We prepared a 50-mM solution of betulinic acid (purity >98%; Alexis, San Diego, CA) in ethanol, stored the solution as small aliquots at −20°C, and then diluted the solution as needed in cell culture medium. We purchased Hoechst 33342, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Tris, glycine, NaCl, sodium dodecyl sulfate (SDS), and bovine serum albumin from Sigma-Aldrich (St. Louis, MO), and we obtained Roswell Park Memorial Institute (RPMI) medium 1640, fetal bovine serum (FBS), 0.4% trypan blue vital stain, and antibiotic-antimycotic mixture from Life Technologies (Grand Island, NY). We obtained rabbit polyclonal STAT3 antibodies and mouse monoclonal antibodies against phospho-STAT3 (Tyr705), bcl-2, bcl-xL, SHP-1, cyclin D1, procaspase-3, and poly(ADP-ribose) polymerase (PARP) from Santa Cruz Biotechnology (Santa Cruz, CA), and we purchased goat anti-rabbit horseradish peroxidase (HRP) conjugate from Bio-Rad (Hercules, CA). We also purchased antibodies to phospho-specific Src (Tyr416), Src, phospho-specific JAK1 (Tyr1022/1023), JAK1, and JAK2 from Cell Signaling Technology (Beverly, MA), goat anti-mouse HRP from Transduction Laboratories (Lexington, KY), and goat anti-rabbit Alexa 594 from Molecular Probes (Eugene, OR). We obtained bacteria-derived recombinant human IL-6 from Novartis Pharmaceuticals (East Hanover, NJ). We obtained bortezomib (PS-341) from Millennium (Cambridge, MA) and thalidomide from Tocris Cookson (Ellisville, MO). The glutathione S-transferase (GST)-JAK2 substrate was provided by Dr. Zhizhuang Joe Zhao (Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK). The constitutive active STAT3 construct was kindly provided by Dr. John DiGiovanni from The University of Texas MD Anderson Cancer Center, Smithville, Texas.
We obtained the human MM cell lines U266 and MM.1S (melphalan-sensitive) from the American Type Culture Collection (Manassas, VA). The U266, MM.1S, prostate carcinoma PC-3 and Du-145 cells were cultured in RPMI 1640 medium containing 1X antibiotic-antimycotic solution with 10% FBS. Human breast carcinoma MCF-7 and MDA-MB-231 were cultured in DMEM/F12 supplemented with 10% FBS. Human embryonic kidney A293 cells were cultured in DMEM with 10% FBS. Human squamous cell carcinoma SCC-4 cells were cultured in DMEM containing 10% FBS, 100 μmol/L nonessential amino acids, 1 mmol/L pyruvate, 6 mmol/L L-glutamine, and 1× vitamins. Cells were maintained at 37°C in an atmosphere of 5% CO2-95% air.
Electrophoretic mobility shift assay for STAT3-DNA binding
We analyzed STAT3-DNA binding with the electrophoretic mobility shift assay (EMSA) using a 32
P-labeled high-affinity sis-inducible element (hSIE) probe (5'-CTTCATTTCCCGT AAATCCCT AAA GCT-3' and 5'-AGCTTTAGGGATTTACGGGAAATGA-3') as previously described12
. Briefly, we prepared the nuclear extracts from betulinic acid-treated cells and incubated the extracts with the hSIE probe. We separated the DNA-protein complex formed from the free oligonucleotides on 5% native polyacrylamide gels. We then visualized the dried gels and quantitated the radioactive bands using Storm 820 and Image quant software (Amersham Biosciences, Piscataway, NJ).
Western blot analysis
To detect various proteins, we treated the U266 cells (2 × 106/mL) with betulinic acid. The cells were then washed and extracted by incubation for 30 min on ice in buffer containing 20 mM HEPES (pH 7.4), 2 mM ethylenediaminetetraacetic acid, 250 mM NaCl, 0.1% NP-40, 2 μg/mL leupeptin, 2 μg/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride, 0.5 μg/mL benzamidine, 1 mM dithiothreitol (DTT), and 1 mM sodium vanadate. The lysate was centrifuged, and the supernatant was collected. Whole-cell extract protein (30 μg) was resolved on 7.5%–12% SDS-polyacrylamide gel electrophoresis (PAGE), electro transferred onto a nitrocellulose membrane, blotted with antibodies, and then detected by electrochemiluminescence (Amersham Biosciences).
