Anti-inflammatory and anticancer effects of polysaccharides from Antrodia cinnamomea: A review

Abstract

Antrodia cinnamomea (Ac), also known as “Niu-Chang-Chih” in Chinese, is a valuable fungus that has been widely used as medicine and food among indigenous people in Taiwan. Ac is rich in polysaccharides (Ac-PS), making it a promising candidate for adjunctive therapy in cancer and inflammation conditions. There are two types of Ac-PS: general (non-sulfated) PS (Ac-GPS) and sulfated PS (Ac-SPS). This review highlights that both Ac-GPS and Ac-SPS possess immunomodulatory, anti-inflammatory, and anticancer properties. Each type influences interleukin signaling pathways to exert its anti-inflammatory effects. Ac-GPS is particularly effective in alleviating inflammation in the brain and liver, while Ac-SPS shows its efficacy in macrophage models. Both Ac-GSP and Ac-SPS have demonstrated anticancer effects supported by in vitro and in vivo studies, primarily through inducing apoptosis in various cancer cell lines. They may also synergize with chemotherapy and exhibit antiangiogenic properties. Notably, Ac-SPS appears to have superior anticancer efficacy, potentially due to its sulfate groups. Furthermore, Ac-SPS has been more extensively studied in terms of its mechanisms and effects on lung cancer compared with Ac-GPS, highlighting its significance in cancer research. In addition, Ac-SPS is often reported for its ability to activate macrophage-mediated responses. Clinically, Ac-GPS has been used as an adjunctive therapy for advanced lung cancer, as noted in recent reports. However, given the numerous studies emphasizing its anticancer mechanisms, Ac-SPS may exhibit greater efficacy, warranting further investigation. This review concludes that Ac-derived Ac-GPS or Ac-SPS have the potential to be developed into functional health supplements or adjunctive therapies, providing dual benefits of anti-inflammatory and anticancer effects.

1. INTRODUCTION

Antrodia cinnamomea (Ac), also known as Niu-chang-chih in Chinese, as well as red camphor mushroom, camphor mushroom, blood Ganoderma, and divine mushroom, is an edible indigenous fungus species in Taiwan. It belongs to the order Polyporales of the Polyporaceae family, and can be classified under the genus Taiwanofungus or Antrodia. The naming and classification of Ac have undergone several revisions; in 2010, the Dictionary of Fungi and MycoBank officially revised the name Niu-chang-chih to Taiwanofungus camphoratus, while also recognizing alternative names such as Ganoderma comphoratum, Antrodia camphorata, and Ac. Specifically, the Bioresource Collection and Research Center (BCRC) in Taiwan has confirmed Ac as the scientific name for this species.¹ Ac grows on Cinnamomum trees and is typically found in forests at elevations between 400 and 2000 m above sea level. Owing to its slow growth rate, Ac is cultivated using two primary techniques: submerged fermentation and solid-state fermentation.² These methods enable extensive cultivation to meet commercial demand.

Native Taiwanese have traditionally used Ac to alleviate fatigue-like symptoms. Both the fruiting bodies and mycelia of Ac are utilized in folk remedies for various ailments, including inflammatory liver disease, food and drug poisoning, diarrhea, abdominal pain, hypertension, itchy skin, and tumor-related diseases, suggesting that Ac has significant potential for development as a complementary medicinal agent.³

Research has demonstrated that Ac exhibits a wide range of bioactivities, such as anti-inflammatory, anti-metastatic, and anti-angiogenesis effects.^3,4 Over the past 20 years, numerous studies have identified its key constituents: triterpenoids, 17% to 18%; polyphenols, 13% to 14%; and flavonoids, 11% to 12%.² Notably, polysaccharides (Ac-PS) are recognized as important active components of Ac. Recent advancements in the extraction and chemical characterization of these PS have led to an increase in Ac-PS-related studies. Currently, two types of Ac-PS have been identified: general (non-sulfated) PS (GPS) and sulfated PS (SPS). Modern cultivation methods have successfully produced various GPS and SPS with a range of biological activities. Despite these advancements, there remains a need for a comprehensive review to consolidate existing knowledge and establish a foundation for further research. This study aims to review the anticancer, anti-inflammatory, and immunomodulatory effects of both Ac-GPS and Ac-SPS reported since 2000 (Fig. 1). Literature was retrieved from databases, including PubMed and ScienceDirect.

Fig. 1.

Compilation of literature on GPS and SPS published over the years. The asterisk(*) indicates that the Ac-SPS has a specific structure. 6-OHDA = 6-hydroxydopamine; A431 = human epidermoid carcinoma; A549, H1975, CL1-5 = human non-small-cell lung cancer cell line; BAECs = bovine endothelial aortic cells; BALB/c = an albino, laboratory-bred strain of the house mouse; BxPC-3 = human pancreatic ductal adenocarcinoma cell line; CE = endothelial cells; CEM = T lymphoblast cell line; CLP = cecal ligation and puncture; CT-26 = mouse colon carcinoma; DC = dendritic cell; D-GalN = D-galactosamine; H22 = Hep G2, hepatocellular carcinoma cell line; HeLa = human uterus/cervix adenocarcinoma; HL-7702 = human normal liver cell line; HMEC-1 = human microvascular endothelial cells; HT-29 = human colorectal adenocarcinoma cell line; HUVEC = human umbilical vein endothelial cells; ICR = CD-1 outbred mice; J774.1 = mouse monocyte macrophage; KCs = Kuffer cells; LIH = lincomycin hydrochloride; LLC-1 = Lewis lung carcinoma cells; LO2 = human liver cells; LPS = lipopolysaccharide; MCF-7 = human breast cancer cell line; ME23.5 = Parkinson dopamine-secreting cells; Mφ = macrophages; NCI-H460 = human large cell lung cancer cell line; OVA = ovalbumin; p- = phosphorylated; RAW264.7 = murine macrophage cell line; S180 = sarcoma 180 cell line; THP-1 = human leukemia monocytic cell; U937 = human myeloid leukemia cell line; VEGF = vascular endothelial growth factor.

