scholarly journals Assessing a Novel 3D Assay System for Drug Screening against OS Metastasis

2021 ◽  
Vol 14 (10) ◽  
pp. 971
Author(s):  
Natalie Koons ◽  
Nicole Amato ◽  
Scott Sauer ◽  
David Warshawsky ◽  
Dalit Barkan ◽  
...  
Keyword(s):  
Mesenchymal Cell ◽  
Disseminated Tumor Cells ◽  
Janus Kinase ◽  
Drug Candidate ◽  
Proliferating Cells ◽  
Conventional Drug ◽  
Molecule Inhibitor ◽  
Basement Membrane Extract ◽  
Definitive Surgery ◽  
Long Term Prognosis

Osteosarcoma (OS) is an aggressive mesenchymal cell tumor that carries a poor long-term prognosis. Despite definitive surgery for the primary tumor and adjuvant chemotherapy, pulmonary metastasis is common and is the primary cause of morbidity. To improve outcomes for patients, we have developed and optimized a phenotypic screen for drugs that may target OS disseminated tumor cells (DTCs) and inhibit their metastatic outbreak rather than merely screening for cytotoxic activity against proliferating cells, as is commonly conducted in conventional drug discovery approaches. We report on the validation of a previously described 3D reconstituted basement membrane extract (3D BME) model system for tumor dormancy and metastatic outgrowth adapted to clonal pairs of high and low metastatic OS cells. A post-hoc validation of the assay was possible by comparing the activity of a drug in our assay with early evidence of activity in human OS clinical trials (regorafenib and saracatinib). In this validation, we found concordance between our assay and human clinical trial experience We then explored an approved veterinary small molecule inhibitor of Janus kinase-1 (oclacitinib) as a potential drug candidate to take advantage of the high prevalence of OS in pet dogs and its translational value to humans. Despite the biological rationale, we found no evidence to support the use of oclacitinib as an antimetastatic agent in OS. The findings support our 3D BME assay as a highly efficient method to examine drugs for activity in targeting OS DTCs.

scholarly journals Rapid Generation of Single-Tumor Spheroids for High-Throughput Cell Function and Toxicity Analysis

2006 ◽  
Vol 11 (8) ◽  
pp. 922-932 ◽  
Author(s):  
Andrea Ivascu ◽  
Manfred Kubbies
Keyword(s):  
Tumor Cell ◽  
Suspension Culture ◽  
Cell Lines ◽  
Cell Function ◽  
Tumor Cell Lines ◽  
Proliferating Cells ◽  
Basement Membrane Extract ◽  
Rapid Generation ◽  
Size Heterogeneity

Spheroids are widely used in biology because they provide an in vitro 3-dimensional (3D) model to study proliferation, cell death, differentiation, and metabolism of cells in tumors and the response of tumors to radiotherapy and chemotherapy. The methods of generating spheroids are limited by size heterogeneity, long cultivation time, or mechanical accessibility for higher throughput fashion. The authors present a rapid method to generate single spheroids in suspension culture in individual wells. A defined number of cells ranging from 1000 to 20,000 were seeded into wells of poly-HEMA-coated, 96-well, round-or conical-bottom plates in standard medium and centrifuged for 10 min at 1000 g. This procedure generates single spheroids in each well within a 24-h culture time with homogeneous sizes, morphologies, and stratification of proliferating cells in the rim and dying cells in the core region. Because a large number of tumor cell lines form only loose aggregates when cultured in 3D, the authors also performed a screen for medium additives to achieve a switch from aggregate to spheroid morphology. Small quantities of the basement membrane extract Matrigel, added to the culture medium prior to centrifugation, most effectively induced compact spheroid formation. The compact spheroid morphology is evident as early as 24 h after centrifugation in a true suspension culture. Twenty tumor cell lines of different lineages have been used to successfully generate compact, single spheroids with homogenous size in 96-well plates and are easily accessible for subsequent functional analysis.

Characterization of BMS-911543, a Functionally Selective Small Molecule Inhibitor of JAK2

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4112-4112 ◽  
Author(s):  
Ashok V Purandare ◽  
Animesh Pardanani ◽  
Theresa McDevitt ◽  
Marco Gottardis ◽  
Terra Lasho ◽  
...  
Keyword(s):  
Small Molecule ◽  
Cell Types ◽  
Small Molecule Inhibitor ◽  
Janus Kinase ◽  
Jak Inhibitors ◽  
In Vivo Models ◽  
Molecule Inhibitor ◽  
Transcriptional Changes ◽  
Dose Levels

