Cell cycle arrest and apoptosis induction by Juniperus communis extract in esophageal squamous cell carcinoma through activation of p53‐induced apoptosis pathway

Abstract Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers. It has a high mortality rate and requires novel effective drugs and therapeutic approaches. Juniperus communis (JCo), used to flavor gin and food, has been documented to have anti‐tumor activity. The aim of this study was to investigate the antitumor activity of JCo extract against ESCC and its possible mechanisms. JCo extract suppressed cell growth in ESCC and showed higher selection for ESCC cells than normal cells compared to the clinical drug 5‐fluorouracil (5‐FU). JCo extract induced cell cycle arrest at the G0/G1 phase by regulating the expression of p53/p21 and CDKs/cyclins, triggering cell apoptosis by activating both the extrinsic (Fas/FasL/Caspase 8) and intrinsic (Bcl‐2/Bax/Caspase 9) apoptosis pathways. Moreover, a combination treatment of JCo and 5‐FU synergistically inhibited proliferation of ESCC cells. These results suggest that JCo extract is a potential natural therapeutic agent for esophageal cancer, as it could induce cell cycle arrest and apoptosis in ESCC cells.


| INTRODUC TI ON
Human esophageal carcinoma is one of the most frequently diagnosed cancers, and the sixth leading cause of cancer-related deaths worldwide, causing the deaths of over 400,000 people annually (Pennathur et al., 2013;Siegel et al., 2016). Esophagus squamous cell carcinoma (ESCC) is an aggressive cancer and the most common histological type seen in China, South America, and Western Europe (Chen et al., 2016;Siegel et al., 2016). In recent years, the overall 5-year survival rate for late-stage ESCC has been less than 15%, and the prognosis remains poor for patients, although the development of therapeutic approaches, such as surgery, chemotherapy and radiotherapy (Liang et al., 2017;Wu et al., 2013;Wu et al., 2012). 5-fluorouracil (5-FU) has been the first-line treatment for ESCC therapy for several decades and is also widely used in the treatment of head and neck carcinomas (Longley et al., 2003). However, single agent chemotherapy with 5-FU is no longer appropriate for ESCC therapy because of its toxicity, drug resistance, and side effects with increasing doses. Therefore, combination therapy with other drugs, such as irinotecan and oxaliplatin, is used to improve prognoses and reduce the side effects of 5-FU (Ferte et al., 2011;Wainberg et al., 2011). Combination therapy improves the response rate and survival rate, but the side effects and drug resistance are still unknown.
Natural products and natural health products (extracts or essential oils) from plants are complex mixtures containing various organic compounds that are important for the development of new drugs for anticancer, antibacterial, anti-inflammatory, and antioxidant purposes (Mann, 2002;Padumadasa et al., 2016;Rong et al., 2016;Soukand et al., 2015). In clinical cancer therapy, there are at least 73 approved anticancer drugs from plants, such as topotecan, etoposide, and paclitaxel (Lin et al., 2015;Pan et al., 2010). Moreover, these natural products can also be used in combination with chemotherapy or radiotherapy, resulting in an enhanced therapeutic efficacy and decreased side effects in cancer treatments (Mileo & Miccadei, 2016). Therefore, an increasing number of researchers are now focusing on investigations of the anti-cancer potential of plants.
In traditional medicine, Juniperus plants are widely used to relieve colds, headaches, respiratory diseases, asthma, and digestive and gynecological disorders (Leporatti & Ivancheva, 2003). Recent studies report that Juniperus (essential oils or extracts) shows antioxidant, anti-microbial, anti-inflammatory, nephroprotective, and hepatoprotective effects (Al-Attar, Alrobai, & Almalki, 2016Carpenter et al., 2012;Orhan et al., 2011). Furthermore, the cytotoxic effects of Juniperus species have been investigated in different cancer types, including melanoma, neuroblastoma, leukemia, lung, breast, and colon cancers (Bayazit, 2004;Gao et al., 2019;Lantto et al., 2016;Pollio et al., 2016;Yaman et al., 2019). In this study, we evaluated the anti-cancer potential of JCo extract by investigating its effects on anti-proliferation, the cell cycle, and apoptosis in the human esophageal squamous cell carcinoma cell line CE81T/VGH. Medium supplemented with 10% fetal bovine serum (Gibco BRL), 10 mM HEPES (Gibco), 1 mM pyruvate (Gibco), P/S (100 μg/ml penicillin, and 100 μg/ml streptomycin; Gibco), and non-essential amino acids (Gibco, CE81T/VGH and CE48T/VGH only). Cells were grown in 10 cm 2 culture dishes in a humidified atmosphere with 5% CO 2 at 37°C. The TP53 in the CE81T/VGH cells was mutated, which was detected using automated extraction of nucleic acids (AccuBioMed Co., Ltd.) and Femtopath Human Primer Sets (HongJing Biotech). JCo was purchased from the PHOENIX company and extracted using steam distillation. The clinical drug 5-Fluorouracil (5-FU; Sigma) was prepared in dimethyl sulfoxide (DMSO) in each in vitro experiment. Cells were treated with equivalent amounts of DMSO in the control and treatment groups, and the final concentration of DMSO in each experiment was 0.01%-0.5%.

