*Article* β**-Caryophyllene in the Essential Oil from** *Chrysanthemum Boreale* **Induces G1 Phase Cell Cycle Arrest in Human Lung Cancer Cells**

**Kyung-Sook Chung 1,**†**, Joo Young Hong 1,2,**†**, Jeong-Hun Lee 1,2, Hae-Jun Lee 1,2, Ji Yeon Park 1, Jung-Hye Choi 2,3, Hee-Juhn Park 4, Jongki Hong 1,\* and Kyung-Tae Lee 1,2,\***


Academic Editor: Igor Jerkovi´c

Received: 5 October 2019; Accepted: 17 October 2019; Published: 18 October 2019

**Abstract:** *Chrysanthemum boreale* is a plant widespread in East Asia, used in folk medicine to treat various disorders, such as pneumonia, colitis, stomatitis, and carbuncle. Whether the essential oil from *C. boreale* (ECB) and its active constituents have anti-proliferative activities in lung cancer is unknown. Therefore, we investigated the cytotoxic effects of ECB in A549 and NCI-H358 human lung cancer cells. Culture of A549 and NCI-H358 cells with ECB induced apoptotic cell death, as revealed by an increase in annexin V staining. ECB treatment reduced mitochondrial membrane potential (MMP), disrupted the balance between pro-apoptotic and anti-apoptotic Bcl-2 proteins, and activated caspase-8, -9, and -3, as assessed by western blot analysis. Interestingly, pretreatment with a broad-spectrum caspase inhibitor (z-VAD-fmk) significantly attenuated ECB-induced apoptosis. Furthermore, gas chromatography–mass spectrometry (GC/MS) analysis of ECB identified six compounds. Among them, β-caryophyllene exhibited a potent anti-proliferative effect, and thus was identified as the major active compound. β- Caryophyllene induced G1 cell cycle arrest by downregulating cyclin D1, cyclin E, cyclin-dependent protein kinase (CDK) -2, -4, and -6, and RB phosphorylation, and by upregulating p21CIP1/WAF1 and p27KIP1. These results indicate that β-caryophyllene exerts cytotoxic activity in lung cancer cells through induction of cell cycle arrest.

**Keywords:** β-caryophyllene; essential oil from *Chrysanthemum boreale*; apoptosis; cell cycle; human lung cancer

### **1. Introduction**

Lung cancer has the fourth highest incidence among cancers, and is the leading cause of cancer death worldwide [1]. The mortality rate is high due to signs of lung cancer generally not appearing until the disease is already at an advanced stage. The two main types of lung cancer consist of small cell lung cancer (SCLC), accounting for a minor percentage of all cases, and non-small cell lung cancer (NSCLC), representing more than 80 to 85% of cases [2]. When the cancer grade is higher than stage I, chemotherapeutic treatment is recommended for NSCLC [3]. Despite substantial progress in the oncology field as a whole, the outcomes following treatment for lung cancer are still poor [4]. However, chemotherapy is associated with significant side effects, and its use can lead to chemoresistance, which

is often fatal. Hence, research and development of novel and less hazardous anti-cancer regimens for NSCLC are required [5–7].

Traditionally, botanical medicines have been used in developing countries as the major source of medical treatment [8]. Anti-cancer natural products or their derivatives included 49% of a total 175 FDA approved small molecules [9]. As an example, vinca alkaloids, isolated from *Catharanthus roseus* (Apocynaceae), cause microtubule disruption and induce cell cycle arrest at metaphase, resulting in apoptosis of cancer cells. SB365, a saponin D extracted from the roots of *Pulsatilla koreana* exhibits anti-proliferative effects in various cancer cell lines. In pancreatic cancer, SB365 induces apoptosis and inhibits angiogenesis, contributing to a rise in patient survival rate to 54%, with no reports of side effects [10]. Currently, the efficacy of intravenous SB365 treatment is being investigated in clinical trials [11].

*Chrysanthemum boreale* (Asteraceae) is a perennation. It is around 1–1.5 m tall, and has yellow flowers that are typically 1.5 cm in diameter. *C. boreale* is mainly distributed along the Korean Peninsula, and has spread to the Manchuria region. *C. boreale* and similar species, for instance *C. indicum* and *C. lavandulaefolium*, have been utilized in conventional eastern treatments for stomatitis, pneumonia, carbuncle, fever, and vertigo [12]. Moreover, the guaianolide derivative 8-acetoxy-4,10-dihydroxy-2,11(13)-guaiadiene-12,6-olide, isolated from *C. boreale*, has been shown to have cytotoxic activity against five human cancer cell lines [13]. However, the mechanisms that underlie the anti-proliferative effects of the essential oil from *C. boreale* (ECB) in NSCLC have not been thoroughly reported. A previous study described the isolation of 87 compounds from the ECB. Among them, the most represented were: monoterpenes camphor (17.93%), α-thujone (13.13%), cis-chrysanthenol (12.80%), 1,8-cineole (3.95%), α-pinene (3.83%), and sesquiterpene β-caryophyllene (3.04%) [12]. In this study, we investigated the cytotoxic potential of ECB in lung cancer cell lines. To determine the active ingredient of ECB, we tested six of its components (1,8-cineole, thujone, β-caryophyllene, camphor, endo-borneol, and 2-isopropyl-5-methyl-3-cyclohexen-1-one) for their anti-proliferative effects, and delineated the underlying molecular mechanisms associated with the cytotoxicity.

