Cell culture

Human gallbladder cancer cell lines GBC-SD, NOZ, and SGC-996 were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences. These cell lines were cultured in a mixture consisting of 89% RPMI 1640 medium (Gibco), 10% fetal bovine serum (Gibco), and 1% penicillin/ /streptomycin (Gibco), respectively. The cells were maintained at a temperature of 37 °C in a cell culture incubator (Thermo Fisher Scientific Inc.) with 5% CO2 and saturated humidity. For subsequent experiments, tumor cells in the logarithmic growth phase were utilized.

Western blot

GBC-SD cells were exposed to varying concentrations of MGCD-265 (#S1361, Selleck) for 24 h. RIPA lysate (#PC101, Epizyme Biotech, Shanghai, China), supplemented with Protease Inhibitor Cocktail (#P8340, Sigma-Aldrich LLC), was employed for cell lysis, followed by a 30-minute incubation on ice and subsequent centrifugation at 4 °C for 15 min at 12,000 g. The resulting supernatant was collected and assessed for protein concentration using the BCA method (#P0012, Beyotime Biotechnology, China). Gel electrophoresis was executed utilizing an SDS-PAGE separator gel (#PG112 and #PG113, Epizyme Biotech, Shanghai, China), with each sample loaded containing an equal protein quantity of 30 µg. Subsequently, proteins were electrophoretically separated and transferred to a PVDF membrane (#IPVH00010, Millipore, Sigma-Aldrich LLC). These membranes were then blocked using a 5% skimmed milk solution (#A600669-0250, Sangon Biotech (Shanghai) Co., Ltd., China) at room temperature for 2 h. Thereafter, the membranes were horizontally cut to probe proteins with different molecular weights. Overnight incubation at 4 °C with the respective primary antibodies was carried out, followed by a triple wash with TBS/Tween Buffer (PS103, Epizyme Biotech, Shanghai, China) and subsequent co-incubation with the secondary antibody at room temperature for 2 h. Immunoblots were tested using an BeyoECL Plus Kit (#P0018S, Beyotime Biotechnology, China), and protein quantification was conducted utilizing Image J software. GAPDH and β-actin were employed as the internal reference for normalization.

The antibodies used in this study are listed below. Anti-p21 (Santa Cruz Biotechnology, #sc-6246, working dilution 1:2000), anti-HSP90AA1 (Abcam, ab303516l, working dilution 1:500), anti-Cyclin D1 (Abcam, ab226977, working dilution 1:2000), anti-Cyclin B1 (Abcam, ab181593, working dilution 1:2000), anti-CDK4 (Abcam, ab137675, working dilution 1:2000), anti-JMJD6 (Abcam, ab307654, working dilution 1:500), anti-CDC25A (Abcam, ab2357, working dilution 1:500), anti-SLC1A5 (Abcam, ab237704, working dilution 1:500), anti-SLC7A11 (Abcam, ab307602, working dilution 1:500), anti-GAPDH (Abcam, ab8227, working dilution 1:5000), anti-Cleaved-caspase3 (Cell Signaling Technology, #9664, working dilution 1:1500), anti-Caspase3 (Cell Signaling Technology, #9662, working dilution 1:1500), anti-Cleaved-PARP (Cell Signaling Technology, #5625, working dilution 1:1500), anti-β-actin (Cell Signaling Technology, #4970, working dilution 1:5000), Goat Anti-Rabbit IgG H&L (HRP) secondary antibody (Abcam, ab6721, working dilution 1:8000), and Goat Anti-Mouse IgG H&L (HRP) secondary antibody (Abcam, ab205719, working dilution 1:8000).

