Research Progress in the Regulation of Tumor Cells and Tumor Stem Cells at Multiple Targets by Antrodia camphorata

Qingfa CHEN, Yan XU, Xiaodong SHI, Chuanfei WEI, Haitao XIE, Ruxi LV

1. The Institute of Tissue Engineering and Regenerative Medicine, Liaocheng People’s Hospital, Liaocheng 252000, China; 2. Department of Pain, Liaocheng People’s Hospital, Liaocheng 252000, China; 3. Department of Neurosurgery, Weifang Yidu Central Hospital, Weifang 262500, China; 4. Centre for Research, Xiankangda Biotech Corporation, Dongguan 523000, China; 5. Department of Gastroenterology, Liaocheng People’s Hospital, Liaocheng 252000, China

Abstract Extensive in vitro and in vivo research reveals multiple intracellular molecular targets of Antrodia camphorata, and these targets affect growth, apoptosis, angiogenesis, invasion and metastasis of cells. These targets include tumor suppressor, cell cycle regulator, transcription factor, angiogenesis and metastasis factor, apoptosis and survival regulator, etc. Additionally, more and more attention has been paid to the molecular mechanism of A. camphorata on the regulation of tumor stem cells. Meanwhile, there is evidence that the immunoregulation of A. camphorata is enhanced, which may lead cell cycle arrest or apoptosis. In this paper, molecular mechanism of tumor cells and tumor stem cells regulated at multiple targets by A. camphorata in vitro and in vivo in the past decade is summarized.

Key words Antrodia camphorata, Tumor stem cell, Cell cycle regulation, Apoptosis, Transcription factor

1 Introduction

Antrodiacamphoratais a kind of medicinal fungi found only in camphor trees grown in Taiwan, and belongs to the mushroom genus of polypores inBasidiomycetes. Its mycelium and fruiting body are edible, and it contains abundant bioactive compounds, including triterpenoids, flavonoids, polysaccharides, maleic acid/succinic acid, benzophenones and benzoquinone derivatives[1].A.camphoratahas extremely high medicinal value, with anti-cancer, liver protection, anti-inflammatory, antiviral, anti-oxidative stress, immune regulation, anti-hyperlipidemia and other functions[2].

In China, it was estimated that there was a case of invasive cancer every six minutes in 2012 (Annual Report of China Cancer Registration System, 2012). Medicinal mushroom is one of the most promising chemical prevention and treatment options for cancer treatment. The ideal cancer chemotherapy or therapeutic reagent should be able to regulate abnormal signal pathway or induce apoptosis, restore control on normal growth of pre-tumor cells or malignant tumor cells, and target a variety of biochemical and physiological pathways of tumor development[3]. As a kind of medicinal fungi,A.camphoratahas low toxicity, and strong targeting ability of signal molecules for survival and growth of tumor cells. The survival of tumor cells depends on their ability to adapt to changes in their microenvironment, escape the growth inhibition of adjacent normal cells and resist signals of apoptosis and growth inhibition (leading to tissue invasion and metastasis). The abnormal regulation of many molecules and signaling pathways contributes to tumor development. The abnormal regulation of many molecules and signaling pathways contains mutation and activation of oncogeneRasand MYC maladjustment induced by mutation or amplification; overexpression of AP-1 transcription factors c-Fos and c-Jun; amplification, overexpression or mutation of cyclin D/E and Cdks2/4; mutation of apoptotic regulators Fas and Bax; mutation or deletion of tumor suppressor genesp53,PTENandRb; mutation of DNA damage response regulators Chk1/2 and ATM/ATR; mutation, amplification or overexpression of survival kinase Akt; mutation of cell cycle inhibitors p21WAF1, p27KIP1, p14ARF, p16INK4A and p15INK4B and translocation of anti-apoptotic protein Bcl-2. A lot of research shows thatA.camphoratacould regulate these cancer targets, inhibit growth of cancer cells or induce apoptosis, and enhance apoptosis effect of cytokines (such as TRAIL, chemotherapeutic agent and γ ray). So far, hundreds of literatures have revealed the therapeutic effects ofA.camphorataand its derived pure compounds. In this paper, the multiple molecular pathways, pharmacokinetics, bioavailability ofA.camphorataare elaborated by reviewing a large number ofinvitrotumor cell culture andinvivoresearch, and the perspective of its clinical application is prospected.

