Thidiazuron decreases epithelial‐mesenchymal transition activity through the NF‐kB and PI3K/AKT signalling pathways in breast cancer

Abstract Breast cancer is the major type among the women population globally. The treatment of cancer metastasis has made modest progress due to multiple factors. Thidiazuron (TDZ) is a novel plant growth regulator that has been shown to have anticancer effects. Therefore, we explored the anti‐metastatic potentials of TDZ in cell lines by assessing its potential to suppress the epithelial‐mesenchymal transition (EMT). We pretreated the BEAS‐2B and breast cancer (MDA‐MB‐231) cells with TDZ and deliberated alteration in a cell viability, mammosphere, migration, NF‐кB signalling, PI3K/AKT signalling and matrix metalloproteinase (MMP) expression and analysed the EMT induction by TGF‐β/TNF‐α‐stimulated BEAS‐2B cells. Treatment with TDZ (5‐50 μmol) diminished the migration and invasion of the extremely metastatic MDA‐MB‐231 cells. Additionally, TDZ treatment led to down‐regulation of uPAR, uPA, VEGF and MMP‐2/‐9 expression and up‐regulation of TIMP‐1/2 expression in these cells. Furthermore, TDZ treatment blocked invasion and EMT in non‐tumorigenic BEAS‐2B epithelial cells stimulated with TGF‐β/TNF‐α.TDZ prevents EMT and may thus block metastasis of breast cancer cells.


| INTRODUC TI ON
Breast cancer is a recurrently identified cancer type in the world, causing women 523 000 deaths and 15.1 million illness-attuned lifetimes in 2015. 1 Advances made in the field of breast cancer over the last few years have augmented the 5-year survival rate of victims worldwide. 2 Despite this significant progress, the efficient treatment approaches for metastatic breast cancer remain as an imperative cause of deaths worldwide. 3 It might seem the elevation of the expectation to treat the metastatic breast cancer, an ailment that is obstinately resistant to finest efforts of modern drug. The epithelial-mesenchymal transition (EMT) was identified as a decisive process for carcinoma development and metastases. 4 Epithelial-mesenchymal transition is a mechanism in that the polarized epithelial cells in tumours drop their polarity and ability to form cell adhesions and become mesenchymal stem cells, which are characterized by increased migratory ability, invasiveness, metastatic potential and drug resistance. 4,5 The Snail and Slug signalling cascades are in the EMT of cancer cells. 6,7 In various cancers, Snail expression is associated with AKT/GSK or NF-кB activity and promotes cell invasion and migration. 8 Specifically, Snail and Slug are transcription factors, which mediate the EMT via binding to the E-boxes in EMT target genes like the classical epithelial cell adhesion molecule E-cadherin, entirely absent in mesenchymal cells. 9 Besides decreasing the E-cadherin expression, the binding of Slug and Snail to E-boxes in a E-cadherin augments the matrix metalloproteinase-9 (MMP-9) activity, a protease that promotes cell invasion. 10,11 MMP-9 performs a vital function in metastasis via destroying extracellular matrix (ECM) and thus enabling the migration as well as invasion of cancer cells. EGF induces the expression in cancer cells via stimulating the signalling pathways PI3K/AKT and NF-kB. [12][13][14] Numerous preceding researches have explored the effects of inhibiting PI3K on cancer metastasis. 12,15 LY294002 and wortmannin are strong PI3K inhibitors, which are normally utilized in laboratory and preclinical researches but have been prohibited from clinical purpose utilization because of their unsteadiness, reduced solubility and/or augmented toxicity. However, few studies on cancer treatments have focused on the suppression of suppression of EMT-associated pathways and/or the mechanisms responsible for EMT inhibition.
Bioactive substances display a number of anticancer features, like repression of cell multiplication, activation of apoptosis and cancer metastasis. Several studies have demonstrated their significant potential to treat the lung cancer or triple-negative breast cancer (TNBC) via inhibiting the tumour and metastasis. 16,17 The less toxicity of these compounds makes them as promising agents for the treatment of cancer.
Thidiazuron (TDZ) is a phytochemical with cytokinin-like activity that has been extensively used as an herbicide and a plant growth regulator. [18][19][20] In a study of the cytotoxic effects of TDZ in HeLa cervical cancer cells, Enkhtaivan et al mentioned that the toxicity of TDZ in cancer cells was increased when evaluated with normal cells. 21 This effect was accompanied via the presence of DNA breaks, loss of mitochondrial membrane potential, and altered several apoptosis-related gene expression. 21 Importantly, TDZ was found to target caspase-3 in molecular docking simulation studies. 21 Based on the above study, we need to inspect whether TDZ could inhibit EMT and metastases in MDA-MB-231 cells.
In this investigation, we aimed to ascertain the potential of TDZ to inhibit metastasis, EMT and associated changes in breast cancer. In that order, we used a validated EMT and metastasis MDA-MB-231 cell-based model. EMT and the chief molecular markers concerned were inspected to characterize the anti-EMT and anti-metastatic properties of TDZ.

