Cell culture and Reagents
Human lung cancer cell lines, NCI-H446 (SCLC) and NCI-H661 (NSCLC), were purchased from Keygen Biotechnology Company (Nanjing, China). Cells were cultured in RPMI 1640 (Hyclone, LA, USA) medium containing 10% fetal bovine serum (Hyclone, LA, USA) and antibiotics (penicillin and streptomycin). Cells were maintained in an incubator with 5% CO2 at 37 °C and saturated humidity. Hoechst 33258 fluorescent dye and Mito-tracker red probe were purchased from Sigma (St. Louis, MO, USA). CellTiter 96 aqueous non-radioactive cell proliferation assay (MTS) cell activity test kit was purchased from Promega (WI, USA). Annexin V-PI apoptosis detection kit was purchased from BD Biosciences (San Diego, CA, USA). Anti-Bak, anti-Bax, anti-Bcl-2, anti-Survivin, anti-Cytochrome C, anti-cleaved Caspase 3 polyclonal primary antibodies, anti-β-actin monoclonal antibody, and anti-rabbit/mouse horseradish peroxidase (HRP) conjugated secondary antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). ABT-263 was purchased from MedChemExpress (NJ, USA).
EEFS Preparation
Fennel dry seeds (250 g) were washed quickly with sterile distilled water once to remove dust and impurities. Then, they were soaked in 75% ethanol at 4 °C for 48 h and the leach solution was obtained by filter paper filtration. The leach solution was transferred to a rotary evaporator and evaporated and concentrated at a low temperature and reduced pressure to obtain a dark green extract. The extract (100 mg) was re-dissolved in 75% ethanol and sterilized with a 0.2 µm needle filter to produce 50 mg/ml EEFS, which was aliquoted into equal parts and stored at 4 °C, without additional substances added. In this study, except for the special statement, all the control groups contained 75% alcohol as the vehicle group.
Hoechst Staining
The cytotoxicity of EEFS was determined by observing changes in nuclear morphology (chromatin condensation or DNA fragmentation). NCI-H446 and NCI-H661 cells were seeded in 6-well plates at a density of 5 × 105 cells/well, and cells in logarithmic growth were treated with EEFS (0–0.8 mg/ml for NCI-H446 cells, 0–1 mg/ml for NCI-H661 cells) for 48 h. Following treatment, Hoechst 33258 (1 µg/ml) was directly added to the culture medium and incubated in the dark for 15 min at room temperature. To measure apoptosis, the morphological changes of the nucleus were observed via inverted fluorescence microscope (Nikon, Japan); six fields were randomly selected and photographed.
Cell Viability Test
Relative cell viability was evaluated by MTS assay. NCI-H446 and NCI-H661 cells were seeded in 96-well plates (5 × 103 cells/well), and when cell density reached 60%, cells were treated with different concentrations of EEFS (0, 0.1, 0.2, 0.4, and 0.8 mg/ml) for 48 h or the same concentration (NCI-H446 cells: 0.8 mg/ml and NCI-H661 cells: 1 mg/ml were set up as effective dose) for different time periods (12, 24, and 48 h). Following treatment, the relative viability (to vehicle control group) of the cells was detected according to the kit manufacturer’s instructions. The relative cell viability was calculated using the following formula: relative cell viability (%) = (1 – (ODcontrol - ODtreated)/ ODcontrol) × 100% [16].
Detection Of Apoptosis By Flow Cytometry
Apoptosis was assessed via the detection of phosphatidylserine (PS) exposure and the binding of Annexin V to the surface of the cell membrane, as well as PI nuclear dye exclusion. NCI-H446 and NCI-H661 cells were grown in 6-well plates at a density of 5 × 105 cells/well. When the cells were in the logarithmic growth stage, they were treated with different concentrations of EEFS (0–0.8 mg/ml for NCI-H446 cells, 0–1 mg/ml for NCI-H661 cells) for 48 h or 0.8 mg/ml for 12, 24, or 48 h. After treatment, the cells were collected and stained according to the instructions of the Annexin V-PI apoptosis detection kit. A FACSCalibur (BD Biosciences, San Diego, CA, USA) was used to detect apoptosis. Cell quest software (BD Biosciences, San Jose, CA, USA) was used to analyze the data and calculate the apoptotic ratio.
