The human Ut-LMS cancer cell lines SK-LMS-1 (cat. no. HTB-88) and SK-UT-1B (cat. no. HTB-115) were purchased from the American Type Culture Collection. The cells were cultured in a humidified incubator maintained in Minimum Essential Medium (cat. no. LM007-07; Welgene, Inc.) containing 10% fetal bovine serum (cat. no. SH30919.03; Hyclone; Cytiva) and 1% streptomycin-penicillin (cat. no. 15140122; Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂. The human uterine smooth muscle cells (Ut-SMCs; cat. no. C-12575) were purchased from PromoCell GmbH and were cultured in smooth muscle cell growth medium 2 (cat. no. C-39267; PromoCell GmbH) supplemented with 1% streptomycin-penicillin. 2-D08 was obtained from MilliporeSigma (cat. no. SML1052-5MG). Dimethyl sulfoxide (cat. no. DMS555.500; BioShop Canada Inc.) was used as a control. Cycloheximide (CHX) solution (cat. no. C4859-1ML), N-acetyl-L-cysteine (NAC; cat. no. A7250), and catalase (CAT; cat. no. C1345) were purchased from Sigma-Aldrich; Merck KGaA.

The cells (5×10³ cells per well) were seeded in 96-well plates for 24 h and then treated with different concentrations of 2-D08 for 24 or 48 h. MTT stock solution (cat. no. M2128; Sigma-Aldrich) was added to each well, before incubation at 37°C for 2 h 30 min and addition of dimethyl sulfoxide (150 µl per well) to all wells after removing the medium. Cell viability was measured at 570 nm using an INNO microplate spectrophotometer (LTEK Co., Ltd.) and normalized to that of the control group.

SK-LMS-1 and SK-UT-1B cells were seeded in 6-well plates (1×10³ cells per well) for 24 h. After removing the medium, 2-D08 was added to a fresh medium containing different concentrations (10, 20, 50, and 100 µM). The cells were cultured at 37°C for 7 days until colonies were visible. To observe colony growth, a crystal violet assay kit (cat. no. ab232855; Abcam) was employed following the manufacturer's protocol. In brief, the cells were fixed with 100% ice-cold methanol for 20 min at −20°C, stained with 2% crystal violet solution for 20 min at room temperature, and colonies containing ≥50 cells were counted. Bright-field microscopy images of the formed colonies were acquired using the EVOS FL Cell Imaging System (Thermo Fisher Scientific, Inc.). The surviving fraction was calculated as the mean number of colonies/[number of inoculated cells × (plating efficiency/100)]. Plating efficiency was defined as 100× (mean number of colonies/number of inoculated cells for control). Survival curves were determined using a linear-quadratic model with the GraphPad Prism 6.0 software (GraphPad; Dotmatics).

LDH was quantified using the Dyne LDH PLUS Cytotoxicity Assay Kit (cat. no. GBL-P500; Dyne Bio Inc.) according to the manufacturer's instructions. Briefly, the cells were treated with 2-D08 at 37°C for 24 or 48 h, as indicated. Subsequently, 100 µl of supernatant was transferred from each well to a new 96-well plate, followed by the addition of 100 µl of LDH PLUS Reaction Mixture to each well and a gentle mix. The plates were incubated at room temperature, protected from light, for 30 min before adding 10 µl of stop solution to each well and being gently mixed. The LDH concentration was measured at 490 nm and the treated groups were compared with the untreated.

Flow cytometric measurement of ROS production was performed using a Muse Oxidative Stress Kit (cat. no. MCH100111; Luminex) according to the manufacturer's instructions. Briefly, SK-LMS-1 cells were seeded in 6-well plates (5×10⁴ cells/well) and allowed to grow at 37°C for 24 h. The cells were then pretreated with 0, 20, 50, or 100 µM 2-D08, diluted in fresh medium at 37°C, for 24 and 48 h. Next, the cells were detached and resuspended in 1X Assay Buffer, and Muse Oxidative Stress Reagent working solution was added. The samples were incubated for 30 min at 37°C before analysis using the Guava Muse Cell Analyzer (cat. no. 0500-3115; Luminex), and further analyzed using Muse analysis software (version 1.5; Luminex).

