Figure 1 Thidiazuron (TDZ) inhibit cell growth and induced apoptosis in MDA‐MB‐231 cancer cells. A, The structure of TDZ is shown. B, MDA‐MB‐231, MCF‐7, A549, HepG2 and normal lung epithelial BEAS‐2B cells were supplemented with TDZ (0‐100 μmol) or a vehicle control (0.1% DMSO) for different time periods, and cell viability was assessed with MTT assays. C, MDA‐MB‐231 cells were supplemented with TDZ (0‐50 μmol) for 24 h, and cell morphology was examined beneath the phase‐contrast microscope. D, MDA‐MB‐231 cells were supplemented with 50 μmol TDZ. DNA fragmentation was analysed by measuring photometric at various time‐points (0‐72 h). E, MDA‐MB‐231 Cells were supplemented with TDZ (50 μmol) and harvested at different time‐points (0‐48 h). Then, the levels of apoptosis protein markers were analysed by Western blotting. β‐actin was utilized as the internal control. The data are articulated as the mean ± SD of triplicate measurements, *P < .05 when evaluated with control cells

Figure 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. C, D, Alterations in the status of metastasis‐associated proteins in respond to the TDZ supplementation were inspected via Western blot. MDA‐MB‐231 cells were supplemented with TDZ (0, 5, 10, 25, or 50 μmol) for 24 h. Β‐actin was utilized as controls. C, Effects of TDZ supplementation on the status of MMP‐2, MMP‐9, uPA, uPAR and VEGF. D, Effects of TDZ supplementation on the statuses of the endogenous inhibitors of the proteins shown in (C), namely TIMP‐1, TIMP‐2 and PAI‐1. The data are articulated as the mean ± SD of triplicate values. *P < .05, when evaluated with control

Figure 3 TDZ down‐regulation of NF‐kB signalling in MDA‐MB‐231 cells. A, Cytosolic fractionation and Western blot analysis of TDZ effects on I‐κB and p‐IKK levels. B, TDZ effects on p65 in nuclear extracts. MDA‐MB‐231 cells were supplemented with 0‐50 μmol TDZ for 2 h. Cytoplasmic and nuclear extracts were prepared by described as methods sections. Lamin B and β‐actin were utilized as the internal controls for the cytoplasmic and nuclear fractions. The data are articulated as the mean ± SD of triplicate values. *P < .05, when evaluated with control. C, Cells were grown on four‐chambered slides, exposed to TDZ (50 μmol) for 2 h, mounted and permeabilized. The cells were incubated with an anti‐p65 antibody subsequently, FITC‐labelled secondary antibody. The subcellular localization of p65 was examined through the confocal microscope at 40× magnification. The data are articulated as the mean ± SD of triplicate measurements. *P < .05 when evaluated with control

Figure 4 TDZ down‐regulation of PI3K/AKT signalling in MDA‐MB‐231. A, Phosphorylated PI3K (p‐PI3K) and AKT (p‐AKT) status was inspected via Western blotting. Cells were supplemented with TDZ (0‐50 μmol) for 24 h. β‐actin, total PI3K, and AKT statuses were used as internal controls. The data are articulated as the mean ± SD of triplicate measurements. *P < .05 when evaluated with control

Figure 5 TDZ inhibits EMT through down‐regulation of MMP9 expression and NF‐κB signalling in MDA‐MB‐231 cells. A, TDZ effects on MMP‐9 expression and NF‐κB signalling are shown. Nuclear p65 levels (6 h) and MMP‐9 expression (24 h) were evaluated following treatment with TDZ (50 μmol) for 6 and 24 hours, respectively, in the absence or presence of the NF‐κB inhibitor Butein (50 μmol), and Western blot analysis was performed. β‐actin and Lamin B were utilized as internal controls. B, TDZ effects on NF‐κB/MMP‐9 expression after down‐regulation of PI3K/AKT signalling pathway activity. Nuclear p65 levels and MMP‐9 expression were evaluated following treatment with TDZ (50 μmol) for 6 and 24 h, respectively, in the absence or presence of the PI3K/AKT inhibitor LY294002 (30 μmol/L), and Western blot analysis was performed. β‐actin and Lamin B were utilized as controls. The data are articulated as the mean ± SD of triplicate measurements. *P < .05 when evaluated with control

Figure 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‐β‐stimulated 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