Propofol (cat. no. HY-B0649) and TAM (cat. no. HY-13757A) were purchased from MedChemExpress. For the experiments, propofol and TAM were dissolved in dimethyl sulfoxide (DMSO) and further diluted in culture medium for storage. Cell Counting Kit-8 (CCK-8) (cat. no. P-CA-001) and phenol red-free high-glucose Dulbecco's Modified Eagle Medium (DMEM) (cat. no. PM150223) were purchased from Wuhan Pricella Biotechnology Co., Ltd. Certified charcoal-stripped fetal bovine serum (FBS; cat. no. 04-201-1A) was purchased from Biological Industries; Sartorius AG.

The human breast adenocarcinoma cell line MCF7 was purchased from American Type Culture Collection (cat. no. HTB-22). The cells were grown in complete DMEM supplemented with 10% charcoal-stripped FBS, penicillin (100 U/ml) and streptomycin (100 µg/ml) under standard conditions at 37°C with 5% CO₂. MCF7-TR cells were prepared as described in previous reports with slight modifications (18,19). Briefly, the induction of TAM resistance was performed using culture medium containing 10 nM TAM for 48 h. Subsequently, the drug was removed and the cells underwent routine culturing, while continuously eliminating susceptible non-viable cells. Once the cell confluence reached 70–80%, the operation was repeated twice in succession. The selection process entailed iterative cycles, each time increasing the drug concentration incrementally. Specifically, the dosage of the drug was doubled with each iteration. This step-by-step adjustment continued until the cells could proliferate stably in a selection culture medium supplemented with 1 µM TAM. The parental control cells were treated with DMSO and underwent identical processing procedures as previously described (18,19). This entire process lasted ~12 months. To maintain TAM resistance, a concentration of 1 µM TAM was continuously supplemented to the MCF7-TR cell culture medium. In addition, the expression levels of two commonly studied TR-associated genes: ER 1 (ESR1) and ATP binding cassette subfamily B member 1 (ABCB1; also called P-glycoprotein), were detected in MCF7 and MCF7-TR breast cancer cells by reverse transcription-quantitative PCR (RT-qPCR). As shown in Fig. S1A, the expression of ESR1 was downregulated, whereas ABCB1 was upregulated in MCF7-TR cells as compared with in non-resistant parental MCF7 controls, which confirmed the successful construction of TAM resistance (20,21).

RT-qPCR was conducted as described in a previous study (22). Briefly, MCF7-TR cells and parental non-resistant control cells were lysed in 1 ml TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) on ice for 15 min. Subsequently, 200 µl chloroform was added to each sample to fully dissociate the nucleoprotein complex. Following centrifugation (12,000 × g, at 4°C for 15 min), the upper aqueous phase was transferred to a new microcentrifuge tube and mixed with an additional 500 µl isopropyl alcohol to precipitate the RNA. After further centrifugation (12,000 × g, at 4°C for 10 min), the RNA pellet was washed twice with 1 ml 75% ethanol. After air-drying, the RNA samples were resuspended in 100 µl diethyl pyrocarbonate-treated deionized water, and their concentrations were determined using a microspectrophotometer (NanoDrop 2000; Thermo Fisher Scientific, Inc.). For RT, first-strand cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (cat. no. K1622; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Subsequently, qPCR was carried out 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 instructions. The thermocycling conditions were as follows: 95°C for 30 sec for cDNA pre-denaturation; 95°C for 10 sec for cDNA denaturation; 60°C for 30 sec for primer annealing and new strand extension. With the exception of the pre-denaturation step, the denaturation, annealing and extension steps were repeated for a total of 40 cycles. GAPDH was used as a housekeeping gene. The primer sequences are listed in Table SI.

Cell cycle progression and the induction of apoptosis were analyzed using a Gallios flow cytometer (Beckman Coulter, Inc.) according to the manufacturer's instructions.

