Evaluation of anticancer effects of Juniperus communis extract on hepatocellular carcinoma cells in vitro and in vivo

Abstract Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and accounts for the fourth leading cause of all cancer deaths. Scientific evidence has found that plant extracts seem to be a reliable choice due to their multitarget effects against HCC. Juniperus communis has been used for centuries in traditional medicine and its anticancer properties have been reported. As a result, the purpose of the study was to investigate the anticancer effect and mechanism of J. communis extract (JCo extract) on HCC in vitro and in vivo. In the present study, we found that JCo extract inhibited the growth of human HCC cells by inducing cell cycle arrest at the G0/G1 phase, extensive apoptosis and suppressing metastatic protein expressions in HCC cells. Moreover, the combinational treatment of JCo and VP-16 was found to enhance the anticancer effect, revealing that JCo extract might have the potential to be utilized as an adjuvant to promote HCC treatment. Furthermore, in vivo study, JCo extract significantly suppressed HCC tumor growth and extended the lifespan with no or low systemic and pathological toxicity. JCo extract significantly up-regulated the expression of pro-apoptotic proteins and tumor suppressor p53, suppressed VEGF/VEGFR autocrine signaling, down-regulated cell cycle regulatory proteins and MMP2/MMP9 proteins. Overall, our results provide a basis for exploiting JCo extract as a potential anticancer agent against HCC.


Introduction
Hepatocellular carcinoma (HCC) is the sixth most common cancer, making it a major health concern worldwide. Prevalent treatments for HCC include surgery, chemotherapy, radiation therapy, and targeted therapy [1]. Chemotherapy is usually used to promote the patients' survival. However, chemodrugs are often associated with toxicity and strong side effects that impair therapeutic efficacy, resulting in high death rates [2]. Consequently, the discovery of novel antitumor agents with better therapeutic efficacy and less toxicity has become an attractive avenue for preventing and treating HCC. Natural products from plants may be a safer choice because use alone or a combination of medicinal plants and conventional therapies has a beneficial effect on survival, immune regulation, and quality of life [3].
The recent trends in drug development focus on natural resources, mainly medicinal plant parts, including leaves, roots, bark, fruits, and seeds are used in herbal medicine. Dietary intake of specific chemical compounds can reduce inflammation, oxidization, and the risk of cancer [4]. In particular, some aliments can affect cancer cells via modified regulation of the cell cycle, cell death, angiogenesis, and metastasis [5,6]. The induction of cell cycle arrest and apoptosis is the most promising approach for curbing tumor cell survival and progression. Indeed, several bioactive components have been reported to target these processes in order to produce anticancer or chemopreventive effects [7].
Juniperus communis has been extensively used in traditional medicine. Extract deriving from different parts of the J. communis tree have been proven to have anti-inflammatory [8,9], antidiabetic [10][11][12], antioxidant [13,14], antimicrobial activities [15,16] as well as anticancer properties [17][18][19][20]. Currently, the antitumor properties of J. communis extract (JCo extract) and the underlying molecular mechanisms remain unknown in the case of HCC. In the present study, we utilized JCo extract to determine their effects and mechanisms of action in the growth inhibition of human HCC cells.

Preparation of J. communis plant extract
Extract of J. communis fruits which was obtained from Nepal was generated via steam distillation by PHOENIX (New Jersey, U.S.A.). After extraction, two layers were generated, one was an aqueous layer and another was an oil layer also called essential oil which we used throughout experiments. The JCo extract was preserved at 4 • C in a brown glass bottle and sealed with parafilm to save the extract from moisture and light. The JCo extract was freshly dissolved in dimethyl sulfoxide (DMSO) to be used for the experiments. The drug concentration was calculated using the following formula: JCo extract (μg)/volume (ml).

TUNEL assay
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was performed using the TUNEL in situ cell death detection kit (Sigma, Missouri, MO) according to the manufacturer's instructions. Cells were fixed with 10% formalin and exposed to 0.01% Triton X-100 for 2 min on ice. After that, cells were incubated with TUNEL reaction buffer. The nuclei of the cells were counterstained with PI (10 μg/ml) and examined under a fluorescence microscope. For tissue TUNEL staining, the slides were rehydrated, incubated with H 2 O 2 , and blocked with 10% bovine serum albumin (BSA, Sigma, Missouri, MO, U.S.A.). After blocking, the slides were incubated with TUNEL reaction buffer and PI (1 μg/ml) for 30 min. The number of TUNEL-positive cells was quantified upon observation of ten random fields under 200× magnification and analyzed via Photoshop (Adobe.Photoshop.CS3.Extended v10.0).

