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Abstract Background Methuosis is a unique form of non-apoptotic cell death triggered by alterations in the trafficking of clathrin-independent endosomes, ultimately leading to extreme vacuolization and rupture of the cell. Results Here we describe a novel chalcone-like molecule, 3-(2-methyl-1H- indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MIPP) that induces cell death with the hallmarks of methuosis. MIPP causes rapid accumulation of vacuoles derived from macropinosomes, based on time-lapse microscopy and labeling with extracellular fluid phase tracers. Vacuolization can be blocked by the cholesterol-interacting compound, filipin, consistent with the origin of the vacuoles from non-clathrin endocytic compartments. Although the vacuoles rapidly acquire some characteristics of late endosomes (Rab7, LAMP1), they remain distinct from lysosomal and autophagosomal compartments, suggestive of a block at the late endosome/lysosome boundary. MIPP appears to target steps in the endosomal trafficking pathway involving Rab5 and Rab7, as evidenced by changes in the activation states of these GTPases. These effects are specific, as other GTPases (Rac1, Arf6) are unaffected by the compound. Cells treated with MIPP lose viability within 2-3 days, but their nuclei show no evidence of apoptotic changes. Inhibition of caspase activity does not protect the cells, consistent with a non-apoptotic death mechanism. U251 glioblastoma cells selected for temozolomide resistance showed sensitivity to MIPP-induced methuosis that was comparable to the parental cell line. Conclusions MIPP might serve as a prototype for new drugs that could be used to induce non-apoptotic death in cancers that have become refractory to agents that work through DNA damage and apoptotic mechanisms.

Methuosis is a form of nonapoptotic cell death characterized by an accumulation of macropinosome-derived vacuoles with eventual loss of membrane integrity. Small molecules inducing methuosis could offer significant advantages compared to more traditional anticancer drug therapies that typically rely on apoptosis. Herein we further define the effects of chemical substitutions at the 2- and 5-indolyl positions on our lead compound 3-(5-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one (MOMIPP). We have identified a number of compounds that induce methuosis at similar potencies, including an interesting analogue having a hydroxypropyl substituent at the 2-position. In addition, we have discovered that certain substitutions on the 2-indolyl position redirect the mode of cytotoxicity from methuosis to microtubule disruption. This switch in activity is associated with an increase in potency as large as 2 orders of magnitude. These compounds appear to represent a new class of potent microtubule-active anticancer agents. [ABSTRACT FROM AUTHOR]

Although macropinocytosis is widely recognized as a distinct form of fluid-phase endocytosis in antigen-presenting dendritic cells, it also occurs constitutively in many other normal and transformed cell types. Recent studies have established that various genetic or pharmacological manipulations can hyperstimulate macropinocytosis or disrupt normal macropinosome trafficking pathways, leading to accumulation of greatly enlarged cytoplasmic vacuoles. In some cases, this extreme vacuolization is associated with a unique form of non-apoptotic cell death termed "methuosis," from the Greek methuo (to drink to intoxication). It remains unclear whether cell death related to dysfunctional macropinocytosis occurs in normal physiological contexts. However, the finding that some types of cancer cells are particularly vulnerable to this unusual form of cell death has raised the possibility that small molecules capable of altering macropinosome trafficking or function might be useful as therapeutic agents against cancers that are resistant to drugs that work by inducing apoptosis. Herein we review examples of cell death associated with dysfunctional macropinocytosis and summarize what is known about the underlying mechanisms. [ABSTRACT FROM AUTHOR]

