Reagents and antibodies
Osthole and cisplatin were purchased from Meilunbio (purity≥98%, Dalian, China). Dicoumarol (DIC), Z-VAD-FMK, Z-DEVD-FMK and 3-Methyladenine (3-MA) were purchased from MedChem Express (Princeton, NJ, USA). N-Acetyl-L-cysteine (NAC) and JC-1 were purchased from Beyotime (Shanghai, China). Dimethyl sulfoxide (DMSO) and 2′, 7′-Dichlor-ofluorescin diacetate (DCFH-DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-rabbit IgG, anti-mouse IgG and antibodies against β-actin were purchased from ZSGB-BIO (Beijing, China). Anti-LC3A/B antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-NQO1, anti-caspase-3, anti-cleaved caspase-3, anti-PARP-1 and anti-GSDME antibodies were purchased from Abcam (Cambridge, MA, USA). FITC Annexin V Apoptosis Detection Kit (BD Pharmingen, CA, USA)
Cell cultures
The human cervical cancer HeLa cell line and normal liver cell line LO2 were obtained from Conservation Genetics CAS Kunming Cell Bank (Kunming, China). The cells were cultured in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Auckland, New Zealand), 100 U/mL penicillin and 100 μg/mL streptomycin (Solarbio, Beijing, China), and maintained in a humid environment with 5% CO2 at 37℃.
Cell viability assay
Hela cells were seeded into 96-well plates at 4000 cells/well and incubated overnight. After that, cells were treated with osthole at indicated concentrations for indicated times. Then, 20 μL MTT (Solarbio, Beijing, China) was added to each well and the plate was incubated for 4 h at 37 °C. The formed formazan crystals were dissolved with 150 μL DMSO, and the absorbance was detected at 490 nm with a microplate reader(TECAN,Switzerland).
Lactate dehydrogenase (LDH) release assay
The LDH release was detected by LDH assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). According to the instruction, cell culture supernatant was collected and added the corresponding reagents. The absorbance was measured at 450 nm with a microplate reader.
Flow cytometry analysis of Annexin V-FITC/PI staining
Osthole treated cells were harvested and washed by cold phosphate-buffered saline (PBS) thrice. The cells were suspended in 200 μL 1×binding buffer containing 5 μL Annexin V-FITC and 5 μL PI and incubated for 30 min in the dark. After that, 100 µL 1×binding buffer was supplemented to each sample, and the cells were detected by flow cytometer (BD Biosciences, CA, USA).
Measurement of ROS
Intracellular ROS levels were determined using the fluorogenic probe DCFH-DA. After treatment with osthole, the cells were washed with PBS and dyed by DCFH-DA (2 μM) for 30 min at 37 °C in the dark. The cells were harvested and the level of ROS was measured by flow cytometry (BD Biosciences, USA).
Hoechst 33342 and PI staining
HeLa cells were cultured in 6-well plates for 24 h and pretreated with osthole for 18 h. Then, cells were washed with PBS and stained by Hoechst 33342 (1 μg/ml) for 5 min, following incubation with PI (5 μg/ml) for 15 min at room temperature. The cells were observed under cell imaging station (ThermoFisher Scientific, Bonn, Germany) after washed with PBS.
Measurement of MMP
Mitochondrial membrane potential was determined by the fluorescent probe JC-1 (Beyotime Biotech, Nanjing, China). The cells were suspended in PBS containing 0.2 μM JC-1. After incubated for 20 min in the dark, the samples were analyzed by flow cytometry. JC-1 existed as a polymer when the MMP is high otherwise exists as a monomer, and the two groups of cells can be distinguished by flow cytometry (BD Biosciences, USA).
MDC staining assay
MDC staining assay was used to detect autolysosome. After treatment with osthole, the cells were incubated with 50 μM MDC for 30 min at 37 °C. Then, the cells were washed three times with PBS and observed under fluorescence microscopy straightway (Olympus, Tokyo, Japan).
Label-Free Quantitative Proteomics
Protein extraction and peptide separation were performed as previous described method[14]. MS experiments were performed on a Q Exactive mass spectrometer that was coupled to Easy nLC (ThermoFisher Scientific, Bonn, Germany). MS data was acquired using a data-dependent top 10 method dynamically choosing the most abundant precursor ions from the survey scan (350–1800 m/z) for HCD fragmentation. The instrument was run with peptide recognition mode enabled. MS experiments were performed triply for each sample.
Bioinformatics analysis of differentially expressed proteins
The MS raw data were processed using MaxQuant software version 1.5.5.1 and quantified by label free quantitation (LFQ)[15]. Gene Ontology (GO) enrichment analysis with biological process, molecular function and cellular components of potential targets were carried out for biological function annotation based on a bioinformatics database (http://geneontology.org/). KOALA (KEGG Orthology and Links Annotation) software was used to analyze KEGG Pathway database. Fisher’s Exact Test was used to the distribution of GO classification or KEGG pathway in target protein collection and total protein collection.
Western blotting analysis
The cells were harvested and lysed with RIPA lysate buffer for 30 minutes on ice. After centrifuged, the supernatant was obtained and quantified using BCA kit. The protein was separated by SDS-PAGE electrophoresis and transferred to a NC membrane. These membranes were blocked with 5% skim milk for 2 h and were incubated with primary antibody at 4 °C overnight. After that, membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:4000) for 1 h at room temperature. Protein bands were detected by Western ECL substrates (Bio-Rad, CA, USA) and gray values were analyzed by Image J.
Xenograft assay
The use of all mice in this study was approved by the Institutional Animal Care and Use Committee of Guilin Medical University. Female BALB/c-nude mice (4 weeks old) were purchased from Hunan SJA Laboratory Animal Co., Ltd. Hela cells (1´107) were injected subcutaneously into the right flanks of the mice. When the tumor volume reached 50 mm3, nude mice were intraperitoneally injected with osthole (150 mg/kg, dissolved in corn oil), and control group mice were injected with corn oil (Sigma, St. Louis, MO, USA). The length and width of the tumor was measured every two days using vernier calipers (Volume= length ´ width2/2). Following treatment for 21 days, the mice were sacrificed, and the weights of xenograft tumors were measured. After that, the total protein from the excised tumor tissues was extracted by RIPA lysis buffer. The expression of proteins was detected by western blotting analysis.
Statistical analysis
All data represent at least three independent experiments and was analyzed by GraphPad Prism 8 software (GraphPad, San Diego, CA). The results were expressed as mean ± standard deviation (SD). Differences among groups were compared using a two-tailed Student’s t-test or one-way ANOVA. P < 0.05 was considered statistically significant.