C57BL/6 female mice weighing 20-23g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The mice were housed in barrier facilities with a 12-hour light/dark cycle. Before performing the experiments, one-week period allowed the mice to adapt to the laboratory environment, during which sufficient food and water were provided. Lapatinib and saikosaponin A were dissolved in 0.5% sodium carboxymethyl cellulose to obtain stock solution. The mice were treated with 950 mg/kg lapatinib or 20 mg/kg saikosaponin A or lapatinib plus saikosaponin A in experimental group through intragastric administration for 28 days. Correspondingly, the mice in control group were treated with the same volume of 0.5% sodium carboxymethyl cellulose (CMC-Na). The entire procedure was performed under an approved anesthetic protocol with pentobarbital sodium (45 mg/kg) to adequately sedate the mouse for the process.
Skin samples were obtained from the abdomen of mice were ﬁxed in 10% phosphate-buffered formalin (pH = 7.4) (Sigma-Aldrich, F8775), embedded in parafﬁn and sectioned at 5 μm intervals. After dewaxing and rehydration, the sections were stained with hematoxylin and eosin (H&E) on glass slides (Beyotime, C0105) according to the instructions. For mast cell toluidine blue staining, 0.5% toluidine blue staining solution (1 g of toluidine blue (Sigma-Aldrich, 198161) in 200 mL 70% ethanol and filtered before use) was prepared. Paraffin embedded sections were mounted onto slides and dewaxed to water and subjected to 95% ethanol for 30 s, then the sections were incubated with toluidine blue staining solution at room temperature for 60 min. After that, the acid alcohol (0.5% HCl in 100 mL 95% ethanol) was used to rinse until sections were colorless. Finally, histopathological analysis was examined by microscopy.
Apoptosis for formalin-fixed and paraffin-embedded skin sections was determined using a One Step TUNEL apoptosis assay kit (Beyotime, C1088) according to the manufacturer's instructions. Nuclei were stained with DAPI (Dojindo, D212) and the TUNEL signals were observed with fluorescence microscopy (Olympus, IX81-FV1000). Cell apoptosis was determined as the ratio of the number of TUNEL-positive nuclei to that of DAPI-positive nuclei.
Human immortal keratinocyte line (HaCaT) was purchased from the Institute of Biochemistry and Cell Biology (Shanghai, China). Normal human epidermal keratinocytes (NHEK) from single donor (C-12003) were purchased from Promocell (Heidelberg, Germany). The cells were maintained in DMEM (Gibco, 10569010) supplemented with 10% fetal bovine serum (Gibco, 10099141) with 100 U/mL penicillin and 100 μg/mL streptomycin in a humid atmosphere of 5% CO2 and 95% air at 37℃. After 3–5 passages, keratinocytes were used for subsequent experiments. HaCaT and NHEK cells used in this study were routinely tested to be negative for mycoplasma contamination.
Regents and antibodies.
Lapatinib (231277-92-2) was purchased from TargetMol (Boston, USA). For in-vitro assay, the concentration of the lapatinib stock solution was 8 mM prepared with dimethyl sulfoxide (DMSO; Sigma-Aldrich, D4540). Saikosaponin A (20736-09-8) was purchased from FEIYU Bio (Nantong, China), and 30 mM stock solution was prepared using DMSO in accordance with the manufacturer's recommendations. Z-VAD-FMK (187389-52-2) was obtained from Selleck Chemicals (Houston, USA). Antibodies against c-PARP (ET1608-10), c-CASP3 (ET1608-64) and HMGB1 (ET1601-2) were purchased from HuaBio (Hangzhou, China). Antibodies against phospho-histone H2A.X (G-H2A.X; #9718S), EGFR (#8504) and ERBB2 (34290) were purchased from Cell Signaling Technology Inc (Beverly, USA). Antibodies against ACTB (db7283) was purchased from diagbio (Hangzhou, China). Cytokeratin 5 (AF0194) was purchased from Affinity Biosciences Inc (Cincinnati, USA). Horseradish peroxidase (HRP)-labeled secondary antibody (GAR007) against primary antibodies was purchased from MultiSciences (Lianke) Biotech (Hangzhou, China).
Cell survival fraction assay.
Sulphorhodamine B (SRB; Sigma-Aldrich, S1402) assay of cellular protein biomass was used to assess cell viability as previously described (Xu, Jin et al., 2018). In brief, cells (5 × 103/well) were plated and treated in 96-well plates and left for 24-hour growth in 5% carbon dioxide incubator at 37°C. After 24-hour drug treatment, cells were fixed with 10% (wt/vol) trichloroacetic acid for over 2 h at 4℃ and stained with SRB (dissolved in 1% acetic acid aqueous solution) for 30 min. Multiskan Spectrum (Thermo Electron Corporation, Marietta, USA) was used for absorbance measurement at 510 nm. The inhibition rate of cell proliferation was calculated for each well as the follow: (the absorbance of control cells - the absorbance of treated cells)/the absorbance of control cells) × 100%.
