Expression of miR-34 is lost in colon cancer which can be re-expressed by a novel agent CDF

Abstract Background Colorectal Cancer (CRC) is one of the leading causes of death worldwide. Numerous cellular events, including deregulated expression of microRNAs (miRNAs), specifically the family of miR-34 consisting of miR-34a, b and c, is known to regulate the processes of growth and metastasis. Methods We evaluated the expression of miR-34 in formalin-fixed paraffin-embedded (FFPE) human colon cancer tissue specimens compared to normal colonic mucosa. Moreover, we also assessed the expression of miR-34 in colon cancer cell lines treated with our newly developed synthetic analogue of curcumin referred as difluorinated curcumin (CDF) compared to well known inhibitor of methyl transferase. Results We found that the expression of miR-34a and miR-34c was down-regulated in colon cancer specimens compared to normal colonic mucosa and the loss of expression was also consistent with data from colon cancer cell lines. This down-regulation was attributed to promoter hypermethylation, because we found that the treatment of colon cancer cells with 5-aza-2´-deoxycytidine, a methyltransferase inhibitor, markedly induced the levels of miR-34a and miR-34c expression. Likewise, CDF was very effective in the re-expression of miR-34a and miR-34c, which was consistent with inhibition of cell growth of both chemo-sensitive and chemo-resistant colon cancer cells. The re-expression of miR-34 led to a marked reduction in the expression of its target gene, Notch-1. Conclusion The loss of expression of miR-34 in colon cancer is in part due to promoter hypermethylation of miR-34, which can be re-expressed with our novel agent CDF, suggesting that CDF could be a novel demethylating agent for restoring the expression of miR-34 family, and thus CDF could become a newer therapeutic agent for the treatment of colon cancer

Cd30 activation causes cell cycle arrest of the cd30+ anaplastic large cell lymphoma cell lines

Amaç: CD30 aktivasyonu sistemik anaplastik büyük hücreli lenfoma hücrelerinde büyümeyi durdurmaktadır. Bu büyüme inhibisyonunun hangi mekanizmalarla meydana geldiği bilinmemektedir. CD30 reseptör molekülü tümör nekrozis faktör ailesinin bir ferdi olmakla birlikte bir ölüm bölgesi (deaş domain) içermez. Bu nedenle hücre çoğalmasını nasıl olup da durdurabildiği tartışmalı bir konudur. Yöntem: Bu çalışmada bir nodal anaplastik hücre lenfoma hücre serisi olan Karpas 299.u, CD30 aktivasyonunun çoğalma üzerindeki baskılayıcı etkilerini araştırmak amacıyla kullandık. CD30 aktivasyonu bir CD30 agonist antikoru olan HeFi-1 ile sağlandı. CD30 aktivasyonunu takiben Annexin V immun boyası ve akım sitometrik yöntemle apoptoz tespiti yapıldı. 72 saatlik bir gözlem süresince apoptoz gözlenmedi. Bulgular: Apoptoz gözlenmemesi üzerine, hücreler hücre siklusu açısından senkronize edilerek CD30 aktivasyonunu takiben DNA içeriği akım sitometrik yöntemle tespit edildi. Bu yöntemin uygulanmasıyla G1 fazında bir kısmi hücre siklus arresti saptandı. Hücrelerin p21 ve retinoblastoma protein düzeyleri Western blot analizi ile ölçüldü. Bu ölçümler sonunda CD30 aktivasyonuyla birlikte p21 düzeyi artışı ve retinoblastoma proteini hipo-fosforilasyonu tespit edildi. Bu bulgular G1 fazında bir hücre siklusu arresti varlığını ve aracı moleküllerin Rb ve p21 olduğunu ortaya koymaktadır. Sonuç: CD30 aktivasyonunun CD30+ nodal anaplastik lenfoma hücre serilerinde hücre çoğalması üzerindeki baskılayıcı etkilerinin zannedildiği gibi apoptoz yoluyla değil, hücre siklusunu düzenleme yoluyla olduğu ortaya çıkarılmıştır. Bu bulgular nodal anaplastik hücreli lenfomaların tedavisinde yeni tedavi stratejilerinin geliştirilmesine yardımcı olabilir.Aims: CD30 activation causes growth inhibition of the systemic anaplastic large cell lymphoma cell lines. The mechanism of growth inhibition is not well characterized. Since CD30 does not have a death domain like the other tumor necrosis factor receptor superfamily members Fas and TNFRI, the mechanism of growth inhibition is a matter of controversy. Methods: In this study, we analyzed the growth inhibitory effects of CD30 activation on the systemic ALCL cell line Karpas 299 by using a CD30 activating antibody HeFi-1. We first investigated the presence of apoptosis by Annexin V and propidium iodide staining using flow cytometric analysis. We did not observe any apoptotic changes or cell death during the 72 hours following CD30 activation, despite a decrease in thymidine incorporation, which prompted us to investigate the possibility of cell cycle arrest caused by CD30 activation. Results: Upon synchronization of the cells and incubation with the HeFi-1 antibody, we observed a partial arrest at G1 phase demonstrated by propidium iodide staining of the permeabilized cells. We then investigated the expression of cell cycle inhibitory proteins p16, p21, p27 and retinoblastoma protein (Rb) by Western blotting. We observed expression of p21 and hypo-phosphorylation of Rb protein secondary to CD30 activation. Significance: These results are consistent with cell cycle arrest in the G1 phase of the cell cycle following CD30 activation. This finding is contrary to the accepted belief that CD30 activation causes apoptosis. These findings may help the development of new therapeutic strategies against nodal anaplastic large cell lymphomas