2. ANTI-INFLAMMATORY PROPERTIES OF GPS AND SPS DERIVED FROM Ac

2.1. Role of GPS in anti-inflammatory responses

The GPS derived from Ac, were initially recognized for their antiviral effects against hepatitis virus B, but subsequent research has revealed their anti-inflammatory and anti-tumor activities (Tables 1 and 2). Cheng et al⁵ first characterized the chemical properties of GPS, B86-PS, and demonstrated its anti-inflammatory effects. B86-PS significantly reduced lipopolysaccharide (LPS)-stimulated inflammation in bovine aortic endothelial cells (BACEs) and human leukemia monocytic (THP-1) cells by decreasing adhesion markers (I-CAM1) and monocyte adhesion.⁵ Further studies found that Ac-GPS, GF2, alleviated inflammation in an ovalbumin-induced asthma mouse model by suppressing the production of IgE, IgG1, and IgG2a. It also diminished the infiltration of monocytes and eosinophils into lung air sacs and reduced the expression of CD4+ T cells in peripheral blood.⁶ In addition, Ac-GPS from the fruiting body was shown to interfere with the innate immune system by lowering the CD11b/CD18 ratio in leukocytes, and decreasing NF-κB expression in lung tissue, along with reducing the levels of various cytokines in serum and splenocytes.⁷ β-glucan derived from Ac mycelia, Ac β-glucan, was also found to mitigate the immune inhibitory effects of the lung tumor microenvironment in Lewis lung carcinoma (LLC)-bearing mice. Oral intake of Ac β-glucan led to increased levels of interleukin-12 (IL-12) and interferon‐gamma (IFN-γ) in tumor lesions while decreasing serum transforming growth factor β (TGF-β) concentrations. Specifically, Ac β-glucan reduced serum TGF-β levels, promoting the transformation of tumor-associated macrophages (TAMs) from the M2 phenotype to the M1 phenotype.⁸ Following these findings, LPS-stimulated inflammation in mouse macrophages has become a widely used model for exploring the anti-inflammatory effects of Ac-GPS.

Table 1.

Anti-inflammation properties of Ac-GPS

Year	Name	Neutral sugar ratio	Cells	Biomedical markers	Functions	Ref.
2004	B86 PS	Glc: Gal: Fuc: Sor	LPS-stimulated BAECs	I-CAM1	Anti-inflammation Inhibition of monocyte adhesion	5
		0.03: 1.00: 0.15: 0.04	LPS-stimulated THP-1	Adhesion
2007	AC-2	Glc: Gal: Man: Rha: Ara: Lyx	LPS-stimulated RAW264.7	NO, iNOS, IL-6, IL-10, MCP-5, Rantes	Anti-inflammation	9,10
		0.85: 1.00: 0.64: 1.02: 14.75: 18.46
2010	GF2	N/A	OVA-induced BALB/c	Airway eosinophilia (OVA-BALF, Eos, Mon, Serum IgG IgE)	Airway inflammation	6
			LPS-stimulated DCs	TGF-β, IFN-γ	Anti-inflammation
2012	Ac-PSs	N/A	Cecal ligation and puncture: BALB/c (lung)	Lung: NF-κB (p65)	Anti-inflammation	7
				Spleen: mRNA (Il-6, IL-10, TNF-α)
				Serum, peritoneal fluid: Il-6, IL-10 TNF-α, IFN-γ, MCP-1
				Leukocytes: CD11a/CD18
2015	A. C. beta-glucan	N/A	LLC-1-bearing mice	Serum (TGF-β, IL-12, GM-CSF, IFN-γ)	Anti-inflammation	8
				IL-10
2016	AC-P	Glc, Gal, Fuc, Man, Rha, Xyl	LPS-stimulated RAW264.7	NO	Anti-inflammation	11
		28.97: 1.00: 0.69: 2.48: 0.48: 0.83
2017	ACP-1	N/A	Human hepatocyte HL-7702	ROS	Anti-inflammation	15
			LPS-stimulated LO2 (HL-7702)	NOX1/4, pERK, pP38, pAKT, TNF-α
2018	ACP	Gal: Man	J774A.1, mouse abdominal macrophage	Activation	TLR4 activation	12
				TNF-α, IL-6, IL-1β	Anti-inflammation
				TNF-α, PKC-α, PKC-δ, COX-2	Endotoxin tolerance
		1: 3	Mouse peritoneal macrophages (ex vivo)	Activation	TLR4 activation
			Human monocyte-derived dendritic cells	TNF-α, IL-6, IL-1β	Anti-inflammation
2019	ACP	N/A	Parkinson disease mice	Mouse striatum (NLRP3)	Parkinson inflammation	13
2019	ACP-2	Glc: Gal: 6-DG	LPS-stimulated LO2	IL-1β, IL-6, COX-2, TNF-α	Hepatic inflammation	16
		5.00: 2.00: 1.00		pP38/NF-κB
2020	ACP	N/A	6-OHDA-stimulated MES23.5 Parkinson disease mice	Mouse striatum (ROS-NLRP3)	Parkinson inflammation	14
				NLRP3, ASC, caspase-1, IL-1β
2022	ACP	N/A	D‐GalN/LPS-treated mice	Apoptosis	Liver-inflammation	17
				ROS-TLR4/NF-κB
			LPS-stimulated KCs	IL-1β, IL-18, TNF-α, ROS, CD68
2022	ACP	N/A	LPS-stimulated KCs	LC3, NLRP3, IL-1β, IL-18, TNF-α	Liver-inflammation	18
			Mouse	Liver-NLPR3
2022	AEPS	Glc: Gal: GlcA: GalA: Man	Lincomycin hydrochloride-stimulated ICR mice	Serum (IL-6, TNF-α)	Intestinal-inflammation	19
		10.81: 1.00: 0.10: 0.18: 0.67