Western blot analysis.
After treatment, cells and skin tissues were harvested by resuspended in lysis buffer (compose of 50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 25 mM NaF, 25 mM B-Sodium Glycerophosphate, 0.3% NP-40, 0.3% Triton X-100, 0.3% Leupeptin, 0.1% PMSF and 0.1% NaVO3). Protein samples (10-20 μg per sample) with loading buffer were boiled for 20 min and separated on 12% SDS-PAGE gels, and then transferred to PVDF membrane (Millipore Corporation, USA) in Tris-Glycine transfer buffer at 330 mA for 1 h. Primary antibodies were then added and incubated on a shaking table at 4℃ overnight followed by the incubation with secondary antibodies for another 2 h at room temperature. Finally, ECL-Plus Kit (PerkinElmer, MA, USA) was used to detect the bands and visualized on autoradiography film.
2 × 104 per well cells were seeded in 6-well plates and left for 24-hour growth in 5% CO2 incubator at 37°C. The cells were then exposed to the drug for indicated times and concentrations. At the end of the incubation period, the culture medium was removed and the cells were washed twice with cold phosphate-buffered saline (PBS; Gibco, 10010023) and harvested through trypsinization. FACS Calibur cytometer (BD, San Jose, USA) was used to analyze the apoptosis rate using a FITC Annexin V Apoptosis Detection Kit I (MultiSciences (Lianke) Biotech, AP101) according to the manufacturer’s instructions.
Mitochondrial membrane potential detection.
Cells were treated and harvested as mentioned above, and then resuspended in 1mL PBS and moved to 1.5 mL centrifuge tube. 10 μg/mL JC-1 (Sigma-Aldrich, T4069) was added and the cells were incubated for 30 min at 37°C without light. After the incubation period, the cells were washed twice with PBS to remove the unbinding JC-1 and then resuspended in 500 μL PBS. FACS Calibur cytometer (BD, San Jose, USA) was used to analyze the change of mitochondrial membrane potential.
ROS was analyzed by using ROS detection kit (Beyotime, Shanghai, China) in accordance with its instruction. Briefly, cells were treated and harvested as mentioned above and resuspend in DCFH-DA working solution and incubated for 20 min at 37°C without light. After the incubation period, the cells were washed three times with FBS-free DMEM medium to remove the free DCFH-DA and then resuspended in 500 μL PBS. FACS Calibur cytometer (BD, San Jose, USA) was used for further analyzing and the signals were observed with fluorescence microscopy.
ELISA (enzyme linked immunosorbent assay).
Supernatants from C57BL/6 mice were used to determine the secretion of TNF-A by mouse precoated TNFA ELISA kit (DAKEWE, 1217202), and supernatants from cells were used to detect the secretion of TNFA or IL6 using human precoated TNFA ELISA kit (DAKEWE, 1117202) or human precoated IL6 ELISA kit (DAKEWE, 1110602) according to the manufacturer’s protocol. Briefly, culture medium or whole blood sample was centrifuged at 1000 × g for 20 min to collect the supernatant. Supernatant samples or serial dilutions of standard recombinant TNFA or IL6 were added at 50 μL/well in 96-well ELISA plate. After that, 100 μL of HRP-labeled detection antibody was added. Subsequently, the liquid in each well was discarded, and the plate was washed five times with washing buffer. Then, chromogenic solution and stop solution was added to each well successively (the interval is about 15 min), and the absorbance was read at a wavelength of 450 nm within 15 min. The absorbance values were converted to corresponding values in pg/mL based on the linear regression transformation of the standard curve.
Plasmid construction and infection.
HA-HMGB1 was generated by restriction digestion of the plasmids with EcoR1 plus NOT1, followed by ligations. Cells were transfected with plasmids using the Lipofectamine 2000 transfection reagent (Invitrogen, 11668-019) according to the manufacturer’s instructions.
siRNA oligonucleotides transfection.
Cells were seeded into 6-well plates at 1.5 × 104 cells per well and grown to 50-60% confluence. Transfection was performed using jetPRIME (Polyplus-transfection, 114-15) according to the manufacturer’s protocol. siRNA oligonucleotides were transfected at a final concentration of 12 nM. Cell culture solution was changed to fresh DMEM medium for further study. The following oligonucleotides were obtained from GenePharma (Shanghai, China) as siRNAs targeting the indicated genes:
Negative Control: 5’-UUCUCCGAACGUGUCACGUTT-3’;
ERBB2 siRNA#1: 5’-GUGCCAAUAUCCAGGAGUUTT-3’;
EGFR siRNA#1: 5’-CAAAGUGUGUAACGGAAUATT-3’;
EGFR siRNA#2: 5’-GCAAAGUGUGUAACGGAAUAGGUAU-3’;
HMGB1 siRNA#1: 5’-CCGTTATGAAAGAGAAATGAA-3’.
