Bufothionine induced the mitochondria-mediated apoptosis in H₂₂ liver tumor and acute liver injury

Male Sprague–Dawley rats weighing 170–200 g and male Kunming mice weighing 20–22 g were purchased from Shanghai Laboratory Animals Ltd (China). The animals were maintained at constant temperature (21 − 25°C) and humidity (50 − 70%) on a 12-h/12-h light/dark cycle with free access to food and water. The animals were allowed to adapt to the laboratory environment for at least 7 days prior to the experiments. All procedures were performed according to the guidelines of the National Animal Welfare Law of China. The Experimental Animal Ethical Committee of Shanghai University of Traditional Chinese Medicine care guidelines were complied with to ensure that the mice received human care (Ethics Approval Number: SCXK2012–0002, 2012.1.30).

For rats in hepato-protective experiments, 8.4 mg of bufothionine were dissolved in 200 μL of DMSO and diluted with 140 mL normal saline. The final concentration of bufothionine was 58.57 μg/mL. For H₂₂-tumor-bearing mice in the anti-hepatocellular carcinoma experiments, 4.5 mg of bufothionine were dissolved in DMSO and diluted with 70 ml normal saline. Obtained concentration of bufothionine was 64.29 μg/mL.

Fifty rats were randomly divided into 5 groups (10 rats each group) by random digit table generated by SPSS 17.0 software. (SPSS Inc., USA). Both the normal group (Nor) and the model control group (Mod) were treated with intraperitoneal injection of normal saline (including 0.3% DMSO). The rats in the three drug groups were intraperitoneally injected with the corresponding drugs as follows: compound glycyrrhizin injection (9.14 mL/kg) (Gly); cinobufacini injection (3.42 mL/kg) (InjA) and bufothionine (9.77 mL/kg) (BufoA).

All rats were treated once daily at a fixed time (09:00) for 8 days. The weight, appearance and behaviors of each rat including activities, diets and secretions were observed and recorded daily. On day 8 ^th, CCl₄ was administered at the dosage of 1.5 mL/kg based on Yokohama et al. [17] at 15:00 o’clock to induce acute liver injury in each group except for the normal control group. The rats were fasted with free access to water for 18 h. On the following day (day 9th) at 09:00, all rats were anesthetized with diethylether to collect blood. The biochemical assays of aspartate aminotransferase (AST); alanine aminotransferase (ALT); alkaline phosphatase (ALP); and total bilirubin (TBIL) were performed by a modular autoanalyzer (Roche, Switzerland) [18]. The livers were also collected and weighed. The obtained specimens were fixed in 10% neutral-buffered formalin, dehydrated, and embedded in paraffin for histo-chemical studies.

Mouse H₂₂ hepatoma cells were purchased from American Type Culture Collection (ATCC, USA). The SMMC-7721 cell line was obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (China). The two cell lines were cultured in RPMI 1640 medium (Gibco, USA) containing 10% heat-inactivated fetal bovine serum (FBS) (Gibco, USA), penicillin (100 U/mL) (Gibco, USA) and 100 mg/L streptomycin (Gibco, USA) at 37°C in a humidified atmosphere of 5% CO₂ in air. The cells were subcultured every 2–3 days. All cell lines used for experiments remained in the logarithmic growth phase.

H₂₂ tumor cells were obtained from in vivo passages. The stocked murine hepatoma H₂₂ cells were subcultured in RPMI 1640 containing 12% FBS and then collected into blank RPMI 1640 culture medium (1 × 10⁸ cells/mL). About 0.2 mL of the cell suspension (approximately 2 × 10⁷ cells) was injected into the abdominal cavity of a mouse. At 5–7 days after the inoculation, ascites occurred in the mice and the fluid was removed using a plastic needle tube (5 mL) under aseptic conditions. About 10–12 mL of fluid was collected from 2–3 mice. The ascites fluid was diluted with an equal amount of normal saline, and centrifuged (Hettich Rotina 420 centrifuge, Germany) at 900 x g for 5 min under 4°C. The pellet was mixed with 20 mL of normal saline. Approximately 0.2 mL of the mixture (1 × 10⁷ H₂₂ cells) was subcutaneously injected into the right flank of each mouse. Cell mixture (10 mL) could be roughly inoculated into 50 mice.

