scholarly journals In Vitro and Ex Vivo Evaluation of Smart Infra-Red Fluorescent Caspase-3 Probes for Molecular Imaging of Cardiovascular Apoptosis

2011 ◽  
Vol 2011 ◽  
pp. 1-13 ◽  
Author(s):  
Manuelle Debunne ◽  
Christophe Portal ◽  
Bruno Delest ◽  
Ebba Brakenhielm ◽  
Françoise Lallemand ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Caspase 3 ◽  
Near Infrared ◽  
Ex Vivo ◽  
Annexin V ◽  
Fluorescent Signal ◽  
Infra Red ◽  
Infrared Probes

Purpose. The aim of this paper is to develop new optical bioprobes for the imaging of apoptosis. Procedure. We developed quenched near-infrared probes which become fluorescent upon cleavage by caspase-3, the key regulatory enzyme of apoptosis. Results. Probes were shown to be selectively cleaved by recombinant caspase-3. Apoptosis of cultured endothelial cells was associated with an increased fluorescent signal for the cleaved probes, which colocalized with caspase-3 and was reduced by the addition of a caspase-3 inhibitor. Flow cytometry demonstrated a similar profile between the cleaved probes and annexin V. Ex vivo experiments showed that sections of hearts obtained from mice treated with the proapoptotic drug doxorubicin displayed an increase in the fluorescent signal for the cleaved probes, which was reduced by a caspase-3 inhibitor. Conclusion. We demonstrated the capacity of these novel probes to detect apoptosis by optical imaging in vitro and ex vivo.

Inhibition of Fas-mediated Apoptosis in Yac-1 Cell via Anti-Fas Ribozyme

2004 ◽  
Vol 36 (3) ◽  
pp. 199-205
Author(s):  
Min Zhang ◽  
Fang Liu ◽  
Wei He ◽  
Yong You ◽  
Ping Zou ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Proteolytic Activity ◽  
Caspase 3 ◽  
Hammerhead Ribozyme ◽  
Annexin V ◽  
In Vitro Transcription ◽  
Rt Pcr ◽  
Fas Mrna

Abstract To detect a new and more effective way against apoptosis mouse lymphomatic cell line-Yac-1 in which fas gene was expressed highly was used as a model for studying the effects of anti-Fas ribozyme on Fas-mediated apoptosis. A hammerhead ribozyme gene targeting the fas mRNA was synthesized and its in vitro transcription vector was constructed, which was transfected into Yac-1 cells using electroporation. Rz596 expression was detected using RT-PCR, and Fas expression in Yac-1 cells was detected using RT-PCR, Western blot and flow cytometry. After treated with anti-Fas antibody (JO2), Yac-1 cell viability was measured with MTT assay, caspase-3 proteolytic activity was detected, and cell apoptosis was measured according to annexin V apoptosis detecting kit. Anti-Fas ribozyme could cleave fas mRNA efficiently in vivo and in vitro. Fas expression in Yac-1 cells transfected with anti-Fas ribozyme was decreased remarkably and correlated with resistance to Fas-mediated apoptosis as determined by flow cytometry and caspase-3 proteolytic activity. Anti-Fas ribozyme was detected in cells transfected with pU6-RZ596 and pU6-dRZ596 and could remarkably decrease the Fas expression in Yac-1 cells, which made Yac-1 cells get rid of Fas-mediated apoptosis. Because of wide expression of fas in organs and tissues, our research was very useful for studying the inhibition of apoptosis of many organs and tissues in the future.

Thrombospondin-1 Induces Apoptosis in Megakaryocytic Leukemia Via CD36 and Caspase-3 Signaling

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5209-5209
Author(s):  
Mo Yang ◽  
Enyu Liang ◽  
Lijing Mao ◽  
Beng H Chong ◽  
Chunfu Li
Keyword(s):  
Endothelial Cells ◽  
Tumor Cells ◽  
Propidium Iodide ◽  
Caspase 3 ◽  
Annexin V ◽  
K562 Cells ◽  
Leukemia Cells ◽  
Cd36 Expression ◽  
Dose Dependent

