Reagents and cell culture. Chemical reagents, including DMSO, SDS, NaCl, and Tris base, were purchased from Sigma-Aldrich (St. Louis, MO). MG132 and cycloheximide (CHX) were obtained from Thermo Fisher Scientific (Waltham, MA). The DMEM, RPMI-1640 medium, and Fetal Bovine Serum (FBS) for cell culture were purchased from Invitrogen (Grand Island, NY). Human oral squamous cell carcinoma (OSCC) cells, including CAL27, SCC15, SCC25, and SCC9 were purchased from American Type Culture Collection (ATCC, Manassas, VA). The cells were maintained according to ATCC protocols in a 37°C humidified incubator with 5% CO2. The ionizing radiation acquired resistance cell lines CAL27-IR and SCC25-IR were established in our laboratory by exposing CAL27 and SCC25 cells to gradually increasing dose of ionizing radiation for approximately 6 months. Briefly, CAL27 and SCC25 cells were serially irradiated with 2Gy of X-rays to a final dose of 80 Gy. Cells were cultured with DMEM medium and maintained at a 37°C humidified incubator with 5% CO2. The irradiated cells were passaged into new culture flask when growing to approximately 80% confluence. Reirradiation of Xrays was repeated over a period of 6 months (starting at 2 Gy and ending with 8 Gy for each irradiation, the dose was gradually increased for each month by 2 Gy), for a total dose of 80 Gy. Immortalized oral epithelial cell hTERT-OME was purchased from Applied Biological Materials (ABM) Inc. (Richmond, BC, Canada). The HaCat cells were purchased form Thermo Fisher Scientific (Waltham, MA). All cells were subjected to mycoplasma analysis every two months. Primary antibodies against Survivin (#2808), cleaved-PARP (#9532), VDAC1 (#4661), Bax (#14796), cleaved-caspase 3 (#9664), cytochrome C (#11940), ubiquitin (#43124), β-actin (#3700), ubiquitin (#3936), p-Survivin Thr34 (#8888), α-tubulin (#3873), p-Akt Ser473 (#4060), p-Wee1 Ser642 (#4910), His-tag (#12698), HA-tag (#2367), p-CDK1 Tyr15 (#4539), and p-CDK1 Thr161 (#9114) were obtained from Cell Signaling Technology, Inc. (Beverly, MA). Antibodies against Ki67 (ab16667) and FbxL7 (#ab59149) were purchased from Abcam (Cambridge, UK). Flag-tag (F3165) antibody was obtained from Sigma Aldrich (St. Louis, MO). Lipofectamine® 2000 (Thermo Fisher Scientific) was used for plasmid transfection following the manufacturer’s instructions.
MTS assays. MTS assay was performed as described previously (17). Briefly, human OSCC cells were seeded in 96-well plates at a concentration of 2×103/well. Cells were treated with DMSO control or xanthohumol for various time points (0, 24, 48, and 72 h), cell viability was determined by MTS assay (G3581, Promega, Madison, WI).
Anchorage-independent cell growth. The anchorage-independent cell growth assay was performed as described previously (18). Briefly, the Eagle’s basal medium containing 10% FBS, 0.6% agar, and different concentration of xanthohumol was loading to a six-well plate as an agar base. Human OSCC cells were counted at the concentration of 8,000 cells/ml and seeded into 6-well plates with 0.3% Basal Medium Eagle agar containing 10% FBS and xanthohumol. The cultures were maintained at 37°C in a 5% CO2 incubator for 2 weeks and colonies were counted.
Western blotting. Whole-cell extract (WCE) was prepared with RIPA buffer (#PI89901, Thermo Fisher Scientific) supplemented with protease inhibitors and concentrated by BCA protein assay. Western blotting was performed as previously described (19). Briefly, A total of 30 μg WCE was mixed with loading buffer and boiled at 95°C for 5 min, followed by SDS-PAGE electrophoresis and electrotransfer. The non-fat milk (5%) was used for membrane blocking at room temperature for 30 min and the membrane was incubated with the primary antibody at 4°C overnight. After incubation with anti-rabbit/mouse IgG HRP second antibody, the target protein was visualized by chemiluminescence.
Immunohistochemical staining (IHC). The tissues from xenograft tumors were fixed in 10% neutral-buffered formalin and subjected to immunohistochemical staining as described previously (20). First, the tissue slides were deparaffinized, followed by rehydration using various concentrations of ethanol. Antigen retrieval was performed by boiling the slides with the sodium citrate buffer (10 mM, pH 6.0) for 10 min. The slides were incubated with 3% H2O2 in methanol for 10 min to deactivate endogenous peroxidase. After washing with PBS, slides were blocked with 50% goat serum albumin in PBS for 1 h at room temperature, followed by incubation with primary antibody (4°C, overnight) and second antibody (room temperature, 45 min) in a humidified chamber. The target protein was visualized by DAB substrate and hematoxylin was used for counterstaining.
Natural compound screening. The 70 compounds of interest were selected from the Natural Product Library (Cat. No. L1400-01/02) from Selleck Chemicals (Houston, TX). CAL27 cells were seeded in a 96-well plate. After overnight incubation, cells were treated with 1 μM of DMSO (control) or natural compounds for 48 h. Cell viability was examined by the MTS assay. Tested compounds are listed in Supplementary Table 1.
