Eckol, a potential inhibitor of aryl hydrocarbon receptor, inhibits proliferation and induces apoptosis in breast cancer cells

The present study was designed to explore phlorotannin eckol protein targets and evaluate the anti-cancer effects of eckol against human breast cancer cells. ProTox-II server and protein-ligand docking analysis deter-mined the aryl hydrocarbon receptor (AhR) as a putative target of eckol. The AhR has been determined as a potential target for breast cancer treatment. The effect of eckol treatment on proliferation, apoptosis, and cell cycle activities was detected in MDA-MB-231 and SK-BR-3 breast cancer cell lines which were selected dependent on AhR expression profiles. Consistent with the AhR expression profile, MDA-MB-231 cells were more sensitive to eckol treatment compared to SK-BR-3 cells in proliferation, apoptosis and cell cycle results. Eckol treatment shows a significant antiproliferative and apoptotic response in breast cancer cells. Overall, these results indicated that the action of eckol may be related to the AhR gene regulation in different breast cancer cell lines

Improved targeting for photodynamic therapy via a biotin-phthalocyanine conjugate: synthesis, photophysical and photochemical measurements, and in vitro cytotoxicity assay

In this study, the peripherally biotin-substituted zinc(ii) phthalocyanine (Pc2) was synthesized as a photosensitizer for the treatment of cancer by photodynamic therapy. The photophysico-chemical properties of the zinc(ii) phthalocyanine-bearing mono-biotin and three branched polyoxyethylene groups were studied in DMSO. The photodynamic activities of this compound were tested on HeLa cervical carcinoma cells and HuH-7 human liver carcinoma cells. The dark toxicity and photosensitizing effect of the conjugate were compared to those of amino-functionalized zinc(ii) phthalocyanine (Pc1) to determine the effect of the biotin group on the photodynamic activity. According to the results, Pc1 showed good photodynamic activity reaction against HeLa and HuH-7 cancer cells. Although the results of the photochemical and photophysical data of Pc1 and Pc2 were close, the in vitro studies of these compounds (1 and 2) have shown that the biotin-substituted conjugate (Pc2) more effectively decreased cell survival than the non-biotin-bearing derivative (Pc1) due to the targeting effect of the biotin. Furthermore, the apoptosis ratio of 2 by the HeLa and HuH-7 cells was detected by flow cytometry via Annexin V/PI double-staining assay. The percentages of the late apoptotic cells were 3.5% and 0.4% in the control groups of HuH-7 and HeLa cells, respectively. The late apoptotic cells increased under light conditions in both HeLa (63.9%) and HuH-7 (83.6%) cells compared to dark conditions (HuH-7, 1% and HeLa, 0.4%). The cellular uptake of Pc2 was relatively high (34.5-fold in HuH-7 and 459 fold in HeLa cells) and localized in the cytoplasm in different cancer model cells. Moreover, the Pc2 treatment and illumination strictly reduced the cell colony forming capacity. The photodynamic activity of the biotin conjugate can be related to the high cellular uptake and/or singlet oxygen generation yield of this photosensitizer

Hepatocellular Carcinoma Cells with Downregulated ZEB2 Become Resistant to Resveratrol by Concomitant Induction of ABCG2 Expression

In hepatocellular carcinoma (HCC), the presence of cancer stem cells (CSCs) have been linked to drug resistance, epithelial-mesenchymal transition (EMT), and cancer relapse. This study investigates the expression profile of ZEB1, ZEB2, ABCG2 in HCC-CSCs, and the role of EMT promoter ZEB2 in cells treated with resveratrol. The expression of ZEB1, ZEB2 and ABCG2 transcripts were analyzed in CD133(+)/CD44(+) cells isolated from the PLC/PRF/5 cell line. ZEB2-dependent ABCG2 gene expression and the effects of resveratrol on proliferation, cell cycle and apoptosis were explored in SNU398 cell clones. An inverse correlation between ZEB1/ZEB2 and ABCG2 levels were observed both in CSCs and in ZEB2-knockdown cells. The resveratrol treatment significantly decreased cell viability, while promoting cell cycle arrest in ZEB2-independent manner. Interestingly, resveratrol-treated cells with low levels of ZEB2 were resistant to apoptosis. The interplay of expression levels of ABCG2 and ZEB family EMT transcription factors may play a role in establishing CSC-like phenotype in HCC cells resistant to resveratrol