6-OHDA = 6-hydroxydopamine; ASC = apoptosis-associated speck-like protein; BALF = bronchoalveolar lavage fluid; CD = cluster of differentiation; COX-2 = cyclooxygenase-2; DC = dendritic cell; Eos = eosinophil; ERK = extracellular signal-regulated kinase; ICAM-1 = intercellular adhesion molecule 1; IFN-γ = interferon gamma; Ig = immunoglobulin; IL = interleukin; iNOS = nitric oxide synthase; LC3 = microtubule-associated proteins 1A/1B light chain 3B; LPS = lipopolysaccharide; MCP-1 = monocyte chemoattractant protein-1; MCP-5 = monocyte chemoattractant protein-1; Mon = monocyte; mRNA = messenger ribonucleic acid; NF-κB = nuclear factor kappa-light-chain-enhancer of activated B cells; NO = nitric oxide; NOX1/4 = NADPH oxidase; NLRP3 = nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3; OVA = ovalbumin; p- = phosphorylated; PKC-α = protein kinase C alpha; PKC-δ = protein kinase C delta; RANTES = Regulated on activation, normal T-cell expressed and secreted; ROS = reactive oxygen species; TGF-β = transforming growth factor beta; TLR4 = toll-like receptor 4; TNF-α = tumor necrosis factor alpha.

Table 2.

Anticancer properties of Ac-GPS

Year	Name	Neutral sugar ratio	Cells	Biomedical markers	Functions	Ref.
2004	Ac-PS	N/A	S180 tumor-bearing mice	Tumor weight	Anti-tumor progression	29
			U937	Cell viability (Ac-PS-MNC-CM)	Anticancer
2005	Fra-3	Gal: Fuc: myo-Ino: Sor	VEGF-stimulated BAECs	CD-1 tube formation	Anti-angiogenesis	30
		1.00: 0.12: 0.06: 0.02		Receptor binding
2005	B86 #3	Glc: Gal: Fuc: GlcN: GalN: myo-Ino	VEGF-stimulated BAECs	Tube formation	Anti-angiogenesis	34
		0.002: 1.00: 0.56: 0.79: 0.32: 0.04
2009	PMAC	Glu: Gal: Fuc: Fru: Xyl	HUVECs	Tube forming (PMAC-MNC-CM)	Anti-angiogenesis	31
		0.10: 1.00: 0.01: 0.01: 0.01	HL-60	VEGF (PMAC-MNC-CM)
			EGG	Neovascularization
2009	PS	Glu: Gal: Fuc: Ino	VEGF-stimulated BAECs	Endothelial cell	Anti-angiogenesis	32
		0.25: 1.00: 0.28: 0.05
2014	ACE	N/A	BxPC-3 (pancreatic adenocarcinoma)	Cell cycle, apoptotic molecules	Inhibition of cell viability and migration	37
2015	ACE	N/A	Hep G2	Cell cycle, apoptotic molecules	Inhibition of cell viability	36
2017	ACW0	N/A	HMEC-1	pFAK, pERK	Anti-angiogenesis	33
2018	ACPS-1	Fuc: Man: Rha: Xyl	HeLa, A431, H22, S180	Apoptosis TOP1/TDP1-mediated DNA repair	Induction of apoptosis	38
		1.44: 31.27: 1.00: 1.77
2020	SqualenePS	Glc: Gal: Fuc: GalN: Man: Sor: Mannitol	LLC-1, CT-26	GLUT1, AKT	Inhibition of cell viability	35
		1.38: 1.00: 0.002: 0.001: 0.01: 0.003: 0.004		Glucose and galactose uptake

Ac-PS = Antrodia camphorata polysaccharides; A431 = epidermoid carcinoma cell line; BAECs = bovine endothelial aortic cells; CT-26 = mouse colon carcinoma; ERK = extracellular signal-regulated kinase; FAK = focal adhesion kinase; GLUT1 = glucose transporter 1; H22 = Hep G2, hepatocellular carcinoma cell line; HL-60 = human promyelocytic leukemia cell line; HMEC-1 = human microvascular endothelial cells-1; HUVECs = human umbilical vein endothelial cells; LLC-1 = Lewis lung cancer 1 cell line; MNC-CM = mononuclear cells conditioned medium; N/A = Not applicable; PMAC = polysaccharides from mycelia of Antrodia cinnamomea; S180 = sarcoma 180 cell line; p- = phosphorylated; TOP1/TDP1 = DNA topoisomerase 1/tyrosyl-DNA phosphodiesterase 1; U937 = human myeloid leukaemia cell line; VEGF = vascular endothelial growth factor.

Many studies have used the in vitro LPS-stimulated RAW264.7 macrophage model to investigate the anti-inflammatory effects of various Ac-GPSs. For instance, Chen et al⁹ reported that AC-2, the highest fraction extracted from Ac mycelia significantly reduced nitric oxide (NO) production and NO synthase (iNOS) expression in LPS-stimulated RAW264.7 cells. AC-2 was also found to inhibit the LPS-induced production of pro-inflammatory mediators such as granulocyte-colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein-1 (MCP-1), and monocyte chemoattractant protein-5 (MCP-5) in RAW264.7 cells.¹⁰ Moreover, Su et al¹¹ demonstrated that AC-P inhibited the LPS-stimulated production of NO manufacturing, marking the first study to detail the composition and branching structure of AC-P’s (1,3;1,6)-β-D-glucans. Furthermore, an Ac-derived galactomannan, the cold water-soluble polysaccharide of A. cinnamomea (galactomannan-repeated; MW>70 kDa) ACP, was found to exhibit immunostimulatory activity in mouse abdominal macrophages (in vivo and ex vivo) by binding to toll-like receptor 4 (TLR4). Pretreatment with ACP enhances immune responses against invading bacteria and mitigates the risk of severe inflammation, suggesting that ACP may serve as a potential adjuvant in immunotherapy for a wide range of diseases.¹²

