Questo lavoro è stato sostenuto dai Cavalieri di Colombo capitolo 9671 (Windsor, Ontario), e un CIHR Frederick Banting e Best Charles Canada Graduate Scholarship assegnato a Ma Dennis. Grazie a Hodge Robert ed Elizabeth Fidalgo da Silva per la loro assistenza con la microscopia time-lapse. Grazie a voi Katie Facecchia per la modifica dei time-lapse video microscopia. Vorremmo anche ringraziare Sudipa giugno Chatterjee e Phillip Tremblay per la revisione critica di questo manoscritto. Questo lavoro è dedicato in memoria di Kevin Couvillon che ha perso la sua battaglia contro il cancro nel 2010.

<ol>
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J. Oncol. 38, 1549-1556 (2011).</li><li>Collins, J., Rinner, U., Moser, M., Hudlicky, T., Ghiviriga, I., Romero, A. E., Kornienko, A., Ma, D., Griffin, C., Pandey, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoenzymatic+synthesis+of+Amaryllidaceae+constituents+and+biological+evaluation+of+their+C-1+analogues.+The+next+generation+synthesis+of+7-deoxypancratistatin+and+trans-dihydrolycoricidine.">Chemoenzymatic synthesis of Amaryllidaceae constituents and biological evaluation of their C-1 analogues. The next generation synthesis of 7-deoxypancratistatin and trans-dihydrolycoricidine.</a> J. Org. Chem. 75, 3069-3084 (2010).</li><li>Zhang, G., Gurtu, V., Kain, S. R., Yan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+detection+of+apoptosis+using+a+fluorescent+conjugate+of+annexin+V.">Early detection of apoptosis using a fluorescent conjugate of annexin V.</a> Biotechniques. 23, 525-531 (1997).</li><li>Madesh, M., Hajn&#243;czky, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VDAC-dependent+permeabilization+of+the+outer+mitochondrial+membrane+by+superoxide+induces+rapid+and+massive+cytochrome+c+release.">VDAC-dependent permeabilization of the outer mitochondrial membrane by superoxide induces rapid and massive cytochrome c release.</a> J. Cell Biol. 155, 1003-1015 (2001).</li><li>Simon, H. U., Haj-Yehia, A., Levi-Schaffer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+reactive+oxygen+species+(ROS)+in+apoptosis+induction.">Role of reactive oxygen species (ROS) in apoptosis induction.</a> Apoptosis. 5, 415-418 (2000).</li><li>Batandier, C., Leverve, X., Fontaine, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opening+of+the+mitochondrial+permeability+transition+pore+induces+reactive+oxygen+species+production+at+the+level+of+the+respiratory+chain+complex+I.">Opening of the mitochondrial permeability transition pore induces reactive oxygen species production at the level of the respiratory chain complex I.</a> J Biol Chem. 279, 17197-17204 (2004).</li><li>Lefranc, F., Sauvage, S., Goietsenoven, G. V. a. n., Mégalizzi, V., Lamoral-Theys, D., Debeir, O., Spiegl-Kreinecker, S., Berger, W., Mathieu, V., Decaestecker, C., Kiss, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Narciclasine,+a+plant+growth+modulator,+activates+Rho+and+stress+fibers+in+glioblastoma+cells.">Narciclasine, a plant growth modulator, activates Rho and stress fibers in glioblastoma cells.</a> Mol. Cancer Ther. 8, 1739-1750 (2009).</li><li>Warburg, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+origin+of+cancer+cells.">On the origin of cancer cells.</a> Science. 123, 309-314 (1956).</li><li>Heerdt, B. G., Houston, M. A., Augenlicht, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+properties+of+colonic+tumor+cells+are+a+function+of+the+intrinsic+mitochondrial+membrane+potential.">Growth properties of colonic tumor cells are a function of the intrinsic mitochondrial membrane potential.</a> Cancer Res. 66, 1591-1596 (2006).</li><li>Green, D. R., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathophysiology+of+mitochondrial+cell+death.">The pathophysiology of mitochondrial cell death.</a> Science. 305, 626-629 (2004).</li><li>Casellas, P., Galiegue, S., Basile, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+benzodiazepine+receptors+and+mitochondrial+function.">Peripheral benzodiazepine receptors and mitochondrial function.</a> Neurochem. Int. 40, 475-486 (2002).</li><li>Mathupala, S. P., Rempel, A., Pedersen, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+glycolytic+metabolism+of+cancer+cells:+a+remarkable+coordination+of+genetic,+transcriptional,+post-translational,+and+mutational+events+that+lead+to+a+critical+role+for+type+II+hexokinase.">Aberrant glycolytic metabolism of cancer cells: a remarkable coordination of genetic, transcriptional, post-translational, and mutational events that lead to a critical role for type II hexokinase.</a> J. Bioenerg. Biomembr. 29, 339-343 (1997).</li></ol>

