Расторопши: семена ранней потенциальных
Mechanistic details of silibinin anti-metastatic efficacy
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- Silibinin targets proteases
- Silibinin targets MAPK signaling
- Silibinin targets tumor microenvironment
Mechanistic details of silibinin anti-metastatic efficacySilibinin targets most of the metastatic events shown in Fig. 1, and in this section we have detailed possible mechanisms that contribute to strong anti-metastatic efficacy of silibinin. Silibinin targets EMT in cancer cellsEMT is a dynamic, multistep and highly coordinated process that includes the loss of inter-cellular junctions, disruption of the tumor basement membrane, activation and rearrangement of the cytoskeleton resulting in increased motility, and the release of cells from parent epithelial tissue [8,15]. The molecular bases of EMT is very complex and involve several interconnected pathways and signaling molecules including growth factors, receptor tyrosine kinases, Notch, NF-κB, Ras superfamily small GTPases, catenins and integrins [8,14,15]. However, most of these pathways converge together to down-regulate the expression of epithelial molecule E-cadherin [104]. E-cadherin is a transmembrane glycoprotein with an extracellular domain that interacts with the E-cadherin molecule on adjacent cells, and an intracellular domain associated with a multi-protein complex comprising three catenins (α, β and p-120) [104]. β-catenin binds tightly to the cytoplasmic domain of E-cadherin and through α-catenin to the actin microfilament network of the cytoskeleton [104]. In general, this complex is important in regulating the cell-cell adhesion, cell polarity and cell shape. The loss of E-cadherin frees β-catenin from the membranous pool, thus making it available for nuclear signaling, which is known to promote cancer cell proliferation, invasiveness and EMT [105]. Therefore, downregulation of E-cadherin expression represents the determinant step in the destabilization of epithelial architecture, and is regulated through a combination of genetic, epigenetic, transcriptional and post-transcriptional mechanisms [14]. Major transcriptional repressors of E-cadherin are zinc finger family members Snail and Slug, the basic helix-loop-helix factors E47 and Twist, and two-handed zinc factors ZEB1 and SIP1 [14]. These transcriptional factors are considered inducers of EMT and are important in cancer cell metastasis [14]. The loss of epithelial characteristics during EMT is also accompanied by increased expression of mesenchymal markers such as cyotskeletal proteins vimentin, smooth muscle actin, γ-actin, β-filamin, and extracellular matrix components such as fibronectin and collagen precursors [15]. The upregulation of these proteins facilitate pseudopod formation and cyotskeletal remodeling for cancer cell motility and invasion [15]. Due to critical importance of EMT in cancer metastasis, targeting EMT has been considered an exciting opportunity towards preventing cancer cells from acquiring motility and invasiveness. Silibinin has been widely reported to target EMT through promoting epithelial characteristics while significantly inhibiting the expression of mesenchymal markers (Fig. 3) [80,90,92,93]. We reported that in TRAMP mice with increasing age and disease stage prostate cancer cells increasingly express mesenchymal markers/regulators while loosing the expression of epithelial markers [80]. For example, at PIN stage, E-cadherin expression was high and it was localized on the intercellular junctions, while the expression of its transcriptional repressor Snail-1 was low; however, with the disease progression to poorly-differentiated stage, E-cadherin expression was lost, while nuclear expression of Snail-1 was increased, suggesting EMT during prostate tumor progression in this model [80]. Importantly, the expression of E-cadherin was significantly higher in tumor tissues from the mice fed with 1% silibinin in diet and that corresponded to low-pathological grade of the disease in these mice [80]. Furthermore, silibinin treatment significantly decreased Snail-1 and fibronectin expression in the prostate tumor tissues [80]. In another study, where silibinin was fed for 11 weeks to TRAMP mice, we observed an increase in E-cadherin expression, and a strong decrease in Snail-1 and vimentin expression [90]. These studies clearly suggested the strong in vivo potential of silibinin to inhibit EMT and disease progression, which might contribute to its strong anti-metastatic efficacy. There are several cell culture studies that also support strong anti-EMT potential of silibinin [92,93]. Wu et al. reported that the anti-invasive and anti-migratory properties of silibinin are due to its strong inhibitory effect on vimentin expression in ARCaP(M) cells [93]. In a recent study, the same group showed that silibinin treatment up-regulates cytokeratin-18 (an epithelial marker) and down-regulates vimentin expression in