Regents and antibodies. Cycloheximide (5087390001), MG-132 (474791), wortmannin (W3144), 3-methyladenine (M9281), brefeldin A (B5936), CDDP (PHR1624) was purchased from Sigma-Aldrich. LysoTracker Red DND-99 (L7528) and Lipofectamine 3000 (L3000015) were from Invitrogen. Anti-p53 (sc-126, Mouse mAb), anti-ATG5 (sc-133158) and anti-SQSTM1 (sc-28359) antibodies were purchased from Santa Cruz Biotechnology. Anti-ubiquitin (58395S) and anti-K63- polyubiquitin (12930S) antibodies were purchased from Cell Signaling Technology. Anti-LC3 antibody (NB100–2220) was purchased from Novus Biologicals. Anti-GAPDH antibodies (AB9132) was purchased from Chemicon. Goat anti-rabbit (Alexa Fluor 488, A-31566) and goat anti-mouse (Alexa Fluor 647, A-21242) secondary antibodies were from Invitrogen.
Cells. Cell culture reagents were purchased from Gibco (Carlsbad, CA). ES-2 and SK-BR-3 were grown in McCoy's 5A Medium. NCI-H1299 and HUVEC were grown in RPMI 1640 medium. MDA-MB-231, MIA PaCa-2 and HEK 293 were grown in DMEM medium. All mediums were supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Gibco) except for MIA PaCa-2, and MIA PaCa-2 were supplemented with 10% fetal bovine serum (Gibco), 2.5% horse serum and 1% penicillin–streptomycin (Gibco). All cell lines were from ATCC and were cultured at 37°C under 5 % CO2.
Preparation of NRs nanoparticles: A mixture containing polymer (Mal-PEG2000-PLA2000, 10.0 mg), DOTAP (2.0 mg) was dissolved in 1.2 mL THF solution and then gradually added to 10 mL PBS (pH=7.4, 0.01 M) with stirring. After additional stirring for 2 h, the nanoparticles solution evaporated by rotary evaporator to remove THF for 2 h, and obtained nanoparticles denoted as dNRs. Then, MBP (5 mg, 0.0025 mmol) was added into the mNRs solution and sustained stirring for 8 h (pH=7.4, 0.01 M). Then, the solution was transferred into a dialysis bag (MWCO 15000 Da) to remove free MBP against PBS (2.0 L, pH=7.4, 0.01 M) for 12 h. After ultrafiltration and concentration using Amicon YM-30 centrifugal filter devices (Millipore, MWCO 5000 Da), the obtained nanoparticles denoted as NRs. mNRs were also prepared by a similar method without added DOTAP during the preparation process.
Preparation of Pt(IV)-loaded NRs nanoparticles: Firstly, the Pt(IV) prodrugs were synthsiszed by previous method32 Then, a mixture containing polymer (Mal-PEG2000-PLA2000, 10.0 mg), DOTAP (2.0 mg) and Pt(IV) prodrug (1.5 mg) was dissolved in 1.4 mL THF solution and then gradually added to 10 mL PBS (pH=7.4, 0.01 M) with stirring. After additional stirring for 2 h, the nanoparticles solution evaporated by rotary evaporator to remove THF for 2 h, and obtained nanoparticles denoted as dNRs/Pt(IV). Then, MBP (5 mg, 0.0025 mmol) was added into the dNRs/Pt(IV) solution and sustained stirring for 8 h (pH=7.4, 0.01 M). Then, the solution was transferred into a dialysis bag (MWCO 14000 Da) to remove free MBP and free Pt(IV) prodrug against PBS (2.0 L, pH=7.4, 0.01 M) for 12 h. After ultrafiltration and concentration using Amicon YM-30 centrifugal filter devices (Millipore, MWCO 5000 Da), the obtained nanoparticles denoted as NRs/Pt(IV). dNRs/Pt(IV) were also prepared by a similar method without added MBP during the preparation process.
