Suppressive Effects of Plumbagin on Invasion and Migration of Breast Cancer Cells via the Inhibition of STAT3 Signaling and Down-regulation of Inflammatory Cytokine Expressions

Abstract

Objective: The aim of this study was to investigate the effects of plumbagin (PL), a naphthoquinone derived from the medicinal plant plumbago zeylanica, on the invasion and migration of human breast cancer cells.

Methods: Human breast cancer MDA-MB-231SArfp cells were treated with different concentrations of plumbagin for 24 h. The effects of plumbagin on the migration and invasion were observed by a transwell method. The expressions of IL-1α, IL-1β, IL-6, IL-8, TGF-β, TNFα, MMP-2 and MMP-9 mRNA in MDA-MB-231SArfp cells were detected using Real-Time PCR. MDA-MB-231SArfp cells were treated with plumbagin at different concentrations for 45 minutes. The activation of STAT3 was detected by western blot. Following this analysis, STAT3 in MDA-MB-231SArfp cells was knocked out using specific siRNA. mRNA levels of IL-1α, TGF-β, MMP-2 and MMP-9 were then detected. Consequently, MDA-MB-231SArfp cells were injected intracardially into BALB/c nude mice to construct a breast cancer bone metastatic model. The mice were injected intraperitoneally with plumbagin. Non-invasive in vivo monitoring, X-ray imaging and histological staining were performed to investigate the effects of plumbagin on the invasion and migration of breast cancer cells in vivo.

Results: The in vitro results showed that plumbagin could suppress the migration and invasion of breast cancer cells and down-regulate mRNA expressions of IL-1α, TGF-β, MMP-2 and MMP-9. Western blotting demonstrated that plumbagin inhibited the activation of STAT3 signaling in MDA-MB-231SArfp cells. The inactivation of STAT3 was found to have an inhibitory effect on the expressions of IL-1α, TGF-β, MMP-2 and MMP-9. In vivo studies showed that plumbagin inhibited the metastasis of breast cancer cells and decreased osteolytic bone metastases, as well as the secretion of MMP-2 and MMP-9 by tumor cells at metastatic lesions.

Conclusions: Plumbagin can suppress the invasion and migration of breast cancer cells via the inhibition of STAT3 signaling and by downregulation of IL-1α, TGF-β, MMP-2 and MMP-9.

Keywords: breast cancer; invasion; migration; plumbagin.

Figure 1

Effects of plumbagin on the invasion and migration of breast cancer cells. (A and C) Plumbagin (2.5-10 μmol·L⁻¹) suppresses in vitro migration of MDA-SA-231SArfp cells at different doses. (B and D) Plumbagin (2.5-10 μmol·L⁻¹) decreases the invasion of MDA-SA-231SArfp cells and tumor cells show little penetration into matrigel at a concentration above 5 μmol·L⁻¹.

Figure 2

Effects of plumbagin on mRNA expressions of cytokines secreted from breast cancer cells. After treatment of plumbagin at 5 μmol·L-1 and 10 μmol·L-1 for 24 h, mRNA expressions of IL-1α and TGF-β secreted from MDA-SA-231SArfp as well as MMP-2 and MMP-9. mRNA expression of IL-6, however, showed a decrease without statistical significance.

Figure 3

PL inhibits the activation of STAT3 in breast cancer cells. (A) MDA-SA-231SArfp cells were exposed to 7.5 μmol·L⁻¹ PL for 45 min and STAT3 activation was detected by western blotting. (B) Western blot analysis of p-STAT3 and STAT3 protein levels in extracts from MDA-SA-231SArfp treated with PL for the indicated times. (C) MDA-SA-231SArfp was treated with STAT3 siRNA or control siRNA for 48 h, and the expressions of IL-1α, TGF-β, MMP-2 and MMP-9 mRNAs were detected by Real-Time PCR. The results are expressed as the mean ± SD at *P<0.05.

Figure 4

Effects of plumbagin on metastases of mammary cancer in nude mice. (A) Few mice exhibited weak fluorescence signals in the plumbagin treatment group during the fourth week. An increasing number of metastatic sites in systematic skeletons and organs were observed from the fourth to seventh weeks per vehicle group. However, in PL treatment cases, the number and intensity of fluorescence signals of metastatic sites were both less than the vehicle group. (B) Quantification of luminescence activities in the two groups at *P<0.05.

Figure 5

Effects of plumbagin on osteolytic lesions of breast cancer bone metastases. (A) Systematic skeleton X-ray imaging. In the vehicle group, a number of osteolytic lesions were observed in nude mice; in the PL treatment group, fewer osteolytic lesions were exhibited with lesion areas smaller than the vehicle. (B) HE staining of knee joints of nude mice. In the vehicle group, destruction of bone tissue in knee joints was obvious with the disappearance of joint structures and few remaining articular cartilage areas in the tibia. However, intact articular structure and fewer destruction of bone tissues were observed in the PL treatment group with small lumps of tumor tissue merely visible in the canal.

Figure 6

Effects of plumbagin on secretions of MMP-2 and MMP-9 in breast cancer cells. (A) immunohistochemistry staining, where fewer numbers of MMP-2 staining-positive breast cancer cells are shown in the PL treatment group than the vehicle. Tumor cells without treatment expressed high levels of MMP-9, which accounts for approximately 50% of all tumor cells. By contrast, MMP-9 staining-positive breast cancer cells by PL treatment decreased markedly. (B-C) Quantification of MMP-2 and MMP-9 positive cells in the tumor tissues from the two groups.