Chemicals and reagents
TPP was purchased from Bidepharm (Shanghai, China), while Dulbecco’s modified Eagle’s medium (DMEM) and Penicillin-Streptomycin solution was purchased from BasoMedia (Shanghai, China). Fetal bovine serum (FBS) was obtained from Gibco (Beijing, China), while Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Chemical technology (Shanghai co. LTD, Shanghai, China). In addition, EdU (5-Ethynyl-2'-deoxyuridine) Cell Proliferation Kit with Alexa Fluor 488 and Enhanced ATP Detection kit was obtained from Beyotime (Guangzhou, China). All the experiments were carried out at least in triplicate.
Cell culture and treatment
Hep3B was obtained from iCell Bioscience Inc (Shanghai, ATCC), and were cultured in DMEM media, supplemented with 10% FBS and antibiotics (100 units/mL penicillin and 0.1mg/mL streptomycin). The cells were incubated at 37°C under a humidified 5% CO2 atmosphere. The cells were passaged every 3 days and then used for experiments in exponential growth phase. Before exposure to TPP, the cells were seeded into 6-well plates, 12-well plates, or 96-well plates (Corning), and then cultured for 24 h. Then fresh culture medium with different concentrations of TPP was added and incubated for the same period (unless otherwise indicated). All the compounds were dissolved in DMSO with a final concentration of 0.1% (v/v). Controls were treated with the corresponding vehicle alone.
Cell Counting Kit-8 Assay
Cell viability was measured using the CCK-8 assay by counting the number of viable cells after various treatments. Briefly, five replicates of the Hep3B cells were seeded in 96-well plates at a cell density of 3,000 cells per well for 24 h. The cells were exposed to fold-ratio dilution concentrations of TPP for 48 or 72 h. After the TPP treatment, 10μL of CCK-8 reagent in 90μL DMEM per well was added to the cell culture medium, and then incubated at 37°C for 2 h. Absorbance was measured 6 times/well at 450 nm using an automatic microplate reader (Synergy4; BioTek, Winooski, VT, USA).
ATP detection assay
The metabolic activity and functional status of the cells were measured by the ATP expression assay using different processes. Firstly, the Hep3B cells were seeded in 6-well plates at a cell density of 200,000 cells per well for 24h. Thereafter, the cells were exposed to various concentrations of TPP (0, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8μM) for 48h, followed by addition of 200μL lysate solution to each well. Pipettes were used to repeatedly blow or shake the culture plate to lyse the cells. The cell lysates were centrifuged at 12000g for 5min, and the supernatant was extracted for analysis. 100μL ATP detection liquid and 20μL of the supernatant samples were added to a 96-well blackboard (Corning) with five replicates for 3min. Luminescence (RLU) was measured by luminoskanTM Ascent Chemiluminescence instrument (Thermo Fisher technology Co. LTD, Shanghai, China).
EdU cell proliferation assay
Cell proliferation was measured by the EdU assay by counting the ratio of proliferated cells in total cells. Hep3B cells were seeded in 12-well plates at a cell density of 30,000 cells per well for 24h. The cells were then exposed to various concentrations of TPP (0, 0.025μM, 0.05μM, 0.1μM and 0.2μM) for 48h. Afterwards, the plated cells were washed three times with PBS and then 1μL of EdU reagent (10mM) in 1mL DMEM with 10% FBS was added. The mixture was incubated at 37°C for 2 h and then fixed in 4% formaldehyde for 30 min at room temperature. After washing 3 times with 3% BSA solution, the cells were permeabilized in 0.3% Triton X-100 for 30 min at room temperature and then washed three times with 3% BSA, 5min per wash. The samples were mixed with click additive solutions, such as Click Reaction Buffer, CuSO4, Azide 488 and Click Additive Solution, then incubated at room temperature for 30min and in darkness. The nuclei were labeled with Hoechst33342. All immunofluorescence images were acquired using a Zeiss Axiovert A1 inverted fluorescence microscope and ImageJ 1.48k software was used for image processing. Changes in brightness or contrast during processing were applied equally across the entire images.
To examine the invasive ability of the tumor cells, 8.0μm pore-size Transwell chambers were added into a 24-well plate, which was divided into the upper and lower chambers. Hep3B cells were seeded in 6-well plates at a cell density of 200,000 cells per well for 24h. The cells were then exposed to various concentrations of TPP (0, 0.025μM, 0.05μM, 0.1μM and 0.2μM) for 48h. Thereafter, 600μl of media with 10%FBS was added to the lower chamber, while 200 serum-free conditioned medium with 30,000 cells was added to the upper chamber. The cells were incubated at 37°C for 36 h, and then the upper chamber cells were wiped with cotton swabs. Invasion cells were fixed and stained with crystal violet for counting.
Wound healing assay
Before exposure to TPP, Hep3B cells were seeded in 6-well plate and cultured in DMEM media containing 10% FBS for 24h. At a cell confluence of 100%, wounds were scratched on the cell monolayer using a 200μL pipette tip. The plates were washed twice with PBS, and then the cells were incubated in DMEM with 1% FBS containing various concentrations of TPP (0, 0.025μM, 0.05μM, 0.1μM and 0.2μM) for 72-96h. The wounded areas were imaged with ICX41 inverted bright field microscope at the same period. The area difference was calculated using ImageJ software (NIH, USA). Three different areas were selected and used to estimate the distance of the migrating cells in each assay, and the assay was carried out as previously described.
