Curcumin potentiates the potent antitumor activity of ACNU against glioblastoma by suppressing the PI3K/AKT and NF-κB/COX-2 signaling pathways.

Glioblastoma (GBM) is a highly invasive and challenging primary tumor of the central nervous system (CNS), and currently available treatments provide limited benefits to patients with this disease. Therefore, the development of novel therapeutic targets and effective treatment strategies is essential. Nimustine hydrochloride (ACNU) is widely used as the standard chemotherapeutic agent and is frequently administered together with other chemotherapeutic agents in clinical studies. Curcumin, a natural polyphenolic compound, could potentially be combined with chemotherapeutics for cancer treatment; however, there are no reports of studies where ACNU and curcumin were combined for GBM treatment, and the mechanisms underlying their activity remain poorly understood. In the present study, we investigated the effects of combined treatment with curcumin and ACNU on GBM cells and found that it significantly enhanced the inhibition of cell proliferation, colony formation, migration, and invasion. In addition, co-treatment with curcumin increased ACNU-induced apoptosis through enhancing the release of cytochrome c from the mitochondrial intermembrane space into the cytosol. Further, curcumin and ACNU acted synergistically in their antitumor effects by targeting N-cadherin/MMP2/9, PI3K/AKT, and NF-κB/COX-2 signaling. These results indicate that curcumin can enhance the anti-proliferation, anti-migration, and proapoptotic activities of ACNU against GBM, and provide strong evidence that combined treatment with curcumin and ACNU has the potential to be an effective therapeutic option for GBM.

Introduction

Glioblastoma (GBM) is the most common primary tumor of the central nervous system (CNS), accounting for 60%–70% of primary brain tumors.1 The majority of GBM tumors become invasive growths without clear boundaries within the surrounding normal brain tissue.2 Consequently, it is hard to achieve full resection, and GBMs are relatively insensitive to radiotherapy and chemotherapy, resulting in high recurrence rates after surgery. For these reasons, the median survival time of patients with GBM is only 12–15 months.3,4 Therefore, the development of innovative therapeutic strategies and more effective agents by researchers and clinicians is urgently required to prolong patient survival and improve quality of life.

Alkylator-based chemotherapy is an important part of standard therapeutic strategies for the treatment of GBM. Nimustine hydrochloride (ACNU) is a nitrosourea with a key role in the treatment of high-grade gliomas.5 In Central Europe and most Asian regions, ACNU is widely used as the standard chemotherapeutic agent6 because of its efficacy and availability. In clinical studies, ACNU is usually administered with other chemotherapeutic agents, such as cisplatin or teniposide, or with radiotherapy, to increase its anticancer effects;7–9 however, it is not sufficiently powerful, and high doses induce side effects, such as myelosuppression, interstitial pneumonia, pulmonary fibrosis, and digestive tract reactions. Therefore, additional novel forms of combination therapy including ACNU are required that use natural antitumor compounds, rather than chemotherapeutic agents, and the detailed molecular mechanisms underlying such combination therapies deserve deeper investigation.

Curcumin, the major constituent of turmeric (Curcuma longa) powder,10 has been a frequent component of traditional medicine remedies for centuries in many Asian countries, owing to its anti-inflammatory and chemotherapeutic properties.11 Previous studies have demonstrated that cur-cumin possesses various physiological and pharmacological characteristics, including antioxidant, anti-inflammatory, immunomodulatory, and antidiabetic activities.12–14 In particular, curcumin can potentially modulate the growth of tumor cells through regulation of various molecular targets and signaling pathways.15–17 Furthermore, curcumin has the potential to be combined with chemotherapeutics for GBM.18,19 Curcumin is a pharmacologically safe and effective candidate for anticancer therapy; however, its utility is greatly hindered because of its poor bioavailability, including poor absorption, rapid metabolism, and rapid systemic elimination;20 therefore, increasing attention should be paid to combination treatment regimens using curcumin with other antitumor agents, particularly natural antitumor compounds, and the detailed molecular mechanisms that mediate the effects of such combinations warrant further investigation.