Abstract Objective: Thrombospondin-1 (TSP-1) is a multi-modular glycoprotein synthesized by megakaryocytes, endothelial cells, smooth muscle cells, fibroblasts and some tumor cells, found in the platelet a-granules and extracellular matrix (ECM) of numerous tissues. TSP-1 has been identified as one of the few naturally occurring anti-angiogenic agents. It inhibited endothelial cells in vitro by the activation of the CD36-p59fyn-Caspase-3-p38MAPK apoptotic cascade. TSP-1 induced in vitro apoptosis of micro-vascular endothelial cells as well as tumor cells. The objectives of our study were to investigate the in vitro effects of TSP-1 on the apoptosis of megakaryocytic leukemia cells and the possible mechanism involving CD36. Methods: We investigated CD36 expression and the effect of TSP-1 on megakaryocytic leukemia cells (Meg-01 and CHRF-288-11), with and without thrombopoietin (TPO), and with and without blocking TSP-1 binding with receptor CD36 on megakaryocytic leukemia cells. The determination of apoptotic markers were used the Annexin V-FITC/Propidium Iodide (PI) and Caspase-3 detection kit by flow cytometry.Results: Our data showed that TSP-1 induced a dose dependent growth inhibition in human CFU-MK assays and significantly counteracted the mitogenic effect from TPO. Moreover, the growth suppression induced by TSP-1 was correlated with CD36 expression in megakaryocytic leukemia cells, where growth inhibition was demonstrated in CD36 positive (Meg-01 and CHRF-288-11) but not in CD36 negative (K562) cells. More importantly, the inhibitory effect of TSP-1 on both human CFU-MK and Meg-01 cells was partially but significantly reversed by the addition of FA6-152 (anti-CD36), a functionally blocking antibody which blocks the access of TSP-1 to CD36 receptor, suggesting that the TSP-1 induced inhibition of megakaryocytopoiesis is probably mediated in part by the binding of TSP-1 to CD36 expressed on the megakaryocytic progenitors. Thus, our findings represent the demonstration that TSP-1 inhibits in vitro megakaryocytopoiesis via interaction with CD36. Our results further demonstrated that TSP-1 induced apoptosis in CD36 positive cells CHRF-288-11 and Meg-01, but not CD36 negative K562 cells, at a dose-dependent manner as demonstrated by DNA Annexin V and propidium iodide (PI) stainings. The addition of anti-CD36 antibody FA6-152 or TPO significantly nullified the effects of TSP-1. TSP-1-mediated apoptosis was consistently associated with the up-regulation of active Caspase-3. Responses of CD36 positive primary AML samples (n=3) to TSP-1 and FA6-152 were similar with those of leukemia cell lines. CD36 negative AML cells appeared less susceptible to TSP-1 and FA6-152.Conclusions: Our data provided evidence that TSP-1 exerted direct apoptotic effects on megakaryocytic leukemia cells and could be developed as an adjunct to conventional therapy, particularly for leukemia cells that express CD36 or other TSP-1 receptors. Thus, our findings represent the demonstration that TSP-1 induces apoptosis in megakaryocytic leukemia cells via CD36 and Caspase-3. Disclosures Yang: National Natural Science Foundation of China: Other: National Natural Science Foundation of China(81270580).

Sirolimus and propranolol inhibit endothelial proliferation while detergent sclerosants induce endothelial activation, microparticle release and apoptosis in vitro

2020 ◽  
Vol 35 (8) ◽  
pp. 566-575 ◽  
Author(s):  
David E. Connor ◽  
Jessica Gerbelli ◽  
An-Ning Chew ◽  
Osvaldo Cooley-Andrade ◽  
Dulani Goonawardhana ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
Endothelial Cells ◽  
Caspase 3 ◽  
Umbilical Vein ◽  
Endothelial Activation ◽  
Endothelial Microparticles ◽  
Sodium Tetradecyl Sulphate ◽  
Umbilical Vein Endothelial Cells