3D Spheroid Invasion Assay
This scheme provides an overview for the measurement of 3D Tumor Single Spheroid cell invasion into surrounding extracellular matrix (corning) to compare the invasion ability of cells under different treatment conditions. Hep3B cells were seeded into 96-well round bottom, ultra-low attachment plate (Corning® Cat. No. 7007) at a density of 3,000 cells per well and centrifuged at 125g for 10 min. The cells were then cultured in 200μL DMEM containing 10% FBS and incubated at 37°C for 24-48h. At the formation of spheroids, the supernatant was removed and placed on a pre-chilled Coolsink 96F within a Coolbox 96F box for about 5-10 minutes. Matrigel stock was diluted to 2x final assay concentration (prepared 8mg/ml Matrigel for a final assay concentration of 4mg/ml) in DMEM medium with 10%FBS in a cold polypropylene tube. Using pre-chilled pipette tips and reverse pipetting technique, 90 µL of the diluted Matrigel was slowly added into appropriate wells. This method utilized IncuCyte® Live-Cell Analysis System (Essen BioScience, A Sartorius Company) for kinetic, image-based bright field and fluorescence measurements of spheroid invasion (invading cells and whole spheroid areas). After incubation at 37°C for 30min, and at matrix polymerization, 100μl/well of DMEM complete culture media containing various concentrations of TPP (0, 0.025μM, 0.05μM, 0.1μM and 0.2μM) or media alone was added into the plates and then returned to the IncuCyte® to monitor spheroid invasion (every day for 12 days). The dimension of the spheroids was calculated using ImageJ software (NIH, USA).
Male Balb/c nude mice (4–5-weeks-old) were purchased from the Animal Experimental Center of Southern Medical University. The animal study was carried out in strict accordance with the Guidelines for the Care and Use of Laboratory Animals, China. All experimental protocols involving animals were approved by the Laboratory Animal Ethics Committee of Zhujiang Hospital of Southern Medical University ( approval document number:LAEC-2021-196), The Hep3B cells at density of 1×107 in 1ml of PBS were inoculated subcutaneously into the left flank of the nude mice. When the mice were housed for one weeks, the mice were randomly assigned to the vehicle control(n=3) and treated groups(n=3), and they were treated with 5% glucose solution (control) and 1mg/kg TPP solution by intraperitoneal administration, three times in one week, with one or two days intervals, respectively. After treatment for 14 days, the mice were sacrificed after the final therapy. Tumors were removed and their mean diameter was measured.
RNA isolation and quantitative real-time RT-PCR
The genes related to cell migration and invasion were validated using real-time PCR (RT-PCR). Briefly, RNA was extracted by TRIzol regent (ThermoFisher technology (China) co. LTD, Shanghai, China), following the manufacturer’s instruction. We then used Nanodrop 2000 (Thermo Fisher Scientific) to quantify the extracted RNA ensuring the ratio of 260/280 between 1.8 and 2.0, and then cDNA was synthesized using reverse-transcription. Next, the cDNA was quantified by real-time PCR with SybrGreen qPCR master Mix (Thermo Fisher technology co. LTD, Shanghai, China). Results were normalized to the expression of GAPDH. The 2-△△Ct method was used to calculate the expression levels.
Next generation mRNA-Sequencing (mRNA-seq) was performed for Hep3B cells. Briefly, after one week of treatment with 0.05uM and 0.1uM TPP, the cells were collected and underwent RNA extraction. The sequencing library was prepared and then sequenced in Illumina HiSeq X10 instrument (Guangzhou Ruijie Biological Co. Ltd). We used FPKM to evaluate the mRNA expression level. In addition, DESeq R package was used to conduct differential expression analysis. Corrected p-value was set at 0.05 and the absolute value of log 2 FC (fold change) was set at ≥ 1 as the threshold for differentially expressed genes (DEGs).
Enrichment analysis of TPP-related genes
After screening for the differentially expressed genes, we conduct further analysis based on the biological and molecular functions of the DEGs. Four packages, “clusterProfiler”, enrichplot”, “org.Hs.eg.db” and “ggplot2”, in R were used to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. In GO enrichment analysis, we analysed the Biological process (BP), molecular function (MF) and cellular component (CC). A P < 0.05 was used as the threshold in the analysis.
Protein-protein interaction (PPI) network
To explore the roles, if any, of the proteins encoded by the DEGs, STRING (https://www.string-db.org/) was used to construct a PPI network. Thereafter, Cytoscape 3.8.2 was used to analyze and visualize the PPI network. Genes with over 20 degrees were considered pivot genes.
The results were presented in form of average±standard deviation, and all the statistical analysis was performed in GraphPad Prism 9.0. One-way or Two-way ANOVA was used to evaluate the differences among the study groups. Statistical analysis between two groups was performed using the two-tailed unpaired Students’ T-test. The symbols used for statistical significance were ****(p<0.0001), ***(p<0.001), **(p<0.01) and *(p<0.05). Six replicates were performed for the statistical analysis, and the data obtained were from three independent and repeated groups.