We hypothesized that curcumin could increase the efficacy of ACNU against GBM. To test this hypothesis and establish an effective alternative approach for GBM treatment, we determined the effects of combined treatment with ACNU and curcumin on GBM cancer cell proliferation, migration, and apoptosis. Moreover, we measured changes in key proteins to determine the molecular mechanisms underlying the effects of such combination treatment. The findings of this study reveal that curcumin has potential for use as an agent in combination therapy to sensitize GBM to the chemotherapeutic effects of ACNU through simultaneous modulation of multiple signaling pathways, indicating that combination treatment with ACNU and curcumin may be an effective alternative approach in GBM therapy.

Materials and methods

Chemicals and reagents

ACNU and curcumin were purchased from Sigma-Aldrich (St Louis, MO, USA). All reagents were dissolved in dimethyl sulfoxide (DMSO) as initial concentrates, and diluted with cell culture medium before use; the final concentration of DMSO was <0.1%. Control cultures received the carrier solvent (0.1% DMSO).

Antibodies and other materials

Antibodies specific for cleaved caspase-3, COX-2, p-IκB-α, IκB-α, p-PI3K, p-AKT, p65, and β-actin as well as all secondary antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Antibodies specific for cytochrome c (cyt c) and p50 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies specific for CDK1, cyclinB1, CDK2, PI3K, AKT, BCL-2, BAX, N-cadherin, vimentin, and lamin B1 were purchased from Proteintech Group Inc. (Rosemont, IL, USA). Antibodies specific for MMP2/9 were purchased from Abcam (Cambridge, UK). RPMI 1640 media, fetal bovine serum (FBS), and trypsin were purchased from Thermo Fisher Scientific (Waltham, MA, USA). All other chemicals were purchased from Sigma Chemical Co. (St Louis, MO, USA), unless otherwise specified.

Cell culture

Human GBM cell lines, U118MG, U87MG, and U251MG, were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI 1640 media, supplemented with 10% FBS, and grown at 37°C in a humidified atmosphere with 5% CO2. The authenticity of all cell lines was verified by genomic short tandem repeat profiling, carried out by Shanghai ZhongQiao Xin Zhou Biotechnology Co., Ltd. (Shanghai, China), and cells were confirmed as free of mycoplasma using a Mycoplasma Detection Kit-Quick Test (Biotools, Jupiter, FL, USA).

CCK-8 assay

Cell viability was measured using CCK-8 assay. Briefly, 6×103 cells were counted and seeded into 96-well culture plates, allowed to adhere overnight, and then treated with appropriate concentrations of ACNU, with or without curcumin. Each concentration was tested five times. After incubation for 48 h, 10 μL CCK-8 was added to each well and, after incubation for 2–4 h at 37°C, absorbance was measured at 450 nm using an EnSpire® Multimode Plate Reader (PerkinElmer, Waltham, MA, USA). Each experiment was repeated at least three times.

Colony formation assay

GBM cells were treated with ACNU or curcumin for 48 h, trypsinized to form single-cell suspensions, and seeded into six-well plates (1,500 cells/well). After incubation at 37°C in 5% CO2 for 10 days until colonies were large enough to be visualized, cells were washed with PBS, fixed in methanol:glacial acetic:ddH2O (1:1:8) for 10 min, and stained with 0.1% crystal violet for 30 min.

Wound healing assay

Wound healing (scratch) assays were conducted to detect cell migration. Briefly, U118MG and U87MG cells were grown to full confluence in six-well culture plates. After 6 h of serum starvation, the confluent cell monolayer was scraped with a sterile 100 μL pipette tip and treated with appropriate doses of ACNU or curcumin.

Confocal immunofluorescence analysis

For immunofluorescence analysis, U87MG cells were seeded on coverslips and treated with different concentrations of ACNU or curcumin for 48 h. Subsequently, cells were fixed with 4% paraformaldehyde at room temperature, permeabilized with 0.2% TritonX-100, and then blocked in PBS containing 5% BSA. Subsequently, the cells were incubated with diluted primary antibodies against cyt c, p50, and p65 overnight at 4°C. Next, cells were incubated with fluorescein isothiocyanate or rhodamine isothiocyanate-conjugated secondary antibodies for 60 min at room temperature in a darkroom. Finally, DAPI was added to each sample for nuclear counterstaining, and fluorescent images were examined using a confocal microscope (Leica SP8, Germany).

Western blot analysis

The concentration of proteins in cell lysates was determined using the BCA method; aliquots were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes. Protein bands were visualized by enhanced chemiluminescence, and the integrated optical densities of bands were quantitated using Image Quant (GE Healthcare). Similar experiments were conducted at least three times.

