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Silibinin and angiogenesis

We have first reported that silibinin feeding inhibits micro-vessel density (MVD) in growing prostate carcinoma DU145 tumors in athymic nude mice, which was associated with a decrease in VEGF expression in the tumors [74]. Similar anti-angiogenic efficacy of silibinin was also observed in prostate tumors growing in the prostate microenvironment in nude mice [70]. Extensive studies in TRAMP (transgenic adenocarcinoma of the mouse prostate) revealed that silibinin targets the onset of ‘angiogenic switch’ in prostate tumors [44, 73]. Huss et al. have shown that angiogenic switch in TRAMP involves an increase in VEGF, VEGFR2 and HIF-1α expression accompanied with increased intra-ductal microvessels, and disease progression from low-grade prostate intraepithelial neoplasia (LGPIN) stage to high-grade PIN (HGPIN), adenocarcinoma and metastasis [13]. Our completed studies showed that silibinin feeding decreased the VEGFR2, VEGF, and HIF-1α expression, and strongly inhibited the MVD in TRAMP prostate tissues [44]. Silibinin treatment also decreased the levels of circulating angiogenic factors VEGF and bFGF in TRAMP mice [73]. These anti-angiogenic effects of silibinin were associated with a potent inhibition of tumor grade as well as metastasis confirming its strong angiopreventive efficacy against prostate cancer [44, 73].

Silibinin has also been extensively tested for its anti-angiogenic efficacy in several animal models of colorectal cancer [69, 71, 72, 75]. We have reported that silibinin feeding inhibited the angiogenesis in HT29 tumor via down-regulating iNOS (inducible nitric oxide synthase), COX2, HIF-1α, and VEGF expression [71]. Silibinin also exerted sustained growth suppressive effects in human colorectal cancer SW480 tumors via decreasing MVD, VEGF and iNOS expression [75]. In transgenic APC^min+ mice, silibinin feeding inhibited the nestin-positive microvessels selectively in the small intestinal polyps by down-regulating the expression of HIF-1α, VEGF, and eNOS (endothelial nitric oxide synthase) in the polyps [69]. Importantly, silibinin feeding did not affect the expression of these molecules in the crypt-villus region in the small intestine of APC^min+ and wild type C57BL/6J mice confirming the polyp specific effect of silibinin [69]. These angiopreventive effects of silibinin were associated with the prevention of spontaneous intestinal polyposis in APC^min+ mice and we observed a decrease in both the number of polyps as well as size of the polyps formed [69]. Similarly, silibinin treatment inhibited VEGF and iNOS expression in azoxymethane-induced colon tumors in A/J mice [76]. Earlier, Yang et al. reported that silibinin treatment inhibits the vascular density index induced by human colorectal cancer LoVo cells in CAM (chicken chorioallantoic membrane) assay through inhibiting VEGF expression [77]. Overall, silibinin has shown strong angiopreventive efficacy against colorectal cancer in xenografts, chemical carcinogenesis, CAM and transgenic models.

Sustained NO (nitric oxide) generation positively correlates with lung cancer development and progression; and our completed studies in lung tumorigenesis models suggest that silibinin’s chemopreventive and angiopreventive effects could be through targeting iNOS expression [43, 68, 78]. In urethane-induced lung tumorigenesis model, silibinin feeding strongly decreased the MVD, VEGF and iNOS levels in lung tumors [43]. The angiopreventive effects of silibinin resulted in a significant decrease in the lung tumor multiplicity as well as tumor size [43]. Using iNOS−/− mice we confirmed that silibinin exerts its chemopreventive and angiopreventive effects against lung tumorigenesis via inhibiting iNOS expression [78]. Our in vitro mechanistic studies also showed that silibinin targets multiple signaling molecules [STATs (1 and 3), AP1, NF-κB, MAPKs and HIF-1α], and that it inhibits cytokine mixture (IFN-γ + IL-1β + TNF-α)-induced iNOS expression in human lung epithelial carcinoma A549 cells [79]. Lung tumor analyses supported these in vitro observations and silibinin was found to inhibit the expression of IFNγ, interleukins, and TNFα in lung tumors [68]. Silibinin also decreased HIF-1α, NF-κB and phosphorylated STAT3 expression in lung tumors [68]. These results suggested that silibinin targets multiple signaling pathways regulating iNOS expression in lung tumor cells, and thereby, it inhibits the angiogenesis and overall tumor progression in lung tumors.

