumin down-regulated the NE-induced secretion and expression of MMPs in glioma cells
To

| Curcumin down-regulated NE-induced expression of MMP-2 and MMP-9 by blocking the ERK/MAPK signalling pathway
To investigate the mechanism of curcumin inhibiting the expression of MMP-2 and MMP-9 induced by NE, LN229 and U87 MG cells were pre-treated with the β-blocker propranolol, and it was found that propranolol could inhibit NE-induced MMP-2 and MMP-9 expression ( Figure 6A

| D ISCUSS I ON
As a natural pharmaceutical compound, curcumin can penetrate the blood-brain barrier, so it has a good therapeutic effect on primary  Abnormal activation of the ERK signalling pathway may trigger the CD147 signalling pathway in tumour cells. 43,44 CD147 is highly expressed in a variety of tumours and, when activated by certain molecules, induces the production of downstream MMP-2 and MMP-9, and is involved in cell migration and invasion. 45 The findings of the present study also confirmed that NE induced CD147 expression by activating ERK1/2 in LN229 and U87 MG cells. In addition, NE-induced MMP-2 and MMP-9 up-regulation can be restored by siRNA-CD147. These results suggest that CD147 is involved in the regulation of the ERK1/2/ MMP-2 / MMP-9 signalling pathway by NE. However, the mechanism by which NE activates ERK1/2 and CD147/MMP-2/MMP-9 needs further study.
In conclusion, these data suggest that curcumin effectively alleviated the invasion of glioma promoted by adverse psychological stress through inhibiting the MAPK/ERK signalling pathway which reduced the expression of CD147 and MMP-2/9. Furthermore, curcumin induced cell cycle changes and increased apoptosis and then inhibited cell proliferation. Therefore, curcumin may be a promising drug for preventing and treating the progression of glioma due to adverse psychological stress.

ACK N OWLED G EM ENTS
The present study was supported by the National Nature Science

CO N FLI C T O F I NTE R E S T
All authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.