MTH-3 was synthesized and designed as previously described (21). Its chemical structure is shown in Fig. 1A. Hsieh et al (21) initially nominated the novel curcuminoid derivative as compound 9a, and the nomenclature was revised to MTH-3 in the study by Chang et al (22). L-glutamine, fetal bovine serum (FBS), streptomycin, Leibovitz's L-15 medium, penicillin G, and trypsin-EDTA were purchased from Thermo Fisher Scientific, Inc. Matrigel was obtained from Corning, Inc. Antibodies were purchased from Cell Signaling Technology, Inc. All other chemicals were purchased from Merck KGaA.

The MDA-MB-231 human breast adenocarcinoma cell line was obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan). MDA-MB-231 cells were cultured with 90% Leibovitz's L-15 medium, 1% penicillin-streptomycin and 10% FBS in 75 cm² culture flasks in an incubator with a humified 5% CO₂ atmosphere at 37°C (23).

MTT assay was conducted to evaluate the cytotoxicity of MTH-3 in MDA-MB-231 cells. The initial concentration of tumor cells was 1×10⁵ cells/ml in a 96-well cell culture plate. Tumor cells were treated with various concentrations of MTH-3 (0, 1, 2, 3, 4 and 5 µM) at 37°C. After 24 h of cell culture, MTT solution (0.5 mg/ml) was added, and the cells were incubated for an additional 4 h at 37°C. Next, the formazan crystals were dissolved in DMSO following the removal of the medium. The formazan product was analyzed spectrophotometrically at a wavelength of 490 nm (24). This analysis was performed in triplicate.

MDA-MB-231 cells were cultured in 12-well plates and treated with different concentrations of MTH-3 (1, 2, 3, 4 and 5 µM) or with 0.1% DMSO in Leibovitz's L-15 medium at 37°C for 24 h. Cells were collected and fixed with absolute ethanol. These cells were subsequently stained with DAPI solution to detect DNA breakdown using the In Situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics GmbH) as previously described (25). This analysis was performed in triplicate.

For the wound healing assay, MDA-MB-231 cells were incubated untill they reached ~90% confluence in a tissue culture plate. Next, to creat a 1 mm wound area, each well of the culture plate was scratched using a micropipette tip. The tumor cells were subsequently cultured in serum-free Leibovitz's L-15 medium with MTH-3 at different concentrations (1, 2, 3 and 4 µM) or with 0.1% DMSO at 37°C for 24 h. Tumor cells and the denuded zones were photographed under a phase-contrast microscope (magnification, ×100) (26). This analysis was performed in triplicate.

To investigate tumor cell invasion, a Transwell assay with a Matrigel^®-coated invasion chamber was performed. Firstly, to form a genuine reconstituted basement membrane, the Transwell insert (polycarbonate filters with an 8-µm porosity) was coated with 30 µg Engelbreth-Holm-Swarm sarcoma tumor matrix (Matrigel^®). Next, 1×10⁶ MDA-MB-231 cells were seeded in serum-free Leibovitz's L-15 medium in a T-75 culture plate. After 24 h of incubation, these tumor cells were suspended, and 5×10⁴ cells/chamber were subsequently added in the upper chamber of the Transwell insert with serum-free medium. The lower chamber contained Leibovitz's L-15 medium with 10% FBS. The tumor cells were treated with different concentrations of MTH-3 (1, 2, 3 and 4 µM) or with 0.1% DMSO at 37°C for 24 h. After incubation for cancer cell invasion, the samples were fixed with 4% formaldehyde for 15 min. Next, 2% crystal violet was used for staining. Finally, the tumor cells in the upper chamber were removed, and the invading cells in the lower chamber were visualized under a light microscope (Leica Microsystems GmbH; magnification, ×200) (26). This analysis was performed in triplicate.

Gelatin zymography assay was performed to investigate the protein activity of matrix metalloproteinase (MMP)-2 and MMP-9. Firstly, MDA-MB-231 cells were treated with different concentrations of MTH-3 (1, 2, 3 and 4 µM) or with 0.1% DMSO in serum-free Leibovitz's L-15 medium at 37°C for 24 h. These tumor cells were then suspended in zymography sample buffer. Next, the samples were added in loading buffer, and subsequently electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel containing 0.1% gelatin. Subsequently, 2.5% Triton X-100 in double-distilled H₂O was used to wash the gels. The gels were then stored in development buffer (0.02% Briji-35, 1 mM ZnCl₂, 5 mM CaCl₂, 50 mM Tris, and 200 mM NaCl at pH 7.5) at 37°C. After 18 h, the gels were stained with 0.5% Coomassie blue G-250. Then, the gels were de-stained. The non-staining bands indicated proteolytic activities. These results were analyzed using ImageJ software (National Institutes of Health) (27). This analysis was performed in triplicate.

Following the manufacturer's instructions, MDA-MB-231 cells (5×10³ cells/total) were seeded into 96-well round-bottom ultralow attachment plates for 3 days. After the spheroid diameter reached 200-µm, MDA-MB-231 cells were treated with different concentrations of MTH-3 (1, 2, 3 and 4 µM) or with 0.1% DMSO. Matrigel solution was added, and the plate was placed in a 37°C incubator for 30 min to polymerize the Matrigel. The system IncuCyte S3 ZOOM System instrument (Essen BioScience) was used to monitor spheroid invasion (28,29). This analysis was performed in triplicate.

