The dietary bioflavonoid quercetin synergizes with epigallocathechin gallate (EGCG) to inhibit prostate cancer stem cell characteristics, invasion, migration and epithelial-mesenchymal transition

Abstract Background Much attention has been recently focused on the role of cancer stem cells (CSCs) in the initiation and progression of solid malignancies. Since CSCs are able to proliferate and self-renew extensively due to their ability to express anti-apoptotic and drug resistant proteins, thus sustaining tumor growth. Therefore, the strategy to eradicate CSCs might have significant clinical implications. The objectives of this study were to examine the molecular mechanisms by which epigallocathechin gallate (EGCG) inhibits stem cell characteristics of prostate CSCs, and synergizes with quercetin, a major polyphenol and flavonoid commonly detected in many fruits and vegetables. Results Our data indicate that human prostate cancer cell lines contain a small population of CD44+CD133+ cancer stem cells and their self-renewal capacity is inhibited by EGCG. Furthermore, EGCG inhibits the self-renewal capacity of CD44+α2β1+CD133+ CSCs isolated from human primary prostate tumors, as measured by spheroid formation in suspension. EGCG induces apoptosis by activating capase-3/7 and inhibiting the expression of Bcl-2, survivin and XIAP in CSCs. Furthermore, EGCG inhibits epithelial-mesenchymal transition by inhibiting the expression of vimentin, slug, snail and nuclear β-catenin, and the activity of LEF-1/TCF responsive reporter, and also retards CSC's migration and invasion, suggesting the blockade of signaling involved in early metastasis. Interestingly, quercetin synergizes with EGCG in inhibiting the self-renewal properties of prostate CSCs, inducing apoptosis, and blocking CSC's migration and invasion. These data suggest that EGCG either alone or in combination with quercetin can eliminate cancer stem cell-characteristics. Conclusion Since carcinogenesis is a complex process, combination of bioactive dietary agents with complementary activities will be beneficial for prostate cancer prevention and/ortreatment.Peer Reviewe

Filamin A regulates focal adhesion disassembly and suppresses breast cancer cell migration and invasion

The actin cross-linking protein filamin A reduces migration, invasion, and metastasis of breast cancer cells

Epigenetic profile of the euchromatic region of human Y chromosome

The genome of a multi-cellular organism acquires various functional capabilities in different cell types by means of distinct chromatin modifications and packaging states. Acquired during early development, the cell type-specific epigenotype is maintained by cellular memory mechanisms that involve epigenetic modifications. Here we present the epigenetic status of the euchromatic region of the human Y chromosome that has mostly been ignored in earlier whole genome epigenetic mapping studies. Using ChIP-on-chip approach, we mapped H3K9ac, H3K9me3, H3K27me3 modifications and CTCF binding sites while DNA methylation analysis of selected CpG islands was done using bisulfite sequencing. The global pattern of histone modifications observed on the Y chromosome reflects the functional state and evolutionary history of the sequences that constitute it. The combination of histone and DNA modifications, along with CTCF association in some cases, reveals the transcriptional potential of all protein coding genes including the sex-determining gene SRY and the oncogene TSPY. We also observe preferential association of histone marks with different tandem repeats, suggesting their importance in genome organization and gene regulation. Our results present the first large scale epigenetic analysis of the human Y chromosome and link a number of cis-elements to epigenetic regulatory mechanisms, enabling an understanding of such mechanisms in Y chromosome linked disorders

PDGF-C Induces Maturation of Blood Vessels in a Model of Glioblastoma and Attenuates the Response to Anti-VEGF Treatment

Recent clinical trials of VEGF inhibitors have shown promise in the treatment of recurrent glioblastomas (GBM). However, the survival benefit is usually short-lived as tumors escape anti-VEGF therapies. Here we tested the hypothesis that Platelet Derived Growth Factor-C (PDGF-C), an isoform of the PDGF family, affects GBM progression independent of VEGF pathway and hinders anti-VEGF therapy.We first showed that PDGF-C is present in human GBMs. Then, we overexpressed or downregulated PDGF-C in a human GBM cell line, U87MG, and grew them in cranial windows in nude mice to assess vessel structure and function using intravital microscopy. PDGF-C overexpressing tumors had smaller vessel diameters and lower vascular permeability compared to the parental or siRNA-transfected tumors. Furthermore, vessels in PDGF-C overexpressing tumors had more extensive coverage with NG2 positive perivascular cells and a thicker collagen IV basement membrane than the controls. Treatment with DC101, an anti-VEGFR-2 antibody, induced decreases in vessel density in the parental tumors, but had no effect on the PDGF-C overexpressing tumors.These results suggest that PDGF-C plays an important role in glioma vessel maturation and stabilization, and that it can attenuate the response to anti-VEGF therapy, potentially contributing to escape from vascular normalization

Targeting Epigenetic Regulation of miR-34a for Treatment of Pancreatic Cancer by Inhibition of Pancreatic Cancer Stem Cells

