IR-780 iodide, 2-thenoyltrifluoroacetone (TTFA), diphenyleneiodonium chloride (DPI), H2O2, and N-acetylcysteine (NAC) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cis-diamminedichloroplatinum (DDP) and doxorubicin hydrochloride (DOX) were obtained from Aladdin (Shanghai, China). MitoTracker Green, mitoSOX, and the mitochondrial membrane potential indicator TMRM were purchased from Invitrogen (Carlsbad, CA, USA). A Complex I Enzyme Activity Microplate Assay was purchased from Abcam (Cambridge, MA, USA). DiBAC4(3) was purchased from MedChem Express(Monmouth Junction, NJ, USA). A Reactive Oxygen Species (ROS) Assay Kit, Hoechst 33258, Crystal Violet Staining Solution, and an ATP Assay Kit were obtained from Beyotime (Shanghai, China). Primary antibodies were Rb anti-Cytochrome C (1:1000, CST, 11940T), Rb anti-Ndufs1 (1:1000, abcam, ab169540), Rb anti-Caspase-9 (1:1000, abcam, ab202068), Rb anti-Caspase 3 Antibody (1:1000, proteintech, 19677-1-AP), Rb anti-cleaved Caspase 3 Antibody (1:1000, abcam, ab13847), Rb anti-PARP (1:1000, proteintech, 13371-1-AP) and Ms anti-β-action (1:5000, proteintech, 6008-1-Ig).
Human bladder cell lines (T24, 5637, and TCCSUP) were purchased from the ATCC (Manassas, VA,
USA), and human normal cell lines (HK2 and SV-HUC-1) were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China). A mouse bladder cell line (MB49) was donated by Dr. Chunmeng Shi from the Institute of Rocket Force Medicine, Third Military Medical University (Chongqing, China). All cells were cultured in their recommended medium supplemented with 10% FBS (Gibco, USA) and 1% penicillin/streptomycin (Beyotime). All cells were passaged at 1:3 ratios and cultured in an incubator at 37 °C with 5% CO2. T24/DDP, a cisplatin-resistant bladder cancer cell line, was established by a concentration gradient method.
An HBO exposure experiment was performed in a temperature-adjustable hyperbaric chamber provided by the neurosurgery department of Southwest Hospital (Chongqing, China). The main parameters for HBO administration were 2.5 atmospheres absolute (ATA) of pure oxygen and a time of 120 min, and the program included 15 min of compression and decompression at a constant velocity. The temperature for cells was 37 °C, while that for animals was 25 °C. Cells in different plates were removed from the chamber after HBO exposure and cultured routinely in an incubator.
MB49 cancer cells grown to 70% confluence were suspended in phosphate-buffered saline (PBS). MB49 transplanting tumor model were established in female C57 BL/6 mice (6-8 weeks old and weighing 17-20 g) by subcutaneously implanting each mouse with 1*107 cells. T24, T24/DDP tumor xenografts were established in female nude mice in the same way.
In vivo fluorescence imaging mice bearing T24, MB49 transplanted tumors were sacrificed at 24 h after intraperitoneal injection with IR-780 at a dose of 1 mg/kg. The dissected organs were subjected to NIR imaging with a Kodak In-Vivo FX Professional Imaging System (New Haven, CT). Cellular dye uptake and the subcellular localization cells of different cell lines (T24, MB49, HK2, and SV-HUC-1) were seeded at the same densities in culture dishes and cultured for 24 h. Then, the cells were incubated with 2 μM IR-780 in 1640 culture medium for 15 min in the incubator before being incubated for 10 min with 1 μM MitoTracker Green. Hoechst was used to stain the nuclei. Finally, these cells were imaged under a confocal microscope.
Cell viability evaluation
Bladder cancer cell lines of different grades (T24, 5637, TCCSUP, T24/DDP and MB49) were treated with the indicated doses of IR-780 with or without HBO exposure, and a CCK-8 kit (Dojindo Kumamoto, Japan) was used to measure cell proliferation after 48 h. T24 cells after treatment with or without different inhibitors were placed in 7.5 μM IR-780+HBO, and cell viability was detected after 48 h.