The apoptotic profiles were studied using the Muse Annexin V & Dead Cell Kit (cat. no. MCH100105; Luminex). Annexin V-PE detects phosphatidylserine on the external membrane of apoptotic cells, while 7-AAD permeates late-stage apoptotic and dead cells by being excluded from live and healthy cells. Ut-LMS cells were treated with 2-D08 at different concentrations (20, 50, or 100 µM) for 24 or 48 h in 6-well plates at 5×10⁴ cells per well. The cells were trypsinized, harvested, and resuspended in a fresh medium. Then, 100 µl of Muse Annexin V & Dead Cell reagent was added. After staining for 20 min at room temperature in the dark, the apoptotic profiles of the cells were recorded using the Guava Muse Cell Analyzer following the manufacturer's instructions and further analyzed using Muse analysis software (version 1.5; Luminex).

SK-LMS-1 cells were fixed with IC Fixation Buffer (cat. no. 00-8222-49; Invitrogen; Thermo Fisher Scientific, Inc.) for 20 min at room temperature and permeabilized with 0.2% Triton X-100 (cat. no. T8787-50ML; Sigma-Aldrich) in 1X phosphate-buffered saline (cat. no. LB 001–02; Welgene, Inc.) for 5 min at room temperature. The cells were then blocked with 2% normal goat serum (cat. no. 5425; Cell Signaling Technology, Inc.) in phosphate-buffered saline and incubated with the anti-Ki67 antibody (1:250; cat. no. MA5-14520; Invitrogen; Thermo Fisher Scientific, Inc.) at 4°C overnight. The anti-Ki67 antibody was detected by incubating the cells with an anti-rabbit IgG antibody (Fab2 Alexa Fluor 594 Conjugate; 1:500; cat. no. 8889S; Cell Signaling Technology, Inc.) for 1 h at room temperature. The slides were mounted using ProLong Gold Antifade Mountant with DAPI (cat. no. P36935; Invitrogen; Thermo Fisher Scientific, Inc.). Fluorescence images were obtained using the EVOS FL Cell Imaging System.

To determine the percentage of proliferating cells based on Ki67 expression, the Muse Ki67 Proliferation Kit (cat. no. MCH10014; Luminex) was used. Briefly, cells expressing Ki67 were harvested, fixed with 1X fixation solution for 15 min at room temperature, and washed with 1X assay buffer. After resuspension, the cells were treated with permeabilization solution for 15 min at room temperature and washed again. Next, the cells were incubated with either Muse Hu IgG1-PE or Muse Hu Ki67-PE antibody for 30 min at room temperature and analyzed using the Guava Muse Cell Analyzer, followed by further analysis using Muse analysis software (version 1.5; Luminex).

The BrdU Cell Proliferation Assay Kit (cat. no. 6831S; Cell Signaling Technology, Inc.) was used to study the proliferating cells. In brief, cells (5×10³ cells/well) were seeded into 96-well plates and treated with 2-D08 at the indicated concentrations for 24 or 48 h. The cells were then exposed to BrdU at 37 °C for 24 h, fixed at room temperature for 30 min, and incubated with mouse anti-BrdU antibody at room temperature for 1 h, and treated according to the manufacturer's protocol. Lastly, the absorbance was measured at 450 nm using an INNO microplate spectrophotometer.

The Muse Cell Cycle Kit (cat. no. MCH100106; Luminex) was used to determine the cell cycle phases of the samples. The cells were centrifuged and fixed for 3 h in 70% ethanol at −20°C. After fixation, the cells were stained with the Muse Cell Cycle Reagent and incubated in the dark at room temperature for 30 min. The samples were analyzed using the Guava Muse Cell Analyzer, followed by further analysis using Muse analysis software (version 1.5; Luminex).

