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. 2023 Sep 26;18(9):e0291693. doi: 10.1371/journal.pone.0291693

Suppression of migration and invasion by taraxerol in the triple-negative breast cancer cell line MDA-MB-231 via the ERK/Slug axis

Yu-ting Xia 1,2, Yu-qin Zhang 1,2, Lu Chen 2, Liangliang Min 2, Da Huang 2, Yulu Zhang 2, Cong Li 2, Zhi-hua Li 2,*
Editor: Salman Shakil3
PMCID: PMC10522031  PMID: 37751436

Abstract

As one of the triterpene extracts of Taraxacum, a traditional Chinese plant, taraxerol (TRX) exhibits antitumor activity. In this study, we evaluated the effects of TRX on the migration and invasion of MDA-MB-231 cells, analyzed the molecular mechanism through network pharmacology and molecular docking, and finally verified it by in vitro experiments. The results showed that TRX could inhibit the migration and invasion of MDA-MB-231 cells in a time- and concentration-dependent manner, while MAPK3 was the most promising target and could stably combine with TRX. In addition, the relative protein expression levels were detected by Western blot, and we observed that TRX could inhibit the migration and invasion of MDA-MB-231 cells via the ERK/Slug axis. Moreover, an ERK activator (tert-butylhydroquinone, tBHQ) partially reversed the suppressive effect of TRX on MDA-MB-231 cells. In conclusion, TRX inhibited the migration and invasion of MDA-MB-231 cells via the ERK/Slug axis.

Introduction

Breast cancer (BC) is a highly heterogeneous malignant tumor. Its incidence rate ranks first among female cancers. TNBC is a subtype with no expression of estrogen receptor (ER), progesterone receptor (PR) or human epidermal growth factor receptor-2 (HER-2). It has a high rate of metastasis and recurrence [1], accounting for approximately 20% of all BCs [2]. Due to negative receptor expression, TNBC is insensitive to endocrine or targeted therapy and is the subtype with the most limited treatment, the worst prognosis and the shortest survival period. Therefore, it is necessary to find new treatments and drugs for TNBC.

Meanwhile, traditional Chinese medicine (TCM) has unique antitumor advantages and tends more to the overall treatment of patients. As a traditional antipyretic and antidote medicine with a bitter taste and cold properties, Taraxacum has a great effect on detoxification, detumescence and lump dissipation and is commonly used in the treatment of breast disease. Modern pharmacological studies have shown that the extracts of Taraxacum can inhibit the occurrence and development of BC. TRX, a pentacyclic triterpene, is one of the most active ingredients and not only has anti-inflammatory [3] and antiviral effects [4] but also inhibits cell proliferation in various tumor cell lines. Previous studies have shown that TRX could inhibit cell metastasis through the Hippo and Wnt signaling pathways in gastric cancer cells [5], promote cervical cancer cell apoptosis by the mitochondrial pathway [6] and inhibit cell proliferation of bladder cancer [7] via the Akt pathway. In addition, TRX was verified to promote autophagy by suppressing mTOR phosphorylation in the BC cell line MCF-7 [8]. However, the mechanism of TRX in the treatment of TNBC is still unclear.

In this study, we evaluated the effects of TRX on the migration and invasion of MDA-MB-231 cells, analyzed the potential targets and pathways through network pharmacology and molecular docking, and finally verified them by in vitro experiments.

Materials and methods

Materials

MDA-MB-231 human TNBC cell line was obtained from the Key Laboratory of Breast Diseases in Jiangxi Province and cultured in high glucose DMEM with 10% fetal bovine serum (FBS). DMEM was purchased from Solarbio (Beijing, China), while FBS was purchased from Gibco (New York, USA). TRX (purity>98%, CAS:127-22-0) was purchased from Herbest Biochemical Technology Co., Ltd. (Baoji, China) and then dissolved in dimethyl sulfoxide (DMSO) from Macklin (Shanghai, China) to prepare a 20 mmol/L (mM) stock solution for storage at -40°C. tBHQ (purity>99%, CAS:1948-33-0) was purchased from MedChemExpress (Shanghai, China) and dissolved in DMSO to prepare a 10 mM stock solution for storage at -40°C.