| Flow cytometric cell cycle analysis
The effect of JCo extract on the cell cycle distribution of CE81T/ VGH was determined by flow cytometry. Cells were seeded at a density of 2 × 10 6 per 100 mm culture dish, incubated overnight, and treated with 70 μg/ml JCo extract for 0, 6, 12, 24, or 48 hr for

| TUNEL assay
Apoptosis was detected using the Situ Cell Death Detection Kit (Roche, Mannheim, Germany). Cells were treated with 70 μg/ml JCo extract for 48 hr, collected, and fixed with 10% formaldehyde for 10 min. The cells were smeared and dried on silane-coated glass slides and rehydrated with PBS. After using 3% H 2 O 2 in methanol to inactivate endogenous peroxidase for 10 min, the cells were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate on ice for 2 min. Cells were incubated with TUNEL solution for 2 hr at 37°C and stained with PI. The TUNEL-positive cells showed green fluorescence, and cell images were taken using a fluorescence microscope (ZEISS AXioskop2) at 400× magnification.

| Western blot analysis
CE81T/VHG cells were seeded in 100 mm culture dishes for incubation overnight, and then treated with 70 μg/mL JCo extract for 0, 6, 12, 24, or 48 hr. Cells were lysed with ice-cold RIPA buffer containing protease inhibitor (BIO BASIC INC.) and phosphatase inhibitor (Bionovas) and incubated at 4°C for 30 min. The resulting lysates were centrifuged at 12,000 × g for 30 min at 4°C, and the protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA). Cell extracts (20 μg protein/lane) were separated using 8%-12.5% SDS-PAGE gel electrophoresis and transferred to polyvinylidene difluoride membranes (FluoroTrans, PALL). The membrane was blocked with 10% skim milk and incubated with primary antibodies or β-actin (iReal Biotechnology) in 1% Bovine serum albumin (in 1× TBS with 0.1% tween-20) overnight, followed by incubation with biotin conjugated secondary antibodies for 2 hr. Finally, membranes were incubated with horseradish peroxidase-conjugated streptavidin (Dianova) for 1 hr and proteins were detected using an ECL kit (T-Pro Biotechnology) and chemiluminescence imaging analyzer (GE LAS-4000, GE healthcare Life. Sciences). Protein bands were quantified using the software ImageJ 1.47t and normalized to the mean values of the untreated control and β-actin.

| Caspase activation analysis
CE81T/VGH cells were seeded at a density of 5 × 10 5 in 6 well culture plates and incubated overnight. After pretreatment with 1 μM Z-DEVD-FMK (Biosciences, USA) for 2 hr, cells were treated with 70 μg/ml JCo extract for 24 hr. Cells were harvested and the protein level of pro-caspase-3 were detected by western blotting.

| Immunocytochemistry staining
Cells were cultured in 100 mm culture dishes overnight and treated with JCo extract for 24 hr. The treated cells were collected, fixed with 4% paraformaldehyde, smeared, and dried on silane-coated glass slides. Endogenous peroxidase was inactivated via treatment with 3% H 2 O 2 for 10 min, and 10% BSA in PBS was used to block nonspecific binding. The target proteins were detected by immunostaining with anti-p-p53, anti-p-RB, anti-Caspase 3, anti-Caspase 8, and anti-Caspase 9 (1/200 dilution) and anti-p21 (iReal Biotechnology Co., Ltd.), and visualized with the HRP-DAB detection system (HK542-XAK, BioGenex).

