Supplementary MaterialsAdditional file 1: Number S1. derived from macropinosomes and late endosomes, followed by metabolic failure and rupture of the plasma membrane. However, not all IPPs that cause vacuolization are cytotoxic. The main goals of the present study were to identify important signaling pathways that contribute to methuosis induced by cytotoxic IPPs and to evaluate the anti-tumor potential of a prototype IPP in vivo. Methods We utilized metabolic flux analysis, glucose uptake, immunoblotting, and selective pharmacological inhibitors to compare the effects of closely related cytotoxic and non-cytotoxic IPPs in cultured glioblastoma cells. To determine whether the use of methuosis-inducing IPPs might be feasible inside a restorative context, we quantified the distribution of our lead IPP compound, MOMIPP, in mouse plasma and mind, and tested its ability to inhibit tumor growth in an intracerebral glioblastoma xenograft model. Results The cytotoxic IPP compound, MOMIPP, causes early disruptions of glucose uptake and glycolytic rate of metabolism. Coincident with these metabolic changes, MOMIPP selectively activates the JNK1/2 stress kinase pathway, resulting in phosphorylation of c-Jun, Bcl-2 and Bcl-xL. At the same concentration, the non-cytotoxic analog, MOPIPP, does not activate these pathways. Pharmacologic inhibition of JNK activity promotes survival, even when cells are extensively vacuolated, but suppression of c-Jun transcriptional activity gives no protection. MOMIPP readily penetrates the blood-brain barrier and is moderately effective in suppressing progression of intracerebral glioblastoma xenografts. Conclusions The results suggest that interference with glucose uptake and induction of JNK-mediated phosphorylation of pro-survival users of the Bcl-2 family represent key events in the methuosis death process. In addition to providing fresh insights into the underlying molecular mechanism of methuosis, the results indicate that compounds of the cytotoxic IPP class may have potential for further development as restorative agents for mind tumors. Electronic supplementary material The online version of this article (10.1186/s12885-019-5288-y) contains supplementary material, which is available to authorized users. A-769662 inhibition the phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) [10]. The product of PIKfyve, PI(3,5)P2, is known to play a critical role in late endosome trafficking [11, 12]. Since our initial description of methuosis, a number of other reports possess noted related cell death phenotypes advertised by a variety of chemical agents and natural products [13C15]. Features of methuosis have also been explained in cells responding to overexpression of miR-199a-3p [16], co-expression of mutant EGFR and K-Ras [17], immunotargeting of CD99 [18], treatment with an oligonucleotide aptamer [19], or NGF-stimulation of TrkA [20]. Despite the growing recognition of the morphological hallmarks of methuosis, the specific molecular mechanisms that link vacuolization of endocytic compartments to loss of cell viability remain poorly recognized. Our structure-activity studies of MOMIPP and several analogs in GBM cells have provided valuable chemical tools to address this question. Specifically, we found that small structural modifications of the indole ring yielded a functionally unique sub-group of IPPs that retained the ability to induce powerful morphological vacuolization, with greatly reduced cytotoxicity [21, 22]. By comparing the effects of MOMIPP with one of the non-lethal analogs (MOPIPP; with propyl substituted for methyl in the 2-position of the A-769662 inhibition indole ring), we mentioned that cells treated with MOMIPP experienced more severe inhibition of endolysosomal degradation pathways for EGF and LDL receptors [5]. Coincidentally, MOMIPP shows stronger binding affinity (lower Kd) for PIKfyve than the non-lethal analogs [10], A-769662 inhibition despite the fact that the cells treated with these compounds possess related vacuolated morphologies. In the present study, the objective was to expand the comparative analysis of cytotoxic versus non-cytotoxic vacuole-inducing IPPs in GBM cells, with the goal of defining pathways essential for triggering cell death. The results indicate that early impairment of glucose uptake and glycolytic IL6R rate of metabolism, with attendant activation of JNK signaling and Bcl-2 phosphorylation, are key elements A-769662 inhibition in the methuosis death program. Methods Cell culture Human being glioblastoma cell lines, U251 (deposited by Darrell Bigner), SF295 (deposited by Paul Kornblith), and SNB19 and SNB75 (deposited by M.L. Rosenblum), were from the Developmental Therapeutics System (DTP) Tumor Repository, NCI Division of Malignancy Treatment and Analysis (DCTD) (operated by Charles River Laboratories for the National Tumor Institute, Frederick, MD). The A172 (Cat. No. CRL-1620), LN229 (Cat No. CRL-2611), T98G (Cat No.CRL-1690), and U87MG (Cat No. HTB-14) cell lines were purchased from A-769662 inhibition your American Type Tradition Collection (Manassas, VA). Normal human being pores and skin fibroblasts were originally derived from a pores and skin biopsy as explained previously [23]. All cell lines were managed in Dulbeccos revised Eagle medium (DMEM; ThermoFisher, Walthham, MA), supplemented with 10% (contamination by periodic staining with DAPI or use of the PlasmoTest assay.