Primary antibodies against epithelial-cadherin (E-cadherin, ab40772), neural-cadherin (N-cadherin, ab76011), Vimentin (ab92547), and Slug (ab85936) were purchased from Abcam (Cambridge, UK), while phospho-ERK1/2 (28733-1-AP), ERK1/2 (11257-1-AP), and GAPDH (60004-1-lg) were purchased from Proteintech (Chicago, USA).

MTT assay

MDA-MB-231 cells in logarithmic growth phase were seeded in 96-well plates (4 x 103 cells/well) and exposed to TRX at 0, 40, 80, and 120 μmol/L(μM). After culturing for 24, 48, and 72h, 20 μL of MTT solution (5 mg/ml) was added to each well and cultured for 4 h. After removing the supernatant solution, 110 μL of DMSO was added and vibrated to dissolve the formazan crystals. The optical density (OD) value was detected by a multifunctional microplate reader at 490 nm. The cell number (% of control): OD value (experimental group) / OD value (control group) * 100%.

Wound healing assay

MDA-MB-231 cells in logarithmic growth phase were seeded in 12-well plates (4 x 105 cells/well). When the cells had adhered and reached 90% confluence, a 20 μL tip was used to scratch the bottom of the plate vertically. Then, the plates were washed twice with PBS after the medium was removed, and the cells were cultured in serum-free medium with 0, 40, or 80 μM TRX. The wounds were photographed by microscopy (OLYMPUS CKX53, 10x) after culture for 0, 24, and 48h, and the wound areas were measured by ImageJ software.

Transwell assay

MDA-MB-231 cells in logarithmic growth phase were resuspended in serum-free medium and seeded in transwell inserts (24-well, 8.0 μM) with 200 μL serum-free medium (8 x 104 cells/insert). Then, 600 μL of medium with 10% FBS was added to the lower chambers. TRX solution was added to final concentrations of the transwell inserts and lower chambers of 0 and 80 μM. After culture for 48 h, 4% paraformaldehyde and 0.1% crystal violet were added successively to the insert to fix and stain the cells. Five images of different visual fields for each insert were taken randomly under the microscope, and the migrated or invaded cells were counted. For invasion rather than migration assays, it is necessary to dilute the premelted Matrigel to 0.3 mg/ml with serum-free medium. Then, 100 μL of diluted Matrigel was added to the Transwell insert and incubated for 4 h. Cells could not be seeded until the Matrigel became solid.

Western blot

MDA-MB-231 cells in logarithmic growth phase were seeded in 6-well plates (4 x 105 cells/well) and exposed to TRX at 0, 40, and 80 μM for 48 h. Total cellular proteins were extracted by RIPA buffer with protease inhibitor, and the concentrations were determined by the BCA protein concentration quantitative method. All samples were separated by 10% SDS-PAGE gel and transferred to PVDF membranes. The membranes were blocked in 5% skim milk at room temperature for 1 h, incubated in primary antibodies at 4°C overnight, washed three times with TBST solution for 10 min each and then incubated in secondary antibodies at room temperature for 1.5 h. The protein bands were developed by using hypersensitive enhanced chemiluminescence (ECL), and the gray values were analyzed by ImageJ software.

Network pharmacology

The 2-dimensional (2D) chemical structure of TRX was obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and then imported into SwissTargetPrediction [9] to predict targets, and those with a “probability>0.1” were selected as TRX targets. TNBC targets were obtained from GeneCards(https://www.genecards.org/), DrugBank [10], Therapeutic Target Database (TTD) [11] and OMIM (https://www.omim.org/) after removing duplicates. TRX targets and TNBC targets were imported into the Bioinformatics online website (http://www.bioinformatics.com.cn/) to obtain common targets and a Venn diagram. These common targets were imported into the STRING database (https://www.string-db.org/) to obtain the protein-protein interaction (PPI) network with the options "the high confidence>0.4" and "hide the disconnected nodes of the network". Meanwhile, GO and KEGG enrichment analyses were carried out in the DAVID database [12] with common targets, and the results were visualized through Micro Bioinformatics, an online bioinformatics analysis, visualization cloud platform.