Neuroinflammation plays a critical role in the pathogenesis and progression of Parkinson disease (PD). A study showed that ACP derived from Ac mycelia can improve movement coordination, gait maintenance, and cognitive abilities in a PD mouse model. In the striatum, ACP was found to upregulate dopamine levels and its derivatives, while simultaneously suppressing the activity of nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome. This suppression is achieved by significantly reducing the expression of NLRP3, IL-1β, caspase-1, and procaspase-1. In the 6-hydroxydopamine-induced damage to dopamine-secreting cells ME23.5, ACP exhibited neuroprotective properties by preserving cell viability and mitigating the oxidative effects of reactive oxygen species (ROS). On the molecular level, ACP was shown to attenuate the levels of pro-inflammatory cytokines IL-1β and IL-18 as well as pro-inflammatory molecules such as NLRP3, apoptosis-associated speck-like protein (ASC), and caspase-1.^13,14

Ac-GPS has a positive impact on the liver through immunomodulatory mechanisms. ACP-1, derived from Ac powder, reduces ROS by increasing the expression of NADPH oxidases 4 (NOX1-4) and attenuating inflammatory cascades via the mitogen-activated protein kinase/AKT serine/threonine kinase 1 (MAPKs/AKT) signaling pathway. Moreover, ACP-1 exhibits protective effects on HL-7702 human liver cells by lowering levels of aspartate aminotransferase (AST) and alanine transaminase (ALT), thereby alleviating hepatotoxicity through the upregulation of superoxide dismutase (SOD), a potent anti-oxidation enzyme.¹⁵ In addition, ACP-2 has been shown to improve the survival rate of human liver cells (LO2) in vitro. It downregulates the TLR4-MyD88 signaling pathway and its downstream targets, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), IL-1β, cyclooxygenase-2 (COX-2), TNF-α, and IL-6.¹⁶ Following acute liver injury, administering ACP to Kuffer cells (KCs; liver macrophages) reduces cell apoptosis and mitigates the ROS-TLR4/NF-kB signaling associated with macrophage activation.¹⁷ In LPS-stimulated KCs, treatment with ACP enhances cell viability while lowering levels of transferases and inflammatory molecules, including AST, ALT, IL-1β, IL-18, and TNF-α, as well as ROS and CD68 expression.^17,18 In brief, specific Ac-GPSs can exert protective effects on liver KCs by regulating immune responses and the anti-oxidation system.^16–18

Gastroenteric diseases, particularly functional bowel syndromes, have become a significant public health concern, prompting increased research into the relationship between inflammation and gut microorganisms. In a model of lincomycin hydrochloride-induced gut inflammation, Ac-derived exopolysaccharides (AEPS; a galactose-modified glucan) were shown to decrease both the incidence and severity of diarrhea while reducing inflammatory cytokines such as IL-6 and TNF-α.¹⁹ Additionally, AEPS was found to enhance the growth of the Lactobacillus species while inhibiting the proliferation of disease-causing organisms.¹⁹ Overall, various Ac-GPSs exhibit anti-inflammatory effects both in vitro and in vivo through multiple pathways, including TLR4, ICAM-1, and TGF-β-mediated signaling (Fig. 2A).

Fig. 2.

Potential mechanisms of Ac-PS (A) and Ac-SPS (B) on anti-inflammation. Ac-GPS = Antrodia cinnamomea polysaccharides; Ac-SPS = Antrodia cinnamomea sulfated polysaccharides; COX-2 = cyclooxygenase-2; DC = dendritic cell; ECs = epithelial cells; EGFR = epidermal growth factor receptor; Eos = eosinophil; ERK = extracellular signal-regulated kinase; FAK = focal adhesion kinase.

2.2. Role of SPSs in anti-inflammatory responses

Ac-PSs can be partially modified with sulfate, resulting in sulfated polysaccharides (Ac-SPSs). Fucoidan, derived from seaweed, is a well-known sulfated polysaccharide recognized for its significant biological activity.²⁰ Consequently, many studies have introduced additional sulfates during the cultivation of Ac mycelia to enhance the production of Ac-SPSs and improve their biological efficacy (Tables 3 and 4). One study found that SPSs extracted from Ac mycelia cultured with sodium sulfate or potassium sulfate, effectively inhibited the inflammatory response by reducing the secretion of TNF-α and IL-6 in LPS-stimulated RAW264.7 cells.²¹ Large-scale preparation and isolation of SPSs, SPSs-K3, from Ac mycelia cultured with potassium sulfate demonstrated their ability to inhibit the production of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) in this model.²² Furthermore, AM-SPS extracted from Ac mycelia cultivated with ammonium sulfate (AM) at 1 mM exhibited anti-inflammatory effects by inhibiting the phosphorylation of AKT and extracellular signal-regulated kinase (ERK), leading to decreased TNF-α secretion in LPS-stimulated RAW264.7 cells.²³ In addition, sulfated polysaccharides, Zn250, extracted from Ac mycelia cultivated with zinc sulfate, also demonstrated anti-inflammatory properties by inhibiting the phosphorylation of P38, AKT, and ERK, thereby suppressing LPS-induced secretion of TNF-α and IL-6 in RAW264.7 cells.²⁴

Table 3.