Epithelial-to-Mesenchymal Plasticity in Circulating Tumor Cell Lines Sequentially Derived from a Patient with Colorectal Cancer

Metastasis is a complicated and only partially understood multi-step process of cancer progression. A subset of cancer cells that can leave the primary tumor, intravasate, and circulate to reach distant organs are called circulating tumor cells (CTCs). Multiple lines of evidence suggest that in metastatic cancer cells, epithelial and mesenchymal markers are co-expressed to facilitate the cells’ ability to go back and forth between cellular states. This feature is called epithelial-to-mesenchymal plasticity (EMP). CTCs represent a unique source to understand the EMP features in metastatic cascade biology. Our group previously established and characterized nine serial CTC lines from a patient with metastatic colon cancer. Here, we assessed the expression of markers involved in epithelial–mesenchymal (EMT) and mesenchymal–epithelial (MET) transition in these unique CTC lines, to define their EMP profile. We found that the oncogenes MYC and ezrin were expressed by all CTC lines, but not SIX1, one of their common regulators (also an EMT inducer). Moreover, the MET activator GRHL2 and its putative targets were strongly expressed in all CTC lines, revealing their plasticity in favor of an increased MET state that promotes metastasis formation

Effect of vascular endothelial growth factor on sperm motility and survival

Vascular endothelial growth factor (VEGF) and its receptors are present in both male and female reproductive Systems. In this experimental study.. the effect of different concentrations of VEGF on sperm motility and survival in vitro was investigated. Human spermatozoa, collected from voluntary, proven fertile donors, were incubated in sperm washing medium containing different concentrations of VEGF (5, 10, 15, 20 ng/ml) for 24 h in a university reproductive endocrinology laboratory setting. Assessment of VEGF action on sperm motion characteristics was evaluated using a computer-assisted semen analyser. Sperm survival was determined by hypo-osmotic swelling and eosin-Y dye tests. VEGF had a positive effect on some parameters of sperm motility in a concentration-dependent manner. Maximal effect was observed at a concentration of 15 ng/ml; motility, progression, straight-line velocity and curvilinear velocity of VEGF-exposed spermatozoa were significantly increased (P < 0.05) at this concentration. However, sperm viability was not prolonged at any concentration of VEGF as shown by hypo-osmotic swelling and eosin-Y dye tests. VEGF may increase some sperm motility parameters, but not survival, in a concentration-dependent manner in vitro

Novel triazole containing zinc(II)phthalocyanine Schiff bases: Determination of photophysical and photochemical properties for photodynamic cancer therapy

A simple and efficient synthesis of novel zinc(II) phthalocyanines bearing triazole moieties was described. The synthetic approach for preparation of the phthalocyanines 1 and 13 was achieved by Schiff base condensation reaction of phthalocyanine tetracarbaldehydes 3 and 12 with 4-amino-4H-1,2,4-triazole 5 in tetrahydrofuran in reasonable yields. The photophysical and photochemical properties of the compounds 1, 3, 12 and 13 were recorded only in DMSO. The singlet oxygen generation ability of the targeted phthalocyanine Schiff bases were investigated in an attempt to understand their potential for photodynamic therapy (PDT) activity. Moreover, in vitro PDT application was performed against the MCF-7 and MDA-MB-231 invasive breast carcinoma cell lines. Preliminary assay showed that the compounds 1 and 13 possessed the phototoxicity and cytotoxicity, with a maximum of 30% MCF-7 cells dead following light irradiation. It was also revealed that the targeted phthalocyanines possess promising characteristic as photosensitizer towards tumor cells

Genome-wide analysis of endogenously expressed ZEB2 binding sites reveals inverse correlations between ZEB2 and GalNAc-transferase GALNT3 in human tumors

ZEB2 is a transcriptional repressor that regulates epithelial-to-mesenchymal transition (EMT) through binding to bipartite E-box motifs in gene regulatory regions. Despite the abundant presence of E-boxes within the human genome and the multiplicity of pathophysiological processes regulated during ZEB2-induced EMT, only a small fraction of ZEB2 targets has been identified so far. Hence, we explored genome-wide ZEB2 binding by chromatin immunoprecipitation-sequencing (ChIP-seq) under endogenous ZEB2 expression conditions

ETS1 is coexpressed with ZEB2 and mediates ZEB2-induced epithelial-mesenchymal transition in human tumors

