ELAS1 induces apoptotic death in adenocarcinoma DU145 and squamous-cell carcinoma SAS cancer cells, but not in normal KD cells

We previously reported that an ELAS1 peptide containing 29 amino acids induces apoptotic death in U2OS human osteosarcoma cells following DNA double-strand break insults. Here, we show that ELAS1 also caused apoptosis in prostate adenocarcinoma DU145 cells and tongue squamous-cell carcinoma SAS cells. ELAS1 appears to be safe because it induced apoptosis only in cancer cells, not in normal KD cells. Because the effect of ELAS1 is dependent on increased stability of p53 and enhanced phosphorylation of p53-S46, we exogenously expressed wild-type p53 protein to fully promote ELAS1-mediated induction of apoptosis in SAS cells. Interestingly, simultaneous expression of Myc-ELAS1 and FLAG-p53 mediated by an internal ribosome entry site efficiently induced apoptosis in SAS cells. Moreover, we prepared a recombinant adenovirus that simultaneously expressed Myc-ELAS1 and FLAG-p53. This adenovirus also killed SAS cells, as determined by a cell viability assay, in the presence of camptothecin, an inducer of DNA double-strand breaks. Moreover, nude mice harboring Myc-ELAS1-expressing SAS cells lived longer than mice harboring Myc-vector-expressing SAS cells, suggesting the usefulness of ELAS1 in vivo. Notably, Cy5-tagged ELAS1-t, which contained only ten amino acids, also efficiently induced apoptosis in both DU145 and SAS cells, suggesting the usefulness of ELAS1-t as a peptide. Taken together, our results suggest that ELAS1 is therapeutically useful as a peptide drug.


Preparation of DU145 and SAS Adv cells and their Tet-ON inducible cell lines
To prepare tetracycline-inducible DU145 (DU145/ Tet-On cells) or SAS (SAS/Tet-On cells) cells, DU145 or SAS cells were transfected with the pTet-On Advanced Vector plasmid (Clontech) and incubated in DMEM supplemented with 10% FBS or Tet system-approved FBS (Clontech, 631101), penicillin/streptomycin, and 0.8 mg/ ml G418. Thereafter, several single colonies were selected for further examination.
The pTRET3-6Myc vector was constructed by inserting a multi-cloning site linker containing 6Myctag (BamHI-HindIII-ClaI-6Myc-AscI-EcoRV-NotI-SalI) into the BamHI and SalI sites of the pTRE-Tight vector (Clontech). DU145/Tet-On and SAS/Tet-On cells were co-transfected with each of the pTRET3-6Myc plasmids (including the empty Myc-vector and Myc-ELAS1) together with Linear Hygromycin Marker (Clontech) using Lipofectamine and PLUS reagents according to the manufacturer's instructions (Invitrogen). Transfected cells were serially diluted and selected with culture medium containing hygromycin (0.2 mg/ml). Single colonies were isolated and positive clones were confirmed by examining the expression patterns in the presence or absence of Dox (1 μg/ml).

γ-IR and drug treatment
Cells were cultured on coverslips and treated with 10 Gy γ-IR using a Gammacell 40 Exactor (Best Theratronics) or the following drugs: 20 μM irinotecan, 100 μg/ml 5-FU, or 10 μM CPT. After treatment with these drugs for 48 h, the culture medium was removed, the cell plate was washed with calcium-and magnesiumfree PBS [PBS (-)], and cells were subjected to FC and Wb analyses. Irinotecan and CPT were purchased from Sigma-Aldrich.

Peptide transfection
The Cy5-labeled ELAS1-t peptide was introduced into DU145_Adv or SAS_Adv cells plated on glass coverslips with a JBS Protein Transduction Kit (Jena Bioscience) and incubated for 24 h (NT sample). Atto488-BSA was used as a positive control for successful transduction. At 3 h after peptide transfection, IR48 samples were treated with 10 Gy IR and then incubated for 48 h. The cells on coverslips were washed two times with PBS (-), three times with glycine buffer, and two times with PBS (-), and then fixed by sequential incubation with 4% formaldehyde prepared in PBS (-), 0.1% Triton X-100 prepared in PBS (-), and 0.05% Tween-20 prepared in PBS (-). After washing, apoptotic cells were detected by the TUNEL assay and observed using a laser scanning confocal microscope (FV10i; Olympus).

Wb analysis
Total cell lysates and immunoprecipitates were resolved by SDS-PAGE and then transferred to PVDF membranes. The membranes were blocked with TBS-T (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 0.05% Tween-20) containing 5% skimmed milk, and then probed with the relevant antibodies. Immunoreactive protein bands were visualized using Western Lightning Plus-ECL (PerkinElmer, NEL105001EA) or Western Lightning ECL Pro (PerkinElmer, NEL122001EA).

GST pull-down and inhibition assay
To prepare GST-purified protein baits, GSTfused CycG1 and B'γ proteins expressed in the E. coli PR745 strain were lysed by sonication and purified using Glutathione Sepharose 4B (GE Healthcare). An in vitro transcription/translation system (TnT T7 Quick Coupled System, Promega) was used to prepare lysate prey, in which pCMV6myc-CycG1 or PP2A_B'γ was added to rabbit reticulocyte lysate (TnT Quick Master Mix) containing 20 μM methionine and incubated at 30°C for 90 min. Protein-protein interactions were examined by adhering the GST-fusion proteins to Glutathione Sepharose 4B, followed by incubation with the TnT lysates expressing 6Myc-tagged proteins by rotating at 4°C overnight. The bound proteins were eluted by boiling in Laemmli buffer after washing with 1 ml of N04 buffer containing 5-fold-concentrated protease inhibitors and then analyzed by SDS-PAGE and western blotting. For the peptide inhibition assay, the GST pull-down assay was conducted in the presence or absence of the indicated peptides ( Figure  6), followed by Wb using an anti-Myc or anti-GST (loading control) antibody.

