d-Borneol enhances cisplatin sensitivity via p21/p27-mediated S-phase arrest and cell apoptosis in non-small cell lung cancer cells and a murine xenograft model

Cell lines and reagents

Human lung cancer A549 (CAT: FH0045) (Additional file 1) and H460 (CAT: FH0050) (Additional file 2) cells were kindly provided by Dr. Cao (Chengdu University of Traditional Chinese Medicine, Chengdu, China), and H460/CDDP cells were purchased from FuHeng Biology (CAT: FH1207, FuHeng Cell Center, Shanghai, China). At 37 °C in a humidified environment with 5% CO2, A549, NCI-H460, and H460/CDDP cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, Australia) with 10% fetal bovine serum (FBS; Gibco, Australia) and 1% penicillin/streptomycin solution (Gibco, Australia) added. CDDP was obtained from Sigma-Aldrich (St. Quentin Fallavier, France). d-Borneol (#T5734) with purity (99.6%) was purchased from Shanghai Topscience Co., Ltd. DMSO (D8371) was purchased from Solarbio Science & Technology Co., Ltd. MTT reagent was purchased from BioFroxx. RT EasyTM I and Real Time PCR Easy SYBR Green One-Step Kit were purchased from Foregene Co., Ltd. Anti-Bax (T40051), Bcl-2 (T40056), and caspase-3 (M005851) were purchased from Abmart (Shanghai, China). Anti-cyclinA2 (ab181591), CDK2 (ab32147), and p27 (ab32034) were purchased from Abcam (Paris, France). Anti-p21 (AF6290), cyclinD3 (AF6251), CDK6 (DF6448), P-gp (AF5185) and tubulin (AF7011) were obtained from Affinity Biosciences.

Determination of borneol type and cell line

The cytotoxicity of borneol on H460 and A549 cells was determined by MTT. Cells were seeded at a density of 5 × 103 cells per well in 96-well plates and incubated for 24 h or 48 h. Then, cells were treated with different doses of d-borneol, l-borneol, and SB (0, 1, 2, 4, 8, 16, and 32 μg/ml) prepared in DMSO and diluted in RPMI-1640 with 1% FBS. After 24 h or 48 h, 20 μl MTT reagent was added to each well. Then, the reagent was removed after incubation for 4 h at 37 °C, and 100 μl DMSO was added. Absorbance at 570 nm was measured by Bio-Tek microplate reader after incubation for 10 min (BioTek, Winooski, VT, USA). Cell viability = (treated viable cells)/(control viable cells) × 100%.

Cell viability assays of combined treatment

To evaluate the concentration of CDDP, H460 and H460/CDDP cells (5 × 103 per well) were seeded in a 96-well plate and treated with CDDP (0.625–160 μg/ml) for 24 h. Then H460/CDDP cells were exposed to CDDP (0.625–160 μg/ml) for 24 h and 48 h to determine the suitable time. Next H460/CDDP cells was exposed to three borneol forms (0.5–16 μg/ml) for 24 h to determine the concentration and type of borneol. Finally for combined treatment, H460/CDDP cells were pretreated with d-borneol (0.5, 1, 2, and 4 μg/ml) for 12 h and co-incubated with CDDP (10 μg/ml) for 24 h. MTT assay, as described in “Determination of borneol type and cell line” section, was used to determine cell viability. The morphology of the cells was observed and photographed by inversion fluorescence microscopy (Leica DMI3000B, Germany).

Evaluation of P-gp function assay

H460/CDDP cells were incubated with CDDP (10 μg/ml) plus d-borneol (0.5, 1, 2, and 4 μg/ml) for 24 h. The cells were resuspended in the medium, stained with rhodamine 123 (Rho 123) solution (Beyotime Biotechnology, Shanghai, China), and incubated for 30 min at 37 °C in 5% CO2. The cells were centrifuged for 5 min at 2000 rpm, then resuspended and incubated for 120 min after being washed twice with medium. Then the cells were centrifuged again and washed twice with PBS. A fluorescence spectrophotometer was used to measure the fluorescence of Rho 123 with emission wavelength of 529 nm and excitation wavelength of 507 nm (BioTek, Winooski, VT, USA).

