Reagents and chemicals
Thiazolyl blue tetrazolium bromide (MTT) (M8180) was purchased from Solarbio (Beijing, China). Triptolide (HY-32735, 99.8% purity), ferrostatin-1 (Fer-1) (HY-100579), Z-VAD (OMe)-FMK (Z-VAD) (HY-16658), 3-methyladenine (3-MA) (HY-19312), necrostatin-1 (Nec-1) (HY-15760), acetylcysteine (NAC) (HY-B0215), and JC-1 (HY-15534) were obtained from MedChemExpress (Shanghai, China). Triptolide was dissolved in dimethyl sulfoxide to make a stock solution, which was stored at − 80 °C. A Cu2+ fluorescent probe (JY0299) was purchased from BIOFOUNT (Beijing, China). Dimethyl sulfoxide (DMSO), stripping buffer (P0025), and the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488 (C0071S) were purchased from Beyotime Biotechnology (Shanghai, China). A reduced glutathione (GSH) assay kit (A006-2-1) was purchased from Nanjing Jiancheng Institute of Biological Engineering (Nanjing, China). Tetrathiomolybdate (TTM) (323446) was purchased from Sigma Aldrich (Darmstadt, Germany). The primary antibodies FDX1 (ab108257), POLD1 (ab186407), ACO-2 (ab129069), SDHB (ab178423), and CTR1 (ab129067) were purchased from Abcam (Shanghai, China). Lipoic acid (cat# 437695) was obtained from Millipore. ATP7A (sc-376467) and ATP7B (sc-373964) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). DLAT (12362), XIAP (2042), β-Actin (4970) and GAPDH (5174) were purchased from Cell Signaling Technology (Beverly, MA, USA). LIAS (11577-1-AP) and vinculin (66305-1-Ig) were purchased from Proteintech (Wuhan, China). COMMD1 (H00150684-M01) was obtained from Abnova. The secondary antibodies goat anti-rabbit IgG (ZB-2301) and goat anti-mouse IgG (ZB-2305 and ZF-0312) were purchased from Beijing Zhong Shan-Golden Bridge Biological Technology (Beijing, China).
Cell lines
The human cervical carcinoma cell lines HeLa (ATCC CCL-2) and SiHa (ATCC HTB-35) were purchased from the American Tissue Culture Collection (ATCC).
Cell viability assays
Cell viability was assessed using MTT assays. A total of 3 × 103 HeLa or SiHa cells per well were added to 96-well plates. Then, triptolide (20, 40, 80, and 160 nmol/l) was applied to the cells for 48 h, 5 mg/ml MTT solution (20 µl/well) was added, and the cells were incubated for 4 h. To dissolve the formazan crystals, 150 µl of DMSO was added after the supernatants were removed. The optical density (OD) was determined using a microplate reader (Multiskan™ MK3; Thermo Fisher Scientific, Inc., US) at a wavelength of 490 nm. The half maximal inhibitory concentration (IC50) values were calculated using GraphPad Prism 9.0 software (GraphPad Software, Inc.). In the chemical rescue experiments, Fer-1, Z-VAD-FMK, 3-MA, Nec-1, and NAC were added 2 h before the addition of triptolide. Cell viability was measured 48 h after the addition of drugs.
EdU cell proliferation assay
The BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488 was used to assess cell proliferation according to the manufacturer’s instructions. A fluorescence microscope (BZ-X810, Keyence) was ultimately used to capture the images.
Cell migration assay
A 24-well transwell chamber system (Corning, 3422) was used to conduct the cell migration assay. The Transwell apparatus was separated into the upper and lower compartments by a special filter. In the upper compartment, HeLa and SiHa cells were suspended in 200 μL of growth medium. Then, 600 μL of growth medium supplemented with 10% fetal bovine serum (FBS) was added to the lower chamber. Triptolide was then added to the upper chamber. Cells on the upper filter surface were removed by wiping with a cotton swab following a 24-h incubation at 37 °C in an incubator. The filters were then fixed in 4% paraformaldehyde and stained with 0.4% crystal violet. The number of cells that migrated to the lower filter surface was recorded using a microscope (BZ-X810, Keyence).
