Supplementary MaterialsFigure S1: Coibamide A is cytotoxic to wild-type MEFs

Supplementary MaterialsFigure S1: Coibamide A is cytotoxic to wild-type MEFs. LC3 expression in vehicle (control) or coibamide A (30 nM)-treated cells in standard nutrient-rich medium. GAPDH served as a loading control. Results are representative of at least four impartial experiments.(TIF) pone.0065250.s002.tif (342K) GUID:?45EAC0F8-3A1F-4629-BB4C-8A7856E05EA8 Figure S3: Coibamide A induces mTOR-independent autophagy in human U251 glioblastoma cells. Human U251 glioblastoma cells were incubated in EBSS starvation medium, or GSK 2334470 treated with or without coibamide A (30 nM) in standard nutrient-rich medium for 4 h. Following treatment cells were lysed and subjected to immunoblot analysis. (A) Immunoblot analysis of LC3 expression, phospho-p70 S6 Kinase (Thr-389) relative to total S6K1, and 4-E binding protein. GAPDH served as a loading control. (B) Immunoblot analysis of endogenous LC3 in U251 cells treated with vehicle (control), coibamide A (30 nM), or EBSS starvation medium for 4 h, each in the presence or absence of bafilomycin (10 nM) for the final 1 h of treatment. GAPDH served as a loading control. Results are representative of three impartial experiments.(TIF) pone.0065250.s003.tif (334K) GUID:?129CF092-1ACC-4D73-89F7-C02F464D469C Physique S4: Expression of apoptotic markers B2M in wild-type MEFs and human U87-MG glioblastoma cells in response to coibamide A treatment. Immunoblot analysis of PARP1 and caspase-3 in wild-type MEFs and U87-MG cells GSK 2334470 after treatment with coibamide A (30 nM). Adherent and detached (Det) cells were harvested 24 h (MEFs) and 72 h (U87-MG) after treatment and examined for expression of the large 89 kDa fragment of PARP1, full length and cleaved caspase-3, and GAPDH as a loading control. Immunoblot is usually representative of an experiment repeated at least three times with similar results.(TIF) pone.0065250.s004.tif (322K) GUID:?8C4C5AC1-6044-493C-9226-7EBA63B0653A Abstract Coibamide A is an 60 cancer cell line panel revealed a potent anti-proliferative response and COMPARE-negative profile indicative of a unique mechanism of action. We report that coibamide A is usually a more potent and efficacious cytotoxin than was previously appreciated, inducing concentration- and time-dependent cytotoxicity (EC50 100 nM) in human U87-MG and SF-295 glioblastoma cells and mouse embryonic fibroblasts (MEFs). This activity was lost upon linearization of the molecule, highlighting the importance of the cyclized structure for both anti-proliferative and cytotoxic responses. We show that coibamide A induces autophagosome accumulation in human glioblastoma cell types and MEFs via an mTOR-independent mechanism; no change was observed in the phosphorylation state of ULK1 (Ser-757), p70 S6K1 (Thr-389), S6 ribosomal protein (Ser-235/236) and 4EBP-1 (Thr-37/46). Coibamide A also induces morphologically and biochemically distinct forms of cell death according to cell type. SF-295 glioblastoma cells showed caspase-3 activation and evidence of apoptotic cell death in a pattern that was also seen in wild-type and autophagy-deficient (ATG5-null) MEFs. In contrast, cell death in U87-MG glioblastoma cells was characterized by extensive cytoplasmic vacuolization and lacked clear apoptotic features. Cell death was attenuated, but still triggered, in Apaf-1-null MEFs lacking a functional mitochondria-mediated apoptotic pathway. From the study of ATG5-null MEFs we conclude that a conventional autophagy response is not required for coibamide A-induced cell death, but likely occurs in dying cells in response to treatment. Coibamide A represents a natural product scaffold with potential for the study of mTOR-independent signaling and cell death mechanisms in apoptotic-resistant cancer cells. Introduction There is high demand for new small molecules that can strategically target the dysregulated signaling pathways that underlie aggressive solid cancers such as glioblastoma. Glioblastoma multiforme (GBM), classed by the World Health Organization (WHO) as a high-grade IV astrocytoma-like tumor, is the most common malignant primary tumor of the central nervous system (CNS) and is associated with a particularly poor prognosis. Present therapeutic strategies have had little impact on the overall survival rate, with median patient survival times remaining at 14 to 19 months depending on the treatment regimen [1], [2], [3]. Collective efforts to classify the pathogenesis of gliomas have shown that GBM frequently harbors a signature of mutations that tend to attenuate the function of tumor suppressor genes, such as p53 and PTEN, or enhance activation of receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) (reviewed in [3],[4]). In turn, cell signaling driven by growth factors, such as the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways, is dramatically GSK 2334470 enhanced. Together these aberrant signaling networks tend to promote cell survival and lend GBM a natural resistance to apoptosis, rendering conventional chemotherapeutic drugs that typically induce apoptosis ineffective for the treatment of this condition [3]. Consequently, there is a great need for new pharmacologic tools that cause cell death.