Real-time quantitative PCR

Total RNA was extracted using TRIzol reagent according to the kit protocol (TaKaRa Bio, Dalian, People’s Republic of China). cDNA was reverse-transcribed using the Prime Script RT Reagent Kit (TaKaRa Bio), according to the manufacturer’s instructions. Real-time quantitative PCR (qPCR) was conducted in a Rotor-Gene Q (Qiagen, Germany) machine using SYBR Premix Ex Taq II (Takara, Japan). Primer pairs were as follows: COX-2, 5′-TCACAGGCTTCCATTGACCAG-3′ and 5′-CCGAGGCTTTTCTACCAGA-3′; β-actin, 5′-GGCACCCAGCACAATGAA-3′ and 5′-TAGAAGCATTTGCGGTGG-3′. After normalization to levels of β-actin, the relative amount of COX2 transcripts in treated cells compared with controls were calculated as means ± standard error (SE).

Flow cytometric analysis

To determine the distribution of cells in the cell cycle and the proportion of apoptotic cells, flow cytometric analysis was conducted using a BD FACS Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA). Briefly, treated cells were collected and fixed with ice-cold 70% ethanol at 4°C for 4 h, and then stained according to the protocol specified for the Cell Cycle Kit (Beyotime). For examination of apoptosis, treated cells were stained with an Annexin V-FITC Apoptosis Detection Kit, according to the manufacturer’s protocol. The cell-cycle distribution and fraction of apoptotic cells were determined using a FACS analysis system.

Statistical analyses

All data are presented as means ± SD of at least three independent experiments. Statistical analyses were conducted using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). One-way analysis of variance or Student’s t-tests were used to evaluate the statistical significance of differences between control and treated groups. Results were considered statistically significant at p<0.05.

The concentrations that induced 50% cell growth inhibition (IC50) values were determined by applying nonlinear regression curve fit analysis, using CVXPT32 software. Effects of combination treatment with curcumin and ACNU were assessed using a combination index (CI) – a quantitative representation of the pharmacological interaction between two compounds.21 The CI theorem of Chou–Talalay allows quantitative definition of the additive effects (CI =1), synergism (CI <1), and antagonism (CI >1) of drug combinations.

Results

Treatment with curcumin in combination with ACNU enhances inhibition of cell proliferation

To determine whether curcumin could potentiate the inhibitory effects of ACNU on GBM cell proliferation, we, first, quantitatively analyzed the effects of curcumin and ACNU alone, or in combination, on the proliferation and morphology of human GBMU118MG, U87MG, and U251MG cells. As shown in Figure 1A, treatment with curcumin or ACNU alone dose-dependently suppressed GBM cell viability from 5 to 50 μM and 10 to 250 μM, respectively; however, combined treatment with ACNU and curcumin (20 μM) significantly enhanced ACNU-mediated inhibition of U118MG, U87MG, and U251MG cell viability compared with ACNU alone. Interestingly, this treatment model showed no obvious cytotoxicity at the same concentrations in a normal human glial cell line (SVG p12; Figure 1A). Next, the IC50 values of ACNU – alone or in combination with curcumin – for inhibition of proliferation of the three cell lines were calculated. As shown in Figure 1B, combined treatment with ACNU and curcumin (20 μM) resulted in a marked reduction of IC50 values, compared with cells treated with ACNU alone; U87 MG cells were more sensitive to the combined treatment than the other two cell lines (Figure 1B).

Figure 1

Effects of combination treatment with curcumin and ACNU on cell proliferation, colony formation, and morphology.

Notes: (A) U118MG, U87MG, U251MG, and SVG p12 cells were treated with curcumin or ACNU alone or in combination at the indicated doses. After treatment for 48 h, cell viability was determined by CCK-8 assay. Cells treated with DMSO were used as the reference group (viability set at 100%). (B) IC50 values of ACNU for inhibition of the viability of cells treated with or without curcumin and CI of these drugs. (C) Changes in cell morphology and spreading of U118MG and U87MG cells treated with ACNU (100 μM), curcumin (20 μM), or ACNU and curcumin combined for 48 h were observed. Cells were photographed using a microscope fitted with a digital camera. (D) U118MG and U87MG cells were treated with ACNU (100 μM) or curcumin (20 μM) alone or in combination. The colony formation of cells was photographed. Data are presented as means ± SD of three independent experiments. The level of significance versus the DMSO-treated group is indicated by *p<0.05, **p<0.01, or ***p<0.001.