Scratch-wound healing recovery assays

A monolayer of H460/CDDP cells was scratched with the tip of a 100 μl pipette for the wound healing experiment, and the floating cells were rinsed off with PBS. Under a microscope, the gap widths were measured. After that, the cells were treated with different doses of CDDP (10 μg/ml) and d-borneol (0.5, 1, 2, and 4 μg/ml), and photographs of the wounds were taken in four random areas by microscope (Leica, Germany) at 0 h and 24 h. ImageJ was used to measure the scratch areas.

Colony-formation assay

H460/CDDP cells were planted at a density of 2 × 105 cells per well in six-well plates. The cells were next treated with CDDP (10 μg/ml) for 24 h, or with CDDP (10 μg/ml) with d-borneol (0.5, 1, 2, and 4 μg/ml) for 24 h. Cells were then digested with trypsin, and 500 cells were reseeded onto six-well plates and cultured for a further 8–10 days. Finally, crystal violet staining solution was used to stain the colonies (Beyotime Biotechnology, Shanghai, China). The number of colonies was quantified by ImageJ. The proportion of control was used to normalize the results.

Flow cytometry cell cycle analysis

H460/CDDP cells were logarithmically cultivated and seeded into 6-cm dishes at a density of 2 × 104 cells/ml. After treatment with the combination of d-borneol (0.5, 1, 2, and 4 μg/ml) and CDDP (10 μg/ml) for 24 h, H460/CDDP cells were collected, washed with PBS, and fixed with ethanol for 24 h. Then the cells were stained with propidium iodide (PI) for 30 min and protected from light. Flow cytometry was utilized to detect changes in cell cycle distribution (BD Biosciences, CA, USA) and analyzed by FlowJo V10 software [28].

Apoptosis analysis by flow cytometry

Cells were treated with CDDP (10 μg/ml), or CDDP (10 μg/ml) plus d-borneol (0.5, 1, 2, and 4 μg/ml), for 24 h. H460/CDDP cells were harvested, washed, and stained with 5 μl FITC-annexin V and 5 μl PI (Keygen, Nanjing, China) according to the manufacturer’s protocol. The cells were incubated in the dark for 15 min at room temperature. The degree of cell apoptosis was assessed by flow cytometry (BD Biosciences, CA, USA).

Hoechst 33258 staining

Hoechst 33258 (Beyotime Biotechnology, Shanghai, China) was used to evaluate the apoptosis in H460/CDDP cells after treatment with CDDP or d-borneol plus CDDP. In brief, cells were fixed with paraformaldehyde–glutaraldehyde fixing solution for 30 min. After being washed in PBS, the cells were further stained with Hoechst 33258 for 20 min at room temperature. The cells were then washed three times in PBS before being examined using a BSF-40 fluorescence microscope (Leica, Germany), and the level of cell apoptosis in each group was calculated.

Mouse xenograft assays

Male Balb/c nude mice (4-week-old) were bought from SPF (Beijing) Biotechnology Co., Ltd. and kept in the Experimental Animal Research Center of Chengdu University of TCM. Mice were maintained in a temperature-controlled facility of 22 ± 2 °C with a 12-h/12-h light/dark cycle and a relative humidity of 55 ± 5% in specific pathogen-free facility. All mice were given free access to water and food. After 1 week of adaptable feeding, H460/CDDP cells (5 × 106 cells in 0.1 ml phosphate-buffered saline) were subcutaneously injected into the right dorsal flank of male BALB/c nude mice. When tumor formation was confirmed, the mice were randomly divided into the following four treatment groups: a control group (Con, saline only, n = 6), a vehicle group (Vehicle, 2% tween), a d-borneol group (Bor, 200 mg/kg, n = 6), a CDDP group (CDDP, 3 mg/kg, n = 6), and a combination treatment group (CDDP plus d-borneol, CDDP + Bor). The mice in the CDDP group were injected with CDDP (3 mg/kg) every 3 days. The mice in the CDDP + Bor group were treated with 200 mg/kg of d-borneol and 3 mg/kg of CDDP. Tumor volume and body weight were recorded every 2 days after tumor injection. At the end of the test, the mice were sacrificed, and the tumors were harvested.