Non ci sono conflitti di interesse dichiarati.

Composti PST e simili hanno dimostrato di avere proprietà anti-cancro 11-15,21. Abbiamo precedentemente riportato PST naturale di destabilizzare selettivamente i mitocondri nelle cellule tumorali, che in tal modo induce apoptosi mediante il rilascio di fattori citotossici 12,14. E &#39;molto probabile che JCTH-4 agisce attraverso lo stesso meccanismo; JCTH-4 ha causato il collasso MMP in MCF7 come si è visto con colorazione TMRM (Fig. 5a), e una maggiore generazione di ROS nei mi...

<table border="1"><tbody><tr><td> Nome del materiale </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> SH-SY5Y linea cellulare </td><td> ATCC </td><td> CRL-2266 </td></tr><tr><td> Dulbecco Modified Eagles Medio F-12 HAM </td><td> Sigma-Aldrich </td><td> 51448C </td></tr><tr><td> Siero fetale bovino </td><td> Gibco BRL </td><td> 16000-044 </td></tr><tr><td> MCF7 linea cellulare </td><td> ATCC </td><td> HTB-22 </td></tr><tr><td> Mezzo RPMI-1640 </td><td> Sigma-Aldrich </td><td> R 0883 </td></tr><tr><td> A quanto pare umano normale linea cellulare di fibroblasti fetali (NFF) </td><td> Coriell Institute for Medical Research </td><td> AG04431B </td></tr><tr><td> Media Dulbeccòs Modified Eagle, medio alto glucosio </td><td> Thermo Scientific </td><td> SH30022.01 </td></tr><tr><td> Tamoxifene sale </td><td> Sigma-Aldrich </td><td> T9262 </td&gt; </tr><tr><td> 35 mm vetro di fondo della cultura piatti </td><td> MatTek </td><td> P35G-014-C </td></tr><tr><td> Leica DMI6000 B microscopio invertito </td><td> Leica Microsystems </td><td> N / A </td></tr><tr><td> Colorante Hoechst 33342 </td><td> Molecular Probes </td><td> H3570 </td></tr><tr><td> Leica DM IRB invertito microscopio a fluorescenza </td><td> Leica Microsystems </td><td> N / A </td></tr><tr><td> Annessina V AlexaFluor-488 </td><td> Invitrogen </td><td> A13201 </td></tr><tr><td> Trypan Blu soluzione </td><td> Sigma-Aldrich </td><td> T8154-20ML </td></tr><tr><td> Emocitometro </td><td> Fisher Scientific </td><td> 267110 </td></tr><tr><td> WST-1 reagente </td><td> Roche Applied Science </td><td> 11644807001 </td></tr><tr><td> Wallac Victor 3 1420 Multilabel contatore </td><td> PerkinElmer </td><td> 1420-011 </td></tr><tr><td> Tetrametilrodamina metilestere (TMRM) </td><td> Gibco BRL </td><td> T-668 </td></tr><tr><td> Glass tessuto grinder </td><td> Fisher Scientific </td><td> K8885300-0002 </td></tr><tr><td> BioRad proteine ​​test </td><td> Bio-Rad Laboratories </td><td> 500 0001Bottom della Forma </td></tr><tr><td> Amplex Red </td><td> Invitrogen </td><td> A12222 </td></tr><tr><td> Perossidasi di rafano (HRP) </td><td> Sigma-Aldrich </td><td> P8125 </td></tr><tr><td> Spectramax Gemini XPS </td><td> Molecular Devices </td><td> 3126666 </td></tr><tr><td> Anti-LC3 anticorpo cresciuto in coniglio </td><td> Novus Biologicals </td><td> NB100-2220 </td></tr><tr><td> Anti-mouse HRP-coniugato anticorpo secondario </td><td> Abcam </td><td> ab6728 </td></tr><tr><td> Anti-coniglio HRP-anticorpo secondario coniugato </td><td> Abcam </td><td> ab6802 </td></tr><tr><td> Chemiluminescenza perossidasi substrato </td><td> Sigma-Aldrich </td><td> CPS160 </td></tr><tr><td> Monodansylcadaverine </td><td> Sigma-Aldrich </td><td> 30432 </td></tr></tbody></table>