For cell cycle analysis, MCF7-TR cells were seeded in a 12-well plate at a density of 8×10⁴ cells/well and were incubated overnight. The cells were treated with 20 µM propofol for 24 h at 37°C in an incubator with 5% CO₂, whereas the control cells were treated with an equal volume of DMSO. Subsequently, the cells were collected and centrifuged at 300 × g at 4°C for 5 min. The cells were fixed with ice-cold 70% ethanol for 15 min on ice, washed with PBS, incubated with PI staining solution containing 50 µg/ml PI, 0.1 mg/ml RNase A and 0.05% Triton X-100 at room temperature for 1 h, and detected using a flow cytometer. The data were processed using the cell cycle fitting software ModFit LT 5.0 (Verity Software House, Inc.).

For apoptosis analysis, MCF7-TR cells were seeded in a 12-well plate at a density of 8×10⁴ cells/well and were incubated overnight. The cells were treated with 10 or 20 µM propofol for 24 h at 37°C in an incubator with 5% CO₂, and the control cells were treated with an equal volume of DMSO. Subsequently, the cells were collected and centrifuged at 300 × g at 4°C for 5 min. Apoptosis detection was performed using a fluorescein isothiocyanate Annexin V/PI kit (Becton, Dickinson and Company) according to the manufacturer's instructions. The samples were analyzed by fluorescence-activated cell sorting using a flow cytometer within 1 h of staining. The data were analyzed using Kaluza v. 1.2 (Beckman Coulter, Inc.), and the percentages of early apoptotic cells (Annexin V⁺ and PI⁻) and late apoptotic cells (Annexin V⁺ and PI⁺) were calculated.

Cell proliferation was assessed using the CCK-8 assay according to the manufacturer's instructions. Briefly, MCF7-TR cells and their parental cells were seeded at a density of 6×10³ cells/well and were cultured in 96-well plates overnight. Subsequently, 2.5, 5 or 10 µM propofol was added to the cells and incubated for 0, 24, 48, 72 or 96 h. A total of 10 µl CCK-8 solution was then added to each well and incubated for 1 h. The absorbance value was detected at 450 nm using a spectrophotometer (Synergy HT; BioTek; Agilent Technologies, Inc.).

The colony formation assay was performed as described in a previous study (23). Briefly, MCF7-TR cells were seeded in 6-well plates at a density of 1×10³ cells/well and were cultured overnight. Subsequently, 2.5 µM propofol was added to each well and the control cells were treated with an equal volume of DMSO Subsequently, the medium was replaced with fresh complete medium on day 3. On day 6, the cell colonies were fixed with 4% paraformaldehyde (cat. no. P0099; Beyotime Institute of Biotechnology) for 10 min at room temperature. After being washed twice with distilled water for 2 min each time, the colonies were stained with 2 ml/well crystal violet solution (cat. no. C0121; Beyotime Institute of Biotechnology) for 10 min at room temperature. After washing twice with distilled water (5 min/wash), the plates were scanned using a scanner (HP ScanJet Pro 2600 f1; HP Development Company, L.P.) and the colonies were calculated manually using an inverted light microscope (CKX53; Olympus Corporation). Colonies with cell numbers >20 were counted.