prostate cancer cells; and these molecular events were associated with morphological reversal of EMT phenotype [92]. This study also suggested that the inhibitory effect of silibinin on EMT could be through targeting various transcriptional factors (NF-κB, ZEB1 and Slug) [92]. In our recently completed in vitro study, we observed that silibinin targets EMT in PCA cells through enhancing E-cadherin expression at cellular membranes [94]. Further, silibinin treatment inhibited the levels of cytoplasmic and nuclear β-catenin without affecting the β-catenin present at cellular membranes [94]. These in vitro studies clearly suggest that silibinin targets EMT in cancer cells through multiple mechanisms (Fig. 3). Silibinin targets proteasesProteases have been implicated in many cancer-related biological activities, mainly because of their ability to break down components of the extracellular matrix, allowing cancer and endothelial cells to spread. Among various types of proteases, family of zinc-dependent endopetidases known as ‘matrix metalloproteinases (MMPs)’ is considered most important as biomarker for cancer prognosis and therapeutic [106,107]. There are direct evidences suggesting the role of MMPs in tumor growth and invasion. For example, MMP-9 deficient mice show reduced formation of melanoma metastasis; MMP-2 knockout mice show reduced melanoma tumor progression [108,109]. The expression of MMPs has also been used to predict the risk of metastasis in many cancers [107]. The expression of MMP-2 and MMP-9 in papillary thyroid carcinoma is well correlated with their invasive capacity and lymph node metastasis [107]. High serum level of MMP-2 has been correlated with the presence of metastases in lung cancer [110]. MMPs like MMP-3 and MMP-7 cleave extracellular domain of E-cadherin, and the ratio of MMPs and E-cadherin has been used to predict metastasis and tumor recurrence in different cancers [107,111]. Therefore, inhibition of MMP activity is considered an exciting approach to target growth and invasiveness of neoplastic cells, and currently several MMP inhibitors are in clinical trial [107,112]. Another attractive approach to target MMPs is through promoting the expression of endogenous inhibitors of MMPs known as tissue inhibitors of metalloproteinases (TIMPs) [113]. In this regard, silibinin has been extensively studied for its efficacy against MMPs. We reported that in TRAMP mice, silibinin feeding significantly decreases expression of MMPs (MMP-2, MMP-3 and MMP-9) but increases TIMP-2 expression in prostate tumor tissue [80,90]. Silibinin treatment has also been shown to significantly decrease expression of MMPs and to increase expression of TIMP-2 in vitro in wide variety of cancer cells [93,100–102,114]. Silibinin treatment decreased MMP-2 expression and increased TIMP-2 protein expression in highly metastatic lung cancer A549 cells and tongue cancer SCC-4 cells [101,102,114]. Further, silibinin treatment strongly inhibited MMP-2 expression in prostate cancer ARCaP(M) cells, osteosarcoma MG-63 cells and human hepatocellular carcinoma HepG-2 cells [93,103,115]. Further, silibinin treatment decreased the activity-, gene- and proteinexpression of PMA-induced MMP-9 expression in MCF-7 breast carcinoma cells [100]. Momeny et al. reported the inhibitory effect of silibinin on MMP-9 expression in human glioblastoma U87MG cells [116]. These studies clearly suggest that silibinin inhibits MMPs while increases TIMP-2 expression in a wide variety of cancer cells. The serine protease urokinase-type plasminogen activator (uPA) and its receptor (uPAR) have been implicated in several tumor processes including adhesion, migration, proliferation and angiogenesis through their interactions with integrins and vitronectin, and through activation of intracellular signaling pathways [117–119]. uPA is known to catalyze the conversion of plasminogen to plasmin, which in turn, exerts strong proteolytic effects including the activation of metalloproteinases and growth factors [117]. The high expression of uPA/uPAR has been correlated with tumor progression and, in some cases, poor patient post-operative survival [119]. We have reported that uPAR level is up-regulated during prostate tumor progression, but silibinin significantly inhibits uPAR expression [80]. Further, silibinin treatment has been reported to inhibit uPA and uPAR expression in several cancer cell lines in vitro [101–103,116]. Cathepsin B is another important cysteine protease whose expression is reported to be higher in cancer cells and is known to mediate cancer cells dissemination through ECM degradation and activation of other proteinases [117]. Silibinin treatment has been reported to decrease cathepsin B expression in highly invasive human glioma cells [116]. Overall, these studies suggest that anti-migratory/anti-invasive efficacy of silibinin could be through its inhibitory effects on a wide-range of proteases. Silibinin targets MAPK signalingMAPKs (ERK1/2, JNK1/2 and p38) belong to widely conserved family of serine/threonine kinases that are activated through reversible phosphorylation and mediate signal transduction of a wide variety of extracellular stimuli (cellmatrix adhesion, growth factors etc.) into intracellular cascades. Recent studies have implicated their role in the regulation of cancer cell motility and invasiveness [120,121]. The sustained activation of MAPK signaling relies mainly on the cross talks between integrins and receptor tyrosine kinases (RTKs) [122]. These signals converge on to activate non-receptor kinases Src and FAK, which eventually leads to activation of MAPKs (Fig. 4) [122]. MAPKs are reported to regulate several transcriptional factors such as AP-1, NF-κB as well as expression of migration-related genes such as uPA, MMPs, β(3)-integrin, cathepsin (Fig. 4) [103,122,123]. ERK has also been reported to regulate the dynamics of focal adhesion and cyotskeletal reorganization through phosphorylating specific cyotskeletal and focal adhesion proteins such as paxillin, focal adhesion kinase (FAK), myosin light chain kinase (MLCK) and microtubule associated protein (MAP) [122]. Overall, inhibition of MAPK signaling might have the potential to prevent cancer cell proliferation, migration, invasion and metastasis. The efficacy of silibinin to target MAPK-mediated mitogenic signaling has been extensively reported [86,124–126]. There are now reports suggesting that silibinin also targets MAPK signaling towards inhibiting the migration and invasion of cancer cells (Fig. 4) [101,103,114,115]. Chen et al. showed that silibinin-mediated decrease in MMP-2, uPA and invasiveness of A549 cells is mainly through inhibition of ERK1/2 [114]. Lee et al. reported in MCF-7 cells that specific inhibition of MAPK signaling is directly involved in the regulation of PMA-induced MMP-9 expression [100]. Study by Hsieh et al. showed that silibinin targets FAK and ERK1/2 expression, suppresses c-Jun levels and AP-1-binding activity and strongly decreases MMP-2 and uPA expression in MG-63 cells [103]. Importantly, inhibitory effect of silibinin on the invasiveness of MG-63 cells was compromised when these cells were transfected to over-express active MEK1 [103]. In our unpublished in vitro studies, we have also observed that silibinin strongly inhibits Src activation in prostate cancer cells. These results clearly support a critical role of MAPK inhibition in silibinin anti-invasive efficacy (Fig. 4). Silibinin targets tumor microenvironmentAs mentioned earlier, tumor microenvironment is an intrinsic part of tumor at all stages of tumor development including metastasis. Therefore, in addition to existing preventive/therapeutic efforts directed at tumor cells, it is essential to develop new measures targeted toward tumor microenvironment. Tumor microenvironment is also considered an attractive target for cytostatic drugs as it is (relatively) genetically stable and chances of it developing chemoresistance are remote. Till date, detailed effect of silibinin has been studied only on one tumor microenvironment component i.e. tumor angiogenesis. Neo-angiogenesis refers to formation of new blood vessels within tumors and is considered important constituent of tumor microenvironment. Formation of these vessels is necessary to provide nutrients and oxygen to the growing tumor cells and also to remove waste products. Furthermore, malignant cells exit from primary tumors into blood circulation only after tumor become neo-vascularized. Furthermore, at distant sites, metastatic cancer cells must again induce angiogenesis to grow beyond micro-metastasis. Therefore, targeting angiogenesis is an important strategy to target cancer cells growth and metastasis. There is plethora of reports suggesting the strong anti-angiogenic efficacy of silibinin in various cancer models [80,81,90,127,128]. Silibinin treatment has been reported to inhibit tube formation and invasive capability of endothelial cells in in vitro assays [79]. Further, silibinin treatment has been reported to significantly decrease microvessels (established and newly formed) density in animal models for variety of cancers [81,127,129]. Silibinin treatment is reported to modulate expression of signaling molecules involved in angiogenesis regulation such as VEGF, VEGF receptors, FGF, HIF-1α, iNOS, COX2, NF-κB, STATs, interleukins, angiopoietin-2, Angreceptor tyrosine kinase [80,81,89,127,128,130,131]. Recently, we also showed that silibinin treatment decreases the population of tumor associated macrophages (TAMs) towards targeting the angiogenic microenvironment in lung tumors [127]. These studies suggest that silibinin might be effective in altering the immediate tumor microenvironment, but further studies are required to understand its effects on other components of tumor microenvironment involved in cancer metastasis. жүктеу/скачать 2.54 Mb. Достарыңызбен бөлісу:
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