Abstract

Background. Glioblastoma multiforme (GBM) is a lethal brain tumor with high mortality and morbidity. Nerolidol (NRD) is a sesquiterpene alcohol sequestered from the essential oils of aromatic florae with potent antioxidant, antiviral, anticancer, cardioprotective, and neuroprotective activity.

Objectives. The aim of the study was to investigate the underlying cell-cycle mechanisms of NRD-mediated antiproliferative and apoptosis activities in GBM using human U-251 cells.

Materials and methods. The current research investigated the antiproliferative and apoptotic activities of NRD on U-251 cells. The effects of NRD were measured using a Cell Counting Kit-8 (CCK-8) assay, 4’,6-diamidino-2-phenylindole (DAPI) staining, messenger ribonucleic acid (mRNA) level assessment, and western blot assay.

Results. Nerolidol decreased U-251 viability in a dose-dependent manner, as well as induced apoptotic activity, reduced B-cell lymphoma-2 (BCL-2) levels, and increased mRNA expression of BCL-2-associated X (Bax), caspase-3 and caspase-9. The attenuation of the cyclin-D1, cyclin-dependent kinase 4 (CDK4) and CDK6 mRNA expression confirmed cell cycle regulation. Western blot analysis of CDK1 indicated reductions in cyclin-B1 and p21. Furthermore, NRD prompted apoptosis through p38 amelioration and increased phosphorylated extracellular signal-related kinase 1 (p-ERK1) and phosphorylated c-Jun N-terminal protein kinase 1 (p-JNK1) levels.

Conclusions. Nerolidol inhibited GBM cell viability and induced apoptosis through the regulation of cell-cycle proteins via p38 mitogen-activated protein kinase (MAPK) signaling pathways. Thus, NRD could be developed as a potential natural therapeutic agent for GBM.

Key words: glioblastoma, p38 MAPK, apoptosis, nerolidol, U-251 cells

Background

Glioblastoma multiforme (GBM) is a fatal cancer of the central nervous system; this primary brain malignancy has an annual prevalence of 3.19/100,000^{1, 2} and a high mortality rate, with a survival rate fewer than 12 months.³ Owing to its rapid progression and infiltrative features, GBM is incurable despite the current treatment modalities of surgery, chemotherapy, c-irradiation, and immunotherapy.^{4, 5} Clinical research suggests that the quick propagation and high invasiveness of GBM cells make it incurable and cause relapse.⁶ Treatment failure and continuous disease progression result in an average lifespan of 1–1.5 years.⁷ The malignant glioma appears to proliferate incessantly. Hence, innovative approaches are urgently required to manage these devastating tumors.

Recent research suggests that novel drug development may entail targeting cell signaling pathways, with multiple signaling networks, cell cycle protein regulation, and apoptosis-associated proteins likely critical to cancer prevention.⁸ The cell cycle is tightly controlled by regulatory proteins, such as cyclins and cyclin-dependent kinases (CDKs) that facilitate the checkpoint switches between G1/S, S and G2/M phases.^{9, 10} Anomalies in cell division or apoptosis lead to irregular cell growth, eventually causing tumor development. As such, apoptosis, a form of programmed cell death, is crucial for the growth and maintenance of healthy tissues and is regulated by specific caspases and proteases.^{11, 12}

Mitogen-activated protein kinases (MAPKs) are involved in intracellular signaling through propagation, disparity and apoptosis.¹³ It is thought that glioma cell incursion and metastasis require the triggering of precise signaling cascades, particularly the p38 MAPK pathway.^{14, 15} Therefore, inhibiting p38 MAPK signaling is a central goal of GBM management.

Numerous natural phytochemicals act as anticancer agents by regulating the proliferation, invasion and metastasis of diverse cancer cells.¹⁶ Nerolidol (NRD) is a sesquiterpene alcohol (3,7,11-trimethyl-1,6,10-dodecatrien-3-ol) extracted as an essential oil from aromatic florae, such as ginger, neroli, lemongrass, tea tree, and lavender.¹⁷ It has anti-inflammatory, antioxidative, anticancer, and apoptotic properties.¹⁸ Recently, NRD was shown to mitigate oxidative stress, apoptosis and inflammation in cardiotoxicity prompted by the chemotherapeutic mediator cyclophosphamide.¹⁹ Furthermore, NRD blocked inflammatory responses in lipopolysaccharide (LPS)-induced acute lung injury (ALI) by modulating antioxidants and the adenosine 5’-monophosphate-activated protein kinase (AMPK)/nuclear factor erythroid 2-related factor 2 (Nrf-2)/heme oxygenase-1 (HO-1) signaling pathway.²⁰ Moreover, NRD is a beneficial antitumor compound due to its efficacy in targeting cell survival and proliferation molecules²¹ that act as chemosensitizers in tumors.^{22, 23} Nerolidol improves the effectiveness of doxorubicin (DOX) in mammary carcinogenesis²² and enhances its efficacy in ovarian cancer and lymphoblast cells.²⁴