Abstract Abstract 4112 We report the characterization of BMS-911543, a potent and functionally selective small molecule inhibitor of the Janus kinase family (JAK) member, JAK2. BMS-911543 is a reversible inhibitor of JAK2 with a biochemical IC50 of 0.001 μ M and Ki of 0.48 nM. It has over 74- and 350-fold selectivity against the other JAK family members, JAK3 and JAK1, respectively. Further, examination of > 450 other kinases did not reveal significant inhibitory activity for this JAK2 inhibitor. Functionally, BMS-911543 displayed potent anti-proliferative and pharmacodynamic (PD) effects in mutated JAK2-expressing cell lines dependent upon JAK2-STAT signaling and had little activity in cell types dependent upon other pathways such as JAK1 and JAK3. BMS-911543 was evaluated in colony growth assays using primary progenitor cells isolated from patients with JAK2V617F-positive myeloproliferative disease (MPD) and resulted in an increased anti-proliferative response in MPD cells as compared with those from healthy volunteers. Similar to these in vitro observations, BMS-911543 was also highly active in in vivo models of JAK2-pSTAT signaling in multiple species (mouse, rat, dog and monkey) with sustained pathway suppression being observed after a single oral dose. Additionally, BMS-911543 was evaluated for effects in a JAK2V617F-expressing SET-2 xenograft model system and displayed a minimally effective dose of <2 mg/kg on pSTAT5 pathway suppression, which lasted up to 8 hours. BMS-911543 was also compared to pan-JAK inhibitors in a mouse model of immunosuppression. At low dose levels active in JAK2-dependent PD models, no effects were observed on antigen-induced IgG and IgM production whereas a pan-JAK family inhibitor showed pronounced effects at all dose levels tested. The mechanistic selectivity of BMS-911543 to pan-JAK family inhibitors was extended through comparative analysis of these inhibitors in whole genome gene expression profiling experiments performed in sensitive cell types. In this comparison, BMS-911543 modulated a distinct subset of transcriptional changes as compared to pan-JAK inhibitors, thereby defining a minimal set of transcriptional changes underlying the pharmacologic effects of JAK2 inhibition. Collectively these results define the mechanistic basis for a differential therapeutic index between selective JAK2 and pan-JAK family inhibition pre-clinically and suggest a therapeutic rationale for the further characterization of BMS-911543 in patients with MPD and in other disorders characterized by constitutively active JAK2 signaling. Disclosures: Purandare: Bristol-Myers Squibb: Employment. McDevitt:Bristol-Myers Squibb: Employment. Gottardis:Bristol-Myers Squibb: Employment. You:Bristol-Myers Squibb: Employment. Lombardo:Bristol_Myers Squibb: Employment. Penhallow:Bristol-Myers Squibb: Employment. Vuppugalla:Bristol-Myers Squibb: Employment. Trainor:Bristol-Myers Squibb: Employment. Lorenzi:Bristol-Myers Squibb: Employment.

scholarly journals Discovery of a Potent PLK1-PBD Small-Molecule Inhibitor as an Anticancer Drug Candidate through Structure-Based Design

Molecules ◽  
2019 ◽  
Vol 24 (23) ◽  
pp. 4351 ◽  
Author(s):  
Yunjiang Zhou ◽  
Fang Yan ◽  
Xiangyun Huo ◽  
Miao-Miao Niu
Keyword(s):  
Cell Proliferation ◽  
Small Molecule ◽  
Hela Cells ◽  
Anticancer Agent ◽  
Pharmacophore Modeling ◽  
Drug Candidate ◽  
The Poor ◽  
Molecule Inhibitor ◽  
Screening Protocol ◽  
Anticancer Target

Polo-box domain of polo-like kinase 1 (PLK1-PBD) has a pivotal role in cell proliferation and could be implicated as a potential anticancer target. Although some small-molecule inhibitors have been developed, their clinical application has been restricted by the poor selectivity. Therefore, there is an urgent need to develop effective PLK1-PBD inhibitors. Herein, we have developed a virtual screening protocol to find PLK1-PBD inhibitors by using combination of structure-based pharmacophore modeling and molecular docking. This protocol was successfully applied to screen PLK1-PBD inhibitors from specs database. MTT assay indicated that five screened hits suppressed the growth of HeLa cells. Particularly, hit-5, as a selective PLK1 inhibitor targeting PLK1-PBD, significantly inhibited the progression of HeLa cells-derived xenograft, with no obvious side effects. This work demonstrates that hit-5 may be a potential anticancer agent.

scholarly journals Combination Trimodality Therapy Using Vismodegib for Basal Cell Carcinoma of the Face