Effects of activation of cd30 signaling pathway on cd30+ cutaneous anaplastic large cell lymphoma cell lines

Amaç: CD30 aktivasyonu çeşitli CD30+ lenfoma hücre kültürleri üzerinde farklı etkilere sahiptir; nodal T hücreli CD30+ anaplastik büyük hücreli lenfoma hücrelerinde proliferasyon hızında azalmaya ve sitolize, Hodgkin lenfoması hücre kültürlerinde ise proliferasyona yol açar. Mac-1 ve Mac-2A aynı hastanın erken ve geç evre CD30+ cilt lenfomasından elde edilmiş hücre kültürü serileridir. Bu iki hücre serisi klonal olarak ilişkilidir. Mac- 1 hücre serisinin büyümesi transforming growş factor-beta (TGF-b) tarafından inhibe edilebilmektedir. Mac-2A ise TGF-b tarafından inhibe edilememektedir. Bu hücre serilerinin CD30 aktivasyonuna nasıl yanıt verdikleri bilinmemektedir. Yöntem: CD30 aktivasyonunun Mac hücre serilerinin büyüme özelliklerine etkilerini araştırmak amacıyla hücreler CD30 aktive edici antikor HeFi-1 ile inkübe edilmiş, 3H-şymidine inkorporasyonu, Tümör nekrozis faktör reseptör assosiye faktör (TRAF1) ekspresyonu ve nükleer faktör-bB (NF-kB) aktivitesi değerlendirilmiştir. Bulgular: Mac-1 ve Mac-2A HeFi-1 ile inkübasyon sonucu artmış bir proliferatif yanıt göstermişlerdir. Kontrol olarak kullanılan nodal anaplastik büyük hücreli lenfoma hücreleri ise büyüme inhibisyonu göstermişlerdir. Mac- 1 hücreleri HeFi- ve TGF-b nötralize edici antikorla inkübe edildiklerinde, proliferatif yanıt tek başına HeFi-1 inkübasyonuna oranla ciddi şekilde artmıştır. TGF-b ya rezistan hücre kültürü Mac-2A ise aynı yanıtı göstermemiştir. Mac-1 and Mac-2A HeFi-1 aracılığıyla CD30 aktivasyonuna NF-kB nükleer bağlanma aktivitesi artımı ve artmış TRAF1 ekspresyonu ile yanıt vermiştir. Sonuç: Bu çalışma CD30+ anaplastik büyük hücreli deri lenfomalarında CD30 iletişim yolunun işlevselliğinin gösterildiği ilk çalışmadır. Mac-1 hücrelerinden otokrin salınan TGF-b CD30 aktivasyonundan doğan proliferatif yanıtı kısmen inhibe etmektedir. Bu sonuçlar TGF-b nın CD30 iletişim sistemi ile interaksiyonu olduğunu ve bunun CD30+ deri lenfomaları patojenezinde bir rol oynayabileceğini de düşündürmektedirAims: CD30 activation has pleiotropic effects on different cell lines representing CD30+ lymphomas. In nodal T-cell anaplastic large cell lymphomas (ALCL), CD30 activation causes cytolysis and decreased proliferation while in Hodgkin s disease there is either no change or proliferation depending on the cell lines. Mac-1 and Mac- 2A are two clonally related cutaneous CD30+ anaplastic large cell lymphoma cell lines developed from early (Mac-1) and advanced (Mac-2A) disease from the same patient. Mac-1 is sensitive to transforming growth factor-b (TGF-b) mediated growth inhibition while Mac-2A is resistant. Both cell lines secrete an activated form of TGF-?. Our aim was to investigate the effects of CD30 activation on Mac cell lines. Methods: To understand the effects of CD30 activation, Mac cell lines were incubated with a CD30 agonistic antibody (HeFi-1). 3H-thymidine incorporation, tumor necrosis factor receptor associated factor 1 (TRAF1) expression and nuclear factor-kB activity was determined. Results: Mac-1 and Mac-2A showed increased proliferation with HeFi-1 while the nodal ALCL cell lines were inhibited. When Mac-1 cells were incubated with HeFi-1 and TGF-b neutralizing antibody, the proliferative rate markedly increased compared to HeFi-1 only. TGF-b resistant cell line Mac-2A did not show the same increase Mac-1 and Mac-2A had activation of NF-kB binding activity and increased expression of TRAF1 in response to CD30 activation by HeFi-1. Significance: This is the first demonstration of functionality of the CD30 signaling pathway in cutaneous CD30+ ALCLs. Autocrine TGF-b secreted from Mac-1 cells partially inhibits the proliferative signal from CD30 activation. These results suggest that TGF-b may interact with the CD30 signaling pathway in the pathogenesis of cutaneous ALCL

