Open Access

Next‑generation sequencing analysis reveals that MTH‑3, a novel curcuminoid derivative, suppresses the invasion of MDA‑MB‑231 triple‑negative breast adenocarcinoma cells

  • Authors:
    • Yu-Jen Chiu
    • Fuu-Jen Tsai
    • Da-Tian Bau
    • Ling-Chu Chang
    • Min-Tsang Hsieh
    • Chi-Cheng Lu
    • Sheng-Chu Kuo
    • Jai-Sing Yang
  • View Affiliations

  • Published online on: May 19, 2021     https://doi.org/10.3892/or.2021.8084
  • Article Number: 133
  • Copyright: © Chiu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Triple‑negative breast cancer (TNBC) behaves aggressively in the invasive and metastatic states. Our research group recently developed a novel curcumin derivative, (1E,3Z,6E)-3-hydroxy-5-oxohepta-1,3,6-triene-1,7-diyl)bis(2‑methoxy-4,1‑phenylene)bis(3-hydroxy2-hydroxymethyl)-2‑methyl propanoate (MTH‑3), and previous studies showed that MTH‑3 inhibits TNBC proliferation and induces apoptosis in vitro and in vivo with a superior bioavailability and absorption than curcumin. In the present study, the effects of MTH‑3 on TNBC cell invasion were examined using various assays and gelatin zymography, and western blot analysis. Treatment with MTH‑3 inhibited MDA‑MB‑231 cell invasion and migration, as shown by Transwell assay, 3D spheroid invasion assay, and wound healing assay. The results of the gelatin zymography experiments revealed that MTH‑3 decreased matrix metalloproteinase‑9 activity. The potential signaling pathways were revealed by next‑generation sequencing analysis, antibody microarray analysis and western blot analysis. In conclusion, the results of the present study show that, MTH‑3 inhibited tumor cell invasion through the MAPK/ERK/AKT signaling pathway and cell cycle regulatory cascade, providing significant information about the potential molecular mechanisms of the effects of MTH‑3 on TNBC.

Introduction

Breast cancer is the most common primary cancer in woman, affecting approximately 12% of women worldwide (1). Breast cancer cases have markedly increased since the 1970s, which has been attributed to the modern lifestyle (2). There are various subtypes of breast cancer, including triple-negative breast cancer (TNBC), which lacks the expression of the genes encoding human epidermal growth factor receptor-2, progesterone receptor, as well as estrogen receptor. The majority of hormone and targeted therapies target one of the three aforementioned receptors, resulting in treatment resistance (2). In addition, TNBC is more common in young women, demonstrating relapse and aggressive behaviors during the invasive and metastatic states. Cancer relapse and distant metastasis markedly increase morbidity and mortality, and are the most formidable obstacles to successful treatment (3). Current medical treatments, such as treatment with doxorubicin and toxoids, frequently lead to drug resistance and cause severe side effects (4). As a result, researchers are exploring novel treatment strategies for TNBC.

Natural products and their derivatives have recently become an important source of drug and therapeutic candidates (5). Between 1981 and 2010, approximately 34% of US Food and Drug Administration-approved novel-marketed drugs were derived from natural products (6). Curcumin, a golden color chemical derived from Curcuma longa plants, has been widely used in Traditional Chinese medicine in East Asia for centuries. Curcumin has diverse pharmacological properties, including anti-bacterial (7), anti-inflammatory (8), anti-oxidant (9), anti-depressant (9), anti-viral (10), anti-diabetes (11) and anticancer properties (1214). During the past 10 years, numerous studies have reported that curcumin and its derivatives can effectively inhibit tumor cell growth, and induce apoptosis, autophagy and cell cycle arrest (4,12). Currently, numerous phase II and III clinical trials have advocated for the application of curcumin in patients with multiple myeloma, myelodysplastic syndromes, pancreatic cancer, head and neck cancer and colon cancer (15). In a previous clinical study, curcumin was proven to be safe even at doses of up to 8 g per day (16). Curcumin is, by all accounts, an ideal medication for the inhibition of cancer growth through various signaling pathways. However, certain animal and human pharmacokinetic studies have reported a poor absorption of curcumin in the gastrointestinal tract. The low systemic bioavailability of curcumin prevents an adequate concentration from reaching the target tissues to achieve pharmacological effects (1720).