Anti-inflammation properties of Ac-SPS

Year	Name	Glc: Gal: Fuc: GlcN: Man	Sulfate content of SPS (µmol/g)	Cells	Biomedical markers	Functions	Ref.
2016	SPSs (Na2SO4, 1 mM)	1.72: 1.00: 0.03: 0.00: 0.09	1215.56	LPS-stimulated RAW264.7	TNF-α, IL-6	Anti-inflammation	21
	SPSs (K2SO4, 0.5 mM)	2.26: 1.00: 0.07: 0.00: 0.12	448.31
2018	SPSs-K3	16.73: 1.00: 0.09: 0.24: 0.56	1590	LPS-stimulated RAW264.7	TNF-α, IL-6, IL-1β	Anti-inflammation	22
2020	AM-SPS	4.79: 1.00: 0.07: 0.07: 0.23	1820	LPS-stimulated RAW264.7	TNF-α, IL-6, pAKT/pERK, NF-κB	Anti-inflammation	23
2020	Zn250	2.26: 1.00: 0.00: 0.15: 0.02	2419	LPS-stimulated RAW264.7	TNF-α, IL-6, TGF-β, pERK, pAKT, pP38	Anti-inflammation	24
2021	Na10_SPS-F3	12.14:1.00:0.08:0.12:0.59	458.04	LPS-stimulated RAW264.7	TNF-α, IL-6, TGFβRII, IκB-α	Anti-inflammation	25
2023	3-SS (sulfated galactoglucan)	5.08:1.00:0.04:0.13:0.40	572.4	LPS-stimulated RAW264.7	TNF-α, IL-6, TGFβRII, IκB-α, pERK/pP38/pAKT	Anti-inflammation	26
2024	Na 500 F3	3.89: 1.00: 0.02: 0.13: 0.06	2256	LPS-stimulated RAW264.7	TNF-α, IL-1β, IL-6, TGF-β, p38, pAKT, pERK, TGFβRII	Anti-inflammation	27
2024	SPS N10	3.14: 1.00: 0.95: 0.16: 0.30	810	LPS-stimulated RAW264.7	TNF-α, IL-6, TGF-β, p38, pAKT, pERK, TGFβRII	Anti-inflammation	28
	SPS K5	3.00: 1.00: 1.00: 0.17: 0.24	999

ERK = extracellular signal-regulated kinase; IL = interleukin; IκB-α = inhibitor of nuclear factor kappa B-alpha; LPS = lipopolysaccharide; NF-κB = nuclear factor kappa-light-chain-enhancer of activated B cells; p- = phosphorylated; TGF-β = transforming growth factor beta; TGFβRII = transforming growth factor beta receptor 2; TNF-α = tumor necrosis factor alpha.

Table 4.

Anticancer properties of Ac-SPS

Year	Name	Glc: Gal: Fuc: GlcN: Man	Sulfate content of SPS, µmol/g	Cells	Biomedical marker	Functions	Ref.
2009	SPSs (MgSO4, 0.5%)	4.06: 1.00: 0.56: 0.00: 0.00	426.5	VEGF-stimulated BAECs	Tube formation	Anti-angiogenesis	32
	SPSs (MnSO4, 0.5%)	0.22: 1.00: 0.03: 0.00: 0.00	1075.11
2012	SPSs	2.12: 1.00: 0.12: 0.03: 0.11	607.2	MCF-7, NCI-H460, HT-29, CEM VEGF-stimulated BAECs	Cell viability Tube formation	Combination therapy, anti-angiogenesis	39
2017	SPS (Na2SO4, 1 mM)	1.72: 1.00: 0.03: 0.00: 0.09	1215.56	A549, LLC-1	TGFβ-induced pSmad2/3, FAK, AKT	Induction of apoptosis and cell cycle arrest, anti-migration	41
2017	B86-III (1,4-β-D-galactoglucan)	3.62:1.00:0.62:0.00:0.00	741.68	A549, H1975	TGFβIR-mediated FAK/Slug axis	Induction of apoptosis and cell cycle arrest, anti-migration	43
2018	AC-SPS-F3 (1-4-β-D-glucan)	23.85:1.00:0.18:0.45:0.80	1060	A549, H1975	EGFR and mTOR	Inhibition of cell viability	44
2018	SPSs-K3	16.73: 1.00: 0.09: 0.24: 0.56	1590	VEGF-stimulated BAECs	Tube formation	Anti-angiogenesis	22
2018	SPSs (ZnSO4, 0.1 µM)	4.16: 1.00: 0.08: 0.13: 0.13	1722	A549	pEGFR/pFAK/pAKT	Inhibition of cell viability	42
2019	SGA (AC-SPS-F3)	23.85:1.00:0.18:0.45:0.80	1060	A549, H1975 LLC-1-bearing mice	TGFβRI/AKT/GSK3β and Slug	Anti-tumor progression Inhibition cell viability Anti-migration	45
2019	ST-SPSs	4.23: 1.00: 0.00: 0.00: 0.13	2500	H1975, CT-26	Apoptotic molecules, EGFR, and AKT	Inhibition of cell viability Combination therapy	40
2020	Zn250	2.26: 1.00: 0.00: 0.15: 0.02	2419	H1975	TGFβRI, FAK, Slug, pERK, pAKT, pP38	Inhibition of cell viability	24
2021	Na10_SPS-F3	12.14: 1.00: 0.08: 0.12: 0.59	458.04	A549, CL1-5, H1975	Caspase-3, PARP, P21, P27, pEGFR, pERK, pAKT, Slug, TNF-α, IL-6, TGFβRII	Inhibition of cell viability	25
2023	ZnF3	15.07: 1.00: 0.00: 0.52: 0.67	1350	A549, H1975, LLC-1, RAW264.7	Apoptotic molecules, M1-polarization markers	Induction of apoptosis, activation of Mφ-mediated anticancer	46
2023	3-SS (sulfated galactoglucan)	5.08: 1.00: 0.04: 0.13: 0.40	572.4	H1975	pEGFR, Slug, pERK, pAKT	Inhibition of cell viability	26
2024	Na 500 F3	3.89: 1.00: 0.02: 0.13: 0.06	2256	H1975	pEGFR, pFAK, pERK, TGβRII	Inhibition of cell viability	27
2024	ASZ-10	2.96: 1.00: 0.09: 0.09: 0.16	1031	A549, LLC-1, RAW264.7	Apoptotic molecules, M1-polarization markers	Induction of apoptosis, activation of Mφ-mediated anticancer	47

A549, H1975, CL1-5 = human non-small-cell lung cancer cell line; BAECs = bovine endothelial aortic cells; CEM = T lymphoblast cell line; CT-26 = mouse colon carcinoma; EGFR = epidermal growth factor receptor; ERK = extracellular signal-regulated kinase; FAK = focal adhesion kinase; H22 = Hep G2, hepatocellular carcinoma cell line; HT-29 = human colorectal adenocarcinoma cell line; LLC-1 = Lewis lung cancer 1 cell line; IL = interleukin; mTOR, mechanistic target of rapamycin; MCF-7 = human breast cancer cell line; NCI-H460 = human large cell lung cancer cell line; PARP = poly(ADP-ribose) polymerase; P21, P27 = major targets of p53 activity; S180 = sarcoma 180 cell line; p- = phosphorylated; TGF-β = transforming growth factor beta; TGFβRI = transforming growth factor beta receptor 1; TGFβRII = transforming growth factor beta receptor 2; TNF-α = tumor necrosis factor alpha; U937 = human myeloid leukaemia cell line; VEGF = vascular endothelial growth factor.

