Roswell Park Memorial Institute 1640 (RPMI-1640) medium (cat. no. PM150110), Iscove's Modified Dulbecco Medium (IMDM; cat. no. PM150510) and fetal bovine serum (FBS; cat. no. 164210) were purchased from Wuhan Pricella Biotechnology Co., Ltd.Isoimperatorin (cat. no. HY-N0286) was purchased from MedChemExpress. For the experiments, isoimperatorin was dissolved in DMSO (5 mM) and further diluted to 100 µM in RPMI-1640 medium for storage. Cell Counting Kit-8 (CCK-8; cat. no. P-CA-001), TRIzol™ (cat. no. 15596018), RevertAid First Strand cDNA Synthesis Kit (cat. no. K1622) and PowerTrack™ SYBR-Green Master Mix (cat. no. A46012) were purchased from Thermo Fisher Scientific, Inc.

The human Tohoku Hospital Pediatrics-1 (THP-1; cat. no. TIB-202) and AML-193 (cat. no. CRL-9589) cell lines were purchased from the American Type Culture Collection. THP-1 cells were cultured in RPMI-1640 medium, while AML-193 cells were cultured in IMDM. Both media were supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. Cell cultures were maintained under standard conditions at 37°C with 5% CO₂.

THP-1 and AML-193 cells were seeded into 96-well plates at a density of 5×10³ cells per well. The cells were then cultured overnight to allow for cell adherence. Subsequently, the cells were treated with 5, 10, 20, 40, 80, 160 and 320 µM isoimperatorin for 24 h. In parallel, a control group was set up, where an equivalent volume of DMSO was added. After incubation, 10 µl CCK-8 solution was added to each well, and the plates were then incubated for an additional 2 h. Subsequently, a spectrophotometer (Synergy HT; BioTek; Agilent Technologies, Inc.) was used measure the absorbance value at 450 nm, which was then used to calculate the IC₅₀ values using nonlinear regression analysis performed on the GraphPad Prism (version 7; Dotmatics) software.

THP-1 and AML-193 cells were seeded into 12-well plates at a density of 1×10⁵ cells per well. The cells were then incubated overnight to allow for cell adherence. Based on the IC₅₀ values for the two cell lines, THP-1 cells were treated with 12.5, 25 and 50 µM isoimperatorin, while AML-193 cells were exposed to 15, 30 and 60 µM isoimperatorin. All treatments were performed at 37°C in a humidified incubator with 5% CO₂ for 24 h. In parallel, a control group was set up, where an equivalent volume of DMSO was added. After the treatment, the cells were carefully collected and then subjected to centrifugation at 300 × g for 5 min at 4°C to pellet the cells. Subsequently, apoptosis detection was performed using a fluorescein isothiocyanate annexin V/PI kit (cat. no. 556547, Becton, Dickinson and Company) according to the manufacturer's instructions. Within 1 h of staining, the samples were analyzed using a flow cytometer (BD FACSCelesta™; BD Biosciences). The acquired data were processed and analyzed using FlowJo™ (version 10.8.1; BD Biosciences) software. The percentages of early apoptotic cells (characterized by being annexin V⁺ and PI⁻) and late apoptotic cells (characterized by being annexin V⁺ and PI⁺) were next calculated.

Cell proliferation assay was conducted using the CCK-8 assay according to the manufacturer's protocol. Briefly, THP-1 and AML-193 cells were seeded into 96-well plates at a density of 5×10³ cells per well. The cells were cultured overnight to allow for cell adherence at 37°C with 5% CO₂. Subsequently, THP-1 cells were treated with 6.25, 12.5 and 25 µM isoimperatorin, while AML-193 cells were exposed to 7.5, 15 and 30 µM isoimperatorin. Cells were incubated at 37°C for 0, 24, 48, 72 and 96 h to observe the time-dependent effects of isoimperatorin on cell proliferation. Subsequently, 10 µl CCK-8 solution was added to each well. The plates were then incubated at 37°C with 5% CO₂ for an additional 2 h. Finally, a spectrophotometer (Synergy HT; BioTek; Agilent Technologies, Inc.) was utilized to measure the absorbance values at 450 nm, which was then used to assess the cell proliferation status, since such absorbance values are directly proportional to the number of viable cells.