To overcome its poor bioavailability and increase its absorption in vivo, a novel curcumin derivative (1E,3Z,6E)-3-hydroxy-5-oxohepta-1,3,6-triene-1,7-diyl)bis(2-methoxy-4,1-phenylene)bis(3-hydroxy-2-hydroxymethyl)-2-methylpropanoate (MTH-3), was designed and developed. Fig. 1A includes a schematic of MTH-3. In a previous study it was demonstrated that, MTH-3 has superior hydrophilicity than curcumin. The log P, calculated logarithmic partition coefficient, of MTH-3 is 1.73, and of curcumin it is 3.38 (21,22). Furthermore, previous findings showed that MTH-3 inhibits tumor proliferation and induces apoptosis in TNBC in vitro and in vivo through cell cycle arrest and the autophagic pathway (21). Chang et al (22) revealed that MTH-3 has a greater inhibitory effect against TNBC cells compared with curcumin. That study also reported a 10-fold higher potency of MTH-3 compared to curcumin against doxorubicin-resistant MDA-MB-231 cell proliferation.

In the present study, the ability of MTH-3 to inhibit invasiveness in TNBC and the potential molecular signaling pathways were investigated.

Materials and methods

Chemicals

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.

Cell culture

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).

Cell viability assay

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.

TUNEL assay

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.

Wound healing assay

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.

Transwell assay

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

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.

3D spheroid invasion assay

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.

Whole transcriptome sequencing (next-generation sequencing analysis)

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.

Antibodies microarray analysis

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.

Western blot analysis

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.

Statistical analysis

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).

Results

MTH-3 inhibits the proliferation in MDA-MB-231 human breast adenocarcinoma cells

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.

MTH-3 inhibits invasion and migration in MDA-MB-231 human breast adenocarcinoma cells

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.

MTH-3 suppresses the activity of MMP-9 in MDA-MB-231 human breast adenocarcinoma cells

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).

MTH-3 inhibits invasion through the MAPK/ERK/AKT signaling pathway and causes cell cycle arrest in MDA-MB-231 human breast adenocarcinoma cells

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.

Table I.

Summary of antibody microarray analysesa.

Table I.

Summary of antibody microarray analysesa.

Antibody codesTarget protein namePhospho site (Human)% CFC (MTH-3 from CTL)Z-ratio (MTH-3, CTL)
NN024CDC42Pan-specific−44−1.76
NK026-3CDK2Pan-specific−34−1.26
NN028Cyclin APan-specific−51−2.03
NN030-1Cyclin D1Pan-specific−71−3.49
PK170-PK171ERK1/2T202+T185−35−1.31
NK120-2p38/MAPKPan-specific−36−1.31
NK120-4p38a/MAPKPan-specific−43−1.68
PK049-2MEK2T394−52−2.18
PN062Proline-rich Akt substrate 40 kDa (PRAS40)T246−47−1.88

a Significant downregulation of phosphorylated ERK1/2, MAPK p38, MEK2, PRAS40, cyclin A, cyclin D1, CDC42 and CDK2. % CFC indicates the percentage change in the normalized intensity of the MTH-3-treated sample compared with the control. A Z ratio of ±1.2 was considered significant. MEK2, MAPK/ERK protein-serine kinase; PRAS40, proline-rich AKT substrate 40 kDa; CDC42, cell division control protein 42; CDK2, cyclin-dependent protein-serine kinase 2; CTL, control.

Discussion

MTH-3 is a novel bis(hydroxymethyl) alkanoate curcuminoid derivative, designed by Hsieh et al (21). Findings of that study revealed that MTH-3 was effective against numerous breast cancer cell lines and induced limited toxicity to normal tissues in an established xenograft nude mouse model of MDA-MB-231 cells. In addition, Chang et al (22) identified a synergistic activity of MTH-3 combined with doxorubicin in the inhibition of MDA-MB-231 cell growth. MTH-3 treatment induced cell cycle arrest, as well as the apoptotic and autophagic pathways in MDA-MB-231 cells. To the best of our knowledge, the present study was the first to demonstrate that MTH-3 inhibited the invasion of MDA-MB-231 cells and elucidate the potential signaling pathways. In anti-metastasis drug discovery, the compounds not only exhibit the cytotoxic effect at high concentrations but also possess anti-metastasis activity at low concentrations (36). Our previous findings demonstrated that MTH-3 inhibited cell proliferation and induced G2/M arrest, cell autophagy and apoptosis at a high concentration (>5 µM) in MDA-MB-231 cells. Results of the present study revealed that a low concentration (2, 3 and 4 µM) of MTH-3 predominantly inhibited MDA-MB-231 tumor cell migration and invasion (Figs. 2 and 3), but did not induce cell apoptosis by TUNEL assay (Fig. 1C and D).

Curcumin has been proven to be safe and effective in inhibiting cancer cell growth (16). However, poor gastric absorption and low systemic bioavailability prevent the pharmacological properties of curcumin from reaching the target tissues. Researchers have developed several methods to overcome the poor hydrophilicity of curcumin, including nanoparticles, liposomes, phospholipid complexes, micelles, and cyclodextrin encapsulation (37). Furthermore, the half-life of curcumin is short, resulting in low bioavailability. Several novel derivatives of curcumin have been developed to delay the metabolism into glucuronides and sulfates through a phase II transformation, replacing its phenolic OH groups with ester (38,39). MTH-3, a novel curcumin derivative designed by Hsieh et al (21), was proven to have a superior solubility in water and alcohol than curcumin in the previous study, with a 10-fold higher potency.