Generation of survivin knockout stable cell lines. Two different single-guide RNAs (sgRNAs) were used to generate CRISPR-Cas9-based survivin knockout constructs (sgSurvivin#1forward, 5’- CAGTTTGAAGAATTAACCCT-3’, reverse, 5’-AGGGTTAATTCTTCAAACTG-3’, sgSurvivin#2 forward, 5’- GAACATAAAAAGCATTCGTC-3’, reverse, 5’-GACGAATGCTTTTTATGTTC-3’). The sgSurvivin plasmid was transiently transfected to the OSCC cells. Puromycin (1 μg/mL) was added to cell culture medium and maintained for 3 weeks for signal clone selection. The Fbxl7 siRNA (sc-62306), Akt1/2 siRNA (sc-43609), and siCtrl (sc-37007) were purchased from Santa Cruz Biotechnology (Dalla, TX). The primers for survivin qRT-PCR analysis is forward: CCACTGAGAACGAGCCAGACTT, reverse: GTATTACAGGCGTAAGCCACCG.
Ubiquitination analysis. The Ubiquitination analysis was performed as described previously (21). Briefly, for endogenous ubiquitination detection, cells were lysed with modified RIPA buffer (20 mM NAP, pH7.4, 150 mM NaCl, 1% Triton, 0.5% Sodium-deoxycholate, and 1% SDS) supplemented with 10 mM N-Ethylmaleimide (NEM) and protease inhibitors. The lysates were sonicated for 30 s, boiled at 95°C for 15 min, and diluted with 0.1% SDS containing RIPA buffer, then centrifuged at 16000×g for 15 min at 4°C. The supernatant was subjected to IP with survivin antibody, survivin ubiquitination was determined by IB analysis. For Nickel pull-down assay, cell lysates were prepared with lysis buffer (6 M guanidine-HCl, 0.1 M Na2HPO4/NaH2PO4, 0.01 M Tris/HCl, pH 8.0, 5mM imidazole, and 10mMβ-mercaptoethanol) supplemented with protease inhibitors and 10 mM N-Ethylmaleimide (NEM). The lysates were sonicated for 30 s, followed by incubation with 50 ml Ni-NTA-agarose (QIAGEN Inc, Valencia, CA) for 4 h at room temperature. The beads were washed with specific washing buffer and boiled with 2×SDS loading buffer containing 200 mM imidazole and subjected to western blotting.
Immunofluorescence (IF). Cells (5×103/well) seeded in a chamber slide were treated with vehicle control, XN (5 μM, 24 h), IR (4 Gy), or a XN and IR combination. Cells were maintained in the incubator for another 72 h. Cells were fixed with 4% paraformaldehyde and permeabilized in 0.5% Triton X-100 for 20 min, followed by blocking in 5% BSA for 1 h and overnight incubation with γ-H2AX antibody at 4°C in a humidified chamber . Alexa Fluor 488 dye-labeled anti-rabbit IgG was used as the secondary antibody. Nuclei were counterstained with DAPI (cat. #P36935, Thermo Fisher Scientific). The following antibodies were used for IF: γ-H2AX (cat. #ab11174, 1:100), goat anti-rabbit IgG Alexa Fluor 488 (cat. #ab150077, 1:500).
Plate colony formation assay. The cells were treated with vehicle control, XN, IR, or a XN and IR combination and seeded into a 6-cm plate (400 cells/well). The cultures were maintained for 2 weeks at 37°C in a 5% CO2 incubator. The colonies were fixed with 4% paraformaldehyde, stained with 0.5% crystal violet, and counted under a microscope. Three independent experiments were performed as indicated.
In vivo tumor growth assay. The in vivo animal study was approved by the Institutional Animal Care and Use Committee (IACUC) of Central South University (Changsha, China). (2×106) cells were s.c.injected into the 6-week-old athymic nude mice (n=6) at the right flank to generate the xenograft mouse model. Compound treatment was initiated when tumor volume reached at 100 mm3. Xanthohumol (10 mg/kg) was administrated by i.p. injection every two days, whereas the control mice were treated with vehicle control. For irradiation treatment, the tumor bearing mice were randomly divided into four groups (n=6): 1,vehicle control (0.5% dimethyl sulfoxide, 100 mL/every two days, i.p.); 2, local ionizing radiation (2 Gy/ twice per week, irradiated with X-rays using X-RAD 320, Precision X-ray, Inc.,); 3, Xanthohumol (10 mg/kg/ every two days, i.p.); 4, Xanthohumol (10 mg/kg/ every two days, i.p.) + local ionizing radiation (2 Gy/ twice per week). Tumor volume was recorded with and determined with the formula: length × width × width/2. Tumor mass was subjected to IHC staining.
Blood analysis. The EDTA-coated tubes were used for Mouse blood collection by cardiac puncture. The red blood cells (RBC), hemoglobin (Hb), white blood cells (WBC), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) were analyzed at the Laboratory of the Third Xiangya Hospital of Central South University (Changsha, Hunan, China).
Statistical analysis. The statistical analysis was conducted with GraphPad Prism 5 (San Diego, CA). The Student's t-test or one-way ANOVA was used to evaluate the difference between tested groups, and a probability value of p< 0.05 was used as the criterion for statistical significance. The experiment was performed triplicate, and all quantitative data are expressed as mean ± sd.