Extracts and components of Ficus carica leaves suppress survival, cell cycle, and migration of triple-negative breast cancer MDA-MB-231 cells

Abstract

Background: Products from Ficus carica have been used in traditional medicine to treat many diseases. This study aimed to analyze anticancer effects of extracts of F. carica leaves on the triple-negative breast cancer cell line MDA-MB-231.

Materials and methods: The human breast cancer cell line MDA-MB-231 was used to evaluate effects of F. carica extracts. Effects of F. carica on cell viability were evaluated using MTT assays. Cell-cycle distribution was examined using cell-cycle analysis. Wound-healing assays were used to evaluate migration of MDA-MB-231. Quantitative reverse-transcription polymerase chain reaction was used to detect levels of Bax, p53, p21, GATA3, ELF5, cyclin-dependent kinases, MMP2, and tissue inhibitors of metalloproteinase.

Results: We investigated the mechanism of anti-growth effects, and found that the expressions of genes that promote apoptosis were increased. In addition, the treated cells illustrated increased portion at S phase and changed expression of cyclin-dependent kinases, demonstrating cell-cycle arrest at the S phase. Furthermore, treated cells showed decreased cell mobility, which is essential for metastasis. Two of the active components of F. carica leaves, bergapten and psoralen, had similar anticancer effects as F. carica leaf extracts, indicating that these two components might play important roles in anticancer effects of F. carica leaves.

Conclusion: Our findings suggest that F. carica leaves might be a good source to develop drugs for suppressing cancer-cell growth and migration to treat triple-negative breast cancers.

Keywords: Ficus carica; MDA-MB-231; breast cancer; migration.

Figure 1

Extracts of Ficus carica leaves, psoralen, and bergapten inhibited the proliferation of MDA-MB-231 cells. Notes: (A) MDA-MB-231 and (B) MCF10A cells were treated with extracts of F. carica leaves (0, 1, 2, 4, 8 mg/mL) at different time points (24, 48, 72 hours). Viability of treated cells evaluated by MTT assays. (C) Chemical structure of psoralen. (D) Chemical structure of bergapten. (E) MDA-MB-231 cells were treated with different concentrations of psoralen (0, 20, 40, 60, 80, 100 μg/mL) and (F) bergapten (0, 5, 10, 15, 20, 25, 30 μg/mL) at different time points (24 hours, 48 hours, 72 hours). Viability of treated cells evaluated by MTT assays. Viability of control cells at 24 hours represented as 100%. Data presented as mean ± SD (n=3). *P<0.01; **P<0.05; ***P<0.001.

Figure 2

Extracts of Ficus carica leaves affected the expression of genes that regulate cell viability. Notes: (A, B) MDA-MB-231 cells were treated with different concentrations of extracts of F. carica leaves (0, 4, 8 mg/mL) at different time points (48, 72 hours). Expression of key genes involved in regulation of cell viability measured by quantitative polymerase chain reaction. (C, D) MDA-MB-231 cells were treated with different concentrations of extracts of F. carica leaves (0, 4, 8 mg/mL) at different time points (48, 72 hours). Expression of key genes of breast cancer biomarkers measured by quantitative polymerase chain reaction. Data presented as mean ± SD (n=3). *P<0.01; **P<0.05; ***P<0.001.

Figure 3

Extracts of Ficus carica leaves induced cell-cycle arrest. Notes: (A) Cells were incubated with extracts of F. carica leaves at concentrations of 0 or 4 mg/mL for 48 hours or 72 hours, stained with propidium iodide, and numbers of cells at different phases of cell cycle analyzed by flow cytometry. (B) Percentage of cells at different phases of cell cycle shown. Reverse-transcription polymerase chain-reaction analysis showed decreased expression of genes in CDK family in MDA-MB-231 upon treatment with extracts of F. carica leaves for 48 hours (C) or 72 hours (D). Data presented mean ± SD (n=3). *P<0.01; **P<0.05.

Figure 4

Extracts of Ficus carica leaves, psoralen, and bergapten suppressed migration of MDA-MB-231 cells. Notes: (A) MDA-MB-231 cells were treated with 1 mg/mL extracts of F. carica leaves and mobility of MDA-MB-231 cells evaluated by wound-healing assays. After treatment for indicated times (0, 24, or 48 hours), cell migration in the denuded zone was photographed. Representative images of migrated cells shown. Scale bars represent 100 μm (B) Relative migration of the treated MDA-MB-231 cells was analyzed. Data presented as mean ± SD (n=9, ***P<0.001). (C) MDA-MB-231 cells were treated with 2 or 8 mg/mL extracts of F. carica leaves for 48 hours and expression levels of MMP2, TIMP1, and TIMP2 measured by reverse-transcription polymerase chain reaction. Data presented as mean ± SD (n=3, *P<0.05, **P<0.01, ***P<0.001). (D) MDA-MB-231 cells were treated with 20 μg/mL psoralen for indicated time points (0, 24, or 48 hours), and cell migration in the denuded zone was photographed. Representative images of migrated cells are shown. Scale bars represent 100 μm (E) Relative migration level of psoralen-treated MDA-MB-231 cells was analyzed. Data presented as mean ± SD (n=9, *P<0.05, **P<0.01). (F) MDA-MB-231 cells were treated with 5 μg/mL bergapten for indicated times (0, 24, or 48 hours) and cell migration in the denuded zone photographed. Representative images of migrated cells are shown. Scale bars represent 100 μm (G) Relative migration level of bergapten-treated MDA-MB-231 cells was analyzed. Data presented as mean ± SD (n=9, *P<0.05).

Figure 4

Extracts of Ficus carica leaves, psoralen, and bergapten suppressed migration of MDA-MB-231 cells. Notes: (A) MDA-MB-231 cells were treated with 1 mg/mL extracts of F. carica leaves and mobility of MDA-MB-231 cells evaluated by wound-healing assays. After treatment for indicated times (0, 24, or 48 hours), cell migration in the denuded zone was photographed. Representative images of migrated cells shown. Scale bars represent 100 μm (B) Relative migration of the treated MDA-MB-231 cells was analyzed. Data presented as mean ± SD (n=9, ***P<0.001). (C) MDA-MB-231 cells were treated with 2 or 8 mg/mL extracts of F. carica leaves for 48 hours and expression levels of MMP2, TIMP1, and TIMP2 measured by reverse-transcription polymerase chain reaction. Data presented as mean ± SD (n=3, *P<0.05, **P<0.01, ***P<0.001). (D) MDA-MB-231 cells were treated with 20 μg/mL psoralen for indicated time points (0, 24, or 48 hours), and cell migration in the denuded zone was photographed. Representative images of migrated cells are shown. Scale bars represent 100 μm (E) Relative migration level of psoralen-treated MDA-MB-231 cells was analyzed. Data presented as mean ± SD (n=9, *P<0.05, **P<0.01). (F) MDA-MB-231 cells were treated with 5 μg/mL bergapten for indicated times (0, 24, or 48 hours) and cell migration in the denuded zone photographed. Representative images of migrated cells are shown. Scale bars represent 100 μm (G) Relative migration level of bergapten-treated MDA-MB-231 cells was analyzed. Data presented as mean ± SD (n=9, *P<0.05).