Resibufogenin solution
Resibufogenin is a tovena lactone compound extracted from toad. The molecular formula is C24H32O4 with a molecular weight of 384.50 g/mol. Resibufogenin is a fat-soluble monomer, we used corn oil to dissolved or 5% DMSO plus normal saline on the oscillator to apply maximum amplitude overnight to form a drug suspension. Resibufogenin were purchased from Herbest (baoji, china), HPLC > 98%.
Cell culture
Human CRC cell lines (SW480, HCT-116) and RIP3+/+ and RIP3−/− (MEF) were obtained from American Type Culture Collection (ATCC; Rockville, MD, USA). SW480 and HCT-116 were incubated in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) and RIP3+/+and RIP3−/− were incubated in Dulbecco’s modified eagle medium (DMEM; Invitrogen). All cell lines were supplemented with 10% (v/v) fetal bovine serum (FBS; Invitrogen) and 1% (v/v) penicillin–streptomycin (Invitrogen) at 37 °C in a humidified atmosphere with 5% CO2.
Xenograft CRC model
All animal research procedures were conformed to the guidelines for The Care and Use of Laboratory Animals published by the National Institutes of Health and were approved by The Laboratory Animals Care and Use Committee of Southern Medical University. SW480-eGFP (1 × 107) cells were injected subcutaneously to the groin of nude mice. Ten days later, the tumors were reached to the volume of ~ 50 mm3 which randomly divided into groups. Animals were kept in a sterile environment. Their body weights and tumor volumes were measured every 5 days throughout the treatment period. The mice were euthanized at the end of the experiments. Tumor xenografts were weighed and photographed.
Liver metastasis model
C57BL6/j mice (6 weeks old) were purchased from the Guangdong Experimental Animal Center (Guangdong, China). Animal study protocols were approved by The Animal Care and Experiment Committee of Southern Medical University. The liver metastasis model was created by intrasplenic injection of eGFP-MC38 cells. Briefly, the cells were slowly injected into the spleen with an insulin syringe and the blood was compressed after 3 min of hemostasis. After 2 weeks of intraperitoneal injection of resibufogenin, the fluorescence images was taken to observe the metastatic foci in liver. Tumor were weighed and photographed.
Lentiviral preparation, viral infection, and stable cell generation
The GV248-shRNA plasmids encoding shRNAs with sequences targeting human RIP3 were purchased from the GenePharma Facility (Suzhoui, China). The shRNA-RIP3 sequence contained 5′-ACTCTCGTAATGATGTCAT-3′. The shRNA 5′-TTCTCCGAACGTGTCACGT-3′ was incorporated as a control. Cells were infected with the collected viruses over 24 h in the presence of polybrene, followed by selection in medium containing puromycin (0.5 mg/ml) for 7–9 days.
Cell proliferation assays
Cell proliferation was measured by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide), according to the manufacturer’s protocol. Briefly, cancer cells (4 × 103) were plated onto each well of a 96-well flat-bottomed plate and grown in RPMI medium containing 10% FBS. After 24 h, cells were treated with different concentrations of resibufogenin (0.1, 1, 2.5, 5, 10 μM) and the cells were incubated for an additional 24–48 h. The MTT (Sigma, USA, 2.5 mg/ml, 10 μl) solution was added to each well and incubated for a further 4 h. When the medium was discarded, 100 μl dimethyl sulfoxide (DMSO) was added to dissolve the formazan dye. The absorbance was read at 490 nm using multiscan spectrum.
Cell invasion analysis
Cells from the serum-free medium (1 × 105 cells/100 μl) were added to the top chamber of each 8-mm–pore transwell chamber (Corning Star; Cambridge, MA, USA). The bottom chamber was prepared using 20% FBS as a chemoattractant. Cells were allowed to migrate through the porous membrane for 48 h at 37 °C. The cells that stuck to the lower surface of the membrane were treated with a fixation/staining solution (0.1% crystal violet, 1% formalin, and 20% ethanol) for visualization. The cells were counted under a microscope in five randomly selected fields (original magnification, 200×). At least four chambers from three different experiments were analyzed.
Cell migration analysis (wound scratch assay)
HCT116 cells were plated onto six-well culture plates in RPMI-1640 medium containing 2% FBS (2 × 106 cells/well). After 24 h, the cell monolayer was scraped with a sterile 20 μl micropipette tip to create a wound, washed with PBS and photographed using Nikon inverted microscope, Thereafter, the cells were treated with resibufogenin (5 μM). After 36 h treatment period, the plates were photographed using the camera system.
