*2.3.* β*-Caryophyllene Regulates G1 Cell Cycle Progression in Human Lung Cancer Cells*

Next, we performed gas chromatography-mass spectrometry (GC/MS) of the ECB. This analysis revealed six major constituents (Figure 3), and their cytotoxicity was determined in A549 and NCI-H358 cells by MTT assay. As shown in Table 1, β-caryophyllene showed the strongest cytotoxicity among these compounds.

**Figure 3.** Total ion chromatogram of ECB. Peaks represent: 1. 1,8-cineole; 2. thujone; 3. β-caryophyllene; 4. camphor; 5. endo-borneol; 6. 2-isopropyl-5-methyl-3-cyclohexen-1-one.


**Table 1.** Cytotoxicity (IC50) of major compounds isolated from ECB, assessed by MTT assay in A549 cells and NCI-H358 cells

In addition, flow cytometry analysis showed that β-caryophyllene treatment induced the accumulation of A549 and NCI-H358 cells in the G1 phase in a time- and dose-dependent manner (Figure 4a,b). Based on these results, we examined the effect of β-caryophyllene on the expression levels of G1 phase-regulatory proteins by western blot analysis. Our results revealed that β-caryophyllene decreased the expression of cyclin-dependent protein kinase (CDK) 2, CDK4, CDK6, cyclin D1, and cyclin E (Figure 4c,d). In addition, β-caryophyllene decreased the levels of phosphorylated retinoblastoma (p-RB) protein, but did not alter total RB levels. This is indicative of a reduced cyclin/CDK activity, which is consistent with the induction of cell cycle arrest. Moreover, β-caryophyllene increased the levels of p21CIP1/WAP1 and p27KIP1, two CDK inhibitors (CDKIs) that play key roles in the establishment of G1 phase progression (Figure 4e). All together, these results indicate that β-caryophyllene promotes cell cycle arrest in G1 phase in A549 and NCI-H358 cells.

**Figure 4.** Effect of β-caryophyllene on G1 cell cycle arrest and cell cycle-related proteins in human lung cancer. (**a**) Cells were treated with β-caryophyllene (A549, 40 μg/mL; NCI-H358, 60 μg/mL) for the indicated times; (**b**) or with the indicated concentrations for 24 h. Cells were stained with PI for 30 min and then subjected to flow cytometry analysis to determine cell cycle progression. (**c** and **d**) A549 and NCI-H358 cells were treated with β-caryophyllene (A549, 40 μg/mL; NCI-H358, 60 μg/mL) for the indicated times and concentrations, and cells were collected for western blot analysis to measure cyclin/CDK protein expression. (**e**) Cells were treated with β-caryophyllene (A549, 40 μg/mL; NCI-H358, 60 μg/mL) for the indicated times, and then subjected to western blot analysis to determine the protein expression of p-RB, RB, p21CIP1/WAP1, and p27KIP1.

#### **3. Discussion**

Cancer chemoprevention is one of the crucial approaches to reduce or delay the occurrence of malignancy after the chronic administration of a synthetic, natural or biological agent [17]. The potential value of this approach has been demonstrated with trials in breast, prostate, and colon cancer [18]. Because of low cytotoxicity to normal cells, minimal side effects, and a wide margin of safety, medicinal herbs can be used to develop new pharmaceutical drugs. Fruits, leaves, and flowers are rich in essential oils, many of which contain important chemopreventive agents. Several essential oil extracts have been shown to exert anti-proliferative, anti-mutagenic, cytotoxic, anti-oxidant, pro-apoptotic, and anti-neoplastic effects. For example, the essential oil of *Eucalyptus benthamii* (Myrtaceae) contains limonene, which has cytotoxic and anti-proliferative effects in cancer cell lines [19].