| In vitro wound-healing activity by Scratch assay
The wound-healing assay was administered to test cell migration.
MDA-MB231 cell monolayers were scratched and treated with CLG according to the previously described protocol {Hseu, 2019 #87}.

| Cell invasion assay
The invasion assay was carried out using BD Matrigel invasion chambers

| Mammosphere formation assay
The mammospheres were seeded in a low adherence with CnT-27, serum-free medium and growth factors (CellnTEC Advanced Cell Systems, Bern, Switzerland) as mentioned by Dontu et al. The selfrenewal capacity of MDA-MB-231 cells was inspected via generating additional mammospheres. At the every process, spheres were estranged into single cells and re-plated beneath the previously used low-adherence situations. Shortly, mammospheres were gathered and centrifuged. Later than the supernatant aspiration, 0.1% trypsin/EDTA was amalgamated to cell pellet, then pellet was resuspended, and the mixture was maintained for 1 minute at 37°C. The suspension was then sifted via a 70-µm sifter, and the cells were counted and re-plated in the medium and then mammospheres were being inspected through the microscopy.

| Western blotting study
Cells were loaded at 1 × 10 6 cells/6-cm plate and subsequently pretreated with TDZ at concentrations ranging from 5 to 50 μmol for 24 hours. After the administration, the cells were scraped and cleansed in a chilled PBS; afterwards, the nuclear, cytoplasmic and total extracts were prepared as mentioned earlier. 56 For the specified concentrations and time-points, the cells were treated with TDZ and Western blotting was performed as stated earlier {Lee, 2020 #88}.

| Immunofluorescence staining
Cells were grown in 8-well plate (1 × 10 4 cells per well density), and the cells were subjected to TDZ (50 μmol) pre-supplementation for the 24 hours. Then, cells were processed for 15 minutes with 2% of paraformaldehyde, permeabilized via Triton X-100 (0.1%) for 10 minutes, cleansed and blocked through 10% of FBS/PBS. After that, they were maintained with anti-p65 primary antibodies in a 1.5% FBS solution. Then, cells were sustained with FITC-conjugated secondary antibodies diluted in 6% BSA/PBS for 1 hour. Afterwards, the cells were stained with 1 μg/mL DAPI for 5 mins. Later than two cleanses with PBS for 5-10 minutes each, the cell arrangements were cleansed with distilled water, fixed in ethanol for 5 minutes and fixed with Fluoromount G (Southern Biotech; attained via Biozol Diagnostica, Eching, Germany). Lastly, immunofluorescence microscopy images were obtained with a Leica D6000 fluorescence microscope (Leica, Germany) outfitted with an AxioCam HR (Carl Zeiss).

| Gelatin zymography assay
The activities of MMP-2 and −9 in the MDA-MB-231 cells were inspected through the gelatin zymography protease technique. Briefly, 3 × 10 5 MDA-MB-231 cells was loaded to 12-well plates consisting of DMEM supplemented with FBS (10%) and grown to a near-confluency.
Subsequently, cells were trypsinized and reseeded in DMEM and incubated with TDZ (25-50 μmol) for 24 hours. Then, a right amount of the gathered medium was assorted with SDS sample buffer but not subjected to either a boiling or reducing step. For the analysis of MMPs, the SDS-PAGE (8%) gel was supplemented with 1 mg/mL gelatin (casein), and the sample was resolved by electrophoresis.

| Actin distribution and cell morphology analysis
Unstimulated BEAS-2B cells at 1 × 10 4 cells/well population were seeded in DMEM medium that restrained FBS (10%). Later than 24 hours, the cells were pre-supplemented with TDZ (25 μmol) for 1 hour and subsequently triggered with TGF-β/TNF-α (10 ng/mL) for 24 hours. Then, the cells were mounted on 3.7% of paraformaldehyde, blocked in 3% of BSA and stained for visualization of F-actin by TRITC-conjugated phalloidin. Then, cells were stained with DAPI (1 μg/mL), as described in section 2.9. Lastly, immunofluorescence microscopy pictures were monitored with a Leica D6000 fluorescence microscope (Leica, Germany) outfitted with an AxioCam HR (Carl Zeiss).

| DNA fragmentation assay
To evaluate the DNA fragmentation in MDA-MB −231 cells upon TDZ treatment was carried out through Cell Death Detection ELISA PLUS kit (Roche Applied Science) by the procedures of the manufacturer and as mentioned earlier. 22

| Statistical analyses
Experimental data were articulated as a mean ± SD. ANOVA was utilized to analyse every investigational result, and Dunnett's test was used for pairwise comparisons. Variations among the test groups were regarded as significant at P < .05.