The anti-angiogenic effects of silibinin have also been observed in other cancers such as skin cancer and bladder cancer [80, 81] suggesting the broad-spectrum angiopreventive efficacy of silibinin. Additionally, several in vitro studies have supported the anti-angiogenic effects of silibinin and provided detailed insight into the mechanisms for silibinin’s angiopreventive action. Silibinin was reported to inhibit the growth of HUVEC (human umbilical vein endothelial cells) and HMVEC (human microvascular endothelial cells) at pharmacologically achievable doses in cell culture [67]. Silibinin also inhibited capillary tube formation on matrigel as well as inhibited HUVEC invasion and migration [67]. Molecular analyses revealed that silibinin induces G1 arrest in endothelial cells via promoting the expression of CDKIs (cyclin dependent kinase inhibitors) and p53 [67]. Furthermore, silibinin treatment induced apoptotic death involving both caspases-dependent and –independent mechanisms [67]. Silibinin also targeted Akt, NF-κB and survivin as well as MMP-2 activity (Figure 3) [67]. Yang et al. have reported similar effects of silibinin on the growth as well as the differentiation of endothelial EA.hy 926 cells [82]. Silibinin also inhibited the chemotactic migration of EA.hy 926 cells towards LoVo colon cancer cells [82]. Yoo et al. have also shown that silibinin suppresses growth and induces apoptotic death in human endothelial ECV304 cells by modulating NF-κB, Bcl-2 family members and caspases [83].

Along with targeting endothelial cells, silibinin has also been reported to target cancer cells towards inhibiting the secretion of pro-angiogenic factors (Figure 3). HIF-1α has emerged as a master regulator of angiogenesis, tumor metabolism and metastasis [84]. Together with other regulators (such as ERK, NF-κB, STATs), HIF-1α controls the expression and secretion of several pro-angiogenic growth factors (Figure 3), that promote chemotactic movement, survival, proliferation and differentiation of endothelial cells. Garcia-Maceira et al. showed that silibinin strongly inhibits hypoxia-induced HIF-1α accumulation and VEGF release in human cervical HeLa and hepatoma Hep3B cells [85]. This effect was correlated with silibinin’s inhibitory effect on the HIF-1α translation through targeting the mTOR-p70S6K and 4E-BP1 pathways [85]. Similarly, Jung et al. have reported that silibinin inhibits HIF-1α protein expression via targeting its synthesis in human prostate cancer cells [86]. This study also showed that silibinin inhibits global protein synthesis via decreasing the levels of eIF4E-associated with eIF4F complex, increasing the levels of eIF4E associated with 4E-BP1 and promoting the eIF2α phosphorylation [86]. Kim et al. (2009) have shown that silibinin treatment inhibits 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced MMP9 and VEGF expression via suppressing the RAF/MEK/ERK pathway in MCF-7 breast cancer cells [87]. Overall, it is clear that silibinin targets both cancer and endothelial cells to effectively inhibit angiogenesis. Specifically, on the one hand silibinin targets multiple signaling cascades in cancer cells to inhibit the secretion of pro-angiogenic factors in the microenvironment, and on the other hand it targets endothelial cell responses (motility, proliferation, survival, and differentiation) to pro-angiogenic stimuli (Figure 3).

Silibinin and metastasis

Metastasis is an extremely complex, multi-step and multi-functional biological event that is responsible for high mortality and morbidity in cancer patients [58, 88–90]. Successful metastasis is dependent on the cumulative ability of cancer cells to suitably respond to the distinct microenvironment at each step in the metastatic cascade starting from primary tumor growth to final metastatic site [58]. Over a century ago, Stephen Paget first reported a non-random pattern of metastasis of cancer cells to certain organs [91, 92]. He proposed “seed and soil hypothesis”, in which he compared the metastasis of cancer cells to the dispersal of seeds by plants. He postulated that seeds (‘cancer cells’) could grow only in a congenial soil (‘specific microenvironment’). For example, osteotropic cancer cells possess certain intrinsic properties that enable them to grow in the bone; and the bone microenvironment provides a fertile soil for their growth. This theory, which placed main emphasis on the compatibility between metastatic cancer cells and their microenvironment, is still relevant, and the metastatic microenvironment has now become an important drug target to treat or prevent metastasis. There are several reports that suggest that silibinin targets the multiple interactions between tumor cells and their microenvironment and prevents/inhibits metastasis [58].