A total of 1×10⁷ THP-1 cells were initially seeded in a 6-cm cell culture dish and cultured overnight. Taking into account the apoptosis experiment results, to investigate signaling pathways without eliciting excessive apoptosis, 12.5 µM isoimperatorin was added to each culture plate and the cells were further incubated for 24 h. Meanwhile, the control cells were treated with an equivalent volume of DMSO. Subsequently, total RNA was isolated from both the isoimperatorin-treated and non-treated THP-1 cells using TRIzol reagent (Thermo Fisher Scientific, Inc.), following the manufacturer's instructions. Next, a NanoDrop 2000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.) was utilized to determine the quality and concentration of the extracted total RNA. Purified total RNA from three replicate samples of both solvent-controlled and isoimperatorin-treated THP-1 cells was then used to prepare an mRNAseq library using the Illumina Stranded Total RNA Prep in combination with Ribo-Zero Plus rRNA Depletion kit (cat. no. 20040529, Illumina, Inc.). Ribosomal RNAs were eliminated through a poly-(A)-containing mRNA selection process, in order to reduce their presence in the subsequent sequencing results. Following ribosomal RNA removal, the remaining mRNAs underwent a series of procedures, including cDNA strand synthesis, purification, end-repair, A-tailing adaptor ligation and PCR amplification. The quality of the constructed libraries was analyzed using an Agilent Bioanalyzer 2100 (Agilent Technologies, Inc.) and the library concentrations were determined through qPCR analysis as described below. Subsequently, each library was adjusted to a concentration of 20 pM and then sequenced using an Illumina NextSeq500 instrument (Illumina, Inc.) equipped with NextSeq System Suite software (version 2.2.0; Illumina, Inc.). Finally, paired-end reads with a length of 150 bp were acquired using the NextSeq 550 System High-Output Sequencing Kit (Illumina, Inc.).

Differential gene expression analysis and Principal Component Analysis (PCA) were conducted using the DESeq package (https://bioconductor.org/packages//2.10/bioc/html/DESeq.html) within the R environment (version 4.4.2; https://www.r-project.org/; Posit Software, PBC). The resultant data were then visualized using the FactoMineR package (https://cran.r-project.org/web/packages/FactoMineR/index.html). Genes were classified as differentially expressed when |log2 fold-change| >1 and P<0.05 (adjusted P-value following false discovery rate correction). Subsequently, a two-way cluster analysis on all the identified DEGs was performed using the pheatmap package (https://cran.r-project.org/web/packages/pheatmap/index.html). Furthermore, volcano plots were generated using the ggplot2 package (https://cran.r-project.org/web/packages/ggplot2/index.html) to visually display the differential gene expression data. For gene expression data analysis, the Database for Annotation, Visualization and Integrated Discovery (https://davidbioinformatics.nih.gov/) software was employed. The DEGs underwent Gene Ontology (GO) enrichment analysis (http://www.geneontology.org/) to identify significantly enriched biological processes, cellular components and molecular functions. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (http://www.genome.jp/kegg/) was utilized to explore the associated signaling pathways. Additionally, Gene Set Enrichment Analysis (GSEA) was carried out with GSEA software (https://www.broadinstitute.org/gsea/).