Nerolidol is hydrophobic in nature, with an XlogP3 value of 4.6, and can readily cross the blood–brain barrier,²⁵ where it exerts neuroprotective effects through its anti-inflammatory and antioxidant properties. Baicalein was determined to be a viable treatment for GBM in studies similar to the current research, because it reduced the viability of GBM cells and caused apoptosis by inhibiting nuclear factor kappa B (NF-κB)-p65 activity.²⁶ However, the anti-glioma action of NRD through MAPK signaling remains uncertain.

Objectives

We assessed the protective influence of NRD on U-251 human GBM cells by examining proliferation, cell cycle protein regulation and apoptosis.

Materials and methods

Chemicals

Nerolidol, fetal bovine serum (FBS), antibiotics (penicillin-streptomycin), phosphate-buffered saline (PBS), 4’,6-diamidino-2-phenylindole (DAPI), dimethyl sulfoxide (DMSO), and sodium dodecyl sulfate (SDS) were purchased from Bio-Rad Laboratories (Hercules, USA). The antibodies were obtained from Roche Diagnostics (Risch, Switzerland).

Cell lines

The U-251 human GBM cells were purchased from Shanghai Aiyan Biotechnology Co., Ltd. (Shanghai, China), and grown in a Dulbecco’s modified Eagle’s medium (DMEM) consisting of FBS (10%), streptomycin (100 μg/mL) and penicillin (100 U/mL) under a CO₂ atmosphere (5%) with less than 95% humidified air at 37°C.

Investigation of cell viability

Cell proliferation was examined using the Cell Counting Kit-8 (CCK-8) assay (Alpha Diagnostics International, San Antonio, USA). The U-251 cells were seeded onto a 96-well plate at 1×10⁵ cells per well, incubated and supplemented with different NRD dosages (10–70 μM). The CCK-8 solution (10 μL) was added separately to all wells. After 1 h of incubation, the optical density (OD) was determined at 450 nm using a microplate absorbance reader, model No. 1681130 (Bio-Rad Laboratories).

Apoptosis exploration by DAPI staining

Human GBM U-251 cells were seeded into 6 wells at 1×10⁵ cells/well. Each well was then supplemented with NRD (30 μM and 40 µM). Treated U-251 cells were stained with DAPI and observed under a fluorescence microscope (Eclipse TS100; Nikon, Tokyo, Japan) with a digital camera (4500 Coolpix; Nikon) to assess cellular apoptotic changes, according to a previously described method.²⁷

Determination of messenger ribonucleic acid expression

Total ribonucleic acid was isolated from human GBM cells U-251 as per the manufacturer’s protocol using TRIzol® reagent (Abcam, Cambridge, USA). The isolated messenger RNA (mRNA) was reverse-transcribed into complementary DNA (cDNA) using a cDNA reverse transcription kit (Abcam), according to the manufacturer’s instructions. Then, SYBR Green Real-Time PCR Master Mixes (Thermo Fisher Scientific, Waltham, USA) examined the cDNAs using the company’s protocols. The band intensity was observed after electrophoresis using 1.5% agarose gels. The band intensity was measured using ImageJ v. 1.48 software (National Institutes of Health, Bethesda, USA). Primer sequences used included B-cell lymphoma-2 (BCL-2), BCL-2 associated X (Bax), caspase-3, cyclin-D1, cyclin-dependent kinase 4 (CDK4), CDK6, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as outlined below in Table 1:

Western blot study

Human U-251 GBM cells were treated with NRD (30 µM and 40 µM) and cultured for 24 h. Cell lysates were prepared for western blot analysis using an ice-cold lysis buffer containing protease inhibitors. Total protein content was measured using Protein BCA Assay Kit (Pierce Chemical, Rockford, USA). The proteins were electrophoretically dispersed and transferred to a polyvinylidene difluoride (PVDF) film, which was blocked with a probe for 60 min at room temperature and incubated with primary antibodies (1:1000 dilutions) overnight at 4°C. Thereafter, the secondary antibodies were added and visualized using an enhanced chemiluminescence protein detection kit (Pierce Chemical).