Ut-LMS cells were exposed to various concentrations of 2-D08 (0, 20, or 50 µM) for 48 h. The cells were then lysed in NP-40 lysis buffer (cat. no. MBS355472; MyBiosource, Inc.) containing a protease inhibitor cocktail (cat. no. P8340; Sigma-Aldrich; Merck KGaA). The Pierce BCA Protein Assay kit (cat. no. 23225; Thermo Fisher Scientific, Inc) was used to measure the protein concentration of lysate. Equal amounts of protein (10–50 µg from cell lysate), denatured by heating, were subjected to 12–15% SDS-PAGE. After electrophoresis, the separated proteins were transferred from the gel to PVDF membranes (cat. no. IPVH00010; Merck KGaA), which were blocked with 5% skimmed milk (cat. no. 262100; BD Biosciences) for 1 h at room temperature, and subsequently incubated with the primary antibodies overnight at 4°C with gentle shaking. The primary antibodies used in the present study were RIP (1:1,000; cat. no. 3493T), LC3B (1:1,000; cat. no. 2775S), p21 (1:1,000; cat. no. 2947S), p27 (1:1,000; cat. no. 3686T), p53 (1:1,000; cat. no. 2527S), Caspase-3 (1:1,000; cat. no. 9665S), cleaved Caspase-3 (1:1,000; cat. no. 9664S), Caspase-9 (1:1,000; cat. no. 9502S), PARP (1:1,000; cat. no. 9542S) and β-Actin (1:1,000; cat. no. 4967S) (all from Cell Signaling Technology, Inc.); and α-smooth muscle (SM) actin (1:1,000; cat. no. ab5694), TAGLN (1:5,000; cat. no. ab14106) and Calponin 1 (1:1,000; cat. no. ab46794) (all from Abcam). After washing with the TBST (Tris-buffered saline, 0.05% Tween 20) solution, the membranes were incubated with anti-rabbit (1:5,000; cat. no. 7074S; Cell Signaling Technology, Inc.) and anti-mouse horseradish peroxidase-conjugated secondary antibodies (1:5,000; cat. no. 7076S; Cell Signaling Technology, Inc.) for 1 h at room temperature. The signals were detected using the Immobilon ECL Ultra Western HRP Substrate (cat. no. WBKLS0100; Merck KGaA) and quantified using the Azure c280 chemiluminescent image system. Densitometric analyses of the resulting images were performed using ImageJ 1.53a software (National Institutes of Health).

Total RNA was extracted from SK-LMS-1 cells using NucleoZOL (cat. no. 740404.200; Macherey-Nagel GmbH & Co. KG). RNA samples were reverse transcribed into cDNA using a ReverTra Ace qPCR RT Kit (cat. no. FSQ-101; Toyobo Life Science). In brief, RNA was denatured at 65°C for 5 min, then placed on ice. The reaction solution was prepared with nuclease-free water, 5X RT buffer, RT enzyme mix, primer mix, and RNA (0.5 pg-1 µg) to a total volume of 10 µl. The mixture was incubated at 37°C for 15 min, heated at 98°C for 5 min, and then stored at 4°C or −20°C. RT-qPCR was performed using a QuantiTect SYBR Green RT-PCR Kit (cat. no. 204243; Qiagen GmbH) and the primer pairs listed in Table SI. The cycling conditions included an initial denaturation step at 95°C for 10 min. Amplification was carried out for 40 cycles with denaturation at 95°C 15 sec, followed by annealing at 50°C for 20 sec to achieve optimal annealing temperatures, and extension at 72°C for 20 sec. The RNA level was expressed as a relative result via the 2^−ΔΔCq method (26) against the endogenous standard control β-actin.

Total RNA was extracted from SK-LMS-1 cells using NucleoSpin RNA/Protein (cat. no. 740933.50; Macherey-Nagel GmbH & Co. KG) according to the manufacturer's instructions. RNA quality was assessed with a 4200 TapeStation System with an RNA Screen Tape (Agilent Technologies, Inc.), and RNA was quantified using a Qubit fluorometer (Thermo Fisher Scientific, Inc.). Libraries with total RNAs were constructed using the KAPA RNA HyperPrep Kit with RiboErase (cat. no. 08098140702; Roche Sequencing Solution, Inc.) according to the manufacturer's instructions. The loading concentration of the final library was measured using the TapeStation 4200 System (Agilent Technologies, Inc.) and qPCR was performed on the LightCycler 480 System (Roche Diagnostics) with the Kapa Library Quantification Kit (cat. no. 07960298001; Roche Sequencing Solution, Inc.), resulting in the final library being loaded at 2 nM. High-throughput sequencing was performed in paired-end 150 sequencing runs using a NovaSeq 6000 (Illumina, Inc.) at Genome Insight, Inc. Raw reads were assembled and low-quality reads were filtered using Cutadapt (v.3.4; National Bioinformatics Infrastructure Sweden). Filtered reads were aligned on a reference genome downloaded from Ensembl (GRCh38; Homo sapiens; http://www.ensembl.org) using STAR (v.2.7.8a) (27) and the gene-level expression for each sample was calculated using RSEM (v.1.3.1) (28). Differentially expressed genes were analyzed using a two-slided Wald test under each condition with the DESeq2 (version 1.26.0) (29) and edgeR (version 3.28.1) (30) packages.