Inflammatory pseudotumor of lymph nodes (a case report)

Yedi yaşında erkek çocukta servikal bölgede lenf düğümü büyümesi, ateş, sedimentasyon artışı bulguları mevcuttur. Boyutları 3x2.5x1.5 cm olan lenf düğümünün seri kesitlerinde kapsül altında, hilus bölgesinde iğsi hücrelerle birlikte inflamatuar hücreler ve damar yapılarında artış izlendi. ımmunhistokimyasal çalışmada iğsi hücreler, kas spesifik aktin (+), vimentin (+), LCA (-), CD3 (-), CD79(-), FVIII (-), desmin (-) olarak değerlendirildi. Histokimyasal yöntemle Ziehl-Neelsen (-) di. Histopatolojik ve klinik bulgularla birlikte lenf düğümünün inflamatuar psödotümörü (ıPT) tanısı konulmuştur. Lenf nodunun inflamatuar psödotümörü lenfadenopatinin benign nedenleri arasında son zamanlarda tanımlanmıştır. Ayırıcı tanı diğer reaktif durumları, Castleman hastalığını, malign lenfoma (Hodgkin lenfoma, periferikşücreli lenfoma) ve malign fibröz histiositomayı içerir. Olgu seyrek görülmesi nedeniyle literatür eşliğinde sunulmuştur.A 7 year-old boy presented with cervical lymph node enlargement, fever and elevated erythrocyte sedimentation rate. A neck mass measuring 3x2.5x1.5 cms was surgically removed. Histologically, the process showed a proliferation of spindle cells, associated with a mixture of inflammatory cells and small blood vessels involving the connective tissue framework (hilum, trabecula, capsule) of the lymph node. Immunohistochemical analysis showed spindle cells positive for muscle-spesific actin and vimentin, but negative for desmin. Histochemical, Ziehl-Neelsen stain was negative. With these clinical and histopathologic findings a diagnosis of “ inflammatory pseudotumor of lymph nodes” was made. Inflammatory pseudotumor of the lymph nodes is a rare, recently described benign cause of lymphadenopathy. The differential diagnosis includes other reactive processes, Castleman's dissease, malignant lymphoma (Hodgkin's lymphoma, peripheral T cell lymphoma) and malignant fibrous histiocytoma. This case is presented due to its rare occurrence along with areview of literature

CARP-1 Functional Mimetics Are a Novel Class of Small Molecule Inhibitors of Malignant Pleural Mesothelioma Cells

