Autophagy and Angiogenesis Inhibition

Angiogenesis, the process by which new blood vessels are formed is critical for embryonic development and physiological functioning of normal tissues. Angiogenesis also plays a critical role in the pathology of many diseases including cancer, wherein the supply and demand for blood vessels determines the rate of cancer growth. A number of therapeutic strategies are being developed to inhibit pathological angiogenesis. Kringle domains of plasminogen such as kringle 5 (K5) and a proteolytic fragment of collagen type XVIII (endostatin) are well-characterized, potent angiogenesis inhibitors. These inhibitors activate different intracellular signaling pathways to induce apoptosis and inhibit cell proliferation. Recent studies from our group have shown that K5 and endostatin can also induce autophagy in addition to apoptosis in endothelial cells. A common feature of the two treatments was the upregulation of Beclin 1 levels leading to alterations in the Beclin 1-Bcl-2 complex. Angiogenesis inhibitor-induced autophagy in endothelial cells was independent of nutritional or hypoxic stress and initiated even in the presence of endothelial-specific survival factors such as vascular endothelial growth factor (VEGF). Interfering with the autophagic response by knocking down Beclin 1 levels dramatically increased apoptosis of endothelial cells. These findings identify the autophagic response as a novel target for enhancing the therapeutic efficacy of angiogenesis inhibitors.

Addendum to:

Kringle 5 of Human Plasminogen, an Angiogenesis Inhibitor, Induces Both Autophagy and Apoptotic Death in Endothelial Cells

T.M.B. Nguyen, I.V. Subramanian, A. Kelekar and S. Ramakrishnan

Blood 2007; 109:4793-802     

Mitotic Arrest in Tobacco Caused by the Phosphoprotein Phosphatase Inhibitor Okadaic Acid

Okadaic acid blocks the cell cycle at early mitosis in suspensioncultures of Nicotiana plumbaginifolia. Nuclear DNA content wasmeasured in treated cells by propidium iodide staining, fluorescencemicroscopy and quantitative analysis of the video image. NuclearDNA levels in inhibited populations showed that cells continuedto progress from G1 phase through S phase and accumulated inG2 phase. Arrested cells in 12 µM okadaic acid had a condensedchromatin configuration and persisting nucleolus similar tonormal early prophase. Progress to early prophase was also indicatedby development of the preprophase band (PPB) of microtubules.PPB microtubules disassembled in 95% of the inhibited cellswith the same timing as in control cells, although the treatedcells did not progress to prometaphase mitotic spindle assemblythat normally precedes PPB breakdown, therefore okadaic acidcan disrupt the normal dependence of PPB disassembly on prometaphasenuclear events and indicates that the normal signal for disassemblymay be an increase in phosphorylation of PPB associated proteins.Okadaic acid at 12 µM caused increased levels of phosphorylatedproteins, in particular those of 108 kDa, 49 kDa, 36 kDa, 33kDa, 31 kDa, but more complex effects on some phosphoproteinswere indicated by reductions in a phosphoprotein of 41 kDa andone of approximately 190 kDa. It is concluded that the mitoticphase of the plant cell cycle is more sensitive than precedingcycle phases to the disruption of protein phosphorylation levelsby okadaic acid and it is proposed that the inhibitor blocksdivision by interfering with essential changes in the phosphorylationstate of proteins at mitosis. This conclusion is discussed inrelation to genetical and biochemical evidence that proteinkinases and phosphatases are involved in the cell division ofplants and other eukaryotes. (Received November 26, 1991; Accepted April 20, 1992)     

Okadaic Acid-Induced Inhibition of B-50 Dephosphorylation by Presynaptic Membrane-Associated Protein Phosphatases