2 Major regulation functions

2.1RegulationofcellcycleIn tumors, upregulation of cell cycle kinase is induced by overexpression of participant or inactivation of dependent kinase inhibitor. It is found that unbalance of cyclinD1-Rb axis is as common as cyclin-D1 accumulation in hepatoma, breast cancer, skin cancer and lung cancer, and affects regulation of cell cycle[4]. Mycelium and fruiting body ofA.camphorataand their extracts of ethanol, methanol and ethyl acetate all have potential anti-tumor activity[2].

A.camphorataand its extracts have the effects of inducing cell cycle arrest to cause apoptosis for hepatoma, leukemia, lung cancer, rectal cancer, breast cancer and other tumor cells. Via PI3K/AKT/mTOR signaling pathway,A.camphoratainhibits the expression of cell cycle associated proteins of breast cancer T47D, further promoting apoptosis[5]. Chenetal. further find that ethanol extract ofA.camphorata(EEAC) interrupts cell cycle in G1 stage and declines expression of related proteins in G1 stage via AKT/FOXO1 signaling pathway to induce apoptosis[6]. Active ingredient ofA.camphorata(Antroquinonol) causes G1 cell cycle arrest and subsequent apoptosis by inhibiting AMPK activation and mTOR way, further playing anti-tumor activity to hepatoma cells[7]. Active ingredient ofA.camphorata(4-acetylantroquinonol B, 4AAQB) inhibits PI3K/Akt/mTOR way of human hepatoma cell line HuH-7 to induce cell cycle arrest, and controls phosphorylation of ERK/PI3K/mTOR signaling pathway to restrain growth of tumorinvitro[8].

2.2RegulationofapoptosisApoptosis causes a series of biochemical events of characteristic cell morphological changes, containing blistering, cell shrinkage, nuclear fragmentation, chromatin concentration, chromosome DNA fragmentation and death. There are many ways of apoptosis, and it is mostly apoptotic pathway mediated by exogenous death receptor and intrinsic mitochondria at molecular level[9]. Most cytotoxic chemotherapy drugs are used to induce apoptosis.A.camphoratahas huge anti-cancer potential, and potential cytotoxic effect on lung cancer, hepatoma, breast cancer, prostate cancer, rectal cancer, oral cancer and other cancer cells[4]. Active ingredients ofA.camphorataSY-1 derivative and triterpenes generate direct cytotoxicity on colon cancer Colo 205 and HT-29 cells by inducing apoptosis[10]. Ethanol extract ofA.camphoratafruiting body (ACEE) increases the expression of p53 and Bax in lung cancer LLC cell, promotes the cracking of poly ADP ribose polymerase (PARP) and caspase-3, and declines the expression of survivin and Bcl-2. ACEE declines simultaneously the expression of JAK2 and phosphorylation of STAT3 in lung cancer LLC cell[11]. Triterpenoid (MAA) isolated fromA.camphoratasignificantly inhibits proliferation of hepatoma cell lines Huh7, HepG2 and Hep3B when compared with normal cell. MAA triggers mitochondrial apoptosis pathway by increasing the expression of Bax, Bak and PUMA, declining the expression of Bcl-XL and Bcl-2, and promoting the increase of DNA fragment, the cracking of PARP, the increase of cell number in G1 stage, chromosome condensation, disruption of mitochondrial membrane potential, release of mitochondrial cytochrome c and the activation of caspases-2, caspases-3 and caspases-9[12]. Active ingredient ofA.camphorataAntrocin inhibits the expression of β-catenin mediated by the acceptor of type 1 insulin-like growth factor 1 (IGF-1R), and regulates cell cycle and apoptosis of prostatic cancer PCa cell by down regulating signaling pathways of PI3K/AKT and MAPK[13]. Co-culture of ethanol extract ofA.camphorata(EAC) and ginger greatly induces the generation of triterpenoids, and induces apoptosis of Huh-7 and HepG2 cells by promoting accumulation in G2/M stage and caspase-3 disruption. Therefore, co-culture of EAC and ginger could be taken as a kind of method effectively curing hepatoma[14].

