Cell Lines and Drug Sources
All cells are purchased from the American Type Culture Collection (ATCC). All cell lines were examined by DAPI DNA staining, and the mycoplasma test was negative. The human NSCLC cell lines H1299, A549, H1650, H460, H1975 or Human normal lung epithelial cells BEAS-2B, HBE were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and antibiotics. The reagents used were as follows: Rolapitant (S5476, Selleck), Estradiol Cypionate (S4046, Selleck), Recombinant Human TRAIL/Apo2L (HY-P7306, MCE), Tunicamycin (HY-A0098, MCE).
Computational virtual screening
The OTUD3 (pdb:4bou) PDB files was downloaded from the PDB (http://www.rcsb.org/). All heterogeneous atoms and the OTUD3 ligand were removed and 4bou chain A was selected for subsequent molecular docking. The OTUD3 docking grid was maximized for rolapitant docking.
PDB file (4bou chain A) were converted to the PDBQT format as macromolecules before virtual screening. The grid (ligand docking search space) was located as described above. Then, Autodock Vina 1.1.2 was used for the subsequent molecular docking.
Protein–ligand interactions were visualized using Pymol version 126.96.36.199. The amino acid residues of OTUD3 protein close to the hit ligands (≤1 Å) were highlighted as potential interactive residues involved in the protein–ligand interaction.
Antibodies used were anti-GRP78 (cat. no. 11587-1-AP, 1:3000, Proteintech), anti-PERK (cat. no. 20582-1-AP, 1:1000, Proteintech), anti-IgG (mouse. no. sc2025, 1:400, Santa Cruz Biotechnology), anti-P-PERK (cat. no.YP1055, 1:1000, Immunoway), anti-Ubiquitin (cat. no. sc8017, Santa Cruz Biotechnology), anti-OTUD3 (cat. no. CSBPA719399LA01HU, 1:1000, CUSABIO), anti-GAPDH (mouse. no. ab8245, 1:10000, Abcam), anti-CyclinA2 (cat. no. ab181591, 1:2000, Abcam), anti-P21 (cat. no. 2947s, Cell Signaling Technology), anti-DR5 (cat. no. 8074s, Cell Signaling Technology), anti-Cleaved-Caspase-8 (cat. no. 9496s, Cell Signaling Technology), anti-Caspase-8 (cat. no. 4790s, Cell Signaling Technology), anti-Cleaved-Caspase-3 (cat. no. 9661s, Cell Signaling Technology), anti-IRE1 (cat. no. 3294, 1:1000, Cells Signaling Technology, Inc.), anti-ATF6 (cat. no. 65880, 1:1000, Cells Signaling Technology, Inc.), anti-ATF4 (cat. no. 11815, 1:1000, Cells Signaling Technology, Inc.), anti-p-eIF2 (cat. no. 5324, 1:1000, Cells Signaling Technology, Inc.), anti-XBP-1s (cat. no. 12782, 1:1000, Cells Signaling Technology, Inc.), anti-PARP (cat. no. 9532, 1:1000, Cells Signaling Technology, Inc.), anti-CHOP (mouse.no.2895,1:1000, Cells Signaling Technology, Inc.), DR4 (cat.no.42533, 1:1000, Cells Signaling Technology, Inc.), anti-FADD (cat. no. 2782, 1:1000, Cells Signaling Technology, Inc.). The secondary antibodies used were anti-rabbit lgG, HRP-linked Antibody (7074s), anti-mouse lgG, HRP-linked Antibody (7076s). All these antibodies were dissolved with 5% skimmed milk in Tris Buffered saline Tween (TBST).
Colony formation assays
1000 cells per well of A549, H1299, H460 and H1975 and normal lung epithelial cells of BEAS-2B were planted in a 6-well plate for 24h. Then cells were treated with different concentrations (0-25 μM) of Rolapitant for 10-14 days until clones could be clearly visible. Cell culture plates were gently washed by PBS twice, fixed with 4% paraformaldehyde phosphate buffer for 15 minutes and then stained with crystal violet for 15minutes. Washing the excess stain with ddH2o and drying them at room temperature for several hours.