Animal study
HepG 2 cancer cells (1 × 10 6 ) were injected subcutaneously into the right flank of female BALB/c nude mice, which were purchased from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan). The experiment was performed at Laboratory Animal Center of Chung Shan Medical University (CSMU) and the procedure was approved by the Institutional Animal Care and Use Committee (IACUC) of CSMU (approval number: CSMU-IACUC-1662). After 5 days from the injection, the mice were randomly divided into two groups (five mice/group). Both groups were treated every 2 days with a vehicle (mineral oil, 100 μg/ml) by subcutaneous (s.c.) administration, while only the experimental group was also treated with JCo extract (200 mg/kg). After tumor size reached approx. 1500 mm 3 (length × width × height), the mice were killed by carbon dioxide asphyxiation, and the collected tumors and organs were fixed in 10% formalin, embedded, and stained with Hematoxylin and immunohistochemical staining was performed. The quantitative score was calculated upon observation of ten random fields under 200× magnification and the pictures were analyzed using Photoshop (Adobe.Photoshop.CS3.Extended v10.0). The methods of staining and scoring were operated according to a previously published protocol [23].

Statistical analysis
All results are expressed as the mean + − standard deviation (SD). Statistically significant differences between groups were determined via Student's t test or one-way ANOVA. A P-value <0.05 was considered as the threshold for statistical significance.

JCo extract significantly inhibited the growth of HCC cells
To assess the anticancer properties of JCo extract, their inhibitory effects on the growth of HCC cells were first determined by MTT assay. HCC cells were treated with various concentrations of JCo extract (0-100 μg/ml) for different time spans. As shown in Figure 1, treatments with JCo extract caused dose-dependent growth inhibition of HCC cells. To determine the toxicity of JCo extract on normal cells, their growth inhibitory effects on kidney epithelial and vascular endothelial cells were determined by MTT assay. As shown in Table 1, treatments with JCo extract did not lead to significant growth inhibition in either of the two normal cell lines. Furthermore, the inhibitory effects of VP-16, a clinical drug, were also tested. The results showed that VP-16 had greater inhibitory effects on normal cells than JCo extract (Table 1). These results indicated that the JCo extract strongly inhibited the growth of HCC cells and had higher selection against tumor cells than normal cells.

JCo extract enhanced the growth inhibition of VP-16 in HepG 2 cells
To determine the effect of combined treatment with JCo extract and VP-16, the cell viability was determined by MTT assay for both drugs alone and their combination, and a CI was calculated. HepG 2 cells were seeded in 96-well plates  treated with one drug alone (JCo extract or VP-16) and a drug combination for 48 and 72 h, and the rate of growth inhibition was compared between each drug alone and the drug combination. As shown in Figure 2, 0.625-10 μg/ml of VP-16 plus 30 μg/ml of JCo extract significantly reduced the cell viability, ranged from 54.39 + − 3.29 to 33.86 + − 1.52% and 20-60 μg/ml of JCo extract plus 1.25 μg/ml of VP-16 statistically decreased cell viability, ranged from 42.98 + − 2.16 to 18.24 + − 0.33%. The results revealed that combined VP-16/JCo extract treatment showed stronger inhibitory effects at 48 and 72 h than VP-16 or JCo extract alone. After that, the CI was less than 1 while JCo extract (≤40 μg/ml) combined with VP-16 (≤2.5 μg/ml), suggesting that the JCo extract and VP-16 acted synergistically in cell growth inhibition and the combination formulas reduced the concentration of VP-16 and JCo extract. It indicated that the combination of JCo extract and VP-16 not only enhanced the inhibitory effect of VP-16 but also might be reduced side effects potentially.