FC analysis
Incubated cells were washed with PBS (-), collected by trypsinization and centrifugation, resuspended, washed twice in PBS (-), and fixed with 70% ethanol. Then, cells were treated with propidium iodide (20 µg/ml) and RNase A (200 µg/ml). Cell sizes and DNA contents were measured using a FACSCalibur instrument (Becton Dickinson) and analyzed using CellQuest software (BD Bioscience).

Cell viability assay
To examine the frequency of cell viability, we performed the methyl tetrazolium (MTT) assays. SAS or KD cells were plated at 96-well plates (1.0×10 4 cells/ well) and incubated overnight. Then, 40 ng/well of DNA were transfected into SAS or KD using lipofectamine (Thermo Fisher Scientific). After 24 h of transaction, Dox was added and incubated for further 48 h. Then, cells were exposed to 10 Gy γ-IR. After additional 48 h of incubation, MTT assays were performed according to the manufacturer's protocol (Dojinbo). Briefly, 10 μl of cell counting Kit-8 (Dojindo, http://www.dojindo.com/ store/p/456-Cell-Counting-Kit-8.html) was added into cells, which were incubated for an additional 3 h. Then, absorbance at 450 nm was measured with a plate reader. The cell viability was determined by (absorbance of cells)/ (absorbance of non-treated vector cells) × 100. Nontreated vector cells were used as a standard.

TUNEL assays
DU145 and SAS cells plated and incubated on cover glasses were fixed by sequential incubation with 4% formaldehyde prepared in PBS (-) for 25 min at 4°C, 0.2% Triton X-100 prepared in PBS (-) for 5 min, and 0.05% NaN 3 prepared in PBS (-) for 10 min at room temperature. The DeadEnd Colorimetric TUNEL System (Promega), which end-labels fragmented DNA, was used to detect apoptotic cells. Then, the fixation solution was washed out and cells were incubated with recombinant terminal deoxynucleotidyl transferase in the presence of biotinylated nucleotides for 1 h at 37°C. Next, cells were washed with PBS (-), treated with 2× SSC for 15 min at room temperature in the dark, stained with Hoechst 33258 (Sigma), and observed using a BX51 microscope (Olympus).

Preparation of adenoviruses and infection of SAS cells
A recombinant adenovirus expressing the Kozak+6Myc-ELAS1+IRES+3Flag-p53 construct under the control of the CAG promoter was prepared by TaKaRa Inc., sent to Unitech Inc. (http://www.uniqtech.co.jp/), and amplified in 293 cells (ATCC, CRL-1573). SAS cells were infected with the amplified adenovirus at a multiplicity of infection of 1.0. A cell viability assay was conducted using crystal violet according to the protocol shown in the bottom panel of Figure 4A. These experiments were performed by Unitech Inc.

Confluency analysis
Extraction and measurement of the areas of adherent culture cells in the cell viability assay ( Figure 4B) were performed using GIMP software, ver. 2.8.14 (GIMP Development Team).Quantification of confluency and statistical significance was performed using Microsoft Excel 2013 software (Microsoft). Briefly, parameters for "Select by Color Tool" in GIMP software were selected as follows: Antialiasing, OFF; Feather edges, OFF; Select transparent areas, OFF; Sample merged, OFF; Threshold, 30.0; Selt by: Saturation. Then, vacant area without cells in every well of the plate was selected for confluency analysis. Here, we selected the vacant area of the plate ( Figure 4B) as a whole, but not for each independent well, in order to attain equal extraction conditions between the wells by eliminating the influence of subjectivity upon counting. Subsequently, the quantity of extracted area (number of pixels) were transferred to Excel software in accordance with the instructions of GIMP (Colors > Info > Histogram) to create a bar graph ( Figure 4C).

Tumorigenesis in nude mice
All animal experiments were performed with the approval of the Animal Experiments Committee of Osaka University (permission number: BikenA-H24-17-0). SAS cells that expressed Dox-inducible Myc-vector or Myc-ELAS1 were injected into the left side of the tongue of BALB/c Slc-nu/nu female nude mice (5-6 weeks old; Japan SLC, Inc.). Each mouse was injected with 2 × 10 6 cells ( Figure 5A and Figure S10A) or 1 × 10 6 cells ( Figure  S10C) prepared in 50 μl of DMEM. The body weight of each mouse was measured every 2 or 3 days. Dox (1.6 mg per mouse) was intraperitoneally injected about twice per week (on the days indicated by orange arrows in Figure 5). In some cases ( Figure 5A and Figure S10A), each mouse was intraperitoneally injected with 0.1 mg of irinotecan prepared in 100 μl of saline once per week (on the days indicated by red arrows in Figure 5 and Figure S10). IHC images were obtained using the implanted tumors of nude mouse sacrificed at 14 days (this mouse was derived from an independent experiment) or sacrificed at 40 days after implantation.