Whole cells were digested with trypsin (Gibco) and lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific, Shanghai, China) on ice. The ratio of phosphatase inhibitor and protease inhibitor to RIPA lysis buffer was 1:1:100. Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany). These membranes were blocked with 5% skimmed milk for 1 h and then incubate overnight with specific antibody. Washing the membranes three times with Tris Buffered saline Tween (TBST) for 10 minutes each. PVDF membranes were incubated with secondary antibodies for 1h at room temperature and then washed with TBST four times for 10 minutes each. Finally, Using the ECL Western blotting system (Thermo Scientific, Waltham, MA, USA) to detect the expression of target proteins.
Cell viability assays
Cell viability assays was performed using the CCK-8 (APEXBIO) test. 1000 cells were seeded in 96-well plates and allowed to grow for 24h. Then each well was treated with dimethyl sulfoxide (DMSO) or different drug dose of Rolapitant for 0h, 24h, 48h,7 2h and 96h. Next, the medium was removed, and 10 μl of CCK-8 which was dissolved in 90 μl of RPMI 1640 medium (Gibco). The optical density (OD) values of each well were measured at 450 nm using a SpectraMax spectrophotometer (Molecular Devices, San Jose, CA, USA) after being incubated at 37°C for 1h.
siRNA and plasmid transfection
Cells (1× 105 cells/well) were seeded in 6-well plates and allowed to grow to 50% confluent at the time of transfection. Diluting 8μl of siRNA or 8μg of plasmid into 200 µl of jetPRIME buffer. Then 4 µl of jetPRIME reagent (Polyplus transfection) was added into the buffer. The buffer was mixed and incubated for 10 minutes at room temperature. Finally, the transfection mix was added into the cells. The sense and anti-sense strands of siRNAs were as follows:
DR5 siRNA sence：5′-GACCCUUGUGCUCGUUGUCTT-3′ and siRNA antisense: 5′-GACAACGAGCACAAGGGUCTT-3′.
CHOP siRNA sense: 5′-CAGUAUCUUGAGUCUAAUATT-3′ and and siRNA antisense: 5′-UAUUAGACUCAAGAUACUGTT-3′.
A549 and H1299 cells treated with DMSO and Rolapitant respectively were lysed in IP Lysis containing phosphatase inhibitors and protease inhibitors on ice for 30 minutes and then purified via centrifugation for 15 minutes, 12000 g at 4 °C. The supernatants were incubated with diluted anti-lgG antibody, anti-GRP78 antibody and the magenetic beads (Thermo Fisher Scientific, MA, USA) overnight at 4 °C. The proteins were eluted with 2×SDS-PAGE Sample Loading Buffer (Beyotime Biotechnology,Jiangsu, CHina) for 10 minutes at 100°C after being washed by the lysis buffer. Immunocomplexes were determined by western blot analysis.
In vivo GRP78 ubiquitylation assay
For in vivo ubiquitylation assays, A549 and H1299 cells were treated with 10 µM of the Rolapitant for 48h. Cells were lysed with NP40 lysis buffer after treated with 20 µM of proteasome inhibitor MG132 (Calbiochem) for 8 h and incubated with anti-GRP78 antibody for 3 h and protein A/G agarose beads (Santa Cruz) for a further 6 h at 4 °C. Then the beads were washed three times with PBS buffer. The proteins were released from the beads by boiling in SDS-PAGE sample buffer and analysed by immunoblotting with anti-Ub monoclonal antibody.
Flow cytometry-based apoptosis detection
Flow cytometry-based apoptosis assay was performed using the Annexin V-FITC Apopotosis Detection Kit (KeyGEN BioTECH) according to the manufacturer’s instructions. A549,H1299 and BEAS-2B cells were seeded in 6-well plates at a concentration of 2×105 cells/well. Cells were lysed by Trypsin-EDTA (0.25%) (GIBCO) after being treated with different doses of Rolpitant for 24h. 500 µl of binding buffer with 5 µl of Annexin V-FITC propidium iodide were added into the plate after being washed twice by PBS. Then 5 µl of Propidium iodide (PI) was added after 10 minutes and keeping the cells in the dark. Analysis was performed on a flow cytometer (BD Biosciences) in 1h and data were processed by Modfit software.