The neuronal tissue-specific protein kinase C (PKC) substrate B-50 can be dephosphorylated by endogenous protein phosphatases (PPs) in synaptic plasma membranes (SPMs). The present study characterizes membrane-associated B-50 phosphatase activity by using okadaic acid (OA) and purified 32P-labeled substrates. At a low concentration of [gamma-32P]ATP, PKC-mediated [32P]phosphate incorporation into B-50 in SPMs reached a maximal value at 30 s, followed by dephosphorylation. OA, added 30 s after the initiation of phosphorylation, partially prevented the dephosphorylation of B-50 at 2 nM, a dose that inhibits PP-2A. At the higher concentration of 1 microM, a dose of OA that inhibits PP-1 as well as PP-2A, a nearly complete blockade of B-50 dephosphorylation was seen. Heat-stable PP inhibitor-2 (I-2) also inhibited dephosphorylation of B-50. The effects of OA and I-2 on B-50 phosphatase activity were additive. Endogenous PP-1- and PP-2A-like activities in SPMs were also demonstrated by their capabilities of dephosphorylating [32P]phosphorylase a and [32P]casein. With these exogenous substrates, sensitivities of the membrane-bound phosphatases to OA and I-2 were found to be similar to those of purified forms of these enzymes. These results indicate that PP-1- and PP-2A-like enzymes are the major B-50 phosphatases in SPMs.     

Farnesylated RhoB Prevents Cell Cycle Arrest and Actin Cytoskeleton Disruption Caused by the Geranylgeranyltransferase I Inhibitor GGTI-298

Here we demonstrate that the geranylgeranyltransferase-I inhibitor GGTI-298 inhibits the RhoB pathway and disrupts stress fiber and focal adhesion formation in NIH-3T3 cells. Farnesylated V14RhoB-CAIM (resistant to GGTI-298), but not geranylgeranylated V14RhoB (-CLLL), prevented inhibition of actin stress fiber and focal adhesion formation, underlining the critical role of RhoB. In contrast, farnesylated, V14RhoA (-CVLS) was unable to prevent effects of GGTI 298 on cytoskeleton organization. Furthermore, the ability of GGTI-298 to induce p21WAF and to block cells in the G0/G1 phase of the cell cycle was also prevented by farnesylated V14RhoB but not by farnesylated V14RhoA. Moreover, treatment with GGTI-298 of cells expressing farnesylated RhoB results in accumulation of these cells in the G2/M phase. Therefore, the RhoB pathway is a critical target of GGTI-298.     

Disruption of Autophagy Results in Constitutive Oxidative Stress in Arabidopsis

Plant cells frequently encounter oxidative stress, leading to oxidative damage and inactivation of proteins. We have recently demonstrated that oxidative stress induces autophagy in Arabidopsis seedlings in an AtATG18a-dependent manner and that RNAi-AtATG18a transgenic lines, which are defective in autophagosome formation, are hypersensitive to reactive oxygen species. Analysis of protein oxidation indicated that oxidized proteins are degraded in the vacuole after uptake by autophagy, and this degradation is impaired in RNAi-AtATG18a lines. Our results also suggest that in the absence of a functional autophagy pathway, plants are under increased oxidative stress, even under normal growth conditions.

Addendum to:

Degradation of Oxidized Proteins by Autophagy during Oxidative Stress in Arabidopsis

Y. Xiong, A.L. Contento, N.Q. Phan and D.C. Bassham

Plant Physiol 2007; 143:291-9     

Inhibition of Autophagy by Chloroquine Enhances the Antitumor Efficacy of Sorafenib in Glioblastoma

Glioblastoma multiforme (GBM) is the most aggressive and common brain tumor in adults. Sorafenib, a multi-kinase inhibitor, has been shown to inhibit cell proliferation and induce apoptosis through inhibition of STAT3 signaling in glioblastoma cells and in intracranial gliomas. However, sorafenib also induces cell autophagy. Due to the dual roles of autophagy in tumor cell survival and death, the therapeutic effect of sorafenib on glioblastoma is uncertain. Here, we combined sorafenib treatment in GBM cells (U373 and LN229) and tumors with the autophagy inhibitor chloroquine. We found that blockage of autophagy further inhibited cell proliferation and migration and induced cell apoptosis in vitro and in vivo. These findings suggest the possibility of combination treatment with sorafenib and autophagy inhibitors for GBM.     