In 2021, a structural analysis identified the anti-inflammatory Ac-SPS, Na10_SPS-F3, derived from Ac mycelia cultured with 10 mM sodium sulfate (Na₂SO₄). This compound is characterized as a sulfate-rich 1,4-β- and α-glucan with partial 3-O-malonyl substitution. Na10_SPS-F3 exerts its anti-inflammatory effects by downregulating TGFβRII and inhibiting IκB-α degradation, thereby suppressing the secretion of TNF-α and IL-6 in LPS-stimulated RAW264.7 cells.²⁵ Another study reported a galactoglucan, 3-SS, extracted from Ac mycelia cultured with 50 mM sodium sulfate, which features a main chain composed of 1,3/1,4-α/β-galactoglucan with 1,6-β-Glc side branches and 2-O-sulfate substitutions. It demonstrated anti-inflammatory activity mediated through LPS-mediated p38/AKT/TGFβRII signaling pathways.²⁶ Many other studies have also highlighted the anti-inflammatory effects of Ac-SPSs on LPS-stimulated macrophages, even in the absence of structural analyses. For example, Na500 F3, extracted from Ac mycelia cultured with 500 mM sodium sulfate, was shown to suppress LPS-macrophage signaling pathways, specifically the ERK/p38/AKT/TGFβRII pathways, along with reducing the secretion of cytokines such as TNF-α and TGF-β.²⁷ Similarly, Ac-SPS: N10 and Ac-SPS: K5, extracted from Ac mycelia cultivated respectively with 10 mM AM ([NH₄]₂SO₄) and 5 mM potassium sulfate (K₂SO₄), inhibited LPS-induced TNF-α expression by blocking the p38/AKT/JNK or TGFβRII/AKT signaling pathways.²⁸

Despite being tested only in vitro on mouse macrophages, various Ac-SPSs were found to exhibit anti-inflammatory responses by targeting TLR4 and TGFβRII-mediated pathways (Fig. 2B). Moreover, similar to Ac-GPSs, Ac-SPSs exhibit anti-inflammatory activity even at high levels of sulfation, suggesting that sulfation may not directly influence the anti-inflammatory effects of Ac-SPSs. Further research is necessary to clarify the distinct anti-inflammatory mechanisms of Ac-GPSs and Ac-SPSs.

3. ANTICANCER PROPERTIES OF GPS AND SPS EXTRACTED FROM Ac

3.1. Role of Ac-GPS in anticancer effects

In view of the immunomodulatory activity of Ac-GPSs, many studies have focused on exploring their anticancer effects through immune system regulation. Liu et al²⁹ were the first to utilize Ac-GPS, AC-PS, in leukemia (U937) and sarcoma180 (S180)-ICR mouse models to demonstrate the immune-based properties of anticancer therapy. AC-PS exhibited anti-tumor activity in sarcoma-bearing mice and showed anti-leukemia effects through mononuclear-mediated cancer inhibition. Specifically, AC-PS significantly inhibited U937 cells when administered alongside mononuclear-stimulated cells.²⁹ Wang et al⁸ demonstrated that oral administration of Ac-derived β-glucan in lung tumor-bearing mice altered the immune cell composition in the tumor microenvironment, particularly by shifting the polarization of TAMs. The Ac-derived β-glucan increased M1 type markers such as IL-12 and IFN-γ in tumor tissues but decreased TGF-β levels in the tumor microenvironment, resulting in suppression of lung tumorigenesis.⁸

Amid the development of immune-based cancer therapies, anti-angiogenesis stands out as a key strategy of Ac-GPSs due to their ability to influence neoangiogenesis in the tumor microenvironment, which is crucial for supplying oxygen and nutrients. Cheng et al³⁰ elucidated the antiangiogenic effects of Ac-GPSs through the vascular endothelial growth factor (VEGF) signaling pathway in endothelial cells. Ac-GPSs inhibit vascular proliferation via the VEGF-Kinase insert domain receptor/fetal liver kinase 1 (KDR/Flk-1) pathway.³⁰ Another study showed that an Ac-GPS with a molecular weight greater than 100 kDa, PMAC, effectively inhibited VEGF production in human leukemia HL-60 cells and prevented matrigel tube formation in human umbilical vein endothelial cells (HUVEC). In addition, PMAC was shown to impede vessel plexus formation in an ex vivo egg model.³¹ Ac-GPS also hindered VEGF-induced tube formation in PC12 cells.³² However, compared with Ac-GPS, Ac-SPS may exhibit an even stronger effect on inhibiting vascular proliferation. In studies using human microvascular endothelial cells (HMEC-1), endothelial migration was significantly impeded with various doses of Ac-SPS, ACW0-sul, while Ac-GPS, ACW0, showed a minimal impact.³³ When compared with other fungi, such as Antrodia malicola, Antrodia xantha, Antrodiella liebmannii, Agaricus murrill, and Rigidoporus, Ac-GPS demonstrated only a modest effect against neovasculogenesis.³⁴

In addition to immune regulation and anti-angiogenesis, Ac-GPS has been reported to use anticancer strategies that include direct inhibition of cancer cells and the promotion of cancer cell death (Fig. 3A). For instance, squalenePS, derived from squalene-treated Ac mycelia, demonstrates anticancer activities by inhibiting cell viability and colony formation. This effect is primarily attributed to a decrease in glucose transporter (GLUT1) on the cell membrane, which results from the inhibition of phosphorylated AKT. The reduced GLUT1 expression leads to decreased glucose uptake and lactate secretion.³⁵ In HepG2 cell models, fruiting body-derived ACE, a crude Ac-GPS, significantly induced apoptosis via membrane damage and mitochondria self-destruction, effectively halting the cell cycle at the G1 checkpoint. Mechanistically, ACE upregulates apoptosis-related proteins, including Fas/APO-1, caspase-3, 8, and 9.³⁶ In addition, research showed that the crude Ac-GPS reduced cell viability and inhibited the migration of pancreatic cancer cells. ACE accelerated cell cycle arrest in the G2/M and subG1 phases and triggered mitochondria-regulated apoptotic responses.³⁷

Fig. 3.