Molecular docking

The crystal structure of the candidate target as the receptor was obtained from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (https://www.rcsb.org/) and imported into open-source PyMOL and AutoDockTools 1.5.7 software for pretreatment, which consisted of removing ligands, removing water and adding hydrogen bonds. Meanwhile, the 3D structure of TRX as the ligand was obtained from the PubChem database, converted into format by OpenBabel 2.4.1 software, and then imported into AutoDockTools for pretreatment, which included adding charges and limiting the ligand conformation. The processed receptor and ligand were subjected to molecular docking by AutoDock Vina. The results of molecular docking were imported into the Protein-Ligand Interaction Profiler (PLIP) web tool to obtain noncovalent interaction information and visualize it in PyMol.

tBHQ experiment

MDA-MB-231 cells were divided into control, TRX, tBHQ, and TRX+tBHQ groups and treated with medium, 80 μM TRX, 10 μM tBHQ, 80 μM TRX and 10 μM tBHQ, respectively. Then, the cell migration and invasion abilities were detected by wound healing and Transwell assays, and the relative protein expression levels were detected by Western blot.

Statistical analysis

All data statistical analysis and charts were provided by GraphPad Prism software, and the results are presented as the mean and standard deviation (mean ± SD). Student’s t test was used to compare differences between two groups, and one-way ANOVA was used to compare multiple groups. A P value<0.05 was considered statistically significant.

Results

TRX inhibited the migration and invasion of MDA-MB-231 cells

To investigate the effect of TRX on MDA-MB-231 cells, we treated cells with different concentrations of TRX. The results of the MTT assay showed that TRX inhibited cellular viability, and the effect of 80 μM TRX was slightly obvious (Fig 1A). Compared to control area, the wound area of 0mM, 40mM and 80mM were 69.6%, 83.2% and 89.7% at 24h, the wound area of 0mM, 40mM and 80mM were 50.4%, 70.5% and 83.8% at 48h. The results of the wound healing assay showed that the migration of MDA-MB-231 cells was inhibited by TRX in a time- and concentration-dependent manner (Fig 1B and 1C). According to the Transwell assay (Fig 1D and 1E), we found that cells migration and invasion were decreased significantly after treatment with 80 μM TRX for 48 h. The inhibition rate of cell migration was 35.3%, and the inhibition rate of cell invasion was 46.3%. Epithelial mesenchymal transformation (EMT) is the key process of cancer cell metastasis, and cell migration and invasion are greatly enhanced when epithelial cells acquire the characteristics of mesenchymal cells. Thus, we observed the expression of EMT markers by Western blot. The expression of the epithelial cell marker E-cadherin was upregulated, and the expression of the mesenchymal cell marker N-cadherin and Vimentin was downregulated (Fig 1F and 1G), suggesting that the migration and invasion of MDA-MB-231 cells were suppressed. These results indicated that TRX inhibited the migration and invasion of MDA-MB-231 cells.

Fig 1. TRX inhibited the migration and invasion of MDA-MB-231 cells.

Fig 1

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A) Cell viability was assessed by MTT assay, and cell numberwere counted by GraphPad software 5.0. B) Cell migration was evaluated by wound healing assay. C) Statistical analysis of the wound healing area ratio. D) Cell migration and invasion were evaluated by Transwell assay. E) Statistical analysis of cells per field. F) The expression of EMT-related proteins was determined by Western blot. G) Statistical analysis of the relative protein expression. These results are representative of at least 3 independent experiments. Data are presented as the mean ± SD.*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared with the 0 μM group.

Prediction that MAPK3 was the critical target by network pharmacology and molecular docking

Fifty-four TRX targets with a probability>0.1 were predicted by importing the 2D chemical structure of TRX (Fig 2A), and 1751 TNBC targets were predicted from the GeneCards, DrugBank, TTD and OMIM databases. Then, we obtained 20 common targets by intersecting TRX targets and TNBC targets (Fig 2B), suggesting that more than 1/3 of TRX targets are closely related to TNBC. The PPI network (Fig 2C) was constructed through the STRING website and Cytoscape software with 20 common targets, in which the size and color depth of nodes had a direct correlation with the importance of targets. Thus, we considered MAPK3 to be the most promising candidate target. GO enrichment analysis included biological process, cellular component, and molecular function, and the results with P<0.01 are shown in Fig 2D. Notably, many biological processes and molecular functions were related to tyrosine phosphorylation, which was just right one of major phosphorylation forms of ERK1, a protein that was encoded by MAPK3.

Fig 2. Prediction that MAPK3 was the critical target by network pharmacology and molecular docking.