To evaluate the possible signaling pathways of MTH-3 in MDA-MB-231 cells, RNA sequencing analysis of MTH-3-exposed and control groups was performed. Total RNA extraction, quantification, as well as sequencing cluster generation and high throughput sequencing were performed using Illumina (New England Biolabs), according to the manufacturer's instructions. Every step was performed under strict monitoring and quality control. After mixing libraries based on the effective concentration and the required sequencing data volume, high throughput sequencing was conducted using a high throughput sequencing platform to capture and sequence the entire mRNA pool. Bioinformatics analysis was performed after obtaining the original sequence data, as previously described (30). These individual libraries were converted to the FASTQ format. The raw sequencing data eliminated the adapter sequences. Short-read alignment was performed using Hisat2 (v2.0.1) with the default parameters (31). Using the pipeline on the human reference genome from the UCSC Genome Browser, the paired-end reads were mapped. Differential mRNA expression analysis was conducted to identify over-represented functional terms presenting in the background. Benjamini-Hochberg false discovery rate correction and a Student's t-test were performed to determine significantly differentially expressed genes (DEGs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) database with the David/EASE tool was used for pathway analysis. Rich factor and q-value were applied to evaluate the degree of KEGG enrichment. The ratio of the number of DEGs to the number of total annotated genes in a certain pathway is shown by Rich factor, and Q value (0–1) is a multiple hypothesis-corrected P-value, which closer to 0 indicate a greater enrichment. Differentially expressed genes (DEGs) between MTH-3-exposed and control groups were selected using three criteria: P<0.005, adj P<0.05 and absolute fold change of ≥1.2). The DEGs were selected by multiple systemic analysis (30,32). This analysis was performed in duplicate.

To further evaluate the correlation between mRNA and protein expression levels, Kinex Antibody Microarray was used for subsequent analysis. Briefly, MDA-MB-231 cells were seeded in serum-free Leibovitz's L-15 medium at 37°C with 2 µM MTH-3 or with 0.1% DMSO for 24 h. Tumor cells were then collected. From each sample, lysate proteins were labeled with proprietary fluorescent dye combination. After blocking the non-specific binding sites, the unbound proteins were washed away, and the chambers were displayed on the microarray. Images were captured using a Perkin-Elmer ScanArray Reader laser array scanner (Thermo Fisher Scientific, Inc.). ImaGene 9.0 (BioDiscovery) was used for spot segmentation and background correction. The overall average intensity of all spots within the samples was subtracted from the raw intensity of each spot to calculate the Z scores (33). Z ratios were calculated by dividing the difference between the averages of the observed protein Z scores by the standard deviations of all the differences for a particular comparison. Z ratio values of ±1.2 were considered statistically significant (33). This analysis was performed in triplicate.

To further analyze the protein expression levels, western blot analysis was performed. Briefly, MDA-MB-231 cells were cultured in Leibovitz's L-15 medium at 37°C with 2 µM MTH-3 or with 0.1% DMSO for 24 h. Tumor cells were then collected to obtain the protein lysate. Bio-Rad protein assay system (Bio-Rad Laboratories, Inc.) was used for determination of protein concentrations. These samples (35 µg per lane) were separated via 10–12% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skimmed dry milk at room temperature for 2 h. The membranes were incubated overnight at 4°C with primary antibodies purchased from Cell Signaling Technology, Inc., including AKT (cat. no. GTX121937; dilution, 1:5,000), p-AKT (phospho Ser473, cat. no. GTX28932; dilution, 1:400), ERK (cat. no. GTX59618; dilution, 1:10,000), p-ERK (phospho Thr202/Tyr204, cat. no. GTX59568; dilution, 1:10,000), p38/MAPK (cat. no. GTX110720; dilution, 1:1,000), p-p38/MAPK (cat. no. GTX48614; dilution, 1:500), JNK (cat. no. GTX52360; dilution, 1:500), p-JNK (phospho Thr183/Tyr185, cat. no. GTX52326; dilution, 1:500), and β-actin (cat. no. GTX109639; dilution, 1:10,000). Subsequently, the membranes were incubated for 4 h at room temperature with horseradish peroxidase-conjugated secondary antibodies, including anti-rabbit IgG (cat. no. 7074; dilution, 1:10,000) and anti-mouse IgG (cat. no. 7076; dilution, 1:10,000). Finally, the protein bands were visualized using ECL reagents (Cytiva) and scanned on a UMAX powerLook Scanner (UMAX Technologies). The National Institutes of Health ImageJ 1.52v program was used for quantitative analysis of the intensity of the band signal. This analysis was performed in quadruplicate.

Data are presented as the mean ± standard deviation. One-way analysis of variance followed by Dunnett's test and Tukey's post hoc test was conducted to analyze the differences between two groups and among multiple groups, respectively, using SPSS software version 25.0 (IBM, Corp.). P<0.05 was considered to indicate a statistically significant difference (34).

