Yaakov Egozi

Selective Inhibition of Ras-dependent Cell Growth by Farnesylthiosalisylic Acid

S-trans,trans-Farnesylthiosalicylic acid (FTS) is a novel farnesylated rigid carboxylic acid derivative. In cell-free systems, it acts as a potent competitive inhibitor (K = 2.6 μM) of the enzyme prenylated protein methyltransferase (PPMTase), which methylates the carboxyl-terminal S-prenylcysteine in a large number of prenylated proteins including Ras. In such systems, FTS inhibits Ras methylation but not Ras farnesylation. Inhibition of the PPMTase by FTS in homogenates or membranes of a variety of tissues and cell lines is inferred from a block in the methylation of exogenously added substrates such as N-acetyl-S-trans,trans-farnesyl-L-cysteine and of endogenous substrates including small GTP-binding proteins. FTS can also inhibit methylation of these proteins in intact cells (e.g. in Rat-1 fibroblasts, Ras-transformed Rat-1, and B16 melanoma cells). Unlike in cell-free systems, however, relatively high concentrations of FTS (50-100 μM) are required for partial blocking (10-40%) of protein methylation in the intact cells. Thus, FTS is a weak inhibitor of methylation in intact cells. Because methylation is the last step in the processing of Ras and related proteins, FTS is not likely to affect steps that precede it, e.g. protein prenylation. This may explain why the growth and gross morphology of a variety of cultured cell types (including Chinese hamster ovary, NIH3T3, Rat1, B16 melanoma, and PC12) is not affected by up to 25 μM FTS and is consistent with the observed lack of FTS-induced cytotoxicity. Nevertheless, FTS reduces the levels of Ras in cell membranes and can inhibit Ras-dependent cell growth in vitro, independently of methylation. It inhibits the growth of human Ha-ras-transformed cells (EJ cells) and reverses their transformed morphology in a dose-dependent manner (0.1-10 μM). The drug does not interfere with the growth of cells transformed by v-Raf or T-antigen but inhibits the growth of ErbB2-transformed cells and blocks the mitogenic effects of epidermal and basic fibroblast growth factors, thus implying its selectivity toward Ras growth signaling, possibly via modulation of Ras-Raf communication. Taken together, the results raise the possibility that FTS may specifically interfere with the interaction of Ras with a farnesylcysteine recognition domain in the cell membrane. This drug, and perhaps other farnesylated rigid carboxylic acid analogs, may be used for in vitro characterization of the PPMTase and for the identification of a putative Ras farnesylcysteine recognition domain in cell membranes.

A new functional Ras antagonist inhibits human pancreatic tumor growth in nude mice

Constitutively active Ras proteins, their regulatory components, and overexpressed tyrosine kinase receptors that activate Ras, are frequently associated with cell transformation in human tumors. This suggests that functional Ras antagonists may have anti-tumor activity. Studies in rodent fibroblasts have shown that S-trans, transfarnesylthiosalicylic acid (FTS) acts as a rather specific nontoxic Ras antagonist, dislodging Ras from its membrane anchorage domains and accelerating its degradation. FTS is not a farnesyltransferase inhibitor, and does not affect Ras maturation. Here we demonstrate that FTS also acts as a functional Ras antagonist in human pancreatic cell lines that express activated K-Ras (Panc-1 and MiaPaCa-2). In Panc-1 cells, FTS at a concentration of 25 – 100 μM reduced the amount of Ras in a dose-dependent manner and interfered with serum-dependent and epidermal growth factor-stimulated ERK activation, thus inhibiting both anchorage-dependent and anchorage-independent growth of Panc-1 cells in vitro. FTS also inhibited tumor growth in Panc-1 xenografted nude mice, apparently without systemic toxicity. Daily FTS treatment (5 mg/kg intraperitoneally) in mice with tumors (mean volume 0.07 cm3) markedly decreased tumor growth (after treatment for 18 days, tumor volume had increased by only 23±30-fold in the FTS-treated group and by 127±66-fold in controls). These findings suggest that FTS represents a new class of functional Ras antagonists with potential therapeutic value.

On the interaction of drugs with the cholinergic nervous system--V. Characterization of some effects induced by physostigmine in mice: in vivo and in vitro studies.

Abstract Four systemic effects induced by s.c. injection of physosthmine and neostigmine to mice were investigated by the “quadro-test” procedure, a novel design in which salivation, tremor, hypothermia and the rotarod-effects are simultaneously and continuously recorded. The time profiles of these effects were used to construct dose-response curves, which were compared to those of brain acetylcholinesterase (AcChE) inhibition by the drug. A good relationship was found between the dose and time dependency of these pharmacological and biochemical parameters, as well as between the relative potencies of physostigmine and neostigmine and their affinity to the enzyme. Using scopolamine. Hbr and its quaternary analogue, as well as tertiary cholinergic agonists and other cholinesterase agents, it was found that the physostigmine-induced hypothermia is mostly a central-muscarinic response, while the tremor is most probably a non-muscarinic peripheral effect. The peripheral-muscarinic origin of the salivation was confirmed. The effects measured in the rotarod test were found to be muscarinic and mixed central-peripheral for physostigmine, and non-muscarinic for neostigmine. The lethality caused by physostigmine seems to be partially centrally mediated, while that of neostigmine is peripheral.