Wound healing assay
In 6-well plates, HeLa and SiHa cells were plated until a monolayer developed. A line was scratched using a 200 µl pipette tip. The cells were washed 3 times with PBS to remove suspended cells, growth medium supplemented with 2% FBS was added to the 6-well plates, and the scratch data were recorded using a microscope (BZ-X810, Keyence). Then, the cells were treated with triptolide and cultured in an incubator for 48 h. The suspended cells were removed, and the scratch data were recorded using a microscope (BZ-X810, Keyence). The wound healing region was examined using ImageJ software. The difference between the 0 h and 48 h areas was determined.
GSH determination
The levels of glutathione (GSH) were measured using a reduced GSH assay kit according to the manufacturer’s instructions.
Mitochondrial membrane potential
The mitochondrial membrane potential was examined using JC-1 staining. In a 6-well plate, 1 × 105 HeLa and SiHa cells were cultured overnight. Then, the culture medium was replaced with fresh medium containing 80 nM or 40 nM TPL, followed by a 48-h incubation; then, the culture medium was discarded and the cells were treated with JC-1 working solution (prewarmed serum-free cell culture medium containing 10 μg/mL JC-1) for 20 min at 37 °C in the dark. The cells were examined under a fluorescence microscope (BZ-X810, Keyence) after being washed with serum-free cell culture media.
Cu2+ fluorescence staining
HeLa and SiHa cells were treated with triptolide for 48 h. A Cu2+ fluorescence probe was added to the culture medium at a working concentration of 10 μM, and the HeLa and SiHa cells were incubated at 37 °C in the dark for 20 min. The dye working solution was removed, the cells were washed twice with growth medium, and the cells were observed under a fluorescence microscope (BZ-X810, Keyence).
Immunofluorescence
HeLa and SiHa cells were fixed with 4% paraformaldehyde for 15 min. After treatment with 0.3% Triton X-100 for 15 min, the cells were blocked with 5% bovine serum albumin (A6020, Biotopped) for 30 min at room temperature. An anti-DLAT antibody (1:100, 12362, Cell Signaling) was used as the primary antibody for incubation. The secondary antibody utilized was FITC-conjugated goat anti-mouse IgG (ZF-0312). The nuclei were labeled with DAPI staining solution (Beyotime).
Inductively coupled plasma‒mass spectrometry (ICP-MS)
The collected cell precipitate was resuspended in 1 ml of PBS, and the cell suspension was crushed with an ultrasonic crusher. One milliliter of 1% nitric acid was added to 1 ml of the crushed cell suspension, which was then incubated at 60 °C for one hour. Another 10 µl of the cell suspension was removed for protein quantification. The Cu content of the cell sample was measured by inductively coupled plasma‒mass spectrometry (ICP-MS, Perkin Elmer, Nexion 350, Waltham, MA, USA). The copper concentration was normalized to the protein concentration.
Western blot analysis
The culture medium of cell samples was removed, and the cells were washed twice with PBS. After RIPA lysis buffer containing 1 mM PMSF was added, the cells were lysed on ice for 30 min, and the cell samples were collected with a cell scraper. For tissue samples, RIPA buffer was added to the tissue, which was subsequently ground, crushed via ultrasonication, and lysed on ice for 30 min. After centrifuging the samples at 12,000 rpm for 20 min at 4 °C, the pellet was removed, and Pierce™ BCA Protein Assay Kits (23227, Thermo Fisher Scientific) were used to measure the protein concentration in the supernatant. Loading buffer was added, and the mixture was heated at 95 °C for 10 min. Proteins were first separated by 8–12% SDS‒PAGE at 80 V for 30 min and then at 120 V for 1 h. After the electrophoresis was stopped, the protein was transferred to a 0.2 μm PVDF membrane, the membrane was blocked with 5% skim milk powder for 2 h at room temperature and incubated with the corresponding primary antibody overnight at 4◦C. Then, after washing with TBST, the corresponding HRP-conjugated secondary antibody was incubated with the membrane for 1 h at room temperature. Following TBST washes, specific protein bands were detected with Immobilon Western HRP Substrate (WBKLS0500, Millipore) and a chemiluminescence imaging system (FluorChem HD2, ProteinSimple or Tanon 4800, Tanon).