The extracellular matrix (ECM) and integrins interact to regulate a variety of cellular functions including adhesion, survival, and motility [3, 93, 94]. Fibronectin, a matrix glycoprotein, is one such ECM component, that has been reported to be up-regulated in several malignant tumors and its expression positively correlates with an invasive and metastatic phenotype [94–96]. We have reported that fibronectin expression increases with tumor progression in prostate tumors in TRAMP mice, and that fibronectin expression was significantly decreased by silibinin treatment [44]. Fibronectin-integrin interaction activates several signaling pathways (FAK, Src, Akt, and GTPase) involved in cell survival and actin-remodeling. In our unpublished studies, we have observed that silibinin targets the fibronectin-prostate cancer cell interaction and inhibits integrins expression as well as down-stream signaling involved in actin-remodeling; thereby inhibits the formation of motile structures. Besides fibronectin, silibinin has been reported to significantly decrease the adhesion of prostate cancer cells to type I collagen [97]. It is important to highlight here that bones are rich in type I collagen and prostate cancer cells generally metastasize to bones. Silibinin treatment also inhibited the adhesion of human prostate cancer PC3M cells with ECM proteins hyaluronan and fibronectin by targeting the expression of transmembrane protein CD44 and its variant form CD44v7-10 [98]. Furthermore, silibinin treatment inhibited the adhesive capability of human osteosarcoma MG-63 cells towards type IV collagen [99]. These studies suggest that silibinin treatment significantly attenuates the interaction of cancer cells with their ECM components, which could adversely affect their motility and invasiveness.

Proteinases have been implicated in many cancer-related biological activities (angiogenesis, metastasis etc.), mainly because of their ability to break down components of the ECM, allowing cancer and other cells to migrate [58]. Silibinin treatment has been shown to significantly inhibit the expression of MMPs (matrix metalloproteinases) and to increase expression of TIMP-2 (tissue inhibitors of metalloproteinases-2) in vitro in a wide variety of cancer cells [58, 100–104]. In vivo, we have observed in TRAMP mice that silibinin feeding significantly decreased the expression of MMP-2, MMP-3 and MMP-9, but increased the TIMP-2 expression in prostate tumor tissue [44, 73]. Furthermore, silibinin treatment has been reported to inhibit serine protease uPA and its receptor uPAR expression in several cancer cell lines in vitro and in vivo [58, 99, 103–105]. Silibinin has also been reported to decrease the expression of cysteine proteinases cathepsin B in highly invasive human glioma cells [105].

During metastasis, several cancer cells undergo a phenomenon known as ‘epithelial to mesenchymal transition’ (EMT). EMT refers to 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 cytoskeleton elements resulting in increased motility and invasiveness, and the release of cells from parent epithelial tissue [58, 106, 107]. EMT is regulated by a multitude of factors located in the TME. For example, CAFs have been reported to promote EMT by secreting MMPs [8]. In our unpublished studies we have also observed that CAFs promote the invasiveness of human prostate cancer LNCaP cells, which is associated with increased vimentin and Akt phosphorylation; and silibinin treatment inhibits the CAFs-induced invasiveness of LNCaP cells as well as strongly decreases the Akt phosphorylation and vimentin expression. Silibinin has also been reported to inhibit EMT through promoting the E-cadherin expression and inhibiting the expression of EMT transcriptional regulators [58, 108]. These results have been accompanied with a strong decrease in the migratory, invasive and metastasis properties of cancer cells both in vitro and in vivo [44, 58, 73, 108]. We have reported that silibinin feeding strongly inhibits the local invasion of prostate cancer cells to the seminal vesicle as well as distant metastasis to liver, lung and kidney in TRAMP mouse model [44, 73].

