Juniperus indica Bertol Extract Combined with 5-Fluorouracil Synergistically Suppresses the Growth of Esophageal Squamous Cell Carcinoma Cells

Abstract

Background: Esophageal cancer is a prevalent malignancy of the digestive tract with a high incidence rate in Asia. Esophageal squamous cell carcinoma (ESCC) is the most common histological type, accounting for more than 90% of cases. JIB is a plant extract of Juniperus indica Bertol that has been shown to have anti-proliferation effects in nasopharyngeal carcinoma, oral cancer, and melanoma. Objectives: The aim of this study was to investigate the anti-cancer effects and underlying mechanisms of action of JIB in combination with 5-FU in ESCC cells. Methods: The effects of the combination of JIB and 5-FU on cytotoxicity, cell cycle arrest, apoptosis and drug resistance development were explored using CE81T/VGH human ESCC cells. Results: The results showed that JIB synergized the inhibitory effects of 5-FU on cell proliferation via downregulating the AKT/mTOR pathway in CE81T/VGH cells harboring the TP53 exon 8 R273C mutation. The combination treatment of JIB and 5-FU induced cell cycle arrest at G0/G1 and S phases via mediating the levels of CDK4/cyclin D1 and CDK2/cyclin A. Furthermore, JIB combined with 5-FU was more effective in inducing apoptosis by activation of extrinsic (Fas/caspase-8) and intrinsic (Bax/caspase-9) pathways than the use of a single drug. Additionally, JIB attenuated the development of resistance to 5-FU in long-term culture and exhibited enhanced cytotoxicity in bile acid environments. Conclusion: JIB combined with 5-FU synergistically inhibited the ESCC growth and triggered cell cycle arrest and apoptosis. These findings suggest that the combination of JIB and 5-FU may be a potential strategy for the treatment of esophageal cancer.

Keywords

esophageal squamous cell carcinoma 5-fluorouracil synergistic effect apoptosis

Introduction

Esophageal cancer is one of the most aggressive malignant cancers, with around 604 100 new cases and 544 000 deaths occurring globally every year.¹ In East Asia and sub-Saharan Africa, esophageal squamous cell carcinoma (ESCC) is the major histological subtype, accounting for more than 90% of all esophageal cancer cases.^2,3 Although surgical procedures, chemoradiotherapy, and chemoimmunotherapy regimens have improved, ESCC still exhibits different degrees of resistance to various chemotherapeutic drugs, with a 5-year overall survival rate of 20%-40%.^4–6 Furthermore, according to a recent epidemiological study, the number of patients with ESCC after middle and late treatment is very high, and with 5-year survival rate is less than 20%.^7,8 Consequently, there is an urgent need to develop novel strategies for ESCC treatment.

5-Fluorouracil (5-FU), a derivative of uracil, undergoes metabolic processes to convert into its active form and is then inserted into DNA or RNA within cells.⁹ This subsequently diminishes the function of thymidylate synthases, thereby impeding DNA synthesis. 5-FU is extensively used in clinical therapy for various types of cancers, including colorectal, gastric, breast, head, and neck malignancies.⁹ Previous studies have indicated adverse effects associated with 5-FU, including decreased appetite, nausea, vomiting, oral ulcers, hair loss, and inhibition of bone marrow function.¹⁰ Moreover, a meta-analysis revealed a frequency of 40.2% for 5-FU resistance in head and neck squamous cell carcinoma.¹¹ However, its clinical application against cancer is restricted by its adverse effects and drug resistance. Therefore, 5-FU is frequently used alongside other drugs, such as cisplatin, paclitaxel, docetaxel, and irinotecan, in cancer therapy. Nevertheless, the effectiveness of this combination treatment is limited, with response rates of only 30% to 40%.^12,13 Therefore, it is critical to identify other agents or adjuvants that can be combined with 5-FU to enhance the sensitivity of cancer cells, while decreasing their toxicity to normal cells.

Natural products are commonly derived from plants, animals, and microorganisms and exhibit a variety of pharmacological activities, including antioxidant, anti-inflammatory, antiviral, and antitumor properties.¹⁴ Recent research reveals that approximately 80% of individuals in developing countries rely on traditional medicines for their basic healthcare requirements, whereas over 60% of patients diagnosed with cancer choose plant-derived products instead of chemotherapy.¹⁵ A significant number of effective chemotherapeutic drugs for cancer treatment are derived from plants, including vincristine, camptothecin, and taxel, which possess various anti-cancer properties such as anti-proliferation, regulation of the cell cycle, induction of apoptosis, and anti-angiogenic effects.¹⁶ Importantly, plant-based ingredients have multiple targets and lower toxicity than conventional agents,¹⁷ and synergistically sensitize tumors to traditional chemotherapy and radiotherapy to enhance their therapeutic effectiveness.¹⁸ Plant-derived natural products with anti-cancer effects have received increasing attention in recent years.