Characteristic of these nanoparticles: The zeta potential and size distribution of mNRs, dNRs, and NRs measured by Malvern ZS90 dynamic light scattering (DLS) instrument (Malvern Instruments Ltd., England). The nanoparticles mNRs, dNRs and NRs were dispersed in pH 7.4 PBS (0.01 M) containing 10% FBS at the concentration of 1.0 mg/mL and incubated at 37 °C. Then, the samples were taken at a designated time point, and the diameter of NPs was measured by Malvern ZS90 dynamic light scattering (DLS) instrument (Malvern Instruments Ltd., England). Then, 10 µL mNRs, dNRs or NRs were added dropwise to copper gauze with a carbon film (400 mesh) and dried overnight, respectively. The morphology and aggregation of the mixed particles were observed by TEM. To confirm that the MBP was bonding to the NRs surface and the Mutp53 could immobilize onto NRs via specific recognition. The NRs core was labeled with Rhodamine B (RhoB) by incorporated RhoB-PEG-b-PLA during the NPs preparation process, then the Aleax fluorTM 488 (Succinimidyl ester) labeled MBP was added and reacted with NPs through click reaction. After that, the Aleax fluorTM 647 (Succinimidyl ester) labeled Mutp53 protein was added to the above bifluorescence labeled NRs and continue stirring for 4 h. Then, the fluorescence-labeled NRs or mixtures (NRs plus Mutp53) were dropped on the coverslip and precipitated for 10 min, and images were acquired using an Abbelight STORM Super-resolution System (Quantum Design China, Ltd.), which is equipped with 488 nm, 530 nm and 647 nm super-resolution compatible laser excitation, enables industry-leading multi-color simultaneous STORM acquisition.
Circular Dichroism Spectroscopy: Ultraviolet CD spectra were obtained using a Chirascan CD-spectrometer with a 1 mm path length cuvette (Applied photophysics limited, England). For the measurement of the combination, the previously prepared mutp53 protein stock solution was diluted to concentrations of 1.25 mM and mixed with mNRs, dNRs and NRs ([NPs] = 5 mg/mL, [MBP] = 1.25 mM) in a volume ratio of 1/1. Spectra of solutions with and without protein were recorded between 195 and 260 nm at 65 °C. The free mutp53 protein and MBP plus mutp53 protein were used as a control group.
Surface plasmon resonance: The GE BIAcore 8K instrument was operated at a constant temperature of 25 °C and the CM5 sensor chip (GE Healthcare) was used in this study. Each CM5 sensor chip consists of 8 identical experimental channels and each channel was divided into two flow cells. In our experimental setup, flow-cell 1 (Fc1) was always kept blank as a reference, while flow-cell 2 (Fc2) was functionalized with the nanocarrier for interaction studies with mutant p53 protein. Specifically, the system was first equilibrated with PBS-T buffer (20 mM Na-phosphate, 150 mM NaCl, and 0.05 % Tween 20, pH 7.4). One experiment channel was activated for 6 min with a mixture of EDC (0.2 M) and NHS (0.05 M). In Fc2, this was followed by 7 min injection of dNRs in 10 mM acetate buffer (pH 4.0). In parallel, PBS-T buffer was injected in Fc2. Finally, 1M ethanolamine-HCl solution was injected onto both Fc1 and Fc2 to block the remaining NHS-ester groups. To observe the interactions between mutp53 and NRs, 5.21 μM mutp53 in PBS solution was injected into the nanocarrier modified sensor surface. The injection time is 3 min at a flow rate of 30 μl/min, followed by a dissociation step of 5 min. To calculate the KD value, a new channel was prepared using identical conditions. 5 different concentrations (4 nM, 20 nM, 100 nM, 500 nM and 2500 nM) of mutp53 in PBS solution were injected onto the channel with the response units measured at single cycle. All signals were subtracted from the reference signal. The binding kinetics was calculated using a 1:1 binding model with BIAcore 8K Evaluation software. The interactions between mutp53 and dNRs were measured as a control group.