Statistical analyses

All statistical analyses employed GraphPad Prism v. 8.0.1 (GraphPad Software, San Diego, USA) and IBM Statistical Package for Social Sciences (SPSS) v. 25 (IBM Corp., Armonk, USA) software. The measurement data were reported as medians and quartiles. The normality of the distribution was tested using the Kolmogorov–Smirnov test (Supplementary Table 1), and although all data displayed a normal distribution, a nonparametric test was used due to small sample sizes. Comparisons among groups (control (n = 6), 30 µM (n = 6) and 40 µM (n = 6)) utilized the Kruskal–Wallis test followed by Dunn’s test. When the test standard had a value of p < 0.05, the difference was considered statistically significant.

Results

All variables had a normal distribution. Table 2 displays the results of comparing variables between groups.

Effects of nerolidol on U-251 cytotoxicity

The U-251 GMB cell proliferation was assessed using multiple NRD treatments (10, 20, 30, 40, 50, and 60 μM/mL) for 24 h. Nerolidol significantly (p < 0.05) reduced cell proliferation in a concentration-dependent manner. The optimum dosages of 30 μM and 40 μM, identified as the IC₅₀ values, were used for further experiments (Figure 1). Nerolidol treatment at 30 μM and 40 μM significantly (p < 0.05 and p < 0.01, respectively) inhibited U-251 growth, indicating that NRD is a potential bioactive remedy for GBM.

Effects of nerolidol on U-215 apoptosis

The DAPI-stained control U-251 cells remained viable, while NRD-treated cells (30 μM and 40 μM) showed significant apoptosis induction (p < 0.05). The shrinkage of cytoplasm, nuclear membrane loss and nucleus disintegration were observed with NRD usage. The administration of 40 μM NRD caused an increased apoptotic effect (Figure 2).

Effects of nerolidol on apoptotic mRNA expression

The untreated U-251 GMB cells had decreased caspase-3 and Bax mRNA levels, whereas the levels of BCL-2 were elevated (Figure 3). Nerolidol (30 μM and 40 μM) treatment enhanced the levels of Bax and caspase-3, and diminished BCL-2 significantly (p < 0.05). These findings indicate that NRD reduced U-251 viability through apoptosis.

Effects of nerolidol on cell cycle gene expression

The mRNA levels in NRD-treated U-251 GMB cells are shown in Figure 4. The untreated cells had augmented mRNA levels of cyclin-D1, CDK4 and CDK6. Nerolidol (30 μM and 40 μM) significantly (p < 0.05) reduced the mRNA levels, with 40 μM NRD causing greater reductions than 30 μM.

Effects of nerolidol on cyclin-B1, CDK1 and p21 protein levels

Nerolidol (30 μM and 40 μM) reduced cyclin-B1 and CDK1, whereas it augmented p21 in a dosage-reliant way. The increase in protein expressions indicates that NRD controlled the cell cycle and apoptosis in U-251 cells (Figure 5).

Effects of nerolidol on MAPK protein levels

Administration of NRD (30 μM and 40 μM) significantly enhanced phosphorylated p38 MAPK protein levels (p < 0.05) in U-251 cells. Meanwhile, phosphorylated extracellular signal-related kinase 1 (p-ERK) and phosphorylated c-Jun N-terminal protein kinase 1 (p-JNK) were also slightly increased (Figure 6). The enhanced MAPK protein expression indicates that NRD-induced apoptosis in U-251 cells is associated with p38 MAPK signaling.

Discussion

The GBM is the most aggressive primary brain malignancy and has high mortality and morbidity.^{1, 2} Moreover, GBM patients have a poor prognosis, with a median survival time of 10–12 months.^{3, 7} Chemopreventive agents treat all phases of tumor evolution and prevent cancer cell propagation, incursion and metastasis,¹⁶ and several phytochemicals act by inhibiting proliferation, invasion, metastasis, angiogenesis, and stimulating apoptosis.^{14, 15} The anticancer effects of NRD have been extensively explored in numerous cancers, including breast cancer,²² hepatocellular carcinomas,²⁸ osteosarcomas,²⁹ oral cancer,³⁰ and ovarian cancer.²⁴ The current research evaluated the anti-GBM effects of NRD by assessing its anti-proliferative, cell-cycle protein regulation and apoptotic properties. Our results showed that NRD inhibited U-251 cell multiplication and cell cycle proteins and induced apoptosis in a dose-dependent manner.