The GraphPad Prism software (v.6.0; Graphpad; Dotmatics) was used for the statistical analyses. The results are expressed as the mean ± standard error of the mean (SEM), and the data were analyzed using one-way analysis of variance, followed by Tukey's post hoc test for comparison among multiple groups. *P<0.05 was considered to indicate a statistically significant difference.

The cytotoxic effect of 2-D08 treatment (Fig. 1A) on Ut-LMS cells was first investigated using the well-established SK-LMS-1 and SK-UT-1B cell models. The viability of Ut-LMS cells was determined using MTT assays followed by treatment with 2-D08 at different concentrations (0–100 µM) for 24 or 48 h. As revealed in Fig. 1B, the viability of SK-LMS-1 cells significantly decreased to 21% in the 100-µM 2-D08-treated group at 24 h, although different doses did not lead to statistically significant changes (Fig. 1B, left graph). After 48 h of treatment, SK-LMS-1 cell viability decreased significantly to 80 and 17% at 50 and 100 µM 2-D08 concentrations, respectively (Fig. 1B, right graph). Treatment of SK-UT-1B cells with 2-D08 at concentrations >10 µM for 24 and 48 h significantly inhibited cell viability (Fig. 1C). By contrast, treatment of normal Ut-SMCs with 2-D08 for 24 and 48 h had a slight inhibitory effect on viability (Fig. 1D). These results indicated that the viability of Ut-LMS cells was significantly affected by 2-D08 treatment.

It was investigated whether 2-D08 affects the proliferation of Ut-LMS cells using a colony formation assay. As demonstrated in the crystal violet-stained (upper panels) and bright-field images (lower panels) in Fig. 1E, the size of a single colony in the 2-D08-treated groups (20, 50 and 100 µM) after 7 days was smaller than that in the control group. Compared with control cells, treatment of Ut-LMS cells with 2-D08 (10, 20, 50, and 100 µM) for 7 days induced a dose-dependent decline in survival fraction (Fig. 1F). Furthermore, plating efficiency was measured to assess the colony-forming ability of Ut-LMS cells compared with that of untreated cells. The mean plating efficiency of Ut-LMS cells decreased significantly (Fig. 1G). These findings indicated that 2-D08 plays a key role in inhibiting colony formation and is a potential drug for clinical applications in Ut-LMS cells.

To determine whether the aforementioned reductions in cell viability induced by 2-D08 in SK-UT-1B cells were associated with apoptotic cell death and cell cycle arrest, a flow cytometric analysis was performed. Apoptotic cells were identified using Annexin V. As revealed in Fig. 2A, the percentage of apoptotic cells slightly increased in 2-D08-treated SK-UT-1B cells compared with that in untreated cells. Additionally, SK-UT-1B cells were stained with propidium iodide, and the cell cycle profiles were evaluated using the Guava Muse Cell Analyzer. The results indicated that treatment with 2-D08 did not affect the progression of the cell cycle in SK-UT-1B cells (Fig. 2B). To investigate whether 2-D08 regulates necrosis or autophagy in SK-UT-1B cells, the expression of RIP (a necrosis marker) and LC3B (an autophagy marker) were analyzed. Cells were treated with 2-D08 at the indicated concentrations, and the expression of RIP1 and LC3B was assessed using western blot analysis. The level of RIP1 was significantly higher in 2-D08-treated cells than in untreated cells, while that of LC3B remained unchanged (Fig. 2C). Furthermore, 2-D08-treated SK-UT-1B cells were evaluated using LDH assays to confirm necrosis. As shown in Fig. 2D, 2-D08 significantly increased the levels of LDH release in SK-UT-1B cells. These results suggested that 2-D08 significantly induces apoptosis and necrosis in SK-UT-1B cells.