Potential mechanisms of Ac-PS (A) and Ac-SPS (B) on inhibition of cancer cells. A431 = human epidermoid carcinoma; A549, H1975 = human non-small-cell lung cancer cell line; Ac-GPS = Antrodia cinnamomea polysaccharides; Ac-SPS = Antrodia cinnamomea sulfated polysaccharides; Bax/Bcl-2 = Bcl-2-associated X protein/B-cell lymphoma 2 protein; BxPC-3 = human pancreatic ductal adenocarcinoma cell line; CEM = T lymphoblast cell line; CEs = endothelial cells; CL1-5 = human lung adenocarcinoma cells; Cle-caspase-3 = cleaved caspase-3; Cle-caspase-9 = cleaved caspase-9; Cle-PARP = cleaved poly (ADP-ribose) polymerase; CT-26 = mouse colon carcinoma; Cyt-c = cytochrome c; EGFR = epidermal growth factor receptor; ERK1/2 = extracellular signal-regulated kinase1/2; FAK = focal adhesion kinase; GLUT1 = glucose transporter 1; H22 = Hep G2, hepatocellular carcinoma cell line; HeLa = human uterus/ cervix adenocarcinoma; HL-60 = human promyelocytic leukemia cell line; HT-29 = human colorectal adenocarcinoma; ICAM-1 = intercellular adhesion molecule 1; IFN-γ = interferon gamma; IL = interleukin; iNOS = nitric oxide synthase; IκB-α = inhibitor of nuclear factor kappa B-alpha; JNK = Jun N-terminal kinase; KC = Kupffer cells; LC3 = microtubule-associated proteins 1A/1B light chain 3B; LLC/LLC-1 = Lewis lung carcinoma cells; LPS = lipopolysaccharide; MAPKs = mitogen-activated protein kinase; MCF-7 = human breast cancer cell line; MCP-1 = monocyte chemoattractant protein-1; MCP-5 = monocyte chemoattractant protein-1; mTOR = mechanistic target of rapamycin; MyD88 = myeloid differentiation primary response 88; NCI-H460 = human large cell lung cancer cell line; NF-κB = nuclear factor kappa-light-chain-enhancer of activated B cells; NO = nitric oxide; p- = phosphorylated; P21, P27 = major targets of p53 activity; RANTES = regulated on activation, normal T-cell expressed and secreted; ROS = reactive oxygen species; S180 = sarcoma 180 cell line; Slug = zinc-finger transcription factor; TDP1 = tyrosyl-DNA phosphodiesterase 1; TGF-β = transforming growth factor beta; TGFβRI = transforming growth factor receptor; TGFβRII = transforming growth factor beta receptor 2; TLR4 = toll-like receptor 4; TNF-α = tumor necrosis factor alpha; TOP1 = DNA topoisomerase 1; VEGF = vascular endothelial growth factor.

Although Ac-GPS has shown potential as a supplement for the direct inhibition of cancer cells in various contexts, there are currently no reports elucidating the correlation between its specific structure and inhibition of cancer cells. Zhang et al³⁸ isolated a mannose-rich polysaccharide with a molecular weight of 23 kDa, ACPS-1, from Ac mycelium. ACPS-1 demonstrated concentration-dependent inhibition of cell survival across various cancer cell lines through the induction of apoptosis. Notably, ACPS-1 revealed a novel anticancer mechanism by regulating the topoisomerase 1/tyrosyl-DNA phosphodiesterase 1 (TOP1/TDP1)-mediated DNA repair pathway.³⁸ This study is the first report of Ac-GPS exhibiting a direct anticancer effect while also analyzing its structural aspects. This milestone suggests that future research on Ac-GPS should increasingly focus on understanding the relationship between its structural characteristics and anticancer activity, paving the way for more targeted therapeutic applications.

3.2. Role of Ac-SPS in anticancer effects

The bioactivity of Ac-SPSs has been notably linked to their anticancer properties. In 2009, Lu et al first identified that Ac-SPS derived from Ac mycelia cultured with manganese sulfate (MnSO₄) or magnesium sulfate (MgSO₄) exhibits significant antiangiogenic activity. These Ac-SPSs were shown to inhibit VEGF-induced tube formation in BACEs. Importantly, their study revealed that Ac-SPSs exhibited a stronger antiangiogenic effect compared with Ac-GPSs.³² The stronger antiangiogenic effect of Ac-SPSs was further confirmed by Liu et al,³³ who chemically modified an Ac heterogalactan through sulfation to produce ACW0-sul, which exhibited even greater antiangiogenic activity. Subsequent research indicated that Ac-SPSs extracted from Ac mycelia cultured without the addition of potassium sulfate (K₂SO₄), also exhibited antiangiogenic effects.^22,39 These findings collectively suggest that Ac-SPSs may possess adjunctive anticancer properties through the inhibition of angiogenesis.

Recent studies have increasingly focused on the direct inhibition of cancer cells by Ac-SPSs. Cheng et al³⁹ demonstrated that Ac-SPS exhibited synergistic activity with doxorubicin (DOX), enhancing the sensitivity of various cancer cells to the drug. Similarly, ST-SPS derived from Ac mycelia cultured with sodium thiosulfate (Na₂S₂O₃), not only inhibited cancer cell lines in vitro on its own but also exhibited significant effects when combined with chemotherapy agents such as cisplatin, gefitinib, and 5-fluorouracil.⁴⁰ These studies indicated that cancer cell survival decreased when ST-SPS was used alone or in combination with chemotherapeutic drugs, suggesting that Ac-SPSs can lower the sensitivity threshold of tumors to chemotherapy, thus potentially reducing adverse events associated with treatment. As such, utilizing Ac-SPS as an adjuvant or anticancer dietary supplement could offer a more effective strategy for cancer treatment. This breakthrough marks a significant advancement in the exploration of Ac-SPSs for combination cancer therapies.