JCo extract induced cell cycle arrest and apoptosis, and reduced expression of proteins involved in tumor autocrine signaling
The cell cycle can be divided into G-G 1 , S and G 2 -M stages, which are considered to be of great significance for controlling tumor growth. Next, to investigate the molecular mechanisms by which JCo extract inhibited the growth of HepG 2 cells, cell cycle analysis was performed. HepG 2 cells were treated with JCo extract at 50 μg/ml for the indicated time. As shown in Figure 3A, drug treatment increased the percentage of cells in G 0 /G 1 phase while reducing the percentage of cells in the S-phase as compared with mock treatment. In addition, the cell population in the sub-G 1 phase significantly increased upon drug treatment in a time-dependent manner ( Figure 3B). Apoptosis plays a vital role in eliminating mutant or excessively proliferating cells in the system to balance the natural environment and interfere with tumor growth. Therefore, it is considered to be a protective mechanism to prevent tumor progression. Next, to assess changes in the apoptotic morphology of cells treated with JCo extract, a TUNEL assay was performed.
The results showed that JCo extract induced apoptosis in HepG 2 cells ( Figure 3C). The results suggested that JCo extract induced cell cycle arrest at G 0 /G 1 phase and cell apoptosis in HCC cells. To further understand the specific mechanism of JCo extract in causing cell cycle arresting in HepG 2 cells, the expression of cell cycle-associated proteins was evaluated using Western blotting. After treatment with JCo extract, the levels of p-p53 increased along with p21, while the level of p-Rb was reduced; besides, the levels of cell cycle regulators, including CDK2, CDK4, cyclin A, cyclin B1, and cyclin D1 decreased in a time-dependent fashion ( Figure  4A). In order to further clarify the molecular mechanisms underlying the induction of apoptosis in HepG 2 cells by JCo extract, the total protein extract was collected and analyzed by Western blot. The levels of FAS, FASL, and procaspase 8 were reduced after treatment with JCo extract. Moreover, the levels of BAX and AIF increased while those of BCL2 and procaspase 9 decreased, and were accompanied by a reduction in procaspase 3 levels and increment of cleaved caspase-3 levels ( Figure 4B). It indicated that JCo extract activated extrinsic and intrinsic caspase cascades, leading to cleavage of caspase-3 and the induction of cell apoptosis. Furthermore, the effects of JCo extract on the modulation of autocrine signaling were also examined by Western blot. The results showed that JCo extract inhibited the expression of VEGFA, VEGFR1 and VEGFR2, suggesting that JCo extract inhibited autocrine signaling ( Figure  4C). Taken together, our results demonstrated that JCo extract induced cell cycle arrest, apoptosis, and inhibited tumor autocrine signaling, resulting in growth inhibition of HCC cells.

JCo extract suppressed tumor growth of HCC in vivo
The above experiments demonstrated that JCo extract inhibited the proliferation of HCC cells and promoted apoptosis in vitro. Based on these findings, we studied the effect of JCo extract on tumor growth in BALB/c (nu/nu) nude mice injected s.c. with HepG 2 cells. The experimental group was treated with JCo extract every 2 days, while the control group was treated only with a mock vehicle until tumor size reached approximately 1500 mm 3 . Mice were then killed, and collected tumors and organs were subjected to histological analysis. Treatment with JCo extract suppressed HCC tumor growth ( Figure 5A) and significantly prolonged the lifespan of treated mice (51 days) as compared with control mice (37 days) ( Figure 5B). To verify the antitumor properties of JCo extract in vivo, the effects of JCo extract on apoptosis and proliferation of mice tumor cells were elucidated. Tumor mass staining via TUNEL revealed a great number of apoptotic cells in organs of mice treated with JCo extract (51 + − 15.23%), indicating that JCo extract led to apoptosis of tumor cells ( Figure 6A). Tumor morphology was observed after H&E staining and showed that JCo extract caused the death of tumor tissue accompanied by increased accumulation of cleaved caspase 3 protein ( Figure 6B). Moreover, the levels of PCNA, VEGFR1 and VEGFR2 were reduced, indicating that JCo extract inhibited tumor proliferation and autocrine signaling ( Figure 6C). These results demonstrated that JCo extract suppressed tumor growth in vivo via inhibition of proliferation and induction of apoptosis, consistent with in vitro results.

JCo extract reduced the expression of angiogenesis and metastasis-related proteins in vivo
To examine the effect of JCo extract on angiogenesis and metastasis, the levels of VEGFA, MMP2 and MMP9 in the tumor tissue of treated mice were evaluated by IHC staining. The results showed that JCo extract not only inhibited VEGFA expression but also reduced vessel numbers ( Figure 7A). On the other hand, JCo extract possessed antimetastatic potential since it reduced MMP2 and MMP9 expression ( Figure 7B).

The toxicity of JCo extract in vivo
To evaluate the tolerance to the toxicity of JCo extract in an HCC established animal model, we measured body weight and evaluated pathological toxicity in control mice as well as mice treated with JCo extract. JCo extract did not significantly affect the body weight of mice ( Figure 8A). Besides, JCo extract also exhibited low or no pathological toxicity in vivo ( Figure 8B). These results suggested that JCo extract showed low or no short-term acute toxicity or long-term accumulative toxicity in vivo, suggesting the therapeutic course of JCo extract was well-tolerated.