Objectives To investigate the effects of detergent sclerosants, sodium tetradecyl sulphate and polidocanol, on endothelial cell activation and microparticle release and the effects of detergent sclerosants, sirolimus and propranolol, on apoptosis in vitro. Methods Cultured human umbilical vein endothelial cells and murine haemangioendothelioma (EOMA) cell lines were incubated with different concentrations of sodium tetradecyl sulphate and polidocanol, as well as sirolimus and propranolol. Endothelial activation was assessed using flow cytometry for CD62e (E-Selectin), CD54 (ICAM-1), CD105 (endoglin), CD144 (VE-Cadherin), CD146 (MCAM) and the release of endothelial microparticles. Cell proliferation was assessed using [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] and carboxyfluorescein succinimidyl ester assays. Apoptosis was assessed using flow cytometry for lactadherin/propidium iodide staining and for Caspase-3 expression. Results Sublytic concentrations of sodium tetradecyl sulphate and polidocanol (0.075%–0.3%) increased the expression of the activation markers CD62e and CD54. The expression of CD105 decreased in sclerosant treated cultured human umbilical vein endothelial cells. Both sodium tetradecyl sulphate and polidocanol induced the release of endothelial microparticles. All agents inhibited cell proliferation. Sodium tetradecyl sulphate and polidocanol-induced apoptosis as evidenced by increased phosphatidylserine exposure and caspase-3 expression, whereas sirolimus and propranolol increased caspase-3 expression only. Conclusion Sublytic concentrations of detergent sclerosants induce endothelial activation and the release of endothelial microparticles. All agents were anti-proliferative in EOMA cell lines, with sodium tetradecyl sulphate and polidocanol inducing cellular apoptosis.

scholarly journals Minyak Cengkeh (Syzygium aromaticum) Menginduksi Apoptosis pada Sel Kanker Servik HeLa melalui Peningkatan Kadar Protein p53

2019 ◽  
Vol 30 (3) ◽  
pp. 185
Author(s):  
Happy Kurnia Permatasari ◽  
Ihda Dian Kusuma ◽  
Elly Mayangsari
Keyword(s):  
Flow Cytometry ◽  
Caspase 3 ◽  
Annexin V ◽  
Syzygium Aromaticum ◽  
Protein P53

<p>Kanker serviks merupakan kanker yang paling sering dijumpai pada wanita setelah kanker payudara. Kanker ini terkait dengan infeksi persisten virus yaitu Human Papillomovirus (HPV). Virus ini mengekspresikan protein onkogenik virus yaitu protein E6 dan E7 yang terkait dengan proses karsinogenesis. Salah satu mekanisme onkogenik virus ini adalah pengikatan protein p53 yang menginduksi degradasi oleh protein E6, mengakibatkan efek anti-apoptosis dan proliferasi sel secara terus menerus. Minyak cengkeh yang mengandung senyawa aktif eugenol telah dilaporkan memiliki efek anti kanker pada beberapa kanker. Namun, mekanisme minyak cengkeh yang terkait dengan penghambatan kanker servik masih belum jelas. Penelitian ini, untuk mengkaji efek minyak cengkeh dari Syzygium aromaticum dalam efek pro-apoptosis sel kanker servik HeLa terkait dengan kadar protein p53. Tingkat apoptosis diamati dengan pewarnaan dengan Annexin-V dan PI dan dilakukan dengan metode flow-cytometry. Dilakukan pengecatan immunohistokima untuk melihat ekspresi caspase-3 aktif untuk mengonfirmasi sel yang apoptosis, sedangkan kadar protein p53 dievaluasi dari lisat sel menggunakan ELISA protein p53. Hasil studi menunjukkan bahwa minyak cengkeh memiliki efek pro-apoptosis, berkaitan dengan kadar protein p53, pada sel kanker serviks secara in vitro.</p>

Human Cytomegalovirus Inhibited Megakaryocytic Differentiation, Maturation and Induced Apoptosis in Vitro.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2413-2413
Author(s):  
Jian Liang Chen ◽  
Godfrey ChiFung Chan ◽  
Jie Yu Ye ◽  
Zheng Xian He ◽  
Qing Wen Wang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Infection Control ◽  
Cell Population ◽  
Human Cytomegalovirus ◽  
Late Stage ◽  
Caspase 3 ◽  
Annexin V ◽  
Megakaryocytic Differentiation ◽  
Mock Infection