Beyond their adjunctive anticancer effects, Ac-SPSs have been shown to inhibit cancer cells through various mechanisms (Fig. 3B). For example, SPS derived from Ac mycelia cultured with AM ([NH₄]₂SO₄) directly regulated the viability and migration of lung cancer cells by downregulating TGFβR and its associated signaling pathways,⁴¹ marking the first elucidation of Ac-SPS’s anti-lung cancer mechanisms. In addition, ST-SPS reduces cell viability of lung cancer cells by inhibiting the epidermal growth factor receptor (EGFR) activity, while simultaneously upregulating apoptotic markers such as cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP).⁴⁰ Subsequent studies have further explored the anti-lung cancer effects of Ac-SPSs cultivated with various trace elements including cupric sulfate, ferric sulfate, and zinc sulfate. These Ac-SPSs exhibited anticancer activity primarily by inhibiting the EGFR/AKT signaling pathway.⁴²

In view of the demonstrated anticancer effects of Ac-SPSs, researchers have increasingly focused on their structural characteristics and mechanisms of action. In 2017, the first structural analysis of an Ac-SPS, B86-III, revealed it to be a 1,4-β-D-galactoglucan with 1, 6-branching. B86-III was shown to inhibit the viability and migration of lung cancer H1975 cells by inducing apoptosis and suppressing the TGFβR/FAK/Slug signaling axis.⁴³ In 2018, another Ac-SPS known as a sulfated glucan of A. cinnamomea (SGA) (formerly AC-SPS-F3) was identified as a sulfated β-(1→4)-D-glucan with long β-(1→6)-Glcp branches. SGA was found to reduce the viability of lung cancer cells by inhibiting EGFR activity and the mammalian target of rapamycin (mTOR).⁴⁴ It also modulated intracellular signaling through the TGFβ/AKT/GSK3β axis, promoting the degradation of Slug in H1975 cells, thereby affecting both cell viability and migration.⁴⁵ Notably, SGA exhibited a synergistic enhancement of cisplatin-induced cytotoxicity in lung cancer cells,⁴⁵ and it is the only Ac-SPS to have its anti-tumor efficacy verified in tumor-bearing animal experiments. Further studies have highlighted additional Ac-SPSs, such as Na10_SPS-F3 and 3-SS, which also exhibit anti-lung cancer activity despite their structural differences.^25,26 Both of these Ac-SPSs inhibit oncogenic proteins such as EGFR and Slug in lung cancer cells.

Recent research has shifted focus towards the potential of Ac-SPSs in cancer immunotherapy strategies. In 2023, a pivotal study identified an Ac-SPS, ZnF3, derived from Ac mycelia cultivated with zinc sulfate, which exhibited immune-activating properties in macrophages. ZnF3 not only inhibited the viability of lung cancer cells and reduced the levels of pro-cancer-related proteins like EGFR and TGFβRI, but also activated macrophages via the AKT/mTOR signaling pathway.⁴⁶ This activation resulted in the expression of M1 phenotype surface proteins such as CD86, CD80, MHCII, and CD64 as well as the secretion of M1 cytokines and immunomodulatory substances like NO and TNF-α. The ZnF3-activated macrophages showed an ability to suppress lung cancer cells, marking a significant advancement in understanding how Ac-SPS can simultaneously exert inhibitory effects on lung cancer and mediate macrophage-driven anti-lung cancer responses, thus opening new avenues for exploring the immuno-activating and anticancer properties of Ac-SPS.⁴⁶ A similar study conducted in 2024 explored further the optimal conditions for cultivating Ac mycelia with zinc sulfate. It confirmed that a crude Ac-SPS, ASZ-10, could activate macrophage M1 polarization through the AKT and mTOR signaling pathways. ASZ-10-stimulated macrophages effectively suppressed cancer cell viability both in vitro and ex vivo.⁴⁷ Current clinical cancer treatments often fail to achieve the desired outcomes due to immune suppression within the tumor microenvironment,⁴⁸ where TAMs play a crucial role. This has led to numerous clinical studies that aimed at targeting macrophage activation for anticancer therapies.^49,50 The demonstrated ability of Ac-SPSs to activate macrophages and mediate anti-lung cancer effects positions them as a promising strategy in future immunotherapy for lung cancer.

4. IMPLICATIONS AND FUTURE RESEARCH

Findings from cell and mouse models indicate that Ac-GPS and Ac-SPS, in addition to their anti-inflammatory properties, could potentially aid conventional anticancer therapy. These Ac-derived polysaccharides exhibit multifunctional anticancer mechanisms, including antiangiogenic effects, promotion of apoptosis, and immunomodulation. They can influence cytokine and chemokine expression in immune cells, helping to balance inflammatory reactions and anti-inflammatory responses. Nevertheless, there is little evidence of Ac examined in human studies. A pilot double-blinded randomized trial in 2016 suggested that Ac extraction might improve survival in patients with high-grade cancers (lung, breast, liver, stomach, and colon) after two months of use. Although the results were not statistically significant, the extraction did alleviate chemotherapy-related adverse events without negatively impacting blood, liver, and kidney functions.⁵¹ Moreover, it was deemed safe and tolerable for human use at high doses, up to 2988 mg/d.⁵² Given these preliminary findings, there is a compelling need for large-scale clinical studies to more thoroughly assess the efficacy of Ac-GPS and Ac-SPS, particularly concerning their roles in immunomodulation and cancer immunotherapy.

ACKNOWLEDGMENTS

This work was supported by grants from the National Science and Technology Council (Taiwan) to T.-Y. Lin (NSTC 112-2320-B-A49-010-MY3, 113-2320-B-A49-028-MY3, and 113-2314-B-532-001-MY3).