Electron microscopy. After harvesting, ES-2 cells were fixed in suspension with 4% glutaraldehyde in 0.1 Mcalcodyate buffer (pH 7.3) overnight at 4°C. Then, cells were post-fixed in 2% osmium tetroxide in 0.1 M osmium acid for 1 hour at room temperature. After dehydration with gradient ethanol and propylene oxide, the cells were embedded in epoxy resin, and then the area containing the cells was cut into ultra-thin sections, stained with uranyl acetate and lead citrate, and observed under a transmission electron microscope (JEOL-1230, Japan).
Establishment of EGFP-LC3/MDA-MB-231 cells. For establishing EGFP-LC3/MDA-MB-231 cells, MDA-MB-231 cells were transfected with pEGFP-LC3 using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer's protocol. 24 h later transfection cells were transferred to a new plate and underwent selection in DMEM medium containing 0.6 mg/ml of G418 (Promega, USA). About 10 days post-transfection and expansion, cell colonies exhibiting strong green fluorescence were selected under a fluorescence microscope.
Immunofluorescence. Cells cultured on coverslips were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.1% Triton X-100 in PBS for 15 min, followed by blocking in 1% BSA for 1 h. After an overnight incubation with the primary antibodies at 4°C, the coverslips were washed three times with PBS and incubated with fluorescent secondary antibodies (Invitrogen, 1:2000) for 1 h in the dark at room temperature. The coverslips were then washed three times in PBS and stain the nucleus with DAPI (Roche). Images were acquired using a confocal microscope (Nikon, Ti-E A1). For immunofluorescence of parafﬁn-embedded tissues, the slices were dewaxed and rehydrated in xylene and graded alcohol solutions first, followed by the steps of normal immunofluorescence. For detecting p53 degradation, anti-p53 antibody (sc-126, 1:100 dilution) and goat anti-mouse (Alexa Fluor 647, A-21242,1:2000 dilution) secondary antibody were used.
Western blotting. Cells were harvested and lysed with radoimmunoprecipitation assay (RIPA) buﬀer containing protease inhibitor cocktail (C600386, Sangon) on ice. An equal volume of 2 × SDS sample buffer was added, and boiled for 10 min. Cell lysate containing 20-100 µg proteins were separated by electrophoresis on a SDS polyacrylamide gel and transferred to PVDF membrane (Millipore, IPVH00010). After blocking with 5% nonfat dry milk for 1 h, the PVDF membrane was incubated 3 h with a primary antibody against specific proteins at room temperature, extensively washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution) for 1 h, and finally visualized with an enhanced chemiluminescence (ECL) kit and imaged by the GE Amersham Imager 600 imaging system.
Cell viability assay. Cells were grown in 96-well plates at a density of approximately 10,000 cells per well. Twenty-four hours later, cells were exposed to indicated treatment for 24 h, followed by MTT assay. Briefly, MTT (thiazoyl blue tetrazolium bromide, Bio Basic) was added to the growing cultures at a ﬁnal concentration of 0.5 mg/mL and incubated for 4 h at 37 ℃. Then, the medium was replaced with DMSO to dissolve blue formazan crystals, and plates were shaken for 15 min in the dark. Results were measured with a spectrophotometer (Elx800, BioTek, Winooski, VT, USA) at 570 nm.
Quantitative PCR analysis. The mRNA expression level of mutp53 was measured by q-PCR. According to the manufacturer's instructions, total RNA from ES-2 cells was extracted using AG RNAex Pro Reagent (AG21101), and purified total RNA was reverse-transcribed to cDNA using the Evo M-MLV RT Kit (AG11711). Then, 2 μL of cDNA was used as template in subsequent quantitative PCR reactions conducted on an ABI7300 using SYBR Green Super Mix kit (Roche). The primers were P53-F (GCGTGAGCGCTTCGAGAT), P53-R (AGCCTGGGCATCCTTGAG), GAPDH-F (GTCGTACCACAGGCATTGTGATGG), and GAPDH-R (GCAATGCCTGGGTACATGGTGG).