Silibinin has also been reported to affect the TME at metastatic site in prostate cancer model. Prostate cancer cells have a high propensity to metastasize to bones [58, 109]. During bone metastasis, prostate cancer cells even express genes like osteocalcin, bone sialoprotein, osteopontin, RANKL, whose expression is normally restricted to bone cells [58, 91, 110]. This phenomenon is termed ‘osteomimicry’ and is considered as an effort by cancer cells to adapt to their microenvironment, helping cancer cells to settle in the bones. Our unpublished data has shown that silibinin inhibits the expression of many osteomimicry related proteins such as RANKL, PTHrP, osteocalcin, and RunX2 in prostate cancer cells both in vitro and in vivo. Once settled in the bones, prostate cancer cells alter the delicate balance of bone remodeling orchestrated by two types of bone cells namely osetoclasts (involved in bone degradation) and osteoblasts (involved in bone formation) [58, 91, 110, 111]. Prostate cancer cells secrete factors that are involved in osetoclast maturation and activation, thereby promoting bone mineralization and the liberation of various growth factors [58, 91, 110, 111]. Bone degradation provides prostate cancer cells the initial space to expand, and the released growth factors promote prostate cancer cell survival and proliferation. These growth factors secreted by bone degradation and those secreted by prostate cancer cells like endothelin-1, BMPs (bone morphogenetic proteins), Wnts, promote osteoblasts maturation and formation of new bone [58, 91, 110, 111]. Mature osteoblasts also secrete growth factors which further promote prostate cancer cell growth in bone [91, 110]. Overall, this vicious cycle involving prostate cancer cells, osteoclasts and osteoblasts promotes bone degradation as well as deposition of new ‘woven type bone’ (uneven/immature/embryonic), and thereby compromises bone health and leads to bone complications in prostate cancer patients. Silibinin has been reported to affect both the components (osteoclasts and osteoblasts) of the bone microenvironment [112, 113]. Kim et al. have reported that silibinin treatment inhibited the formation of TRAP-positive multinuclear osteoclasts in bone marrow-derived macrophage cells cultured in the presence of M-CSF and RANKL or with osteoblasts and 1,25(OH)₂D₃ [113]. Silibinin also inhibited osteoclast differentiation in murine monocyte/macrophage cell line RAW264.7 stimulated with RANKL [113]. However, silibinin treatment did not affect osteoclast function when mature osteoclasts were treated with silibinin [113]. Silibinin also inhibited TNFα-induced osteoclastogenesis in bone marrow-derived macrophage cells treated with M-CSF and TGF-β [113]. Silibinin effect on osteoclast differentiation seems to be through targeting the fusion of TRAP+ mononuclear pre-osteoclasts forming TRAP+ multinucleate mature osteoclasts [113]. In our unpublished studies, we have observed that prostate cancer cells promote osteoclast activation in RAW264.7 cells, while silibinin treatment inhibits the prostate cancer cells potential to induce osteoclastogenesis. Silibinin seems to target multiple RANKL-induced signaling pathways such as NF-κB, MAPKs, Akt, AP1, and NFATc1 and also to inhibit NFATc1 regulated genes (TRAP, OSCAR and Cathepsin K) (our unpublished results) that are important in osteoclastogenesis (Figure 4) [114, 115]. In another study, the bone-forming and osteoprotective effects of silibinin were studied in cell culture in murine osteoblastic MC3T3-E1 cells [112]. Silibinin treatment increased the bone nodule formation by enhancing calcium deposits [112]. Silibinin also increased the induction of osteoblastogenic biomarkers alkaline phosphatase (ALP), collagen type 1, connective tissue growth factor (CTGF), and BMP-2 (Figure 4) [112]. Silibinin treatment also inhibited RANKL secretion by differentiated MC3T3-E1 cells (Figure 4) [112]. But the effect of silibinin on cancer cell-induced osteoblastogenesis remains unknown (Figure 4). Overall, these studies confirmed that silibinin targets several TME components towards lowering the metastatic growth of cancer cells.