RNA extraction, reverse transcription and quantitative real-time polymerase chain reaction (RT-qPCR) analysis

Following drug treatment, cancer cells underwent a pre-cooled PBS (Gibco) wash, and total RNA extraction from the cells was executed using TRIzol™ LS Reagent (#10296010, Invitrogen, Thermo Fisher Scientific Inc.). Next, the RNA concentration was assessed utilizing a NanoDrop spectrophotometer (NanoDrop Technologies Inc.). Reverse transcription of RNA into cDNA was carried out HiScript II Q Select RT SuperMix for qPCR (+ gDNA wiper) according to manufacturer instructions (#R233-01, Nanjing Vazyme Biotechnology Co., LTD, China). RT-qPCR was conducted employing a LightCycler 96 fluorescent quantitative PCR instrument using 2×AceQ qPCR SYBR Green Master Mix (#Q111-02, Nanjing Vazyme Biotechnology Co., LTD, China). The expression of β-actin or GAPDH served as the internal control. The relative transcript levels of genes were statistically analyzed using the 2−ΔΔCT method.

The primers used are listed below. AKT2 forward: 5’-ACCACAGTCATCGA GAGGACC-3’, reverse: 5’-GGAGCCACACTTGTAGTCCA-3’; AKT3 forward: 5’-TGAAGTGGCACACACTCTAACT-3’, reverse: 5’-CCGCTCTCTCGACAAATG GA-3’; CDK2AP1 forward: 5’-ATGTCTTACAAACCGAACTTGGC-3’, reverse: 5’-GCCCGTAGTCACTGAGCAG-3’; CDKN1A forward: 5’-CGATGGAACTTCGACT TTGTCA-3’, reverse: 5’-GCACAAGGGTACAAGACAGTG-3’; MAP2K6 forward: 5’-GAAGCATTTGAACAACCTCAGAC-3’, reverse: 5’-CCTGGCTATTTACTGT GGCTC-3’; MMP14 forward: 5’-GGCTACAGCAATATGGCTACC-3’, reverse: 5’-GATGGCCGCTGAGAGTGAC-3’; MMP2 forward: 5’-TACAGGATCATTGGCTA CACACC-3’, reverse: 5’-GGTCACATCGCTCCAGACT-3’; CDK4 forward: 5’-CTGGTGTTTGAGCATGTAGACC-3’, reverse: 5’-GATCCTTGATCGTTTCGGC TG-3’; MMP24 forward: 5’-GCCGGGCAGAACTGGTTAAA-3’, reverse: 5’-CCCGTAAAACTGCTGCATAGT-3’; MMP26 forward: 5’-TCGGAATGGGACAG ACCTACT-3’, reverse: 5’-TCAAAGGGGTCACATTGCTCC-3’; MMP7 forward: 5’-GAGTGAGCTACAGTGGGAACA-3’, reverse: 5’-CTATGACGCGGGAGTTTAA CAT-3’; PIK3AP1 forward: 5’-GAGCCAGAGACCTACGTGG-3’, reverse: 5’-TGTCATCCAGCTTACATCTCACA-3’; PIK3R1 forward: 5’-ACCACTACCGGA ATGAATCTCT-3’, reverse: 5’-GGGATGTGCGGGTATATTCTTC-3’; PIK3CG forward: 5’-GGCGAAACGCCCATCAAAAA-3’, reverse: 5’-GACTCCCGTGC AGTCATCC-3’; PIK3CD forward: 5’-AAGGAGGAGAATCAGAGCGTT-3’, reverse: 5’-GAAGAGCGGCTCATACTGGG-3’; PIK3CB forward: 5’-TATTTGGACTT TGCGACAAGACT-3’, reverse: 5’-TCGAACGTACTGGTCTGGATAG-3’; PIK3C2B forward: 5’-TCAGGGCAATGGGGAACAC-3’, reverse: 5’-CGTAACAGCTTGA GGTCGGTC-3’; ODC1 forward: 5’-TTTACTGCCAAGGACATTCTGG-3’, reverse: 5’-GGAGAGCTTTTAACCACCTCAG-3’; CACYBP forward: 