2.3RegulationoftumorinvasionandmetastasisTumor metastasis is the fact that cancer cells move from the primary site to other sites and continue to grow through lymphatic channels, blood vessels or body cavities. Tumor migration requires two different functions: strong mobility and invasion of cancer cells, and the migrating cells are basically the same as the primary cancer. Besides cadherin, matrix metalloproteinase (MMP) and urokinase plasminogen activator (uPA) also play important role in invasion and migration of tumor[15]. Anti-cancer and anti-migration activities ofA.camphorataare mainly from its polysaccharides, triterpenoids, benzenes, lignans, diterpenoids, steroids,etc[4]. Antroquinonol (ANQ) isolated from mycelium ofA.camphoratacould inhibit significantly migration and invasion of breast cancer MDA-MB-231 cell, and the invasion of MCF7 cell induced by TPA. Specific mechanism is that ANQ could inhibit the phosphorylation of ERK and AKT, further inhibiting gene expression and enzyme activity of MMP-9[16]. Active ingredient ofA.camphorataAntcin-A (ATA) could block obviously epithelial-mesenchymal transition (EMT) of breast cancer cells MCF-7 and MDAMB231. The process is realized by up regulating epithelial cell markers (E-cadherin and occludin), down regulating mesenchymal cell markers (N-cadherin and vimentin) and the activity of miR-200c mediated by p53[17]. The extract ofA.camphoratafruiting body could inhibit the activities of MMP-9 and uPA, further inhibiting invasion and migration of breast cancer cell MDA-MB-231. Additionally, extract ofA.camphoratafruiting body also declines the expression of MMP-2, MMP-9, uPA, uPAR and VEGF related to migration[18]. Active ingredient ofA.camphorata4AAQB shows good anti-tumor generation and anti-metastasis effects in tumor xenotransplantation and lung metastasis model. In the process, 4AAQB declines the production of VEGF and inhibits the activation of GTPase via Rho and Rac1, and 4AAQB could reduce pulmonary metastases in the model of lung metastasisinvivo[19]. 2,3,5-trimethoxy-4-cresol (TMC) obtained from mycelium ofA.camphorataby solid culture could decline migration and invasion of lung cancer cells. It is found that TMC increases the levels of E-cadherin and TIMP-1 and declines the levels of Akt, MMP-2 and MMP-9, to inhibit migration of lung cancer cells[20]. Head and neck cancer is resistant to traditional therapy. The ethanol extract ofA.camphorataand lyophilized mycelium powder ofA.camphoratacould effectively inhibit growth and migration of head and neck cancer cellsinvivo, without side effects[21]. EEAC and its active ingredients (adenosine, zhankuic acid A, and cordycepin) could change migration of hepatoma cells by inhibiting the ERp57 and PGK-1, PI3K/Akt and MAPK signaling pathways[22].

2.4RegulationoftumorangiogenesisAngiogenesis refers to the process of new blood vessels forming from existing blood vessels, and angiogenesis plays an important role in the development of tumor.A.camphorataand other medicinal mushrooms have the function of inhibiting angiogenesis[23]. Recent clinical studies find that the treatment effect of tumor is the most obvious when combining antiangiogenic therapy with chemotherapy or radiotherapy[24]. ACEE could inhibit cell migration and angiogenesis in human umbilical vein endothelial cells (HUVEC). ACEE inhibits the phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) and expression of angiogenic kinase in HUVECs induced by vascular endothelial growth factor (VEGF), and decreases the expression of Janus kinase 2 (JAK2) and the activation of transcription activator 3 (STAT3). ACEE can inhibit VEGF-induced microvascular formationinvivo, and ACEE declines obviously the expression of VEGFR2 in Lewis lung cancer cell, and downregulates the expression of CD31 and VEGFR2 in lung cancer transplanted mice[25]. The anti-tumor effect ofA.camphoratais not limited to its direct cytotoxicity to tumor cells, but also shown as the indirect way of closing nutrition supply and escape channel of solid tumor or inhibiting angiogenesis, migration and expression of hypoxia inducible factor-α (HIF-α) related to cancer stem cells. Biological efficacy of extract ofA.camphoratafruiting body (AC+3E) is verified in tamoxifen resistant breast cancer cell (ER+T47D).Invitrostudy shows that AC+3E could inhibit vitality and migration of ER+T47D breast cancer cell. AC+3E inhibits significantly PI3K/Akt and cyclin signaling pathway. Meanwhile, AC+3E extract also changes Jak/Stat3 signaling pathway of tumor cell. AC+3E therapy inhibits vitality and migration of tamoxifen resistant breast cancer cells by upregulating the expression of E-cadherin and downregulating the expression of β-catenin, Rac1 and RhoA.Invivostudy shows that AC+3E extract inhibits effectively tubular structure of endothelial cells and tumor growth. AC+3E extract could carry out simultaneous targeted therapy on tamoxifen resistant breast cancer cells and tumor related vascular cells[5].

