Antibodies and reagents
Antibodies against cleaved-PARP was purchased from Proteintech (Chicago, IL). Antibodies specific to phospho-H2AX and phospho-CHK2 were purchased from Cell Signaling (Danvers, MA). Antibodies against β-actin, proliferating cell nuclear antigen (PCNA), and rabbit horseradish peroxidase (HRP)-conjugated antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against CD44 was purchased from Novus Biologicals (Centennial, CO). Other reagents and chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
Purification of CDT subunits
Recombinant His-tagged CdtA, CdtB and CdtC subunits were constructed as described in our previous study [23]. A single colony of Escherichia coli BL21-DE3 containing cdtA, cdtB, cdtC expression plasmids, respectively, were picked and cultured in LB broth with ampicillin (100 μg/mL) at 37°C. E. coli harboring cdtA, cdtB, cdtC recombinant plasmids were then induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37°C for 5 h for which containing cdtA and cdtB, and at 16°C for 16 h for which containing cdtC, respectively. Recombinant proteins were purified by metal affinity chromatography (Clontech, CA, USA) and analyzed by SDS-PAGE and western blot assay.
Preparation of HA-decorated nanoparticle-encapsulated CdtB
Recombinant CdtB was purified according to the protocol as described above. To prepare HA-decorated nanoparticle-encapsulated CdtB, a simple ionic gelation with magnetic was stirred at room temperature. The determination of the optimal preparation conditions was performed by examining different proportions of HA/arginine-chitosan (HA/Arg-CS). In brief, aqueous HA (4.00 mg/mL, 0.50 mL) was added into aqueous Arg-CS with various concentrations (1.00, 2.00, 3.00 or 4.00 mg/mL, 0.50 mL), and then the solutions were gently shaken for 30 min at 37°C. After collecting the prepared HA/Arg-CS NPs produced by centrifugation, a Zetasizer instrument was used to measure the particle size, polydispersity index, and zeta potentials. Meanwhile, the NP suspension was placed onto a mesh copper grid and positively stained with osmium tetroxide to observe their morphology under transmission electron microscopy.
Next, different concentrations of CdtB were encapsulated in nanoparticles to determine the optimal condition for preparing CdtB-loaded HA/Arg-CS NPs. CdtB dissolved in deionized water (3.00, 2.00, 1.00 mg/mL; 0.25 mL) was mixed with HA solution (8.00 mg/mL; 0.25 mL) to form CdtB/HA aqueous mixed solutions (1.50:4.00, 1.00:4.00, and 0.50:4.00 mg/mL, 0.50 mL). The CdtB/HA solutions (0.50 mL) were added to the optimal concentration of Arg-CS in deionized water (0.50 mL) and stirred at 37°C for 30 min. After centrifugation, the amount of free CdtB in the supernatant was detected by a protein concentration assay (Bio-Rad, CA, USA) and protein-loading efficiency of the NPs calculated from the following equation:
Cell culture
Human prostate epithelial cell line (PZ-HPV-7) was derived from the peripheral zone of a benign prostate and cultured as described previously [24]. The methods that used to knockdown endogenous DOC-2/DAB2 interactive protein (DAB2IP) in PCa cells were described previously [25]. PC3 knockdown DAB2IP (PC3-KD) cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Hyclone, Logan, UT) with 5% fetal bovine serum (FBS) (Hyclone) containing puromycin (0.4 μg/mL), 1× penicillin/streptomycin, and G418 (800 μg/mL) in the environment of 37°C and 5% CO2.
Analysis of cell viability
PC3-KD cells were seeded in 96-well plates and treated with 0 nM, 10 nM, 50 nM, 100 nM, 200 nM, 500 nM, and 1,000 nM of HA/Arg-CS (HA-NPs), CDT holotoxin and HA-CdtB-NPs, respectively. After incubation for 48 h, cells were treated with 100 μL of 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution in 37°C for 2 h. Cell viability were analyzed by measuring the ability of viable cells reducing MTT (Sigma-Aldrich) to formazan [26].