Abstract Abstract 2413 Poster Board II-390 Human cytomegalovirus (HCMV) can cause life-threatening infection in immunocomprimised individuals, such as patients undergoing intensive chemotherapy or bone marrow transplantation. Thrombocytopenia is one of the manifestations in active HCMV infection, which may be a consequence of viral suppression on megakaryopoiesis. The exact underline mechanisms remain uncertain. Our previous studies suggested that HCMV directly infects megakaryocytic progenitors and inhibits their proliferation. Colony-formation of HCMV-infected CFU-MK decreased in a dose-dependent manner (Blood, 2003, abstract). In present study, we explored the mechanisms further by using a phorbol 12-myristate 13-acetate (PMA) stimulating polyploidization to mimic the late stage of megakaryocytic differentiation and maturation in vitro. After co-culture of a megakaryocytic cell line CHRF-288-11 with HCMV AD169 experimental strain from day 0 to day 3 (multiple of infection, MOI=1), the polyploidization of megakaryocyte was determined by DNA content analysis using flow cytometry. Compared with negative control, the proportion of polyploidy (ploidy N ≥ 8) megakaryocytes decreased by 52%, 32% and 16% in HCMV-infected cell at day 3, day 6 and day 9 respectively. As a specific receptor for megakaryopoietic differentiation, the c-Mpl protein (TPO receptor) was also examined in CHRF-288-11 cell line. The proportion of c-Mpl positive cells showed a 23% decrease in HCMV-infected group in compared to the mock infection control (using ultralviolet treated HCMV) at day 5. In addition, apoptotic signals from megakaryocytic surface, cytoplasma and mitochondria were detected in HCMV-infected cells by flow cytometry with Annexin V, Caspase-3 and JC-1 assay. Compared to mock infection control at day 5, annexin-V positive cells population increased by 57%; active caspase-3 signal increased by 125% in viable cell population; and cell population with damaged mitochondial membrane showed a 5-times increase. In conclusion, our data demonstrated that: (1) HCMV inhibited megakaryocytic differentiation and maturation at late stage; (2) HCMV reduced c-Mpl positive cell population; (3) HCMV induced megakaryocytic apoptosis through intrinsic apoptotic pathway as shown by the functional alteration of mitochodial membrane, activation of caspase-3 and structural damage of outer cellular membrane. HCMV-induced thrombocytopenia is the consequence of multiple processes involving inhibition of megakaryocytic proliferation, differentiation, maturation and also increased megakaryocytic apoptosis. Disclosures: No relevant conflicts of interest to declare.

FAS Mutations Are Associated with Therapeutic Resistance in Follicular Lymphoma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1549-1549
Author(s):  
Nathalie Johnson ◽  
Denis Gaucher ◽  
Hawley Rigsby ◽  
Ryan D Morin ◽  
Joseph M. Connors ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Caspase 3 ◽  
Annexin V ◽  
Therapeutic Resistance ◽  
Wild Type ◽  
Fasl Expression ◽  
Treatment Refractory