Juniperus Indica Bertol, also referred to as the Black Juniper, belongs to the Cupressaceae family and is found in various countries, including Bhutan, China, India, Nepal, and Pakistan.¹⁹ Traditionally, Juniperus Indica Bertol extract (JIB) has been used in food flavoring as well as in herbal medicine treatment to alleviate symptoms such as headache, fever, cough, and cold.^20,21 Furthermore, JIB has been shown to possess antimicrobial properties and cytotoxic effects against SiHa (human cervical cancer), A549 (human lung carcinoma), and A431 (human skin carcinoma) cells.²² The combination of JIB and Cisplatin effectively suppressed the growth of oral cancer and melanoma cells by inducing cell cycle arrest and apoptosis.^23,24 However, there is a lack of knowledge regarding the effects of JIB combined with 5-FU on ESCC. The CE81T/VGH ESCC line derived from a Taiwanese patient in East Asia, a region with a high incidence rate of ESCC, was commonly used in ESCC studies.²⁵ In present study, we selected the CE81T/VGH cells and investigated the anti-cancer effects and mechanisms of action of JIB in combination with 5-FU. This study proposes that the combined use of JIB and 5-FU produces synergistic anti-cancer effects in and may potentially serve as a novel therapeutic approach for the treatment of ESCC.

Materials and Methods

Cell Culture and Reagents

CE81T/VGH and CE48T/VGH (human ESCC cell lines) were obtained from the Food Industry Research and Development Institute (Hsinchu City, Taiwan). Normal cells, including NIH/3T3 (mouse fibroblast cells), MDCK (canine kidney epithelial cells), and SVEC (mouse vascular endothelial cells) were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured in Dulbecco's modified Eagle's medium (Gibco) augmented with 10% fetal bovine serum (FBS; Gibco BRL, Gaithersburg, Maryland), HEPES (10 mM; Gibco), pyruvate (1 mM; Gibco), and penicillin/streptomycin solution (100 U/mL penicillin and 100 µg/mL streptomycin; Gibco) and MEM non-essential amino acid solution (Gibco), and maintained in a humidified 5% CO₂ atmosphere at 37 °C. JIB extract was prepared using the same method as previously described,²⁴ and the major components included δ-3-Carene (46.43%), δ-cadinene (8.73%), D-Limonene (8.31%), α-Pinene (7.92%) and β-Cubebene (5.11%), identified by GC-MS analysis. JIB and 5-FU (Sigma-Aldrich, St Louis, MO, USA) were dissolved in dimethyl sulfoxide (DMSO) and stored at 4 °C or −20 °C until use in each in vitro experiment, and the final concentration of DMSO was 0.5% in the medium.

TP53 Exon 8 Analysis

DNA was extracted using a LabPrepTM Genomic DNA Mini Kit (TAIGEN Bioscience Corporation, Taiwan) and quantified using a spectrophotometer (GeneQuantTM 1300; Biochrom, UK). A Femtopath TP53 Exon 8 Primer Set (Hong Jing Biotech, Taiwan) was used to amplify TP53 exon 8. DNA concentration was 100 ng/μl and the final volume was 20 μL in each sample. All procedures were performed according to the manufacturer's instructions. The PCR products were separated on 2% agarose gel in Tris-borate EDTA solution (TBE) buffer and photographed by AlphaImager HP System (ProteinSimple, California, USA). Subsequently, a band with a size range of 300 kb–400 kb was collected and sequenced by the Genomics Company (Taipei, Taiwan).

Cytotoxicity Assay

Cells were seeded in 96-well culture plate at the density of 5 × 10³ cells and incubated for 24 h. Cells were then treated with JIB (0-100 μg/mL) or 5-FU (0-10 μg/mL) for 24 h, 48 h or 72 h. Following this, the medium was replaced with 100 μL MTT solution (0.5 mg/mL; Sigma) and incubated for 4 h. After removing the supernatant, formazan crystals were dissolved in DMSO and the optical density (O.D.) was measured at 550 nm using a microplate reader (SpectraMax M5, Molecular Devices, USA). Cell viability (%) = O.D. (treated cells) / O.D. (control cells) × 100. The half-maximal inhibitory concentration (IC₅₀) values were calculated from the dose-response curves. Besides, selectivity index (SI) was defined as IC₅₀ of normal cells/IC₅₀ of ESCC cells.

To analyze the combined effects, CE81T/VGH cells (5 × 10³ cells/well) were cultured in 96-well plates and the procedure described above was used to estimate the viability of ESCC cells treated with JIB plus 5-FU. The experiment was divided into two parts, one was treatment with JIB (0-80 μg/mL) and/or 5-FU (0.5 μg/mL) for 48 h, and the other was treatment with 5-FU (0-10 μg/mL) and/or JIB (40 μg/mL) for 48 h. The cell viabilities of the treated cells were determined using the MTT assay, which was subsequently employed to calculate the IC₅₀ values. Combination index (CI) was calculated by following [IC₅₀ (JIB + 5-FU) / IC₅₀ (JIB)] + [IC₅₀ (5-FU + JIB) / IC₅₀ (5-FU)]. The combined effects of JIB and 5-FU were evaluated based on the CI, including synergism (CI < 1), additivity (CI = 1), and antagonism (CI > 1).²⁶

