2.1. Materials
Triptonide, tripterifordin, wilforgine, wilforine, oryhosphenic acid, triptophenolide, wilforlide A, triptolide, celastrol and demethylzeylasteral were obtained from Chengdu Must Biotech, Ltd, with a purity > 98%. Triptonide was dissolved in dimethyl sulfoxide (DMSO) for in vitro studies or in normal saline for in vivo experiments. Bafilomycin A1 and a p38 MAPK inhibitor (SB203580) were purchased from MCE (Shanghai, China) and dissolved in DMSO. Primary antibodies against p53, phospho-p53, p38, phospho-p38, cleaved PARP, GAPDH, CDK2, CDC25A, cyclin A, LC3, p62/SQSTM1, Beclin1 and others were purchased from Cell Signaling Technology (Boston, MA). The secondary antibodies goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
2.2. Cells and cell culture
The human ovarian cancer cell Line A2780 was obtained from Sunncell Biotechnology (Wuhan, China). The cells were maintained in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin–streptomycin (KeyGEN, Jiangsu, China). The cells were cultured at 37°C in an atmosphere containing 5% CO2.
2.3. Cell viability assay
The cells were seeded into 96-well plates at 3× 103 cells/well. After 24 h, the cells were incubated with different concentrations of triptonide (0, 4, 8, 16, 32 or 64 nM) for 48 h. Then, 20 µL of MTT solution (5 mg/mL) (Beyotime Biotechnology Shanghai, China) was added to each well, and the plates were incubated for another 4 h at 37°C before the optical density was measured at 570 nm by a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).
2.4. Colony formation assay
A2780 cells were seeded in six-well plates at a density of 500–800 cells/well, allowed to attach for 24 h, and subsequently exposed to different concentrations of triptonide (0, 0.25 and 0.5 nM) for 7–14 days. The cells were fixed with 4% paraformaldehyde for 20 min and subsequently stained with 0.5% crystal violet solution for 30 mins. The stained colonies were counted and photographed after washing and air drying.
2.5. Flow cytometry analysis
A2780 cells were seeded in six-well plates, allowed to attach for 24 h and exposed to different media for 24 h. Then, the cells were harvested to prepare cell suspensions. The cells were stained with PI according to the instructions of the Cell Cycle Analysis Kit (KeyGEN, Jiangsu, China). Apoptosis was assessed according to the instructions of the Annexin V-FITC/PI apoptosis detection kit (KeyGEN, Jiangsu, China). The expression of both were analyzed by flow cytometry (Becton, Dickinson and Company, VT).
2.6. EdU staining assay
A2780 cells were treated with different concentrations of triptonide for 24 h after they were seeded in 96-well plates at a density of 5000 cells per well. EdU staining was subsequently performed to detect cell proliferation activity using a KitkFluor555 Click-iT EdU Kit (KeyGEN, Jiangsu, China) according to the manufacturer’s instructions. The number of fluorescent dots represents the cell proliferation efficiency.
2.7. Quantitative real-time polymerase chain reaction (qRT–PCR)
Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen, CA, USA) and reverse-transcribed using a cDNA reverse transcription kit. (Vazyme Biotech, Nanjing, China). qPCR assays were performed with ChamQ SYBR Color qPCR Master Mix (Vazyme Biotech, Nanjing, China). The sequences of the PCR primers used were as follows: p53-Forward: 5′-CAGCACATGACGGAGGTTGT-3′, reverse: 5′-TCATCCAAATACTCCACACGC-3′; 18S-Forward: 5′-GTAACCCGTTGAACCCCATT-3′, reverse: 5′-CCATCCAATCGGTAGTAGCG-3′.
2.8. siRNA transfection
P53 siRNA was purchased from GenePharma (Jiangsu, China).
Cells were transfected with P53 siRNA plasmids using Lipofectamine 2000 reagent (Invitrogen) and Opti-MEM (Gibco). The experiment was conducted in three groups. The sequences of the P53 siRNA primers used were as follows:
si-P53 (sequence 1 is 5’-AAUAUUCUCCAUCCAGUGGTT-3’, and sequence 2 is 5’-CACCATCCACTACAACTACAT-3’). si-Control (5’-UUCUCCGAACGUGUCACGUTT-3’).
2.9. Western blot analysis
After the cells were exposed to drugs for 24 h, they were homogenized in 1× RIPA buffer (Cell Signaling Technology, MA, USA), and the protein supernatant was separated by centrifugation at 12,000 × g for 15 min at 4°C. The protein concentrations were determined by using a BCA Protein Assay Kit (KeyGEN, Jiangsu, China). The protein samples were separated via SDS‒PAGE and transferred to 0.45- and 0.22-µm PVDF membranes (Millipore, Billerica, MA, USA). After they were blocked with 5% skim milk in 1× TBST for 1 h at room temperature, the membranes were incubated with primary antibody overnight at 4°C followed by incubation with secondary antibody for 1 h at room temperature. The immunoreactive bands were visualized with a gel imaging system (Bio-Rad, CA, USA) using an enhanced chemiluminescence (ECL) kit (Bio-Rad, CA, USA) and analyzed using ImageJ software.
2.10. In vivo study
Subcutaneous injection of tumor cells was used to establish a xenograft tumor model in nude mice. Approximately 400x104 A2780 ovarian cancer cells were subcutaneously inoculated in the right flanks of the BALB/6 mice. Mice were purchased from Vital River (Beijing). When the tumor size reached 100 mm3, the mice were divided into a control group (normal saline), a low concentration of triptonide (3 mg/kg) group and a high concentration of triptonide (5 mg/kg) group. The mice were intraperitoneally injected with drugs every day to observe tumor formation, and the tumor volume was measured every 2 days. The longest diameter a and the shortest diameter b of the tumor were measured, the tumor volume V was calculated according to the Formula a*b2/2, and a tumor growth curve was drawn. On the 20th day, the animals were euthanized according to experimental animal ethics, the tumors were removed and weighed, and the tumor inhibition rate was calculated. The effects of triptonide on liver, lung, and heart tissues were detected via HE staining. All experimental procedures followed the requirements of the Laboratory Animal Welfare and Ethics Committee of Jinan University (Approval No. IACUC-20230913-02).
2.11. Statistical analysis
The data were derived from 2 or 3 independent experiments and are presented as the mean ± standard deviation (SD). Student’s t test and one-way ANOVA were performed to analyze the significance of the difference between two groups or among multiple groups, respectively. All the statistical analyses were performed using SPSS 16.0 and GraphPad Prism 7.0. Figures were drawn with Adobe Illustrator 2021 and FigDraw. P values < 0.05 were considered to indicate statistical significance.