Induction of Autophagy by Amino Acid Starvation in Fish Cells

Autophagy is well established as a starvation-induced process in yeast and mammalian cells and tissues. To elucidate the cellular mechanisms induced by starvation in fish, we characterized the induction of autophagy in cultured zebrafish cells under starvation conditions. As an autophagic marker protein, the microtubule-associated protein 1-light chain 3B protein (MAP1-LC3B) was cloned from the fish cells, and its expression and localization were characterized. In zebrafish embryonic (ZE) cells, posttranslational modifications produced two distinct forms of MAP1-LC3B, i.e., a cytosolic form and a membrane-bound form (types I and II, respectively). Immunofluorescence microscopy revealed fluorescently labeled autophagosomes in cells stably transfected with a green fluorescent protein (GFP)–MAP1-LC3B fusion protein and showed that this protein accumulated in punctate dots in a time-dependent manner in response to amino acid starvation. Starvation also induced the degradation of long-lived proteins. Treatment with 3-methyladenine and wortmannin, two class-III inhibitors of phosphoinositide 3-kinase (PI3K), repressed autophagy under starvation conditions, indicating that the PI3K class-III pathway regulates starvation-induced autophagy in fish.     

Okadaic Acid Induces Cycloheximide and Caspase Sensitive Apoptosis in Immature Neurons

Previous studies have shown that okadaic acid (OA) evokes tau phosphorylation and neurofibrillary changes in vivo, and in cultured neurons, that resemble Alzheimers disease pathogenesis. In order to investigate the mechanism of OA-neurotoxicity, we treated cultured rat neurons with OA and examined nuclear morphology, phosphatidylserine (PS) externalization, -fodrin cleavage, and the effects of cell death inhibitors. Our results demonstrated that cycloheximide (CHX) and the broad-spectrum caspase inhibitor, ZVAD, significantly reduced cell death in a dose-dependent manner. Nuclear fragmentation, a hallmark of apoptosis, occurred after OA treatment and was inhibited by CHX or ZVAD. PS externalization was apparent in 6–12 h in neurites and in cell bodies, and peaked at 24 h after OA treatment. Cleavage of -fodrin as visualized by the appearance of 150- and 120-kDa bands appeared with a time course similar to PS externalization. These results suggest that OA induce CHX and caspase sensitive neuronal apoptosis.     

Neurotoxic and Synaptic Effects of Okadaic Acid,an Inhibitor of Protein Phosphatases

Protein phosphorylation and dephosphorylation reactions, catalyzed by kinases and phosphatases, are involved in the regulation of a wide variety of physiological processes. In the nervous system, such reactions seem to modulate the function of several proteins crucial in synaptic transmission, including voltage-gated and ligand-gated channels, neurotransmitter release, and neurotransmitter transporters. On the other hand, hyperphosphorylation of certain cytoskeletal proteins or receptors may lead to neuronal death. In the present work we review the neurotoxic effect of okadaic acid (OKA), a potent and specific inhibitor of the serine/threonine protein phosphatases 1 and 2A, as well as its action on synaptic function. We analyze recent findings demonstrating that the microinjection of OKA in rat hippocampus induces neuronal stress, hyperexcitation and neurodegeneration, and discuss their possible relationships to alterations of protein phosphorylation-dephosphorylation observed in Alzheimer's disease brain. These results suggest that protein hyperphosphorylation due to inhibition of phosphatases in vivo induces neuronal stress and subsequent neurodegeneration.     

Okadaic Acid and Cultured Frog Sciatic Nerves: Potent Inhibition of Axonal Regeneration in Spite of Unaffected Schwann Cell Proliferation and Ganglionic Protein Synthesis

Abstract: Okadaic acid (OA) is a frequently used phosphatase inhibitor that by inhibiting dephosphorylation increases the net phosphorylation level in various systems. In the present study OA was used to assess the role of balanced phosphorylation-dephosphorylation reactions for successful regeneration of peripheral nerves. To achieve this, the effects of OA on phosphorylation levels, neurite outgrowth, injury-induced support cell proliferation, and neurofilament stability, respectively, were investigated in the in vitro regenerating, adult frog sciatic sensory nerve. OA at a moderate concentration (20 n M ) increased phosphorylation levels and almost completely inhibited the in vitro regeneration in a reversible way. The effect on regeneration was not due to induced neurofilament instability and was only seen when the drug was applied in the outgrowth region. The latter and the absence of effects on support cell proliferation indicate that OA acts locally at the level of newly formed axons. However, the inhibition of regeneration was not a consequence of reduced delivery of proteins by axonal transport, because this process in fact was increased by OA. Altogether, the study suggests that properly balanced phosphorylating-dephosphorylating reactions are critical for regeneration of peripheral nerves.     