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Alec M. Block ◽  
Fiori Alite ◽  
Aidnag Z. Diaz ◽  
Richard W. Borrowdale ◽  
Joseph I. Clark ◽  
...  
Keyword(s):  
Local Excision ◽  
Basal Cell ◽  
Locally Advanced ◽  
Extensive Resection ◽  
Trimodality Therapy ◽  
Excellent Outcome ◽  
Molecule Inhibitor ◽  
The Face ◽  
Prolonged Recovery ◽  
Definitive Surgery

Background. For large basal cell carcinomas (BCCs) of the head and neck, definitive surgery often requires extensive resection and reconstruction that may result in prolonged recovery and limited cosmesis. Vismodegib, a small-molecule inhibitor of the hedgehog pathway, is approved for advanced and metastatic BCCs. We present a case of advanced BCC treated with combination of vismodegib, radiotherapy, and local excision resulting in excellent response and cosmesis.Case Presentation. A 64-year-old gentleman presented with a 5-year history of a 7 cm enlarging right cheek mass, with extensive vascularization, central ulceration, and skin, soft tissue, and buccal mucosa involvement. Biopsy revealed BCC, nodular type. Up-front surgical option involved a large resection and reconstruction. After multidisciplinary discussion, we recommended and he opted for combined modality of vismodegib, radiotherapy, and local excision. The patient tolerated vismodegib well and his right cheek lesion decreased significantly in size. He was then treated with radiotherapy followed by local excision that revealed only focal residual BCC. Currently, he is without evidence of disease and has excellent cosmesis.Conclusions. We report a case of locally advanced BCC treated with trimodality therapy with vismodegib, radiotherapy, and local excision, resulting in excellent outcome and facial cosmesis, without requiring extensive resection or reconstructive surgery.

RGB 286638, a Novel Multi-Targeted Small Molecule Inhibitor, Induces Multiple Myeloma (MM) Cell Death through Abrogation of CDKDependent and Independent Survival Mechanisms

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2759-2759 ◽  
Author(s):  
Diana Cirstea ◽  
Teru Hideshima ◽  
Samantha Pozzi ◽  
Sonia Vallet ◽  
Hiroshi Ikeda ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Small Molecule ◽  
Small Molecule Inhibitor ◽  
Janus Kinase ◽  
Transcriptional Networks ◽  
Molecule Inhibitor

Abstract Uncontrolled proliferation and development of drug resistance in multiple myeloma (MM) cells are consequences of the numerous genetic aberrations which are further stimulated in the context of bone marrow microenvironment. Thus, inhibition of complementary pro-survival signaling and transcriptional networks rather than individual pathway is required for induction of optimal cytotoxicity in MM cells. Recent studies have shown that cyclin-dependent kinases inhibitors (CDKIs), designed to block cell cycle progression through inhibition of CDK/cyclin complexes, block transcription through suppression of RNA polymerase II phosphorylation at its C-terminal domain (CTD), resulting in downregulation of cell proliferation (cyclins: D, A, B1, pRb) and anti-apoptotic proteins (i.e. Mcl-1, survivin, XIAP). Here we examined the anti-MM activity of RGB 286638, a novel multi-targeted small molecule inhibitor, originally designed to induce broad cell cycle suppression via multiple CDK inhibition. Treatment with RGB 286638 triggered a dose-dependent cytotoxicity in conventional drug-sensitive (MM.1S, RPMI 8226, U266, OPM2), and resistant (MM.1R, Dox-40, LR5) MM cell lines, as well as primary tumor cells from MM patients. Induction of apoptosis was evidenced by Annexin V/PI staining, and confirmed by PARP and caspase cleavage. Additionally, RGB 286638 overcame the proliferative advantage conferred by MM patient-derived bone marrow stromal cells (BMSCs) and cytokines (IL-6, IGF-1) on MM cells. To determine molecular mechanisms responsible for RGB 286638-induced cytotoxicity, we assessed the cell cycle profile, which revealed G2/M arrest followed by increased sub-G1 phase. Importantly, RGB 286638 inhibited phosphorylation of RNA polymerase II in a dose- and a time-dependent fashion, followed by suppression of CDK1/cyclin B, CDK4, 6/Cyclin D1, D3, and CDK2/Cyclin E complexes associated with rapid down-regulation of Rb phosphorylation. Interestingly, RGB 286638 effectively reduced IL-6-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation (Tyr705) and Janus kinase 2 (JAK2) phosphorylation (Tyr 1007/1008), suggesting that RGB 286638 is a possible JAK2 inhibitor. Based on sufficient in vitro cytotoxicity, we examined anti-tumor activity of RGB 286638 in vivo using a human MM cell xenograft model in SCID mice and demonstrated that RGB 286638 inhibited tumor growth and prolonged survival. In conclusion, our data demonstrate preclinical activity and provide the rational to test RGB 286638 in the treatment of MM.