Abbreviations: ACNU, nimustine hydrochloride; Curc, curcumin; CI, combination indices; DMSO, dimethyl sulfoxide; IC50, concentrations that induced 50% cell growth inhibition.

In addition, assessment of the effects of combination treatment with curcumin and ACNU was determined by calculation of CI values, which provide a quantitative representation of pharmacological interactions between two compounds.21 CI values calculated for the growth-suppressive effects of combined curcumin and ACNU treatment indicated a high level of synergism, with values <1 for both U118MG (CI =0.76) and U87MG (CI =0.81) cells; however, this treatment model exhibited an additive effect in U251MG cells (CI =1.08; Figure 1B).

Considering these results, we next conducted experiments using U118MG and U87MG cells to investigate the molecular mechanism underlying the synergistic effects of combined treatment of GBM cells with curcumin and ACNU. Changes in the morphology and spreading of U118MG and U87MG cells after combined treatment with curcumin and ACNU were determined. As shown in Figure 1C, shrunken cells and plasma membrane blebs were present after combination treatment with curcumin and ACNU or ACNU alone. Moreover, combined treatment with curcumin and ACNU significantly enhanced the inhibition of colony formation in both U118MG and U87MG cell lines, as compared to treatment with the single agents (Figure 1D). These results demonstrate that the addition of curcumin can enhance the antitumor activity of ACNU in GBM.

Combination treatment with curcumin and ACNU promotes apoptosis and cell-cycle arrest

We next determined whether the synergistic inhibition of cell proliferation induced by combined treatment with ACNU and curcumin is associated with enhanced activation of apoptotic pathways in GBM cells. As shown in Figure 2A, treatment with ACNU (100 μM) or curcumin (20 μM) alone for 48 h induced apoptosis of 10.3% and 12.6% of U87 cells, respectively; however, combination treatment with both agents markedly enhanced apoptosis, resulting in 22.7% apoptotic cells. Furthermore, we determined the levels of the apoptosis-related proteins – cleaved caspase-3, BAX, and BCL-2 – by Western blot analysis after treatment of cells for 48 h. As shown in Figure 2B, co-treatment with curcumin and ACNU resulted in increased levels of cleaved caspase-3 and elevated the BAX/BCL-2 ratio.

Figure 2

Effects of combination treatment with curcumin (Curc) and ACNU on caspase-dependent apoptosis and cell-cycle progression.

Notes: Human U118MG and U87MG cells were treated with ACNU (100 μM) or curcumin (20 μM) alone or in combination. (A) After treatment for 48 h, levels of apoptosis were determined by FACS analysis, and the percentage of apoptotic cells was calculated. (B) The expression of C-casp3 and BCL-2/BAX proteins in U118MG and U87MG cells was analyzed by Western blotting. (C) The process of release of cyt c from the mitochondria into the cytoplasm was observed by immunofluorescence imaging analysis in U87MG cells. (D) U87MG cell-cycle analysis was carried out after 48 h of treatment with ACNU (100 μM) or curcumin (20 μM), alone or in combination, using a BD Accuri C6 Flow Cytometer (BD Biosciences, San Jose, CA, USA). (E) Expression levels of CDK1, cyclin B1, and CDK2 proteins were analyzed by Western blot. Densitometric measurements were normalized to β-actin, expressed relative to the control group (set as 1), and are reported beneath the Western blot images. Data are presented as means ± SD of three independent experiments. The level of significance is indicated by *p<0.05 or **p<0.01.

Abbreviations: ACNU, nimustine hydrochloride; Curc, curcumin; C-casp3, cleaved-caspase 3; cyt c, cytochrome c.

The release of cyt c from mitochondria into the cytosol can induce apoptosis; therefore, we next conducted immunofluorescence imaging analysis to determine the subcellular localization of cyt c to investigate whether curcumin can enhance ACNU-mediated cyt c release. The results demonstrate that treatment with ACNU alone can effectively induce the release of cyt c from the inter-mitochondrial space into the cytosol in U87MG cells, whereas co-treatment with curcumin greatly enhanced this cyt c release (Figure 2C). These results indicate that the combination of curcumin and ACNU promotes apoptosis induction by triggering cyt c release and facilitating caspase activation in the cytosol.