MicroRNA-34a (miR-34a) is a transcriptional target of p53 and is down-regulated in pancreatic cancer. This study aimed to investigate the functional significance of miR-34a in pancreatic cancer progression through its epigenetic restoration with chromatin modulators, demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-dC) and HDAC inhibitor Vorinostat (SAHA).Re-expression of miR-34a in human pancreatic cancer stem cells (CSCs) and in human pancreatic cancer cell lines upon treatment with 5-Aza-dC and SAHA strongly inhibited the cell proliferation, cell cycle progression, self-renewal, epithelial to mesenchymal transition (EMT) and invasion. In pancreatic CSCs, modulation of miR-34a induced apoptosis by activating caspase-3/7. Treatment of pancreatic CSCs with the chromatin-modulating agents resulted in the inhibition of Bcl-2, CDK6 and SIRT1, which are the putative targets of miR-34a. MiR-34a upregulation by these agents also induced acetylated p53, p21(WAF1), p27(KIP1) and PUMA in pancreatic CSCs. Inhibition of miR-34a by antagomiR abrogates the effects of 5-Aza-dC and SAHA, suggesting that 5-Aza-dC and SAHA regulate stem cell characteristics through miR-34a. In CSCs, SAHA inhibited Notch pathway, suggesting its suppression may contribute to inhibition of the self-renewal capacity and induction of apoptosis. Interestingly, treatment of pancreatic CSCs with SAHA resulted in the inhibition of EMT with the transcriptional up-regulation of E-Cadherin and down-regulation of N-Cadherin. Expression of EMT inducers (Zeb-1, Snail and Slug) was inhibited in CSCs upon treatment with SAHA. 5-Aza-dC and SAHA also retard in vitro migration and invasion of CSCs.The present study thus demonstrates the role of miR-34a as a critical regulator of pancreatic cancer progression by the regulating CSC characteristics. The restoration of its expression by 5-Aza-dC and SAHA in CSCs will not only provide mechanistic insight and therapeutic targets for pancreatic cancer but also promising reagents to boost patient response to existing chemotherapies or as a standalone cancer drug by eliminating the CSC characteristics

EGCG Enhances the Therapeutic Potential of Gemcitabine and CP690550 by Inhibiting STAT3 Signaling Pathway in Human Pancreatic Cancer

Background: Signal Transducer and Activator of Transcription 3 (STAT3) is an oncogene, which promotes cell survival, proliferation, motility and progression in cancer cells. Targeting STAT3 signaling may lead to the development of novel therapeutic approaches for human cancers. Here, we examined the effects of epigallocathechin gallate (EGCG) on STAT3 signaling in pancreatic cancer cells, and assessed the therapeutic potential of EGCG with gemcitabine or JAK3 inhibitor CP690550 (Tasocitinib) for the treatment and/or prevention of pancreatic cancer. Methodology/Principal Findings: Cell viability and apoptosis were measured by XTT assay and TUNEL staining, respectively. Gene and protein expressions were measured by qRT-PCR and Western blot analysis, respectively. The results revealed that EGCG inhibited the expression of phospho and total JAK3 and STAT3, STAT3 transcription and activation, and the expression of STAT3-regulated genes, resulting in the inhibition of cell motility, migration and invasion, and the induction of caspase-3 and PARP cleavage. The inhibition of STAT3 enhanced the inhibitory effects of EGCG on cell motility and viability. Additionally, gemcitabine and CP690550 alone inhibited STAT3 target genes and synergized with EGCG to inhibit cell viability and induce apoptosis in pancreatic cancer cells. Conclusions/Significance: Overall, these results suggest that EGCG suppresses the growth, invasion and migration of pancreatic cancer cells, and induces apoptosis by interfering with the STAT3 signaling pathway. Moreover, EGCG furthe

O-Glycosylation Regulates Ubiquitination and Degradation of the Anti-Inflammatory Protein A20 to Accelerate Atherosclerosis in Diabetic ApoE-Null Mice

Background: Accelerated atherosclerosis is the leading cause of morbidity and mortality in diabetic patients. Hyperglycemia is a recognized independent risk factor for heightened atherogenesis in diabetes mellitus (DM). However, our understanding of the mechanisms underlying glucose damage to the vasculature remains incomplete. Methodology/Principal Findings: High glucose and hyperglycemia reduced upregulation of the NF-κB inhibitory and atheroprotective protein A20 in human coronary endothelial (EC) and smooth muscle cell (SMC) cultures challenged with Tumor Necrosis Factor alpha (TNF), aortae of diabetic mice following Lipopolysaccharide (LPS) injection used as an inflammatory insult and in failed vein-grafts of diabetic patients. Decreased vascular expression of A20 did not relate to defective transcription, as A20 mRNA levels were similar or even higher in EC/SMC cultured in high glucose, in vessels of diabetic C57BL/6 and FBV/N mice, and in failed vein grafts of diabetic patients, when compared to controls. Rather, decreased A20 expression correlated with post-translational O-Glucosamine-N-Acetylation (O-GlcNAcylation) and ubiquitination of A20, targeting it for proteasomal degradation. Restoring A20 levels by inhibiting O-GlcNAcylation, blocking proteasome activity, or overexpressing A20, blocked upregulation of the receptor for advanced glycation end-products (RAGE) and phosphorylation of PKCβII, two prime atherogenic signals triggered by high glucose in EC/SMC. A20 gene transfer to the aortic arch of diabetic ApoE null mice that develop accelerated atherosclerosis, attenuated vascular expression of RAGE and phospho-PKCβII, significantly reducing atherosclerosis. Conclusions: High glucose/hyperglycemia regulate vascular A20 expression via O-GlcNAcylation-dependent ubiquitination and proteasomal degradation. This could be key to the pathogenesis of accelerated atherosclerosis in diabetes

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery