Phellinus linteus suppresses growth, angiogenesis and invasive behaviour of breast cancer cells through the inhibition of akt signalling

ABSTRACT The antitumour activity of a medicinal mushroom _Phellinus linteus_ (PL), through the stimulation of immune system or the induction of apoptosis, has been recently described.

However, the molecular mechanisms responsible for the inhibition of invasive behaviour of cancer cells remain to be addressed. In the present study, we demonstrate that PL inhibits

proliferation (anchorage-dependent growth) as well as colony formation (anchorage-independent growth) of highly invasive human breast cancer cells. The growth inhibition of MDA-MB-231 cells

is mediated by the cell cycle arrest at S phase through the upregulation of p27Kip1 expression. _Phellinus linteus_ also suppressed invasive behaviour of MDA-MB-231 cells by the inhibition

of cell adhesion, cell migration and cell invasion through the suppression of secretion of urokinase-plasminogen activator from breast cancer cells. In addition, PL markedly inhibited the

early event in angiogenesis, capillary morphogenesis of the human aortic endothelial cells, through the downregulation of secretion of vascular endothelial growth factor from MDA-MB-231

cells. These effects are mediated by the inhibition of serine-threonine kinase AKT signalling, because PL suppressed phosphorylation of AKT at Thr308 and Ser473 in breast cancer cells. Taken

together, our study suggests potential therapeutic effect of PL against invasive breast cancer. SIMILAR CONTENT BEING VIEWED BY OTHERS ANTI-METASTASIS ACTIVITY OF 5,4’-DIHYDROXY

6,8-DIMETHOXY 7-O-RHAMNOSYL FLAVONE FROM _INDIGOFERA ASPALATHOIDES_ VAHL ON BREAST CANCER CELLS Article Open access 29 May 2024 SULFORAPHANE SUPPRESSES METASTASIS OF TRIPLE-NEGATIVE BREAST

CANCER CELLS BY TARGETING THE RAF/MEK/ERK PATHWAY Article Open access 24 March 2022 GRACILLIN SUPPRESSES CANCER PROGRESSION THROUGH INDUCING MERLIN/LATS PROTEIN-PROTEIN INTERACTION AND

ACTIVATING HIPPO SIGNALING PATHWAY Article 07 March 2025 MAIN Despite the fact that breast cancer mortality of breast cancer decreased by 22.9% from 1991 to 2003, breast cancer remains the

leading cause of cancer death among 20- to 59-year-old US women with estimated 40 460 new death in 2007 (Jemal et al, 2007). One of the reasons for such a high mortality is the invasive

behaviour of cancer cells, which results in the metastasis of breast cancer. Cancer metastasis consists of several interdependent processes including uncontrolled cell proliferation,

invasion through surrounding tissues, migration to the distant sites of the human body, and adhesion, invasion and colonisation of other organs and tissues (Price et al, 1997). Tumour growth

and metastasis also require angiogenesis, the formation of blood vessels by capillaries sprouting from pre-existing vessels (Folkman, 1995). Therefore, inhibition of growth, invasive

behaviour and cancer cell-mediated angiogenesis will lead to the suppression of cancer metastasis and would further increase survival of breast cancer patients. _Phellinus linteus_ (PL) is a

basidiomycete fungus, located mainly in tropical America, Africa and Asia, where it gained significant recognition as medicinal mushroom in the traditional Oriental medicine (Dai and Xu,

1998). The biologically active compounds isolated from PL are polysaccharides (Song et al, 1995), acidic proteo-heteroglycans with mixed _α_- and _β_-linkages, and a (1 → 6)-branched type (1

→ 3)-glycan (Kim et al, 2003b). These complex polysaccharides have been detected in a variety of different mushroom species and linked to the immunostimulatory and antitumour activities

(Wasser, 2002). Therefore, PL stimulated proliferation of T lymphocytes and activated B cells (Kim et al, 1996, 2003c), and induced maturation of bone marrow-derived dendritic cells (Park et

al, 2003) and the macrophage response (Kim et al, 2003a). On the other hand, PL demonstrated anti-inflammatory effects in lipopolysaccharide-stimulated macrophages (Kim et al, 2006). The

direct anticancer effect of PL has been demonstrated by the inhibition of invasive melanoma B16BL6 cells through the downregulation of mRNA level of urokinase-plasminogen activator (uPA),

and by the inhibition of pulmonary metastasis in mice (Lee et al, 2005). _Phellinus linteus_ suppressed proliferation by the inhibition of cyclin-dependent kinases cdk2, 4 and 6, and induced

apoptosis through the activation of caspase 3 in lung cancer cells (Guo et al, 2007) and apoptosis of prostate cancer cells (Collins et al, 2006; Zhu et al, 2007). In the present study, we

evaluated the effect of PL on the highly invasive and metastatic human breast cancer cells. Here, we show that PL inhibits proliferation (anchorage-dependent growth) as well as colony

formation (anchorage-independent growth) of highly invasive human breast cancer cells. In addition, PL also suppresses invasive behaviour of MDA-MB-231 cells by the inhibition of cell

adhesion, cell migration and cell invasion. Finally, PL suppressed breast cancer cell-mediated angiogenesis of endothelial cells _in vitro_. Collectively, our study suggests the mechanism(s)

employed for the inhibition of proliferation, invasive behaviour and angiogenesis of invasive breast cancer cells by an extract from medicinal mushroom PL. MATERIALS AND METHODS CELL

CULTURE AND REAGENTS Human breast cancer cells (MCF-7, MDA-MB-231) and human prostate cancer cells (PC-3, LNCaP) were obtained from ATCC (Manassas, VA, USA). MCF-7 and MDA-MB-231 cells were

maintained in DMEM medium, PC-3 cells were maintained in F-12 medium and LNCaP cells were maintained in RPMI 1640 medium. All media contained penicillin (50 U ml−1), streptomycin (50 U 

ml−1), and 10% fetal bovine serum (FBS). Media and supplements came from GIBCO BRL (Grand Island, NY, USA). Fetal bovine serum was obtained from Hyclone (Logan, UT, USA). Aqueus extract of