Fig 2

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A) The 2D chemical structure of TRX. B) Venn diagram of TRX and TNBC targets. C) The PPI network of 20 common targets. D), E) Histogram of GO and KEGG enrichment analysis results. F) The Sankey bubble diagram with common targets and KEGG enrichment analysis results. G) Molecular docking of TRX onto the MAPK3 protein. Molecules are depicted by a stick model, the HBonds, Hydrophobic and salt bridges are depicted by green, red and yellow lines, respectively, and the distances are shown in angstroms.

There were 15 KEGG pathways (Fig 2E) enriched from common targets which could be divided three categories: cellular processes, organic systems and human diseases. Adherens junctions were the only cellular process, and the loss of cell adhesion was the first and most important step in cancer infiltration and metastasis, which was consistent with the results of the cell migration and invasion experiments. Furthermore, we could easily observe that MAPK3 was the critical target by drawing a Sankey bubble diagram (Fig 2F) to visualize the common target and results of KEGG enrichment analysis. According to molecular docking (Fig 2G), we found that MAPK3 could bind to TRX stably, and the noncovalent interactions between them included hydrogen bonds, hydrophobic interactions and salt bridges (Table 1). Therefore, we believe that MAPK3 is a critical target by which TRX inhibits the migration and invasion of MDA-MB-231 cells.

Table 1. Molecular docking for MAPK3 and TRX.

Target PDB ID Affinity Interactions
(Kcal/mol) Type Residue Distance
MAPK3 2ZOQ -9.4 hydrogen bonds ARG87, ARG211 3.6, 3.9, 3.3
hydrophobic interactions VAL205 3.2
salt bridges ARG165 4.1, 3.1
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TRX inhibited the migration and invasion of MDA-MB-231 cells via the ERK/Slug axis

Adherens junction was the only pathway closely related to cell migration and invasion, which ranked high in the KEGG enrichment analysis results. Meanwhile, in this pathway, we found that ERK1 could affect E-cadherin by acting on Slug (Fig 3A). Thus, we speculated that TRX might inhibit the migration and invasion of MDA-MB-231 cells via the ERK/Slug axis. Then, we detected the protein expression levels of p-ERK, ERK and Slug in MDA-MB-231 cells after TRX treatment. The Western blot results showed that TRX significantly suppressed the expression of p-ERK and Slug in a time- and concentration-dependent manner. The total protein expression level of ERK showed no obvious change (Fig 3B and 3C). These results indicated that TRX could inhibit the migration and invasion of MDA-MB-231 cells via the ERK/Slug axis.

Fig 3. TRX inhibited the migration and invasion of MDA-MB-231 cells via the ERK/Slug axis.

Fig 3

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A) Map04520, named the adherens junction, shows that the ERK/Slug axis might be the mechanism. The red of scale indicates a positive correlation between gene and TRX, while the green indicates a negative correlation. B) The expression of p-ERK, ERK and Slug was determined by Western blot. C) Statistical analysis of protein expression in MDA-MB-231 cells. These results are representative of at least 3 independent experiments. Data are presented as the mean ± SD. NS, no significant difference, **p<0.01, ***p<0.001 compared with the 0 μM group.

ERK activator(tBHQ) reversed the TRX-induced suppression of MDA-MB-231 cell migration and invasion

To further confirm that TRX inhibits the migration and invasion of MDA-MB-231 cells via the ERK/slug axis, we observed whether tBHQ could reverse the TRX-induced suppression of MDA-MB-231 cell migration and invasion by wound healing and Transwell assays. In wound-healing assay, compared to control group, the he wound area of TRX group were 91.1% at 24h, 89.6% at 48h. While the wound area of TRX+tBHQ group were 85.6% at 24h, 73.5% at 48h. In the cell migration of transwell assay, the cell number of TRX group was 230 per filed, while the TRX+tBHQ group was 356 per filed. In the cell invasion of transwell assay, the cell number of TRX group was 279 per filed, while the TRX+tBHQ group was 515 per filed. These results indicated that tBHQ could partially reverse the TRX-induced suppression of MDA-MB-231 cell migration and invasion (Fig 4A–4D). Moreover, we detected the expression of EMT-related proteins (E-cadherin, N-cadherin, Vimentin) and axis-related proteins (p-ERK, ERK, Slug) by Western blot and observed that tBHQ could partially reverse the increase in the expression of E-cadherin and the decrease in N-cadherin, Vimentin, Slug and ERK phosphorylation caused by TRX (Fig 4E–4H). Moreover, we detected axis-related proteins with immunofluorescence. The results shown in Fig 4I indicated that TRX could decrease the expression of ERK phosphorylation and Slug. In this case, we believed that TRX could indeed inhibit the migration and invasion of MDA-MB-231 cells via the ERK/slug axis.