A total of 5×10⁶ MCF7-TR cells were seeded in a 6-cm cell culture dish and cultured overnight. Subsequently, propofol (10 µM) was added to each plate and the cells were cultured for 24 h. The control cells were treated with an equal volume of DMSO. Total RNA was obtained from propofol-treated or non-treated MCF7-TR cells using TRIzol reagent according to the manufacturer's instructions. The quality and concentration of mRNA were detected using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). The purified mRNA from three repeat samples of the solvent control and propofol-treated MCF7-TR cells was processed to prepare an mRNAseq library using Illumina Stranded Total RNA Prep with Ribo-Zero Plus or Ribo-Zero Plus Microbiome kit (Illumina, Inc.) (24). Briefly, ribosomal RNAs were eliminated by a poly-(A) containing mRNA selection procedure to minimize their sequencing. Subsequently, the remaining mRNAs were subjected to cDNA strand synthesis, purification, end-repair, A-tailing adaptor ligation and PCR amplification. The library quality was analyzed using Agilent Bioanalyzer 2100 (Agilent Technologies, Inc.) and the concentrations of the libraries were detected by qPCR analysis. Subsequently, 20 pM each library was sequenced using an Illumina NextSeq500 instrument (Illumina, Inc.) equipped with NextSeq System Suite v2.2.0 software (Illumina, Inc.). Paired-end reads with a length of 150 bp were harvested 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) and depicted using the FactoMineR package (https://cran.r-project.org/web/packages/FactoMineR/index.html) in R (version 4.4.2; http://www.r-project.org/). Genes with |log2FoldChange|>1 and P<0.05 were identified as being differentially expressed. Two-way cluster analysis on all DEGs was performed using the pheatmap package (https://cran.r-project.org/web/packages/pheatmap/index.html), based on expression levels across samples and patterns within samples, using Euclidean distance and complete linkage hierarchical clustering. Volcano plots were generated using the ggplot2 package (https://cran.r-project.org/web/packages/ggplot2/index.html). Data analysis for gene expression was performed based on the Database for Annotation, Visualization and Integrated Discovery (https://davidbioinformatics.nih.gov/). DEGs underwent GO enrichment analysis to determine the enriched biological processes, cellular components and molecular functions (http://www.geneontology.org/). The signaling pathways were investigated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (http://www.genome.jp/kegg/). In addition, Gene Set Enrichment Analysis (GSEA) was performed using GSEA software (https://www.broadinstitute.org/gsea/) using the following: Permutation, geneset; metric, Diff_of_classes; metric, weighted; # permutation, 2,500.

Data are presented as the mean ± SD and are representative of three independent experiments. Data analysis was performed using GraphPad Prism 7 (Dotmatics). Statistical significance between two experimental groups was evaluated using an unpaired two-tailed Student's t-test. Multiple sets of data were compared using a one- or two-way analysis of variance with Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Fig. 1A shows the chemical structure of propofol. To investigate the effect of propofol on MCF7-TR cells, the cells were incubated with 10 and 20 µM propofol for 24 h. Apoptosis induction was then analyzed by flow cytometry (Figs. 1B, 1C, S1B and S1C). Notably, 10 µM propofol caused only a slight, but significant, increase in both early and late apoptosis of MCF7-TR cells (Fig. S1C), whereas 20 µM resulted in a significant increase (Figs. 1C and S1C). Therefore, 20 µM propofol was selected for further analysis. Propofol at a concentration of 20 µM induced a marked increase in the percentage of MCF7-TR cells present in the G₀/G₁ phase, and notable decreases in the percentage of cells present in the S and G₂/M phases of the cell cycle compared with in the control group, suggesting that it induced significant cell cycle arrest (Fig. 1D and E). Considering that 20 µM propofol may induce significant apoptosis and cell cycle arrest, lower concentrations of propofol, specifically 2.5, 5 and 10 µM, were selected for the cell proliferation assay. Consistent with these findings, propofol treatment markedly inhibited cell proliferation in a dose-dependent manner. Notably, when the concentration of propofol reached 10 µM, it caused a significant decrease in the proliferation of MCF7-TR cells after 96 h of incubation (Fig. 1F). MCF7-TR cells exhibited greater sensitivity to propofol compared with non-TR control cells, as evidenced by significantly reduced cell proliferation in MCF7-TR cells following propofol treatment (Fig. S1D). Since prolonged exposure to either 5 or 10 µM propofol significantly reduced cell proliferation (Fig. 1F), a concentration of 2.5 µM was selected to investigate its effects on cell colony formation. In the cells treated with 2.5 µM propofol, both the size and number of colonies were markedly reduced compared with in the cells treated with a solvent control, indicating that propofol exerted an inhibitory effect on the colony-forming ability of MCF7-TR cells (Fig. 1G and H). These results indicated that propofol exhibited potential antitumor characteristics in TR breast cancer cells.