Background: Methuosis is a unique form of non-apoptotic cell death triggered by alterations in the trafficking of clathrin-independent endosomes, ultimately leading to extreme vacuolization and rupture of the cell. Results: Here we describe a novel chalcone-like molecule, 3-(2-methyl-1H- indol-3-yl)-1-(4-pyridinyl)-2-propen-1- one (MIPP) that induces cell death with the hallmarks of methuosis. MIPP causes rapid accumulation of vacuoles derived from macropinosomes, based on time-lapse microscopy and labeling with extracellular fluid phase tracers. Vacuolization can be blocked by the cholesterol-interacting compound, filipin, consistent with the origin of the vacuoles from non-clathrin endocytic compartments. Although the vacuoles rapidly acquire some characteristics of late endosomes (Rab7, LAMP1), they remain distinct from lysosomal and autophagosomal compartments, suggestive of a block at the late endosome/lysosome boundary. MIPP appears to target steps in the endosomal trafficking pathway involving Rab5 and Rab7, as evidenced by changes in the activation states of these GTPases. These effects are specific, as other GTPases (Rac1, Arf6) are unaffected by the compound. Cells treated with MIPP lose viability within 2-3 days, but their nuclei show no evidence of apoptotic changes. Inhibition of caspase activity does not protect the cells, consistent with a non-apoptotic death mechanism. U251 glioblastoma cells selected for temozolomide resistance showed sensitivity to MIPP-induced methuosis that was comparable to the parental cell line. Conclusions: MIPP might serve as a prototype for new drugs that could be used to induce non-apoptotic death in cancers that have become refractory to agents that work through DNA damage and apoptotic mechanisms. [ABSTRACT FROM AUTHOR]

The human type III phosphatidylinositol 3-kinase, hVps34, converts phosphatidylinositol (PtdIns) to phosphatidylinositol 3-phosphate [PtdIns(3)P]. Studies using inhibitors of phosphatidylinositide 3-kinases have indicated that production of PtdIns(3)P is important for a variety of vesicle-mediated trafficking events, including endocytosis, sorting of receptors in multivesicular endosomes, and transport of lysosomal enzymes from the trans-Golgi network (TGN) to the endosomes and lysosomes. This study utilizes small interfering (si)RNA-mediated gene silencing to define the specific trafficking pathways in which hVps34 functions in human U-251 glioblastoma cells. Suppression of hVps34 expression reduced the cellular growth rate and caused a striking accumulation of large acidic phase-lucent vacuoles that contain lysosomal membrane proteins LAMP1 and LGP85. Analysis of these structures by electron microscopy suggests that they represent swollen late endosomes that have lost the capacity for inward vesiculation but retain the capacity to fuse with lysosomes. Morphological perturbation of the late endosome compartment was accompanied by a reduced rate of processing of the endosomal intermediate form of cathepsin D to the mature lysosomal form. There was also a reduction in the rate of epidermal growth factor receptor (EGFR) dephosphorylation and degradation following ligand stimulation, consistent with the retention of the EGFR on the limiting membranes of the enlarged late endosomes. By contrast, the suppression of hVps34 expression did not block trafficking of cathepsin D between the TGN and late endosomes, or endocytic uptake of fluid-phase markers, or association of a PtdIns(3)P-binding protein, EEA1, with early endosomes. LAMP1-positive vacuoles were depleted of PtdIns(3)P in the hVps34-knockdown cells, as judged by their inability to bind the PtdIns(3)P probe GFP-2xFYVE. By contrast, LAMP1-negative vesicles continued to bind GFP-2xFYVE in the knockdown cells. Overall, these findings indicate that hVps34 plays a major role in generating PtdIns(3)P for internal vesicle formation in multivesicular/late endosomes. The findings also unexpectedly suggest that other wortmannin-sensitive kinases and/or polyphosphoinositide phosphatases may be able to compensate for the loss of hVps34 and maintain PtdIns(3)P levels required for vesicular trafficking in the early endocytic pathway or the TGN. [ABSTRACT FROM AUTHOR]