2.5Regulationofactivityofnucleartranscriptionfactor

NF-κB (nuclear factor-κB) is a kind of heterodimer proteins, and is composed of different combinations of members from transcription factor Rel family. Rel/NF-κB transcription factor family mainly joins in stress-induced immunity and inflammatory responses. These molecules play important role in the development of hematopoietic cells, keratinocytes and structure of lymphoid organs. Additionally, NF-κB family members are related to the development of tumor and formation of synapses. Activated protein 1 (AP-1) is another transcription factor, and could regulate the expression of several genes joining in cell differentiation and proliferation. Functional activation of AP-1 transcription complex is related to tumor formation and malignant transformation. The complex is composed of homodimer or heterodimer of members of JUN and FOS protein families. p53 is a kind of negative regulators of cell proliferation, which could regulate cell growth and differentiation, and EMT and metastasis of cancer cells via β-catenin signaling pathway.

The extract ofA.camphoratamycelium inhibits the invasion of MDA-MB-231 breast cancer cell by restraining the degradation of extracellular matrix (ECM), and downregulating the expression of MMPs (containing MMP-9 and MMP-2), uPA and uPAR. The extract ofA.camphoratamycelium inhibits the expression of MMP-2, MMP-9, uPA and uPAR in MDA-MB-231 cell, further inhibiting NF-κB nuclear transposition by downregulating MAP kinase cascade ERK1/2, JNK and p38[18]. In MDA-MB-231 breast cancer cell, AP-1 and NF-κB channels induce MMP-9 gene expression and EMT. Active ingredient ofA.camphorataAntroquinonol could inhibit signal transduction of AP-1 and NF-κB. Antroquinonol inhibits expression of EMT related proteins and TPA induced EMT of breast cancer cell MCF-7 by downregulating ERK-AP-1 and AKT-NF-κB signaling cascade. Antroquinonol inhibits aggressiveness of breast cancer by restraining the expression of MMP-9 and EMT depending on ERK-AP-1 and AKT-NF-κB[16]. The extract ofA.camphoratamycelium 4-acetylantroquinonol B could affect the expression of proteins p53, p21 and p27 to inhibit the proliferation of HepG2 cell[26].

2.6RegulationoftumorimmunogenicityAnother anti-tumor mechanism ofA.camphoratais to regulate the immune response of the body. Fruiting body ofA.camphoratacould enhance the functions of macrophagesinvivo, improve proliferative activity of T cells and the ability of B cells to produce antibodies, and restore the proportion of CD4+/CD8+. Oral administration of 172.2, 344.4 and 861.0 mg/kg ofA.camphoratafruiting body in BALB/c mice enhances the activities of natural killer cells (NK) and lymphokine activated killer cells (LAK)invivosignificantly, and increases serum concentrations of IL-2, IFN-γ and immunoglobulin IgG significantly[27]. The life of ATCC BNL IMEA.7R.1 hepatoma mice is prolonged significantly, and the cytotoxicity to hepatoma cells is enhanced after oral administration of high-dose (100 mg/kg) and moderate-dose (33 mg/kg) fruiting body ofA.camphorata. The immune regulation of hepatoma bearing mice is changed by upregulating the generation of IFN-γ, IL-2 and TNF-α, increasing serum level of tumor-specific IgG, and increasing TNF-α and nitric oxide via peritoneal macrophage[28].