PL was supplied by the Maitake Products Inc. (Paramus, NJ, USA). Stock solution was prepared by dissolving PL in sterile water at a concentration 50 mg ml−1 and stored at 4°C. AKT inhibitors

LY294002 and AKT inhibitor III were obtained from Calbiochem (San Diego, CA, USA). CELL PROLIFERATION ASSAY Cell proliferation was determined by the tetrazolium salt method, according to

the manufacturer's instructions (Promega, Madison, WI, USA). Briefly, cancer cells were cultured in a 96-well plate and treated at indicated times with PL (0–1.0 mg ml−1). At the end of

the incubation period, the cells were harvested and absorption was determined with an ELISA plate reader at 570 nm, as described (Jiang et al, 2004b). Data points represent mean±s.d. in the

representative experiment of triplicate determinations. Similar results were obtained in two independent experiments. CELL VIABILITY Cell viability of MCF-7 and MDA-MB-231 cells was

determined after incubation with PL (0–1.0 mg ml−1) for 24, 48 and 72 h by staining with Trypan Blue as described (Sliva et al, 2002a). ANCHORAGE-INDEPENDENT GROWTH MDA-MB-231 cells were

harvested and seeded in six-well plates coated with 1% agarose. Anchorage-independent growth was assessed after incubation for 10–14 days with culture media with or without PL (0–1.0 mg 

ml−1), which were replaced every 4 days. Plates were stained with 0.005% crystal violet, and the colonies were counted manually under a microscope and photographed (Slivova et al, 2004).

CELL CYCLE ANALYSIS MDA-MB-231 cells (0.75 × 106) were seeded and after 24 h treated with PL (0.5 mg ml−1) for the indicated period of time (0–48 h). After incubation, the cells were

harvested by trypsinisation, washed with Dulbecco's phosphate-buffered saline containing 2% FBS, and resuspended in propidium iodine (50 _μ_g ml−1). Cell cycle analysis was performed on

a FACStarPLUS flow cytometer (Becton-Dickinson, San Jose, CA, USA), as previously described (Sliva et al, 2001). Data are the mean±s.d. from three independent experiments. CELL ADHESION,

MIGRATION AND INVASION ASSAYS Cell adhesion was performed with Cytomatrix Adhesion Strips coated with human vitronectin (Chemicon International, Temecula, CA, USA). Briefly, MDA-MB-231 cells

were treated with PL (0–0.5 mg ml−1) for 24 h, harvested and counted. Cell adhesion was determined after 1.5 h of incubation at 37°C (Lloyd et al, 2003). Cell migration of MDA-MB-231 cells

treated with PL (0–1.0 mg ml−1) was assessed in Transwell chambers in the DMEM medium containing 10% FBS (Slivova et al, 2004). Invasion of MDA-MB-231 cells treated with PL (0–1.0 mg ml−1)

was assessed in Transwell chambers coated with 100 _μ_l of Matrigel™ (BD Biosciences, Bedford, MA, USA) diluted 1:4 with DMEM, after 72 h of incubation (Slivova et al, 2004). _IN VITRO_

ENDOTHELIAL CELL MORPHOGENESIS ASSAY (CAPILLARY MORPHOGENESIS) Human aortic endothelial cell (HAEC) differentiation into ‘capillary-like’ structures was observed using a two-dimensional

Matrigel-based assay as we described previously (Harvey et al, 2002). Initially, 200 _μ_l of ice-cold growth factor-reduced Matrigel (Becton Dickinson Labware, Bedford, MA, USA), an

extracellular matrix preparation derived from the Engelbreth–Holm–Swarm tumour, was placed into each well of a 24-well tissue culture-treated plate. Human aortic endothelial cells were

harvested, resuspended in serum-free EBM media and plated at 3.5 × 104 cells per well-coated with Matrigel. Human aortic endothelial cells were further incubated with PL (0–0.5 mg ml−1) or

with conditioned media from MDA-MB-231 cells, which were prepared by the incubation of MDA-MB-231 cells in the presence of PL (0–0.5 mg ml−1) for 24 h. Endothelial HAE cells differentiated

into capillary-like structures within 16 h of incubation at 37°C in the presence of 5% CO2. These structures were examined microscopically ( × 40) using an inverted Olympus CK40 microscope.

To facilitate analysis of the structures, non-adherent cells incorporated in excess medium were removed from each well prior to quantitative analysis. Photomicrographs were taken to assess

the extent of capillary-like structural formation. Quantification of the capillary-like structures was performed counting the number of nodes per field, where a node is defined as an

intersection of at least three cells. Each sample was assayed in triplicate and reproduced in at least two additional experiments. WESTERN BLOT ANALYSIS MDA-MB-231 cells were treated with PL

(0–1.0 mg ml−1) for 24–72 h as indicated in the text and whole-cell extracts prepared as described previously (Sliva et al, 2002a, 2002b). Equal amounts of proteins (20 _μ_g per lane) were

separated on NuPAGE 4–12% Bis-Tris gel (Invitrogen, Carlsbad, CA, USA) and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). The protein expression was detected with the

corresponding primary antibodies: anti-cyclin-D1, anti-cyclin-E, anti-cyclin-A, anti-cdk2, anti-cdk4, anti-p21, anti-p27, anti-_β_-actin, (Santa, Cruz Biotechnology, Santa Cruz, CA, USA),

anti-AKT, anti-phospho-AKT (Thr308) and anti-phospho-AKT (Ser473) (Cell Signaling, Beverly, MA, USA), respectively. Protein expression was visualised using the ECL Western Blotting Detection

System (Amersham Biosciences, Buckinghamshire, UK). UROKINASE-PLASMINOGEN ACTIVATOR SECRETION DMEM media from MDA-MB-231 cells treated with PL (0–1.0 mg ml−1) for 24 h were collected and

concentrated, and the secretion of uPA was detected by western blot analysis with anti-uPA antibody (Oncogene Research Products, Cambridge, MA, USA), as described (Sliva et al, 2002b).