Beclin 1 was originally identified as a novel Bcl-2-interacting protein, but co-immunoprecipitation studies suggest that the major physiological partner for Beclin 1 is the mammalian class III phosphatidylinositol 3-kinase (PI 3-kinase) Vps34. Beclin 1 has been proposed to function as a tumor suppressor by promoting cellular macroautophagy, a process that is known to depend on Vps34. However, an alternative role for Beclin 1 in modulating normal Vps34-dependent protein trafficking pathways has not been ruled out. This possibility was examined in U-251 glioblastoma cells. Immunoprecipitates of endogenous Beclin 1 contained human Vps34 (hVps34), but not Bcl-2. Suppression of Beclin 1 expression by short interfering (si)RNA-mediated gene silencing blunted the autophagic response of the cells to nutrient deprivation or C2-ceramide. However, other PI 3-kinase-dependent trafficking pathways, such as the post-endocytic sorting of the epidermal growth factor receptor (EGFR) or the proteolytic processing of procathepsin D en route from the trans-Golgi network (TGN) to lysosomes, were not affected. Depletion of Beclin 1 did not reduce endocytic internalization of a fluid phase marker (horseradish peroxidase, HRP) or cause swelling of late endosomal compartments typically seen in cells where the function of hVps34 is impaired. These findings argue against a role for Beclin 1 as an essential chaperone or adaptor for hVps34 in normal vesicular trafficking, and they support the hypothesis that Beclin 1 functions mainly to engage hVps34 in the autophagic pathway. [ABSTRACT FROM AUTHOR]

Chemotherapy, a mainstay modality for cancer, is often hindered by systemic toxicity and side effects. With the emergence of nanomedicine, the development of drug therapy has shifted toward targeted therapy. Hyaluronan (HA) is an ideal molecule as a targeted delivery system because many carcinomas overexpress HA receptors. We have conjugated resveratrol, a natural polyphenol, and 3-(5-methoxy, 2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MOMIPP), a chalcone, to HA with the goal of enhancing drug bioavailability and targeting triple negative breast cancers. We demonstrate the ability of HA conjugates to accumulate in the tumor interstitium within 6 h after tail vein injections. In vitro, these conjugates interact with their target receptors, which are overexpressed by triple negative breast cancer cells under static and physiological flow. These interactions result in enhanced uptake and efficacy of the therapeutic, as demonstrated by a reduced IC 50 over that of nonconjugated drugs. Thus, HA offers a platform to solubilize, target, and enhance the efficacy of chemotherapeutics.

Because many cancers harbor mutations that confer resistance to apoptosis, there is a need for therapeutic agents that can trigger alternative forms of cell death. Methuosis is a novel form of non-apoptotic cell death characterized by accumulation of vacuoles derived from macropinosomes and endosomes. Previous studies identified an indole-based chalcone, 3-(5-methoxy-2-methylindol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MOMIPP), that induces methuosis in human cancer cells. Herein, we describe the synthesis of related 2-indolyl substituted pyridinylpropenones and their effects on U251 glioblastoma cells. Increasing the size of the 2-indolyl substituent substantially reduces growth inhibitory activity and cytotoxicity, but does not prevent cell vacuolization. Computational models suggest that the results are not due to steric-driven conformational effects. The unexpected uncoupling of vacuolization and cell death implies that the relationship between endosomal perturbations and methuotic cell death is more complex than previously realized. The new series of compounds will be useful in further defining the molecular and cellular mechanisms underlying methuosis.

Methuosis is a novel caspase-independent form of cell death in which massive accumulation of vacuoles derived from macropinosomes ultimately causes cells to detach from the substratum and rupture. We recently described a chalcone-like compound, 3-(2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (i.e., MIPP), which can induce methuosis in glioblastoma and other types of cancer cells. Herein, we describe the synthesis and structure-activity relationships of a directed library of related compounds, providing insights into the contributions of the two aryl ring systems and highlighting a potent derivative, 3-(5-methoxy, 2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (i.e., MOMIPP) that can induce methuosis at low micromolar concentrations. We have also generated biologically active azide derivatives that may be useful for future studies aimed at identifying the protein targets of MOMIPP by photoaffinity labeling techniques. The potential significance of these studies is underscored by the finding that MOMIPP effectively reduces the growth and viability of Temozolomide-resistant glioblastoma and doxorubicin-resistant breast cancer cells. Thus, it may serve as a prototype for drugs that could be used to trigger death by methuosis in cancers that are resistant to conventional forms of cell death (e.g., apoptosis).