Plasmid construction and transfection. All p53 mutations (S241F, R248W, R175H) were generated by QuickMutation site-directed mutagenesis kit based on pcDNA3.1-p53 plasmid according to the product manual (D0206, Beyotime). All plasmids were sequenced to confirm the designed mutation. For transfection of plasmid DNA, cells were grown to 70–80% confluency and transfected with plasmid DNA using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer's instructions. After 24 hours, the transfected cells were transferred to a new dish and selected in DMEM medium containing 0.6 mg/ml G418 (Promega, USA). About 10 days post-transfection and expansion, the selected cells were used for subsequent experiments.
Gene knockdown. PLKO vector (Sigma-Aldrich) was co-transfected with plasmids encoding gag and vsvg into HEK 293T packaging cells using lipofectamine 3000 (Invitrogen). Then, after 48-hour transfection, viral supernatant was collected and added into the culture medium of MDA-MB-231 cells in the presence of 5 μg/mL polybrene (Sigma-Aldrich). The infected cells were selected by puromycin to establish stable cells. shATG5 in PLKO vector: 5′-CCAGATATTCTGGAATGGAAA-3′.
p53 ubiquitination studies. ES-2 cells after NRs treatment were harvested and lysed with RIPA buﬀer containing protease inhibitor cocktail (Sangon). Cell extracts containing an equal amount of protein were incubated with Protein A/G-Agarose (sc-2003) and p53 antibody (sc-126, 2 μg per sample) overnight. After washing with PBST buffer, the precipitants were resolved on SDS–PAGE for western blotting with antibodies against ubiquitin (58395S, 1:1000 dilution), K63-Ub (12930S, 1:1000 dilution) or p53 (ab32389, 1:5000 dilution).
Sphere-formation assay. Single-cell suspensions of ES-2 cells were plated (1,000 cells per well) into 24-well Ultra Low Attachment plates (Corning) in serum-free RPMI 1640 culture media (Gibco) supplemented with 2% B27 (Invitrogen), basic fibroblast growth factor (20 ng/ml, Invitrogen) and epidermal growth factor (20 ng/ml, Millipore). The cells were grown in humidified atmosphere with 5% CO2 for 7 days, the half of old culture medium was replaced by an equal amount of fresh culture medium every 3 day. The diameters of spheres in different groups were recorded with inverted phase-contrast microscopy (Nikon, DS-Fi3).
Transwell assay. RPMI 1640 media supplemented with 10% FBS was added to the lower chamber. ES-2 cells (1 ×104 per well) were added to the upper chambers in serum-free 1640. After 12 hours incubation with PBS, free peptide, mNRs, dNRs, NRs, cells which migrated to the lower face of the membrane were fixed with methanol and stained with 0.1% of crystal violet. After being washed by PBS for 3 times, cells were imaged with microscopy (Nikon, Ci-L).
Colony formation assay. 24 h after seeding ES-2 cells (500 per well) on a 6-well plate, cells were treated with PBS, free peptide, mNRs, dNRs, NRs for 24 hours and then changed to new medium. After culturing for another 10 days, colonies were fixed with methanol, stained with 0.1% of crystal violet and imaged.
Hoechst/PI double staining. Briefly, ES-2 cells at 5 × 103 cells/well in 96-well plates were incubated with nanoparticles for 24 h. After treatment, cellular nuclei were stained with Hoechst 33342 (10 μg/mL). Propidium Iodide (10 μg/mL) was used to observe cell death by fluorescence microscopy (Nikon, DS-Fi3). Cell death was expressed by the ratio of PI-positive cells to Hoechst positive cells.
CRISPR/Cas9 System. CRISPR/Cas9 system was used to partially knock out p53 in ES-2 cells. Single guide RNA (sgRNA) is generated by the online CRISPR design tool (http://crispr.mit.edu/). The sgRNA sequences were cloned into PX458 gene vector, and transfected into ES-2 cells with Lipofectamine 3000 (Invitrogen, L3000015) in Opti-MEM according to the manufacturer's instructions. The p53-specific gRNA sequence was: sgp53-F:5-CACCGCCATTGTTCAATATCGTCCG, sgp53-R:5-AAACCGGACGATATTGAACAATGGC;
Animals. BALB/c mice (female, 18–20 g, 5–6 weeks), BALB/c nude mice (female, 18–20 g, 5–6 weeks) and NOD/SCID mice (female, 18–20 g, 5–6 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals and maintained at the SPF animal facility at the South China University of Technology (SCUT).