| TDZ suppresses the viability of cell lines
We began our assessment of the anticancer effects of TDZ

| TDZ promotes DNA fragmentation and caspase-3 and PARP cleavage in MDA-MB-231 cells
Apoptosis is a critical mechanism, which helps to reduce cell growth. 22 The augmented DNA fragmentation was noticed in a TDZ (50µmol)-treated MDA-MB-231 cells as determined ( Figure 1D).
Additionally, TDZ treatment resulted in a time-reliant stimulation of caspase-3, manifested as cleaved caspase-3, and cleavage of PARP protein ( Figure 1E). The cleavage of caspase-3 and PARP were statistically significant after 12 and 24 hours of incubation with TDZ.

| TDZ inhibits MDA-MB-231 cell migration and invasion
Wound healing is a critical mechanism in that the skin renews itself followed by the damages, and this process was mentioned to be associated to EMT and tumorigenesis. 23 We inspected the inhibitory potential of TDZ on cell migration by performing a wound-healing analysis. Untreated MDA-MB-231 cells revealed appreciable migratory potential, while wound closure by cells treated with 25 or 50 μmol TDZ was delayed ( Figure 2A). Furthermore, the gap difference was significantly reduced to exposure to 25 or 50 μmol TDZ, whereas wound closure in the control group ( Figure 2A). Next, we measured the ability of TDZ to inhibit MDA-MB-231 cell invasion by performing Matrigel invasion assays. The results showed that at doses F I G U R E 2 TDZ inhibits human breast cancer cell proliferation, cell migration and invasion. A, MDA-MB-231 cell monolayers were scratched, and cells were supplemented with TDZ (25 and 50 µmol) for 24 h. Migration was noticed with an optical microscope (200× magnification) by a wound-healing assay. Commercially available MRI wound-healing tool was used to calculate the area of the wound and assess wound closure. B, MDA-MB-231 cells supplemented with TDZ (0-50 µmol) were loaded to the upper chambers of Matrigel-coated transwells, and invasion was inspected via total cell counting, which had crossed to the lower chamber after 24 h The inhibition percentage of invasion was quantified and is expressed relative to the control (untreated cells), whose level of invasion was set at 100%. Invading cells quantified by using manual counting. for 24 hours. The results showed substantial dose-dependent increases in the status of TIMP-1, TIMP-2 and PAI-1 ( Figure 2D).
Together, these findings indicate that TDZ may inhibit migration and invasion by modulating by down-regulations relevant proteins.

| TDZ attenuates NF-κB activation by suppressing I-κBα down-regulation
The NF-κB signalling cascade is a critical regulator of the MMP, VEGF and uPA expression. 6  to TDZ treatment was confirmed by immunofluorescence analysis ( Figure 3C). I-κBα binds to NF-κB preventing its transfer to the nucleus where it would exert its function. The degradation of I-κBα releases p65 for translocation to the nucleus and therefore we measured the effect of TDZ treatment on I-κBα cytoplasm levels. 12 TDZ-treated cells and found that statistically significantly reduced I-κBα degradation ( Figure 3A). Based on these results TDZ could degradation of the protein, which controls the stability of NF-κB activation. We utilized Western blotting to investigate IKKα phosphorylation to decide if the I-κBα degradation inhibition is an outcome of IKKα phosphorylation inhibition. Phosphorylated IKKα in the cytoplasmic fraction of cells supplemented with diverse dosages of TDZ and found that TDZ treatment appreciably reduced the levels of phosphorylated IKKα ( Figure 3A). Overall these outcomes reveal that TDZ suppresses nuclear stimulation of NF-κB probably by inhibiting I-κBα degradation via a reduction in phosphorylated IKKα levels.

| TDZ inhibits MMP-9 via suppression of the PI3K/AKT and NF-κB pathways in MDA-MB-231 cells
NF-κB performs a major function in the mediation of expression of MMP in cancer cells. 6 The previous results showed that TDZ treatment down-regulated MMP-9 expression as well as NF-κB and PI3K/ AKT signalling. Since MMP-9 expression is arbitrated through the NF-κB that in turn is mediated through the PI3K/AKT, we next examined whether TDZ-mediated down-regulation occurred via NF-κB inhibition and PI3K/AKT cascade. Consequently, first, we evaluated the potential of TDZ treatment on NF-κB stimulation and MMP-9 expression using MDA-MB-231 cells pretreated with butein. The outcomes were proved that TDZ decreased p65 and MMP-9 protein levels and that these TDZ-mediated effects were more pronounced with butein pretreatment ( Figure 5A).
The PI3K/AKT signalling is a critical pathway concerned in NF-κB/MMP-9 stimulation. We have already shown that TDZ attenuates PI3K/AKT activation (see Figure 5B). To investigate the relationship between the PI3K/AKT pathway and TDZ-mediated decreases in p65 and MMP-9 levels, we assessed the impact of treatment with LY294002, MMP-9 expression, on nuclear p65, a PI3K inhibitor, in a presence or absence of TDZ. We observed that in cells treated with LY294002 without TDZ, nuclear p65 levels and expression of MMP-9 were lower than in those treated without LY294002 and TDZ ( Figure 5B). Overall, these outcomes were revealed that TDZ suppresses MMP-9 expression via PI3K/AKT and NF-κB cascade inhibition in MDA-MB-231 cells.