Immunocytochemistry for STAT3 localization
Betulinic acid-treated MM cells were plated on a glass slide by centrifugation using a cytospin 4 (ThermoShendon, Pittsburgh, PA), air-dried for 1 h at room temperature, and fixed with 4% formaldehyde. After a brief washing in phosphate-buffered saline (PBS), slides were blocked with 5% normal goat serum for 1 h and then incubated with a rabbit polyclonal antihuman STAT3 antibody (dilution 1/100). After overnight incubation, the slides were washed and then incubated with goat anti-rabbit immunoglobulin (Ig)G-Alexa 594 (dilution 1/100) for 1 h and counterstained for nuclei with Hoechst 33342 (50 ng/mL) for 5 min. We stained the slides with mounting medium (Sigma-Aldrich) and analyzed them under an epifluorescence microscope (Labophot-2; Nikon, Tokyo, Japan). We used a Photometrics Coolsnap CF color camera (Nikon) and MetaMorph version 4.6.5 software Molecular Devices, Downingtown, PA) to capture the pictures.
STAT3 luciferase reporter assay
A293 cells were plated in six-well plates with 5 × 105 per well in DMEM containing 10% FBS. The STAT3-responsive elements linked to a luciferase reporter gene were transfected with wild-type or dominant-negative STAT3-Y705F (STAT3F). Transfections were done according to the manufacturer's protocols using Fugene-6 (Roche). At 24 h post transfection, cells were pretreated with betulinic acid for 4 h and then induced by IL-6 for additional 24 h before being washed and lysed in luciferase lysis buffer (Promega). Luciferase activity was measured with a luminometer by using a luciferase assay kit (Promega) and was normalized to β-galactosidase activity. All luciferase experiments were done in triplicate and repeated three times. The data show the mean and the SD of the mean of the experiments.
We determined the anti-proliferative effect of betulinic acid against the MDA-MB231, MCF-7, MM cells, Du-145, PC-3 line using the MTT dye uptake method.
We determined the viability of the cells using the Live/Dead assay (Invitrogen, Carlsbad, CA), which measures intracellular esterase activity and plasma membrane integrity.
Flow cytometric analysis
To determine the effect of betulinic acid on the cell cycle, we first synchronized the U266 cells by serum starvation and then exposed the cells to betulinic acid. We washed the cells with PBS, and fixed with 70% ethanol, and then incubated for 30 min at 37°C with 0.1% RNase A in PBS. We then washed the cells again, resuspended and stained them with PBS containing 25 °g/mL propidium iodide for 30 min at room temperature. We analyzed the cell distribution across the cell cycle with a FACS Calibur flow cytometer (BD, Franklin Lakes, NJ).
To determine the effect of betulinic acid on JAK2 activation, we performed an immuno complex kinase assay using GST-JAK2 as the substrate. Briefly, we precipitated the JAK complex from the whole-cell extracts with an antibody against JAK2 with protein A/G-Sepharose beads (Pierce Biotechnology, Rockford, IL). After 2 h, we washed the beads with whole-cell extract buffer and then resuspended the complex in a kinase assay mixture containing 50 mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (pH 7.4), 20 mM MgCl2, 2 mM DTT, 20 μCi [-32P]adenosine triphosphate (ATP), 10 μM unlabeled ATP, and 2 μg substrate GST-JAK2. After incubation at 30°C for 30 min, we terminated the reaction by boiling the complex-kinase mixture with SDS sample buffer for 5 min. Finally, the protein was resolved on 10% SDS-PAGE, the gel was dried, and the radioactive bands were visualized with the Storm 820 imaging system. To determine the total amounts of JAK2 in each sample, we resolved 30 μg of the whole-cell proteins on 10% SDS-PAGE, electro transferred the proteins to a nitrocellulose membrane, and then blotted with anti-JAK2 antibody.
Transfection with SHP-1 siRNA
SCC4 cells were plated either in six-well plates or chamber slides allowed to adhere for 24 h. On the day of transfection, 12 μl of HiPerFect transfection reagent (QIAGEN) was added to 50 nM SHP-1 siRNA in a final volume of 100 μl of culture medium. After 48 h of transfection, cells were treated with betulinic acid for 4 h, and whole-cell extracts were prepared for SHP-1, STAT3, and phospho-STAT3 analysis by Western blot. Cells transfect in the chamber slides were treated with betulinic acid for 24 h and viability of the cells were determined by Live-Dead assay.
Transfections with Constitutive STAT3 Construct
A293 cells were plated in chamber slides in DMEM containing 10% FBS. After 24 h, the cells were transfected with constitutive STAT3-plasmid by FuGene 6 according to manufacturer's protocol (Roche, Indianapolis, IN). Twenty-four hours after the transfection, cells were treated with betulinic acid for 24 h, and viability of the cells was determined by Live/Dead assay.