Quantitative real-time polymerase chain reaction
Total RNA was extracted from HeLa and SiHa cells using TRIzol Reagent (15596018, Invitrogen). The RNA samples were subjected to quantitative PCR with the corresponding primers using the One Step SYBR® PrimeScript™ RT‒PCR Kit (RR066A, Takara) and the Thermal Cycler Dice Real Time System (Takara) according to the manufacturer’s instructions. β-actin and GAPDH were used as the internal controls. The results were analyzed using the 2−ΔΔCT method.
In the experiments, the following primers were used: ATOX1, Forward, 5′-GTGCTGAAGCTGTCTCTCGG-3′, and Reverse 5′-GCCCAAGGTAGGAAACAGTCTTT-3′; COX17, Forward, 5′-TGCGTGTATCATCGAGAAAGGA-3′, and Reverse 5′-GCCTCAATTAGATGTCCACAGTG-3′; CCS, Forward, 5′-GGGGACCTTACAAACAACTGC-3′, and Reverse 5′-GCATCAGCACGGACATTGC-3′; ATP7A, Forward, 5′-CTGAAATCTATGGCCTTAGAAG-3′, and Reverse 5′-CATTGCTACCCGTTTCC-3′; ATP7B, Forward, 5′-CTTGGGATACTGCACGGACTTC-3′, and Reverse 5′-CCTCAGCCACTCACGGTTTC-3′; CTR1, Forward, 5′-TTGGCTTTAAGAATGTGGACCT-3′, and Reverse 5′-GACTTGTGACTTACGCAGCA-3′; β-actin, Forward, 5′-TGTGGCATCCACGAAACTAC-3′, and Reverse 5′-GGAGCAATGATCTTGATCTTCA-3′; and GAPDH, Forward, 5′-CTCTGACTTCAACAGCGAC-3′, and Reverse 5′-CGTTGTCATACCAGGAAATG-3′.
Copper salt stain kit
Analysis was performed using a copper salt stain kit (G3040, Solarbio Life Science, China) according to the manufacturer’s instructions.
Network pharmacology and protein‒ligand docking
The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) was used to retrieve the targets of triptolide. The GeneCards database (https://www.genecards.org/) was used to retrieve potential targets for cervical cancer and copper homeostasis. The three-dimensional structure of the XIAP protein (5oqw) was obtained from UniProt (https://www.uniprot.org/). The mol2 file of triptolide (MOL003187) was obtained from TCMSP. Protein‒ligand docking was carried out on the CB-Dock2 website (https://cadd.labshare.cn/cb-dock2/) [17].
Nude xenograft mouse model
This study was approved by the Institutional Animal Care and Use Committee of the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences. Six- to eight-week-old BALB/c male nude mice were purchased from Beijing Huafukang Biotechnology (Beijing, China). Mice were kept in conventional pathogen-free settings with simulated 12-h light/dark cycles and consistent humidity levels of 50 to 60%. Throughout the trial, the mice had free access to standard laboratory food and tap water. After seven days of adaptive rearing, 5 × 106 SiHa cells in 100 μL of PBS were injected subcutaneously into the dorsal right axilla of nude mice. One week after injection, 24 mice were randomly divided into four groups: (1) blank control, (2) low-dose triptolide (0.2 mg/kg), (3) middle-dose triptolide (0.4 mg/kg), and (4) high-dose triptolide (0.6 mg/kg). All treatments were given every day for two weeks. Every two days, the body weights of mice were recorded, and the tumor volume was determined using the following formula: [ (length × width × width)/2]. After 15 days of treatment, the mice were euthanized. The tumor tissues and relevant organs were collected.
Statistical analysis
All the statistical analyses were performed using GraphPad Prism 9.0. Two-tailed unpaired Student’s t tests were used for comparisons of two groups. One-way ANOVA was used to determine the statistical significance of multiple comparisons. p < 0.05 was considered to indicate statistical significance.
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