Western Blotting

The cells treated with 40 μg/mL JIB and/or 0.5 μg/mL 5-FU were harvested and lysed with RIPA buffer containing protease (Bio Basic Inc., Canada) and phosphatase inhibitors (Bionovas, Toronto, Canada). Proteins (20 μg) were separated by SDS-PAGE and subsequently transferred onto a PVDF membrane (PALL Corporation, USA). The membrane was blocked with 5% skimmed milk and incubated with primary antibodies, included anti-AKT, anti-p-AKT, anti-mTOR, anti-p-mTOR, anti-P70S6K, anti-p-P70S6K, anti-NFκB, anti-p-p53, anti-p-Rb, anti-CDK4, anti-Cyclin D1, anti-CDK2, anti-Cyclin A, anti-Fas, anti-caspase 8, anti-Bax, anti-caspase 9, anti-caspase 3, anti-PARP (Santa Cruz, CA, USA) and anti-β-actin (iReal Biotechnology Co., Ltd, Hsinchu, Taiwan) overnight at 4 °C. The next day, the membrane was incubated with biotin-conjugated secondary antibodies (Santa Cruz, CA, USA). This was followed by incubation with peroxidase-conjugated streptavidin (Jackson ImmunoResearch Inc., USA). Finally, the antibody-reactive proteins were incubated with enhanced chemiluminescence (ECL, T-Pro Biotechnology, Taiwan) and detected by ImageQuant LAS-4000 (GE Healthcare Life. Sciences, NJ, USA). The band intensity of the specific proteins was quantified using software ImageJ (National Institutes of Health, version 1.47t, Bethesda, MD) and normalized against β-actin signal.

Cell Cycle and subG1 Phase Analysis

CE81T/VGH cells were seeded in a 10 cm dish at a density of 2 × 10⁶ cells and incubated for 24 h. On achieving 60% confluence, they were treated with 40 μg/mL JIB and/or 0.5 μg/mL 5-FU for 0 h, 6 h, 12 h, 24 h or 48 h. After harvesting and washing with PBS, cells were incubated in solution containing RNase A (0.1 mg/mL, Sigma) and PI solution (40 μg/mL, Sigma) at 4 °C overnight. The distribution of cells in different phases, including the G0/G1, S, and G2/M phases, as well as the percentage of cells in the subG1 phase, were assessed using FACScan (Beckton Dickinson, USA) and FlowJo software (FlowJo, Treestar Inc., Ashland, OR, USA).

Caspase Activity

Caspase activity was detected using the Cell Meter Generic Fluorometric Caspase Activity Assay Kit (AAT Bioquest, Sunnyvale, CA, USA). CE81T/VGH cells were seeded in 10 cm dish at the density of 2 × 10⁶ cells, incubated for 24 h, and treated with JIB (40 μg/mL), 5-FU (0.5 μg/mL) or drug combination for 3 h, 6 h, 12 h and 24 h. Treated cells were harvested, reacted with 0.1% NP-40, and incubated with TF2-VAD-FMK solution for 2.5 h. Fluorescent signaling was detected using FACScan (Beckton Dickinson, USA).

TUNEL Assay

Apoptosis was determined using terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL kit, Roche, Germany). After 48 h of treatment with JIB (40 μg/mL), 5-FU (0.5 μg/mL) or JIB combined with 5-FU, the cells were collected to identify the apoptotic cells. The treated cells were fixed with 10% formaldehyde, permeabilized using 0.1% Triton X-100 in 0.1% sodium citrate on ice, and incubated with TUNEL solution for 2 h at 37 °C. Propidium iodide (PI, 10 μg/mL) was used to stain the nuclei. TUNEL-positive cells were observed and photographed using a fluorescence microscope (ZEISS AXioskop2, Carl Zeiss, Germany) at 400× magnification.

In Vitro Resistance Assay

Cells were seeded in 96-well culture plate at a density of 5 × 10³ per well and subjected to continuous treatment for 5 days and 10 days with JIB (40 μg/mL), 5-FU (0.5 or 1 μg/mL), or a combination of both drugs. Every three days throughout the experiment, the supernatants were replaced with a fresh medium containing the drugs. The treated cells were then photographed after being stained with 0.1% crystal violet for 10 min and washing with PBS. The absorbance was measured at 510 nm after dissolution in 10% acetic acid, and the percentage of cell survival was calculated. The survival rate of control (day 5) was assumed to be 100%.

Bile Acids Assay

Glycocholic acid, taurocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, deoxycholic acid, and hydrochloric acid (Sigma-Aldrich) were used to imitate acidic and bile acid-containing environments.²⁷ JIB (40 μg/mL) and 5-FU (0.5 μg/mL) were incubated for 1 h in different environments, including (1) pH7; (2) pH4; (3) pH7 + bile acids; (4) pH4 + bile acids, in incubator with 5% CO₂ at 37 °C. Cells were plated in 96-well culture plates and treated with the incubated drugs for 24 h, 48 h, and 72 h, and cell viability was measured using the MTT method as described above.

Statistical Analysis

The data were expressed as means ± standard deviation (SD). Statistical analysis was performed using SPSS v16.0 software or Excel 2016 software, and the differences between two or multiple groups were determined using unpaired Student's t-test or one-way analysis of variance (ANOVA), respectively. A P-value of less than .05 was set at be statistically significant. The experiments were conducted in duplicate or triplicate at least three times.