Abstract Abstract 1549 Introduction: We recently reported on recurrent genomic alterations present in genome, transcriptome or exome data from 127 cases of NHL (Morin et al. Nature. 2011 Jul 27;476:298–303). Our data suggests FAS acts as a tumor suppressor in B cells based on a selection bias towards mutations that would result in truncated or altered protein, most of which were in the functional death domain. FAS is the prototypical death receptor involved in the extrinsic apoptotic pathway. Germline mutations in FAS have a dominant-negative phenotype, leading to dysfunctional FAS-mediated apoptosis, accumulation of lymphocytes and autoimmune lymphoproliferative syndrome. The role of FAS in lymphomagenesis is unclear. The FAS mutations in our study were limited to 6 cases of germinal center B lymphomas, including 3 cases of aggressive follicular lymphoma (FL). Herein we investigate the role of FAS in therapeutic resistance in FL. Methods: One of the FL cases had treatment-refractory disease. Biopsies taken at diagnosis and after progression following 2 cycles of cyclophosphamide, vincristine, prednisone and rituximab (CVP-R) were available for transcriptome sequencing. Owing to our discovery of FAS mutations in this and other FL patients, we sequenced exon 7–9 of FAS on an extended cohort of 214 clinically-annotated FL samples to determine its potential for association with clinical outcome. To functionally validate our findings we transfected two lymphoma cell lines that are sensitive to FAS-induced apoptosis with either FAS wild-type, FAS mutant (Y232*) or empty vector control. Single clones (wild type and Y232*) with similar expression of FAS protein by flow cytometry were compared for their ability to undergo apoptosis after exposure to the FAS agonist antibody CH-11 and therapeutic levels of cyclophosphamide, adriamycin, vincristine and etoposide. We defined apoptotic cells as those expressing annexin V+/propidium iodine-negative and activated caspase 3 by flow cytometry and cells demonstrating cleaved caspase 8 by western blotting. We further determined if etoposide, chosen because it has no background fluorescence, could change the expression of FAS or FAS ligand in primary lymphocytes using multi-color flow cytometry. Results: We demonstrate that FAS mutations are associated with clinically-aggressive FL. In a patient with treatment-refractory FL, only 3 mutations were confirmed to be somatic and significantly enriched in the post-treatment biopsy: CTSS, RPS24 and FAS Y232* (p values <0.05), where CTSS and RPS24 have no known function in lymphocytes. In an extended cohort of FL samples, 6/214 (3%) had non-synonymous mutations in FAS predicting for a severely altered or truncated protein. Coding FAS mutations were associated with a trend towards an earlier time to progression (1 year versus 2.8 years, p=0.08) and an increased risk of histological transformation to DLBCL (n=4/6, p=0.036). Indeed, 5 of the 6 developed early resistance to primary rituximab-based chemotherapy. In vitro studies demonstrated that the FAS Y292* impairs extrinsic apoptosis induced by CH-11 (decrease in cleaved caspase 8, activated caspase 3 and Annexin V+/PI- cells). However, there was no difference in apoptosis between FAS Y232* mutants and FAS wild type cells after exposure to chemotherapy in vitro. These results led us to speculate that other factors are involved in resistance to chemotherapy. Using primary tonsilar lymphocytes in culture, we observed a significant increase in both FAS expression on primary B cells and the cognate ligand FASL expression in primary T cells following a 24-hour exposure to etoposide. Conclusion: Mutations in FAS are associated with clinical therapeutic resistance in FL. FAS mutations can impair FAS-mediated apoptosis. Our results suggest that chemotherapy may potentiate an immune response in part by increasing FAS expression in B cells and FASL expression in T cells. This may enhance killing of FAS wild type but not FAS mutant expressing tumor cells. Thus the role the tumor microenvironment in chemotherapy-induced cell death may be under appreciated in FL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A critical role for increased labile zinc in reducing sensitivity of cultured sheep pulmonary artery endothelial cells to LPS-induced apoptosis

2012 ◽  
Vol 302 (12) ◽  
pp. L1287-L1295 ◽  
Author(s):  
Kalidasan Thambiayya ◽  
Karla Wasserloos ◽  
Valerian E. Kagan ◽  
Detcho Stoyanovsky ◽  
Bruce R. Pitt
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Caspase 3 ◽  
Critical Role ◽  
Annexin V ◽  
Zinc Homeostasis ◽  
No Donor ◽  
Vitro Assay ◽  
Induced Apoptosis