#### **2. Results**

#### *2.1. ECB Induces Apoptosis in Human Lung Cancer Cells*

To determine the anti-proliferative effects of ECB, we examined its cytotoxic potential in human lung carcinoma (A549), pancreatic adenocarcinoma (AsPC-1), and colon adenocarcinoma (HT-29) cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were treated with various concentrations of ECB for 48 h. The IC50 values for A549, AsPC-1, and HT-29 were 28.18 ± 1.96, 30.86 ± 2.32, and 55.21 ± 3.06 μg/mL, respectively, indicating that ECB was more cytotoxic in A549 compared to other cell lines. For this reason, we decided to focus on lung carcinoma cells, using A549 and NCI-H358 as cell line models of NSCLC in our study. Similarly to A549, ECB showed dose-dependent cytotoxicity in NCI-H358 cells (IC50: 31.19 ± 2.01 μg/mL,) compared to L132 normal lung epithelial cells (IC50: > 100 μg/mL) (Figure 1a). Additionally, treatment with ECB induced a time-dependent increase in the sub-G1 cell death population in A549 and NCI-H358 cells (Figure 1b). In order to understand if ECB-induced cell death was apoptotic nature, we further examined whether ECB could induce exposure of phosphatidylserine (PS) in A549 and NCI-H358 cells by biparametric flow cytometry analysis, using PI and annexin V to stain DNA (dead cells) and PS (cells undergoing apoptosis), respectively. As shown in Figure 1c, treatment with ECB significantly increased the percentage of PI-annexin V double-positive cells in a concentration-dependent manner. These results suggest that ECB can induce A549 and NCI-H358 lung cancer cells death via apoptosis, rather than non-specific necrosis.

**Figure 1.** The effects of the essential oil from *C. boreale* (ECB) on cell viability and apoptosis in human lung cancer cells. (**a**) A549 cells and NCI-H358 cells were treated with increasing amounts of ECB for 48 h. To determine cell viability, MTT assay was performed. (**b**) Cells were treated with 30 μg/mL of ECB for the indicated times. The cell cycle progression was determined by staining with PI and flow cytometry. Results are representative of three independent experiments. (**c**) Cells treated with different concentrations of ECB (10, 20, or 30 μg/mL for 48 h) were double-stained with PI and annexin V, which specifically detects the externalization of phosphatidylserine (PS), and examined by flow cytometry. Data are presented as means ± SD of three independent experiments. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. the control group.

#### *2.2. ECB-Induced Apoptosis is Mediated by Caspase Activation and Mitochondrial Pathway in Human Lung Cancer Cells*

The apoptotic process begins in response to intrinsic or extrinsic death signals, and several proteins are involved in this process, including caspases. Procaspases are the precursors of caspases. When cleaved, they become active, promoting apoptotic cues. Caspase-8 plays a pivotal role in the extrinsic apoptotic pathway [14]. By contrast, caspase-9 is activated as result of Bcl-2 proteins reducing the MMP in the intrinsic pathway. Finally, caspase-3 is activated through both the intrinsic and extrinsic pathways, and apoptosis occurs [15]. To examine the effect of ECB on the apoptotic process, we assessed the expression of apoptosis-related proteins by western blot analysis. Cancer cells treated with 10, 20, or 30 μg/mL ECB for 48 h exhibited reduced levels of procaspase-3, -8, and -9 (Figure 2a), suggesting activation of both the intrinsic and extrinsic apoptotic pathways, ultimately leading to apoptosis. Besides, analysis of PI-annexin V double-positive cells by flow cytometry showed that treatment with the pan-caspase inhibitor z-VAD-fmk could attenuate ECB-induced cell death in A549 and NCI-H358 cells (Figure 2b). Next, we measured the MMP of A549 and NCI-H358 cells after treatment with ECB, using the fluorescent dye DiOC6. As shown in Figure 2c, ECB increased mitochondrial membrane depolarization at 48 h compared to that at 0 h. Because Bcl-2 family proteins can regulate MMP [16], we evaluated levels of these proteins in the presence or absence of ECB. Western blot analysis revealed that the presence of ECB decreased anti-apoptotic Bcl-2 and Bcl-XL protein levels, and increased the expression of pro-apoptotic Bad (Figure 2d). Thus, these data indicate that ECB activates apoptosis by regulating the expression of Bcl-2 family proteins and reducing MMP, which leads to caspase activation in A549 and NCI-H358 cells.

**Figure 2.** Effects of ECB on apoptosis-related proteins. (**a**) A549 and NCI-H358 cells were treated

with different doses of ECB for 48 h. Western blot analysis was conducted to determine the protein expression levels of procaspase-3, -8, -9, and cleaved caspase-9. (**b**) Cells were pretreated with or without z-VAD-fmk (25 μM) for 30 min, treated with ECB (30 μg/mL) for 48 h, and PI-annexin V double staining was performed to determine the fraction of apoptotic cells. (**c**) A549 and NCI-H358 cells were stained with DiOC6 at 0 and 48 h and flow cytometry was performed to measure mitochondrial membrane polarization. (**d**) A549 and NCI-H358 cells were treated with different concentrations of ECB for 48 h, and western blot analysis was performed to determine the protein expression levels of Bcl-2, Bcl-XL, and Bad. Data are presented as means ± SD of three independent experiments. \*\*\* *p* < 0.001 vs. the control group; # *p* < 0.001 vs. the ECB-treated group.