2.7RegulationoftumorstemcellsMany tissue-specific cancer stem cells (CSC) have the ability of self-renewal and differentiation, and the resistance to chemotherapy and radiotherapy, and CSC cannot be eliminated by conventional anti-cancer drugs and therapies[29]. CD44, CD133 and acetaldehyde dehydrogenase 1 (ALDH1) are recognized CSC marker protein, and CD34+/CD133+CSC could be isolated from non-CSC tumor cells via flow cytometry[4]. As traditional Chinese drug with multiple biological functions,A.camphoratahas anti-CSC tolerance to chemotherapy and radiotherapy. Fermented products ofA.camphoratamycelium (AC-MFB) could inhibit viability of endothelial cells, cell migration and angiogenesis in EA.hy926 and SVEC4-10 tumor cells. The extracted active ingredient ofA.camphoratamycelium 4-Acetyl-antroquinonol B (4-AAQB) has obvious chemical sensitivity and effect on CSC cell of colon cancer. 4-AAQB could effectively disturb tumor signal pathway, inhibit acquisition of CSC phenotype, downregulate stem cell related genes, weaken tumor invasiveness, and reverse chemical tolerance of colon cancer cells[30]. 4-AAQB also could inhibit catenin/LEF1/Stat3 signaling pathway, further inhibiting tumorigenic activity of U87MG and DBTRG-05MG glioma cellsinvitroandinvivoinduced by CSC[31].A.camphoratacould upregulate tumor suppressor miR-142-3p to enhance chemical sensitivity of 5-FU, and inhibit occurrence and canceration of colon tumor[32].A.camphoratamycelium and its ethyl acetate extract could inhibit CD133+CSC of multiple tumors, such as glioma (GBM 8401), lung cancer (A549), breast cancer (MDA-MB-231), hepatoma (HepG2), rectal cancer (SW620) and squamous cell carcinoma of head and neck (SAS).A.camphorataimproves anti-proliferation ability of CD133+CSC for all tumors, especially anti-proliferation ability of CSC for squamous cell carcinoma of head and neck, breast cancer and lung cancer. The research also shows thatA.camphorataenhances radiotherapy or chemotherapy to treat tumor CSC by inhibiting the expression of a variety of microRNAs in CSC of breast cancer and glioma[33].

3 Conclusions and prospects

As a rare medicinal fungus in Taiwan,A.camphoratahas been highly concerned for 30 years since it was found in the 1990s. Researchers have shifted from identifying the biological function of the total extract ofA.camphoratato identifying specific functional targets of single active compounds[34]. Many animal model tests have been used to study the activity and function of different compounds ofA.camphorata, such as anti-tumor, hepatoprotective, anti-inflammatory, immunomodulatory, and antioxidant effects.

Similar to gallic acid (EGCG), quercetin, genistein, curcumin and resveratrol,A.camphoratacould improve greatly human health.A.camphoratahas low toxicity and the ability to target a variety of signal molecules related to survival and growth of tumor cells. The mechanism of anti-tumor activity ofA.camphoratais not single, containing inducing apoptosis, activating calcium-calpain-mitochondria pathway, inhibiting tumor angiogenesis and nuclear transcription factor, and regulating immunity. It still needs a large number ofinvivoandinvitroexperiments to further illustrate anti-tumor mechanism ofA.camphorata. Additionally,A.camphoratacontains many ingredients, and the function of single compound is not clear, which needs further research.

The pharmacological mechanism ofinvivotest ofA.camphoratahas been applied in clinical research. Via the clinical trial method, pharmacokinetics, pharmacodynamics and safety of Antroquinonol (a kind of active compounds fromA.camphorate) are studied, and it is in clinical phase II currently[35]. Due to lack of chemotherapy drugs for malignant tumors, active compounds ofA.camphorataare particularly urgent for the potential treatment of triple-negative breast cancer that estrogen receptor is negative, mesothelioma, acute lymphocytic leukemia, acute myelocytic leukemia, Hodgkin’s lymphoma, malignant astrocytoma,etc. Current research data could provide new insights for potential treatment of tumors byA.camphorata, and useful suggestions for designing antifungal drugs.