Fig 1.
Hierarchical clustering analyses and heatmaps to show differentially regulated gene expression in SKOV3 cells.
SKOV3 cells were treated with 10 ng/ml TGFβ1 with or without 3 mg/ml CFG for 24 h. (A) 43 significantly up-regulated and down-regulated genes were identified in the gene expression profiles. 27 cellular proliferation or apoptosis associated genes (B) and 28 EMT-related genes (C) were significantly differentially regulated in the CFG-treated cells. Each gene is presented as the mean ± SD of three independent experiments. (D) Gene Ontology Analysis to indicate the biological process regulated by CFG.
Fig 2.
CFG suppresses ovarian cancer cell proliferation in vitro.
SRB assays (A) and MTT experiments (B) to show the inhibitory effect of CFG on HEY and SKOV3 cell growth after treatment with different concentration of CFG. (C) Crystal violet staining to show the colony formation ability of CFG-treated HEY and SKOV3 cells compared with control. Representative images are presented in the panel. The data represent mean ± SD of three experiments.
Fig 3.
CFG changes cell cycle distribution and cell cycle-related protein expression in ovarian cancer cell lines in vitro.
HEY (A) and SKOV3 (B) cell lines were treated with CFG 3 mg/ml for 24 h and the cell cycle distribution was analyzed by flow cytometry. (C and D) Quatitative PCR to detect the mRNA expression of cell cycle assoicated protein. HEY cells (C) and SKOV3 cells (D) were treated with 3 mg/ml CFG for 24 h. (E) HEY cells were treated with 3 mg/ml CFG for 24h and 48 h and then stained with immunofluorescen labeled antibodies against Cdt1 (red, G1 phase) and Geminin (green, G2 phase). Representative images are presented. (F) Cell cycle-related proteins were detected by western blot in HEY and SKOV3 cells treated with 3 mg/ml CFG for 24h.
Fig 4.
CFG induces apoptosis and senescence in ovarian cancer cells in vitro.
(A) HEY and SKOV3 cell lines were treated with 3 mg/ml CFG for 24 h. The percentage of apoptotic cells were measured by flow cytometry using the tunnel apoptosis detection assay. (B and C) Apoptosis-related genes, including Bad, Bcl-Xl, Caspase 7 were detected by quantitative PCR and western blot. (D) β-gal Staining was performed to demonstrate the effect of 3 mg/ml CFG treatment on HEY and SKOV3 cell senescence.
Fig 5.
CFG inhibits the mobility of HEY and SKOV3 cells.
(A and B) The wound healing assay was used to demonstrate the migration ability of HEY and SKOV3 cells, treated with 3mg/ml CFG only or in combination with 10 ng/ml TGF β1. Cell migration rate was quantitatively evaluated by measuring the cell covered area (C and D). Data are presented as mean ± SD. The experiments were repeated at least three times. *p <0.05 compared with the control. **p <0.01 compared with the control.
Fig 6.
CFG decreases TGF- β 1-induced invasion and migration of HEY and SKOV3 cells in vitro.
HEY cells (A) and SKOV3 cells (B) were treated with 3mg/ml CFG only or in combination with 10 ng/ml TGF β1 for 24 h prior to use and the invasion and migration assays were then performed. Crystal violet OD values represent the amounts of invated and migrated HEY cells (C) and SKOV3 cells (D). Data is presented as mean ± SD. The experiments was repeated at least three times. * p <0.05 compared with the control. **p <0.01 compared with the control.
Fig 7.
CFG downregultates AKT/GSK3β signal pathway and EMT markers.
Cells were treated with 3 mg/ml CFG only or in combination with 10 ng/ml TGF β1 for 24 h. The cellular proteins, including E-cadherin, N-Cadherin, Vimentin, Fibronectin, β-Catenin, pAKT, AKT, pGSK3β, GSK3β, SNAIL, and SLUG, were detected by Western blot.
Fig 8.
CFG treatment reduced tumor growth and metastasis in vivo xenograft mouse.
(A) The photos of the tumor tissues dissected from the mice on day 44 after injection and with aforementioned treatment. (B) The tumor dynamic growth curves of different groups. (C) Tunel assay to show the apoptotic cells in the tumor tissues with indicated treatment at 200× magnification. Lower layer shows the percentage of apoptotic cells in each group. For A, B, and C, SKOV3: SKOV3 control group of subcutaneous injection; CFG: CFG group of subcutaneous injection; Cisplatin: Cisplatin group of subcutaneous injection. (E) H&E staining showed the lung metastases and immunochemistry staining to show the expression of E-Cadherin and N-Cadherin in each group. SKOV3: SKOV3 control group of tail intravenous injection; CFG: CFG group of tail intravenous injection; Normal lung: Normal group for tail intravenous injection. *p < 0.05 and **p < 0.01
Table 1.
The composition of Compound Fuling Granule (CFG).