| Statistical Analysis
The data are presented as the mean ± standard deviation (SD). Oneway ANOVA or Student's t-test were used to analyze the differences between each group, and p values <.05 were considered statistically significant. All experiments were repeated at least 3 times in duplicates or triplicates.
To explore the growth-inhibitory effect of JCo extract on esophageal cancer cells, cell viability was measured by an MTT assay. CE81T/ VGH and CE48T/VGH cells, treated with different concentrations of JCo extract or 5-FU, were significantly inhibited in a time-and dose-dependent manner (Figure 1a,b)   Values are mean ± SD (μg/ml). a A significant difference between the JCo group compared with the 5-FU group. The flow cytometry results showed the average proportion of the G 0 /G 1 phase after 1, 3, 6, 12, and 48 hr was 55.44 ± 1.06%, 61.21 ± 1.11%, 66.31 ± 0.47%, 61.56 ± 0.46% and 57.97 ± 1.00% after 70 μg/ml treatment, respectively (Figure 2a). For dosage analysis, the average proportion of the G 0 /G 1 phase after treatment with 0, 50, 70, and 90 μg/ml of JCo extract for 24 hr was 55.44 ± 1.06%, 68.96 ± 0.76%, 67.72 ± 0.26% and 65.22 ± 0.23%, respectively ( Figure 2b). Moreover, the percentage of cells in the subG 1 phase increased in a time-dependent and dose-dependent manner after JCo extract treatment (Figure 2a,b). The results suggest that JCo extract blocks CE81T/VGH cells in the G 0 /G 1 phase, triggers cell death, and increases the percentage of cells in the subG 1 phase.

| Effect of JCo extract on apoptosis detected by a TUNEL assay
To determine the effect of JCo extract on apoptosis induction, apoptotic morphology was observed via the TUNEL assay kit. Cells

| Anti-cancer molecular mechanism of JCo extract in CE81T/VGH
Next, to examine the molecular mechanisms of the cell cycle in JCo extract treated cells, the expression levels of cell cycle regulatory proteins, including p53, Rb, p21, CDK2, CDK4, cyclin A, cyclin B1, and cyclin D1 were assessed by western blotting. p53, p-p53, and p21 were increased, but Rb and p-Rb were decreased, indicating that the cell cycle was regulated by JCo extract treatment

| D ISCUSS I ON
Natural products from plants contain various components that are important in the development of new drugs for cancer, infection, and inflammation (Mann, 2002;Padumadasa et al., 2016;Rong et al., 2016;Soukand et al., 2015). Moreover, the therapeutic activity of natural products in traditional medicine and herbal medicine is attributed to functional group structures, especially polyphenolic compounds (Soobrattee et al., 2005). Juniperus communis (JCo) is a coniferous evergreen coniferous shrub that is widely spread throughout the Northern Hemisphere across the Himalayas from the Kumaun region (Khare, 2007;Moein et al., 2010;Nakanishi et al., 2004). Juniperus communis oil contains monoterpene hydrocarbons, such as α-pinene, myrcene, sabinene, and β-pinene (Bais et al., 2014;Cabral et al., 2012;Hajdari et al., 2015), most of which are polyphenolic compounds, indicating their anti-cancer potential.
Studies have shown that Juniperus communis L. berry water extracts induces p53-associated cell apoptosis in human SH-SY5Y neuroblastoma cells (Lantto et al., 2016), and increases the effect of 5-FU in non-small lung cancer A549 cells through regulation of AKT phosphorylation (Raasmaja et al., 2019). In addition, juniper berry oil from Juniperus communis is a chemo-preventive dietary agent that inhibits cell proliferation and inflammation and induces apoptosis, resulting in suppression of colon tumor formation in azoxymethane-treated rats (Yaman et al., 2019). In our previous study, JCo extract inhibited cell proliferation and induced cell cycle arrest and apoptosis in melanoma B16/F10 cells (Gao et al., 2019). In this study, we investigated the anti-cancer potential of JCo extract in ESCC cells. The results demonstrate that JCo extract reduces cell viability, induces cell cycle arrest at the G0/G 1 phase and cell apoptosis via regulation of relative protein expression, and has a synergistic effect when combined with

