Investigating the Potential to Deliver and Maintain Plasma and Brain Levels of a Novel Practically Insoluble Methuosis Inducing Anticancer Agent 5-Methoxy MOMIPP Through an Injectable In Situ Forming Thermoresponsive Hydrogel Formulation.

A new indole based chalcone molecule MOMIPP induced methuosis mediated cell death in gliobastoma and other cancer cell lines. But the drug was insoluble in water and had a very short plasma half-life. The purpose of this work was to develop a formulation that can provide sustained levels of MOMIPP in vivo. Initial studies established drug solubility in various solvents. N-methyl pyrrolidone (NMP) was determined as an excellent solvent for the drug. Subsequently a poloxamer-407 based thermoreversible gel containing NMP was used to develop the formulation. Rheological studies were performed via oscillatory temperature mode, continuous shear analysis, and oscillatory frequency mode experiments. The mechanical properties of the formulations were tested using a texture profile analyzer. The gelation temperature and time of formulations increased with increasing amounts of NMP. However, the viscosity at 20 °C and storage modulus decreased as the amount of NMP increased. Characterization studies helped to identify the gel formulation that was used to administer the drug orally, sub-cutaneously, and intra-peritoneally. When the gel was given intraperitoneally the target plasma and brain levels of over 5 µM was maintained for about 8 h. Thus, a thermoreversible gel formulation that can deliver MOMIPP in animal studies was successfully developed.

Dysregulation of Macropinocytosis Processes in Glioblastomas May Be Exploited to Increase Intracellular Anti-Cancer Drug Levels: The Example of Temozolomide.

Macropinocytosis is a clathrin-independent endocytosis of extracellular fluid that may contribute to cancer aggressiveness through nutrient supply, recycling of plasma membrane and receptors, and exosome internalization. Macropinocytosis may be notably triggered by epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR), two well-known markers for glioblastoma aggressiveness. Therefore, we studied whether the expression of key actors of macropinocytosis is modified in human glioma datasets. Strong deregulation has been evidenced at the mRNA level according to the grade of the tumor, and 38 macropinocytosis-related gene signatures allowed discrimination of the glioblastoma (GBM) samples. Honokiol-induced vacuolization was then compared to vacquinol-1 and MOMIPP, two known macropinocytosis inducers. Despite high phase-contrast morphological similarities, honokiol-induced vacuoles appeared to originate from both endocytosis and ER. Also, acridine orange staining suggested differences in the macropinosomes' fate: their fusion with lysosomes appeared very limited in 3-(5-methoxy -2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MOMIPP)-treated cells. Nevertheless, each of the compounds markedly increased temozolomide uptake by glioma cells, as evidenced by LC-MS. In conclusion, the observed deregulation of macropinocytosis in GBM makes them prone to respond to various compounds affecting their formation and/or intracellular fate. Considering that sustained macropinocytosis may also trigger cell death of both sensitive and resistant GBM cells, we propose to envisage macropinocytosis inducers in combination approaches to obtain dual benefits: increased drug uptake and additive/synergistic effects.

The JNK signaling pathway plays a key role in methuosis (non-apoptotic cell death) induced by MOMIPP in glioblastoma.

BACKGROUND: Synthetic indolyl- pyridinyl- propenones (IPPs) induce methuosis, a form of non-apoptotic cell death, in glioblastoma and other cancer cell lines. Methuosis is characterized by accumulation of cytoplasmic vacuoles 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 key 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 in a therapeutic context, we quantified the distribution of our lead IPP compound, MOMIPP, in mouse plasma and brain, 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 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 offers 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 members of the Bcl-2 family represent key events in the methuosis death process. In addition to providing new 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 therapeutic agents for brain tumors.

6-MOMIPP, a novel brain-penetrant anti-mitotic indolyl-chalcone, inhibits glioblastoma growth and viability.