DENSITOMETRIC ANALYSIS Autoradiograms of the western blots were scanned with HP scanjet 5470c scanner. The optical densities of p27, phospho-AKT (Thr308), phospho-AKT (Ser473), AKT,

_β_-actin and uPA proteins on the films were quantified and analysed with the UN-SCAN-IT software (Silk Scientific, Orem, UT, USA). The ratios of p27/_β_-actin and phosphoAkt/Akt were

calculated by standardising the ratios of each control to the unit value. VASCULAR ENDOTHELIAL GROWTH FACTOR SECRETION MDA-MB-231 cells were treated with PL (0–0.5 mg ml−1) for 24 h, cell

media collected and secretion of vascular endothelial growth factor (VEGF) was determined using a respective Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the

manufacturer's instructions. STATISTICAL ANALYSIS Data are presented as means±s.d. Statistical comparison between the control group (0 _μ_g ml−1 of PL) and groups with different PL

doses was carried out using two-sided Student's _t_-tests. The value of _P_<0.05 was considered to be significant. RESULTS _PHELLINUS LINTEUS_ SUPPRESSES PROLIFERATION AND COLONY

FORMATION OF HIGHLY INVASIVE BREAST CANCER CELLS Invasive behaviour of cancer cells is directly linked to their metastatic potential resulting in the high cancer mortality. Therefore, we

evaluated if PL inhibits growth of highly invasive (MDA-MB-231) and poorly invasive (MCF-7) breast cancer cells. As seen in Figure 1, increased concentration of PL (0–1.0 mg ml−1) markedly

suppressed proliferation of MDA-MB-231 as well as MCF-7 cells in a dose- and time-dependent manner. Nevertheless, the effect of PL on poorly invasive cells was more pronounced, because the

concentration 0.25, 0.5 and 1.0 mg ml−1 of PL suppressed proliferation of MCF-7 cells by 60.2, 70.1 and 78.0%, respectively (Figure 1B), whereas the same concentration suppressed

proliferation of MDA-MB-231 cells by 15.5, 21.5 and 43.1%, respectively (Figure 1A), after 24 h of incubation. The same sensitivity of MCF-7 cells was evident also after additional 48 and 72

 h of incubation, where only the highest concentration of PL (1.0 mg ml−1) suppressed proliferation of poorly invasive and highly invasive breast cancer cells with the same potency (Figure

1). To determine if the effect of PL on cancer cells is cytotoxic or cytostatic, we evaluated the cell viability after 24, 48 and 72 h of PL treatment. Although PL decreased the viability of

MDA-MB-231 and MCF-7 cells, the strongest inhibition of cell viability at the highest used concentration of PL (1.0 mg ml−1) after 72 h was only 13.5% for MDA-MB-231 cells (Figure 1C) and

10.6% for MCF-7 cells (Figure 1D), whereas the same concentration suppressed proliferation of MDA-MB-231 cells by 86.6% (Figure 1A) and MCF-7 cells by 90.6% (Figure 1B). Therefore, these

data suggest that the PL inhibits growth of breast cancer cells predominantly through its cytostatic effect. Interestingly, PL also suppressed proliferation of poorly invasive prostate

(LNCaP) and highly invasive prostate (PC-3) cancer cells in a dose- and time-dependent manner, and LNCaP cells were more sensitive to the PL treatment (not shown). In addition to cell

proliferation (anchorage-dependent growth), colony formation (anchorage-independent growth) is one of the typical characteristic of the metastatic potential of cancer cells _in vitro_ and

strongly correlates with tumorigenesis _in vivo_ (Freedman and Shin, 1974). To determine whether PL suppresses colony formation of highly invasive breast cancer cells, we evaluated the

anchorage-independent growth of MDA-MB-231 cells. As seen in Figure 2, MDA-MB-231 cells formed colonies on agar after 14 days of incubation, and the presence of increased concentration of PL

(0–1.0 mg ml−1) resulted in the significant suppression of number of colonies (Figure 2E). Therefore, PL inhibits anchorage-dependent as well as anchorage-independent growth of highly

aggressive breast cancer cells. _PHELLINUS LINTEUS_ INDUCES CELL CYCLE ARREST AT S PHASE To determine whether the inhibition of cell proliferation is associated with cell cycle arrest,

MDA-MB-231 cells were treated for 24 and 48 h with PL (0.5 mg ml−1) and analysed by flow cytometry. Cell cycle analysis demonstrated that PL causes cell cycle arrest at S phase of cell cycle

(Table 1), where the amount of cells in S phase significantly increased from 27% (control – 0 h) to 34% (24 h) and 44% (48 h). In addition, PL treatment did not induce the amount of cells

in sub-G0/G1 phase, further suggesting the cytostatic effect of PL on MDA-MB-231 cells (Table 1). To examine the mechanism responsible for the cell cycle arrest at S phase, we evaluated the

expression of cell cycle regulatory proteins (cyclins) and cdks involved in the progression from G1 phase to S phase (Sherr and Roberts, 1999; Ekholm and Reed, 2000). Therefore, MDA-MB-231

cells were treated with PL (0–1.0 mg ml−1) for 24 h and the expression of cyclin D1, E, A, cdk4, cdk2, p21 and p27 in cell extracts was evaluated by western blot analysis with respective

antibody. Neither of the G1 regulatory proteins cyclin-D1, cdk4 or p21, G1-late phase or G1/S-phase regulatory proteins cyclin E, A and cdk2 demonstrated changes in their expression after

the treatment with PL (Figure 3A). Nevertheless, PL (0.5 and 1.0 mg ml−1) markedly induced expression of a cyclin-dependent kinase inhibitor p27 (Figure 3B). Therefore, PL inhibits

proliferation of breast cancer cells by the upregulation of p27 resulting in S-phase cell cycle arrest. _PHELLINUS LINTEUS_ SUPPRESSES INVASIVE BEHAVIOUR OF BREAST CANCER CELLS The ability

of cancers to metastasise is directly associated to cell adhesion, migration and invasion. Integrin receptor _α_V_β_3 is involved in adhesion of breast cancer cells through its interaction

with extracellular matrix (ECM) protein vitronectin (Wong et al, 1998). To investigate if PL affects adhesion of invasive breast cancer cells, MDA-MB-231 cells were pretreated with PL (0–0.5

 mg ml−1) for 24 h and their adhesion to vitronectin was determined. As seen in Figure 4A, adhesion of MDA-MB-231 cells was markedly suppressed by the PL treatment. Next, we evaluated if PL

also inhibits cell migration. MDA-MB-231 cells were pretreated with PL (0–1.0 mg ml−1) for 1 h and cell migration was determined after additional 5 h of incubation. As seen in Figure 4B, PL

also markedly suppressed migration of breast cancer cells in a dose-dependent manner. Finally, the effect of PL on cell invasiveness was evaluated. MDA-MB-231 cells were plated on the