Western Blotting
Western blotting was performed in NCI-H446 and NCI-H661 cells as well as tumor tissues from xenograft mice to examine protein levels, based on a previously published method with minor modifications [17]. Briefly, after treatment with EEFS, ABT-263 or EEFS + ABT-263 under various conditions, NCI-H446 and NCI-H661 cells were collected and washed in PBS and then re-suspended in lysis buffer with 1% NP-40 containing protease inhibitor cocktail. The tumor tissues were homogenized in an ice bath. After the tumor tissues or cell cultures were fully lysed, they were centrifuged at 12,000 rpm and 4 °C for 10 min. The supernatants were transferred to new EP tubes and the protein concentration was measured using the Bradford method. After sample preparation, an equal amount of protein (20 µg) was loaded onto polyacrylamide gels for electrophoresis. Following electrophoresis, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Stafford, VA, USA) and blocked at room temperature with Tris-Buffered Saline Tween-20 (TBST) containing 5% skim milk for 30 min. Then, the membrane was incubated overnight at 4 °C with bovine serum albumin (BSA) (1:1000) containing a specific first antibody. After washing the membrane 3 times (10 min/time) with PBST, the membrane was incubated with the corresponding second antibody (1:5000) at room temperature for 2 h. In the darkroom, an enhanced chemiluminescence (ECL) luminescent solution (Millipore, Billerica, MA, USA) was used to display the immunoreactive strip, which was further exposed on film.
Mitochondrial Toxicity Test
Mitochondrial toxicity following EEFS treatment was evaluated by membrane potential reduction and Cytochrome C release. Briefly, NCI-H446 cells were seeded in 6-well plates and once in logarithmic growth, the cells were treated with 0.8 mg/ml EEFS for 6, 12, and 24 h. Following treatment, Mito-tracker red probe (500 nM) was directly added into the culture medium and incubated at 37 °C in the dark for 15 min. The changes of mitochondrial membrane potential were observed and evaluated under a fluorescent microscope (Nikon, Japan). In addition, mitochondria were isolated and purified according to the protocol provided by the mitochondrial extract kit (Solarbio Science & technology, Beijing, China). Western blotting was performed to examine Cytochrome C release from mitochondria.
Wound Healing Test
Wound healing (closure) tests were performed to assess the effects of EEFS on lung cancer cell migration. Briefly, NCI-H446 and NCI-H661 cells were seeded in 6-well plates. After scratching with a pipette tip, the floating cells were washed off with PBS and serum-free medium was added. Then, the cells were treated with EEFS (0, 0.1, 0.2, and 0.4 mg/ml for NCI-446 cells and 0, 0.125, 0.25, and 0.5 mg/ml for NCI-H661 cells) for 48 h. Cells were observed under a microscope and images of the scratches were taken at 0 h and 48 h after scratching. The wound closure width was measured by Image J software to calculate the difference in cell migration rate. Wound area was standardized using the following formula: wound closure rate (%) = ((initial wound area - wound area after 48 h ) / initial wound area) × 100% [18].
Clonogenic Assays
The inhibitory effect of EEFS on the tumorigenicity of lung cancer cells was examined by clonogenic assay (colony formation test). NCI-H446 cells were inoculated into 6-well plates at a density of 1 × 103 cells/dish and treated with 0.1, 0.2, and 0.4 mg/ml EEFS for 48 h. Then, the cells were rinsed with PBS and new culture medium was added. After 14 days, culture medium was removed, and cells were rinsed with PBS. Cells were fixed with 4% paraformaldehyde and stained with 0.4 g/L crystal violet at room temperature for 15 min. Photos were taken and the colonies were counted.
Tumor Growth Inhibition Test
All animal experiments were conducted with a protocol approved by the animal care and use Committee of Shengjing Hospital of China Medical University. Male BALB/c-nu/nu nude mice, weighing between 14–16 g and aged from 4 to 6 weeks were reared in SPF (specific pathogen free) conditions with a 12 h light/12 h dark cycle and room temperature of 24–26 °C with a relative humidity of 50–60%. Nude mice were given standard feed and sterile drinking water. The mice were implanted with 5 × 106 NCI-H446 cells in 0.2 ml PBS in the armpit of the right forelimb. Tumor volume was assessed by measuring the length (L) and width (W) with Vernier calipers every other day. Tumor volume was calculated according to the following formula: tumor volume (mm3) = 0.52 × L × W2. When the tumor volume reached 100 mm3, the mice were randomly divided into two groups (n = 4). In the treatment group, 0.1 ml EEFS (200 mg/kg) was dissolved with 0.1% sodium carboxymethylcellulose (CMC-Na) and given by gavage daily for 26 days, the control group was gavaged with CMC-Na. At the end of the experiment, the animals were sacrificed by intraperitoneal injection of 150 mg/kg pentobarbital sodium. After photographing the mice, the tumors were excised and photographed. The tumors were weighed to calculate the average tumor mass. The tumor tissues were stored at − 80 °C for further analysis.
Statistical analysis
Graphpad prism 8.0 (San Diego, CA, USA) was used to analyze data. Student's t test was used to compare means between two groups, and one-way ANOVA followed by Dunnett’s test was used to compare means between multiple groups. A p value less than 0.05 was considered statistically significant.