PURPOSE: 3-(6-Methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one (6-MOMIPP) is a novel indole-based chalcone that disrupts microtubules. The present study aims to define the mechanism through which 6-MOMIPP induces cell death and to evaluate the efficacy of the compound in penetrating the blood-brain barrier and inhibiting growth of glioblastoma xenografts. METHODS: The effects of 6-MOMIPP were evaluated in cultured U251 glioblastoma cells, using viability, flow cytometry, and tubulin polymerization assays. Scintillation proximity and tubulin crosslinking methods were used to identify the binding site of 6-MOMIPP on tubulin, and western blots were performed to define the signaling pathways that contribute to cell death. LC/MS assays were used to study the pharmacokinetic behavior of 6-MOMIPP in mice. Subcutaneous and intracerebral xenograft models were utilized to assess the effects of 6-MOMIPP on growth of U251 glioblastoma in vivo. RESULTS: The findings indicate that 6-MOMIPP targets the colchicine site on ß-tubulin. At concentrations ≥ 250 nm, 6-MOMIPP induces mitotic arrest, caspase activation and loss of cell viability. Cells are protected by caspase inhibitors, pointing to an apoptotic mechanism of cell death. Loss of cell viability is preceded by activation of Cdk1(Cdc2) and phosphorylation of Bcl-2 and Bcl-xL. Inhibition of both events with a Cdk1 inhibitor prevents cell death. 6-MOMIPP has broad activity against the viability of multiple glioblastoma, melanoma and lung carcinoma cell lines. Viability of normal cells, including differentiated neurons, is not significantly affected at a drug concentration (1 µM) that reduces viability in most cancer lines. Pharmacokinetic studies in mice show that concentrations of 6-MOMIPP in the brain mirror those in the plasma, indicating that 6-MOMIPP readily penetrates the blood-brain barrier. Studies with mice bearing human U251 glioblastoma xenografts demonstrate that 6-MOMIPP is effective in suppressing growth of subcutaneous and intracerebral tumors without causing general toxicity. CONCLUSIONS: The results indicate that 6-MOMIPP is a novel microtubule disruptor that targets the colchicine binding site on ß-tubulin to induce mitotic arrest and cell death. The ability of 6-MOMIPP to penetrate the blood-brain barrier and inhibit growth of glioblastoma xenografts suggests that it warrants further preclinical evaluation as potential small-molecule therapeutic that may have advantages in treating primary and metastatic brain tumors.

KRAS mutant allele-specific expression knockdown in pancreatic cancer model with systemically delivered bi-shRNA KRAS lipoplex.

The KRAS oncogene, present in over 90% of pancreatic ductal adenocarcinomas, is most frequently the result of one of three gain-of-function substitution mutations of codon 12 glycine. Thus far, RAS mutations have been clinically refractory to both direct and selective inhibition by systemic therapeutics. This report presents the results of pre-clinical assessment of a lipoplex comprising a plasmid-encoded, modular bi-functional shRNA (bi-shRNA), which executes selective and multi-mutant allelic KRASG12mut gene silencing, encased within a fusogenic liposome systemic delivery vehicle. Using both a dual luciferase reporter system and a Restriction Fragment Length Polymorphism (RFLP) assay, selective discrimination of KRASG12mut from KRASwt was confirmed in vitro in PANC1 cells. Subsequently, systemic administration of the bi-shRNAKRAS fusogenic lipoplex into female athymic Nu/Nu mice bearing PANC1 xenografts demonstrated intratumoral plasmid delivery, KRASG12mut knockdown, and inhibition of tumor growth, without adverse effect. Clinical trials with the bi-shRNA lipoplex have been implemented.

Vacuole-inducing compounds that disrupt endolysosomal trafficking stimulate production of exosomes by glioblastoma cells.

Exosomes are produced from mammalian cells when multivesicular endosomes fuse with the plasma membrane, releasing their intralumenal vesicles. In this study we assessed the effects of MOPIPP, a novel indole-based chalcone, and vacuolin-1, a distinct triazine-based compound, on exosome production in cultured glioblastoma and 293T cells. Both compounds promote vacuolization of late endosome compartments and interfere with trafficking of late endosomes to lysosomes, without significant cytotoxicity. The results show that vacuolated cells treated with these compounds release exosomes with morphologies similar to untreated controls. However, both compounds trigger multi-fold increases in release of exosome marker proteins (e.g., CD63, Alix) in exosome fractions collected from equivalent numbers of cells. Despite the marked increase in exosome production, the profiles of selected miRNA cargoes carried by the exosomes were generally similar in cells treated with the compounds. Insofar as MOPIPP and vacuolin-1 seem able to increase the overall yield of exosomes from cultured cells, they might be useful for efforts to develop exosome-based therapeutics.

Receptor-Mediated Attachment and Uptake of Hyaluronan Conjugates by Breast Cancer Cells.

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 IC50 over that of nonconjugated drugs. Thus, HA offers a platform to solubilize, target, and enhance the efficacy of chemotherapeutics.