Fig 4. ERK activator (tBHQ) reversed the TRX-induced suppression of MDA-MB-231 cell migration and invasion.

Fig 4

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A) Cell migration was evaluated by wound healing assay. B) Statistical analysis of the wound healing area ratio. C) Cell migration and invasion were evaluated by Transwell assay. D) Statistical analysis of the cells per field. E), G) The expression of relative proteins was determined by Western blot. F), H) Statistical analysis of the relative protein expression. I) Immunofluorescence images of p-ERK, ERK and Slug (red) expression following control, TRX, tBHQ, TRX+tBHQ treatment. DAP1 staining was done to visualize nuclei (blue). These results were representative of at least independent experiments. Data are presented as the mean ± SD. NS, no significant difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared with the control group.

Discussion

According to the latest data published by the International Agency for Research on Cancer (IARC) [13], BC has become the most common cancer worldwide. Improving the awareness and ability of BC prevention and treatment is one of the most important measures to ensure women’s health. Although, overall, BC has a better survival than others, postoperative patients with TNBC usually have no appropriate medicine options due to the lack of sufficient receptor expression. Therefore, it is necessary to promote research innovation drugs.

It is well known that the participation of TCM could enhance efficacy and reduce toxicity in a great measure during the process of tumor treatment [14]. Taraxacum, as a pure natural herb, is a heat-clearing TCM. The broad application of different taraxacum extracts has shown that they could inhibit the proliferation and metastasis of TNBC [15], and TRX has been proven to have an inhibitory effect on a variety of tumor cell lines. In this study, we found that the migration and invasion of MDA-MB-231 cells were inhibited by TRX through wound healing and Transwell assays.

EMT, a process in which epithelial cells acquire the characteristics of mesenchymal cells, is considered to be closely related to cell migration and invasion, causing cells to become more invasive with the loss of polarity and adhesive ability [16]. E-cadherin, N-cadherin and Vimentin are classic markers of EMT. The expression level of E-cadherin was negatively correlated with BC cell migration and invasion [17], and in dedifferentiated breast cancer cells, it was significantly lower than that in differentiated cells [18]. N-cadherin with high expression in invasive BC cells could promote cell metastasis by interacting with surrounding stromal cells [19]. Vimentin is highly expressed in invasive BC cells and enhances cell metastasis by promoting the EMT process [20]. Therefore, we detected the protein expression levels of E-cadherin, N-cadherin and Vimentin by Western blot. After TRX treatment, E-cadherin expression was upregulated, while N-cadherin and Vimentin expression was downregulated, indicating that the EMT process in MDA-MB-231 cells was inhibited. Combined with the previous results, we believed that TRX could inhibit the migration and invasion of MDA-MB-231 cells.

Network pharmacology has been widely used in TCM research because it has the same integrity and systematic characteristics as TCM. Based on the theoretical basis of systems biology, it combines bioinformatics with network analysis to study the mechanism of medicine effects at the system level. Meanwhile, molecular docking, as a commonly used method for drug screening, can directly reveal the interactions between drugs and targets and predict their affinity. In this study, we observed that MAPK3 was the critical target by which TRX inhibited the migration and invasion of MDA-MB-231 cells by network pharmacology, and it could stably bind to TRX by molecular docking. Extracellular regulated protein kinase 1 (ERK1), encoded by MAPK3, belongs to the mitogen-activated protein kinase (MAPK) family [21]. Extracellular regulated protein kinase 2 (ERK2), encoded by MAPK1 is highly similar to ERK1 in sequence, protein function, upstream activation pathways and downstream targets. Therefore, ERK1 and ERK2 are often collectively referred to as ERK [22]. ERK is usually located in the nucleus and is transferred to the cytoplasm after being phosphorylated under various stress states [23]. ERK is basically activated by dual phosphorylation at threonine and tyrosine sites [24,25], hence, the protein tyrosine phosphatase activity predicted by GO enrichment analysis is necessary for ERK activation [26]. Previous studies have shown that the phosphorylation level of ERK is closely related to tumor occurrence and development. The inhibition of ERK phosphorylation could suppress cancer-stromal interactions in pancreatic cancer [27] and promote the ULK1 degradation process, leading to an improved invasive phenotype under hypoxia and osteolytic bone metastasis in BC cells induced by ULK1 deficiency [28]. Otherwise, among the 15 pathways obtained from KEGG enrichment analysis, adherens junction was the most significant pathway and closely related to cell migration and invasion. ERK is located in this pathway and acts on Slug to regulate cell adhesion, leading to cadherins switching. The Western blot results showed that TRX significantly reduced the expression levels of ERK phosphorylation and Slug. Therefore, we hypothesized that TRX could inhibit the migration and invasion of MDA-MB-231 cells via the ERK/Slug axis and verified this hypothesis by adding an ERK activator.