enhancement of apoptotic and autophagic induction by a novel synthetic c-1 analogue of 7-deoxypancratistatin in human breast adenocarcinoma and neuroblastoma cells with tamoxifen

Il cancro al seno è uno dei tumori più diffusi tra le donne in Nord America. Molti attuali trattamenti anti-tumorali, incluse le radiazioni ionizzanti, inducono apoptosi attraverso danni al DNA. Sfortunatamente, tali trattamenti non sono selettivi per le cellule tumorali e produrre tossicità simile in cellule normali. Abbiamo riportato l&#39;induzione di apoptosi selettiva in cellule tumorali dal pancratistatin composto naturale (PST). Recentemente, un romanzo PST analogico, un derivato C-1 di 7-acetossimetil deoxypancratistatin (JCTH-4), è stato prodotto mediante sintesi de novo e presenta comparabili selettiva attività inducente l&#39;apoptosi nelle linee cellulari tumorali diverse. Recentemente, autofagia è stato implicato in neoplasie sia come pro-sopravvivenza e pro-morte meccanismi in risposta alla chemioterapia. Tamoxifene (TAM) ha sempre dimostrato l&#39;induzione di pro-sopravvivenza autofagia in numerosi tumori. In questo studio, l&#39;efficacia di JCTH-4 da solo e in combinazione con TAM per indurre la morte cellulare in significatività maternor (MCF7) e neuroblastoma (SH-SY5Y) le cellule è stata valutata. TAM solo indotto l&#39;autofagia, ma la morte delle cellule insignificante, mentre JCTH-4 da sola ha causato significativa induzione di apoptosi con alcuni induzione di autofagia. È interessante notare che il trattamento combinatoria ha prodotto un drastico aumento di induzione di apoptosi e autofagia. Abbiamo monitorato le variazioni morfologiche tempo-dipendenti MCF7 cellule in fase di TAM-indotta autofagia, JCTH-4-indotta l&#39;apoptosi e l&#39;autofagia e morte cellulare accelerata con trattamento combinatoria utilizzando microscopia time-lapse. Abbiamo dimostrato questi composti di indurre apoptosi / autofagia per spese mitocondriale in queste cellule tumorali. È importante sottolineare che questi trattamenti non ha influenzato la sopravvivenza di fibroblasti umani non tumorali. Così, questi risultati indicano che JCTH-4 in combinazione con TAM potrebbe essere usato come un sicuro e molto potente terapia antitumorale contro il cancro al seno e cellule di neuroblastoma.

Watch this Scientific Journal Video about Enhancement of Apoptotic and Autophagic Induction by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin in Human Breast Adenocarcinoma and Neuroblastoma 

Enhancement of Apoptotic and Autophagic Induction by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin in Human Breast Adenocarcinoma and Neuroblastoma Cells with Tamoxifen

Abbiamo sintetizzato un analogo romanzo di pancratistatin paragonabile attività anti-cancro come pancratistatin nativo; interessante, il trattamento con tamoxifene combinatoria ha prodotto un miglioramento drastico induzione apoptotica e autophagic da mitocondriale di mira con effetti minimi sui fibroblasti non tumorali. Così, JCTH-4 in combinazione con tamoxifene potrebbe fornire una sicura terapia antitumorale.