Excessive accumulation of ROS in cells can lead to oxidative stress, resulting in damage to nucleic acids, lipids, proteins, membranes and mitochondria (31). To assess whether 2-D08 increases ROS levels in SK-LMS-1 cells, the cells were cultured with different concentrations of 2-D08 (0, 20, 50 and 100 µM) for 24 and 48 h, followed by analysis with the Guava Muse Cell Analyzer. ROS levels were at 33.0, 43.0, 50.5 and 77.6% in control and 2-D08 treated SK-LMS-1 cells at the indicated concentration for 24 h, respectively (Fig. 3A, upper panels). After 48 h of 2-D08 treatment, the respective results were 43.7, 61.3, 82.55 and 88.48% (Fig. 3A, bottom panels). Consequently, these results indicated that 2-D08 induces ROS generation in SK-LMS-1 cells.

To examine whether cells undergo apoptosis, SK-LMS-1 cells were treated with different concentrations (20, 50, or 100 µM) of 2-D08 for 24 and 48 h. Apoptotic cells were determined in flow cytometry using Annexin V. As demonstrated in Fig. 3B, the percentage of apoptotic cells significantly increased in a dose-dependent manner. After 24 h of treatment, the percentages of apoptotic cells were 7.01% at 20 µM, 15.66% at 50 µM and 90.93% at 100 µM, compared with 5.56% for the control cell culture (Fig. 3B, upper panels). After 48 h of treatment, the percentages of apoptotic cells were 8.81% at 20 µM, 16.77% at 50 µM, and 83.39% at 100 µM, compared with 6.53% for the control cell culture (Fig. 3B, bottom panels). To further identify whether ROS production is involved in 2-D08-induced apoptosis, SK-LMS-1 cells were treated with 2-D08 (100 µM) with or without NAC and CAT. Both NAC and CAT, known as ROS scavengers (32,33), effectively reduced 2-D08-induced apoptosis in SK-LMS-1 cells (Fig. 3C). These results indicated that 2-D08 induces ROS-mediated apoptosis in SK-LMS-1 cells. However, antioxidants, acting as ROS-scavengers, block the anticancer effect of 2-D08 on SK-LMS-1 cells.

The aforementioned experiments demonstrated that a consistent proliferation inhibitory effect occurs with 20–50 µM 2-D08 and that high doses (100 µM) have the potential to induce non-specific cytotoxicity. Based on these findings, subsequent experiments were conducted using 20- and 50-µM doses at 48 h. Cell proliferation was assessed by evaluating Ki67 expression and using a BrdU assay. The expression of the proliferation marker Ki67 is demonstrated in Fig. 4A. To accurately quantify the expression of Ki67 in SK-LMS-1 cells, flow cytometry was utilized. The proportion of Ki67-positive cells decreased from 93.48 to 67.79 (20 µM) and 65.03% (50 µM) after a 48-h treatment with 2-D08 (Fig. 4B). BrdU incorporation in proliferating cells significantly decreased in 50-µM 2-D08-treated cells at 48 h compared with that in the control (n=10) (Fig. 4C). These results indicated that 2-D08 treatment induces antiproliferative effects in SK-LMS-1 cells.

To investigate whether 2-D08-mediated inhibition of cell proliferation is related to cell cycle arrest, SK-LMS-1 cells were treated with varying concentrations of 2-D08 for 24 and 48 h. The cell cycle profiles were assessed using the Guava Muse Cell Analyzer. Interestingly, 2-D08 significantly increased the cell population at the S and G2/M phases, while simultaneously reducing it in the G0/G1 phase after 48 h of treatment (Fig. 4D). This experiment confirmed that 2-D08-mediated cell cycle arrest contributes to inhibition of proliferation in SK-LMS-1 cells.

