7-difluoromethoxyl-5,4'-di-n-octylgenistein inhibits growth of gastric cancer cells through downregulating forkhead box M1

Abstract

Aim: To investigate whether the 7-difluoromethoxyl-5, 4'-di-n-octylgenistein (DFOG), a novel synthetic genistein analogue, affects the growth of gastric cancer cells and its mechanisms.

Methods: A series of genistein analogues were prepared by difluoromethylation and alkylation, and human gastric cancer cell lines AGS and SGC-7901 cultured in vitro were treated with various concentrations of genistein and genistein analogues. The cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were incubated by DFOG at different concentrations. The growth inhibitory effects were evaluated using MTT and clonogenic assay. The distribution of the phase in cell cycle was analyzed using flow cytometric analysis with propidium iodide staining. The expression of the transcription factor forkhead box M1 (FOXM1) was analyzed by reverse transcription-polymerase chain reaction and Western blotting. The expression levels of CDK1, Cdc25B, cyclin B and p27(KIP1) protein were detected using Western blotting.

Results: Nine of the genistein analogues had more effective antitumor activity than genistein. Among the tested analogues, DFOG possessed the strongest activity against AGS and SGC-7901 cells in vitro. DFOG significantly inhibited the cell viability and colony formation of AGS and SGC-7901 cells. Moreover, DFOG efficaciously arrested the cell cycle in G2/M phase. DFOG decreased the expression of FOXM1 and its downstream genes, such as CDK1, Cdc25B, cyclin B, and increased p27(KIP1) at protein levels. Knockdown of FOXM1 by small interfering RNA before DFOG treatment resulted in enhanced cell growth inhibition in AGS cells. Up-regulation of FOXM1 by cDNA transfection attenuated DFOG-induced cell growth inhibition in AGS cells.

Conclusion: DFOG inhibits the growth of human gastric cancer cells by down-regulating the FOXM1 expression.

Keywords: 7-difluoromethoxyl-5,4’-di-n-octylgenistein; Forkhead box M1; Gastric cancer; Genistein; Therapeutic action.

Figure 1

Inhibition of cell viability by genistein and genistein analogues. A: AGS cell line; B: SGC-7901 cell line. ^aP < 0.05 vs treatment with genistein; ^cP < 0.05 vs treatment with genistein or other genistein analogues. 1: Genistein (5,7,4'-trihydroxylisoflavone); 2: 7-difluoromethyl genistein; 4a: 7-difluoromethyl-5,4'-dimethyl genistein; 4b: 7-difluoromethyl-5,4'-diethyl genistein; 4c: 7-difluoromethyl-5,4’-di-n-propyl genistein; 4d: 7-difluoromethyl-5,4’-di-benzyl genistein; 4e: 7-difluoromethyl-5,4'-diheptyl genistein; 4f: 7-difluoromethyl-5,4'-di-n-octyl genistein; 4g: 7-difluoromethyl-5,4'-didecyl genistein; 4h: 7-difluoromethyl-5,4'-diisobutyl genistein.

Figure 2

Decrease of colony number and inhibition of colony formation by 7-difluoromethoxyl-5,4’-di-n-octylgenistein. A: Decrease of colony number by 7-difluoromethoxyl-5,4’-di-n-octylgenistein (DFOG); B: Inhibition of colony formation by DFOG and genistein in AGS cell line. ^aP < 0.05 vs treatment with dimethyl sulfoxide (DMSO); ^cP < 0.05 vs treatment with 10 μmol/L genistein (GEN) or 1 μmol/L DFOG.

Figure 3

Increase of cells in G2/M phase and induction of cell cycle arrest in G2/M phase by 7-difluoromethoxyl-5,4’-di-n-octylgenistein. A: Increase of cells in G2/M phase by 7-difluoromethoxyl-5,4’-di-n-octylgenistein (DFOG); B: Induction of cell cycle arrest in G2/M phase by DFOG and genistein in AGS cell line. ^aP < 0.05 vs treatment with dimethyl sulfoxide (DMSO); ^cP < 0.05 vs treatment with 10 μmol/L genistein (GEN) or 1 μmol/L DFOG.

Figure 4

Down-regulation of forkhead box M1 expression by 7-difluoromethoxyl-5,4’-di-n-octylgenistein and genistein in AGS and SGC-7901. A: mRNA level using reverse transcription-polymerase chain reaction in AGS cell line; B: Protein level using Western blotting in AGS cell line; C: Decrease of forkhead box M1 (FOXM1) protein expression in SGC-7901 cell line. DMSO: Dimethyl sulfoxide; GEN: Genistein; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 5

Modulation of the protein expressions of forkhead box M1 downstream target genes by 7-difluoromethoxyl-5,4’-di-n-octylgenistein and genistein. A: AGS cell line; B: SGC-7901 cell line. DMSO: Dimethyl sulfoxide; GEN: Genistein; DFOG: 7-difluoromethoxyl-5,4’-di-n-octylgenistein.

Figure 6

Forkhead box M1 small interfering RNA enhances the down-regulation of forkhead box M1 protein expression by 7-difluoromethoxyl-5,4’-di-n-octylgenistein and effects of forkhead box M1 small interfering RNA transfection or 7-difluoromethoxyl-5,4’-di-n-octylgenistein in AGS cell line. A: Western blotting analysis; B: The cell viability inhibitory effects of 7-difluoro methoxyl-5,4’-di-n-octylgenistein (DFOG) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte trazolium bromide assay; C: Both on cell cycle distribution in AGS cells. ^aP < 0.05 vs treatment with dimethyl sulfoxide (DMSO); ^cP < 0.05 vs treatment with 5 μmol/L DFOG or forkhead box M1 (FOXM1) small interfering RNA (siRNA) alone.

Figure 7

Forkhead box M1 cDNA transfection reduces the down-regulation of forkhead box M1 protein expression by 7-difluoromethoxyl-5,4’-di-n-octylgenistein in AGS cell line. A: Western blotting analysis; B: The cell viability inhibitory effects of 7-difluoro methoxyl-5,4’-di-n-octylgenistein (DFOG) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte trazolium bromide assay. ^aP < 0.05 vs treatment with dimethyl sulfoxide (DMSO); ^cP < 0.05 vs treatment with 5 μmol/L DFOG or forkhead box M1 (FOXM1) cDNA transfection alone.