Further, we investigated synergistic effects of ACNU and curcumin on cell proliferation and cell-cycle arrest. As shown in Figure 2D, treatment with ACNU alone induced cell-cycle arrest at G2/M phase, whereas combined treatment with ACNU and curcumin led to a higher percentage of cells in G2/M phase. To ascertain the detailed mechanisms underlying these events, the expression of key regulators of the G2 checkpoint (CDK1, CDK2, and cyclin B1) were evaluated by Western blotting in U118MG and U87MG cells 48 h after treatment. Our results indicate that co-treatment with ACNU and curcumin resulted in a dramatic reduction in levels of CDK1, cyclin A, and cyclin B proteins (Figure 2E). These data provide evidence that curcumin can enhance ACNU-mediated inhibition of cell proliferation, at least in part, by inducing cell-cycle arrest at the G2/M phase.

Combination treatment with curcumin and ACNU enhances inhibition of cell migration and invasion

Wound healing and transwell assays were employed to determine the effects of combination treatment with ACNU and curcumin on human GBM cell migration and invasion. Treatment with ACNU or curcumin alone suppressed cell migration and invasion; however, combined treatment significantly enhanced this inhibition (Figure 3A and C). Quantitative analysis of cell migration and invasion was conducted (Figure 3B and D). Further, to ascertain the detailed mechanisms underlying the combination effects on cell migration and invasion, levels of key protein markers, including matrix metalloproteinases (MMP2/9), N-cadherin, and vimentin, were evaluated (Figure 3E). These results confirm that curcumin enhances the ACNU-mediated inhibition of GBM cell migration and invasion.

Figure 3

Effects of combination treatment with curcumin and ACNU on cell migration and invasion.

Notes: (A) Migration of U118MG and U87MG cells was analyzed by scratch assay. After 48 h treatment with ACNU (100 μM) or curcumin (20 μM), alone or in combination, the wound gap was observed and photographed. (B) Percentages of migration cells were calculated relative to the original gap. (C) Cell invasion was analyzed in U87MG cells treated with ACNU (100 μM) or curcumin (20 μM), alone or in combination, for 24 h. Cell invasion was observed and photographed, and the percentage of invasion cells (D) was calculated. (E) Levels of expression of N-cadherin, vimentin, and MMP-2/9 proteins were analyzed by Western blot in the different treatment groups. Densitometric measurements were normalized to β-actin, expressed relative to the control group (set as 1), and are reported beneath Western blot images. Data are presented as means ± SD of three independent experiments. The level of significance versus the DMSO-treated control group is indicated by *p<0.05 or **p<0.01.

Abbreviations: ACNU, nimustine hydrochloride; Curc, curcumin; C-casp3, cleaved-caspase 3; cyt c, cytochrome c; DMSO, dimethyl sulfoxide.

Combination treatment with curcumin and ACNU enhances PI3K/AKT signaling inhibition

The PI3K/AKT signaling pathway regulates cancer cell proliferation and death,22,23 and curcumin can inhibit cancer cell proliferation by targeting PI3K/AKT signaling.24 We used Western blotting to analyze the effect of curcumin and ACNU on the expression of pro-survival proteins associated with the PI3K/AKT pathway in GBM cells. As shown in Figure 4A, combined treatment with curcumin and ACNU markedly suppressed the levels of phosho-PI3K (p-PI3K) and p-AKT proteins, whereas it had almost no effect on total PI3K and AKT protein expression. In addition, quantitative analysis of protein levels was undertaken (Figure 4B).

Figure 4

Effect of combined treatment with curcumin (Curc) and ACNU on the PI3K/AKT signaling pathway.

Notes: (A) Cells were treated with ACNU (100 μM) or curcumin (20 μM), alone or in combination. After 48 h, the levels of p-PI3K, p-AKT, PI3K, and AKT proteins were analyzed by Western blotting. β-actin served as the loading control. (B) Quantitative analysis of the proteins in (A) was undertaken. (C, D) Cells were treated with the PI3K inhibitor, LY294002, or ACNU and curcumin, or their combination (LY294002 + Curc + ACNU). After 48 h, cell viability was determined by CCK-8 analysis. Data are presented as means ± SD of three independent experiments. The level of significance versus the DMSO-treated group is indicated by *p<0.05, **p<0.01, or ***p<0.001.