Results

TP53 Exon 8 R273C Mutation in CE81T/VGH Cells

Mutations in the TP53 gene are associated with cancer development. In particular, mutations in TP53 exon 8 cause tumors to become malignant, greatly reducing patient survival rates. Thus, we determined the status of TP53 exon 8 in CE48T/VGH and CE81T/VGH human ESCC cell lines. As shown in Figure 1A, the results revealed the presence of wild-type TP53 exon 8 in CE48T/VGH cells. In contrast, the signal for codon 273 of TP53 exon8 in CE81T/VGH cells was changed from CGT to TGT by approximately 47%, indicating that approximately half of the cell population harbored the TP53 exon 8 R273C mutation.

Figure 1.

The effects of JIB on cell viability of ESCC and normal cells. (A) The TP53 of CE81T/VGH and CE48T/VGH were amplified by Femtopath TP53 Exon 8 Primer Set and PCR, and then the products were sequenced. Approximately one-third population of the CE81T/VGH exhibited the R273C mutation of TP53. (B–F) CE48T/VGH, CE81T/VGH, NIH/3T3, MDCK and SVEC cells were treated with a serial dilution of JIB for 24 h, 48 h, and 72 h, and the cell viability was assessed using MTT assay. *P < .05, versus control (24 h). ^#P < .05, versus control (48 h). ^&P < .05, versus control (72 h).

JIB Inhibited Cell Proliferation of ESCC

To investigate the anti-proliferation effect of JIB on ESCC cells, we used the MTT assay to measure cell viability. The cells were treated with serial dilutions of JIB for 24 h, 48 h, or 72 h. The results showed that JIB inhibited the growth of both ECSS cell lines in a time- and dose-dependent manner (Figure 1B and C). However, the inhibitory effects of JIB on normal cells were lower than those on ESCC cells at the same dose (Figure 1D–F). In Table 1, the IC₅₀ values of JIB in ESCC cells (41.56-53.19 μg/mL) were lower than normal cells (76.56-84.64 μg/mL) at 48 h, indicating that JIB showed lower cytotoxicity in normal cells compared with ESCC cells. Moreover, selection index (SI) of JIB (1.30-2.00, 24 h; 1.44-2.04, 48 h; 1.37-2.24, 72 h) was higher than that of 5-FU (0.07-0.51, 24 h; 0.02-1.16, 48 h; 0.04-3.44, 72 h), indicating that JIB compared to 5-FU was more selective to ESCC instead of normal cells. The results demonstrated that the proliferation of ESCC cells, with or without TP53 exon8 mutation, was significantly inhibited by JIB.

Table 1.

The IC₅₀ Values of JIB at 24, 48, and 72 h on ESCC and Normal Cells.

Cell line		CE48T/VGH	CE81T/VGH	NIH/3T3	MDCK	SVEC
JIB	24 h	42.45 ± 0.68*	58.31 ± 4.08*	84.96 ± 4.00	81.33 ± 0.77	75.93 ± 1.11
	48 h	41.56 ± 1.21*	53.19 ± 3.09*	78.05 ± 1.79	84.64 ± 0.85	76.56 ± 1.50
	72 h	37.40 ± 3.59*	51.61 ± 5.35*	70.95 ± 2.40	83.96 ± 1.75	72.12 ± 3.25
5-FU	24 h	9.72 ± 0.63	> 10	0.65 ± 0.10	> 5	2.49 ± 0.26
	48 h	4.31 ± 0.48	7.48 ± 0.00	0.12 ± 0.01	> 5	0.57 ± 0.02
	72 h	2.05 ± 0.08	0.43 ± 0.001	0.09 ± 0.002	1.48 ± 1.20	0.30 ± 0.03

Note: The half-maximal inhibitory concentration (IC₅₀) values were calculated from dose–response curves of JIB or 5-FU at 24, 48 and 72 h and presented as means ± SD (μg/mL). *P < .05, versus normal cell groups.

JIB Synergized 5-FU to Suppressed the Growth of ESCC Cells

To determine the effects of the combination of JIB and 5-FU on ESCC growth, CE81T/VGH cells were treated with serial dilutions of JIB or 5-FU combined with IC₃₀ concentrations of 5-FU or JIB. The results showed that cell viability was significantly reduced after treatment with JIB (0-80 μg/mL) combined with 5-FU (0.5 μg/mL) for 48 h, compared to that observed in JIB treatment alone (Figure 2A). Furthermore, the combination of 5-FU (0-10 μg/mL) and JIB (40 μg/mL) reduced the viability of ESCC cells compared to 5-FU alone (Figure 2B). The combination index (CI) of JIB and 5-FU was 0.76, indicating a synergistic effect (CI < 1). Moreover, the AKT/mTOR signaling pathway was analyzed through western blotting to further explore the anti-ESCC mechanism induced by the combination of JIB and 5-FU. The levels and phosphorylation of AKT/mTOR/P70S6K decreased after treatment, especially in the combination group (Figure 2C and D). Additionally, the level of downstream transcription factor NF-κB also reduced. These findings indicated that the proliferation of CE81T/VGH cells was effectively inhibited by the combination of JIB and 5-FU through the regulation of the AKT/mTOR pathway.

Figure 2.

The effects of JIB combined with 5-FU on AKT/mTOR pathway. Cells were treated with (A) JIB (0-80 μg/mL) and/or 0.5 μg/mL 5-FU; (B) 5-FU (0-10 μg/mL) and/or 30 μg/mL JIB for 48 h, and detected the viability using MTT assay. (C) CE81T/VGH cells were subjected to treatment with JIB (40 μg/mL) and/or 5-FU (0.5 μg/mL) for 48 h, and collected and analyzed for proteins expression by western blotting. (D) The band intensity of proteins was quantified using the software ImageJ and then normalized to β-actin. Results were represented as the means ± SD of three independent experiments. *P < .05, versus control. ^#P < .05, versus JIB alone. ^&P < .05, versus 5-FU alone. Con, control.