Valorizzazione dei induzione apoptotica e autofagica da un romanzo sintetico C-1 analogico di 7-deoxypancratistatin in adenocarcinoma del seno umano e cellule di neuroblastoma con Tamoxifene

Breast cancer is one of the most common cancers amongst women in North America. Many current anti-cancer treatments, including ionizing radiation, induce ...

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JoVE Journal

Medicine

18.6K Views.  University of Windsor. Abbiamo sintetizzato un analogo romanzo di pancratistatin paragonabile attività anti-cancro come pancratistatin nativo; interessante, il trattamento con tamoxifene combinatoria ha prodotto un miglioramento drastico induzione apoptotica e autophagic da mitocondriale di mira con effetti minimi sui fibroblasti non tumorali. Così, JCTH-4 in combinazione con tamoxifene potrebbe fornire una sicura terapia antitumorale.

Video: Enhancement of Apoptotic and Autophagic Induction by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin in Human Breast Adenocarcinoma and Neuroblastoma Cells with Tamoxifen

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Differenziazione Isolamento, caratterizzazione e comparata delle cellule umane Dental Pulp staminali derivate da denti permanenti mediante due diversi metodi

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Ricerca

Medicina

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent &gamma;-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered &gamma;-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

The cytotoxic activity of the phenanthridine PJ-34 in cancer cells undergoing mitosis was documented in real time by live confocal imaging. PJ-34 eradicated human breast cancer MDA-MB-231 cells harboring extra-centrosomes in mitosis. Unlike normal bi-focal mitosis, the extra-centrosomes were not clustered in the two spindle poles in the presence of PJ-34.

Cella della morte associata a mitosi anomalo osservato da Imaging confocale in cellule vive di cancro

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Avvio del carcinoma metastatico della mammella dalla Targeting del duttale con Adenovirus-Cre: A Novel modello di topo transgenico di cancro al seno

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Profiling di microRNA regolati dagli estrogeni in cellule di carcinoma mammario

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

Utilizzo del saggio soft Agar Colony Formazione per identificare inibitori di tumorigenicità in cellule di carcinoma mammario

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Studiare cancro del pancreas staminali caratteristiche cellulari per lo sviluppo di nuove strategie di trattamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective mining of the information in the large-scale datasets that are obtained from gene expression microarrays, deep sequencing experiments or metabolic profiling is essential to uncover and then effectively target the critical regulators of diseased cell phenotypes. Estrogen Receptor &#945; (ER&#945;) is one of the master transcription factors regulating the gene programs that are important for estrogen responsive breast cancers. In order to understand to role of ER&#945; signaling in breast cancer metabolism we utilized transcriptomic, cistromic and metabolomic data from MCF-7 cells treated with estradiol. In this report we described generation of samples for RNA-Seq, ChIP-Seq and metabolomics experiments and the integrative computational analysis of the obtained data. This approach is useful in delineating novel molecular mechanisms and gene regulatory circuits that are regulated by a particular transcription factor which impacts metabolism of normal or diseased cells.

Integration of data from genome-wide sequencing experiments and metabolomics experiments is a challenge. In this paper we report, for the first time, generation, analysis and integration of transcriptome, cistrome and metabolome data from breast cancer cells treated with estradiol.

Systems Biology di regolazione metabolica da Estrogen Receptor Signaling nel cancro al seno

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Induzione di aterosclerosi accelerata nei topi: il modello "Wire-Injury"

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

Un metodo di isolamento di romanzo Clinical Grade per rene umano perivascolari cellule stromali

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

In Vitro e In Vivo rilevamento di Mitophagy in cellule umane, C. Eleganse topi

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...