However, we acknowledge that there are still several limitations of this study. First, we simply detected the expression changes of EMT markers but did not show the EMT in more detail by cellular immunofluorescence staining. Second, further experiments in vivo are essential to verify the molecular mechanism. Moreover, due to the high invasiveness of TNBC, MDA-MB-231 cells were selected for this study. However, the other three subtypes of BC should also be studied to determine the therapeutic effect of TRX on BC. MTT assay relies on cell number to determine cell viability, while the experimental results are greatly influenced by the seeding density. Thus, it is necessary to support this conclusion through other experiments.

Conclusion

In summary, we illustrated that TRX could inhibit the migration and invasion of MDA-MB-231 cells, and the mechanism by which TRX inhibited the migration and invasion of MDA-MB-231 cells via the ERK/Slug axis was elucidated by network pharmacology, molecular docking and Western blot. Thus, TRX may be a promising therapeutic strategy for blocking tumorigenesis in TNBC.

Supporting information

S1 Raw images

(ZIP)

Click here for additional data file. (19.9MB, zip)

Data Availability

We have uploaded the raw experimental data to Figshare, a stable public repository. The DOI is 10.6084/m9.figshare.24114909.

Funding Statement

This work was supported by Natural Science Fund in Jiangxi Province (Contract grant number: 20202BAB206046) and Jiangxi Provincial Postgraduate Innovation Special Fund Project (Contract grant number: YC2021-S500).

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Decision Letter 0

Salman Shakil

25 May 2023

PONE-D-23-09759Suppression of migration and invasion by taraxerol in the triple-negative breast cancer cell line MDA-MB-231 via the ERK/Slug axisPLOS ONE

Dear Dr. li,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. The manuscript of Xia et al. is interesting. However, there are many flaws of the current version of the manuscript pointed out by the reviewers. Please address all the reviews and academic editor comments.    Academic Editor comments:  

  1. Please express MTT assay results as viable cells (% of control), change the figure 1a accordingly, and add information about limitations of MTT assay in the discussion part. You can find relevant information here - DOI: 10.1097/CAD.0000000000001131.

  2. Please perform molecular dynamic simulation to validate docking results. 

  3. Manuscript needs significant English improvement.

Please submit your revised manuscript by Jul 09 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Salman Shakil

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: I Don't Know

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This article "Suppression of migration and invasion by taraxerol in the triple-negative breast cancer cell line MDA-MB-231 via the ERK/Slug axis" is interesting, but I feel it's not a completed work. Authors focused on Slug only but there are important transcription factors (e.g., Twist, Snail, Zeb etc), especially Twist1 is a master regulator of cadherins as well as a key factor in EMT associated metastasis. What's the role of taraxerol on Twist in MDA-MB-231? Also, author claims that taraxerol suppress cell migration and invasion by ERK/Slug axis. Is it direct suppression or indirect? May be, it is the secondary effect of other mechanism? May be reporter assay could clarify this. To consider this paper for publishing, I think these issues need to be clarified.

Reviewer #2: The manuscript is intriguing as it explores the mechanistic aspects of taraxerol (TRX)-induced anti-migratory and anti-invasive effects on MDA-MB-231 cells. The study reveals that TRX inhibits the migration and invasion of MDA-MB-231 cells by targeting the ERK/Slug pathway. Nonetheless, prior to considering publication in PLOS ONE, there are several noteworthy issues that need to be addressed.