5-FU in CE81T/VGH cells in vitro.
Esophageal cancer is one of the most frequently diagnosed cancers and the sixth leading cause of cancer-related deaths worldwide, F I G U R E 4 Cell cycle and apoptosis relative protein expression in JCo extract treated cells. CE81T/VGH cells were treated with 70 μg/ml of JCo extract for 6, 12, 24, or 48 hr, and western blotting was used to detect altered expression of the cell cycle (a) and apoptosis (b) relative proteins. (c) Cells pretreated with Z-DEVD-FMK, a caspase 3/7 inhibitor, for 2 hr, and then treated with 70 μg/ml of JCo extract for 24 hr. Protein expression of Pro-Caspase 3 was detected by western blotting causing more than 400,000 deaths each year (Pennathur et al., 2013;Siegel et al., 2016). The overall 5-year survival rate of late-stage ESCC is less than 15%, and there has been little improvement in the past few decades, although therapeutic approaches have been developed (Liang et al., 2017). Presently, drugs from plants, regardless of whether they are crude extracts or isolated bioactive compounds, have received a lot of scientific attention in cancer therapy because of their higher selection for cancer cells, resulting in a lower possibility of side effects (Fulda, 2010;Wang et al., 2018). In this study, These results suggest that JCo extract shows lower cytotoxicity to normal cells compared with 5-FU, which reduces the possibility of causing side effects.
The cell cycle progresses from quiescence (G 0 phase) to proliferation (G 1 , S, G 2 , and M phases), and then returns to a quiescent state. It is well known that cell cycle arrest in response to DNA damage or cell stress is essential to maintaining genomic integrity. Cell cycle checkpoints play a crucial role in controlling the inhibition of cell cycle transitions or induction of signal transduction after cell stress (Flatt & Pietenpol, 2000). The analysis of cell cycle distribution revealed that ECSS cells treated with JCo extract were blocked in the G 0 /G 1 phase of the cell cycle, but there was an increase in the subG 1 population. These results indicate that JCo extract inhibits cell proliferation through G 0 /G 1 phase arrest in ESCC cells. Cyclin-dependent kinases (CDKs) and their activated cyclins play key roles in mammalian cell cycle regulation, and CDK/cyclin complex activity depends on the balance between cyclins and cyclin-dependent kinase inhibitors (CKIs).
In the cell cycle, it has been determined that CDK4/cyclin D and CDK2/cyclin E promote the passage of cells through the G 1 and S phases, while CDK1/cyclin B regulates the transition of cells through late G 2 and mitosis (Morgan, 1995). The phosphorylation of Rb is responsible for the transitions between G 0 /G 1 and G 1 /S (Taya, 1997). In this study, JCo extract upregulated p53, p-p53, and p21, and downregulated Rb, p-Rb, CDK2, CDK4, cyclin A, cyclin B1, and cyclin D1, suggesting that JCo extract treatment blocked the cell cycle via regulation of cell cycle relative proteins, especially in the G 0 /G 1 phase.
Apoptosis is a basic process that is crucial for the development and maintenance of cellular homeostasis within tissues. Apoptosis

5-fluorouracil (5-FU) was the first-line treatment for ESCC
therapy for several decades (Longley et al., 2003), but single agent chemotherapy of 5-FU is no longer appropriate for ESCC therapy because of its toxicity, drug resistance, and side effects. Therefore, the development of combination therapies with other drugs, such as irinotecan and oxaliplatin, is used to improve prognoses and reduce the side effects of 5-FU (Ferte et al., 2011;Wainberg et al., 2011).
Combination therapy enhances the response rate and survival rate,

ACK N OWLED G M ENTS
The present study was supported by grants from the Chung Shan Medical University Hospital Foundation, Taiwan, Republic of China

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.
F I G U R E 6 JCo extract enhanced 5-FU induced cytotoxicity in esophageal cancer cells. Cells were treated with a combination of (a) JCo extract (20, 40, or 80 μg/ml) with or without 0.5 μg/ml 5-FU; 5-FU (0, 0.625, 1.25, 2.5 μg/ml) combined with or without 30 μg/mL JCo extract for 48 hr, and cell viability was measured via an MTT assay. (b) The results were analyzed using CompuSyn to calculate the normalized isobologram

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author by reasonable request.