Myxoma Virus Oncolytic Efficiency Can Be Enhanced Through Chemical or Genetic Disruption of the Actin Cytoskeleton

Myxoma virus (MYXV) is one of many animal viruses that exhibit oncolytic properties in transformed human cells. Compared to orthopoxviruses like vaccinia (VACV), MYXV spreads inefficiently, which could compromise its use in treating tumors and their associated metastases. The VACV F11 protein promotes virus exit and rapid spread by inhibiting Rho signalling, which results in a disruption of cortical actin. We have previously shown that although MYXV lacks an F11 homolog, the F11L gene can be introduced into MYXV promoting the spread of this Leporipoxvirus in natural host cells. Here we show that the F11-encoding (F11L⁺) MYXV strain replicates to higher levels in a number of human cancer cells. We also show that F11L⁺ MYXV induces better tumor control and prolonged survival of mice bearing MDA-MB-231 cancer cells. Furthermore, we show that this virus also spreads more efficiently from the site of growth in one injected tumor, to a second untreated tumor.While we focused mostly on the use of a modified MYXV we were able to show that the effects of F11 on MYXV growth in cancer cells could be mimicked through the use of pharmacological inhibition or siRNA-mediated silencing of key regulators of cortical actin (RhoA, RhoC, mDia1, or LIMK2). These data suggest that it may be possible to increase the oncolytic efficacy of wild-type MYXV using chemical inhibitors of RhoA/C or their downstream targets. Furthermore, since all viruses must overcome barriers to exit posed by structures like cortical actin, these findings suggest that the oncolytic activity of other viruses may be enhanced through similar strategies.     

Inhibition of Bacterial Conjugation by Ribonucleic Acid and Deoxyribonucleic Acid Male-Specific Bacteriophages

Both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) male-specific phages, with an F-specific host range, inhibited the bacterial mating process of Escherichia coli. DNA phages prevented the formation of mating pairs but had no effect on mating pairs once they were formed. A step in RNA phage infection, prior to RNA penetration, prevented the formation of mating pairs and, in addition, prevented a fraction of existing mating pairs from completing the mating process. These findings are compatible with the hypothesis that donor cells have a single surface structure involved in both conjugation and male-phage adsorption and that this element is the F pilus.     

Inhibition of ethylene biosynthesis by salicylic Acid

Salicylic acid inhibited ethylene formation from ACC in self-buffered (pH 3.8) pear (Pyrus communis) cell suspension cultures with a K₁^app of about 10 micromolar after 1 to 3 hours incubation. Inhibition appeared noncompetitive. Among 22 related phenolic compounds tested, only acetylsalicylic acid showed similar levels of inhibition. Inhibition by salicylic acid was inversely dependent on the pH of the culture medium and did not require a continuous external supply of salicylate. When compared to known inhibitors of the ethylene forming enzyme, cobalt, n-propyl gallate, and dinitrophenol, inhibition by salicylic acid most closely resembled that by dinitrophenol but salicylic acid did not produce the same degree of respiratory stimulation. Results are discussed in terms of other known effects of salicylic acid on plants, pH-dependency, and the possible influence of salicylic acid on electron transport.     

Studies of the Inhibition of Stomatal Opening by Naphth-1-ylacetic Acid and Abscisic Acid

Evidence is presented which confirms the inhibitory action ofthe synthetic auxin, NAA, on stomatal opening. In contrast tothe natural auxin IAA, NAA exerts effects which, in some respects,resemble those of ABA. The mode of action of NAA on the guardcells is, however, thought to be different from that of ABA.When NAA and ABA are applied together, their combined actionresults in a greater reduction in stomatal aperture than wouldbe predicted from their separate effects. Key words: NAA, ABA, IAA Stomata, Commelina