Comments:

1. The manuscript is lacking specific information about MDA-MB-231 cells. It is recommended to provide details about MDA-MB-231 cells in their initial mention within the introduction of the manuscript.

2. In Figure 1b, the cell density at 0 hours appears inconsistent across all the wells. Please observe the 80 µM TRX-treated well at 0 hours and the 0 µM TRX-treated treated well at 0 hours, as clear disparities in cell concentration are evident. Are there any representative photographs available that show equal cell density in all the wells at the initial time point?

3. The authors' bioinformatics approach in identifying MAPK3 as the target of TRX is quite fascinating. However, figure 2 is not acceptable to me because of its poor resolution. I was unable to verify the details in the figure due to its blurry appearance. Kindly submit a higher-resolution version of Figure 2 for review.

4. Once again, Figure 3a is deemed unacceptable due to its blurry appearance. Kindly provide an improved figure with a higher resolution.

5. In the text of the Results section, it is mentioned that the Western blot results indicate a significant suppression of p-ERK and Slug expression by TRX in a time- and concentration-dependent manner. However, upon examining Figure 3c, it is apparent that the authors only presented a concentration-dependent effect of TRX, and no demonstration of a time-dependent effect is evident.

6. The mechanistic details of TRX-induced anti-migratory and anti-invasive effects on MDA-MB-231 cells have been supported by Western blotting data. However, to enhance the strength of the findings and improve the manuscript's quality, the authors could consider including immunofluorescence staining or any other supplementary data. Incorporating imaging data would provide additional support and bolster the validity of their conclusions. It is recommended that the authors consider this suggestion.

Reviewer #3: The manuscript by Xia et al. describes the role of Taraxerol (TRX) in inhibiting the migration and invasion of breast cancer cell line, MDA-MB-231. They identified a possible in-vitro mechanism, ERK/SLUG axis, by which TRX executed its anti-tumour function in this cell line. They concluded that TRX can be a potential therapeutic target that acts by restricting the tumorigenesis during Breast cancer. The subject of the study is interesting and timely. The experimental design in this manuscript is sound, and the conclusions are important.

However, I have some concerns. The manuscripts are not written in a proper English standard. Some of the statements are vague and the flow of the sentence is very often interrupted. Also, authors need to be consistent in their way of writing. I would recommend reviewing the manuscript by professional english editors before it gets accepted for publication. Additionally, I would need to see more data and rigorous support for their claims before I can recommend that this manuscript be published.

Introduction:

1. No space between the end of sentence and the references in the introduction section. This has been observed throughout the manuscript.

2. Authors need to elaborate the meaning of TNBC and TRX before they start using it in the sentence.

Materials and methods:

1. Authors need to mention what kind of cell line MDA-MB-231 is?

2. MTT assay: The cell count should be written as 4 x 103 cells.

3. Wound healing assay: Mention the model of microscope and what kind of microscope was used to capture images.

4. Authors need to mention the version of any bioinformatics or statistical software used throughout the manuscript. For example: GraphPad Prism, ImajeJ software etc.

5. Provide catalogue details for reagents such as Matrigel.

6. Western blotting: Authors need to rewrite the last sentence as the photographs cannot be taken by ECL rather the bands were developed using ECL reagents.

7. Network Pharmacology: Why the cut-off of probability0.1 was selected for TRX target?

8. Network pharmacology: Authors need to give a clear explanation about how the results were visualized by bioinformatics in the last sentence.

Results:

In the result section, my major concern is authors need to explain the results before/after giving any interpretation which was missing throughout the manuscript. Also, Authors need to mention how much increase/decrease they have noticed in numbers throughout the result sections.

With all the Western blots figure, the author needs to mention if they have stripped and re-probed the blot. If so, they need to describe the stripping procedure in the method section.

1. Cell viability: Authors should plot the graphs as cell viability (%) since OD value hardly makes any sense here.

3. Figure 1C: Authors need to have one more set of bars with 0h at 0 mm, 40 mM and 80 mM. The statistical analysis for the treated groups should be based on this set.

4. Figure 1C: Authors need to be elaborative in writing down the percentage results in the result section. When it says, decreased/ increased significantly readers need to know to which extent the differences were observed.