MTH-3 significantly inhibited MDA-MB-231 cell invasion and migration in the present study. Cancer metastasis is a complex process. The basement membrane and extracellular matrix are major physical barriers to inhibiting cancer cell invasion and migration (26). MMPs play a key role in degrading the basement membrane and extracellular matrix. Previous findings have shown that tumor cells can specifically produce MMP-2 and −9 to destroy these natural barriers, resulting in the invasion and migration of tumor cells into adjacent tissue and blood vessels (40). MMP-2, which can degrade type V, VI and X collagens, gelatins and IV collagen in the basement membrane, was found to be constitutively expressed in various tissues, including cancer cells, rather than as part of the initial response to invasion (41). MMP-9 can be secreted extracellularly to degrade type IV collagens and fibronectins, which can be stimulated by various inflammatory cytokines and growth factors during pathological processes, including affecting the adhesion ability of tumor cells (42). The results of the present study showed that MTH-3 inhibits MDA-MB-231 cell invasion and migration by decreasing the activity of MMP-9, instead of that of MMP-2. These results were consistent with those reported by Fan et al (43), who demonstrated that casticin inhibits MMP-9 protein expression and activity in breast cancer cells.

Numerous studies have demonstrated the anti-metastasis effects of curcumin in breast cancer cells. Coker-Gurkan et al reported that curcumin inhibits invasion and metastasis by targeting NF-κB signaling in breast cancer cell lines, including MCF-7, MDA-MB-453 and MDA-MB-231 (45). Gallardo and Calaf (46) and Hu et al (47) reported curcumin induces anti-metastasis activity through epithelial-mesenchymal transition. Guan et al (48) reported that curcumin suppresses migration in MDA-MB-231 cells through PI3K/AKT signaling pathway. To the best of our knowledge, the present study was the first to report that MTH-3, a novel curcuminoid derivative, suppresses tumor invasion in breast cancer. The possible signal transduction was further investigated.

Next-generation sequencing (NGS)-based molecular diagnosis and analysis is becoming one of the major tools of personalized treatment and drug development (49). RNA expression profile analysis provides a more accurate analysis of the tumor phenotype compared with genome analysis, which makes it the most powerful tool of high throughput quantitative transcriptomics (50). The expression levels of targets of molecular medicines were analyzed and profiling of the activation of the relevant molecular pathways was used to enable the personalized prescription of a wide range of molecular-targeted therapies (51). To evaluate the possible signaling pathways of MTH-3 in MDA-MB-231 cells, whole transcriptome sequencing analysis of MTH-3-exposed and control groups was performed in the present study. The results showed that MTH-3-altered gene expressions were markedly associated with cell invasion and MAPK/ERK/AKT signaling pathway. The MAPK/ERK/AKT signaling pathway was suppressed in MDA-MB-231 human breast adenocarcinoma cells after MTH-3 treatment, as shown in Fig. 5. Inhibition of the signaling transduction was confirmed by western blot analysis, as shown in Fig. 6. Furthermore, cell cycle-related gene expression was also decreased. Since mRNA expression analysis can be inaccurate and potentially misleading, antibody microarray analysis was subsequently performed to investigate the correlation between mRNA and protein expression levels (52,53). The results in Table I revealed that treatment of MTH-3 significantly downregulated phosphorylated ERK1/2, p38/MAPK, MEK2 and proline-rich Akt substrate 40 kDa (PRAS40). MTH-3 also significantly downregulated phosphorylated cyclins A and D1, CDC42 and CDK2.

The MAPK/ERK and PI3K/AKT signaling pathways play an important role in cancer cell survival, proliferation, apoptosis, invasion and metastasis (54,55). The aberrant activation of this signaling pathway induces the survival, proliferation and metastasis of breast cancer cells (54). Chen et al (56) reported that curcumin inhibits doxorubicin-induced epithelial-mesenchymal transition through the suppression of PI3K/AKT and TGF-β signaling transduction in TNBC. Berrak et al (57) reported that curcumin induced cell cycle arrest and inhibited PI3K signaling transduction in MCF-7 breast cancer cells. Guan et al (48) demonstrated that curcumin suppresses proliferation and migration through autophagy-dependent AKT degradation in MDA-MB-231 cells.