A total of 1×10⁷ THP-1 and AML-193 cells were seeded into 6-cm cell culture dishes and cultured overnight to allow for cell adherence. Subsequently, 12.5 and 15 µM isoimperatorin was added to THP-1 and AML-193 cells, respectively, and further cultured for 24 h. Control cells were treated with an equal volume of DMSO. Total RNA was extracted from both isoimperatorin-treated and non-treated cells using TRIzol reagent, according to the manufacturer's instructions. Specifically, 200 µl chloroform was added to each sample to fully dissociate the nucleoprotein complex. Subsequently, the samples were centrifuged at 12,000 × g for 15 min at 4°C. The upper aqueous phase was then carefully transferred to a new microcentrifuge tube and mixed with 500 µl isopropyl alcohol to precipitate the RNA. After centrifugation at 12,000 × g for 10 min at 4°C, the resulting RNA pellet was washed twice with 1 ml 75% ethanol. Once air-dried, the RNA samples were resuspended in 100 µl diethyl pyrocarbonate-treated deionized water. The concentrations of the resuspended RNA samples were measured using a microspectrophotometer (NanoDrop 2000; NanoDrop Technologies; Thermo Fisher Scientific, Inc.). For RT, first-strand cDNA was synthesized following the manufacturer's instructions of the RevertAid First Strand cDNA Synthesis Kit (cat. no. K1622; Thermo Fisher Scientific, Inc.). Subsequently, qPCR was performed on a Bio-Rad CFX-Touch Real-Time PCR System (Bio-Rad Laboratories, Inc.) using the PowerTrack™ SYBR-Green Master Mix (cat. no. A46012; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The thermocycling conditions were as follows: cDNA pre-denaturation, 95°C for 30 sec; cDNA denaturation, 95°C for 10 sec; and primer annealing and new strand extension, 60°C for 30 sec. The denaturation, annealing and extension steps were repeated for a total of 40 cycles. For each pathway, two most significantly up- and downregulated genes were selected to verify the reliability of RNAseq results. GAPDH was selected as a reference gene and quantification of gene expression was performed using the 2^−ΔΔCq method (23). The primer sequences are shown in Table SI.

Data are presented as the mean ± SD of three independent experiments. Statistical analysis was performed using GraphPad Prism (version 7; Dotmatics) software. Statistical significance was evaluated using a one- or two-way analysis of variance with Tukey's post hoc test for multiple comparisons, or unpaired two-tailed Student's t-test for comparisons between two groups. P<0.05 was considered to indicate a statistically significant difference.

Fig. S1A depicts the chemical structure of isoimperatorin. The IC₅₀ values for isoimperatorin-treated THP-1 and AML-193 cells were 51.93 µM (Fig. S1B) and 67.55 µM (Fig. S1C), respectively. To explore the impact of isoimperatorin on tumor biological progression in AML, THP-1 cells were incubated with 12.5, 25 and 50 µM isoimperatorin for 24 h. Subsequently, induction of apoptosis was analyzed using flow cytometry. A concentration of 12.5 µM isoimperatorin induced a statistically significant increase in both early-stage and late-stage apoptosis within THP-1 cells when compared with control cells (Fig. 1A). Within the 25 and 50 µM groups, the proportions of cells undergoing early and late apoptosis were also significantly increased compared with the control group, which demonstrated that isoimperatorin induced apoptosis in THP-1 cells in a dose-dependent manner (Fig. 1A-C). Given that 50 µM isoimperatorin induced substantial apoptosis within 24 h, lower concentrations of isoimperatorin, specifically 6.25, 12.5 and 25 µM, were selected for cell proliferation assays on THP-1 cells. Treatment with isoimperatorin significantly inhibited cell proliferation in a dose-dependent manner; 25 µM isoimperatorin led to a significant reduction in the proliferation of THP-1 cells following 24, 48, 72 and 96 h of incubation compared with control group (Fig. 1D). Furthermore, in AML-193 cells, isoimperatorin induced significant apoptosis at both early and late apoptotic stages at concentrations of 15, 30 and 60 µM (Fig. 1E-G). In cell proliferation assays with further reduced concentrations, isoimperatorin significantly inhibited AML-193 cell proliferation at a concentration of 7.5 µM, whereas 15 and 30 µM isoimperatorin induced a more pronounced and significant loss of cell proliferation (Fig. 1H). These results suggested that isoimperatorin has the capacity to induce apoptosis and impede cell proliferation within the THP-1 and AML-193 cell lines, and thereby exhibited potential antitumor activity.