In vivo IR-780+HBO evaluation
In vivo, treatment was administered on the fifth day of transplanted tumor establishment through intraperitoneal injection of 3 mg/kg IR-780. The IR-780+HBO group and the HBO-only group were then placed in the hyperbaric chamber 4 h after IR-780 injection. The change in tumor volume was monitored every other day during the experiment. After treatment every two days for a total of 5 interventions, the mice were weighed and sacrificed, and the transplanted tumors from the animals were photographed and weighed. Subsequently, the tumor tissues and organs were dissected and fixed in 4% paraformaldehyde. Hematoxylin and eosin (H.E.) staining was performed to observe the toxicity in normal tissues. To study the long-term development of tumors after IR-780+HBO treatment, tumor volume was monitored in the IR-780+HBO group and DDP group. After 5 injections of 3 mg/kg IR-780 and 3 mg/kg DDP, the mice continued to be fed for 28 days. To compare the anti-tumor effect of IR-780+HBO and DDP on drug-resistant bladder cancer, 3 mg/kg IR-780 and 3 mg/kg DDP were injected into transplanted tumor mice in the IR-780 group and DDP group respectively. After treatment every two days for a total of 5 interventions, the mice were photographed and sacrificed, and the transplanted tumor from the animals were photographed and weighed.
For the apoptosis assay, cells were collected after treatment, and an Annexin V/PI detection kit (BD Biosciences) was used to detect apoptosis by flow cytometry according to the protocol described. For the ROS assay, cells were incubated with the probe DCFH-DA at a concentration of 10 μM after the different treatments for 30 min at 37 °C, and then flow cytometry was used for fluorescence intensity detection. For the mitochondrial ROS assay, cells subjected to the different treatments were incubated with the probe mitoSOX at a concentration of 5 μM for 10 min at 37 °C, and then flow cytometry was used for fluorescence intensity detection. For the plasma membrane potential assay, cells were incubated in KRH (Krebs-Ringer-HEPES) buffer containing 100 nM DiBAC4(3) for 20 min at room temperature before flow cytometry detection. For the mitochondrial membrane potential (MMP) assay, TMRM was also detected by flow cytometry.
Colony formation assay
After intervention for 6 h, cells were seeded in six-well plates at a density of 1000 cells per well and cultured overnight to attach. The medium was replaced with fresh medium after 24 h, and the cells were cultured for 12 days. Subsequently, the cells were fixed with paraformaldehyde for 10 min and subjected to crystal violet (Beyotime) staining for 5 min. The samples were photographed, and the colonies > 0.2 mm in diameter were counted under a microscope.
Transmission electron microscopy (TEM)
After 24 h of treatment, cells were collected and fixed overnight at 4 °C in 2.5% glutaraldehyde. Then, the cells were subjected to secondary fixation in 2% osmium tetroxide followed by embedding in resin. Images of thin sections were obtained by TEM.
Western blot analysis
Cells were harvested after treatment for 24 h, and total proteins were extracted with RIPA lysis buffer (Beyotime). After separation using 12% SDS-polyacrylamide gels, the proteins were transferred onto PVDF membranes (Millipore, USA). The PVDF membranes were blocked with Quick Blocking Buffer (Beyotime) for 15 min and then incubated overnight with primary antibodies at 4 °C. The antibodies described above were used. After incubation for 1 h with secondary antibodies in an incubator at 37 °C, the signals were identified by chemiluminescence detection.
All data were expressed as the means ± standard derivation at least three independent experiments
. Statistical analysis of experimental data was performed using GraphPad Prism 8.0. Comparisons between two groups were performed using the student’ t-test, one-way analysis of variance (ANOVA) or two-way ANOVA were used for multiple groups. P-values <0.05 were considered to indicate statistical significance.