Induced Tamoxifen Resistance is Mediated by Increased Methylation of E-Cadherin in Estrogen Receptor-Expressing Breast Cancer Cells.

Estrogen receptor-positive breast cancers are treated with tamoxifen, a drug that competitively inhibits the binding of estrogen to its receptor. Resistance to tamoxifen is a major hurdle in effective management of target breast cancer patient population. A number of dynamic changes within the tumor microenvironment, including the phenomenon of epithelial to mesenchymal transition (EMT), determine the response to endocrine therapy. EMT is marked by silencing or suppression of epithelial marker, E-Cadherin and we found significantly down-regulated E-Cadherin, among other epithelial markers, and a significantly up-regulated mesenchymal marker, Twist, among other mesenchymal markers, in a model system that comprised of tamoxifen sensitive MCF-7 cells and their tamoxifen-resistant counterparts, MCF-7-TAM, developed by chronic and escalating exposure of parental cells to tamoxifen. Further, E-cadherin, but not Twist, was differentially expressed in MCF-7-TAM cells because of differential methylation. Treatment with demethylating agent 5-azacytidine increased the expression of E-cadherin thus verifying a role of methylation in its silencing and, moreover, 5-azacytidine treatment also re-sensitized MCF-7-TAM cells to tamoxifen, as evaluated by assays for viability, apoptosis and migration potential. The 5-azacytidine effects were similar to effects of E-cadherin overexpression in MCF-7-TAM cells. This work describes novel mechanism of E-cadherin downregulation in tamoxifen resistant breast cancer cells. Further studies are needed to exploit this information for betterment of breast cancer therapy.

Risk factors and therapies in vascular diseases: An umbrella review of updated systematic reviews and meta-analyses.

Aberrant blood vessel functioning and systemic circulation are key causes for vascular disorders; cardiovascular, cerebrovascular, renal artery stenosis, and peripheral artery diseases. Epidemiological and basic science evidence supported genetic reasons, compounded by obesity, hypercholesterolemia, hypertension, diabetes, and smoking as risk factors. This is an umbrella review of risk factors and therapies in vascular disorders, exploring systematic reviews and meta-analyses studies in PubMed, Cochrane, Embase, and Central published in January 2000-May 2018. We made qualitative eligibility gradation of the articles based on inclusion criteria, and independently extracted descriptive and methodologic data to compile their outcomes. We considered 95% confidence interval and the between-study heterogeneity, designated by I 2 . Overall, we extracted 217 studies of impressive quality and at low risk of bias, including 124, 30, 23, 32, and 8, respectively, for the search terms "cardiovascular," "renal," "cerebral," and "limb ischemia" each in combination with "risk factors" and "therapeutics." Our search on genome-wide analyses revealed genes associated with HDL-cholesterol, matrix metalloproteases, angiogenesis, notch3, renin-angiotensin, apolipoprotein E, insulin, and cytokine levels as critical participants in the pathogenesis of vascular diseases. Hypertension and endothelial growth factor-linked polymorphisms were found to contribute to vascular damage. The studies reinforced that lifestyle and dietary patterns influenced susceptibility of circulatory system diseases. Additionally, endovascular medicines, surgical vascularization, angioplasty, and renal artery stenting appeared as major therapeutic approaches in vascular patients. Altogether, our review offers up-to-date information on pathophysiology of vascular diseases and provides insight into existing research, clinical management and clinical gaps in the field.

Cardioprotection and improvement in endothelial-dependent vasodilation during late-phase of whole body hypoxic preconditioning in spontaneously hypertensive rats via VEGF and endothelin-1.

The present study was designed to investigate the effect of late phase of whole body hypoxic preconditioning on endothelial-dependent vasorelaxation and cardioprotection from ischemia-reperfusion injury in spontaneously hypertensive rats (SHR). Hypoxic preconditioning was performed by subjecting rats to four episodes of alternate exposure to low O2 (8%) and normal air O2 of 10 min each. After 24 h, the mesenteric arteries and hearts were isolated to determine the vascular function and cardioprotection from ischemia-reperfusion (I/R) injury on the Langendorff apparatus. There was a significant impairment in acetylcholine-induced relaxation in norepinephrine precontracted arteries (endothelium-dependent function) and increase in I/R-induced myocardial injury in SHR in comparison to Wistar Kyoto rats (WKY). However, hypoxic preconditioning significantly restored endothelium-dependent relaxation in SHR and attenuated I/R injury in both SHR and WKY. Hypoxic preconditioning also led to an increase in the levels of endothelin-1 (not endothelin-2 or -3), vascular endothelial growth factor-A (VEGF-A) and HIF-1α levels. Pretreatment with bevacizumab (anti-VEGF-A) and bosentan (endothelin receptor blocker) significantly attenuated hypoxic preconditioning-induced restoration of endothelium-dependent relaxation and cardioprotection from I/R injury. These interventions also attenuated the levels of VEGF-A and HIF-1α without modulating the endothelin-1 levels. It may be concluded that an increase in the endothelin-1 levels with a subsequent increase in HIF-1α and VEGF expression may possibly contribute in improving endothelium-dependent vasorelaxation and protecting hearts from I/R injury in SHR during late phase of whole body hypoxic preconditioning.

Involvement of Endothelin 1 in Remote Preconditioning-Induced Cardioprotection through connexin 43 and Akt/GSK-3ß Signaling Pathway.