Matrigel-coated filters in the presence of PL (0–1.0 mg ml−1) and the amount of cells invaded through Matrigel counted after 48 h of incubation. As seen in Figure 4C, PL inhibits invasion of

MDA-MB-231 cells in a dose–response manner. Because cancer metastasis and invasiveness are associated with the uPA–uPA receptor (uPAR) system, we evaluated whether PL affects secretion of

uPA from cancer cells. MDA-MB-231 cells were treated with PL (0–1.0 mg ml−1) for 24 h and uPA secretion evaluated by western blot analysis in conditioned media. In agreement with the data

demonstrating inhibition of cell adhesion, migration and invasion PL markedly decreased secretion of uPA from MDA-MB-231 cells (Figure 4D). _PHELLINUS LINTEUS_ INHIBITS CAPILLARY

MORPHOGENESIS OF ENDOTHELIAL CELLS THROUGH THE SUPPRESSION OF VEGF SECRETION Capillary morphogenesis (tube formation) of human endothelial cells is one of the first important steps in

angiogenesis associated with the cancer progression and metastasis. Because cancer microenvironment contains a variety of cells, we also evaluated if the inhibition of capillary

morphogenesis of endothelial cells can be mediated through breast cancer cells. Therefore, we determined if PL itself or conditioned media from breast cancer cells exposed to PL suppress

capillary morphogenesis of HAECs. Human aortic endothelial cells grown on Matrigel were treated directly with PL (0–0.5 mg ml−1) or with the conditioned media from MDA-MB-231 cells treated

with PL (0–0.5 mg ml−1, PL-CM) for 24 h, and tube formation were evaluated. As seen in Figure 5A, PL significantly suppressed capillary morphogenesis of HAECs. Moreover, conditioned media

from MDA-MB-231 cells without PL induced capillary morphogenesis of HAECs (PL _vs_ PL-CM at 0 PL), and conditioned media from cells exposed to PL (PL-CM) also suppressed capillary

morphogenesis of HAECs in a dose–response manner (Figure 5A–C). Because MDA-MB-231 cells express pro-angiogenic VEGF (Basu et al, 2005), we hypothesised that secreted VEGF from these cells

induces capillary morphogenesis of endothelial cells, which can be inhibited by PL. Therefore, we evaluated whether PL inhibits secretion of VEGF from MDA-MB-231 cells treated with PL (0–0.5

 mg ml−1). As seen in Figure 5D, secretion of VEGF from MDA-MB-231 cells was markedly decreased by PL in a dose–response manner. Collectively, our data suggest that PL inhibits capillary

morphogenesis of endothelial cells directly as well as indirectly through the suppression of secretion of VEGF from breast cancer cells. _PHELLINUS LINTEUS_ INHIBITS ACTIVITY OF AKT KINASE

AKT serine-threonine kinase (protein kinase B) regulates a variety of cellular processes through the phosphorylation of a wide spectrum of downstream substrates finally resulting in the

expression of proteins involved in cell proliferation, invasiveness and angiogenesis among others (Woodgett, 2005; Dillon et al, 2007). To determine if PL modulates AKT activity in breast

cancer cells, MDA-MB-231 cells were treated with PL (0–1.0 mg ml−1) for 24 h and the phosphorylation status of AKT evaluated in whole-cell extracts by western blot analysis. As seen in

Figure 6A, PL inhibits phosphorylation of AKT at Thr308 in a dose–response manner. In addition, PL treatment also markedly decreased phosphorylation of AKT at Ser473 (Figure 6B). To evaluate

if the suppression of AKT activity PL is directly responsible for the inhibition of capillary morphogenesis through the downregulation of expression of VEGF in MDA-MB-231 cells, we used a

pharmacological approach with specific AKT inhibitors. Therefore, MDA-MB-231 cells were treated with LY294002 (10 _μ_ M) or AKT inhibitor III (10 _μ_ M) for 24 h, and secretion of VEGF was

evaluated. As seen in Figure 7A, AKT inhibitors markedly suppressed secretion of VEGF from breast cancer cells. Moreover, conditioned media from MDA-MB-231 cells treated with LY294002 and

AKT inhibitor III also inhibited capillary morphogenesis of endothelial cells (Figure 7B). Therefore, these data suggest that the inhibition of angiogenesis _in vitro_ by PL is mediated, to

some extent, through the suppression of AKT activity, which results in the suppression of secretion of VEGF from breast cancer cells. DISCUSSION Regardless of different theories of biology

of breast cancer as (i) a local disease that spreads over time to develop distant metastases; (ii) a systemic disease from the outset, with distant metastases present well before diagnosis

(iii) or the combination of both as a heterogeneous disease, cancer metastasis is one of the major medical problems in breast cancer patients (Punglia et al, 2007). While several

chemotherapeutic agents or their combinations (e.g. taxanes, trastumazab, gemcitabine or capecitabine) demonstrated activity in the metastatic breast cancer setting (Tripathy, 2007), there

is a paucity of natural antiproliferative and anti-metastatic nontoxic agents. As demonstrated practically four decades ago, polysaccharide extracts from basidiomycete fungus PL suppressed

tumour growth _in vivo_ (Chihara et al, 1969). In addition, PL also reduced tumour growth and the frequency of pulmonary metastasis without toxic effects (Han et al, 1999). Recent studies

elucidated some of the molecular mechanism(s) responsible for the inhibition of growth through cell cycle arrest and induction of apoptosis in lung and prostate cancer cells (Collins et al,