*C. boreale* has been reported to possess anti-inflammatory [20] and anti-bacterial properties [21]; and β-caryophyllene, found in this and other plants (including the Asteraceae *Helichrysum gymnocephalum* and the Myrtaceae *Syzygium aromaticum*), retains anti-proliferative activities against various cancer cells through induction of apoptosis, anti-microbial, and antioxidant properties [22–24]. β-Caryophyllene is also contained in the essential oil of *Citrus aurantifolia* (Rutaceae), and studies have shown its ability to induce apoptosis in human colon cancer cells [25]. Furthermore, recent research has revealed an anti-carcinogenic effect for α-thujone, another major component of *C. boreale*. In particular, α-thujone treatment impaired the proliferation of glioblastoma (GBM) cells and angiogenesis, and reduced melanoma metastasis in a GBM rat model [26]. Though it was reported to exert an attenuating effect on the viability of the GBM [27], in the present study α-thujone did not show any cytotoxicity up to 500 μM against human lung cancer cells. Another compound in the ECB, endo-borneol, is a bicyclic organic terpene with the hydroxyl group located in an endo position. Many studies have reported that endo-borneol possesses metabolism-enhancing, anti-inflammatory, and antioxidant activities [28,29]. However, neither endo-borneol nor 2-isopropyl-5-methyl-3-cyclohexen-1-one showed any cytotoxicity in our lung cancer cytotoxicity assay. In the current study, we identified anti-proliferative factors that are required for ECB-induced cell cycle arrest and apoptosis of lung cancer cells. Our findings substantiate the results of published studies, and contribute to establishing novel natural compounds as anti-cancer therapeutics.

During the past decades, suppression of tumor cell proliferation through the induction of apoptosis has been recognized as a strategy for the identification of chemotherapeutic agents [27]. Apoptosis plays a major role in normal physiologic processes and is accompanied by blebbing of the cell membrane, DNA fragmentation, induction of apoptotic bodies, and activation of caspases [14]. Caspases are members of the cysteine-aspartic acid protease family and have crucial roles in triggering and executing apoptosis [30]. The extrinsic and intrinsic pathways form the two branches of caspase-dependent apoptotic signaling. The extrinsic pathway is activated by various receptors, such as TNF-α receptor, FasL receptor, toll-like receptor, and death receptor, which leads to the formation of the 'death-inducing signaling complex' (DISC) and activation of caspase-8 [31]. By contrast, the intrinsic pathway (or 'mitochondrial pathway') is activated by ROS, DNA damage, pro-apoptotic Bcl-2 family proteins, calcium, and some metals. Consequentially, activation of this pathway induces MMP and expedites the release of cytochrome *c*. Released cytochrome *c* forms part of the apoptosome, a complex that contains Apaf-1. The apoptosome triggers procaspase-9 cleavage and activation, which in turn triggers the activation of caspase-3, leading to apoptosis. In this study, we used PI-annexin V double staining to demonstrate that ECB induces apoptosis in human lung cancer cells. In addition, our data showed that ECB reduced MMP, likely due to the downregulation of Bcl-2 and Bcl-XL, and upregulation of Bad protein expression.

Our results indicated that β-caryophyllene induces cell cycle arrest at the G1 phase in human lung cancer cell lines. This assumption is based on the observation that β-caryophyllene promoted downregulation of G1 cell cycle positive regulators, such as CDK2, CDK4, CDK6, cyclin D1, and cyclin E, and upregulation of G1 cell cycle negative regulators p21CIP1/WAF1 and p27KIIP1. Furthermore, β-caryophyllene decreased RB phosphorylation. In a previous study, β-caryophyllene has been shown

to exert a selective anti-proliferative effect in HCT116 colon cancer cells, without affecting normal cell lines [22]. Interestingly, β-caryophyllene-treated HCT116 cells displayed several apoptotic features, such as DNA fragmentation, chromatin condensation, and MMP disruption. Thus, previous studies, together with our data, suggest that β-caryophyllene might have cell-type specific phenotypic effects, and that β-caryophyllene-induced cell cycle arrest might lead to apoptosis.

In summary, our study revealed that ECB exerts its anti-proliferative effect via caspase activation and mitochondria-dependent apoptosis in human lung cancer cells. The active constituent of ECB is β-caryophyllene, which promotes G1 cell cycle arrest in these cell types. Taken together, these data suggest that ECB should be considered as a potential chemotherapeutic agent for the treatment of non-small cell lung cancer.