Silibinin and inflammation

Chronic inflammation is a frequent cause of cancer. Even in cancers that are not necessarily the outcome of chronic inflammation, invariably, there are inflammatory components in their microenvironment. In fact, the constant disruption of homeostasis by proliferating transformed cells produces a local chronic inflammatory environment, which is considered an attempt by the body to re-establish normal homeostasis [1]. However, in the presence of cancer cells, immune cells react paradoxically and promote the survival and proliferation of cancer cells [1]. As a result tumors have been characterized as ‘wounds that do not heal’. These observations clearly suggest that reducing inflammation in the TME should inhibit or prevent cancer growth. There are plenty of evidences now suggesting that silibinin could modify inflammatory or immune components towards preventing carcinogenesis [69, 72, 76, 116–119]. Provinciali et al. have reported that silibinin administration delayed the development of spontaneous mammary tumors, reduced the number and size of tumors and diminished lung metastasis in HER-2/neu transgenic mice [116]. Silibinin treatment affected the leukocytes infiltration into the tumors and there were increased numbers of neutrophils, CD4+ and CD+8 lymphocytes but there was a slight decrease in macrophage number [116]. We have also reported that silibinin treatment inhibits TAMs in the TME, which was correlated with angiopreventive effects of silibinin against lung tumorigenesis (Figure 3) [68]. Meeran et al. have reported that silibinin treatment inhibited UVB-induced local and systemic immuno-suppression [118]. In trinitrobenzene sulfonic acid (TNBS)-induced colitis model, silibinin treatment significantly reduced several components of inflammatory colitis such as NF-κB activity, levels of IL-1β, TNFα, thiobarbituric acid reactive substances (TBARS), protein carbonyl, myeloperoxidase activity, and an improvement in antioxidant capability of the colon tissue [119].

The arachidonic acid pathway is at the core of inflammatory response. In this pathway, COX enzymes are responsible for the formation of prostaglandins (PGE2, PGF2α, and PGD2), prostacyclin and thromboxane, while lipoxygenase generates 5-HPETE which is converted to leukotrienes. COX2 is over-expressed in several cancers and considered an attractive drug target [120]. We have reported that chronic exposure to a physiological dose of UVB strongly increased the COX2 levels in the skin and skin tumors [80]. Pre- or post-topical treatment or dietary feeding of silibinin was reported to strongly inhibit UVB-induced COX2 levels [80]. Silibinin has also been reported to inhibit COX2 expression in colorectal cancer in xenografts, transgenic and chemically-induced colorectal cancer models [69, 71, 72, 76]. Silibinin treatment has been shown to inhibit the formation of cyclooxygenase pathway metabolites (PGE2, prostacyclin, and thromoxanes) by human mononuclear cells, platelets, and endothelial cells stimulated with LPS or A23187 [117]. Silibinin treatment also strongly inhibited the formation of 5-lipoxygenase metabolites by human granulocytes (leukotrienes LTB4, LTC4/D4/E4/F4) when stimulated with A23187, FMLP, or opsonized zymosan. Silibinin was also reported to be a strong scavenger of HOCl (IC50 7 μM) produced by human granulocytes [117]. Together, these studies suggest a strong inhibitory effect of silibinin on arachidonic acid pathway.

Transcriptional factors (AP1, NF-κB, STATs etc.) regulate the expression of several pro-inflammatory cytokines [79, 121–124]. Recently, we reported that silibinin targets several signaling pathways (ERK1/2, STAT1/3, NF-κB, and EGFR) towards inhibiting the TNFα and IFNγ induced expression of pro-inflammatory enzymes COX2 and iNOS [123]. Silibinin has been reported to inhibit TNFα-induced NF-κB activation in prostate cancer and colorectal cancer cells [124, 125]. Silibinin treatment also strongly inhibited the UVB-induced activation of STAT3 and NF-κB in skin and skin tumors in SKH-1 hairless mice [80]. Overall, silibinin targets multiple signaling pathways towards inhibiting the secretion of pro-inflammatory cytokines.

In general, published literature shows that silibinin targets many cellular as well as non-cellular components of the TME (Figure 5) towards inhibiting angiogenesis, metastasis and inflammation. There is also evidence now that silibinin targets the abnormal tumor metabolism as well as insulin signaling pathways (IGF/IGFBP3) [74, 126, 127]. Silibinin is already been tested clinically for its efficacy against several cancers including prostate and colon cancer [58, 128–130]. Considering the important role of TME in carcinogenesis, it is important that we focus on its chemopreventive efficacy not only in terms of effect on cancer cells but also the biomarkers in the TME (such as macrophages, CAFs, angiogenesis, and cytokines).

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