Malignant pleural mesothelioma (MPM) is an asbestos-related thoracic malignancy that is characterized by late metastases, and resistance to therapeutic modalities. The toxic side-effects of MPM therapies often limit their clinical effectiveness, thus necessitating development of new agents to effectively treat and manage this disease in clinic. CARP-1 functional mimetics (CFMs) are a novel class of compounds that inhibit growth of diverse cancer cell types. Here we investigated MPM cell growth suppression by the CFMs and the molecular mechanisms involved. CFM-1, -4, and -5 inhibited MPM cell growth, in vitro, in part by stimulating apoptosis. Apoptosis by CFM-4 involved activation of pro-apoptotic stress-activated protein kinases (SAPKs) p38 and JNK, elevated CARP-1 expression, cleavage of PARP1, and loss of the oncogene c-myc as well as mitotic cyclin B1. Treatments of MPM cells with CFM-4 resulted in depletion of NF-κB signaling inhibitor ABIN1 and Inhibitory κB (IκB)α and β, while increasing expression of pro-apoptotic death receptor (DR) 4 protein. CFM-4 enhanced expression of serine-phosphorylated podoplanin and cleavage of vimetin. CFMs also attenuated biological properties of the MPM cells by blocking their abilities to migrate, form colonies in suspension, and invade through the matrix-coated membranes. Both podoplanin and vimentin regulate processes of cell motility and invasion, and their expression often correlates with metastatic disease, and poor prognosis. The fact that phosphorylation of serines in the cytoplasmic domain of podoplanin interferes with processes of cellular motility, CFM-4-dependent elevated phosphorylated podoplanin and cleavage of vimentin underscore a metastasis inhibitory property of these compounds, and suggest that CFMs and/or their future analogs have potential as anti-MPM agents

A H2AX–CARP-1 Interaction Regulates Apoptosis Signaling Following DNA Damage

Cell Cycle and Apoptosis Regulatory Protein (CARP-1/CCAR1) is a peri-nuclear phosphoprotein that regulates apoptosis via chemotherapeutic Adriamycin (doxorubicin) and a novel class of CARP-1 functional mimetic (CFM) compounds. Although Adriamycin causes DNA damage, data from Comet assays revealed that CFM-4.16 also induced DNA damage. Phosphorylation of histone 2AX (γH2AX) protein is involved in regulating DNA damage repair and apoptosis signaling. Adriamycin or CFM-4.16 treatments inhibited cell growth and caused elevated CARP-1 and γH2AX in human breast (HBC) and cervical cancer (HeLa) cells. In fact, a robust nuclear or peri-nuclear co-localization of CARP-1 and γH2AX occurred in cells undergoing apoptosis. Knock-down of CARP-1 diminished γH2AX, their co-localization, and apoptosis in CFM-4.16- or Adriamycin-treated cells. We found that CARP-1 directly binds with H2AX, and H2AX interacted with CARP-1, but not CARP-1 (Δ600–652) mutant. Moreover, cells expressing CARP-1 (Δ600–652) mutant were resistant to apoptosis, and had diminished levels of γH2AX, when compared with cells expressing wild-type CARP-1. Mutagenesis studies revealed that H2AX residues 1–35 harbored a CARP-1-binding epitope, while CARP-1 amino acids 636–650 contained an H2AX-interacting epitope. Surface plasmon resonance studies revealed that CARP-1 (636–650) peptide bound with H2AX (1–35) peptide with a dissociation constant (Kd) of 127 nM. Cells expressing enhanced GFP (EGFP)-tagged H2AX (1–35) peptide or EGFP-tagged CARP-1 (636–650) peptide were resistant to inhibition by Adriamycin or CFM-4.16. Treatment of cells with transactivator of transcription (TAT)-tagged CARP-1 (636–650) peptide resulted in a moderate, statistically significant abrogation of Adriamycin-induced growth inhibition of cancer cells. Our studies provide evidence for requirement of CARP-1 interaction with H2AX in apoptosis signaling by Adriamycin and CFM compounds

SMAD3 deficiency promotes vessel wall remodeling, collagen fiber reorganization and leukocyte infiltration in an inflammatory abdominal aortic aneurysm mouse model

