Figure 1.
Isoliquiritigenin (ISL) at a noncytotoxic concentration suppresses ovarian cancer cell epithelial-to-mesenchymal transition (EMT) traits. (A) Chemical structure of ISL. (B) The cell viability of SKOV3, OVCAR5, ES2, and TOV21G cells treated with ISL (2, 4, 8, 16, 32, 64, and 100 μM) for 72 h was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (C) Observed cell morphology of cells treated with ISL (10 μM) for 72 h; 100× magnification. Scale bars, 25 μm. (D) Cells were treated with ISL (1, 5, and 10 μM) for 72 h and analyzed by Western blotting. (E) Bar graph shows results of quantitative analysis of Western blotting. Protein expression is presented as fold changes and normalized to β-actin. Data are presented as mean ± SD, n = 3. Student t-test was used for statistical tests. * p < 0.05, ** p < 0.01 compared with the control group.
Figure 1.
Isoliquiritigenin (ISL) at a noncytotoxic concentration suppresses ovarian cancer cell epithelial-to-mesenchymal transition (EMT) traits. (A) Chemical structure of ISL. (B) The cell viability of SKOV3, OVCAR5, ES2, and TOV21G cells treated with ISL (2, 4, 8, 16, 32, 64, and 100 μM) for 72 h was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (C) Observed cell morphology of cells treated with ISL (10 μM) for 72 h; 100× magnification. Scale bars, 25 μm. (D) Cells were treated with ISL (1, 5, and 10 μM) for 72 h and analyzed by Western blotting. (E) Bar graph shows results of quantitative analysis of Western blotting. Protein expression is presented as fold changes and normalized to β-actin. Data are presented as mean ± SD, n = 3. Student t-test was used for statistical tests. * p < 0.05, ** p < 0.01 compared with the control group.
Figure 2.
ISL inhibits the migration and invasion of SKOV3 and OVCAR5 cells. (A) Cells were grown to confluence, followed by treatment with vehicle or ISL (1, 5, and 10 μM) for 24 h. A scratch was made with a fine pipette tip and cells were kept in medium containing 2% FBS with or without ISL. Images were taken at 0 and 24 h under a phase-contrast microscope. (B) Photomicrographs of migration to the lower side of chamber. Cells were pretreated with vehicle or ISL (1, 5, and 10 μM) for 48 h, followed by analysis of cell migration using a transwell assay. Bar graph shows results of quantitative analysis of migration. The number of stained cells in five randomly selected fields was counted. (C) Photomicrographs of invasion to the lower side of chamber. Cells were pretreated with vehicle or ISL (1, 5, and 10 μM) for 48 h, followed by analysis with a cell Matrigel invasion assay. Bar graph shows the results of quantitative analysis of invasion. The number of stained cells in five randomly selected fields was counted. Data are presented as mean ± SD. n = 3. 100× magnification. Scale bars, 25 μm. Student t-test was used for statistical tests. * p < 0.05, ** p < 0.01 compared with the control group.
Figure 2.
ISL inhibits the migration and invasion of SKOV3 and OVCAR5 cells. (A) Cells were grown to confluence, followed by treatment with vehicle or ISL (1, 5, and 10 μM) for 24 h. A scratch was made with a fine pipette tip and cells were kept in medium containing 2% FBS with or without ISL. Images were taken at 0 and 24 h under a phase-contrast microscope. (B) Photomicrographs of migration to the lower side of chamber. Cells were pretreated with vehicle or ISL (1, 5, and 10 μM) for 48 h, followed by analysis of cell migration using a transwell assay. Bar graph shows results of quantitative analysis of migration. The number of stained cells in five randomly selected fields was counted. (C) Photomicrographs of invasion to the lower side of chamber. Cells were pretreated with vehicle or ISL (1, 5, and 10 μM) for 48 h, followed by analysis with a cell Matrigel invasion assay. Bar graph shows the results of quantitative analysis of invasion. The number of stained cells in five randomly selected fields was counted. Data are presented as mean ± SD. n = 3. 100× magnification. Scale bars, 25 μm. Student t-test was used for statistical tests. * p < 0.05, ** p < 0.01 compared with the control group.

Figure 3.
Effects of ISL on the expression of E-cadherin, Vim, N-cadherin, Twist1, ZEB1, and ZEB2 in SKOV3 and OVCAR5 cells. (A) Cells were treated with vehicle or ISL (1, 5, and 10 μM) for 72 h, followed by qRT-PCR analysis. Gene expression is presented as fold changes and normalized to β-actin. (B) Cells were treated with ISL (1, 5, and 10 μM) for 72 h and analyzed by Western blotting. The bar graph shows results of a quantitative data of Western blotting. Protein expression is presented as fold changes and normalized to GAPDH. Data are presented as mean ± SD. n = 3. Student t-test was used for statistical tests. * p < 0.05, ** p < 0.01 compared with the control group.
Figure 3.
Effects of ISL on the expression of E-cadherin, Vim, N-cadherin, Twist1, ZEB1, and ZEB2 in SKOV3 and OVCAR5 cells. (A) Cells were treated with vehicle or ISL (1, 5, and 10 μM) for 72 h, followed by qRT-PCR analysis. Gene expression is presented as fold changes and normalized to β-actin. (B) Cells were treated with ISL (1, 5, and 10 μM) for 72 h and analyzed by Western blotting. The bar graph shows results of a quantitative data of Western blotting. Protein expression is presented as fold changes and normalized to GAPDH. Data are presented as mean ± SD. n = 3. Student t-test was used for statistical tests. * p < 0.05, ** p < 0.01 compared with the control group.
Figure 4.