2006; Guo et al, 2007; Zhu et al, 2007). Nevertheless, the molecular mechanism(s) responsible for the inhibition of invasive behaviour and angiogenesis was not fully addressed. In the

present study, we demonstrate that PL inhibits cell proliferation (anchorage-dependent growth) as well as colony formation (anchorage-independent growth) of highly invasive breast cancer

cells through the S-phase cell cycle arrest mediated by the upregulation of expression of p27. Cell growth, which is the reflection of the progression of cell cycle, is aberrantly regulated

in the majority of cancers. The cell cycle is regulated by a series of checkpoints employing cyclins, cdks and cdk inhibitors (Sherr, 1966). p27 is one of the cdk inhibitors, which binds to

S-phase cyclin–cdk complexes and inhibits their cell cycle stimulatory activities. Because a loss of p27 expression has been linked to the aggressive behaviour in a variety of human

epithelial tumours including breast cancer, the induction of p27 expression could lead to cell cycle arrest and inhibition of tumour growth (Tan et al, 1997; Lloyd et al, 1999; Macri and

Loda, 1999). Our data are in agreement with recent papers also demonstrating the inhibition of growth of prostate cancer and leukaemia cells through the S-phase cell cycle arrest by the

upregulation of expression of p27 (Zhang et al, 2006; Shishodia et al, 2007). Recently, Guo et al (2007) demonstrated that PL suppresses growth of lung cancer cells through G1-phase cell

cycle arrest mediated by the inhibition of cdk2, 4 and 6 activities. Our results show cell cycle arrest at S phase in breast cancer cells through the upregulation of p27. Nevertheless, our

data are not in a disagreement with Guo _et al_ because the passage through G1 phase into S phase is regulated by the activities of cdk2, 4 and 6, which are controlled by cdk inhibitor p27

(Slingerlan and Pagano, 2000). Moreover, cell cycle arrest at S phase can also be interpreted as the arrest at G1-S, because the majority of cells are at G0/G1 and S but not G2 phases (Table

1). Most importantly, PL suppresses growth of cancer cells by the cell cycle arrest. Here, we show that PL inhibits adhesion, migration and invasion through the suppression of secretion of

uPA from highly invasive breast cancer cells. Our data are in agreement with the study by Lee et al (2005) demonstrating the inhibition of adhesion, invasion and expression of uPA in mouse

melanoma cells. Furthermore, our data suggest the mechanism of inhibition of invasiveness by PL. Therefore, secreted uPA from breast cancer cells interacts with uPAR and converts plasminogen

to plasmin (Blasi and Carmeliet, 2002). Plasmin degrades ECM components and stimulates other proteolytic enzymes (MMPs), which through the degradation of ECM contribute to cell invasion

(Blasi and Carmeliet, 2002). Secreted uPA can bind to uPAR and forms a complex with integrin receptor _α_V_β_3, which through its interaction with vitronectin is involved in adhesion and

migration of breast cancer cells (Slivova et al, 2005). Inhibition of uPA secretion will reduce the formation of uPA–uPAR–_α_V_β_3–vitronectin complex, with the consequent suppression of

adhesion and migration of invasive breast cancer cells. Alternatively, PL can also modulate activities of other proteins involved in the invasive behaviour of breast cancer cells (e.g.

matrix metalloproteinases, _β_1 and _β_4 integrins, epidermal growth factor receptors and others (Carraway and Sweeney, 2006; Bissell, 2007)). Nevertheless, in the present study, we propose

that PL suppresses invasiveness through the inhibition of uPA secretion. Finally, we and others have previously demonstrated that inhibition of uPA suppressed invasiveness of breast cancer

cells (Sliva et al, 2002b; Das et al, 2003: Mi et al, 2006). Recently, Song et al (2003) demonstrated anti-angiogenic activity of PL in chorioallantoic membrane (CAM) chick embryo assay.

However, the mechanism of the inhibition of angiogenesis by PL, related to cancer, was not previously addressed. In the present study, we demonstrate that PL inhibits one of the first steps

in angiogenesis – tube formation of endothelial cells. While PL directly suppressed capillary morphogenesis of endothelial cells, our data further suggest that this effect can be mediated by

the inhibition of secretion of VEGF from breast cancer cells. Although in our experimental conditions it was impossible to remove PL from the conditioned media from breast cancer cells

(PL-CM), and therefore their inhibitory effect on capillary morphogenesis of HEACs could be considered as a direct effect of PL on HEACs, our data suggest that both (direct and indirect)

effects are involved. Thus, (i) conditioned media from breast cancer cells without PL significantly increased capillary morphogenesis of endothelial cells, suggesting that proangiogenic

factor is released from breast cancer cells, (ii) PL itself as well as conditioned media containing PL suppressed capillary morphogenesis and (iii) the secretion of VEGF from breast cancer

cells was inhibited by PL. In agreement with our observation, suppression of endothelial capillary morphogenesis through the inhibition of secreted VEGF from a variety of cancer cells was

described recently (Fukumoto et al, 2005; Stanley et al, 2005; Jang et al, 2007; Kong et al, 2007). Therefore, inhibition of specific pro-angiogenic protein within cancer cells will affect

the whole cancer microenvironment (containing different cells) and will finally result in the suppression of tumour angiogenesis. One of the suitable molecular cancer targets is AKT kinase,

which inhibition in breast cancer cells resulted in cell cycle arrest, inhibition of growth and colony formation, inhibition of migration, invasion and suppression of angiogenesis (Das et

al, 2003; Yacoub et al, 2003; Jiang et al, 2004a; Basu et al, 2005; Fukumoto et al, 2005; Jallal et al, 2007). Our data clearly demonstrate that PL suppresses AKT activity through the