We previously noted an important signaling role for decreased labile intracellular zinc ([ Zn ] i) in LPS-induced apoptosis in cultured sheep pulmonary artery endothelial cells (SPAEC) (Tang ZL, Wasserloos KJ, Liu X, Stitt MS, Reynolds IJ, Pitt BR, St Croix CM. Mol Cell Biochem 234–235: 211–217, 2002; Thambiayya K, Wasserloos KJ, Huang Z, Kagan VE, St Croix CM, Pitt BR. Am J Physiol Lung Cell Mol Physiol 300: L624–632, 2011). In the present study, we used small interfering RNA (siRNA) to important contributors of zinc homeostasis [ SLC39A14 or Zrt/Irt-like protein 14 (ZIP14), a zinc importer; metallothionein (MT), a zinc binding protein ] to define molecular pathways by which extracellular zinc or nitric oxide (NO) increase labile [ Zn ] i [ e.g., zinc-sensitive fluorophore (FluoZin-3) detectable and/or chelatable by N, N, N′, N′-tetrakis(2-pyridylmethyl)ethylenediamine ] and reduce the sensitivity of SPAEC to LPS. Addition of 10 μM zinc to serum-free medium of SPAEC increased [ Zn ] i and abolished LPS-induced apoptosis (e.g., increased annexin V binding). The increase in [ Zn ] i and the protective effect of extracellular zinc were sensitive to reduction in ZIP14 expression (by siRNA), but not affected by collectively knocking down major isoforms of sheep MT (sMT-Ia, -Ib, -Ic, and -II). Pretreatment of wild-type SPAEC with 250 μM of the NO donor S-nitroso- N-acetylpenicillamine (SNAP) increased labile zinc in a relatively similar fashion to addition of extracellular zinc and reduced sensitivity of SPAEC to LPS-induced apoptosis (e.g., caspase-3/7 activation) in a N, N, N′, N′-tetrakis(2-pyridylmethyl)ethylenediamine-sensitive fashion. The antiapoptotic effects of SNAP were insensitive to siRNA knockdown of ZIP14, but were abolished (along with SNAP-induced increase in [ Zn ] i) when SPAEC were pretreated with siRNA to sheep MT. Zinc was able to directly inhibit recombinant caspase-3 activity in an in vitro assay. Collectively, these data show that increases in labile [ Zn ] i are an important component of ZIP14- or NO-mediated resistance to LPS-induced apoptosis. Cytoprotection via ZIP14 appeared to be secondary to transcellular movement of extracellular zinc, whereas NO-mediated protection was secondary to S-nitrosation of MT and redistribution of [ Zn ] i.

scholarly journals Isolation, Characterization, and Transplantation of Cardiac Endothelial Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-18 ◽  
Author(s):  
Busadee Pratumvinit ◽  
Kanit Reesukumal ◽  
Kajohnkiart Janebodin ◽  
Nicholas Ieronimakis ◽  
Morayma Reyes
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Biology ◽  
Cell Function ◽  
Ex Vivo ◽  
Enzymatic Digestion ◽  
Clinical Applications ◽  
Endothelial Cell Function

Isolation andex vivoexpansion of cardiac endothelial cells have been a recurrent challenge due to difficulties in isolation, cell heterogeneity, lack of specific markers to identify myocardial endothelial cells, and inadequate conditions to maintain long-term cultures. Herein, we developed a method for isolation, characterization, and expansion of cardiac endothelial cells applicable to study endothelial cell biology and clinical applications such as neoangiogenesis. First, we dissociated the cells from murine heart by mechanical disaggregation and enzymatic digestion. Then, we used flow cytometry coupled with specific markers to isolate endothelial cells from murine hearts. CD45+ cells were gated out to eliminate the hematopoietic cells. CD31+/Sca-1+ cells were isolated as endothelial cells. Cells isolated from atrium grew faster than those from ventricle. Cardiac endothelial cells maintain endothelial cell function such as vascular tube formation and acetylated-LDL uptakein vitro. Finally, cardiac endothelial cells formed microvessels in dorsal matrigel plug and engrafted in cardiac microvessels following intravenous and intra-arterial injections. In conclusion, our multicolor flow cytometry method is an effective method to analyze and purify endothelial cells from murine heart, which in turn can beex vivoexpanded to study the biology of endothelial cells or for clinical applications such as therapeutic angiogenesis.