RNA sequencing

Total RNA was prepared for cDNA libraries using protocol provided by Oxford Nanopore Technologies (ONT). Briefly, SuperScript IV First-Strand Synthesis System (Invitrogen) was used for full-length mRNA reverse transcription following cDNA PCR for 14 circles with LongAmp Tag (NEB). The PCR products were then subjected to formalin-fixed paraffin-embedded (FFPE) DNA repair and end-repair (NEB) steps and following adaptor ligation using T4 DNA ligase (NEB). Agencourt XP beads were used for DNA purification according to ONT protocol. The libraries were sequenced on PromethION platform at Biomarker Technology Company (Beijing, China).

RNA extraction and quantitative real-time PCR

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The optical density (OD) at 260/280 nm was measured using a nucleic acid/protein analyzer for RNA purity testing, and then transformed to cDNA. The following were the RT-PCR reaction conditions: 95 °C for 10 min, 40 cycles of 15 s at 95 °C, and 30 s at 60 °C (Bio-Rad, USA). The primer sequences are presented in Table 1. The internal reference gene (GAPDH) was used to adjust the relative expression levels using the fluorescence quantitative analysis technique of 2−ΔΔCt method.

Table 1 Primer sequences for real-time PCR assaysWestern blot analysis

H460/CDDP cells were treated with CDDP (10 μg/ml) plus d-borneol (2 μg/ml) for 24 h before protein extraction with radioimmunoprecipitation assay (RIPA) buffer supplemented with a complete phosphatase and protease inhibitor cocktail (Beyotime Biotechnology, Shanghai, China). Protein quantification was performed by BCA assay kit (Beyotime Biotechnology, Shanghai, China). SDS–PAGE separated the proteins, which were then transferred to PVDF (Millipore Corporation, Billerica, USA). The membranes were blocked with 5% skim milk for 2 h at room temperature and then washed with TBST. Then the membranes were incubated with the following primary antibodies overnight at 4 °C: anti-P-gp (1:1000), anti-cyclin A2 (1:2000), anti-cyclin D3 (1:1000), anti-CDK2 (1:5000), anti-CDK6 (1:1000), anti-p21 (1:1000), anti-p27 (1:5000), anti-Bax (1:1000), anti-Bcl-2 (1:1000), and anti-caspase-3 (1:1000). Appropriate secondary antibodies conjugated to horseradish peroxidase were used for 1 h at room temperature before exposure to enhanced chemiluminescence (ECL). Polyclonal rabbit tubulin (1:2000) was used as loading control. The relative density of each protein band was normalized to tubulin. A signal was acquired with a ChemiDocTMXRS + imaging system (Bio-Rad, Marnes-la-Coquette, France), and blots were analyzed with Image Lab software (Bio-Rad) and ImageJ.

Histological examination

Tumor tissue was fixed in 10% formalin for 24 h, embedded in paraffin, and sliced every 5 μm. After dewaxing in xylene, the tissue was dehydrated in decreasing concentrations of alcohol and stained with hematoxylin and eosin (H&E). Then, the representative areas were captured by inversion fluorescence microscopy at 200× and 400× magnification (Leica DMI3000B, Germany).

Immunohistochemistry

Tumor tissues were fixed, paraffin-embedded, and sectioned. The deparaffinized and rehydrated sections were subjected to antigen retrieval using sodium citrate buffer. After incubation with 10% normal goat serum for 1 h, the sections were incubated with primary antibody overnight at 4 °C. The subsequent procedures were performed according to the manufacturer’s instructions. Then, the representative areas were captured by inversion fluorescence microscopy at 200× and 400× magnification (Leica DMI3000B, Germany).

Biochemical assay

The blood samples were centrifuged at 3000 rpm (10 min, 4 °C) to collect plasma, and then levels of albumin (ALB), total protein (TP), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CRE) were determined by Roche Automatic biochemical analyzer (cobas c311) according to the respective kits purchased from Roche Diagnostics GmbH (Shanghai, China).

Statistical analysis

Statistical analysis was performed using SPSS statistical package. When there were only two groups, the differences were examined via t-test, and when there were more than two groups, they were assessed by one-way analysis of variance (ANOVA). Data are expressed as mean ± standard deviation (SD). *,# denote P < 0.05.

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