inhibition of AKT phosphorylation at Thr308 and at Ser473 in MDA-MB-231 cells, which demonstrate high levels of constitutively active AKT. Furthermore, inhibition of AKT with LY294002 and

more specific AKT inhibitor III suppressed secretion of VEGF from breast cancer cells resulting in the decrease of capillary morphogenesis of endothelial cells. Our observation is in

agreement with Xia et al (2006) who demonstrated, by using siRNA against AKT, the downregulation of VEGF expression in ovarian cancer cells, and the inhibition of angiogenesis in CAM chick

embryo assay. In conclusion, our study suggests PL as a natural compound possessing antiproliferative, antimetastatic and anti-angiogenic effects, which could be considered for the therapy

of invasive breast cancers. However, further studies are necessary to confirm and evaluate these anticancer effects _in vivo_. CHANGE HISTORY * _ 16 NOVEMBER 2011 This paper was modified 12

months after initial publication to switch to Creative Commons licence terms, as noted at publication _ REFERENCES * Basu GD, Pathangey LB, Tinder TL, Gendler SJ, Mukherjee P (2005)

Mechanisms underlying the growth inhibitory effects of the cyclo-oxygenase-2 inhibitor celecoxib in human breast cancer cells. _Breast Cancer Res_ 7: R422–R435 Article  CAS  Google Scholar 

* Bissell MJ (2007) Modeling molecular mechanisms of breast cancer and invasion: lessons from the normal gland. _Biochem Soc Trans_ 35: 18–22 Article  CAS  Google Scholar  * Blasi F,

Carmeliet P (2002) uPAR: a versatile signaling orchestrator. _Nat Rev Mol Cell Biol_ 3: 932–943 Article  CAS  Google Scholar  * Carraway III KL, Sweeney C (2006) Co-opted integrin signaling

in ErbB2-induced mammary tumor progression. _Cancer Cell_ 10: 93–95 Article  CAS  Google Scholar  * Chihara G, Maeda Y, Hamuro J, Sasaki T, Fukuoka F (1969) Inhibition of mouse sarcoma 180

by polysaccharides from Lentinus edodes (Berk.) sing. _Nature_ 222: 687–688 Article  CAS  Google Scholar  * Collins L, Zhu T, Guo J, Xiao ZJ, Chen CY (2006) _Phellinus linteus_ sensitises

apoptosis induced by doxorubicin in prostate cancer. _Br J Cancer_ 95: 282–288 Article  CAS  Google Scholar  * Dai YC, Xu MQ (1998) Studies on the medicinal polypore, _Phellinus baumii_, and

its kin, _P_. _linteus_. _Mycotaxon_ 67: 191–200 Google Scholar  * Das R, Mahabeleshwar GH, Kundu GC (2003) Osteopontin stimulates cell motility and nuclear factor kappaB-mediated secretion

of urokinase type plasminogen activator through phosphatidylinositol 3-kinase/Akt signaling pathways in breast cancer cells. _J Biol Chem_ 278: 28593–28606 Article  CAS  Google Scholar  *

Dillon RL, White DE, Muller WJ (2007) The phosphatidyl inositol 3-kinase signaling network: implications for human breast cancer. _Oncogene_ 26: 1338–1345 Article  CAS  Google Scholar  *

Ekholm SV, Reed SI (2000) Regulation of G(1) cyclin-dependent kinases in the mammalian cell cycle. _Curr Opin Cell Biol_ 12: 676–684 Article  CAS  Google Scholar  * Folkman J (1995)

Angiogenesis in cancer, vascular, rheumatoid and other disease. _Nat Med_ 1: 27–31 Article  CAS  Google Scholar  * Freedman VH, Shin SI (1974) Cellular tumorigenicity in nude mice:

correlation with cell growth in semi-solid medium. _Cell_ 3: 355–359 Article  CAS  Google Scholar  * Fukumoto S, Morifuji M, Katakura Y, Ohishi M, Nakamura S (2005) Endostatin inhibits lymph

node metastasis by a down-regulation of the vascular endothelial growth factor C expression in tumor cells. _Clin Exp Metastasis_ 22: 31–38 Article  CAS  Google Scholar  * Guo J, Zhu T,

Collins L, Xiao ZX, Kim SH, Chen CY (2007) Modulation of lung cancer growth arrest and apoptosis by _Phellinus Linteus_. _Mol Carcinog_ 46: 144–154 Article  CAS  Google Scholar  * Han SB,

Lee CW, Jeon YJ, Hong ND, Yoo ID, Yang KH, Kim HM (1999) The inhibitory effect of polysaccharides isolated from _Phellinus linteus_ on tumor growth and metastasis. _Immunopharmacology_ 41:

157–164 Article  CAS  Google Scholar  * Harvey K, Siddiqui RA, Sliva D, Garcia JGN, English D (2002) Serum factors involved in human microvascular endothelial cell morphogenesis. _J Lab Clin

Med_ 140: 188–198 Article  CAS  Google Scholar  * Jallal H, Valentino ML, Chen G, Boschelli F, Ali S, Rabbani SA (2007) A Src/Abl kinase inhibitor, SKI-606, blocks breast cancer invasion,

growth, and metastasis _in vitro_ and _in vivo_. _Cancer Res_ 67: 1580–1588 Article  CAS  Google Scholar  * Jang YJ, Kim DS, Jeon OH, Kim DS (2007) Saxatilin suppresses tumor-induced

angiogenesis by regulating VEGF expression in NCI-H460 human lung cancer cells. _J Biochem Mol Biol_ 40: 439–443 CAS  PubMed  Google Scholar  * Jemal A, Siegel R, Ward E, Murray T, Xu J,

Thun MJ (2007) Cancer statistics, 2007. _CA Cancer J Clin_ 57: 43–66 Article  Google Scholar  * Jiang J, Slivova V, Harvey K, Valachovicova T, Sliva D (2004a) _Ganoderma lucidum_ suppresses

growth of breast cancer cells through the inhibition of Akt/NF-kappaB signaling. _Nutr Cancer_ 49: 209–416 Article  CAS  Google Scholar  * Jiang J, Slivova V, Valachovicova T, Harvey K,