After 7 days, inoculated mice with tumor sizes of 100–150 mm³ were selected and randomly divided into different groups (10–12 mice per group) by random digit tablegenerated by SPSS 17.0 software. Twelve mice without inoculation were characterized as the normal group and injected with normal saline. Among the inoculated groups, one group was injected with normal saline as the model group (Mod) and the other groups were respectively injected intraperitoneally with the following drugs: cinobufacini injection (InjB) (5.14 mL/kg), bufothionine (8.02 mL/kg) (BufoB) and 5-fluorouracil (5-Fu) (3.42 mL/kg). The dosages of drugs for the animals were calculated according to the human dosages recommended on the package inserts. All mice were treated once daily between 09:00 and 10:00 continuously for 10 days except for the 5-Fu group, in which the drug was administered twice a week. The appearances and behaviors were carefully observed. Any abnormal situation such as diarrhea, irritation and emaciation should be recorded. Tumor sizes of the animals were also measured with vernier calipers. Tumor weight indices were expressed as the ratio of tumor weight difference (final tumor weight - initial tumor weight) to body weight. Tumor inhibition rate (D) was obtained with mean tumor indices of drug group divided by mean tumor indices of model group.

When the treatments were over, all animals were deprived of food but allowed free access to water for 16 h. On the following morning, the mice were euthanized for collection of the tumors and spleens, which were immediately placed in 4% buffered paraformaldehyde and fixed overnight. The obtained tumor tissues were dehydrated through an ethanol gradient and embedded in paraffin. Tissue sections (5 μm) were then dewaxed and rehydrated. The sections were stained with hematoxylin and eosin (HE) for observation.

Carcinoma cell line SMMC-7721 cells in the logarithmic growth phase were seeded in 35 mm culture dishes at a density of 3 × 10⁵ cells/mL and incubated for 24 h. Subsequently, the adherent cells were incubated with different concentrations of bufothionine (90.62, 18.12, or 3.62 ng/mL) for 48 h,while the control groups were treated with culture medium containing 10% FBS. The adherent cells were washed twice with ice-cold phosphate-buffered saline (PBS), lysed with RIPA buffer (RIPA buffer/protease inhibitors/EDTA = 100:1:1; Thermo Beyotime, China) for 5–10 min on ice, harvested with a scraper, and centrifuged for 15 min at 14,000 × g under 4°C. The resulting supernatants containing the protein fraction were stored at-80°C until analysis.

The protein concentrations were determined with a Bicinchoninic Acid (BCA) Protein Assay Kit (Pierce, USA) according to the manufacturer’s instructions. Afterwards, all protein samples were mixed with Laemmli loading buffer (Beyotime Institute of Biotechnology, China), boiled for 3–5 min, and stored at-20°C until analysis.

Equivalent amounts of proteins were subjected to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinyl lidene difluoride membranes (Beyotime Institute of Biotechnology, China). Subsequently, the membranes were blocked with 5% skim milk (Beyotime Institute of Biotechnology) and shaked at 80 r/min for 1 h under room temperature on a shaker and incubated with specific primary antibodies (1:1000 dilution in PBS) against β-actin, Bcl-2, Bax, caspase-3, caspase-9, cyto-c, Bid, and p53 (Santa Cruz Biotechnology Inc., USA) overnight at 4°C. The membranes were washed three times with western cleaning solution (Beyotime Institute of Biotechnology, China), incubated with a horseradish peroxidase-conjugated secondary antibody (1:15,000 dilution in ultrapure water) for 30 min at room temperature in a dark place, and washed three times with western cleaning solution. Finally, immune-reactive proteins were detected by enhanced chemo-luminescence (Odyssey Infrared Imaging System, LI-COR Co. LTD, USA). In this experiment, β-actin was used as an internal control to confirm that the amounts of loaded protein were equal. All experiments were repeated at least three times.