5’-CTCCCATTAC AACGGGCTATAC-3’, reverse: 5’-GAACTGCCTTCCACAGAGATG-3’; HSPA6 forward: 5’-CAAGGTGCGCGTATGCTAC-3’, reverse: 5’-GCTCATTGATGATCC GCAACAC-3’; AHSA1 forward: 5’-ACGCCACCAACGTCAACAA-3’, reverse: 5’-ACAGTGTTTTCAGCTTATCCGTG-3’; DNAJA1 forward: 5’-AGGAGCAGTAGA GTGCTGTCC-3’, reverse: 5’-TCTCGAACTATCTTCCTTCCGT-3’; BAG3 forward: 5’-TGGGAGATCAAGATCGACCC-3’, reverse: 5’-GGGCCATTGGCAGAGGATG-3’; CHORDC1 forward: 5’-CCTTGCTGTGCTACAACCG-3’, reverse: 5’-CGGAA CACCTGGGTGGTATG-3’; STIP1 forward: 5’-CCTTACAGTGCTACTCCGA AGC-3’, reverse: 5’-ATAGGCAGCAGAACGGTTGC-3’; PSAT1 forward: 5’-TGCCGCACTCAGTGTTGTTAG-3’, reverse: 5’-GCAATTCCCGCACAAGATTCT-3’; HSPD1 forward: 5’-ATGCTTCGGTTACCCACAGTC-3’, reverse: 5’-AGCCCGAGTGAGATGAGGAG-3’; SLC7A11 forward: 5’-TCTCCAAAGGAGG TTACCTGC-3’, reverse: 5’-AGACTCCCCTCAGTAAAGTGAC-3’; HSPA8 forward: 5’-ACCTACTCTTGTGTGGGTGTT-3’, reverse: 5’-GACATAGCTTGGAGTGGT TCG-3’; HSP90AA1 forward: 5’-AGGAGGTTGAGACGTTCGC-3’, reverse: 5’-AGAGTTCGATCTTGTTTGTTCGG-3’; HSPH1 forward: 5’-ACAGCCATG TTGTTGACTAAGC-3’, reverse: 5’-GCATCTAACACAGATCGCCTCT-3’; DNAJB1 forward: 5’-AAGGCATGGACATTGATGACC-3’, reverse: 5’-GGCCAAAGTTCA CGTTGGT-3’; HSPA1B forward: 5’-TTTGAGGGCATCGACTTCTACA-3’, reverse: 5’-CCAGGACCAGGTCGTGAATC-3’; HSPA1A forward: 5’-GCCTTTCCAAGA TTGCTGTT-3’, reverse: 5’-TCAACATTGCAAACACAGGA-3’; FKBP4 forward: 5’-GAAGGCGTGCTGAAGGTCAT-3’, reverse: 5’-TGCCATCTAATAGCCAGCCAG-3’; MTHFD2 forward: 5’-CTGCGACTTCTCTAATGTCTGC-3’, reverse: 5’-CTCGCCAACCAGGATCACA-3’; MRPL18 forward: 5’-GCAGCGAAACCTGAA GTGGA-3’, reverse: 5’-GTGCCAGAACTCACGGGAG-3’; SLC1A5 forward: 5’-GAGCTGCTTATCCGCTTCTTC-3’, reverse: 5’-GGGGCGTACCACATGATCC-3’; CDC25A forward: 5’-GTGAAGGCGCTATTTGGCG-3’, reverse: 5’-TGGTTGCTCATAATCACTGCC-3’; ZFAND2A forward: 5’-GATCATTTTCCATA CGCTGCAC-3’, reverse: 5’-CGTCTGGTATCTGGCCCTTTT-3’; RASSF1 forward: 5’-AGGACGGTTCTTACACAGGCT-3’, reverse: 5’-TGGGCAGGTAAAA GGAAGTGC-3’; JMJD6 forward: 5’-TTGGACCCGGCACAACTACTA-3’, reverse: 5’-TCTGCCCTTTCCACGTTATCC-3’; MICB forward: 5’-TCTTCGTTACAACC TCATGGTG-3’, reverse: 5’-TCCCAGGTCTTAGCTCCCAG-3’; DNAJB4 forward: 5’-GCAGGAGGTACTGATGGACAA-3’, reverse: 5’-ACCACCCATTCGTCTT CCAAA-3’; DDIAS forward: 5’-AGGTTCAGATGCCAGTAACTTCT-3’, reverse: 5’-AGTGATTGTTAGGTGCCTGAGA-3’; BYSL forward: 5’-GGCTGAGCCGAC GGATTTT-3’, reverse: 5’-CCTCGTCATCTGATCCATCCTG-3’; HSPA4L forward: 5’-CGGCTTTCTCAACTGCTACAT-3’, reverse: 5’-ACCTGTCGCTGTACTCATT GG-3’; GAPDH forward: 5’-CAATGACCCCTTCATTGACC-3’, reverse: 5’-TGGAAGATGGTGATGGGATT-3’; β-actin forward: 5’-CCTCGCCTTTGCCGATCC-3’, reverse: 5’-GGATCTTCATGAGGTAGTCAGTC-3’.