Sliva D (2004b) _Ganoderma lucidum_ inhibits proliferation and induces apoptosis in human prostate cancer cells PC-3. _Int J Oncol_ 24: 1093–1099 CAS  PubMed  Google Scholar  * Kong D, Li Y,

Wang Z, Banerjee S, Sarkar FH (2007) Inhibition of angiogenesis and invasion by 3,3′-diindolylmethane is mediated by the nuclear factor-kappaB downstream target genes MMP-9 and uPA that

regulated bioavailability of vascular endothelial growth factor in prostate cancer. _Cancer Res_ 67: 3310–3319 Article  CAS  Google Scholar  * Kim BC, Choi JW, Hong HY, Lee SA, Hong S, Park

EH, Kim SJ, Lim CJ (2006) Heme oxygenase-1 mediates the anti-inflammatory effect of mushroom _Phellinus linteus_ in LPS-stimulated RAW264.7 macrophages. _J Ethnopharmacol_ 106: 364–371

Article  Google Scholar  * Kim HM, Han SB, Oh GT, Kim YH, Hong DH, Hong ND, Yoo ID (1996) Stimulation of humoral and cell mediated immunity by polysaccharide from mushroom _Phellinus

linteus_. _Int J Immunopharmac_ 18: 295–303 Article  CAS  Google Scholar  * Kim GY, Oh YH, Park YM (2003a) Acidic polysaccharide isolated from _Phellinus linteus_ induces nitric

oxide-mediated tumoricidal activity of macrophages through protein tyrosine kinase and protein kinase C. _Biochem Biophys Res Commun_ 309: 399–407 Article  CAS  Google Scholar  * Kim GY,

Park HS, Nam BH, Lee SJ, Lee JD (2003b) Purification and characterization of acidic proteo-heteroglycan from the fruiting body of _Phellinus linteus_ (Berk. & M.A. Curtis) Teng.

_Bioresour Technol_ 89: 81–87 Article  CAS  Google Scholar  * Kim GY, Park SK, Lee MK, Lee SH, Oh YH, Kwak JY, Yoon S, Lee JD, Park YM (2003c) Proteoglycan isolated from _Phellinus linteus_

activates murine B lymphocytes via protein kinase C and protein tyrosine kinase. _Int Immunopharmacol_ 3: 1281–1292 Article  CAS  Google Scholar  * Lee HJ, Lee HJ, Lim ES, Ahn KS, Shim BS,

Kim HM, Gong SJ, Kim DK, Kim SH (2005) Cambodian _Phellinus linteus_ inhibits experimental metastasis of melanoma cells in mice via regulation of urokinase type plasminogen activator. _Biol

Pharm Bull_ 28: 27–31 Article  CAS  Google Scholar  * Lloyd Jr FP, Slivova V, Valachovicova T, Sliva D (2003) Aspirin inhibits highly invasive prostate cancer cells. _Int J Oncol_ 23:

1277–1283 CAS  PubMed  Google Scholar  * Lloyd RV, Erickson LA, Jin L, Kulig E, Qian X, Cheville JC, Scheithauer BW (1999) p27kip1: a multifunctional cyclin-dependent kinase inhibitor with

prognostic significance in human cancers. _Am J Pathol_ 154: 313–323 Article  CAS  Google Scholar  * Macri E, Loda M (1999) Role of p27 in prostate carcinogenesis. _Cancer Metastasis Rev_

17: 337–344 Article  CAS  Google Scholar  * Mi Z, Guo H, Wai PY, Gao C, Kuo PC (2006) Integrin-linked kinase regulates osteopontin-dependent MMP-2 and uPA expression to convey metastatic

function in murine mammary epithelial cancer cells. _Carcinogenesis_ 27: 1134–1145 Article  CAS  Google Scholar  * Park SK, Kim GY, Lim JY, Kwak JY, Bae YS, Lee JD, Oh YH, Ahn SC, Park YM

(2003) Acidic polysaccharides isolated from _Phellinus linteus_ induce phenotypic and functional maturation of murine dendritic cells. _Biochem Biophys Res Commun_ 312: 449–458 Article  CAS

  Google Scholar  * Price JT, Bonovich MT, Kohn EC (1997) The biochemistry of cancer dissemination. _Crit Rev Biochem Mol Biol_ 32: 175–253 Article  CAS  Google Scholar  * Punglia RS, Morrow

M, Winer EP, Harris JR (2007) Local therapy and survival in breast cancer. _N Engl J Med_ 356: 2399–2405 Article  CAS  Google Scholar  * Sherr CJ (1966) Cancer cell cycles. _Science_ 274:

1672–1677 Article  Google Scholar  * Sherr CJ, Roberts JM (1999) CDK inhibitors: positive and negative regulators of G1-phase progression. _Genes Dev_ 13: 1501–1512 Article  CAS  Google

Scholar  * Shishodia S, Sethi G, Ahn KS, Aggarwal BB (2007) Guggulsterone inhibits tumor cell proliferation, induces S-phase arrest, and promotes apoptosis through activation of c-Jun

N-terminal kinase, suppression of Akt pathway, and downregulation of antiapoptotic gene products. _Biochem Pharmacol_ 74: 118–130 Article  CAS  Google Scholar  * Slingerlan J, Pagano M

(2000) Regulation of the Cdk inhibitor and its deregulation in cancer. _J Cell Physiol_ 183: 10–17 Article  Google Scholar  * Sliva D, Harvey K, Mason R, Lloyd Jr F, English D (2001) Effect

of phosphatidic acid on human breast cancer cells exposed to doxorubicin. _Cancer Invest_ 19: 781–788 Article  Google Scholar  * Sliva D, Labarrere C, Slivova V, Sedlak M, Lloyd Jr FP, Ho

NWY (2002a) _Ganoderma lucidum_ suppresses motility of highly invasive breast and prostate cancer cells. _Biochem Biophys Res Commun_ 298: 603–612 Article  CAS  Google Scholar  * Sliva D,