Abbreviations: ACNU, nimustine hydrochloride; Curc, curcumin; DMSO, dimethyl sulfoxide.

To confirm the involvement of the PI3K/AKT pathway in the synergistic inhibition of tumor cell growth by ACNU and curcumin, U118MG and U87MG cells were pretreated with the PI3K-specific inhibitor, LY294002 (75 μM), for 8 h, followed by co-treatment with curcumin and ACNU. After 48 h, cell viability was analyzed using a CCK-8 assay. As shown in Figure 4C and D, treatment with either LY294002 or curcumin and ACNU inhibited cell viability, whereas combined treatment with LY294002 followed by curcumin and ACNU had no significant effect on cell viability. These results indicate that combined treatment with curcumin and ACNU may partially inactivate PI3K/AKT signaling, thereby affecting cell proliferation.

Combined treatment with curcumin and ACNU enhances inhibition of COX-2 expression and NF-κB translocation

High levels of COX-2 expression are associated with cancer cell proliferation, migration, and invasion.25,26 We next evaluated the activities of curcumin and ACNU treatment on COX-2 expression in GBM cells at the protein and mRNA levels by Western blotting and qPCR. As shown in Figure 5A and B, combined treatment with curcumin and ACNU significantly decreased COX-2 expression in U118MG and U87MG cells. Next, U118MG and U87MG cells were pretreated with the COX-2-selective inhibitor, celecoxib (CB; 75 μM) for 8 h, followed by curcumin and ACNU co-treatment. After incubation for 48 h, cell viability was analyzed by CCK-8 assay. As shown in Figure 5C, treatment with either CB or ACNU and curcumin significantly inhibited cell proliferation compared with controls, whereas CB pretreatment followed by ACNU and curcumin co-treatment did not significantly alter cell viability inhibition as compared with monotherapy. These results imply that the enhanced inhibition of proliferation by combined treatment of GBM cells is partially mediated by inactivation of COX-2 signaling.

Figure 5

Treatment with curcumin and ACNU enhances the inhibition of COX-2 expression and NF-κB translocation.

Notes: Expression levels of COX-2 protein were analyzed by Western blot (A) in human GBM U118MG and U87MG cells treated with ACNU (100 μM) or curcumin (20 μM) for 48 h, and its mRNA levels were also analyzed in U118MG cells (B). (C) Cells were treated with ACNU (10 μM) in combination with curcumin (20 μM) for 48 h after pretreatment with the COX-2 selective inhibitor, CB (75 μM) for 8 h, and cell viability was determined by CCK-8 analysis. (D) After treatment for 48 h, levels of p65 and p50 proteins, as well as those of IκBα and p-IκBα, were detected in the cytoplasm and nucleus by Western blot analysis. (E) After treatment for 48 h, subcellular localizations of p50 and p65 in U87 MG cells were examined by confocal microscopic analysis. Densitometric measurements were normalized to β-actin, expressed relative to the control group (set as 1), and are reported beneath Western blot images. Data are presented as means ± SD of three independent experiments. The level of significance is indicated by *p<0.05, **p<0.01, or ***p<0.001.

Abbreviations: ACNU, nimustine hydrochloride; CB, celecoxib; Curc, curcumin; GBM, glioblastoma.

As the NF-κB signaling pathway participates in the regulation of COX-2 expression in cancers,25 we hypothesized that the combination of curcumin and ACNU may regulate COX-2 expression in GBM through its effects on the NF-κB signaling pathway. To test this hypothesis, U118MG and U87MG cells were treated with curcumin and ACNU for 48 h, cytoplasmic and nuclear proteins were extracted, and the expression of key NF-κB signaling proteins was examined. The results showed that the combination treatment notably decreased p-IκBα and p-p65 in the cytoplasm compared with treatment with curcumin or ACNU treatment alone, whereas these treatments had no obvious effects on levels of total IκBα or p65 (Figure 5D). Furthermore, NF-κB, p50, and p65 protein levels in the nucleus decreased. Based on these results, we hypothesized that combination treatment markedly enhanced inhibition of NF-κB p65/p50 dimer translocation from the cytoplasm to the nucleus. To test this hypothesis, immunofluorescence assays were carried out. As expected, treatment with ACNU alone suppressed NF-κB p65/p50 translocation from the cytoplasm to the nucleus, whereas combination treatment enhanced the suppressive effect (Figure 5E). Together, these results support the conclusion that combined treatment with curcumin and ACNU potentiates the suppression of tumor cell growth through modulation of NF-κB/COX-2 signaling.

