latrunculin-b and Glioblastoma

Inhibition of integrins alphavbeta3 and alphavbeta5 in human brain microvascular endothelial cells (HBMECs) by the function-blocking peptide RGDfV induces loss of spreading on vitronectin, cell detachment, and apoptosis. We demonstrate that cell detachment is not required for apoptosis because plating on bovine serum albumin-blocked poly-L-lysine (allows attachment, but not integrin ligation and cell spreading) also induced apoptosis. Latrunculin B (LatB), which inhibits F-actin polymerization, induced transient loss of HBMEC spreading on vitronectin, but not their detachment, and induced apoptosis despite recovery of cell spreading. However, LatB did not cause apoptosis in 5 tumor cell lines. In HBMECs, both LatB and RGDfV induced transient Y412 and Y245 phosphorylation of endogenous c-Abl, a nonreceptor tyrosine kinase that reciprocally regulates F-actin. LatB also induced nuclear translocation of c-Abl in HBMECs. STI-571 (imatinib), a targeted therapy for BCR-ABL1(+) leukemias and inhibitor of c-Abl, platelet-derived growth factor receptor, and c-Kit, decreased endothelial apoptosis. LatB-induced HBMEC apoptosis, and its inhibition by STI-571 also occurred in a 3-dimensional collagen model, supporting physiologic relevance. Last, siRNA to c-Abl (but not nonspecific siRNA) also inhibited RGDfV- and LatB-induced apoptosis. Thus, endogenous c-Abl mediates endothelial apoptosis induced by inhibition of integrins alphavbeta3/alphavbeta5 or by LatB-induced disruption of F-actin.

Topics: Actin Cytoskeleton; Actins; Animals; Apoptosis; Bridged Bicyclo Compounds, Heterocyclic; Cattle; Cell Adhesion; Cell Line, Tumor; Cell Shape; Culture Media; Culture Media, Serum-Free; Endothelial Cells; Fetal Blood; Genes, abl; Glioblastoma; Humans; Integrin alphaVbeta3; Medulloblastoma; Membrane Potential, Mitochondrial; Neuroblastoma; Proto-Oncogene Proteins c-abl; Receptors, Vitronectin; RNA Interference; RNA, Small Interfering; Thiazolidines

Autophagy is a lysosome-dependent cellular catabolic mechanism mediating the turnover of intracellular organelles and long-lived proteins. Reduction of autophagy activity has been shown to lead to the accumulation of misfolded proteins in neurons and may be involved in chronic neurodegenerative diseases such as Huntington's disease and Alzheimer's disease. To explore the mechanism of autophagy and identify small molecules that can activate it, we developed a series of high-throughput image-based screens for small-molecule regulators of autophagy. This series of screens allowed us to distinguish compounds that can truly induce autophagic degradation from those that induce the accumulation of autophagosomes as a result of causing cellular damage or blocking downstream lysosomal functions. Our analyses led to the identification of eight compounds that can induce autophagy and promote long-lived protein degradation. Interestingly, seven of eight compounds are FDA-approved drugs for treatment of human diseases. Furthermore, we show that these compounds can reduce the levels of expanded polyglutamine repeats in cultured cells. Our studies suggest the possibility that some of these drugs may be useful for the treatment of Huntington's and other human diseases associated with the accumulation of misfolded proteins.

Topics: Autophagy; Calcium Channel Blockers; Cell Line, Tumor; Drug Evaluation, Preclinical; Fluspirilene; Glioblastoma; Green Fluorescent Proteins; Humans; Intracellular Membranes; Loperamide; Microtubule-Associated Proteins; Mycotoxins; Peptides; Phagosomes; Phosphatidylinositol Phosphates; Pimozide; Protein Kinases; Recombinant Fusion Proteins; Sirolimus; Small Molecule Libraries; TOR Serine-Threonine Kinases; Trifluoperazine; Zinc Fingers