Combination treatment with platycodin D and osthole inhibits cell proliferation and invasion in mammary carcinoma cell lines

https://doi.org/10.1016/j.etap.2013.03.012Get rights and content

Highlights

  • Combination treatment with platycodin D and osthole.

  • The platycodin D–osthole combination inhibits proliferation and invasiveness of mammary carcinoma cells.

  • The platycodin D–osthole combination exhibits perturbations in TGF-β/Smads signaling pathway.

Abstract

In this study, two invasive mammary carcinoma cells (MDA-MB-231 and 4T1) were utilized to evaluate the inhibitory activities of platycodin D, osthole, and the two in combination. The anti-proliferative effect was tested using the MTT and BrdU assay, and the combination of 15 μM osthole and 75 μM platycodin D was used for subsequent analyses. The anti-invasive effect was evaluated by the transwell assay. The results showed that the combination treatment reduced both cell proliferation and invasion. Western blot and real-time PCR revealed that the platycodin D–osthole combination significantly decreased TβRII, Smad2, Smad3 and Smad4 gene or protein expressions, as well as effectively blocked TGF-β-induced phosphorylation of Smad2 and Smad3. Thus, this study demonstrates that the anti-cancer effects of the platycodin D–osthole combination in breast cancer cells involve proliferation inhibition and invasion blockade, both of which may be mediated by perturbations in the TGF-β/Smads pathway.

Introduction

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among females worldwide, accounting for 23% (1.38 million) of total new cancer cases and 14% (458,400) of total cancer deaths in 2008 (Jemal et al., 2011). It is estimated that 30–75% of breast cancer patients will eventually develop recurrent and metastatic disease (Gamucci et al., 2007). Transforming growth factor-beta (TGF-β) belongs to a family of cytokines that regulate various cell behavior, such as proliferation, differentiation, apoptosis, adhesion, and migration (Massagué, 2008). TGF-β can induce epithelial to mesenchymal transition (EMT), a process associated with invasiveness (Moustakas and Heldin, 2007). TGF-β signals predominantly through a receptor complex that is comprised of the type I receptor ALK5 (activin receptor-like kinase 5) and the type II receptor TβRII. TβRII phosphorylates ALK5 upon TGF-β-induced heteromeric complex formation (Shi and Massagué, 2003). Then, activated ALK5 initiates its intracellular canonical signaling pathway by phosphorylating Smad2 and Smad3. These activated Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and act as transcription factors to regulate target gene expression (Schmierer and Hill, 2007, Moustakas and Heldin, 2009). There is ample experimental evidence to support a role for TGF-β/Smads during progression of various types of carcinomas, including breast cancer. Research has verified that disrupting TGF-β signaling in human breast cancer cells, either by ectopic overexpression of mutated Smad2/3 or Smad2/3-binding site mutation in receptors, inhibits invasion and metastasis to the lungs, as shown in rodent models with tail vein injection (McEarchern et al., 2001, Tian et al., 2003). Wiercinska et al. reported that knockdown of Smad3 or Smad4 results in a severe defect in TGF-β-induced three-dimensional spheroid invasion, suggesting a critical role for Smad3 and Smad4 in TGF-β-induced invasion of breast cancer MCF10CA1a cells (Wiercinska et al., 2011).