The tumor cells were treated with different concentrations of MTH-3 (1, 2, 3, 4 and 5 µM) or with 0.1% DMSO at 37°C for 24 h. The cells were suspended and analyzed by MTT method to detect the cell viability. The results showed a significantly reduced viability after 3, 4 and 5 µM MTH-3 at 91.45±4.69, 89.27±3.34 and 67.83±3.62%, respectively (Fig. 1B).

On the other hand, as shown in Fig. 1C and D, MTH-3 treatment at a concentration of 1–4 µM did not significantly induce apoptotic cell death, revealed by TUNEL assay. As a result, the concentrations of 1, 2, 3 and 4 µM of MTH-3 were subsequently selected to further study the effects on MDA-MB-231 cell invasion and metastasis.

The tumor cells were incubated with various concentrations of MTH-3 (1, 2, 3 and 4 µM) or with 0.1% DMSO at 37°C for 24 h to determine their ability of cell migration and invasion abilities by Transwell, wound healing and 3D spheroid invasion assays. In Fig. 2A and B, the tumor cells were found to be significantly decreased in the lower chamber in the MTH-3 treatment groups. In Fig. 2C and D, the edge distance in MTH-3 treatment groups was significantly wider than that in the control group, indicating that MTH-3 inhibited MDA-MB-231 cell motility in a concentration-dependent manner. To develop a tumor cell invasion and migration model, 3D spheroid invasion assay was applied to recapitulate both biochemical and physical characteristics of the tumor microenvironment. In Fig. 3A and B, MTH-3 significantly inhibited tumor spheroid invasion and migration in the Matrigel-coated area. These results suggested that MTH-3 inhibited MDA-MB-231 cells invasion and migration in a concentration-dependent manner. In combination, these data suggested that a concentration of <5 µM MTH-3 predominantly inhibited tumor cell migration and invasion. However, MTH-3-exhibited toxicity in MDA-MB-231 cells may require higher concentrations or longer incubation times.

Gelatin zymography was performed to further determine the role of MMP-2/-9 activity in MTH-3-treated MDA-MB-231 cells. Following MTH-3 treatment for 24 h, the conditioned media of tumor cells with different concentrations of MTH-3 (1, 2, 3 and 4 µM) or with 0.1% DMSO were collected to measure the gelatinase activity of MMP-2 and −9. The results revealed that MTH-3 decreased MMP-9 activity in a concentration-dependent manner in MDA-MB-231 cells in vitro (Fig. 3C and D).

To gain insight into the biological activity of MTH-3 in MDA-MB-231 cells, RNA sequencing transcriptional profile analysis was performed. As shown in Fig. 4A, three replicates for normalized RNA-sequencing data from MTH-3-treated samples and the control group were clustered separately using unsupervised Principal Component Analysis, indicating a significantly different Gene Expression Omnibus analysis. Genes in blue were highly expressed, and those in red were expressed at low levels. Fig. 4B shows the differential expression of an MA plot. Red dots represent significantly upregulated genes, and blue dots symbolized significantly downregulated genes. Fig. 4C contains a bar graph of significantly up- or downregulated genes between MTH-3-treated and control groups. A total of 315 genes were upregulated and 648 downregulated. To further determine the physiological activities of the genes and associated functions, the KEGG database were used (35). KEGG pathway analysis and hypergeometric tests were performed to identify the pathways of the DEGs and related pathways that were significantly enriched compared to the transcriptome background. In Fig. 4D, the scatter plot is used for the graphical representation of the KEGG pathway enrichment analysis, which is measured by the Rich factor, Q-value, and the number of genes enriched in these pathways. The top 20 KEGG pathways, most significantly enriched for the analysis, were selected and shown. The enriched KEGG pathways are shown in Fig. 5. Genes in red are upregulated and those in blue are downregulated. Gene ontology (GO) enrichment analysis revealed that the MTH-3-altered expression of genes was largely associated with the MAPK/ERK/AKT signaling cascade and cell cycle pathway. The sequencing raw data are shown in Table SI.

Subsequently, the protein expression levels were examined using antibody microarray analysis and western blot analysis to investigate the correlation between mRNA and protein expression levels, since mRNA expression analysis can be inaccurate and potentially misleading (33). The results presented in Table I revealed that MTH-3 treatment significantly downregulated phosphorylated MAPK p38α, MAPK/ERK protein-serine kinase (MEK2), ERK1/2 and proline-rich AKT substrate 40 kDa (PRAS40). Furthermore, MTH-3 significantly downregulated phosphorylated Cyclin A, Cyclin D1, cell division control protein 42 (CDC42) and cyclin-dependent protein-serine kinase 2 (CDK2). As shown in Fig. 6, the protein expression of p-AKT, p-ERK, p-p38 and p-JNK was decreased. In combination, the present results demonstrated that MTH-3 suppressed the MAPK/ERK/AKT signaling pathway and cell cycle-related protein phosphorylation in MDA-MB-231 human breast adenocarcinoma cells. The original images of the integral western blot gels are shown in Fig. S1.