A sulfated glucan from Antrodia cinnamomea reduces Slug expression through regulation of TGFβ/AKT/GSK3β axis in lung cancer
Introduction
The medicinal mushroom (Antrodia cinnamomea) is an abundant source for materials with various biological activities (Lu et al., 2013), and has been used intensively in Asian folk medicine. Polysaccharides from A. cinnamomea are polymers with many therapeutic properties, such as anti-cancer (Chang, Kuo, Chang, & Kong, 2015), antiangiogenic (Chen, Lu, Cheng, & Wang, 2005), and immunomodulatory (Kuo, Chang, Cheng, & Wu, 2008). We have previously defined various sulfated polysaccharides (SPSs) from A. cinnamomea and identified their bioactivities, such as antitumor (Lu, Lin, Chao, Hu, & Hsu, 2017; Lu, Lin, Hu et al., 2017) and anti-inflammatory (Cheng, Chao, Chang, & Lu, 2016). However, the antitumor mechanism of SPS remains unclear. We here report a sulfated glucan of A. cinnamomea (called SGA, formerly termed AC-SPS-F3 (Lu, Lin, & Chang, 2018); Scheme 1) that inhibits lung cancer in vitro and in vivo.
Lung cancer has the highest mortality rate of all cancers. Non-small-cell lung cancer (NSCLC) is the predominant type and presents with metastasis and poor survival (Denlinger, Ikonomidis, Reed, & Spinale, 2010; Sharma, Bell, Settleman, & Haber, 2007). Specifically, adenocarcinoma, a subtype of NSCLC, is common histological type of lung cancer and presents constitutive activity of epidermal growth factor receptor (EGFR) mutations from females who have never smoked. Many EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib (Iressa) and erlotinib (Tarceva), have been used on clinical lung adenocarcinoma (mutant (mut)-EGFR adenocarcinoma) therapy. However, wild type (wt)-EGFR of NSCLC and TKIs-induced secondary mutation are less sensitive to the TKIs (Chan & Hughes, 2015; Shih, Gow, & Yang, 2005). To develop effective new strategies and identify new drugs/adjuvants for the treatment of lung cancer, molecular mechanisms controlling tumor progression and metastasis are urgently needed.
High TGFβ1 expression is an important prognostic parameter for patients with NSCLC (Sterlacci et al., 2012), and it is important for tumor progression and metastasis (Hasegawa et al., 2001). In addition, over-expression of TGFβ1 and/or TGFβ receptors (TGFRs) have been reported as leading to lung tumor progression and pulmonary metastasis, reducing the survival rates of NSCLC patients (Hasegawa et al., 2001; Saji et al., 2003). TGFβ is described as a tumor promoter, with the ability to induce epithelial-to-mesenchymal transition (EMT) (Huber, Kraut, & Beug, 2005; Thiery, Acloque, Huang, & Nieto, 2009). EMT plays a key role in tumor metastasis since it is a process whereby cells change morphology and develop enhanced motility. Specifically, EMT regulators, such as Snail and Slug, mediate the transcriptional repression of E-cadherin, induce tumor metastasis and drug-resistance (Batlle et al., 2000; Cano et al., 2000; Casas et al., 2011). But over-expression of Slug in NSCLC patients is significantly associated with a lower overall survival rate (Wang et al., 2009). In addition, high expression of Slug contributes to the TKI resistance in lung adenocarcinoma cells containing activated EGFR mutations (Chang et al., 2011). Therefore, blockage of TGFβ-mediated Slug expression is a current strategy for combating lung cancer and reducing drug resistance.
This study explores the inhibitory effects of SGA on the TGFβR/AKT/GSK3β/Slug axis as well as its capability to reduce lung tumor progression in both the TKI-resistant lung adenocarcinoma cells and an LLC1-bearing mouse model. Our novel findings suggest that SGA inhibits cell viability and enhances cisplatin-induced cytotoxic effects via induction of lipid raft/lysosomal-dependent TGFβRI degradation, and that it concurrently enhances GSK3β-mediated Slug degradation. This is the first report to reveal a novel function of the sulfated galactoglucan of A. cinnamomea in suppressing lung cancer cell progression.
Section snippets
Cell lines and cell culture
The human lung cancer H1975 and the A549 cell line were obtained from Dr. C.M. Tsai (NYMU and Veteran’s General Hospital-Taipei, Taiwan) and the American Type Culture Collection (ATCC, USA), respectively. The Lewis lung carcinoma LLC1 cell line was purchased from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). Cells were cultured as previously described (Lin, Hsu, Sun, Wu, & Tsao, 2017; Wu et al., 2011).
Reagents and antibodies
Sulfated glucan (SGA) was purified as shown in Supplementary Methods
Preparation and purification of sulfated galactoglucan, SGA
A. cinnamomea strain B86 was maintained on potato dextrose agar (PDA) slants (at 28 ℃) and transferred to fresh medium at 3-week intervals. Mycelia on the media surface were transferred to 800 mL culture flasks containing 100 mL of 24 g/L potato-dextrose-broth, 1.0 mM sodium sulfate, and 20 g/L glucose, pH 5.6. Sulfated polysaccharides (SPSs) were isolated from 49-day-old cultures according to our previous study (Lu, Lin, Chao et al., 2017). To remove contaminating exopolysaccharides, mycelia
SGA suppresses cell viability and synergistically enhanced cisplatin-induced cytotoxic effects in lung cancer cells
Our previous study showed that SPS, isolated from 1 mM sodium sulfate cultivation for 49 days from A. cinnamomea, presents potent anti-cancer and anti-inflammatory activity (Cheng et al., 2016; Lu, Lin, Chao et al., 2017). Herein, we further fractionated the SPS and identified a sulfated glucan (called SGA). The estimated Mn values (average molecular weight) of SGA was determined to be 12.38 kDa using a dextran series (Sigma-Aldrich) with average molecular weights of 69.80, 40.00, 10.50, and
Discussion
Polysaccharides from both mushrooms and seaweeds have shown antitumor and immunoregulatory activities in cancer cells and tumor-bearing mouse models. For example, fucose-rich polysaccharides from G. lucidum suppressed breast and lung cancer tumorigenesis via downregulation of TGFβRs and activation of antibody-mediated cytotoxicity, respectively (Liao et al., 2013; Tsao & Hsu, 2016). In addition, water-soluble glucans, a polysaccharide of d-glucose monomers commonly existing in mushrooms and
Disclosure of potential conflicts of interest
The authors have no potential conflicts of interest to disclose.
Acknowledgements
This work was supported by the Ministry of Science and Technology: MOST107-2636-B-010-003 (Young Scholar Fellowship Program) and the National Yang-Ming University School of Medicine of Development and Construction Program (107F-M01-07M32) to TYL, as well as MOST105-2320-B-077-002-MY3, and the Ministry of Health and Welfare: MOHW107-NRICM-B-325-000202 to MKL. We thank Dr. H.Y. Hsu (NYMU) for the generous gift of experimental chemicals and use of equipment.
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