Figure 1.
SK228 treatment results in the reduced in vitro migratory and invasive potential of breast cancer cells.
Chemical structure of SK228 is shown. Cells were treated with different concentrations of SK228 or DMSO for 24 h. After incubation, cells were transferred to transwells and given 24 h to migrate or invade. (A) Quantitation of the migratory and invasive abilities of MDA-MB-231 cells (**P<0.01). (B) Quantitation of the migratory and invasive abilities of Hs-578 T cells (*P<0.05, **P<0.01). (C) Quantitation of the migratory and invasive abilities of BT-549 cells (**P<0.01).
Figure 2.
SK228 induces the morphological changes in breast cancer cells.
The morphologies of breast cancer cells change from fibroblastoid to epithelial-like after being incubated with SK228 for 24 h (MDA-MB-231 cells: 0.8 µ M of SK228, Hs-578 T cells: 4 µM of SK228 and BT-549 cells: 1.2 µ M of SK228). The effects of SK228 on morphological change were documented by using a light microscopy at the indicated time.
Figure 3.
SK228 increases the expression of E-cadherin and decreases the expression of ZEB1 in breast cancer cells.
SK228 increases the expression of E-cadherin and decreases the expression of ZEB1 as assessed by using Western Blot analysis (A) and RT-PCR (B).
Figure 4.
E-cadherin becomes distributed in the cytoplasm closer to the cell membrane after SK228 treatment.
Immunofluorescence staining results shows that E-cadherin is induced and distributed in the cytoplasm closer to the cell membrane after SK228 treatment.
Figure 5.
miRNA real-time PCR shows that SK228 induces the exoression of miR-200 family in breast cancer cells.
Cells are treated with different concentrations of SK228 for 48-200 family are analyzed by using real-time PCR. (A) MDA-MB-231 cells, (B) Hs-578 T cells, (C) BT-549 cells.
Figure 6.
miR-200c/ZEB1/E-cadherin signal pathway regulates the epithelial-to-mesenchymal transition in breast cancer cells.
Re-expression of miR-200c in breast cancer cells results in up-regulation of E-cadherin expression and the down-regulation of ZEB1 expressions as assessed by using Western Blot analysis (A) and RT-PCR (B). siRNA knockdown of the expressions of ZEB1 results in up-regulation of E-cadherin expression in breast cancer cells (C).
Figure 7.
SK228 inhibits the growth of 4T1-Luc xenografts and consequently delays cancer cell metastasis via reversal of EMT process and miR-200c/ZEB1/E-cadherin signaling pathway.
(A) BALB/c mice bearing the established 4T1-Luc tumors (50 mm3) are treated with SK228 (12.5 mg/kg/mouse/week) via intraperitoneal injection on the first 3 days in the first week, then two shots on day 1 and day 4 at a dose of 6.25 mg/kg/week for another 3 weeks (final dose = 50 mg/kg/mouse), or vehicle (30% PEG400+70% PBS) control. The results of spontaneous metastasis assays show that SK228 treatment delays cancer cell metastasis in animal models. (B) The tumor size indicated in the graph represents the measurement of the primary tumor only. Tumor volumes, measured every other day until day 28, are expressed as mean ± SD; *P = 0.045. (C) The average body weight between vehicle and SK228 groups is not significantly different.