Flow cytometry
Two CRC cell lines (SW480 and HCT-116) were seeded in 6-well plates at a concentration of 1 × 105 cells per well. After the cells adhered to the wall, 2 mL of resibufogenin was added to each well. A blank control group was set, then the plates were incubated for 24 or 48 h. Thereafter, the plates were analyzed by flow cytometry using a Beckman Analytical Flow Cytometer in accordance with the Flow Kit Specification (BD). A quadrant graph consisting of four quadrants was obtained and the number of cells per quadrant was the proportion of the total number of cells examined. The second quadrant represents the early apoptotic cells and the third and fourth quadrants represent the late apoptotic and necrotic cells respectively.
LDH assay
Cell damage was determined by the release of lactate dehydrogenase (LDH) into the cell culture medium. The release of LDH was quantified using the LDH Cytotoxicity Assay Kit (Beyotime Biotechnology, CHINA) according to the manufacturer’s instructions.
Real-time quantitative polymerase chain reaction (qRT-PCR) analysis
Total RNA was isolated from HCT116 and SW620 cells using Trizol reagent (ET101, TransGen Biotech, CHINA), cDNA was synthesized (RR037A, TAKARA BIO, JAPAN) and amplified with a PCR kit (218073, QIAGEN, Germany). qRT-PCR was repeated 3 times. GAPDH was used as the reference control. The primers used for qRT-PCR are summarized as follows: RIP3, Sense: 5-GACCTCAAGCCCTCCAATGTTC-3 and Antisense: 5-AAGTAAGCTAGGGTGCCCCCA-3; GAPDH, Sense: 5-ACCACAGTCCATGCCATCAC-3 and Antisense: 5-TCACCACCCTGTTGCTGTA-3.
Western blot analysis
Protein expression was assessed by immunoblot analysis of cell lysates (30–60 μg) in RIPA buffer in the presence of rabbit antibodies to RIP3 (1:1000; Cell Signaling Technology), E-cadherin, fibronectin (FN), β-actin (1:500; Santa Cruz Biotech, CA, USA), GLUD1, PYGL, GLUL (1:500; Proteintech, Danvers, MA, USA), Cyt-c, and apoptosis-inducing factor (AIF) (1:500; Abcam, Cambridge, UK). The specific protein bands were visualized using an enhanced ECL system (Bio-Rad).
Immunofluorescence
Cells were stained with MitoTracker and incubated with antibody at 4 °C overnight, then incubated with Cy3-labeled goat anti-rabbit or anti-mouse IgG antibody (1:500) in darkness for 60 min at room temperature. The cells were then counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and examined under a confocal microscope (Nikon, Japan) with excitation and emission wavelengths of 550 and 570 nm, respectively, and a 100 × 1.40 NA oil immersion objective. For mitochondrial staining, 100 nM MitoTracker Green (Molecular Probes) was added to cultures 30 min before fixation.
ROS detection
Cells were washed three times with PBS and stained with 20 μM DCFH-DA for 30 min at 37 °C and 5% CO2 in an incubator. The cells were trypsinized, collected by centrifugation, washed again using PBS, and re-suspended in 1 mL PBS. ROS generation was measured using flow cytometry (Beckman Analytical Flow Cytometer).
Immunohistochemistry (IHC) staining
Tumor tissue was fixed with 4% PFA, paraffin embedded, cut into 5 μm samples. Immunohistochemical staining was conducted according to the manufacturer’s protocol. Briefly, endogenous peroxidase was blocked in a peroxidase blocking solution (0.03% hydrogen peroxide containing sodium acid) for 5 min. Tissue sections were washed gently with phosphate buffer saline (PBS, pH 7.2) and subsequently incubated with RIP3 (1:200; Abcam, Cambridge, UK), PYGL (1:50; Abcam, Cambridge, UK) and GLUL (1:200; Abcam, Cambridge, UK) and GLUD1 (1:100; Abcam, Cambridge, UK) antibodies at − 4 °C, overnight. The second day, the slides were incubated with secondary antibodies (Pre-diluted; Zhongshan Golden Bridge, Beijing, China), slides were counterstained with haematoxylin before mounting.
Frozen sections
Tumor cryosections (5 μm-thick) were fixed for 20 min in freshly cold acetone. Sections were washed in phosphate buffered saline with 0.3% Trixton-X100 for 15 min, and then goat serum blocked for 1 h. Sections were incubated overnight at 4 °C with antibodies above. The second day, the sections were incubated for 1 h at room temperature with a goat anti-rabbit Cy2-conjugated secondary antibody (1:200, Thermo Fish, USA), After several washes in PBS the sections were mounted in Vectashield (Vector Laboratories) containing 4′,6-diamidino-2-phenyl indole (DAPI). Stained sections were imaged on confocal laser scanning microscopy.
Statistical analyses
The results were expressed as the mean ± SEM from three independent experiments. The P-values were two-tailed and calculated using one-way ANOVA. Statistical significance was specified as P < 0.05.