Discussion
HCC is the sixth most common type of cancer in the world and the third most common cause of cancer-related deaths worldwide [1]. Systemic chemotherapy, such as sorafenib and lenvatinib, is used to treat patients with advanced HCC related to vascular invasion and metastasis. However, the median survival time of sorafenib treatment is 12.3 months, and the median survival time of levatinib treatment is 13.6 months [24], suggesting the long-term survival rate is still not satisfactory. Therefore, novel treatment strategies for HCC are required to increase the survival rates of patients. Natural products from plants may be a safer choice and have a beneficial effect on survival, immune regulation and quality of life [3]. In this study, we demonstrated the anticancer properties of JCo extract in vitro. JCo extract inhibited the growth of HCC cells in a dose-dependent manner. Also, as compared with the clinical drug VP-16, JCo extract exhibited lower cytotoxicity in normal cells, suggesting that JCo extract might induce fewer side effects after treatment. Moreover, JCo extract not only exerted inhibitory effects on HCC cells but also showed an enhancement of inhibition in combination with VP-16 in vitro, suggesting the potential application of JCo extract as adjuvant agents. Also, our results showed that the anticancer capacity of JCo extract was determined by the induction of cell cycle arrest via the activation of the p53/p21 pathway [25,26], repressed expression of cell cycle regulators and the activation of intrinsic and extrinsic apoptosis pathways via the caspase cascade [27][28][29]. On the other hand, we  also found that JCo extract inhibited the protein expressions of VEGF, VEGFR1 and VEGFR2, which might block the autocrine or paracrine signaling of HCC [30]. Thus, the extracts of J. communis plant exerted antiproliferative effects through the induction of cell cycle arresting and apoptosis in HepG 2 cells might serve as a potential anticancer agent for HCC.
Because it is difficult to cure HCC, prevention medicine might be an effective way to reduce cancer mortality. In our preliminary animal study, JCo extract was given a subcutaneous injection to inhibit HCC tumor growth and the results had shown that subcutaneous injection revealed the best therapeutic effect than oral gavage and intravenous injection. Moreover, JCo extract contains small molecules and lipophilic property compounds that might be slowly released into blood through subcutaneous tissue absorption and maintained drug concentration in the blood to continuously suppress tumor growth. Besides, our previous study showed that JCo extract exhibited well-suppressive effects on the growth of melanoma by subcutaneous treatment in C57/BL6 mice [23]. So, in the present study, we have chosen the subcutaneous injection to administrate JCo extract in vivo. The data showed that JCo extract suppressed tumor growth in vivo, improved the survival rate, and prolonged the lifespan of tumor-bearing mice, suggesting that JCo extract might be exploited as chemopreventive agents for preventing HCC progression. To get insights into the anticancer mechanisms in vivo, we examined tumor morphology after treatment with JCo extract, and we found that JCo extract induced apoptosis in tumor cells. Moreover, it has been found that HCC cells can express VEGF and VEGFR to trigger cell proliferation, survival, migration and invasion; besides, VEGF/VEGFR signaling can affect the prognosis and average survival time of patients [31,32]. Therefore, VEGF/VEGFR signaling can be one of a target for HCC treatment. In the results, JCo extract suppressed VEGF/VEGFR protein expressions in vivo that was consistent with the data found in vitro, suggesting JCo extract might reduce the autocrine and paracrine signaling pathways. Moreover, JCo extract reduced the vessel formation and suppressed MMP2/MMP9 protein expression in Figure 8. Analysis of pathological toxicity after treatment with JCo extract (A) HepG 2 cells were subcutaneously injected into the right flank of female nude mice (n=3). Mice were divided into an experimental and a control group (n=5), and both groups were subcutaneously treated with mineral oil (100 μg/ml), while only the experimental group also received JCo extract (200 mg/kg). Body weights were determined every 2 days. (B) After the killing of mice, organs were collected, fixed with 10% formalin, and stained with H&E to observe the tissue morphology under 400× magnification.
vivo, revealing that JCo extract might exert antimetastatic potential on HCC. Next, we also evaluated the toxicity of JCo extract in vivo by body weight and pathological analysis, and the results showed no significant difference in body weight after treatment with JCo extract. After pathological analysis, organ tissue showed no obvious damage after administration of JCo extract. As a result, JCo extract might display low or no toxicity to organs, suggesting JCo extract exhibited antihepatoma properties with well-tolerance.
In conclusion, JCo extract exhibited anticancer activity with growth inhibition, repression of autocrine and paracrine signaling, and induction of cell apoptosis in vitro and in vivo. In light of the above safe and effective antitumor effects, JCo extract may represent a potential agent for the treatment of HCC patients.

Data Availability
All data generated or analyzed during the present study are included in this article.