5. Figure 1D, 1E: No proper explanation of the results. Authors need to mention clearly which reading (Migration or invasion) was taken from which (upper and lower) well?

6. Figure 2: What are these 20 targets? Include a table as a supplementary file.

7. Figure 2: As authors have mentioned MAPK3 as the most promising candidate, I would like to see the protein expression level of MAPK3 alongside ERK/p-ERK by western blotting in Figure 3B/4G. Also, if authors are specifically saying that MAPK3 is involved then why they are measuring total p-ERK/ERKs instead of p-ERK1/ERK1? This is contradictory as MAPK1 can also be a major candidate here based on the observation of the western blots in figure 3B and 4G.

8. Fig 3A: Did author measure the expression of Cadherin in the blots? If not, why the Cadherin in this figure is marked as red? There is no explanation about cadherin in this section. They also need to explain clearly in the figure legend about the scales drawn on the top right side.

9. Fig 3B,C: Did authors measure the expression of p-ERK1/ERK1 or both ERKs? If the probing for p-ERK, ERK and GAPDH was done on the same blot after stripping, how does the author confirm that p-ERK and ERK does not cross-react? Authors need to describe how did they quantify p-ERK and ERK level?

10. Fig 3B,C: “TRX significantly suppressed the expression of p-ERK and Slug in a time- and concentration-dependent manner”: Wrong statement as authors did not measure the time-dependent effect here.

11. Fig 4A,D: Authors need to give proper explanation for their interpretation?

Discussion:

1. Need reference for this statement: According to the latest data published by the International Agency for Research….

2. This sentence doesn’t make sense: Therefore, it is necessary to promote research no innovation drugs..

3. Is it dedifferentiated? : and in dedifferentiated breast cancer cells, it was significantly..

Figure legend:

All the legends need to be re-written by giving more scientific details of the experiments. Authors need to mention what statistical analysis they have used for each of the experiment. Is it one/two-way ANOVA or t-test?

Figure: 1A: Wrong statement as the OD values cannot be counted by GraphPad software

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Farjana Ahmed

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2023 Sep 26;18(9):e0291693. doi: 10.1371/journal.pone.0291693.r002

Author response to Decision Letter 0

9 Jul 2023

Dear Editor:

Thank you for your and the reviewers’ comments on our manuscript entitled “Suppression of migration and invasion by taraxerol in the triple-negative breast cancer cell line MDA-MB-231 via the ERK/Slug axis”. (EMID: f1702d110dff026b). Our deepest gratitude goes to the anonymous reviewers for their careful work and thoughtful suggestions that have helped improve this manuscript substantially. Meanwhile, thank you for giving us a chance to revise and resubmit our manuscript.

Based on these comments, we have made careful modifications again. All changes made to the manuscript are marked in red. We modified the figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in Supporting Information . These changes will not influence the content and framework of the manuscript. We hope that these revisions are satisfactory and the revised manuscript will be acceptable for publication.

Below you will find our point-by-point responses to your comments/questions. Once again, thank you for your comments and suggestions.

Sincerely,

XIA Yu-ting, ZHANG Yu-qin, CHEN Lu, MIN Liang-liang, HUANG Da, ZHANG Yu-lu, LI Cong, LI Zhi-hua*

*Corresponding author. E-mail address: huazhili0802@163.com

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (3.9MB, docx)

Decision Letter 1

Salman Shakil

4 Sep 2023

Suppression of migration and invasion by taraxerol in the triple-negative breast cancer cell line MDA-MB-231 via the ERK/Slug axis

PONE-D-23-09759R1

Dear Dr. Li,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Salman Shakil

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: No more comments on "Suppression of migration and invasion by taraxerol in the triple-negative breast cancer cell line MDA-MB-231 via the ERK/Slug axis"

Reviewer #2: All the comments, suggestions, and queries have been convincingly addressed by the authors. I recommend accepting the article for publication in PLOS ONE.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

Acceptance letter

Salman Shakil

18 Sep 2023

PONE-D-23-09759R1

Suppression of migration and invasion by taraxerol in the triple-negative breast cancer cell line MDA-MB-231 via the ERK/Slug axis

Dear Dr. Li:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr Salman Shakil

Academic Editor

PLOS ONE

Articles from PLOS ONE are provided here courtesy of PLOS

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