The present study was aimed to explore the role of endothelins in remote preconditioning (RP)-induced myocardial protection in ischemia-reperfusion (IR) injury. RP stimulus was given by subjecting hind limb to four cycles of ischemia and reperfuion (5 minutes each) using blood pressure cuff in male rats. Following RP, hearts were isolated and subjected to 30 minutes of ischemia and 120 minutes of reperfusion on Langendorff apparatus. The extent of myocardial injury was determined by measuring the levels of LDH-1, CK-MB and cardiac troponin T (cTnT) in coronary effluent; caspase-3 activity and Bcl 2 expression in heart (apoptosis); infarct size by triphenyl tetrazolium chloride and contractility parameters including left ventricular developed pressure, dp/dtmax dp/dtmin and heart rate. RP reduced ischemia reperfusion-induced myocardial injury, increased the levels of endothelin 1 (in blood), Akt-P, GSK-3ß-P and P-connexin 43 (in hearts). Pretreatment with ETA receptor antagonist, BQ 123 (1 and 2 mg/kg), ETB receptor antagonist, BQ 788 (1 and 3 mg/kg) and dual inhibitor of ETA and ETB receptor, bonsentan (25 and 50 mg/kg) abolished these effects of RP. However, the effects of bonsentan were more pronounced in comparison to BQ 123 and BQ 788. It is concluded that RP stimulus may release endothelin 1 in the blood, which may activate myocardial ETA and ETB receptors to trigger cardioprotection through connexin 43 and Akt/GSK-3ß pathway.

Cyclin A2 regulates homologous recombination DNA repair and sensitivity to DNA damaging agents and poly(ADP-ribose) polymerase (PARP) inhibitors in human breast cancer cells.

Defects in homologous recombination (HR) repair are found in breast cancers. Intriguingly, breast cancers with defective HR show increased sensitivity to DNA crosslinking agents and poly(ADP-ribose) polymerase (PARP) inhibitors. As such, genes that can affect HR functions have been of high interest in studies aiming to develop biomarkers for predicting response to treatment with these agents. Cyclin A2 is a key component of the core cell cycle machinery. However, whether cyclin A2 dysfunctions could cause HR defect and mediate sensitivity to DNA damaging agents remain unclear. Here we show that loss of cyclin A2 causes high rates of double-strand breaks (DSB) in MCF-7 and MDA-MB-231 cells. The increased DSB was due to defective HR-mediated repair of the breaks, resulting from reduced MRE11 and RAD51 proteins. Cyclin A2 mediates MRE11 abundance through its MRE11 mRNA binding property and RAD51 abundance through inhibition of proteasome degradation of RAD51. Moreover, cyclin A2 depletion hypersensitized the cells to DNA damaging agents, such as cisplatin and melphalan. Our results demonstrate novel roles for cyclin A2 in regulating HR repair and determining sensitivity to DNA cross linkers and PARP inhibitors in breast cancer cells.

Pokemon and MEF2D co-operationally promote invasion of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most deadly human malignancy, and frequent invasion and metastasis is closely associated with its poor prognosis. However, the molecular mechanism underlying HCC invasion is still not completely elucidated. Pokemon is a well-established oncogene for HCC growth, but its contribution to HCC invasion has not been studied yet. In this paper, Pokemon was found to be overexpressed in MHCC-97H HCC cell line, which possesses higher invasiveness. Downregulation of Pokemon abolished the invasion of MHCC-97H HCC cell lines. Pokemon overexpression was able to enhance the invasion of MHCC-97L cells with lower invasiveness. MEF2D, an oncogene promoting the invasion of HCC cells, was further detected to be upregulated and downregulated when Pokemon was overexpressed and silenced, respectively. Online database analysis indicated that one Pokemon recognition site was located within the promoter of MEF2D. Chromatin co-precipitation, luciferase, and qPCR assays all proved that Pokemon can promote the expression of MEF2D in HCC cells. Restoration of MEF2D expression can prevent the impaired invasion of HCC cells with Pokemon silencing, while suppression of MEF2D abolished the effect of Pokemon overexpression on HCC invasion. More interestingly, MEF2D was also found to increase the transcription of Pokemon by binding myocyte enhancer factor 2 (MEF2) sites within its promoter region, implying an auto-regulatory circuit consisting of these two oncogenes that can promote HCC invasion. Our findings can contribute to the understanding of molecular mechanism underlying HCC invasion, and provided evidence that targeting this molecular loop may be a promising strategy for anti-invasion therapy.

AGR2 expression is regulated by HIF-1 and contributes to growth and angiogenesis of glioblastoma.

Glioblastoma multiforme (GBM) tumors are the most common type of brain tumors characterized by extensive angiogenesis that is mostly orchestrated by tumor hypoxia. The hypoxia induced factor-1 (HIF-1) transcriptional complex is the "master control switch" for hypoxia. Dysregulation of anterior gradient protein 2 (AGR2) expression is associated with tumor growth and metastasis. Whether AGR2 is a hypoxia-responsive factor and affects tumor progression via angiogenesis remains unknown. Here, we show that GBM cell lines, U87 and LN18, exhibited enhanced hypoxic responses compared with control normal human astrocytes, and a corresponding HIF-1-dependent increase in AGR2 mRNA and protein. Recombinant AGR2 and conditioned medium from GBM cells induced human umbilical vein endothelial cell (HUVEC) migration and tube formation, which were abrogated by anti-AGR2 neutralizing antibodies. Expression of the HIF-1α oxygen-dependent degradation domain mutant in cells resulted in elevated AGR2 levels and an increased ability to induce HUVEC migration and tube formation in vitro and enhanced growth and vascularity of tumor xenografts in vivo, which were prevented by AGR2 knockdown. Taken together, these results indicate that AGR2 expression is regulated by HIF-1 and plays an important role in control of glioblastoma growth and vascularity. Our findings suggest that inhibiting AGR2 may represent a new therapeutic target for anti-angiogenic cancer treatment.