Animal experiments

Female BALB/c nude mice (6 weeks old, weighing 16–18 g) were housed at constant temperature (23 ± 2 °C) and controlled light (12 h light:12 h dark) under pathogen-free conditions. All experimental procedures strictly adhered to the applicable guidelines and regulations regarding animal research. The animal study protocol was approved by the Animal Care and Use Committee of Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University.

Subcutaneous xenograft model

GBC-SD cells in the logarithmic growth phase were suspended and subcutaneously inoculated into the right hind limb of BALB/c nude mice in a 100 µL sterile PBS, containing 2 × 106 cells. Upon the tumor volume reaching approximately 100 mm3, the mice were randomly assigned to one of three groups: a control group, a low-dose MGCD-265 group (5 mg/kg), and a high-dose MGCD-265 group (10 mg/kg). MGCD-265 was obtained from Selleck (#S1361). The drug was orally administered via gavage daily for 12 consecutive days, with a dosage of 100 µL per dose. Tumor size and body weight were measured at three-day intervals. On day 28 post-treatment, the mice were humanely euthanized using the cervical dislocation method, and the tumors were harvested immediately for subsequent experiments.

Hematoxylin and eosin (H&E) staining

Fresh tissues underwent fixation using a 4% paraformaldehyde solution (#P0099, Beyotime Biotechnology) for 24 h. Subsequently, they were dehydrated through a series of graded ethanol concentrations and then embedded in paraffin. These Sect. (6 μm in thickness) were subjected to H&E staining (#C0105S, Beyotime Biotechnology), enabling the observation of pathological structures under light microscopy (Olympus, Japan).

Immunohistochemistry (IHC)

Paraffin sections were subjected to dewaxing and rehydration processes, followed by immersion in Citrate Antigen Retrieval Solution (#P0081, Beyotime Biotechnology) for 10 min to facilitate antigen repair. Next, to inhibit endogenous peroxidase activity, a 3% hydrogen peroxide solution was applied, and the sections were washed with PBS three times. Blocking of non-specific binding sites was achieved through a 30-minute incubation with a 3% bovine serum albumin solution (#A610903, Sangon Biotech (Shanghai) Co., Ltd., China). Subsequently, the primary antibodies: anti-Cleaved-caspase3 (Cell Signaling Technology, #9664, working dilution 1:200), anti-CDK4 (Abcam, ab137675, working dilution 1:200), anti-CDK6 (Proteintech Group, Inc., #14052-1-AP, working dilution 1:200), anti-JMJD6 (Proteintech Group, Inc., 16476-1-AP, working dilution 1:200), anti-CDC25A (Abcam, ab2357, working dilution 1:100), anti-SLC1A5 (Abcam, ab237704, working dilution 1:100), and anti-SLC7A11 (Abcam, ab307602, working dilution 1:100) were incubated overnight at 4 °C. After washing with PBS, the sections were incubated with Goat Anti-Rabbit IgG H&L (HRP) secondary antibody (Abcam, ab6721, working dilution 1:1000), or Goat Anti-Mouse IgG H&L (HRP) secondary antibody (Abcam, ab205719, working dilution 1:1000) at room temperature for 1 h. Color reaction was performed using Horseradish catalase DAB color kit (#C520017, Sangon Biotech (Shanghai) Co., Ltd.). The sections were counterstained with hematoxylin, followed by a process of dehydration, sealing, and subsequent observation under a light microscope (Olympus, Japan).