Platycodon grandiflorum and Cnidium monnieri are plants that grow in China, and are the two key ingredients of the clinical prescription developed by Professor Hanjun Tang (Longhua Hospital, Shanghai, China) who has substantial experience and success in reducing recurrence risk of breast cancer (Sheng et al., 2011, Han et al., 2012). Platycodin D ([(2S,3R,4S,5S)-3-[(2S,3R,4S,5R,6S)-5-[(2S,3R,4S,5R)-4-[(2S,3R,4R)-3,4-dihydroxy-4-(hydroxymethyl)oxolan-2-yl]oxy-3,5-dihydroxyoxan-2-yl]oxy-3,4-dihydroxy-6-methyloxan-2-yl]oxy-4,5-dihydroxyoxan-2-yl](4aR,5R,6aR,6aS,6bR,8aR,10R,11S,12aR,14bS)-5,11-dihydroxy-9,9-bis(hydroxymethyl)-2,2,6a,6b,12a-pentamethyl-10-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethy)oxan-2-yl]oxy-1,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydropicene-4a-carboxylate, C57H92O28; Fig. 1A) is a major constituent of triterpene saponins found in the root of Platycodon grandiflorum, and has been shown to possess anti-inflammatory, neuroprotective, apoptosis-inducing, and anti-adipogenic properties (Choi et al., 2009, Lee et al., 2011). Osthole (7-methoxy-8-(3-methyl-2-butenyl) coumarin, C15H16O3; Fig. 1B), a natural coumarin derivative, is extracted from the fruit of C. monnieri (L.) Cusson. Osthole exhibits many pharmacological and biological activities, including anti-allergic, anti-osteoporosis, anti-inflammatory, and anti-oxidation (Liao et al., 2010, Chen et al., 2011). Recently, the antitumor activities of platycodin D and osthole have attracted much attention. Micromolar concentrations of platycodin D showed growth-inhibitory cytostatic activity in immortalized keratinocytes and leukemic cells (Ahn et al., 2006, Kim et al., 2008). Osthole has some estrogenic activity, as well, preventing the synthesis and action of estrogens in Her2-overexpressing cancer cells, and which makes it a very promising natural lead compound for breast cancer treatment (Lin et al., 2010).

In the present study, we attempted to evaluate the in vitro efficacy of combination treatment with platycodin D and osthole against cell viability, proliferation and invasion. Two invasive mammary carcinoma cell lines, human MDA-MB-231 and murine 4T1, were used as the model system. The effects of combination treatment on cell viability, proliferation and invasion were evaluated by the methyl thiazolyl tetrazolium (MTT), 5-bromo-2-deoxyuridine (BrdU) and transwell assays, respectively. Since TGF-β/Smads signaling is involved in malignant progression of breast cancer, we further investigated the effects of combination treatment on gene and protein expressions of TβRII, Smad2, Smad3, phospho-Smad2, phospho-Smad3, and Smad4 by real-time reverse transcription (RT)-PCR and Western blotting, respectively.

Section snippets

Compounds

Platycodin D and osthole (purity ≥99%, HPLC grade) were purchased from Nanjing Zelang Medical Technology Co., Ltd (Nanjing, Jiangsu, China). The structures of the compounds are shown in Fig. 1.

Cell culture

The murine 4T1 and human MDA-MB-231 mammary carcinoma cell lines were purchased as mycoplasma-free and source-certified preparations from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The normal breast epithelial Hs 578Bst cells were purchased from American

Inhibition of viability and proliferation by platycodin D and osthole in 4T1 and MDA-MB-231 cells

We first evaluated the cytotoxicities of platycodin D and osthole on 4T1 and MDA-MB-231 cells. As shown in Fig. 2, cells were treated with platycodin D (20, 40, 80, 160 and 320 μM) and osthole (5, 10, 20, 40 and 80 μM) for 24 h, followed by MTT and BrdU analysis. Both platycodin D and osthole reduced cell viability and proliferation in a dose-dependent manner, but osthole showed a higher toxicity than platycodin D (IC50 values of platycodin D on 4T1 and MDA-MB-231 were 73.9 μM and 81.8 μM,

Discussion

Platycodon grandiflorum and C. monnieri are used in a variety of applications in traditional Chinese medicine. Platycodin D and osthole, the major extracts of Platycodon grandiflorum and C. monnieri respectively, exhibit many pharmacological and biological activities. However, the therapeutic role and regulatory mechanism of platycodin D combined with osthole on breast cancer remain poorly understood. In the present study, platycodin D or osthole induced significant decreases in viability and

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgement

The authors are grateful for financial support from the National Natural Science Foundation of China (No. 81173269 & No. 81102597 & No. 81102598) the Foundation of Shanghai Municipal Health Bureau (No. 2010Y119).

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