Disruption of endolysosomal trafficking pathways in glioma cells by methuosis-inducing indole-based chalcones.

Methuosis is a form of non-apoptotic cell death involving massive vacuolization of macropinosome-derived endocytic compartments, followed by a decline in metabolic activity and loss of membrane integrity. To explore the induction of methuosis as a potential therapeutic strategy for killing cancer cells, we have developed small molecules (indole-based chalcones) that trigger this form of cell death in glioblastoma and other cancer cell lines. Here, we report that in addition to causing fusion and expansion of macropinosome compartments, the lead compound, 3-(5-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MOMIPP), disrupts vesicular trafficking at the lysosomal nexus, manifested by impaired degradation of EGF and LDL receptors, defective processing of procathepsins, and accumulation of autophagosomes. In contrast, secretion of the ectodomain derived from a prototypical type-I membrane glycoprotein, ß-amyloid precursor protein, is increased rather than diminished. A closely related MOMIPP analog, which causes substantial vacuolization without reducing cell viability, also impedes cathepsin processing and autophagic flux, but has more modest effects on receptor degradation. A third analog, which causes neither vacuolization nor loss of viability, has no effect on endolysosomal trafficking. The results suggest that differential cytotoxicity of structurally similar indole-based chalcones is related, at least in part, to the severity of their effects on endolysosomal trafficking pathways.

Synthesis and biological evaluation of isomeric methoxy substitutions on anti-cancer indolyl-pyridinyl-propenones: Effects on potency and mode of activity.

Certain indolyl-pyridinyl-propenone analogues kill glioblastoma cells that have become resistant to conventional therapeutic drugs. Some of these analogues induce a novel form of non-apoptotic cell death called methuosis, while others primarily cause microtubule disruption. Ready access to 5-indole substitution has allowed characterization of this position to be important for both types of mechanisms when a simple methoxy group is present. We now report the syntheses and biological effects of isomeric methoxy substitutions on the indole ring. Additionally, analogues containing a trimethoxyphenyl group in place of the pyridinyl moiety were evaluated for anticancer activity. The results demonstrate that the location of the methoxy group can alter both the potency and the mechanism of cell death. Remarkably, changing the methoxy from the 5-position to the 6-position switched the biological activity from induction of methuosis to disruption of microtubules. The latter may represent a prototype for a new class of mitotic inhibitors with potential therapeutic utility.

Non-apoptotic cell death associated with perturbations of macropinocytosis.

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.

Synthesis and biological evaluation of indolyl-pyridinyl-propenones having either methuosis or microtubule disruption activity.

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.

Methuosis: nonapoptotic cell death associated with vacuolization of macropinosome and endosome compartments.

Apoptosis is the most widely recognized form of physiological programmed cell death. During the past three decades, various nonapoptotic forms of cell death have gained increasing attention, largely because of their potential importance in pathological processes, toxicology, and cancer therapy. A recent addition to the panoply of cell death phenotypes is methuosis. The neologism is derived from the Greek methuo (to drink to intoxication) because the hallmark of this form of cell death is displacement of the cytoplasm by large fluid-filled vacuoles derived from macropinosomes. The demise of the cell resembles many forms of necrosis, insofar as there is a loss of metabolic capacity and plasma membrane integrity, without the cell shrinkage and nuclear fragmentation associated with apoptosis. Methuosis was initially defined in glioblastoma cells after ectopic expression of activated Ras, but recent reports have described small molecules that can induce the features of methuosis in a broad spectrum of cancer cells, including those that are resistant to conventional apoptosis-inducing drugs. This review summarizes the available information about the distinguishing morphological characteristics and underlying mechanisms of methuosis. We compare and contrast methuosis with other cytopathological conditions in which accumulation of clear cytoplasmic vacuoles is a prominent feature. Finally, we highlight key questions that need to be answered to determine whether methuosis truly represents a unique form of regulated cell death.

Differential Induction of Cytoplasmic Vacuolization and Methuosis by Novel 2-Indolyl-Substituted Pyridinylpropenones.

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.

Synthesis and evaluation of indole-based chalcones as inducers of methuosis, a novel type of nonapoptotic cell death.

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).

A switch between cytoprotective and cytotoxic autophagy in the radiosensitization of breast tumor cells by chloroquine and vitamin D.