Total RNA isolation, library preparation and RNA transcriptomics sequencing

Tumor cells were cultivated in six-well plates and subjected to treatment with MGCD-265 for 24 h. Total RNA was then extracted from these cells utilizing TRIzol™ LS Reagent (#10296010, Invitrogen, Thermo Fisher Scientific Inc.) as per the manufacturer’s guidelines. The quality of the extracted total RNA was measured using Q9000 Micro-Volune Spectrophotometer (Quawell Ltd.), and the purity of the total RNA samples was assessed based on the ratio of absorbance at 260 nm and 280 nm, with ratios between 1.8 and 2.0 being considered acceptable. The RNA integrity numbers ≥ 7 was used for library preparation.

For library preparation, we employed the TruSeq® Stranded mRNA Library Prep Kit (Illumina, USA) following manufacturer’s instructions. Subsequently, these libraries were utilized for paired-end sequencing, accomplished through the HiSeq X Sequencing Platform (Illumina, USA). To evaluate gene expression levels, RPKM values (Reads Per Kilobase Million) of transcripts and the ratio of transcripts were utilized to calculate the total RPKM values for each gene.

Gene Ontology (GO) and Kyoto Encyclopedia Of Genes And Genomes (KEGG) Analysis

The utilization of GO functional annotation analysis and KEGG analysis is a prevalent approach in conducting extensive investigations on gene functional enrichment, encompassing analyses of biological process (BP), molecular function (MF), and cellular component (CC). KEGG databases serve as valuable resources for examining pertinent genomic data, biological pathways, diseases, and drugs. Pathway-based enrichment of genes is performed, and differential expressed genes (DEGs) are subjected to GO and KEGG analyses utilizing the “cluster profiler” tool within the R package.

Drug synergy calculation

Following the drug intervention tests outlined above, the rate of inhibition was computed using the formula: rate of inhibition = ((1 - OD of experimental group/OD of control group)) x 100%. The half inhibitory concentration (IC50) was determined utilizing Origin Pro 50.7 software (OriginLab, Massachusetts, USA). To evaluate the synergistic effect of MGCD-265 and SKLB325, Q values were calculated based on King’s formula analysis: Q = E(A + B)/(E A + E B -E A XE B), where E(A + B) represents the combined drug inhibition rate, and EA and EB represent the inhibition rates of drugs A and B, respectively. A Q value of 0.85–1.15 indicates a simple summation of the effects of the two drugs, Q greater than 1.15 indicates enhancement (or synergy), and Q less than 0.85 indicates antagonism.

GEO database-based analysis

The gallbladder cancer transcriptome dataset (GSE74948) was obtained from the NCBI GEO database, encompassing six samples: three cancerous tissue samples and three normal tissue samples. Corresponding platform annotation files were also acquired to convert probes into gene symbols. In cases where multiple probes corresponded to the same gene symbol, the average value was computed as the gene’s expression value. Differential expression analysis was carried out using the limma package, employing linear regression and empirical Bayesian methods. This analysis yielded the respective P-values and logFC values for the genes. The threshold for identifying differentially expressed genes was set at P-value < 0.05 and |logFC| > 2.

To further characterize these differentially expressed genes, the clusterProfiler package was utilized for GO and KEGG enrichment analysis. This analysis provided insights into the biological processes and pathways associated with the genes. Both up- and down-regulated genes underwent GO and KEGG analyses to comprehensively understand the alterations in molecular pathways. Additionally, exploring protein-protein interactions (PPIs) among the differentially expressed genes was crucial. Human protein-protein interaction data were obtained from the online STRING database, utilizing a minimum interaction score of 0.9 as the parameter value, thus ensuring high-confidence interactions were considered.

