Clove Essential Oil as a Source of Antitumoral Compounds Capable of Crossing the Blood-Brain Barrier: A Focus on the Effects of β-Caryophyllene and Eugenol in a Glioblastoma Cell Line

Abstract

This study aimed to investigate β-Caryophyllene (BCA) pharmacokinetics as well as the potential antitumor activity and mechanism of action of BCA and eugenol (EU), alone or in combination, in U87 glioblastoma (GB) cells. The BCA pharmacokinetic was studied by evaluating its concentration profiles in rat blood and cerebrospinal fluid after oral and intravenous administration. EU and BCA antitumor mechanisms were assessed by comparing their effects in U87 GB cells and non-tumoral HMC3 cells. Cell death, cell cycle regulation and mitochondrial membrane potential (MMP) were evaluated using flow cytometry. mRNA levels of target genes were evaluated by qPCR. Secreted cytokines were measured by Luminex^®. BCA, as well as EU, permeates the brain. EU and BCA affected the viability and proliferation of U87 cells (up to 50%, p < 0.001) but not HMC3 cells and showed a synergistic effect. BCA and EU induced G0/G1 cell cycle arrest, increasing apoptosis/necrosis. EU and BCA induced the downregulation of mRNAs encoding for key proteins involved in GB angiogenesis (VEGFA decreased op to 60%, p < 0.01), proliferation and progression, and showed anti-inflammatory activity (IL-4 significantly decreased, p < 0.001). EU and BCA demonstrated strong and multitarget antitumor activity in U87 cells. Our results provide a strong rationale for the further evaluation of EU and BCA as possible therapeutic molecules in GB management.

Keywords: antitumor activity; essential oils; eugenol; glioblastoma; pharmacokinetics; β-Caryophyllene.

Figure 1

Elimination profile of BCA after 0.4 mg intravenous infusion to rats (2 mg/kg). Data are expressed as the mean ± SE of four independent experiments. The elimination followed apparent first-order kinetics, confirmed by the semilogarithmic plot reported in the inset (n = 7, r = 0.980, p < 0.001). The half-life of BCA was calculated to be 49.7 ± 2.0 min.

Figure 2

BCA concentrations (μg/mL) detected in the CSF of rats after intravenous administration of a 0.4 mg (2 mg/kg) dose. Data are expressed as the means ± SEM of four independent experiments.

Figure 3

Blood BCA concentrations (μg/mL) within 300 min after oral administration of 10 mg (50 mg/kg) dose to rats. Data are expressed as the means ± SEM of four independent experiments.

Figure 4

CSF BCA concentrations (μg/mL) within 150 min after oral administration of 10 mg (50 mg/kg) dose to rats. Data are expressed as the means ± SEM of four independent experiments.

Figure 5

Antiproliferative/cytotoxic activity of EU and BCA, alone or in a combined treatment, in U87 (A,C,E) and HCM3 (B,D,F) cell lines. Cells were treated with EU (2.5, 25 and 200 µM) and BCA (1.25, 12.5 and 125 µM) alone or in combination and subsequently subjected to Alamar blue^® assay. Metabolic activity is expressed as a percentage of untreated control cells (CTL). Data are presented as means ± SEM and are representative of at least three independent experiments. *** p < 0.001 with respect to the control (CTL) values.

Figure 6

The highest single agent (HSA) synergy scores for EU and BCA were calculated using SynergyFinder software for U87 (A) and HMC3 (B) cell lines. A synergy score value > 10 is considered synergistic; between −10 and +10 was considered additive, while a synergy score < −10 was considered antagonistic.

Figure 7

Evaluation of colony formation after treatment with EU and BCA. U87 cells were treated with EU 200 µM and BCA 125 µM, alone or in combination. After a 14-day incubation, colony formation by U87 cells was evaluated by microscopy and the number of colonies was determined using FIJI/ImageJ. ** p < 0.01 with respect to the control (CTL) values.

Figure 8

Assessment of cell death profiles in U87 (A) and HMC3 (B) cell lines treated with EU (200 µM) and/or BCA (125 µM), alone or in combination after 24 h. Cells were co-stained with AV and PI, and the percentage of non-apoptotic cells or apoptotic cells was determined by flow cytometry. * p < 0.05 and ** p < 0.01 with respect to the control (CTL) values. Data are means ± SEM and are representative of at least three independent experiments.

Figure 9

Impact of EU and BCA treatments on cell cycle progression on U87 (A) and HCM3 (B) cell lines and CDK4 mRNA levels in both cell types (C). Cells were treated with EU (200 µM) and BCA (125 µM) alone or in combination and incubated for 24 h. (A,B) Cells were stained with PI/RNase and the cell cycle was assessed by flow cytometry. The proportion of cells in the G₀/G₁, S and G₂/M cell cycle phases was expressed as a percentage of the total cell population. (C) Relative expression of CDK4 mRNA levels was assessed by qRT-PCR, and the results were normalized to GAPDH expression. * p < 0.05 and *** p < 0.001 with respect to the control (CTL) values. Data are means ± SEM and are representative of at least three independent experiments.

Figure 10

Analysis of the mitochondrial membrane potential in U87 (A) and HCM3 (B) cell lines. Cells were treated with EU (200 µM) and BCA (125 µM) alone or in combination and incubated for 24 h. Increased values in the monomer/aggregate (M/A) ratio indicate a decrease in the mitochondrial membrane potential. Results are expressed as the M/A ratio of JC-1, which was calculated as the fraction of MFI observed for each molecule. *** p < 0.001. The means ± SEM shown are representative of at least three independent experiments.

Figure 11

Assessment of BCL2 (A), BCL2L1 (B), BAK1 (C), BAX (D) and CASP9 (E) mRNA levels in U87 and HMC3 cells treated with EU (200 µM) and BCA (125 µM) for 24 h. Relative expression was assessed by qRT-PCR after normalization with GAPDH expression. * p < 0.05; ** p < 0.01. Data are the means ± SEM of at least three independent experiments.

Figure 12

Assessment of TP53 (A) and MDM2 (B) mRNA levels in U87 and HMC3 cells treated with EU (200 µM) and BCA (125 µM) for 24 h. Relative expression was assessed by qRT-PCR after normalization with GAPDH expression. * p < 0.05; ** p < 0.01. Data are means ± SEM of at least three independent experiments.

Figure 13

Assessment of PTEN (A) and VEGF (B) mRNA levels in U87 and HMC3 cells treated with EU (200 µM) and BCA (125 µM) for 24 h. Relative expression was assessed by qRT-PCR after normalization with GAPDH expression. * p < 0.05; ** p < 0.01; *** p < 0.001. Data are means ± SEM of at least three independent experiments.

Figure 14

Effects of EU and BCA on the concentration of the inflammatory cytokines IL-6 (A), IL-4 (B), IL-8 (C) and TNF-α (D). Cells were treated with EU (200 μM) and BCA (125 μM) alone or in combination and incubated for 24 h. * p < 0.05; *** p < 0.001 compared to the control condition (untreated cells). Data as means ± SEM are representative of at least three independent experiments.

Figure 15

General scheme of the experimental design of the in vitro study. Eugenol and β-Caryophyllene were added to HMC3 (normal) and U87 (glioblastoma) cells, alone or in combination for 24–72 h. After treatment, cell viability, cell cycle analysis, real-time qPCR on different genes involved in tumor progression/angiogenesis, mitochondrial membrane potential (MMP) and cytokines secretion were analyzed.