Ras GTPases are best known for their ability to serve as molecular switches regulating cell growth, differentiation and survival. Gene mutations that result in expression of constitutively active forms of Ras have been linked to oncogenesis in animal models and humans. However, over the past two decades, evidence has gradually accumulated to support a paradoxical role for Ras proteins in the initiation of cell death pathways. In this review we survey the literature pointing to the ability of activated Ras to promote cell death under conditions where cancer cells encounter apoptotic stimuli or Ras is ectopically expressed. In some of these cases Ras acts through known effectors and well defined apoptotic death pathways. However, in other cases it appears that Ras operates by triggering novel non-apoptotic death mechanisms that are just beginning to be characterized. Understanding these mechanisms and the factors that go into changing the nature of Ras signaling from pro-survival to pro-death could set the stage for development of novel therapeutic approaches aimed at manipulating pro-death Ras signaling pathways in cancer.

Methuosis is a unique form of nonapoptotic cell death triggered by alterations in the trafficking of clathrin-independent endosomes, ultimately leading to extreme vacuolization and rupture of the cell. Methuosis can be induced in glioblastoma cells by expression of constitutively active Ras. This study identifies the small GTPases, Rac1 and Arf6, and the Arf6 GTPase-activating protein, GIT1, as key downstream components of the signaling pathway underlying Ras-induced methuosis. The extent to which graded expression of active H-Ras(G12V) triggers cytoplasmic vacuolization correlates with the amount of endogenous Rac1 in the active GTP state. Blocking Rac1 activation with the specific Rac inhibitor, EHT 1864, or coexpression of dominant-negative Rac1(T17N), prevents the accumulation of vacuoles induced by H-Ras(G12V). Coincident with Rac1 activation, H-Ras(G12V) causes a decrease in the amount of active Arf6, a GTPase that functions in the recycling of clathrin-independent endosomes. The effect of H-Ras(G12V) on Arf6 is blocked by EHT 1864, indicating that the decrease in Arf6-GTP is directly linked to the activation of Rac1. Constitutively active Rac1(G12V) interacts with GIT1 in immunoprecipitation assays. Ablation of GIT1 by short hairpin RNA prevents the decrease in active Arf6, inhibits vacuolization, and prevents loss of cell viability in cells expressing Rac1(G12V). Together, the results suggest that perturbations of endosome morphology associated with Ras-induced methuosis are due to downstream activation of Rac1 combined with reciprocal inactivation of Arf6. The latter seems to be mediated through Rac1 stimulation of GIT1. Further insights into this pathway could suggest opportunities for the induction of methuosis in cancers that are resistant to apoptotic cell death.

Expression of activated Ras in glioblastoma cells induces accumulation of large phase-lucent cytoplasmic vacuoles, followed by cell death. This was previously described as autophagic cell death. However, unlike autophagosomes, the Ras-induced vacuoles are not bounded by a double membrane and do not sequester organelles or cytoplasm. Moreover, they are not acidic and do not contain the autophagosomal membrane protein LC3-II. Here we show that the vacuoles are enlarged macropinosomes. They rapidly incorporate extracellular fluid-phase tracers but do not sequester transferrin or the endosomal protein EEA1. Ultimately, the cells expressing activated Ras detach from the substratum and rupture, coincident with the displacement of cytoplasm with huge macropinosome-derived vacuoles. These changes are accompanied by caspase activation, but the broad-spectrum caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethylketone does not prevent cell death. Moreover, the majority of degenerating cells do not exhibit chromatin condensation typical of apoptosis. These observations provide evidence for a necrosis-like form of cell death initiated by dysregulation of macropinocytosis, which we have dubbed "methuosis." An activated form of the Rac1 GTPase induces a similar form of cell death, suggesting that Ras acts through Rac-dependent signaling pathways to hyperstimulate macropinocytosis in glioblastoma. Further study of these signaling pathways may lead to the identification of other chemical and physiologic triggers for this unusual form of cell death.