Synthesis of a Novel Curcumin Derivative as a Potential Imaging Probe in Alzheimer’s Disease Imaging

2019 ◽  
Vol 16 (8) ◽  
pp. 723-731 ◽  
Author(s):  
Alexander Sturzu ◽  
Sumbla Sheikh ◽  
Hubert Kalbacher ◽  
Thomas Nägele ◽  
Christopher Weidenmaier ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Flow Cytometry ◽  
Transgenic Mice ◽  
Ex Vivo ◽  
Amyloid Plaques ◽  
Laser Scanning Microscopy ◽  
Β Amyloid ◽  
Curcumin Derivative

Background: Curcumin has been of interest in the field of Alzheimer’s disease. Early studies on transgenic mice showed promising results in the reduction of amyloid plaques.However, curcumin is very poorly soluble in aqueous solutions and not easily accessible to coupling as it contains only phenolic groups as potential coupling sites. For these reasons only few imaging studies using curcumin bound as an ester were performed and curcumin is mainly used as nutritional supplement. Methods: In the present study we produced an aminoethyl ether derivative of curcumin using a nucleophilic substitution reaction. This is a small modification and should not impact the properties of curcumin while introducing an easily accessible reactive amino group. This novel compound could be used to couple curcumin to other molecules using the standard methods of peptide synthesis. We studied the aminoethyl-curcumin compound and a tripeptide carrying this aminoethyl-curcumin and the fluorescent dye fluorescein (FITC-curcumin) in vitro on cell culture using confocal laser scanning microscopy and flow cytometry. Then these two substances were tested ex vivo on brain sections prepared from transgenic mice depicting Alzheimer-like β-amyloid plaques. Results: In the in vitro CLSM microscopy and flow cytometry experiments we found dot-like unspecific uptake and only slight cytotoxicity correlating with this uptake. As these measurements were optimized for the use of fluorescein as dye we found that the curcumin at 488nm fluorescence excitation was not strong enough to use it as a fluorescence marker in these applications. In the ex vivo sections CLSM experiments both the aminoethyl-curcumin and the FITC-curcumin peptide bound specifically to β- amyloid plaques. Conclusion: In conclusion we successfully produced a novel curcumin derivative which could easily be coupled to other imaging or therapeutic molecules as a sensor for amyloid plaques.

Design, Synthesis and In Vitro Anti-Cancer Evaluation of Novel Derivatives of 2-(2-Methyl-1,5-diaryl-1H-pyrrol-3-yl)-2-oxo-N-(pyridin-3- yl)acetamide

2020 ◽  
Vol 16 (3) ◽  
pp. 340-349
Author(s):  
Ebrahim S. Moghadam ◽  
Farhad Saravani ◽  
Ernest Hamel ◽  
Zahra Shahsavari ◽  
Mohsen Alipour ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Peripheral Neuropathy ◽  
Cell Line ◽  
Normal Cell ◽  
Caspase 3 ◽  
Annexin V ◽  
Normal Cell Line ◽  
Anti Cancer ◽  
Mcf 7

Objective: Several anti-tubulin agents were introduced for the cancer treatment so far. Despite successes in the treatment of cancer, these agents cause toxic side effects, including peripheral neuropathy. Comparing anti-tubulin agents, indibulin seemed to cause minimal peripheral neuropathy, but its poor aqueous solubility and other potential clinical problems have led to its remaining in a preclinical stage. Methods: Herein, indibulin analogues were synthesized and evaluated for their in vitro anti-cancer activity using MTT assay (on the MCF-7, T47-D, MDA-MB231 and NIH-3T3 cell lines), annexin V/PI staining assay, cell cycle analysis, anti-tubulin assay and caspase 3/7 activation assay. Results: One of the compounds, 4a, showed good anti-proliferative activity against MCF-7 cells (IC50: 7.5 μM) and low toxicity on a normal cell line (IC50 > 100 μM). All of the tested compounds showed lower cytotoxicity on normal cell line in comparison to reference compound, indibulin. In the annexin V/PI staining assay, induction of apoptosis in the MCF-7 cell line was observed. Cell cycle analysis illustrated an increasing proportion of cells in the sub-G-1 phase, consistent with an increasing proportion of apoptotic cells. No increase in G2/M cells was observed, consistent with the absence of anti-tubulin activity. A caspase 3/7 assay protocol showed that apoptosis induction by more potent compounds was due to activation of caspase 3. Conclusion: Newly synthesized compounds exerted acceptable anticancer activity and further investigation of current scaffold would be beneficial.

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