Rizzo MT, English D (2002b) Phosphatidylinositol 3-kinase and NF-_κ_B regulate motility of invasive MDA-MB-231 human breast cancer cells by the secretion of urokinase-type plasminogen

activator (uPA). _J Biol Chem_ 277: 3150–3157 Article  CAS  Google Scholar  * Slivova V, Valachovicova T, Jiang J, Sliva D (2004) _Ganoderma lucidum_ inhibits invasiveness of breast cancer

cell. _J Cancer Integr Med_ 2: 25–30 Google Scholar  * Slivova V, Zaloga G, DeMichele SJ, Mukerji P, Huang YS, Siddiqui R, Harvey K, Valachovicova T, Sliva D (2005) Green tea polyphenols

modulate secretion of urokinase plasminogen activator (uPA) and inhibit invasive behavior of breast cancer cells. _Nutr Cancer_ 52: 65–72 Article  Google Scholar  * Song KS, Cho SM, Lee JH,

Kim HM, Han SB, Ko KS, Yoo ID (1995) B-lymphocyte-stimulating polysaccharide from mushroom _Phellinus linteus_. _Chem Pharm Bull (Tokyo)_ 43: 2105–2108 Article  CAS  Google Scholar  * Song

YS, Kim SH, Sa JH, Jin C, Lim CJ, Park EH (2003) Anti-angiogenic, antioxidant and xanthine oxidase inhibition activities of the mushroom _Phellinus linteus_. _J Ethnopharmacol_ 88: 113–116

Article  Google Scholar  * Stanley G, Harvey K, Slivova V, Jiang J, Sliva D (2005) _Ganoderma lucidum_ suppresses angiogenesis through the inhibition of secretion of VEGF and TGF-beta1 from

prostate cancer cells. _Biochem Biophys Res Commun_ 330: 46–52 Article  CAS  Google Scholar  * Tan P, Cady B, Wanner M, Worland P, Cukor B, Magi-Galluzzi C, Lavin P, Draetta G, Pagano M,

Loda M (1997) The cell cycle inhibitor p27 is an independent prognostic marker in small (T1a,b) invasive breast carcinomas. _Cancer Res_ 57: 1259–1263 CAS  PubMed  Google Scholar  * Tripathy

D (2007) Capecitabine in combination with novel targeted agents in the management of metastatic breast cancer: underlying rationale and results of clinical trials. _Oncologist_ 12: 375–389

Article  CAS  Google Scholar  * Wasser SP (2002) Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides. _Appl Microbiol Biotechnol_ 60: 258–274 Article  CAS 

Google Scholar  * Wong NC, Mueller BM, Barbas CF, Ruminski P, Quaranta V, Lin EC, Smith JW (1998) Alphav integrins mediate adhesion and migration of breast carcinoma cell lines. _Clin Exp

Metastasis_ 16: 50–61 Article  CAS  Google Scholar  * Woodgett JR (2005) Recent advances in the protein kinase B signaling pathway. _Curr Opin Cell Biol_ 17: 150–157 Article  CAS  Google

Scholar  * Yacoub A, Han SI, Caron R, Gilfor D, Mooberry S, Grant S, Dent P (2003) Sequence dependent exposure of mammary carcinoma cells to Taxotere and the MEK1/2 inhibitor U0126 causes

enhanced cell killing _in vitro_. _Cancer Biol Ther_ 2: 670–676 CAS  PubMed  Google Scholar  * Xia C, Meng Q, Cao Z, Shi X, Jiang BH (2006) Regulation of angiogenesis and tumor growth by

p110 alpha and AKT1 via VEGF expression. _J Cell Physiol_ 209: 56–66 Article  CAS  Google Scholar  * Zhang Y, Wang Z, Ahmed F, Banerjee S, Li Y, Sarkar FH (2006) Down-regulation of Jagged-1

induces cell growth inhibition and S phase arrest in prostate cancer cells. _Int J Cancer_ 119: 2071–2077 Article  CAS  Google Scholar  * Zhu T, Guo J, Collins L, Kelly J, Xiao ZJ, Kim SH,

Chen CY (2007) _Phellinus linteus_ activates different pathways to induce apoptosis in prostate cancer cells. _Br J Cancer_ 96: 583–590 Article  CAS  Google Scholar  Download references

ACKNOWLEDGEMENTS This work was supported by the Methodist Health Foundation, by the Department of the Army Medical Research and Materiel Command, Breast Cancer Research Training Program and

by Maitake Products Inc. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Cancer Research Laboratory, Methodist Research Institute, 1800 N Capitol Ave, E504, Indianapolis, 46202, IN, USA D

Sliva, A Jedinak, J Kawasaki, K Harvey & V Slivova * Department of Medicine, Indiana University, Indianapolis, IN, USA D Sliva * Indiana University Simon Cancer Center, School of

Medicine, Indiana University, Indianapolis, IN, USA D Sliva Authors * D Sliva View author publications You can also search for this author inPubMed Google Scholar * A Jedinak View author

publications You can also search for this author inPubMed Google Scholar * J Kawasaki View author publications You can also search for this author inPubMed Google Scholar * K Harvey View

author publications You can also search for this author inPubMed Google Scholar * V Slivova View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING

AUTHOR Correspondence to D Sliva. RIGHTS AND PERMISSIONS From twelve months after its original publication, this work is licensed under the Creative Commons Attribution-NonCommercial-Share

Alike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/ Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Sliva, D.,

Jedinak, A., Kawasaki, J. _et al._ _Phellinus linteus_ suppresses growth, angiogenesis and invasive behaviour of breast cancer cells through the inhibition of AKT signalling. _Br J Cancer_

98, 1348–1356 (2008). https://doi.org/10.1038/sj.bjc.6604319 Download citation * Revised: 21 February 2008 * Accepted: 27 February 2008 * Published: 25 March 2008 * Issue Date: 22 April 2008

* DOI: https://doi.org/10.1038/sj.bjc.6604319 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is

not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * _Phellinus linteus_ * invasiveness * angiogenesis *

AKT