JIB Combined with 5-FU Induced Cell Cycle Arrest

Flow cytometry was used to evaluate the cell cycle distribution after treatment with JIB and/or 5-FU. As shown in Figure 3A, the number of cells in the G0/G1 phase increased significantly after JIB treatment (68.21 ± 0.64%), 5-FU (39.35 ± 0.49%) and combination treatment (44.28 ± 1.29%) for 48 h compared with the control (35.33 ± 0.49%). Additionally, treatment with 5-FU (45.40 ± 0.63%) and combination (41.82 ± 1.40%) for 48 h increased the percentage of cells in S phase compared to that in the control (31.87 ± 0.21%). Western blot analysis revealed that treatment resulted in an upregulation of p-p53 protein expression while causing a downregulation of p-Rb expression, with the combined treatment group showing the most significant effect (Figure 3B and C). Moreover, JIB combined with 5-FU reduced the expression of CDK4/Cyclin D1 and CDK2/Cyclin A proteins. These results indicated that the combination of JIB and 5-FU induced cell cycle arrest at the G₀/G₁ or S phase via the downregulation of CDK4/Cyclin D1 and CDK2/Cyclin A, thereby inhibiting cell proliferation.

Figure 3.

The combination of JIB and 5-FU induced cell cycle arrest at G0/G1 and S phases. (A) CE81T/VGH cells were exposed to JIB (40 μg/mL), 5-FU (0.5 μg/mL) or a combination of two drugs for 0 h, 6 h, 12 h, 24 h, and 48 h. After labeling with PI overnight, DNA content of treated cells were evaluated by flow cytometry. The percentage of each phases were quantified using Flowjo software. (B, C) After treatment for 48 h, the levels of cell cycle-related proteins were measured and quantified by western blotting and software ImageJ. Data were represented as the means ± SD of three independent experiments. *P < .05, versus control. ^#P < .05, versus JIB alone. ^&P < .05, versus 5-FU alone. Con, control.

Combination of JIB and 5-FU Induced Cell Apoptosis

Caspase activation is considered an indicator of apoptosis. Therefore, we performed a caspase activity assay to identify drug-induced apoptosis. After 24 h incubation, the percentage of caspase activation increased from 100.00 ± 3.34%, 110.36 ± 4.49%, 153.01 ± 6.81%, and 195.15 ± 6.87% in JIB, 5-FU, and combination groups, respectively (Figure 4A). In comparison with the single drugs, the combination treatment demonstrated greater activation of caspases. Additionally, after 12 h of drug combination treatment, the number of cells in the Sub-G1 phase began to increase. (Figure 4B). At 48 h, the percentages of the sub-G1 phase sequentially were 6.01 ± 0.16%, 12.55 ± 0.76%, 15.39 ± 0.25% and 25.86 ± 1.46%. Furthermore, treated cells showed obvious positive results after treatment of 48 h, and the number of TUNEL-positive cells in JIB, 5-FU and combination groups increased from 2.15 ± 0.24% to 13.54 ± 1.56%, 17.68 ± 2.17% and 29.36 ± 1.82%, respectively (Figure 4C and D). The results of western blotting revealed that both extrinsic (Fas/caspase-8) and intrinsic (Bax/caspase-9) apoptosis proteins were activated, leading to the activation of caspase-3 and poly (ADP-ribose) polymerase (PARP) after treatment, particularly in the combination group. (Figure 4E and F). This finding indicated that the combination of JIB and 5-FU resulted in a more rapid and potent induction of apoptosis than a single drug treatment in ESCC cells.

Figure 4.

JIB combined with 5-FU triggered apoptosis through extrinsic and intrinsic pathway. (A) CE81 T/VGH cells were treated with JIB (40 μg/mL) and/or 5-FU (0.5 μg/mL) for 0 h, 3 h, 6 h, 12 h and 24 h, and activity of caspases was assessed using the Generic Fluorometric Caspase Activity Assay Kit. (B) The percentage of treated cells in Sub-G1 phase was detected by flow cytometry. (C, D) After treatment for 48 h, apoptotic cells were determined using TUNEL assay, and calculated as a percentage relative to the total cells (100%). (E, F) Western blotting analysis was performed to assess the expression of proteins related to apoptosis. β-actin served as the internal control. All data were shown as means ± SD of three independent experiments. *P < .05, versus control. ^#P < .05, versus JIB alone. ^&P < .05, versus 5-FU alone. Con, control. P-Cas, Pro-caspase.

JIB Inhibited the Re-Growth of 5-FU-Treated ESCC Cells

5-FU resistance was determined using an in vitro resistance assay. ESCC cells were treated with JIB, 5-FU, or a combination of the drugs and incubated for 5 days or 10 days for long-term treatment. In the results, 5-FU alone treatment (0.5 μg/mL and 1 μg/mL) effectively decreased cell viability at day 5, and the survival rate were decreased from 100 ± 1.96% to 42.99 ± 4.46% and 33.27 ± 3.44%, respectively (Figure 5). However, the re-growth (49.53 ± 10.58% and 57.57 ± 10.44%) of cells treated with 5-FU (0.5 μg/mL and 1 μg/mL) was observed at day 10, suppressed in combination with JIB (24.83 ± 4.23% and 12.82 ± 2.15%). These results suggested that JIB reduced the development of 5-FU resistance in ECSS cells, thereby enhancing its efficacy against cancer.