Calcitriol or 1,25-dihydroxyvitamin D3, the hormonally active form of vitamin D, as well as vitamin D analogs, has been shown to increase sensitivity to ionizing radiation in breast tumor cells. The current studies indicate that the combination of 1,25-dihydroxyvitamin D3 with radiation appears to kill p53 wild-type, estrogen receptor-positive ZR-75-1 breast tumor cells through autophagy. Minimal apoptosis was observed based on cell morphology by DAPI and TUNEL staining, annexin/PI analysis, caspase-3, and PARP cleavage as well as cell cycle analysis. Induction of autophagy was indicated by increased acridine orange staining, RFP-LC3 redistribution, and detection of autophagic vesicles by electron microscopy, while autophagic flux was monitored based on p62 degradation. The autophagy inhibitors, chloroquine and bafilomycin A1, as well as genetic suppression of the autophagic signaling proteins Atg5 or Atg 7 attenuated the impact of the combination treatment of 1,25 D3 with radiation. In contrast to autophagy mediating the effects of the combination treatment, the autophagy induced by radiation alone was apparently cytoprotective in that either pharmacological or genetic inhibition increased sensitivity to radiation. These studies support the potential utility of vitamin D for improving the impact of radiation for breast cancer therapy, support the feasibility of combining chloroquine with radiation for the treatment of breast cancer, and demonstrate the existence of an "autophagic switch" from cytoprotective autophagy with radiation alone to cytotoxic autophagy with the 1,25 D3-radiation combination.

A chalcone-related small molecule that induces methuosis, a novel form of non-apoptotic cell death, in glioblastoma cells.

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.

Death pathways triggered by activated Ras in cancer cells.

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.

Induction of nonapoptotic cell death by activated Ras requires inverse regulation of Rac1 and Arf6.

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.

Killing of cancer cells by the photoactivatable protein kinase C inhibitor, calphostin C, involves induction of endoplasmic reticulum stress.

Calphostin C (cal-C) is a photoactivatable inhibitor that binds to the regulatory domain of protein kinase C (PKC) and to other proteins that contain diacylglycerol/phorbol ester binding sites. Cal-C is cytotoxic against many types of cancer cells, yet the basis for this activity remains poorly understood. Here, we show that one of the earliest effects of cal-C is an impairment of glycoprotein export from the endoplasmic reticulum (ER), accompanied by formation of ER-derived vacuoles. Vacuolization of the ER is correlated with induction of an ER stress response that includes activation of c-Jun N-terminal kinase and protein kinase R-like ER kinase, as well as increased expression of CCAAT/enhancer binding protein homologous transcription factor (CHOP; GADD153). These effects of cal-C are not mimicked by staurosporine, an inhibitor of PKC catalytic activity, indicating that ER stress is due to interaction of cal-C with targets other than PKC. In conjunction with the induction of ER stress, breast carcinoma cells undergo caspase-dependent cell death with early activation of caspases 9 and 7 and cleavage of poly(ADP-ribose)polymerase. Reduction of CHOP expression by short hairpin RNA decreases the sensitivity of the cells to cal-C, suggesting that induction of apoptosis by cal-C is related, at least in part, to ER stress triggered by disruption of ER morphology and transport function. Antineoplastic drugs that work by inducting ER stress have shown promise in preclinical and clinical trials. Thus, the present findings raise the possibility that cal-C may be useful for photodynamic therapy based on induction of ER stress in some forms of cancer.

MYCN promotes the expansion of Phox2B-positive neuronal progenitors to drive neuroblastoma development.

Amplification of the oncogene MYCN is a tumorigenic event in the development of a subset of neuroblastomas that commonly consist of undifferentiated or poorly differentiated neuroblasts with unfavorable clinical outcome. The cellular origin of these neuroblasts is unknown. Additionally, the cellular functions and target cells of MYCN in neuroblastoma development remain undefined. Here we examine the cell types that drive neuroblastoma development in TH-MYCN transgenic mice, an animal model of the human disease. Neuroblastoma development in these mice begins with hyperplastic lesions in early postnatal sympathetic ganglia. We show that both hyperplasia and primary tumors are composed predominantly of highly proliferative Phox2B(+) neuronal progenitors. MYCN induces the expansion of these progenitors by both promoting their proliferation and preventing their differentiation. We further identify a minor population of undifferentiated nestin(+) cells in both hyperplastic lesions and primary tumors that may serve as precursors of Phox2B(+) neuronal progenitors. These findings establish the identity of neuroblasts that characterize the tumor phenotype and suggest a cellular pathway by which MYCN can promote neuroblastoma development.