Epithelial-mesenchymal transition (EMT) is an embryonic program that is reactivated in cancer and regulates the invasion and metastasis of tumor cells. Zinc finger E-box binding homeobox 2 (ZEB2) induces EMT by upregulating matrix metalloproteinases (MMP), yet MMP genes lack ZEB2 binding motif in their promoters. Recently, expression of MMPs was associated to the activation of ETS1 transcription factor; however, a link between ZEB2 and ETS proto-oncogene 1, transcription factor (ETS1) remains to be elucidated. Hence, we investigated the transcriptional regulation of ETS1 by ZEB2 after our initial observation that ZEB2 and ETS1 are coexpressed in hepatocellular carcinoma cells (HCCs). Chromatin immunoprecipitation and luciferase reporter assays clearly showed that ZEB2 binds to E-box sequences on the promoter of ETS1. Elevated expression of ETS1 was found in DLD-ZEB2 and A431-ZEB2 inducible systems, and knockdown of ZEB2 caused an explicit downregulation of ETS1 in shZEB2-SNU398 and shZEB2-SK-HEP-1 cells. Repression of ETS1 expression in ZEB2-induced conditions substantially impaired the migration and invasive capacities of DLD1 cells. Mechanistically, knockdown of ETS1 in ZEB2-expressing cells resulted in the downregulation of established ZEB2 targets TWIST and MMP9. Correlation analyses in HCC lines, cancer complementary DNA arrays, and The Cancer Genome Atlas RNA-sequencing data set revealed that ZEB2 and ETS1 are coexpressed, and their expressions in human tumors show a highly significant positive correlation. Our results demonstrated that ZEB2 acts as an upstream regulator of ETS1 and, in turn, ETS1 maintains ZEB2-induced EMT. These findings add another level of complexity to the understanding of ZEB2 in the invasion and metastasis of cancer cells, and put ZEB2/ETS1 axis as a novel therapeutic target in human malignancies

Genome-wide analysis of endogenously expressed ZEB2 binding sites reveals inverse correlations between ZEB2 and GalNAc-transferase GALNT3 in human tumors

Background: ZEB2 is among transcriptional repressors that regulate epithelial-to-mesenchymal transition through binding to bipartite E-box motifs in gene regulatory regions. Despite the high frequency of E-boxes on the genome and multiplicity of pathophysiological processes regulated during ZEB2-induced EMT, only a small fraction of ZEB2 targets have been identified so far. Hence, we explored genome-wide ZEB2 binding by Chromatin immunoprecipitation-sequencing study under endogenous-ZEB2 expression conditions. Methods: we designed a ChIP-Seq study by using a homemade anti-ZEB2 monoclonal antibody, clone 6E5, in SNU398 hepatocellular carcinoma cells with high-endogenous ZEB2 expression. We validated ChIP-Seq targets by ChIP-qPCR assays and explored ZEB2-dependent expression of target genes in ZEB2-repressed and –overexpressed cells by RT-qPCR and Western blotting. Changes in gene expression were assessed in human tumor cDNA arrays by RT-qPCR and, differential expression and correlation analyses were performed in expO and Human Protein Atlas datasets.Results: more than 500 genes were annotated and intervals related to these genes were found to include ZEB2 binding motif “CACCTG” according to TOMTOM motif analysis in the MEME Suite database. Assessment of ZEB2-dependent expression of target genes in ZEB2-silenced SNU398 cells and ZEB2-induced DLD1 cells revealed GALNT3 gene as a target with the highest but inversely correlated expression. Remarkably, GALNT3 also displayed the highest enrichment in ChIP validation assays. In our analyses of tumor cDNA arrays and expO datasets significant differential expression and a significant inverse correlation between ZEB2 and GALNT3 transcripts were detected in most of the tumors. Moreover, protein expression of ZEB2 and GALNT3 was explored in "Human Protein Atlas" database and a negative correlation was observed in all analyzed tumor types, except malignant melanoma. In contrast to negative or weak expression of ZEB2, tumor tissues displayed strong or moderate GALNT3 staining.Conclusions: our findings that ZEB2 negatively regulates a GalNAc-transferase involved in O-glycosylation add another level of complexity to the understanding of the role of ZEB2 in EMT and cancer metastasis. This, in turn, implicates that the proteins glycosylated by GALNT3 might be exploited as novel diagnostics and therapeutic targets in human cancers. <br/