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. 2016 Jun;21(2):88-94.
doi: 10.15430/JCP.2016.21.2.88. Epub 2016 Jun 30.

The Inhibitory Effects of Forsythia Koreana Extracts on the Metastatic Ability of Breast Cancer Cells and Bone Resorption by Osteoclasts

Yu Li Kim  1 , Sun Kyoung Lee  2 , Kwang-Kyun Park  1 , Won-Yoon Chung  1
Affiliations

The Inhibitory Effects of Forsythia Koreana Extracts on the Metastatic Ability of Breast Cancer Cells and Bone Resorption by Osteoclasts

Yu Li Kim et al. J Cancer Prev. 2016 Jun.

Abstract

Background: Breast cancer is the most common malignant disease in women. The patients with advanced breast cancer develop metastasis to bone. Bone metastasis and skeletal-related events by breast cancer are frequently associated with the invasiveness of breast cancer cells and osteoclasts-mediated bone resorption. Forsythia koreana is used in oriental traditional medicine to treat asthma, atopy, and allergic diseases. The aim of this study was to evaluate the inhibitory effects of F. koreana extracts on the invasion of breast cancer cells and bone resorption by osteoclasts.

Methods: Cell viability was measured by an MTT assay and the migration and invasion of MDA-MB-231 cells were detected by a Boyden chamber assay. The formation of osteoclasts and pit was detected using tartrate-resistant acid phosphatase staining and calcium phosphate-coated plates, respectively. The activities of matrix metalloproteinases (MMPs) and cathepsin K were evaluated by gelatin zymography and a cathepsin K detection kit.

Results: The fruit and leaf extracts of F. koreana significantly inhibited the invasion of MDA-MB-231 cells at noncytotoxic concentrations. The fruit extract of F. koreana reduced the transforming growth factor β1-induced migration, invasion and MMPs activities of MDA-MB-231 cells. In addition, the fruit, branch, and leaf extracts of F. koreana also inhibited the receptor activator of nuclear factor kappa-B ligand-induced osteoclast formation and osteoclast-mediated bone-resorbing activity by reducing the activities of MMPs and cathepsin K.

Conclusions: The extracts of F. koreana may possess the potential to inhibit the breast cancer-induced bone destruction through blocking invasion of breast cancer cells, osteoclastogenesis, and the activity of mature osteoclasts.

Keywords: Bone metastasis; Bone resorption; Breast neoplasm; Forsythia koreana; Osteoclast.

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Figures

Figure 1.
Figure 1.
The effects of Forsythia koreana extracts on the viability and invasion of MDA-MB-231 cells. MDA-MB-231 cells were treated with indicated concentrations of F. koreana (A) fruit, (B) branch, and (C) leaf extracts for 24 hours. The cell viability was determined by an MTT assay. (D) MDA-MB-231 cells were placed on the Matrigel-coated upper chambers with serum-free Dulbecco’s modified Eagle medium (DMEM) containing F. koreana extracts. The lower chambers were filled with 600 μL of 5% FBS-DMEM and F. koreana extracts. Twenty-four hours later, the invaded cells were counted. Data are expressed mean ± SE. *P < 0.05, **P < 0.01 vs. untreated cells.
Figure 2.
Figure 2.
The effects of Forsythia koreana fruit extract on TGF-β1-induced migration and invasion of MDA-MB-231 cells. (A) MDA-MB-231 cells were placed on the upper chambers with serum-free Dulbecco’s modified Eagle medium (DMEM) containing F. koreana fruit extract and TGF-β1 (10 ng/mL). The lower chambers were filled with 600 μL of 5% FBS-DMEM containing F. koreana fruit extract. The cells were incubated for 6 hours. (B) MDA-MB-231 cells were placed on the Matrigel-coated upper chambers with serum-free DMEM containing F. koreana fruit extract and TGF-β1 (10 ng/mL). The lower chambers were filled with 600 μL of 5% FBS-DMEM containing F. koreana fruit extracts. The cells were incubated for 24 hours. The migrated or invaded cells were fixed, stained, and counted as a described in Materials and Methods. Data are expressed mean ± SE. #P < 0.05, ##P < 0.01 vs. untreated cells, *P < 0.05, **P < 0.01 vs. MDA-MB-231 cells with TGF-β1 alone. (C) MDA-MB-231 cells were cultured with F. koreana fruit extract and 1% FBS-DMEM in the absence or presence of TGF-β1 (10 ng/mL) for 24 hours. The levels of matrix metalloproteinase (MMP)-2 and MMP-9 in the collected medium were examined using gelatin zymography. The clear bands indicate the activities of MMPs.
Figure 3.
Figure 3.
The effects of Forsythia koreana extracts on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast formation. The bone marrow-derived macrophages (BMMs) were treated with M-CSF (30 ng/mL) and indicated concentrations of F. koreana (A) fruit, (B) branch, or (C) leaf extracts for 5 days. The cell viability was determined by an MTT assay. Data are expressed as the mean ± SE. *P < 0.05, **P < 0.001 vs. untreated cells. (D) The BMMs were cultured in α-minimum essential medium with M-CSF (30 ng/mL), RANKL (100 ng/mL) and F. koreana extracts at the indicated concentrations for 5 days. The cells were detected by tartrate-resistant acid phosphatase staining and osteoclasts were counted (× 100, magnification). Data are expressed as the mean ± SE. #P < 0.05 vs. BMMs without RANKL (C, control). *P <0.05, **P < 0.001 vs. BMMs with RANKL alone.
Figure 4.
Figure 4.
The effects of Forsythia koreana extracts on bone resorbing-activity of osteoclasts. Bone marrow-derived macrophages (BMMs) were seeded in Osteo assay plate and treated with M-CSF (30 ng/mL) and receptor activator of nuclear factor kappa-B ligand (RANKL) (100 ng/mL) for 5 days. After induction of osteoclasts, the cells were treated with F. koreana extracts for an additional 2 days. (A) The resorption pits were observed under a light microscopy (× 100, magnification). (B) The activities of matrix metalloproteinase (MMP)-2 and MMP-9 in cultured media of osteoclasts were detected by gelatin zymography as described in Materials and Methods. (C) Cathepsin K levels in cultured media of osteoclasts were measured using the Sensizyme Cathepsin K activity kit. Data are expressed as the mean ± SE. #P < 0.05 vs. BMMs without RANKL (C, control). *P < 0.05, **P < 0.001 vs. BMMs with RANKL alone.
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