Cell culture and treatment
Hep3B cells derived from American Type Culture Collection (ATCC, Rockville, MD, USA) were grown in Dulbecco’s modified Eagle medium (DMEM, Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, Gibco, Australia). The cells were incubated in an incubator containing 95% air and 5% CO2 at 37 ℃. Dihydromyricetin (DHM) obtained from Sigma-Aldrich (St Louis, MO, USA) was dissolved in dimethylsulfoxide (DMSO, Sigma-Aldrich) and then different concentrations of DHM were used to stimulate Hep3B cells. Nedaplatin (NDP, Nichi-Iko Pharmaceutical Co., Ltd.) was solubilized in sterilized H2O. DMSO served as a control group.
Animals and tumor models
The mice were housed under standard animal room conditions (temperature 22±1°C and humidity 55±5%). Mice were anesthetized with 2% isoflurane (RWD, Shenzhen, China) using a Rodent Anesthesia Machine (VetEquip Inc., Pleasanton, Ca). Animals were sacrificed with 2–3 times the anesthetic dose of isoflurane (via inhalation), followed by cervical dislocation, shaven and sterilized with 75% ethanol. After opening the abdominal cavity, the liver and tumor tissue were collected and weighed. All animals had free access to sterile tap water and food during the experiments. The mice were randomly divided into three groups, including control groups (n=8), DHM (n=8) and NDP (n=8) for 3 weeks. Hep3B cells were transplanted into the mouse via subcutaneous injection of 1×107 cells. One week after transplantation, tumors had grown to a volume of approximately 20 mm3 with a model success rate of 100%. All the animal experiments and surgical procedures were approved by the Institutional Animal Care and Use Committee of Guangdong Medical University (GDY1802018).
Cell Viability Measurement
The cell viability was assayed by adding Cell Counting Kit‑8 assays (CCK-8) solution as described (CCK-8, Dojindo Molecular Technologies, Gaithersburg, MD)[19, 20]. The Hep3B cells were seeded in 6-well plates (1×105 cells/ well) and were allowed to adhere for 8 h. The medium was replaced with medium containing different concentrations of DHM (0, 10, 20, 30, 40, 50 µM). DMSO control wells contained 0.1% DMSO. After 12 h, the culture medium of the cells was discarded, 10 μL of CCK-8 solution was added into each well and cells were incubated at 37 °C for another 2 h. Finally, the absorbance was analyzed at 450 nm using a Microplate Reader (Molecular Devices, San Jose, CA, USA). All the assays were performed for three times independently. Absorbance of cells in the absence of treatment was regarded as 100% of cell survival. Cell survival was calculated as: absorbance/absorbance of control ×100%.
Colony formation assay
Cell viability was performed using a colony formation assay . Hep3B cells were seeded into a 6-well plate (3×102 cells/well) for 8 h, followed by treatment with two different concentrations of DHM and NDP for 24 h. Hep3B cells were cultured with drug-containing medium for ten days. The cells were fixed with methanol-glacial acetic acid stationary solution (3:1) at room temperature for 10 min and stained with 1% crystal violet (Amresco, Solon, OH, USA). The following formula was used to calculate the colony formation inhibition rate: Colony formation inhibition rate = (control group -experimental group)/control group ×100%.
Cell apoptosis assay
Cell apoptosis was assessed by flow cytometry assay (BD, FranklinLakes, NJ) . In brief, the cells were seeded in 6‑well plates (1x106 cells/well), followed by 24 h incubation at 37˚C. The cells were then treated with different concentrations of DHM and NDP for 24 h. The assay was performed using the Annexin V‑FITC/PI cell apoptosis detection kit (BD Pharmingen, USA) according to the manufacturer's protocol. Subsequently, the cells were monitored by flow cytometry (FACSCalibur, Becton Dickinson, USA), and the data were analyzed using FlowJo™ software (version 10, FlowJo LLC).
Cell migration and invasion Assay
Cell migration and invasion were detected by using Transwell assay with 8.0 µm porous polycarbonate membranes (Millipore, Bedford, Massachusetts, USA) . In brief, cells were treated with different concentration of DHM and NDP and adjusted cell density to 1×105. The lower transwell contained 600 µl DMEM with 10% FBS. After incubation 24 h at 37 °C, the non-traversed cells in the upper compartment were wiped by a wet cotton swab. Traversed cells on the lower side of the filter were fixed stained with 0.1% crystal violet (Amresco, Solon, OH, USA). Then these cells were stained with 0.5% crystal violet (Merck, Darmstadt, Germany) for 20 min and counted microscopically (Olympus, Tokyo, Japan). The method of cell invasion was similar with cell migration, except that the inserts were coated with BD MatrigelTM Matrix (BD Biosciences, NY, USA).
Wound healing assay
Cell migratory abilities was tested by a wound healing assay. Hep3B cells were seeded in 12-well dishes (5×104 cells/well), and incubated in DMEM with 10% FBS for 24 h at 37 ˚C. The cells were then exposed in the absence or presence of DHM and NDP. Then the cells were scratched across the surface of the well by pipette tip. After an incubation at 37°C of 24 h, the scratches were observed.
Western blot analysis
The effects of DHM and NDP on the expression levels of Bcl-2, Cleaved-Caspase 3, Cleaved- Caspase 9, Bak, Bax and Bad were analyzed using western blot . Protein samples were obtained from Hep3B cells that were treated with different concentrations of DHM and NDP for 24 h using cell lysis buffer (RIPA, Beyotime Biotechnology, Shanghai, China). The proteins were collected and detected by using the BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA). Subsequently, total protein (20 μg) samples were separated using SDS‑PAGE (10% gel) and transferred onto a polyvinylidene fluoride (PVDF) membranes and blocked in 5% skim milk powder for 1 h at room temperature. Following the membranes were incubated with the corresponding antibodies. Primary antibodies of Bcl-2, Cleaved-Caspase 3, Cleaved- Caspase 9, Bak, Bax and Bad were (all from Cell Signaling Technology) were incubated with the membrane at 4 °C overnight. Then, blots were washed three times with TBST and were incubated with secondary anti-bodies for 1 h at room temperature. The blots were detected using enhanced chemiluminescence (ECL) reagents (Super Signal Dura kit, Pierce, IL, USA) according to the manufacturer’s instructions. The blots were quantified by using Image Lab™ Software (Bio-Rad).
Hematoxylin and Eosin staining
Briefly, liver tissues were immersed in 4% paraformaldehyde for 4h and transferred to ethanol (75%, 85%, 95%). Then they were treated with xylene paraffin-embedded according to a previous report . Before immunostaining, 3-µm-thick liver tissue sections were dewaxed in xylene, rehydrated by decreasing concentrations of ethanol (95%, 85%, 75%) and washed in PBS. And then stained with hematoxylin and eosin (H&E). After staining sections were dehydrated through increasing concentrations of ethanol and xylene.
The study was approved by ARRIVE guidelines (http://www.nc3rs.org.uk/arrive-guidelines). Moreover, we confirm that all methods were performed in accordance with the relevant guidelines and regulations.
All data are expressed as the mean ±S.E.M., and at least three independent replicates were used for per group. All statistical procedures were analyzed by SPSS 22.0 (IBM, Manassas, VA, USA), and plots were generated using GraphPad prism 8.0 (GraphPad Software, La Jolla, CA, USA) (https://www.graphpad.com/scientific-software/prism/). The SPSS analysis show that our results are normal distribution, and homogeneity of results between each treatment groups are equal. Significant differences between treatment groups were determined by one-way ANOVA (SPSS 22.0, Chicago, IL, USA).