RNAseq analysis was conducted for THP-1 cells treated with isoimperatorin and untreated control cells. As a 24-h treatment with 25 and 50 µM isoimperatorin led to a prominent increase in the apoptosis of THP-1 cells, while 12.5 µM isoimperatorin induced a slight apoptosis in THP-1 cell, 12.5 µM isoimperatorin was selected to explore its regulatory impacts on gene expression in THP-1 cells. PCA demonstrated a distinct separation between isoimperatorin-treated and untreated THP-1 cells (indicating treatment-induced transcriptional shifts) and tight intragroup clustering (validating experimental reliability), supporting the robustness of analyses (Fig. 2A). In total, 688 DEGs were identified in THP-1 cells subsequent to the administration of isoimperatorin. Among these, the expression of 466 genes was significantly downregulated, while 222 genes were significantly upregulated compared with those in the control cells (Fig. 2B and C). Administration of isoimperatorin significantly altered the gene expression profiles in THP-1 cells. To determine the GO terms and KEGG signaling pathways into which the DEGs were clustered post-isoimperatorin treatment, analyses were performed based on the rich factor and FDR values. The top 20 most significantly altered (based on the FDR value) GO term classifications and KEGG enrichment results are shown. A substantial number of DEGs were involved in processes related to ‘cell communication’ and ‘signaling’ and certain DEGs were clustered into categories such as ‘cell migration’, ‘cell projection’ and ‘cell motility’ in GO enrichment (Fig. 2D). Furthermore, KEGG enrichment analysis demonstrated that numerous significantly enriched pathways in all treated isolates were associated with the nervous system, including ‘cholinergic synapse’, ‘axon guidance’ and ‘GABAergic synapse’. Additionally, numerous DEGs were also significantly enriched in the ‘MAPK signaling pathway’, ‘calcium signaling pathway’ and ‘Rap1 signaling pathway’ (Fig. 2E). Collectively, these results indicated that isoimperatorin exerts a marked regulatory effect on tumor biology-associated signal transduction in THP-1 cells.

DEGs that were significantly enriched and implicated in tumor biology processes were analyzed based on the rich factor and FDR value (Fig. 3). The influence of isoimperatorin on tumor biology was exerted through several key signaling pathways, namely ‘microRNAs in cancer’ (Fig. 3A), ‘prostate cancer’ (Fig. 3B), ‘small cell lung cancer’ (Fig. 3C) and ‘proteoglycans in cancer’ (Fig. 3D). Among these pathways, the DEGs affected by isoimperatorin treatment were predominantly clustered within the ‘microRNAs in cancer’ signaling pathway. In this pathway, nine genes were upregulated, while nine genes were markedly downregulated (Fig. 3A). Regarding the ‘prostate cancer’ signaling pathway, upon the administration of isoimperatorin, four genes were significantly upregulated, while six genes were downregulated (Fig. 3B). In the context of ‘small cell lung cancer’ signaling, four genes were downregulated and five genes were significantly upregulated (Fig. 3C). In the context of ‘proteoglycans in cancer’ signaling, isoimperatorin treatment led to the upregulation of five genes and the downregulation of nine genes (Fig. 3D). To validate the RNAseq results, two most significantly up- and downregulated genes were selected from each of the four biological processes identified in the analysis. These genes were then subjected to RT-qPCR evaluation in the THP-1 (Fig. 3E-H) and AML-193 (Fig. S2A-D) cell lines. RT-qPCR results corroborated the RNA-seq data: the expression trends of selected genes were consistent between the two methods, confirming the reliability of the RNA-seq findings. For key genes in tumor biology-such as MYC and BCL2, which were significantly downregulated by isoimperatorin-in vitro validation revealed that their expression changes following isoimperatorin treatment were associated with altered cell proliferation and apoptosis and supports the conclusion that isoimperatorin regulates tumor-related biological processes through these genes.