Furthermore, several studies have demonstrated that cyclin proteins regulate tumor cell invasion and metastasis. Cell cycle arrest inhibits tumor invasion and metastasis (5861). Fusté et al (58), Body et al (62), and Chen et al (63) reported that cyclin D1 fosters tumor cell invasion and metastasis through cytoplasmic mechanisms. CDC42 knockdown was shown to inhibit tumor cell migration and invasion, and be associated with the downregulation of cyclins A, D1 and E/CDK2 (14,59,60,64). In previous studies, MTH-3 was found to inhibit TNBC cell proliferation and induce apoptosis through the autophagic pathway and cause cell cycle arrest with a higher potency than curcumin (21,22). The present study demonstrated the ability of MTH-3 to inhibit the invasion of TNBC cells through the MAPK/ERK/AKT signaling pathways and cell cycle regulatory cascade.

In conclusion, the present study revealed that MTH-3 inhibits tumor invasiveness via the MAPK/ERK/AKT signaling pathway and cell cycle regulatory cascade in human adenocarcinoma MDA-MB-231 cells. Significant information with respect to the possible signal transduction of MTH-3 in TNBC was provided in the current study, and the results suggested that MTH-3 may be used in the treatment of breast cancer medication in the future.

Supplementary Material

Supporting Data
Supporting Data

Acknowledgements

This study was supported in part through the Medical Research Core Facilities, Office of Research and Development at China Medical University (Taichung, Taiwan), and Yen Tjing Ling Medical Foundation (Taipei, Taiwan). We also thank Mr. Kai-Hsiang Chang and Mr. Chin-Chen Lin (Tekon Scientific Corp.) and Mr. Chang-Wei Li (AllBio Science Incorporated, Taiwan) for their assistance and support with the equipment in this study.

Funding

Funding for this study was provided in part by China Medical University Hospital, Taichung, Taiwan (DMR-109-147), Ministry of Science and Technology, Taiwan (MOST 109-2320-B-039-041-), Taipei Veterans General hospital, Taipei, Taiwan (V110B-038), and Yen Tjing Ling Medical Foundation, Taipei, Taiwan (CI-110-6). Chinese Medicine Research Center, China Medical University from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan also provided partial support.

Availability of data and materials

Data of transcriptome sequencing in this published article have been uploaded to the European Nucleotide Archive. Accession no.: ERP128028.

Authors' contributions

YJC, FJT, SCK and JSY contributed to the study design. YJC, DTB, LCC, MTH and JSY conducted the experiments. YJC, FJT, CCL and JSY analyzed the data. YJC, CCL and JSY confirmed the authenticity of all the raw data. YJC, CCL, JSY, and SCK wrote and revised the paper. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

TNBC

triple-negative breast cancer

ER

estrogen receptor

GO

gene ontology

(HER2/neu)

human epidermal growth factor receptor-2

PR

progesterone receptor

FBS

fetal bovine serum

DEGs

differentially expressed genes

KEGG

Kyoto Encyclopedia of Genes and Genomes

PI3K

phosphoinositide 3-kinase

RPKM

reads per kilobase per million mapped reads

p38/MAPK

mitogen-activated protein-serin kinase p38 α

MEK2

MAPK/ERK protein-serine kinase

PRAS40

proline-rich Akt substrate 40 kDa

CDC42

cell division control protein 42

CDK2

cyclin-dependent protein-serine kinase 2

ERK1/2

extracellular regulated protein-serine kinase 1 and 2

MMP

matrix metalloproteinase

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July-2021
Volume 46 Issue 1

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Spandidos Publications style
Chiu Y, Tsai F, Bau D, Chang L, Hsieh M, Lu C, Kuo S and Yang J: Next‑generation sequencing analysis reveals that MTH‑3, a novel curcuminoid derivative, suppresses the invasion of MDA‑MB‑231 triple‑negative breast adenocarcinoma cells. Oncol Rep 46: 133, 2021
APA
Chiu, Y., Tsai, F., Bau, D., Chang, L., Hsieh, M., Lu, C. ... Yang, J. (2021). Next‑generation sequencing analysis reveals that MTH‑3, a novel curcuminoid derivative, suppresses the invasion of MDA‑MB‑231 triple‑negative breast adenocarcinoma cells. Oncology Reports, 46, 133. https://doi.org/10.3892/or.2021.8084
MLA
Chiu, Y., Tsai, F., Bau, D., Chang, L., Hsieh, M., Lu, C., Kuo, S., Yang, J."Next‑generation sequencing analysis reveals that MTH‑3, a novel curcuminoid derivative, suppresses the invasion of MDA‑MB‑231 triple‑negative breast adenocarcinoma cells". Oncology Reports 46.1 (2021): 133.
Chicago
Chiu, Y., Tsai, F., Bau, D., Chang, L., Hsieh, M., Lu, C., Kuo, S., Yang, J."Next‑generation sequencing analysis reveals that MTH‑3, a novel curcuminoid derivative, suppresses the invasion of MDA‑MB‑231 triple‑negative breast adenocarcinoma cells". Oncology Reports 46, no. 1 (2021): 133. https://doi.org/10.3892/or.2021.8084