Epigenetic effects of the natural flavonolignan silibinin on colon adenocarcinoma cells and their derived metastatic cells.

Epigenetic modifications are important in tumorigenesis. The most frequent epigenetic phenomena in cancer are histone deacetylation and DNA hypermethylation, which lead to gene silencing, particularly of tumor suppressor genes. However, monotherapies with histone deacetylase (HDAC) or DNA methyltransferase (DNMT) inhibitors lack efficacy, hence there is a need to enhance their anticancer action in a safe and effective combination therapy. The present study investigated the epigenetic effects of the natural flavonolignan silibinin in a model of colon cancer progression, the primary adenocarcinoma cells SW480 and their derived metastatic cells SW620. Silibinin did not change the activity of HDACs, but it was able to significantly inhibit DNMT activity in both SW480 and SW620 cells. The clinically used HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), and the broad spectrum HDAC inhibitor, trichostatin A (TSA), combined with silibinin demonstrated synergistic effects on cell death induction, may be related to its DNMT inhibition properties. The present data suggest that treatments combining silibinin and HDAC inhibitors may represent a promising approach, given the non-toxic nature of silibinin and the fact that HDAC inhibitors selectively target cancer cells.

Silibinin inhibits tumor growth in a murine orthotopic hepatocarcinoma model and activates the TRAIL apoptotic signaling pathway.

AIM: The present study investigated the molecular mechanism of silibinin-induced antitumoral effects in hepatocarcinoma Hep-55.1C cells in vitro and in a hepatocarcinoma model in mice. MATERIALS AND METHODS: Cell death was analyzed by flow cytometry. The genetic expression of apoptotic and inflammatory biomarkers was assessed by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR). Orthotopic grafting of Hep-55.1C cells into the liver of C57BL/6J mice was performed, and tumor growth was followed by micro-computed imaging. RESULTS: Silibinin activated the extrinsic apoptotic pathway in Hep55.1C cells, as attested by the up-regulation of TNF-related apoptosis-inducing ligand (TRAIL) and TRAIL Death receptor 5 (DR5) transcripts, and by the activation of caspase-3 and -8. After grafting of Hep-55.1C cells into mouse liver, the oral administration of silibinin at 700 mg/kg body weight for four weeks caused a significant reduction of tumor growth, associated with the down-regulation of inflammatory components [matrix metalloproteinase -7 and -9, (MMP-7, MMP-9), Interleukin-1 beta (IL1ß)], the up-regulation of apoptotic mediators (TRAIL, DR5), and caspase-3 activation. CONCLUSION: Silibinin treatment exerted important anticarcinogenic effects, including the activation of TRAIL death receptor apoptotic signaling pathway in Hep-55.1C hepatocarcinoma cells, both in vitro and in hepatocarcinoma grafts in mice.

Silibinin, a natural flavonoid, modulates the early expression of chemoprevention biomarkers in a preclinical model of colon carcinogenesis.

The flavonolignan silibinin, the major biologically active compound of the milk thistle (Silybum marianum), has been shown to possess anticancer properties in a variety of epithelial cancers. The present study investigated the potential of silibinin as a chemopreventive agent in colon carcinogenesis. The rat azoxymethane (AOM)-induced colon carcinogenesis model was used because of its molecular and clinical similarities to sporadic human colorectal cancer. One week after AOM injection (post-initiation), Wistar rats received daily intragastric feeding of 300 mg silibinin/kg body weight per day until their sacrifice after 7 weeks of treatment. Silibinin-treated rats exhibited a 2-fold reduction in the number of AOM-induced hyperproliferative crypts and aberrant crypt foci in the colon compared to AOM-injected control rats receiving the vehicle. Silibinin-induced apoptosis in the colon mucosal cells was demonstrated by flow cytometry after propodium iodide staining and by colorimetric measurement of caspase-3 activity. Mechanisms involved in silibinin-induced apoptosis included the downregulation of the anti-apoptotic protein Bcl-2 and upregulation of the pro-apoptotic protein Bax, inverting the Bcl-2/Bax ratio to <1. This modulation already takes place at the mRNA expression level as shown by real-time RT-PCR. Furthermore, silibinin treatment significantly (P<0.01) decreased the genetic expression of biomarkers of the inflammatory response such as IL1ß, TNFα and their downstream target MMP7, all of them shown to be upregulated during colon carcinogenesis. The downregulation of MMP7 protein was confirmed by western blot analysis. The present findings show the ability of silibinin to shift the disturbed balance between cell renewal and cell death in colon carcinogenesis in rats previously injected with the carcinogen AOM. Silibinin administered via intragastric feeding exhibited potent pro-apoptotic, anti-inflammatory and multi-targeted effects at the molecular level. The effective reduction of preneoplastic lesions by silibinin supports its use as a natural agent for colon cancer chemoprevention.

The flavonolignan silibinin potentiates TRAIL-induced apoptosis in human colon adenocarcinoma and in derived TRAIL-resistant metastatic cells.