DEGs that were enriched and involved in metabolic processes were analyzed based on the rich factor and FDR value (Fig. 4). The influence of isoimperatorin on metabolism was manifested through several key metabolic pathways, namely ‘glycosaminoglycan biosynthesis-heparan sulfate/heparin’ (Fig. 4A), ‘mucin type O-glycan biosynthesis’ (Fig. 4B), ‘steroid hormone biosynthesis’ (Fig. 4C) and ‘glycosphingolipid biosynthesis-ganglio series’ (Fig. 4D). Among these pathways, the metabolic pathways associated with ‘glycosaminoglycan biosynthesis-heparan sulfate/heparin’ and ‘glycosphingolipid biosynthesis-ganglio series’ were inhibited by isoimperatorin. This was further substantiated as four genes within the ‘glycosaminoglycan biosynthesis-heparan sulfate/heparin’ pathway and two genes in the ‘glycosphingolipid biosynthesis-ganglio series’ pathway were all significantly downregulated upon treatment with isoimperatorin (Fig. 4A and D). In ‘mucin type O-glycan biosynthesis’ signaling, isoimperatorin treatment significantly suppressed the expression of three genes and promoted the expression level of one gene (Fig. 4B). In the ‘steroid hormone biosynthesis’ pathway, isoimperatorin treatment enhanced the expression of three genes while inhibiting that of two genes (Fig. 4C). To validate the RNAseq results, two most significantly upregulated and two most significantly downregulated genes were selected from each of the four biological processes identified in the analysis. These genes were then subjected to RT-qPCR evaluation in THP-1 (Fig. 4E-H) and AML-193 (Fig. S2E-H) cells. RT-qPCR results corroborated the RNAseq data for the expression trends of selected metabolic process-related genes, validating the reliability of RNA-seq findings. These results suggested that isoimperatorin could significantly regulate the expression of genes associated with metabolic processes in THP-1 cells.

Based on the KEGG enrichment analysis, analysis of the DEGs implicated in immune response-associated signaling pathways was performed. According to the rich factor and FDR value, the top four most altered immune response-associated signaling pathways in THP-1 cells treated with isoimperatorin were as follows: ‘Chemokine signaling pathway’ (Fig. 5A), ‘Th1 and Th2 cell differentiation’ (Fig. 5B), ‘NOD-like receptor signaling pathway’ (Fig. 5C) and ‘leukocyte transendothelial migration’ (Fig. 5D). In the ‘chemokine signaling pathway’, treatment with isoimperatorin significantly suppressed the expression of nine genes and increased the expression levels of four genes (Fig. 5A). Additionally, in the context of the ‘Th1 and Th2 cell differentiation’ pathway, isoimperatorin treatment increased the expression of three genes while downregulating four genes (Fig. 5B). Furthermore, in the ‘NOD-like receptor signaling pathway’, isoimperatorin treatment significantly downregulated the expression of three genes and upregulated the expression levels of eight genes (Fig. 5C). The immune response process associated with ‘leukocyte transendothelial migration’ was inhibited by isoimperatorin. This was further corroborated as all seven genes within this pathway were significantly downregulated upon treatment with isoimperatorin (Fig. 5D). To validate the RNAseq results, two most significantly up- and downregulated genes were selected from each of the four biological processes identified in the analysis. These genes were then subjected to RT-qPCR evaluation in THP-1 (Fig. 5E-H) and AML-193 (Fig. S2I-L) cells. The RT-qPCR results corroborated the RNA-seq data for the expression trends of the selected immune-response process-related genes were consistent between the two methods, confirming the reliability of the RNA-seq findings. These findings suggested that isoimperatorin could significantly regulate the expression of genes associated with the immune-response process in THP-1 cells.

To further elucidate the molecular functions implicated in tumor biology, immune response and metabolism, considering the findings derived from the GO and KEGG analyses in isoimperatorin-treated THP-1 cells, GSEA was performed on the RNAseq data (Fig. 6). The positively enriched gene sets were intricately involved in ‘Rig-I-like receptor signaling pathway’ (Fig. 6A) and ‘legionellosis’ (Fig. 6B). By contrast, the negatively enriched gene sets were associated with ‘ribosome’ (Fig. 6C) and ‘glucosaminoglycan biosynthesis-heparan sulfate heparin’ (Fig. 6D). These gene sets were constituted by genes that are closely related to specific immune signaling and metabolites, which indicates that isoimperatorin potentially exerts its functions, at least in part, through the activation or inhibition of these signaling pathways.