Figure 5.

JIB reduced re-growth of ESCC cells in long-term culture with 5-FU. The cells were exposed to JIB (40 μg/mL), 5-FU (0.5, 1 μg/mL), or two drugs combination for durations of 5 and 10 days. The drugs in the media were replaced every three days throughout the experiment. (A) The treated cells were stained with 0.1% crystal violet and then photographed. (B) After dissolution in 10% acetic acid, the absorbance was measured at 510 nm, and the percentage of surviving cells relative to control (day 5) was calculated. Data were presented as the means ± SD in three technical replicates. *P < .05, versus control (100%). ^#P < .05, versus JIB alone. ^&P < .05, versus 5-FU alone. ^$P < .05, versus day 5. N, no JIB treatment.

Anti-Cancer Activity of JIB in Bile Acids Environment

To determine the impact of the bile acid environment on JIB or 5-FU activity, cell viability was used as a parameter to evaluate the anti-cancer activities of drugs incubated in acidic and/or bile acid environments. Four groups were evaluated: (1) pH7, (2) pH4, (3) bile acids at pH7 and (4) bile acids at pH4. Cells were treated with the drugs for 24 h, 48 h, or 72 h, and cell viability was determined using the MTT assay. As shown in Figure 6, cell viability in the 5-FU group showed no significant change between the different environments at the same time. The JIB group showed higher cell viabilities at pH4 with and without bile acids, than at pH 7, indicating that the acidic environment affected the cytotoxicity of JIB. Furthermore, the viability of JIB-treated cells reduced in environments containing bile acids at pH7 (from 65.94% to 36.75% at 72 h) and pH4 (from 68.34% to 42.95% at 72 h) compared to that in environments without bile acids. These findings suggested that the anti-cancer properties of 5-FU were maintained in the bile acid environment; however, the presence of bile acids enhanced the anti-cancer activity of JIB in ESCC cells.

Figure 6.

The anti-proliferative activity of JIB in bile acids environment. The viability of the treated cells was used to determine the anti-cancer activity of JIB incubated in a bile acid environment. JIB (40 μg/mL) and 5-FU (0.5 μg/mL) were incubated in different environments, including (1) pH 7, (2) pH 4, (3) bile acids at pH 7 and (4) bile acids at pH 4, for 1 h and then treated CE81T/VGH cells for 24 h (A), 48 h (B) and 72 h (C). Cell viability was measured using MTT assay. Results were represented as the means ± SD of three independent experiments. *P < .05, versus control. ^#P < .05, versus pH 7. ^&P < .05, versus no bile acids.

Discussion

Plant-derived natural products have been extensively used for the treatment of various diseases, including cancer,¹⁴ and they exhibit lower toxicity than conventional drugs.¹⁷ Thus, there has been growing interest in recent years regarding plant-based anti-cancer products. Juniperus (family Cupressaceae) is a genus of evergreen aromatic shrubs and trees with approximately 75 species worldwide, of which three well-known species are Juniperus communis, Juniperus recurve, and Juniperus indica.²⁸ Juniperus Indica Bertol, also known as Black Juniper, has been reported to have antibacterial activity against gram-positive bacteria, cytotoxic activity against human cervical cancer, lung carcinoma, and skin carcinoma, and synergistic effects when combined with cisplatin.^22–24 In this current study, we demonstrated that Juniperus Indica Bertol extract (JIB) significantly inhibited the growth of human ESCC cells, possibly through the AKT/mTOR signaling pathways, and compared to 5-FU exhibited lower cytotoxicity to normal cells. It is also important to emphasize that the combination of JIB and 5-FU produced synergistic effects on the induction of cell cycle arrest and apoptosis and attenuated the development of 5-FU drug resistance in ESCC cells. It demonstrated that JIB has the potential to be a novel and effective agent or adjuvant for the treatment of ESCC.

TP53 is a common mutation in cancers that encodes the tumor suppressor protein p53 and can be detected early in the precancerous stage.²⁹ TP53 is closely associated with many cancers, including ovarian, colorectal, esophageal, and head and neck cancers.^30,31 Smoking, ultraviolet light, aflatoxins, vinyl chloride, and other factors have been shown to promote mutations in TP53 gene.³² Common mutations are missense mutations between codons 125 and 300, especially codon 273 in exon 8 of TP53, which contains the DNA-binding domain.^29,33 Patients with mutations in exon 8 of TP53 exhibit poorer OS than patients without mutations who have non-small-cell lung cancer and lung adenocarcinoma.³⁴ In ESCC, the mutation rate of TP53 is greater than 50%, and approximately one-third of TP53 mutations are found in exon 8.³⁵ Therefore, it is important to identify effective chemotherapeutics for ESCC with mutations in exon 8 of TP53. In the present study, it showed that presence of wild type TP53 exon 8 in CE48T/VGH cells and mutant type TP53 exon 8 R273C (approximately 47% mutation rate) in CE81T/VGH. Moreover, JIB suppressed the growth of both ESCC cell lines in a time- and dose-dependent manner. This finding demonstrates that JIB has inhibitory activity not only against ESCC cells with wild-type TP53 but also against ESCC cells with mutant TP53, suggesting that JIB has therapeutic potential in ESCC patients with different statuses of TP53 gene.

