Sensitivity of neuroprogenitor cells to chemical-induced apoptosis using a multiplexed assay suitable for high-throughput screening

The study of death receptor family induced apoptosis has gained momentum in recent years with the knowledge that therapeutic antibodies targeting DR4 and DR5 (death receptor's 4 and 5) have proved efficacious in multiple clinical trials. The therapeutic rationale is based on targeting and amplifying a tumour tissues normal cell death programme (apoptosis). While advances in the targeting of DR4 and DR5 have been successful the search for an agonistic antibody to another family member, the Fas receptor, has proven more elusive. This is partly due to the differing in vitro and in vivo characteristics of individual antibodies. In order to induce Fas targeted cell death an antibody must be capable of binding to and trimerising the receptor. It has been shown that antibodies capable of performing this function in vivo, with the assistance of tumour associated cells, do not always induce apoptosis in vitro. As a result the use of current methodologies to detect functional antibodies in vitro may have dismissed potential therapeutic candidates ('false negative'). Here we report a novel high throughput screening technique which artificially cross-links antibodies bound to the Fas receptor. By combining this process with Annexin-V and Prodidium Iodide (PI) staining we can select for antibodies which have the potential to induce apoptosis in vivo.

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Complementary Cell-Based High-Throughput Screens Identify Novel Modulators of the Unfolded Protein Response

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057111414893 ◽

2011 ◽

Vol 16 (8) ◽

pp. 825-835 ◽

Cited By ~ 33

Author(s):

Andrew M. Fribley ◽

Patricia G. Cruz ◽

Justin R. Miller ◽

Michael U. Callaghan ◽

Peter Cai ◽

...

Keyword(s):

Unfolded Protein Response ◽

High Throughput ◽

High Throughput Screening ◽

Luciferase Reporter ◽

Tumor Cell Death ◽

Unfolded Protein ◽

Induced Apoptosis ◽

The Unfolded Protein Response ◽

Protein Response ◽

The University

Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than 2 decades, indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, the authors hypothesized that high-throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small-molecule activators of the apoptotic arm of the UPR to control or kill OSCC. They have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2α/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR subpathways. An ˜66 K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of prefractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80 µM. A series of citrinin derivatives was isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds, the authors examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, it was found that patulin at 2.5 to 10 µM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34, and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis in OSCC cells. These data support the use of this complementary HTS strategy to identify novel modulators of UPR signaling and tumor cell death.

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Identification of Inhibitors for MDM2 Ubiquitin Ligase Activity from Natural Product Extracts by a Novel High-Throughput Electrochemiluminescent Screen

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057108315038 ◽

2008 ◽

Vol 13 (3) ◽

pp. 229-237 ◽

Cited By ~ 39

Author(s):

Christy A. Sasiela ◽

David H. Stewart ◽

Jirouta Kitagaki ◽

Yassamin J. Safiran ◽

Yili Yang ◽

...

Keyword(s):

Natural Product ◽

High Throughput ◽

High Throughput Screening ◽

Antimicrobial Drugs ◽

Screening Programs ◽

Lead Compounds ◽

Ubiquitin Ligase Activity ◽

Testing Complex ◽

Induced Apoptosis ◽

Potential Therapeutics

High-throughput screening technologies have revolutionized the manner in which potential therapeutics are identified. Although they are the source of lead compounds for ~65% of anticancer and antimicrobial drugs approved by the Food and Drug Administration between 1981 and 2002, natural products have largely been excluded from modern screening programs. This is due, at least in part, to the inherent difficulties in testing complex extract mixtures, which often contain nuisance compounds, in modern bioassay systems. In this article, the authors present a novel electrochemiluminescent assay system for inhibition of MDM2 activity that is suitable for testing natural product extracts in high-throughput screening systems. The assay was used to screen more than 144,000 natural product extracts. The authors identified 1 natural product, sempervirine, that inhibited MDM2 auto-ubiquitination, MDM2-mediated p53 degradation, and led to accumulation of p53 in cells. Sempervirine preferentially induced apoptosis in transformed cells expressing wild-type p53, suggesting that it could be a potential lead for anticancer therapeutics. ( Journal of Biomolecular Screening 2008:229-237)

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