| TDZ inhibits the MMP expression stimulated by TNF-α/TGF-β activation of non-tumorigenic cells
Epithelial-mesenchymal transition plays significant functions in a typical development, tissue fibrosis and metastasis. TGF-α is the active EMT inducer and is suspected to lead to tissue fibrosis, which includes hepatic, renal and lung fibrosis. However, there is evidence that TNF-α is related to the EMT. 26 Therefore to inspect the po-  Figure 6C). Taken together, these outcomes were revealed that TDZ may inhibit TNF-α/TGF-β-stimulated MMPs and uPA in BEAS-2B cell lines. cells express E-cadherin. 27 Thus, to determine whether TDZ affects mammosphere formation by MAD-MB-231 cells, we uncovered cells to numerous doses of TDZ for one week, and our findings indicated that TDZ reserved adherent spherical breast cancer cluster formation in vitro, as these cells become unable to generate secondary spheres ( Figure 7C). These results indicate that TDZ is capable of a strongly inhibiting the formation of mammospheres by breast cancer cells.  Figure 8A). Further, we examined the F-actin distribution. Cells administered with TNF-α/TGF-β appeared to have a fibroblastic phenotype rather than an epithelial phenotype, and TDZ pretreatment prevent the TNF-α/TGF-β-stimulated morphological alterations ( Figure 6B). This outcome was evidenced by the clustering of the nuclei in the TNF-α/TGF-β administered group, as seen with a cells edges in with the Phalloidin images ( Figure 8B). These findings confirm that TDZ can attenuate EMT, by inhibiting invasion and F-actin distribution in TNF-α/TGF-β-induced BEAS-2B cells. The NF-kB and PI3K/AKT pathways are concerned in frequent pathological progressions, including adhesion of cancer cells, angiogenesis, inflammation, and metastasis. 32 The inhibition of NF-κB stimulation represses uPA, VEGF, MMPs and tumour metastasis. 9 To invade surrounding tissue, cancer cells must be able to transverse vessel walls, and to assist this event, they produce uPA, MMPs and VEGF, important components of cancer cell metastasis and invasion. [33][34][35] In this exploration, we found that TDZ inhibits con-   50 In this examination, we found that TDZ treatment prevents EMT in metastatic BEAS-2B

cells. Our outcomes indicate the inhibition of EMT or inhibition of
the functions of EMT transcription factors may be an additional process concerned in the antitumour potential of this compound.
In a hepatocellular carcinoma study, TGF-β transforming activation has been shown to facilitate liver cancer lung metastases in mouse models. 28 It remains to be known if the same mechanism is involved in lung metastases of breast cancer. In our examination, we also inspected the anti-EMT and anti-metastatic potentials of TDZ and the related processes in non-tumorigenic BEAS-2B cells stimulated with TNF-α/TGF-β. TGF-β promotes tumour progression via stimulating EMT. 51 TGF-β-stimulated EMT has the characteristics like attainment of a fibroblastic morphology, loss of E-cadherin localization and augmented cellular motility. 52 TNF-α is a pro-inflammatory F I G U R E 6 TDZ inhibits TGFβ/TNF-α induced MMPs and uPA in BEAS-2B cells and un-induced MBA-MDA-231. A, TDZ effects on MMP-2 and MMP-9 activity in a medium from MDA-MB-231 cells were inspected by gelatin zymography. B, TDZ effects on MMP-2 and MMP-9 activity in a medium from TNF-α/TGF-βstimulated BEAS-2B cells were evaluated by gelatin zymography. C, TDZ effects on uPA activity in conditioned medium from TNF-α/TGF-β-induced BEAS-2B cells were evaluated by Western blotting. The data are articulated as the mean ± SD of triplicate measurements. *P < .05 when evaluated with control

This study was supported by Deanship of Scientific Research at King
Faisal University, Al Ahsa, Saudi Arabia, under grant no. 180090.

CO N FLI C T O F I NTE R E S T
No conflicting interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.