To study the detailed mechanism(s) of action of 2-D08, western blotting was employed to detect the protein levels of the cell cycle-regulatory proteins p21, p27 and p53. Application of 2-D08 in the SK-LMS-1 cells significantly increased p21 protein levels at 20 and 50 µM, but not p53 levels. Treatment with the low dose of 2-D08 (20 µM) increased p27 levels, while, at a high dose (50 µM), no significant increase was observed (Fig. 5A). CHX assay was also conducted to inhibit protein synthesis in SK-LMS-1 and SK-UT-1B cells to evaluate the expression of p53. When treated with CHX for the indicated time, the expression of the p53 protein remained unchanged in SK-LMS-1 cells but decreased in SK-UT-1B cells (Fig. S1). The mRNA levels of p21, but not of p27 and p53, significantly increased compared with the group treated with 2-D08 (Fig. 5B). To improve understanding of how 2-D08 triggers apoptosis in SK-LMS-1 cells, western blot analysis was conducted by investigating the expression of Caspase-3, the pro-apoptotic protein cleaved Caspase-3, Caspase-9 and PARP antibodies with cell lysates. Caspase-3 cleavage was detected under these conditions, indicating that 2-D08 activated Caspase-3 at a concentration of 50 µM (Fig. 5C). By contrast, there was no change in the expression of Caspase-9 or PARP (Fig. 5C). Together, these findings suggested that the modulation of cell cycle- and apoptosis-related proteins may contribute to the 2-D08-induced suppression of proliferation and apoptosis in SK-LMS-1 cells.

The contractile apparatus of SMCs is characterized by the presence of high levels of α-SM-actin, calponin 1, TAGLN (SM22α) and SM-MHC, which are specific markers of the stage of cell differentiation (34,35). Various SMC malignancies can modulate the differentiated-to-differentiated phenotype in response to changes in the surrounding environment (36–40). For example, the expression of SM-specific markers in human Ut-LMS cell lines, including SK-LMS-1, is low because these cancerous cells have a dedifferentiated SMC phenotype (40). It was investigated whether 2-D08 affects the expression of SM-specific markers. As demonstrated in Fig. 6A, compared with the control group, treatment of SK-LMS-1 cells with 2-D08 did not significantly affect the expression of α-SM-actin, TAGLN, or calponin 1. Furthermore, the mRNA levels of SM-specific markers, excluding TAGLN, did not change significantly compared with those in the group treated with 2-D08 (Fig. 6B). Taken together, these findings demonstrated that 2-D08 does not directly regulate the phenotypic modulation of SK-LMS-1 cells.

It was aimed to obtain additional insights into the effects of 2-D08 and conducted transcriptome analyses with RNA-seq in both control and SK-LMS-1 cells treated with 50 µM 2-D08 for 48 h. Principal component analysis revealed clear clustering per treatment, indicating significant differences in the transcriptome profiles between the two groups (Fig. 7A). The hierarchical clustering heatmap showed that 259 genes were differentially expressed between these two groups (Fig. 7B, adjusted P-value <1.3 and |log2 fold change (FC)|>2). Of the 259 genes, the expression of 192 was upregulated and that of 67 was downregulated in 2-D08-treated SK-LMS-1 cells. The genes with high FCs are listed in Table I and all genes with significantly altered expression are included in Table SII. To obtain information on 2-D08-specific target genes in SK-LMS-1 cells, DEseq2 was utilized to identify differentially expressed genes (DEGs). This analysis revealed that the expression of 1,031 genes was downregulated and that of 2,907 genes was upregulated in 2-D08-treated SK-LMS-1 cells compared with those in the control (adjusted P-value <0.05 and |log2FC|>1). The distribution of DEGs is illustrated in a volcano plot (Fig. 7C). DEGs between the two groups were also examined using edgeR. As revealed in Fig. S2, 3,827 DEGs were identified, with 2,834 showing upregulated expression and 993 showing downregulated expression in two groups. Using gene ontology (GO) functional analysis, statistically significant enrichment was identified for numerous GO biological processes related to DNA replication (cell cycle) between the two groups (Fig. 7D). Gene set enrichment analysis observed that DNA replication (normalized enrichment score=2.35) and DNA-dependent DNA replication (normalized enrichment score=2.509) were positively regulated in the 2-D08-treated group (Fig. 7E). Additionally, various GO molecular functions related to the apoptotic process were significantly enriched (Fig. 7F). Taken together, these findings suggested that 2-D08 modulates pathways related to the cell cycle and apoptosis in SK-LMS-1 cells.