Toxicity study. BALB/c mice were randomly divided into five groups (5 mice per group) and injected intravenously with 25 mg per kg of dNRs, dNRs/Pt, NRs or NRs/Pt or PBS. 24 h after injection, the blood serum was collected and mouse alanine aminotransferase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN) and serum creatinine (SCR) were measured using Automatic Biochemical Analyzer (3100, Hitachi, Japan) according to the manufacturer's instructions. The major organs of the mice after treatment were also collected and stained by haematoxylin & eosin (H&E).
In vivo biodistribution. DiD-labelled NRs was prepared by label DiD with NRs. The real-time imaging of DiD-NRs (25 mg/kg) was conducted on ES-2 tumor-bearing BABL/c nude mice using the In-Vivo Xtreme (Bruker, German). After 24 h, mice were sacrificed and major organs (heart, liver, spleen, lung, kidney, and tumor) were harvested from mice and imaged in vitro.
Animal model studies. For the ES-2 tumor model, 1×107 of ES-2 cells were subcutaneously injected in the right ﬂank of the BALB/c nude mice. Mice were randomly divided into seven groups (6 mice for each group), and when tumor volumes reached about 100 mm3, the treatment procedures were initiated, with mice receiving the indicated injection into the tail vein every 2 days. The respective treatments were: PBS, CDDP (1mg/kg), dNRs (25 mg/kg), dNRs/Pt (25 mg/kg), NRs (25 mg/kg), NRs/Pt (25 mg/kg). Treatment was terminated on day 13, with the mice sacrificed and the excised tumors photographed and weighed. For the PDX tumor model, a human ovarian carcinoma specimen was obtained from Nanfang Hospital (Guangzhou, Guangdong, China). The mutp53 status (P72R+/+, C141Y+/+ and L350P+/-) for the tumor was verified by whole-exome sequencing conducted at Beijing Genomics Institute as well as by sequencing of the full-length p53 coding sequence after RT-PCR. The experiments using patient-derived materials have complied with all relevant ethical regulations and were performed according to the approved guidelines established by the Institutional Human Research Subjects Protection Committee of the Ethics Committee of the South China University of Technology, with the informed consent from the patients. The tumor tissue obtained after surgery was cut into 2~3 mm3 fragments and orthotopically explanted in the mammary fat pad of the NOD/SCID mice. Once reaching approximately 800 mm3, tumor xenografts were cut into 2~3 mm3 slices and propagated by following the same explanting procedure. The mice bearing xenografts of the third generation were randomly divided into 4 groups containing 6 mice per group and received the indicated treatments when tumor grew to about 100 mm3. Treatment lasted 13 days, following the same procedure as described for the ES-2 model. The body weight and tumor sizes of mice was measured every 2 days, and tumor volume was calculated by the formula V=lw2 2-1, in which l and w indicate the length and width of the tumor, respectively.
TUNEL assay. ES-2 and PDX tumors that received various treatments were used for TUNEL assay (Beyotime, C1088). 4 mm frozen sections were washed with PBS and then incubated with proteinase K for 20 min at room temperature. After washing 3 times with PBS to remove unreacted protease K, the TUNEL reaction mixture was added to rinsed slides and incubated in a humidified chamber for 60 min at 37℃. After washing with PBS, the sections were stained for 10 min with DAPI, washed with PBS, and visualized with a fluorescence microscope (Nikon, DS-Fi3)
Immunohistochemistry (IHC). The slices of parafﬁn-embedded tissues were dewaxed and rehydrated in xylene and graded alcohol solutions. Anti-p53 antibody (sc-126, 1:200 dilution) were used to stain the slices. Cell nuclei were stained with hematoxylin.
Statistical analysis. All data were expressed as Mean ± SEM or Mean ± SD, and analyzed by ANOVA. *P < 0.05, **P < 0.01 and ***P <0.001 were considered statistically significant.