Apoptosis is a well-recognized cell death program triggered by various stimuli.^{11, 12} Certain natural bioactive anticancer agents were shown to eliminate malignant cells by restoring defective apoptosis.³¹ The CCK-8 assay demonstrated that NRD could dramatically inhibit GBM cell viability, indicating that NRD displayed a robust anti-proliferative action on U-251 cells. Furthermore, DAPI staining showed that NRD treatment improved programmed cell death in GBM cells. Likewise, reverse transcription polymerase chain reaction (RT-PCR) results showed reduced BCL-2 and elevated Bax and caspase-3 mRNA levels after NRD treatment. These are well-known as apoptosis-associated proteins, and the ratio of BCL-2 to Bax was markedly reduced by NRD. Similar anti-proliferative and apoptotic properties of NRD were reported in hepatocellular carcinoma²⁷ and osteosarcoma.²⁸ Our findings demonstrate that NRD inhibited proliferation and stimulated apoptosis in U-251 cells.

Cell-cycle checkpoints play a vital role in cellular development and apoptosis,^{9, 10} and NRD is described as an anti-proliferative mediator in leiomyoma cells by diminishing the cyclin-D1 and CDK4/6 G1-S checkpoint proteins. Matus et al. reported that NRD (cis- and trans-) stimulated G1 cell cycle arrest in ELT3 cells.³² Satomi et al. confirmed that NRD triggered G1 cell cycle arrest in human hepatocellular carcinoma cells by increasing cytochrome-P450 enzymes.³³ As such, the underlying anticancer mechanisms of NRD are thought to involve cell cycle arrest,²⁷ oxidative phosphorylation reduction³² and apoptosis.³⁴ In the current research, NRD treatment reduced the mRNA expression of cyclin-D1, CDK4 and CDK6, while it increased p21 expression and reduced CDK1 and cyclin-B1 protein levels. The cell-cycle transition from the S phase to the G2/M phase is largely determined by the CDK1 and cyclin-B1 complexes.^{9, 10} Collectively, these findings suggest that NRD employed anti-proliferative effects in GBM cells by stimulating cell death through cell-cycle arrest.

The MAPKs play a central role in cell development, proliferation and death. Highly conserved MAPKs in mammalian cells consist of p38, ERK and JNK, which are stimulated via a phosphorylation cascade.¹³ The ERK stimulates cellular proliferation, while p38 and JNK induce apoptosis.^{13, 14} This study established that NRD can successfully suppress p38 MAPK phosphorylation via JNK- and ERK-regulated signalling pathway. Previous research showed that NRD essential oil isolated from Lindera erythrocarpa inhibited IκB degradation and NF-κB phosphorylation through MAPK phosphorylation in LPS-stimulated macrophages.³⁵ This study demonstrated that NRD showed anti-proliferative and apoptotic activity on U-251 cells via MAPK signaling.

Limitations

Our study highlights NRD as a pharmacological inhibitor of the p38 signaling pathway in GBM cells; additional details of its underlying anticancer actions need to be examined in the future studies.

Conclusions

Our study revealed that NRD inhibited proliferation and induced apoptosis in U-251 cells. Nerolidol also induced apoptosis by stimulating pro-apoptotic proteins and inhibiting anti-apoptotic proteins. Indeed, NRD triggered apoptosis and cell cycle arrest via p38-MAPK stimulation. This pathway could be intricately linked with the antitumor action of NRD in U-251 cells. Hence, NRD is a promising natural anticancer and chemopreventive agent for GBM. However, in vivo studies are needed to understand the apoptosis mechanism and cell-cycle regulation induced by NRD in GBM cells.

Supplementary data

The supplementary materials are available at https://doi.org/10.5281/zenodo.8202031. The package contains the following files:

Supplementary Table 1. Results of normality tests as presented in Figure 3.

Supplementary Table 2. Results of normality tests as presented in Figure 4.

Supplementary Table 3. Results of normality tests as presented in Figure 5.

Supplementary Table 4. Results of normality tests as presented in Figure 6.