Elsevier

International Immunopharmacology

Volume 67, February 2019, Pages 239-247
International Immunopharmacology

Effects of Platycodin D on S100A8/A9-induced inflammatory response in murine mammary carcinoma 4T1 cells

https://doi.org/10.1016/j.intimp.2018.12.008Get rights and content

Highlights

  • S100A8/A9 was increased and released in the pre-metastatic lungs.

  • S100A8/A9 stimulated migration and invasion ability of 4T1 cells.

  • Pla D attenuated S100A8/A9-induced growth, migration, and invasion of 4T1 cells.

  • Pla D inhibited S100A8/A9-induced inflammatory response in 4T1 cells.

  • Pla D suppressed IL-6, IL-1β, and TNF-α via inhibition of NF-κB pathways.

Abstract

Activation of the inflammatory signaling pathway is the most vital part of the pre-metastatic events of breast cancer. Platycodin D (PlaD) shows favorable pharmacological activities in anti-inflammatory and anti-tumor effect. The main purpose of this study was to survey the effects of PlaD on S100A8/A9-induced inflammation in mouse mammary carcinoma 4T1 cells. S100A8/A9 immunolocalization and expression in pre-metastatic lung tissue were assessed by immunofluorescence staining and ELISA. 4T1 cells were treated with 2.5 μg/mL recombinant S100A8/A9 heterodimer and 7.5, 10, or 12.5 μM of PlaD. After 24 h of incubation, cell viability, migration, and invasion were evaluated by CCK-8, wound-healing, and transwell assay, respectively. Nuclear translocation of NF-κB p65 was determined by immunostaining and western blot. The levels of pro-inflammatory cytokines including IL-1β, IL-6, and TNF-α were detected by ELISA. The results showed that S100A8/A9 was actively increased and released into the extracellular space during the pre-metastatic phase of breast cancer. PlaD treatment attenuated S100A8/A9-induced growth, migration, and invasion of 4T1 cells. Furthermore, PlaD decreased the levels of IL-1β, IL-6, and TNF-α by inhibiting nuclear translocation of NF-κB p65. In conclusion, this study demonstrated that PlaD inhibited S100A8/A9-induced inflammatory response in 4T1 cells by suppressing the expression of IL-6, IL-1β, and TNF-α via inhibition of NF-κB signaling pathways.

Introduction

Breast cancer, a serious threat to women's health, was the most frequently diagnosed cancer in the female all around the world [1,2]. Distant metastasis, the leading cause of death in patients with breast cancer, includes bone, lung, liver, and brain, among which lung metastasis is one of the most frequent organ metastases [3].

Pre-metastatic niche acts a pivotal part in tumor metastasis to specific organs. Primary tumor and target organs work together to establish the pre-metastatic niche, promoting the process of lung metastasis [4,5]. A recent study has shown that neutrophils in bone marrow-derived cells (BMDCs) create an inflammatory microenvironment that promotes metastasis by inhibiting innate and secondary anti-tumor immune responses [6]. It is believed that vascular endothelial growth factor (VEGF), macrophage-colony stimulating factor (M-CSF), and tumor necrosis factor α (TNF-α), which are secreted by the melanoma before the tumor metastases to the lung, induced inflammatory cell aggregation in the pre-metastatic niche [7]. Similarly, our previous study showed that lymphocyte mass existed in lung tissue before metastasis, indicating that lymphocytes were associated with the establishment of the pre-metastatic niche [8].

In summary, activation of the inflammatory signaling pathways is one of the essential pre-metastatic events. S100A8, S100A9, and S100A8/A9 heterodimers are calcium-binding proteins that play extracellular pro-inflammatory functions and participate in the entire process of pre-metastatic events [[9], [10], [11]]. During the pre-metastasis period, the expression of S100A8 and S100A9 in the lung was significantly increased under host effect. These factors recruit a large number of BMDCs to gather in the lung before metastasis and form a pre-metastatic microenvironment similar to an inflammatory state, which is suitable for tumor cell adhesion and infiltration [12]. The stability of S100A8/A9 heterodimer is better than that of S100A8 and S100A9, so the pro-inflammatory effect of S100A8 and S100A9 is often attributed to their heterodimer [13]. Since the abnormal expression of S100A8/A9 in microenvironment before metastasis, it may contribute to the survival, proliferation, migration, and invasion of circulating tumor cells in lung tissue.

According to the literature reported, as a major triterpene saponin extracted from the roots of Platycodon grandiflorum, Platycodin D (PlaD) shows favorable pharmacological activities in anti-inflammatory and anti-tumor effect [14]. PlaD attenuated interleukin 1β (IL-1β)-induced inflammatory response in osteoarthritis chondrocyte by activating LXRα [15]. PLaD prevented inflammation caused by LPS through the LXRα–ABCA1 signaling pathway, which not only disrupted lipid rafts, but also prevented TLR4 translocation into lipid rafts [14]. Wang's results confirmed that PlaD inhibited IL-13-induced the expression of inflammatory cytokines and mucus in nasal epithelial cells by inhibiting the activation of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways [16]. PlaD potentiated proliferation inhibition and apoptosis induction upon AKT inhibition via feedback blockade in non-small cell lung cancer cells [17]. However, the anti-inflammatory effects of PlaD in the pre-metastatic lung of breast cancer have not been deeply investigated. In the present study, we sought to explore the effects of PlaD on S100A8/A9-induced inflammation in murine mammary carcinoma 4T1 cells.

Section snippets

Compounds

PlaD (purity ≥ 98%, HPLC grade) was obtained from Sichuan Weikeqi Biological Technology Co., Ltd (Sichuan, China). The structure of PlaD is presented in Fig. 1.

Cell culture

The murine mammary carcinoma 4T1 cell line was purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 medium (Gibco-Invitrogen, NY, USA) containing 10% fetal bovine serum (FBS, Gibco-Invitrogen) at 37 °C in a 5% CO2 humidified chamber.

Animals and experimental pre-metastatic model

Female

The release of S100A8/A9 from the cytosol into the extracellular space in the pre-metastatic lungs

S100A8/A9 is increased significantly in many inflammatory processes, and this heterodimer has been used as an inflammatory biomarker for many years [19]. In the present study, we found the release of S100A8/A9 into the extracellular space in the lungs of 4T1-tumor-bearing mice (Fig. 2A). Extracellular S100A8/A9 was absent or was present at low levels in lungs from non-tumor mice (Fig. 2A). Moreover, S100A8/A9 levels were increased approximately 2.5-fold in tumor mice (p < 0.01) compared to

Discussion

The increase in vascular permeability and activation of inflammatory signaling pathway are the typical initiation events of microenvironment formation of pre-pulmonary metastasis [12]. Our previous study [8] found that pulmonary vascular permeability and capillary telangiectasia were increased in 4T1-tumor-bearing mice compared with normal mice. Besides, we also observed lymphocytic infiltration in the pre-metastatic lung tissues. In the initial stage of inflammation, the activation of

Acknowledgments

The authors are grateful for financial supports from the Natural Science Foundation of Shanghai (No. 17ZR1430700 and 17ZR1430900) and the 2018–2020 Three-year Action Plan for Traditional Chinese Medicine Further Development in Shanghai (No. ZY (2018-2020)-CCCX-2002-10).

Conflict of interest

The authors declare that they have no competing interests.

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