Protein-protein interaction network

The STRING database is a valuable tool for both identifying known proteins and predicting protein interactions. In our study, we leveraged the capabilities of the STRING database to pinpoint differentially expressed genes (DEGs) that achieved a combined score surpassing 400. These identified DEGs were employed to construct mRNA-associated PPIs, which were further presented visually using Cytoscape (version 3.6.1).

Analysis of the drug gene interaction database (DGIdb)

The DGIdb, a database focused on drug-gene interactions, offers comprehensive information regarding the correlation between genes and their established or potential pharmaceutical agents. In order to identify potential pharmaceutical candidates or small molecule inhibitors for gallbladder cancer, the DGIdb database was utilized to screen for such compounds, employing key target genes as screening criteria.

CCK-8 assay

In the logarithmic growth phase, GBC-SD, NOZ, and SGC-996 cells were seeded in 96-well plates at a density of 2 × 103 cells/well (100 µL/well). After cell attachment, the culture medium was replaced with fresh medium containing varying drug concentrations. Each drug concentration was tested in five replicate wells, with a corresponding set of blank control wells. The incubation continued for 24 h. Post-incubation, the culture medium was replaced with 100 µL of a CCK-8 (#B34304, Selleck) mixed with culture medium. Following a 4-hour incubation in the incubator, absorbance at 450 nm was measured using TECAN F50 Microplate Reader, allowing for the calculation of cell viability and IC50.

Colony formation assay

GBC-SD, NOZ, and SGC-996 cells in logarithmic growth phase were enzymatically dissociated using 0.25% trypsin (Gibco) to obtain single-cell suspensions in culture medium. These single cells were then plated at a density of 400 cells per well in 6-well plates, with each group having 3 replicate wells. Subsequently, cells were exposed to varying concentrations of MGCD-265 for a duration of 10 days. Following this incubation period, the cell culture plates underwent several washes with PBS, fixation with 4% paraformaldehyde for 1 h, and staining with Rapid Giemsa staining kit (#E607314, Sangon Biotech (Shanghai) Co., Ltd.) for 30 min at room temperature. The resultant cell clones were quantified.

Cell cycle analysis

Gallbladder cancer cells in logarithmic growth phase were seeded in six-well plates and treated with different concentrations of MGCD-265 for 24 h. Adherent cells were detached using trypsin digestion, yielding cell suspensions. These suspensions were then fixed in pre-chilled 70% ethanol overnight at 4 °C. Following centrifugation and removal of ethanol, cells were washed twice with cold PBS, resuspended, and treated with 100 µL RnaseA (1 mg/mL) and 400 µL propidium iodide (PI) staining buffer (KeyGEN BioTECH, #KGA9101-100) in accordance with the manufacturer’s instructions. After a 30-minute incubation at 4 °C in the dark, the cells were again washed twice with cold PBS, resuspended, and subjected to cell cycle analysis using flow cytometry (CytoFLEX LX, Beckman).

Cell apoptosis analysis

Gallbladder cancer cells in logarithmic growth phase were cultured in 24-well plates (1.0 × 105 cells/well) and exposed to varying concentrations of MGCD-265 for 24 h. Each concentration was tested in triplicate and a corresponding blank control was established. After a 24-hour incubation period, both adherent and supernatant cells were collected. A cell suspension was then prepared and mixed with 300 µL of Binding Buffer, 5 µL of Annexin V, and 5 µL of PI (KeyGEN BioTECH, # KGA1102-100). This suspension was incubated for 20 min at room temperature, protected from light. Apoptosis was measured using flow cytometry (CytoFLEX LX, Beckman).

Statistical analysis

All data were presented as mean ± standard deviation (SD), with each experiment being replicated three times. Statistical analyses were performed using SPSS 22.0. An independent Student’s t-test was utilized for comparing two groups, while one-way analysis of variance (ANOVA) was employed for evaluating differences among multiple groups. A significance level of p < 0.05 was considered statistically significant.