scholarly journals Src phosphorylation converts Mdm2 from a ubiquitinating to a neddylating E3 ligase

2015 ◽  
Vol 112 (6) ◽  
pp. 1749-1754 ◽  
Author(s):  
Christopher N. Batuello ◽  
Paula M. Hauck ◽  
Jaimie M. Gendron ◽  
Jason A. Lehman ◽  
Lindsey D. Mayo
Keyword(s):  
Precursor Cell ◽  
Tumor Suppressor Protein ◽  
Neural Precursor Cell ◽  
Wild Type ◽  
Proliferating Cells ◽  
Tumor Suppressor Protein P53 ◽  
Phosphorylated Protein ◽  
Molecule Inhibitor ◽  
Double Minute ◽  
Murine Double Minute

Murine double minute-2 protein (Mdm2) is a multifaceted phosphorylated protein that plays a role in regulating numerous proteins including the tumor suppressor protein p53. Mdm2 binds to and is involved in conjugating either ubiquitin or Nedd8 (Neural precursor cell expressed, developmentally down-regulated 8) to p53. Although regulation of the E3 ubiquitin activity of Mdm2 has been investigated, regulation of the neddylating activity of Mdm2 remains to be defined. Here we show that activated c-Src kinase phosphorylates Y281 and Y302 of Mdm2, resulting in an increase in Mdm2 stability and its association with Ubc12, the E2 enzyme of the neddylating complex. Mdm2-dependent Nedd8 conjugation of p53 results in transcriptionally inactive p53, a process that is reversed with a small molecule inhibitor to either Src or Ubc12. Thus, our studies reveal how Mdm2 may neutralize and elevate p53 in actively proliferating cells and also provides a rationale for using therapies that target the Nedd8 pathway in wild-type p53 tumors.

Cardiac myocytes cultured on a basement membrane extract of the ehs tumor

1986 ◽  
Vol 44 ◽  
pp. 270-271
Author(s):  
L. Terracio ◽  
A. Dewey ◽  
K. Rubin ◽  
T.K. Borg
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Cardiac Myocytes ◽  
Normal Tissue ◽  
Cell Spreading ◽  
Collagen Iv ◽  
Basement Membrane Extract ◽  
Membrane Extract ◽  
Growth Development ◽  
Multicellular Organisms

The recognition and interaction of cells with the extracellular matrix (ECM) effects the normal physiology as well as the pathology of all multicellular organisms. These interactions have been shown to influence the growth, development, and maintenance of normal tissue function. In previous studies, we have shown that neonatal cardiac myocytes specifically interacts with a variety of ECM components including fibronectin, laminin, and collagens I, III and IV. Culturing neonatal myocytes on laminin and collagen IV induces an increased rate of both cell spreading and sarcomerogenesis.

Quantitative Model-Based Image Analysis of NuMa Distribution Links Nuclear Organization with Cell Phenotype

2001 ◽  
Vol 7 (S2) ◽  
pp. 578-579
Author(s):  
David W. Knowles ◽  
Sophie A. Lelièvre ◽  
Carlos Ortiz de Solόrzano ◽  
Stephen J. Lockett ◽  
Mina J. Bissell ◽  
...  
Keyword(s):  
Image Analysis ◽  
Mammary Epithelial Cells ◽  
Nuclear Organization ◽  
Critical Role ◽  
Quantitative Model ◽  
Mammary Epithelial ◽  
Cell Phenotype ◽  
Proliferating Cells ◽  
Mitotic Apparatus ◽  
Model Based

The extracellular matrix (ECM) plays a critical role in directing cell behaviour and morphogenesis by regulating gene expression and nuclear organization. Using non-malignant (S1) human mammary epithelial cells (HMECs), it was previously shown that ECM-induced morphogenesis is accompanied by the redistribution of nuclear mitotic apparatus (NuMA) protein from a diffuse pattern in proliferating cells, to a multi-focal pattern as HMECs growth arrested and completed morphogenesis . A process taking 10 to 14 days.To further investigate the link between NuMA distribution and the growth stage of HMECs, we have investigated the distribution of NuMA in non-malignant S1 cells and their malignant, T4, counter-part using a novel model-based image analysis technique. This technique, based on a multi-scale Gaussian blur analysis (Figure 1), quantifies the size of punctate features in an image. Cells were cultured in the presence and absence of a reconstituted basement membrane (rBM) and imaged in 3D using confocal microscopy, for fluorescently labeled monoclonal antibodies to NuMA (fαNuMA) and fluorescently labeled total DNA.

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