Flow cytometry-based cell cycle analysis
A549, H1299 and BEAS-2B cells were seeded in 6-well plates at a concentration of 2×105 cells/well. Cells were collected by Trypsin-EDTA (0.25%) (GIBCO) after being treated with different doses of Rolpitant for 24h. The plates were washed twice by PBS and then adding 500 μl of 70% absolute ethyl alcohol at 4°C for at least 12 h. Finally, cells were resuspended in 500 µl of PI solution in the presence of RNase. A flow cytometer (BD Biosciences) was used to determine the percentage of cell cycle and data were analyzed using Modfit software.
Flow cytometry-baesd cell surface DR5 analysis
For flow cytometric analyses, cells were lysed with Trypsin, blocked by PBS, 10% FBS for 10 min on ice and subsequently incubated with 5 μl of PE-labeled mouse isotype-mached Control lgG1 (eBioscience, invitrogen) and 5 μl of CD262 (DR5) monoclonal antibody conjugated with PE fluorochrome (eBioscience, invitrogen). All stains were performed using PBS, 10% FBS for 30 minutes in the dark at either RT or 4°C. Cells were suspended in 400 µl of PBS after two washing steps by PBS and then analyzed on flow cytometer (BD Biosciences). Data was processed and presented using Modfit software.
Total RNA was collected using Takara MiniBEST Universal RNA Extraction Kit (Takara Biotechnology CO., Ltd) according to the manufacturer’s instructions. The concentration and quality of RNA were detected by 260/280 nm absorbance. Using PrimeScript RT Master Mix (Takara biotechnology Co., Ltd) reverse transcripts RNA into high-volume DNA. Quantitative real-time PCR was performed with ABI 7300 PCR system (Applied Biosystems, CA, USA), using SYBR Green Master Mix (Termo Fisher Scientific, USA). All primers were designed and synthesized by Sangon Biotechnology Co. (Shanghai,China),according to the gene sequence in Genbank. Following an initial denaturation at 95 °C for 30 s, 45 cycles of PCR amplification were performed at 95 °C for 5 s and 60 °C for 30 s. Relative expression of target gene mRNA was normalized to GAPDH as an internal control and calculated using 2−△△CT. The primer sequences are as following: GRP78 forward: 5’-CTGTCCAGGCTGGTGTGCTCT-3’ and GRP78 reverse: 5’-CTTGGTAGGCACCACTGTGTTC-3’, DR5 forward: 5′-GCCCCACAACAAAAGAGGTC-3′ and DR5 reverse: 5′-AGGTCATTCCAGTGAGTGCTA-3′, GAPDH forward: 5′-ACGGATTTGGTCGTATTGGG-3′ and GAPDH reverse: 5′-CGCTCCTGGAAGATGGTGAT-3′,
CHOP forward: 5′- AGCCAAAATCAGAGCTGGAA-3′ and CHOP reverse: 5′- TGGATCAGTCTGGAAAAGCA-3′, p21 forward: 5′- AGTGTGCCGTTGTCTCTTCG-3′ and p21 reverse: 5′- ACACCAGAGTGCAAGACAGC -3′, CyclinA2 forward: 5′-TGATGAAACTATGACCATGATGTCC-3′ and CyclinA2 reverse: 5′- TTCACAGAACGCAGACCACC-3′.
Human Tumor Xenograft Models
The animal experimental protocols were approved by the Animal Care and Use Committee of Nanjing Medical University, China. Female BALB/c nude mice (4-6-weeks-old) were purchased from Charles River Labs (Wilmington, MA, USA). Mice were received a 200µl A549 cells suspensions (2×107 cells per mL) by subcutaneous injection under the armpit. Then mice were randomized into three groups when the tumor size reached approximately 150-200mm3. Rolapitant was dissolved in 10%DMSO, 40%PEG300, 5%Tween-80, 45%saline, 4% Tween-80 at the final concentration of 100mg/ml. Eight mice in treatment group were given 75 or 50 mg/kg/d Rolapitant every three days. Control mice were injected with drug vehicle. The experiment was complete when the tumor size of the control group reached 2000mm3. The weight and tumor size of the mice were monitored every 3 days. Tumor tissues were collected immediately and detected when the mice were killed.
All experiments were performed independently at least three times and in triplicate each time. Data were analyzed with GraphPad Prism 8.0. All Statistical analysis were calculated by Student’s t-test or the analysis of variance (ANOVA). The criterion for statistical significance was P-values < 0.05.