Quantitative real-time PCR (RT-qPCR).
The procedures were referred to the previous study (Luo, Yan et al., 2021). After treatment, cells were harvested with the Trizol reagent (Invitrogen, 15596-026). Equal amounts of RNA were reverse transcribed into complementary DNA with the cDNA reverse transcription kit (Transgene, AT311). RT-qPCR was performed on a 7500 Fast System (Applied Biosystems, Singapore) using the iTaq Universal SYBR Green Supermix (Bio-Rad, 172-5124), and variable ddH2O to generate a volume of 20 μL. The samples underwent two-step amplification with an initial step at 95°C (3 min), followed by 95°C (3 s) and 60°C (31 s) for 39 cycles. The melting curve was analyzed. Fold changes in the expression of each gene were calculated by the comparative threshold cycle (Ct) method using the formula 2-(ΔΔCt). Three independent biological samples were quantified in technical duplicates and expression values were normalized to housekeeper gene.
The primer sequences were as follows:
GAPDH-Reverse : 5’-TCCTTGGAGGCCATGTGGGCCAT-3’;
Cells were treated with or without lapatinib for 24 h and total RNA was isolated using Trizol reagent according to the manufacturer’s protocol. After purification, the mRNA was reverse transcribed to create 1 μg of final cDNA for the construction of sequencing libraries using Illumina TruSeq RNA Sample Prep Kit (FC-122-1001). The average insert size for the cDNA library was 300 ± 50 bp. The cDNA was then sequenced using a HiSeq 4000 system (Illumina) and a 150-bp paired-end run according to the manufacturer's instructions. Differential expression analysis was performed based on adjusted p-values, GO enrichment and KEGG analysis of the differentially expressed mRNAs were also conducted. The RNA-Seq data were deposited in the NCBI’s Gene Expression Omnibus database (GSE195713).
For in-vitro assay, cells were seeded on round cover slips in a 24-well plate one night in advance and treated with the drug as indicated time and concentration. After discarding culture medium, cells were fixed with fresh 4% paraformaldehyde (Sigma-Aldrich, P6148) in PBS for 20 min at room temperature. Next, cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, 9036-19-5) in PBS for 10 min at 4°C and blocked with 1% bovine serum albumin (BSA; Sigma-Aldrich, B2064) in PBS for 30 min at room temperature. For in-vivo assay, skin tissues were collected and fixed with fresh 4% paraformaldehyde overnight. Then the tissues were dehydrated with 30% sucrose (Sinopharm Chemical Reagent, 10,021,418) and embedded with Optimal Cutting Temperature (Sakura Finetek, 4583) to make frozen sections (8 µm). The sections were washed with PBS for 3 times and blocked with 1% BSA in PBS with 0.1% Tween-20 (Aladdin, T104863) for 30 min. Cells or sections were incubated with primary antibodies G-H2AX (diluted at 1: 200) or cytokeratin 5 (diluted at 1: 200) at 4°C overnight. After washed with PBS, the cells were incubated with Alexa Fluor 488- or Alexa Fluor 568-conjugated secondary antibodies (Thermo Fisher Scientific, A21202, A10042, 1:200) for 2 h, stained with DAPI for 5 min and mounted for ﬂuorescence microscopy.
The comet assay was performed as described previously. Firstly, single cell suspension was prepared at a density of about 2 × 104 cells/mL in PBS. Next, preheated 0.5% normal-gelling-temperature agarose were placed onto microscope slide, after ensuring that it has completely solidified, then 85 μL 0.5% low-gelling-temperature agarose with 10 μL single cell suspension and 0.5% low-gelling-temperature agarose were spread successively (placed for over 10 min in turn). The slides were dipped into alkaline solution (1% Triton X-100, 10% DMSO and 89% lysis buffer containing 10 mM Tris, 2.5 M NaCl and 100 mM Na2EDTA with pH = 10) for 1 h at 4℃ to lyse samples. The slides were placed in a horizontal electrophoresis chamber filled with cold alkaline solution (pH=12.3) for 20 min to unwind DNA and electrophoresis was conducted for 20 min at 300 mA (about 0.6 V/cm). Subsequently, the samples were taken out and neutralized with Tris-HCl (pH = 7.5) for 15 min and then dehydrated in 70% ethanol. The samples were then stained with DAPI in dark place for 6 min and captured images by fluorescence microscope. Using Comet Assay Software Project (CASP), the percentage of DNA in tail was scored per sample.
Statistical analysis was performed using GraphPad Prism 9 software. All data were expressed as the mean value ± standard deviation (SD). When comparing two groups, Student’s t test (unpaired, two-tailed) was performed. Dunnett’s multiple comparisons test was used to assess the significance of differences among treatment groups with a single control mean, and a Sidak test was used to assess the significance of differences among various pairs of groups. All experiments were repeated three times and P values were indicated in the figure legends.