HA-CD44 localization and DNA double strand break (DSB) by immunofluorescence
PC3-KD cells were seeded onto cover glasses and incubated for 24 h. For DSB analysis, cells were first treated with 2 Gy ionizing radiation (IR), then exposed to 200 nM of HA-NPs, CDT holotoxin and HA-CdtB-NPs, respectively. Cells were subsequently fixed with 1% paraformaldehyde for 1 h, permeabilized by 0.1% triton X-100 for 15 min. For HA-CD44 colocalization assay, cells were then treated with primary antibody against CdtB. For DSB analysis, cells were treated with anti-pH2AX and anti-53BP1 at the dilution of 1:200, followed by treatment with secondary antibody at the dilution of 1:500 and 4’,6-diamidino-2-phenylindole DAPI (0.2 μg/mL). The stained cells were observed using Laser Scanning Confocal Microscope (LSM780, ZEISS, Germany).
Analysis of CdtB in the nuclear extraction
PC3-KD cells were treated with 100 nM HA-NPs, CDT holotoxin, and HA-CdtB-NPs, followed by incubation for 0, 0.5, 1, 3, and 6 h. The nuclear proteins were isolated using a nuclear extraction kit (Pierce, Rockford, IL), as described previously [23]. CdtB level in the nuclear fraction was then analyzed by western blot assay.
Western blot analysis
PC3-KD cells were seeded onto 60 mm dish and incubated in 37°C for 24 h, then treated with medium (mock), IR, HA-NPs accompanied by IR, CDT holotoxin accompanied by IR, and HA-CdtB-NPs accompanied by IR, respectively, for 24 h and 48 h. Cell lysates were centrifuged at 12,000 rpm for 20 min at 4°C. After quantification, samples were resolved by 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (PVDF, Millipore, MA). The PVDF membrane was blocked and probed with primary antibodies against (ADP-Ribose) P polymerase (PARP), pH2AX, pATM, pCHK2, and β-actin in the condition of gentle shaking. After an overnight incubation at 4°C, the membrane was then probed with horseradish peroxidase-conjugated secondary antibody (Millipore) in room temperature for 1 h. The proteins of interests were detected using ECL Western Blotting Detection Reagents (GE Healthcare, Piscataway, NJ, USA) and were visualized using an Azure c400 system and AzureSpot Analysis Software (Azure Biosystems, USA) by following the manufacturer’s instructions.
Cell cycle analysis by flow cytometry
PC3-KD cells were incubated with ICRF-193 (2 μg/mL), HA-CdtB-NPs (50 nM,100 nM, and 200 nM), and HA-NPs alone for 24 h. The cells were washed with PBS, centrifuged for 1,000 rpm in room temperature for 5 min. Subsequently, the prepared cells were resuspended in hypotonic buffer (0.2 mg/mL RNase A, 20μg/mL propidium iodide, and 0.1% Triton X-100). Analysis of cell cycle were performed by flow cytometry (Becton Dickinson, CA).
Comet assay
Experiment protocols followed instructions by Trevigen CometAssay® Kit (Trevigen, Gaithersburg, MD, USA). PC3-KD cells were seeded in 60 mm dish and respectively treated with medium (mock), HA-NPs, CDT holotoxin, and HA-CdtB-NPs with three of the latter accompanied with IR. After incubation for 24 h, PC3-KD cells (1×105/mL) were first mixed with LMAgarose with the proportion of 1:10 and placed onto CometSlideTM with complete coverage of sample area, then incubated the slide at 4°C in the dark for 30 min. Cells were then immersed with 4°C lysis solution at 4°C for 30-60 min and followed by alkaline unwinding solution at 4°C in the dark for 1 h. CometSlideTM were placed in alkaline electrophoresis solution and set power supply to 21 volts then applied voltage for 30 min and washed by distillation-distillation H2O (ddH2O) and 70% alcohol. After CometSlideTM were dried and stained by CYBR® Gold, cells were observed using Leica CTR 4000 (Leica, Germany).
Statistical analysis
The analysis among multiple groups was carried out by means of one-way analysis of variance. The post-hoc test was used to measure the statistical significance of differences between two groups. Statistical analyses and chart drafting were carried out using the GraphPad Prism 6 software (GraphPad Software Inc, CA, USA).