The PI3K/Akt/mTOR pathway is a highly complex regulatory network in the human body and is regulated by multiple signaling molecules, affecting the proliferation, metastasis, angiogenesis, and chemoradiotherapy sensitivity of ESCC.³⁶ A previous study showed that the Akt/mTOR signaling pathway was downregulated by JIB alone.²³ In present study, JIB synergistically inhibited the expression and phosphorylation of AKT/mTOR/P70S6K in ESCC cells treated with 5-FU. It indicated that the proliferation of CE81T/VGH cells was significantly suppressed by the synergistic application of JIB and 5-FU, mediated through the modulation of the AKT/mTOR pathway. Increasing evidences indicate that numerous plant extracts or mixtures exhibit the properties of chemoprevention and anti-cancer by disrupting the cell cycle and inducing apoptosis.^37,38 Cell cycle arrest is a critical regulatory mechanism that regulates cell proliferation and triggers apoptosis. The cell cycle comprises four sequential stages, G1, S, G2, and M, which are closely regulated by cyclins and cyclin-dependent kinases (CDKs).³⁹ In the present, the combined administration of JIB and 5-FU preserved their individual anti-cancer properties while inducing the cell cycle arrest in the G0/G1 or S phase by regulating the expression of p-p53, p-RB, CDK4/Cyclin D1 or CDK2/Cyclin A. Apart from cell cycle arrest, promoting apoptosis represents an appealing alternative strategy for the treatment of cancer. Apoptosis is a typical cytotoxic phenomenon induced by anti-cancer drugs and exhibits distinct morphological characteristics such as caspase activation, an increased number of cells in the subG1 phase, chromatin condensation, DNA fragmentation, and apoptotic bodies.⁴⁰ Treatment with the combination of JIB and 5-FU synergistically resulted in time-dependent activation of caspases, accumulation of cells in the subG1 phase, and an increase in TUNEL-positive cells compared to treatment with the drug alone. It is well known that apoptosis is triggered by extrinsic (death receptor) and intrinsic (mitochondrial disruption) pathways, in which the activation of caspase-8 and caspase-9 subsequently initiates the cleavage of caspase-3 and PARP, thereby initiating irreversible apoptosis.⁴¹ In this study, JIB synergistically increased the expression of Fas and Bax, resulting in the activation of caspase-8 and caspase-9 in cells exposed to 5-FU, respectively. Subsequently, the extrinsic and intrinsic signaling triggered the cleavage of caspase-3 and PARP and induced apoptosis. These results suggest that JIB synergized the effects of 5-FU on the induction of cell cycle arrest and death receptor- and mitochondrial-mediated apoptosis to suppress the growth of ESCC cells.

Among chemotherapy medications for ESCC, 5-fluorouracil (5-FU) is the first-line treatment option and is widely used to treat colorectal, gastrointestinal, and breast cancers.⁹ The actions of 5-FU are mediated by its three active metabolites, including fluorodeoxyuridine monophosphate (FdUMP), fluorodeoxyuridine triphosphate (FdUTP), and fluorouridine triphosphate (FUTP).⁹ Thymidylate synthase is inhibited by 5-FU, which disrupts DNA synthesis via its metabolite, FdUMP. Furthermore, FdUTP can affect cellular metabolism through multiple pathways, such as transcription, translation, and post-translational modifications, by incorporating into RNA. FUTP replaces uridine triphosphate (UTP) during RNA synthesis resulting in aberrant RNA processing. However, 5-FU has a low selection index because of its high toxicity to normal cells, and recurrent or metastatic ESCC often exhibits resistance to 5-FU-based chemotherapy.^12,13 Therefore, there is an urgent need to identify other drugs or adjuvants used in combination with 5-FU to improve the sensitivity of ESCC cells to 5-FU. Recent studies have shown that the simultaneous use of natural products and conventional chemotherapy and/or radiation therapy can enhance the sensitivity of tumors, improve the effectiveness of treatment, and reduce the required doses while minimizing toxicity to normal cells.¹⁸ JIB was more selective for ESCC cells than for normal cells and showed synergistic growth inhibition in combination with 5-FU, indicating that reduced doses of JIB or 5-FU required for equivalent inhibitory effects could improve cytotoxicity in normal cells. Furthermore, in the in vitro drug resistance assay, cells treated with 5-FU regrew on day 10 compared to day 5, suggesting that the treated cells developed resistance to 5-FU. However, JIB suppressed the growth of 5-FU-treated cells in the combination treatment. Previous studies have reported that upregulation of the CDKN2A gene can inhibit p53 expression and activate the PI3K/Akt pathway, thereby inducing tumor cells to develop resistance to 5-FU.⁴² Therefore, regulating the expression of p53 or PI3K/Akt pathway may help prevent or reverse 5-FU resistance. The combination treatment of JIB and 5-FU not only increased the expression of p-p53 but also reduced the levels and phosphorylation of AKT/mTOR/P70S6K. These findings indicate that JIB has a high potential to attenuate the development of drug resistance in 5-FU-based ESCC treatment.