TGF-beta signaling plays critical roles in the pathogenesis of aneurysms; however, it is still unclear whether its role is protective or destructive. In this study, we investigate the role of SMAD3 in the pathogenesis of calcium chloride (CaCl2)-induced abdominal aortic aneurysms (AAA) in Smad3(-/-), Smad3(+/-) and Smad3(+/+) mice. We find that loss of SMAD3 drastically increases wall thickening of the abdominal aorta. Histological analyses show significant vessel wall remodeling with elastic fiber fragmentation. Remarkably, under polarized light, collagen fibers in the hyperplastic adventitia of Smad3(-/-) mice show extensive reorganization accompanied by loosely packed thin and radial collagen fibers. The expressions of matrix metalloproteinases including MMP2, MMP9, and MMP12 and infiltration of macrophage/T cells are drastically enhanced in the vascular wall of Smad3(-/-) mice. We also observe marked increase of NF-kappaB and ERK1/2 signaling as well as the expression of nuclear Smad2, Smad4 and TGF-beta1 in the vessel wall of Smad3(-/-) mice. In addition, we find that SMAD3 expression is reduced in the dedifferentiated medial smooth muscle-like cells of human AAA patients. These findings provide direct in vivo evidence to support the essential roles of SMAD3 in protecting vessel wall integrity and suppressing inflammation in the pathogenesis of AAAs

Black holes, gravitational waves and fundamental physics: a roadmap

The grand challenges of contemporary fundamental physics—dark matter, dark energy, vacuum energy, inflation and early universe cosmology, singularities and the hierarchy problem—all involve gravity as a key component. And of all gravitational phenomena, black holes stand out in their elegant simplicity, while harbouring some of the most remarkable predictions of General Relativity: event horizons, singularities and ergoregions. The hitherto invisible landscape of the gravitational Universe is being unveiled before our eyes: the historical direct detection of gravitational waves by the LIGO-Virgo collaboration marks the dawn of a new era of scientific exploration. Gravitational-wave astronomy will allow us to test models of black hole formation, growth and evolution, as well as models of gravitational-wave generation and propagation. It will provide evidence for event horizons and ergoregions, test the theory of General Relativity itself, and may reveal the existence of new fundamental fields. The synthesis of these results has the potential to radically reshape our understanding of the cosmos and of the laws of Nature. The purpose of this work is to present a concise, yet comprehensive overview of the state of the art in the relevant fields of research, summarize important open problems, and lay out a roadmap for future progress. This write-up is an initiative taken within the framework of the European Action on 'Black holes, Gravitational waves and Fundamental Physics'

Expression of miR-34 is lost in colon cancer which can be re-expressed by a novel agent CDF

Abstract Background Colorectal Cancer (CRC) is one of the leading causes of death worldwide. Numerous cellular events, including deregulated expression of microRNAs (miRNAs), specifically the family of miR-34 consisting of miR-34a, b and c, is known to regulate the processes of growth and metastasis. Methods We evaluated the expression of miR-34 in formalin-fixed paraffin-embedded (FFPE) human colon cancer tissue specimens compared to normal colonic mucosa. Moreover, we also assessed the expression of miR-34 in colon cancer cell lines treated with our newly developed synthetic analogue of curcumin referred as difluorinated curcumin (CDF) compared to well known inhibitor of methyl transferase. Results We found that the expression of miR-34a and miR-34c was down-regulated in colon cancer specimens compared to normal colonic mucosa and the loss of expression was also consistent with data from colon cancer cell lines. This down-regulation was attributed to promoter hypermethylation, because we found that the treatment of colon cancer cells with 5-aza-2´-deoxycytidine, a methyltransferase inhibitor, markedly induced the levels of miR-34a and miR-34c expression. Likewise, CDF was very effective in the re-expression of miR-34a and miR-34c, which was consistent with inhibition of cell growth of both chemo-sensitive and chemo-resistant colon cancer cells. The re-expression of miR-34 led to a marked reduction in the expression of its target gene, Notch-1. Conclusion The loss of expression of miR-34 in colon cancer is in part due to promoter hypermethylation of miR-34, which can be re-expressed with our novel agent CDF, suggesting that CDF could be a novel demethylating agent for restoring the expression of miR-34 family, and thus CDF could become a newer therapeutic agent for the treatment of colon cancer.</p