RNAseq analysis was performed for MCF7-TR cells treated with or without propofol. Given that a 24-h treatment with 20 µM propofol resulted in a significant increase in the apoptosis of MCF7-TR cells (Figs. 1B, 1C, S1B and S1C), 10 µM propofol was selected to investigate its gene regulatory effects on MCF7-TR cells. PCA indicated optimal intergroup difference and intragroup consistency between MCF7-TR cells treated with or without propofol (Fig. 2A). A total of 1,065 DEGs were identified in MCF7-TR cells following treatment with propofol, in which the expression levels of 685 genes were markedly downregulated and those of 380 genes were upregulated compared with those in the control cells (Fig. 2B-D; Table SII). Overall, propofol treatment markedly altered the gene expression profiles of MCF7-TR cells.

In order to identify the key biological processes in which the DEGs in propofol-treated MCF7-TR cells were involved, the molecular functions they exerted and the pivotal signaling pathways they were involved in, GO term classification and KEGG enrichment analysis were performed. As shown in Fig. 3A, a high number of DEGs in MCF7-TR cells treated with propofol were clustered in the ‘cell periphery’ and located in the ‘plasma membrane’, possessing phosphatase activity, and were involved in the regulation of the immune response and in the phosphorylation processes. Furthermore, KEGG enrichment analysis indicated that various enriched pathways in all treated cells were related to the process of ‘transcriptional misregulation in cancer’ and ‘cell cycle’, which occur in cancer biology (Fig. 3B). In addition, the enriched signaling pathways that were identified were associated with specific immune response processes, as indicated by ‘cytokine-cytokine receptor interaction’ and ‘chemokine signaling pathway’, and the endocrine system, as indicated by ‘estrogen signaling pathway’ and ‘progesterone-mediated oocyte maturation’ according to the rich-factor and false discovery rate (FDR) values (Fig. 3B). These results indicated that propofol treatment could markedly regulate tumor biology-associated processes, immune response and metabolism.

Based on the GO term classification and the KEGG enrichment analysis, the DEGs involved in immune response-associated signaling pathways were further analyzed. As shown in Fig. 4, the top four most dysregulated signaling pathways, according to the rich factor and FDR value, in MCF7-TR cells treated with propofol were as follows: Chemokine signaling pathway (Fig. 4A), IL-17 signaling pathway (Fig. 4B), TNF signaling pathway (Fig. 4C) and leukocyte transendothelial migration (Fig. 4D). Among these pathways, DEGs affected by propofol treatment were mainly clustered into the chemokine signaling pathway, in which 13 genes were upregulated and seven genes were markedly downregulated (Fig. 4A). In addition, for TNF signaling pathway, two genes (MMP9 and TRAF1) were significantly downregulated, and seven genes were upregulated upon propofol administration (Fig. 4C). These results suggested that propofol administration in MCF7-TR cells significantly regulated immune response signals, mainly manifested by the chemokine signaling pathway.

The enriched DEGs involved in the metabolic process, according to the rich factor and FDR value, were analyzed and are shown in Fig. 5. The impact of propofol on metabolism was characterized by riboflavin metabolism (Fig. 5A), fatty acid biosynthesis (Fig. 5B), thyroid hormone synthesis (Fig. 5C) and arachidonic acid metabolism (Fig. 5D). Among these processes, the effect of propofol on metabolic regulation was mainly characterized by inhibition of the expression levels of metabolism-related genes. No upregulated genes were noted in the processes of riboflavin metabolism and fatty acid biosynthesis, and higher numbers of downregulated genes were noted in the thyroid hormone synthesis and arachidonic acid metabolism processes in MCF7-TR cells following treatment with propofol compared with in the control cells. These findings indicated that propofol was involved in regulation of the metabolic process, which was mainly dependent on downregulation of the expression levels of metabolic genes.

To further characterize the molecular functions involved in the immune response and metabolism, based on the findings from the GO and KEGG analyses in propofol-treated MCF7-TR cells, GSEA was performed on the RNAseq data (Fig. 6). The negatively enriched gene sets were involved in ABC transporters (Fig. 6A), the adipocytokine signaling pathway (Fig. 6B), and aldosterone synthesis and secretion (Fig. 6C), while the positively enriched gene set was involved in the aminoacyl-tRNA biosynthesis (Fig. 6D). These gene sets were composed of genes associated with certain metabolites, which indicated that propofol functions partially by activating and inhibiting metabolism-related signaling pathways.