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 (). 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 αVβ3
, 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–αVβ3
–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, β1
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.