pitstop-2 and Neoplasms

Exosomes, small-membrane vesicles, are secreted by cells and include several types of proteins and nucleic acids. Exosomes transfer cellular information derived from donor cells and are involved in various physiological and pathological events, such as organ-specific metastasis. Elucidating the exosome uptake mechanisms is important for understanding the progression processes of organ-specific metastasis. However, whether the exosomes secreted by the donor cells are selectively or non-selectively incorporated into the recipient cells is unknown.. In this study, three human carcinoma cell lines, A549 (lung), HCT116 and COLO205 (colon), were used. The exosome isolation efficiency was compared between three methods: ultracentrifugation, ExoQuick-TC and Total Exosome Isolation kits. Recipient cells were treated with Pitstop 2, an inhibitor of clathrin-dependent endocytosis, or genistein, an inhibitor of caveolae-dependent endocytosis, and then incubated with DiO-labeled exosomes.. Among the three methods examined, ultracentrifugation was the most efficient and reproducible. Exosomes derived from a donor cell line are incorporated into the three cell lines, but the exosome uptake capability was different depending on the recipient cell type and did not depend on the donor cell type. Exosome uptake in COLO205 was inhibited by Pitstop 2 and genistein. Exosome uptake in HCT116 was inhibited by Pitstop 2, but not genistein, while that in A549 cells was not inhibited by these inhibitors. Taken together, these results suggest that the exosomes secreted by donor cells are non-selectively incorporated into recipient cells and that the exosome uptake mechanism is different depending on the recipient cells.. Different recipient cells' exosome uptake capabilities may be involved in organ-specific metastasis.

Topics: A549 Cells; Biological Transport; Endocytosis; Exosomes; Genistein; HCT116 Cells; Humans; Neoplasm Metastasis; Neoplasms; Sulfonamides; Thiazolidines

F-box and WD40 repeat domain-containing 7 (FBXW7) is an E3 ubiquitin ligase involved in the ubiquitination and degradation of multiple oncogenic substrates. The tumour suppressor function is frequently lost in multiple cancers through genetic deletion and mutations in a broad range of tumours. Loss of FBXW7 functionality results in the stabilisation of multiple major oncoproteins, culminating in increased cellular proliferation and pro-survival pathways, cell cycle deregulation, chromosomal instability and altered metabolism. Currently, there is no therapy to specifically target FBXW7-deficient tumours.. We performed a siRNA kinome screen to identify synthetically lethal hits to FBXW7 deficiency.. We identified and validated cyclin G-associated kinase (GAK) as a potential new therapeutic target. Combined loss of FBXW7 and GAK caused cell cycle defects, formation of multipolar mitoses and the induction of apoptosis. The synthetic lethal mechanism appears to be independent of clathrin-mediated receptor endocytosis function of GAK.. These data suggest a putative therapeutic strategy for a large number of different types of human cancers with FBXW7 loss, many of which have a paucity of molecular abnormalities and treatment options.

Topics: Apoptosis; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Clathrin; F-Box Proteins; F-Box-WD Repeat-Containing Protein 7; Humans; Intracellular Signaling Peptides and Proteins; Mitosis; Neoplasms; Protein Serine-Threonine Kinases; RNA Interference; RNA, Small Interfering; Sulfonamides; Synthetic Lethal Mutations; Thiazolidines; Ubiquitin-Protein Ligases