Discussion

In this study, we demonstrated that curcumin enhances ACNU-mediated antitumor activity, including its suppression of human GBM cell viability, clonogenicity, and migration, mainly through induction of apoptosis and cell-cycle arrest. The IC50 value of ACNU was significantly decreased when it was used in combination with curcumin in comparison with that of ACNU monotherapy. Moreover, we determined that curcumin potentiates the effects of ACNU through simultaneous modulation of the cyt c/caspase-dependent apoptotic pathway, inactivation of PI3K/AKT signaling, and inhibition of the NF-κB/COX-2 pathway. To our best knowledge, this is the first report of the synergistic effects of curcumin and ACNU on GBM cells, and includes the demonstration of the mechanisms underlying these observations. Our results may serve as guidance for the use of combinations that include natural antitumor compounds to enhance the efficacy of GBM treatment.

Recently, increasing attention has been paid to chronic inflammation, which can drive increased cancer risk, including that of meningitis-associated malignant brain tumors.27–30 A pivotal factor in the inflammatory processes is COX-2, which is associated with carcinogenesis and resistance to apoptosis,31–34 suggesting that it may mediate the effects of inflammation. In addition, COX-2 overexpression is important in the development of several human tumor types, including gliomas,35 and is associated with high tumor aggressiveness and poor patient prognosis.36,37 It is established that activation of NF-κB p65/p50 contributes to COX-2 overexpression through binding to sites in its promoter.38,39 Our study demonstrates that combined treatment with curcumin and ACNU enhances inhibition of COX-2 expression, likely mediated by repression of translocation of NF-κB dimers from the cytosol to the nucleus, thereby abrogating COX-2 transcriptional activation in GBM cells.

The PI3K/Akt signaling pathway is key to the regulation of glioma cell growth, proliferation, cell cycle, and apoptosis. According to the literature, PI3K/AKT inhibition contributes to the inactivation of NF-κB signaling40 and activation of molecular proapoptotic processes, such as mitochondrial membrane permeabilization and caspase release.41 In the present study, we detected enhanced inhibition of PI3K/AKT-mediated cyt c/caspase-dependent apoptosis, as well as suppression of NF-κB signaling, after co-treatment of GBM cells with ACNU and curcumin, in comparison with single-agent treatment.

Cancer cell migration and invasion result in the diffuse and invasive growth of GBM, which makes these tumors difficult to eradicate using conventional therapeutic methods. GBM cells often gain the ability to migrate, invade, and metastasize through epithelial–mesenchymal transition.42 This implies that strategies focused on the inhibition of cell migration and invasion using specific small-molecule inhibitors could be an effective alternative approach to suppression of GBM growth. MMP-2/9, the important members of the MMP family, can promote cell migration through disruption of the extracellular matrix.43 N-cadherin and vimentin are the markers for cell invasion and were detected in the present study. Our results demonstrate that combination treatment with ACNU and curcumin led to significant downregulation of MMP-2/9, N-cadherin, and vimentin, compared with ACNU or curcumin monotherapy. These results imply that the enhanced inhibition of cell proliferation induced by the combination of ACNU and curcumin is associated with an increased inhibition of both migration and invasion of GBM cells.

In summary, this study demonstrates that combination treatment not only significantly inhibits glioma cell proliferation and invasion but also increases glioma cell apoptosis and cell-cycle arrest through simultaneous targeting of N-cadherin/MMP2/9, cyt c/caspase, PI3K/AKT, and NF-κB/COX-2 signaling (Figure 6), compared with curcumin or ACNU treatment alone. More importantly, the therapeutic efficacy was significantly higher in glioma cells treated with both curcumin and ACNU than those treated with either cur-cumin or ACNU alone. These findings provide new insights into the molecular mechanisms underlying the effects of combination treatment with ACNU and curcumin on GBM cell growth inhibition and provide strong evidence that combined treatment with ACNU and curcumin may be an effective therapeutic option in GBM.

Figure 6

Schematic showing that curcumin and ACNU act synergistically to inhibit GBM growth by targeting multiple signaling pathways.

Abbreviations: ACNU, nimustine hydrochloride; Curc, curcumin; GBM, glioblastoma.