Bile acids are produced from cholesterol in the liver and facilitate the absorption of dietary fatty acids and cholesterol in the gastrointestinal tract. During the 1940s, bile acids were initially considered as tumor promoters owing to the tumorigenic action of deoxycholic acid, a secondary bile acid.⁴³ In recent years, the functions of bile acids have been significantly broadened to include crucial roles as signaling molecules in the gastrointestinal tract and other organs.⁴⁴ Bile acids are involved in cancer cell proliferation, apoptosis, angiogenesis, invasion, metastasis, inflammation, and immune regulation.⁴⁵ It is widely acknowledged that ESCC is associated with tobacco smoking and alcohol consumption. In addition to damaging cellular DNA by decreasing metabolic activity and enhancing oxidation, alcohol also facilitates the penetration of carcinogens within tobacco smoke into the squamous epithelium.⁴⁶ From an alternative point of view, it reported that excessive alcohol consumption and tobacco smoking reduce the motility of the lower esophagus, slow the emptying of the stomach, and increase gastric secretion, resulting in gastroduodenal reflux, which may be associated with the development of ESCC.^47–49 Esophageal cancer cells are frequently exposed to high concentrations of bile acids. In this study, we assessed the activity of JIB in an environment rich in bile acids using a mixture of five bile salts that are common components of reflux in the lower esophagus.⁵⁰ In current study, the anti-cancer activity of JIB against ESCC cells increased in the presence of bile acids, but did not change in the 5-FU group. A possible reason for this phenomenon is that bile acids can promote drug absorption and act as lipophilic drug-solubilizing and permeation-modifying surfactants,⁵¹ improving the bioavailability of JIB components with poor aqueous solubility. Recent research has shown that bile acids are frequently employed as drug carriers to enhance drug absorption across biological membranes and applied for mixed micelle composition, bilosomes, and conjugates with a variety of drugs.⁵² Also, it was demonstrated that the application of bile acids improves the anti-cancer efficacy of natural compounds isolated from plants, such as dihydroartemisinin and camptothecin.⁵³ Together, the findings indicated that JIB effectively combats malignancies frequently exposed to a bile acid-rich environment, resulting in enhanced anti-cancer activity. For patients who undergo surgical resection of the tumor in combination with chemotherapy, JIB can be beneficial to achieve better therapeutic effects and reduce the risk of ESCC recurrence.

It was concluded that JIB combined with 5-FU synergistically inhibits the growth of ESCC potentially through induction of the cell cycle arrest and apoptosis. However, this study also has certain limitations because the conclusions were only confirmed at the cellular level using a single cell line. In future research, it is planned to validate the findings using different ESCC cell lines and further investigate the anti-cancer activity and toxicity in vivo using animal models.

Conclusion

This study demonstrated that the combination of JIB and 5-FU inhibited cell growth, induced cell cycle arrest, and triggered apoptosis more effectively than either JIB or 5-FU alone. Moreover, JIB attenuated the development of drug resistance to 5-FU and showed increased cytotoxicity in an environment rich in bile acids, which improved its therapeutic efficacy against ESCC. These findings suggest that JIB could potentially be used as an adjuvant in combination with 5-FU as a novel strategy for ESCC treatment. Nevertheless, large-scale clinical trials are needed in the future to observe the anti-cancer efficacy of combined treatment of JIB and 5-FU in ESCC patients.

Supplemental Material

sj-pptx-1-npx-10.1177_1934578X251324602 - Supplemental material for Juniperus indica Bertol Extract Combined with 5-Fluorouracil Synergistically Suppresses the Growth of Esophageal Squamous Cell Carcinoma Cells

Supplemental material, sj-pptx-1-npx-10.1177_1934578X251324602 for Juniperus indica Bertol Extract Combined with 5-Fluorouracil Synergistically Suppresses the Growth of Esophageal Squamous Cell Carcinoma Cells by Hong-Wei Gao, Kai-Fu Chang, Xiao-Fan Huang, Peng-Yun Hung, Ming-Chang Hsieh and Nu-Man Tsai in Natural Product Communications

Footnotes

Acknowledgements

Flow cytometry was performed in the Instrument Center of Chung Shan Medical University, which is supported by National Science Council, Ministry of Education and Chung Shan Medical University.

Authors Contributions

Conceptualization: H.-W.G., M.-C.H. and N.-M.T.; Data curation: M.-C.H., K.-F.C. and P.-Y.H.; Formal analysis: K.-F.C. and P.-Y.H.; Methodology: H.-W.G., X.-F.H.; Writing - original draft preparation: H.-W.G., K.-F.C. and X.-F.H.; Writing - review and editing: H.-W.G., M.-C.H. and N.-M.T.; Funding acquisition: M.-C.H. and N.-M.T.; Resources: N.-M.T.; Supervision: N.-M.T.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Approval

Ethical approval is not applicable to the article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Chung Shan Medical University Hospital, Ministry of Science and Technology, Taiwan, (grant number CSH-2023-C-051, MOST 111-2320-B-040-022).

ORCID iDs

Kai-Fu Chang

Nu-Man Tsai

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Supplemental Material

Supplemental material for this article is available online.

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