Silibinin, a flavonolignan, is the major active component of the milk thistle plant (Silybum marianum) and has been shown to possess anti-neoplastic properties. TNF-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent which selectively induces apoptosis in cancer cells. However, resistance to TRAIL-induced apoptosis is an important and frequent problem in cancer treatment. In this study, we investigated the effect of silibinin and TRAIL in an in vitro model of human colon cancer progression, consisting of primary colon tumor cells (SW480) and their derived TRAIL-resistant metastatic cells (SW620). We showed by flow cytometry that silibinin and TRAIL synergistically induced cell death in the two cell lines. Up-regulation of death receptor 4 (DR4) and DR5 by silibinin was shown by RT-PCR and by flow cytometry. Human recombinant DR5/Fc chimera protein that has a dominant-negative effect by competing with the endogenous receptors abrogated cell death induced by silibinin and TRAIL, demonstrating the activation of the death receptor pathway. Synergistic activation of caspase-3, -8, and -9 by silibinin and TRAIL was shown by colorimetric assays. When caspase inhibitors were used, cell death was blocked. Furthermore, silibinin and TRAIL potentiated activation of the mitochondrial apoptotic pathway and down-regulated the anti-apoptotic proteins Mcl-1 and XIAP. The involvement of XIAP in sensitization of the two cell lines to TRAIL was demonstrated using the XIAP inhibitor embelin. These findings demonstrate the synergistic action of silibinin and TRAIL, suggesting chemopreventive and therapeutic potential which should be further explored.

Silibinin triggers apoptotic signaling pathways and autophagic survival response in human colon adenocarcinoma cells and their derived metastatic cells.

Silibinin, a flavonolignan isolated from the milk thistle plant (Silybum marianum), possesses anti-neoplastic properties. In vitro and in vivo studies have recently shown that silibinin inhibits the growth of colorectal cancer (CRC). The present study investigates the mechanisms of silibinin-induced cell death using an in vitro model of human colon cancer progression, consisting of primary tumor cells (SW480) and their derived metastatic cells (SW620) isolated from a metastasis of the same patient. Silibinin induced apoptotic cell death evidenced by DNA fragmentation and activation of caspase-3 in both cell lines. Silibinin enhanced the expression (protein and mRNA) of TNF-related apoptosis-inducing ligand (TRAIL) death receptors (DR4/DR5) at the cell surface in SW480 cells, and induced their expression in TRAIL-resistant SW620 cells normally not expressing DR4/DR5. Caspase-8 and -10 were activated demonstrating the involvement of the extrinsic apoptotic pathway in silibinin-treated SW480 and SW620 cells. The protein Bid was cleaved in SW480 cells indicating a cross-talk between extrinsic and intrinsic apoptotic pathway. We demonstrated that silibinin activated also the intrinsic apoptotic pathway in both cell lines, including the perturbation of the mitochondrial membrane potential, the release of cytochrome c into the cytosol and the activation of caspase-9. Simultaneously to apoptosis, silibinin triggered an autophagic response. The inhibition of autophagy with a specific inhibitor enhanced cell death, suggesting a cytoprotective function for autophagy in silibinin-treated cells. Taken together, our data show that silibinin initiated in SW480 and SW620 cells an autophagic-mediated survival response overwhelmed by the activation of both the extrinsic and intrinsic apoptotic pathways.

Identification of gene expression profiles correlated to tumor progression in a preclinical model of colon carcinogenesis.

The rat azoxymethane (AOM)-induced colon carcinogenesis model provides useful information for understanding human colorectal neoplasia. Here, we used the AOM model to measure the gene expression profiles of biomarkers related to tumor progression. We assessed tumor progression stages by computed tomographic (CT) colonography. Messenger RNAs were isolated from tumors and mucosal samples, and gene expression levels were assessed by real-time quantitative polymerase chain reaction (PCR). We show that early stages of tumor progression are associated with an upregulation of matrix metalloproteinase-7 (MMP-7) and of genes involved in the inflammatory response, including interleukin (IL1beta) and tumor necrosis factor-alpha (TNFalpha). The ratio of B-cell lymphoma/leukemia 2 (Bcl-2)-associated X proteins (Bax) to Bcl-2 transcript (proapototic/antiapoptotic signals) is elevated in early stages of tumor progression (Bax/Bcl-2 >1) and reversed in more advanced stages of tumor development (Bax/Bcl-2 <1). These changes are associated with the reduced expression of TNF-related apoptosis-inducing ligand (TRAIL)-death receptor 5 (DR5) and FAS (also known as CD95) apoptotic receptors. Advanced stages of tumor development are characterized by an increase in MMP-9 expression associated with the upregulation of components of the innate immune system: alpha-defensin 5 (DEF-5) and neutrophil gelatinase-associated lipocalin (NGAL). The identification of specific gene expression profiles that correlate with tumor progression stages, as reported in the present study, may represent an important step in evaluating potential chemopreventive and/or chemotherapeutic agents prior to initiating clinical trials.