Reagents and antibodies
Parthenolide was purchased from MedChemExpress (cat#HY-N0141) and dissolved in DMSO. The Annexin V-FITC/PI staining kit was was purchased from Thermofisher (cat#88-8005-72). The antibodies for western blot are as followings: primary antibodies against phospho-EGFR (cat#3777), total-EGFR (cat#4267), phospho-ERK (cat#4377), total-ERK (cat#4695), phospho-AKT (cat#4060), AKT (cat#9272), cleaved-caspase-3 (cat#9661), PARP (cat#9532), HRP goat anti-rabbit (cat#7074) and-mouse secondary antibodies (cat#5127) and GAPDH (cat#5174) were purchased from CST Cell Signaling Technology.
The human NSCLC cell lines ( H1975, PC-9, HCC827) were obtained from the American Type Culture Collection (ATCC). Cells were cultured in DMEM or RPMI1640 medium which supplemented with 10 % FBS (Gibco, USA), 100 μg/ml streptomycin and 100 U/ml penicillin (Gibco, USA). All the cell lines were cultured at 37°C in a humidified environment containing 5% CO2.
Cell viability assay
Cell viability was determined by cell counting Kit-8 (Dojindo). Briefly, 5000 cells/well were seeded in 96-well plate for 24 h, and treated with increased concentrations of parthenolide for an additional 24 h, then 10 ul CCK8 reagent was added and incubated for 3 h. The absorbance was measured using a microplate reader at 450 nm. The percent of growth is calculated by normalising against the control cells, and the IC50 value was calculated using GraphPad Prism7.
Colony formation assay
Briefly, 1000 cells/well (H1975, PC-9 and HCC827) were seeded in 6-well plate, cells were exposed to various concentration of parthenolide， The culture medium was changed every 3 days until the visible colonies were formed. The colony was washed with cold PBS, fixed with 4% paraformaldehyde for 15 min, then stained with 0.5% crystal violet for 20 min. The colonies were photographed.
Cells were resuspended in 1×100 μl binding buffer, propidium iodide (5 μl) and Annexin-V (5 μl) were added to the solution, mixed well and incubated in dark for 20 min at room temperature. Cells were washed with 1ebinding buffer and centrifuged at 400g at 4°C for 5 minutes. Then cell pellets were resuspended and apoptosis assay was performed on a Beckman CytoFLEX FCM. The percentage of apoptotic cells was identified by both of early apoptotic (PI- Annexin V+) and late apoptic (PI+ Annexin V+).
Cells were harvested in yysis buffer, and supernatant was centrifuged at 12,000 g for 10 min. Extract was quantified with bradford method. Lysate was heated to 100 °C in SDS sample buffer with 50 mM DTT for 5 min. Protein was separated by 10 % SDS-PAGE gel and transferred to PVDF membrane. Blots were blocked for 1 h in TBS-T containing 5 % non-fat dry milk, incubated with primary antibodies at 4°C overnight. After washing, blots were incubated at room temperature with secondary antibodies labeled with conjugated to HRP for 1 h. Finally, blots were detected using the LICOR system for enhanced chemiluminescence.
Download EGFR (PDB ID:4HJO) from the RSCB PDB database (http://www.rcsb.org/) and save it as an PDB format file. Then it was imported into AutoDock 4 software for pretreatment, including extraction of ligand small molecules, removal of water molecules, hydrogenation and so on. The sdf file of parthenolide3D structure downloaded from PubChem database was optimized, and parthenolide was docked with protein by AutoDock 4. PyMol were employed to generate the docking input files and to analyze the docking results.
Construction of protein-protein interaction (PPI) network
The 3D structure of parthenolide was obtained by pubchem (https://pubchem.ncbi.nlm.nih.gov /) and imported into pharmmapper server (http://www.lilab-ecust.cn/pharmmapper/) for parthenolide target prediction . The specific setting is Generate Conformers: yes; Maximum Generated Conformations: 300; Select Target Set: Human Protein Target Only; Number of Reserved Matched Targets: 300. A total of 243 predetermined proteins were obtained. A total of 58 proteins with ZValue > 0.8 were screened. Fifty eight target protein genes were introduced into STRING (https://string-db.org/) database  and Uniprot database for gene correction. Fifty six target protein genes were successfully transformed into gene abbreviations, of which BRAF1_HUMAN and PTGD2_HUMAN were not transformed successfully. The transformed 56 protein genes were further introduced into STRING. Under the condition that the confidence score score > 0.4 and hiding the unconnected nodes in the network, a co-expression network constructed by 51 qualified protein genes was obtained. The co-expression network was analyzed by STRING, downloaded chart information, and beautified by cytoscape software.
Mouse xenograft assay
The animal experiments involved in this experiment were conducted according to the Animal Ethics Committee of Guangdong Medical University (GDY1902062) and and committee-approved protocols. 2.5×106 H1975 cells were resuspended in FBS-free medium and injected subcutaneously in 100 μl volume into the 6-week-old nude mice. The tumor xenografts were allowed to grow to ~60 mm3, and five mice per group were treated with vehicle (2 % DMSO, 40 % PEG400, and 2 % Tween 80 in normal saline) or parthenolide (20 mg/kg) via intraperitoneal (ip) injection daily for 14 days. Body weight was monitored every day. Tumors were measured every three days using a digital caliper, and tumor volumes (mm3) were calculated using the following formula: volume (mm3) = length (mm) × width (mm)2 × π/6.
Histology and immunohistochemistry
Tumor tissues were fixed in 10 % formalin and embedded in paraffin. The sections were dewaxed in xylene and dehydrated in alcohol. The sections were stained with hematoxylin and eosin (H&E) for histology analysis. For immunohistochemistry, the sections were incubated with 0.01M sodium citrate (pH=6.0) for antigen retrieval, endogenous peroxidase was blocked by adding 0.3 % H2O2 and incubated for 30 min in a 10 % goat serum albumin. The sections were incubated overnight at 4 °C with antibodies to p-ERK, p-AKT, p-EGFR, and cleaved caspase-3 from Cell Signaling Technology. Samples were incubated with a HRP-conjugated anti-rabbit secondary antibody for 1h, and signal was visualized by incubation with diaminobenzidine. Finally, the sections were counterstained with Mayer's haematoxylin, dehydrated, cleared in xylene and mounted in permount TM mounting medium.
The results are presented as the mean ±SD. The statistical significance of differences was assessed using the ANOVA and Student’s t-test. P < 0.05 were considered statistically significant.