ISL blocked intraperitoneal xenograft development and prolonged the survival of ovary-tumor-bearing mice. (A) Flowchart of therapy scheme. (B) Luciferase-containing SKOV3 cells (5 × 106 cells/mouse) were injected intraperitoneally into nude mice for 3 weeks, followed by administration of 12.5 or 25 mg/kg of ISL. Tumors were imaged in the Xenogen system. (C) Kaplan–Meier analysis of animal endpoint survival following treatment with ISL. (D) Body weight was measured every week. (E) The mRNA expression of E-cad, Vim, and ZEB1 was detected in tumor tissues. Gene expression is presented as fold changes and normalized to β-actin. (F) Representative pictures of immunohistochemical staining of E-cadherin, Vim, and ZEB1 in tumor tissues. Scale bars, 20 μm. Data are presented as mean ± SD, n = 6. Student t-test was used for statistical tests. * p < 0.05, ** p < 0.01 compared with the vehicle group.
Figure 4.
ISL blocked intraperitoneal xenograft development and prolonged the survival of ovary-tumor-bearing mice. (A) Flowchart of therapy scheme. (B) Luciferase-containing SKOV3 cells (5 × 106 cells/mouse) were injected intraperitoneally into nude mice for 3 weeks, followed by administration of 12.5 or 25 mg/kg of ISL. Tumors were imaged in the Xenogen system. (C) Kaplan–Meier analysis of animal endpoint survival following treatment with ISL. (D) Body weight was measured every week. (E) The mRNA expression of E-cad, Vim, and ZEB1 was detected in tumor tissues. Gene expression is presented as fold changes and normalized to β-actin. (F) Representative pictures of immunohistochemical staining of E-cadherin, Vim, and ZEB1 in tumor tissues. Scale bars, 20 μm. Data are presented as mean ± SD, n = 6. Student t-test was used for statistical tests. * p < 0.05, ** p < 0.01 compared with the vehicle group.
Table 1.
Expression of EMT-Associated Genes in untreated (control) vs ISL-treated SKOV3 cells (ISL).
Gene | Control | ISL | Gene | Control | ISL | Gene | Control | ISL |
---|
AHNAK | 1 | 0.734623 | IL1RN | 1 | 0.418554 | SMAD2 | 1 | 0.572358 |
AKT1 | 1 | 0.924291 | ILK | 1 | 0.785611 | SNAI1 | 1 | 1.566291 |
BMP1 | 1 | 0.404673 | ITGA5 | 1 | 0.535606 | SNAI2 | 1 | 1.131232 |
BMP2 | 1 | 0.743396 | ITGAV | 1 | 0.513642 | SNAI3 | 1 | 2.443003 |
BMP7 | 1 | 0.31247 | ITGB1 | 1 | 0.594326 | SOX10 | 1 | 0.510887 |
CALD1 | 1 | 0.419532 | JAG1 | 1 | 0.435286 | SPARC | 1 | 1.120049 |
CAMK2N1 | 1 | 0.767472 | KRT14 | 1 | 1.161878 | SPP1 | 1 | 4.084098 |
CAV2 | 1 | 0.53588 | KRT19 | 1 | 0.717957 | STAT3 | 1 | 0.572006 |
CDH1 | 1 | 1.068836 | KRT7 | 1 | 0.497813 | STEAP1 | 1 | 0.182371 |
CDH2 | 1 | 0.296297 | MAP1B | 1 | 0.413056 | TCF3 | 1 | 1.037395 |
COL1A2 | 1 | 0.373447 | MMP2 | 1 | 1.518571 | TCF4 | 1 | 0.875013 |
COL3A1 | 1 | 0.374667 | MMP3 | 1 | 0.474877 | TFPI2 | 1 | 0.403746 |
COL5A2 | 1 | 0.668282 | MMP9 | 1 | 0.924883 | TGFB1 | 1 | 0.583945 |
CTNNB1 | 1 | 0.573468 | MSN | 1 | 1.18802 | TGFB2 | 1 | 0.394966 |
DSC2 | 1 | 0.948605 | MST1R | 1 | 0.764398 | TGFB3 | 1 | 1.295126 |
DSP | 1 | 0.463705 | NODAL | 1 | 1.54975 | TIMP1 | 1 | 1.09639 |
EGFR | 1 | 0.396217 | NOTCH1 | 1 | 0.595044 | TMEFF1 | 1 | 0.619747 |
ERBB3 | 1 | 0.34008 | NUDT13 | 1 | 0.524084 | MEM132A | 1 | 0.890626 |
ESR1 | 1 | 0.696109 | OCLN | 1 | 0.350573 | TSPAN13 | 1 | 1.582411 |
F11R | 1 | 0.662682 | PDGFRB | 1 | / | TWIST1 | 1 | 0.811606 |
FN1 | 1 | 0.797309 | PLEK2 | 1 | 1.065871 | VCAN | 1 | 0.987891 |
FOXC2 | 1 | 0.659119 | PPPDE2 | 1 | 0.419386 | VIM | 1 | 1.056343 |
FZD7 | 1 | 0.748245 | PTK2 | 1 | 0.654937 | VPS13A | 1 | 0.762398 |
GNG11 | 1 | 0.770319 | PTP4A1 | 1 | 0.389229 | WNT11 | 1 | 2.137225 |
GSC | 1 | 1.215896 | RAC1 | 1 | 0.782444 | WNT5A | 1 | 1.517202 |
GSK3B | 1 | 0.321123 | RGS2 | 1 | 1.443939 | WNT5B | 1 | 2.762547 |
IGFBP4 | 1 | 0.614624 | SERPINE1 | 1 | 0.598634 | ZEB1 | 1 | 0.602478 |
| | | SIP1 | 1 | 0.928877 | ZEB2 | 1 | 0.506116 |