Growth inhibition of ras-dependent tumors in nude mice by a potent ras-dislodging antagonist

A lipophilic farnesyl moiety attached to the carboxyl terminal cysteine of ras proteins structurally supports their membrane anchorage, required for ras‐dependent growth‐factor signaling and for transforming activity of ras oncoproteins. It has been shown that inhibition of ras farnesylation can block tumor growth in nude mice but that some ras‐dependent tumors escape such blockage as a result of prenylation of ras. S‐trans‐transfarnesylthiosalicylic acid (FTS) is a potent ras‐dislodging antagonist that does not affect ras prenylation but rather acts on the mature, membrane‐bound ras and facilitates its degradation. Here we demonstrate that FTS induces reappearance of stress fibers in H‐ras‐transformed rat‐1 cells (EJ cells) in vitro, inhibits their anchorage‐independent growth in vitro, and blocks EJ‐tumor growth in nude mice. The anchorage‐independent growth of cells expressing ErbB2 (B104), but not that of v‐raf‐transformed cells, is also inhibited by FTS, suggesting specificity towards activated ras. FTS treatment (5 mg/kg i.p. daily) caused inhibition (75–80%) of tumor growth in nude mice implanted with EJ, but not in mice implanted with v‐raf‐transformed cells, with no evidence of systemic toxicity. Moreover, FTS treatment increased the survival rate of EJ‐tumor‐bearing mice from 48 to 68 days. Here we demonstrate anti‐tumor potency in a synthetic, non‐toxic, ras‐dislodging antagonist acting independently of farnesyltransferases. Int. J. Cancer 80:911–918, 1999.

Central muscarinic receptor degeneration following 6-hydroxydopamine lesion in mice

Abstract Adult mice received two 70 μg doses of 6-hydroxydopamine intracisternally 72 hours apart, and the muscarinic binding properties of discrete brain regions were then investigated at various time intervals. Three days after the second injection, 3 H-norepinephrine uptake was drastically reduced in all brain regions studied, and a distinct decrease in muscarinic receptor density was observed in the striatum (−18%), medulla-pons (−17%) and cerebellum (−15%) of lesioned animals as compared with controls. No changes were detected in muscarinic receptor density in the cortex or the hippocampus of treated animals, nor were any changes seen in the affinity of the labelled ligand for its receptor or in the displacement properties of the muscarinic binding by agonists in any of the regions studied. These effects still persisted after 60 days, with a further reduction in striatal muscarinic density to 74% of control values. Data are interpreted with respect to the proposed model for cholinergic modulation of central catecholamine release and cholinergic-catecholaminergic interactions in the striatum.

On the interaction of drugs with the cholinergic nervous system--IV. Tolerance to oxotremorine in mice: in vivo and in vitro studies.

Abstract A new procedure was used to follow continuously and simultaneously four systemic effects induced by oxotremorine in mice: salivation, tremor, hypothermia and those measured in the rotarod test. Using an equipotent dose of methoxotremorine, it was found that apart from salivation, the other systemic effects are centrally originated. By comparing the complete dose-response curves for these systemic effects in naive and tolerant mice, it was found that: (1) Salivation is the most sensitive effect in naive mice; (2) All curves shift in parallel to the right on continuous exposure to oxotremorine, in a manner that seems to be dose-dependent; (3) the tolerance to oxotremorine is reversible. Since oxotremorine-tolerant mice were found to be cross-tolerant to various tertiary anti-cholinesterase agents and cholinergic agonists, the involvement of the muscarinic receptor in the tolerance to oxotremorine was investigated, using two different approaches. (1) Continuous blockade of the receptor by scopolamine in vivo prevented tolerance development to oxotremorine; this effect was found to depend on the time of scopolamine administration relative to oxotremorine injection, and on the systemic effect measured. (2) The amount of receptor and its affinity towards a specific ligand were determined in vitro ; no significant differences were found between naive and oxotremorine-tolerant animals. The significance of these results in the elucidation of possible tolerance mechanisms is discussed.

Acetylcholine rhythm in the preoptic area of the rat hypothalamus is synchronized with the estrous cycle

Acetylcholine levels in the preoptic area of rat hypothalamus were found to change rhythmically, in synchronization with the estrous cycle. During the proestrous critical period, these levels demonstrated a drop followed by a sharp rise. The absence of this phenomena in males indicates sexual dimorphism. The close association between this newly detected hypothalamic acetylcholine rhythm and the estrous cycle suggests the possible involvement of acetylcholine in regulation of the estrous cycle.

Acetylcholine in the rat pituitary: a possible humoral factor

Significant amounts of acetylcholine (ACh) were detected in each of the 3 lobes of the rat pituitary (3-6 pmol/anterior lobe, 3 pmol/intermediate lobe and 1.8 pmol/posterior lobe). In the anterior lobes of cyclic rats the levels of ACh varied with the estrous cycle, with daily peaks being observed on the days of proestrus and estrus. The occurrence of ACh, apparently as a humoral factor, appears to be unique to the anterior pituitary.

On the interaction of drugs with the cholinergic nervous system

Tolerance to physostigmine salicylate was induced in mice using various schedules of s.c. injections. The rate and degree of tolerance development were assessed by comparing the ED50 values (equipotent doses) and by comparing the peak effects induced by a constant dose. These were measured on four systemic responses induced by the drug — hypothermia, tremor, salivation, and the effects measured in the rotarod test. The degree of tolerance development was found to be dose-dependent with a maximal achievable tolerance for every dose. The tolerance development to the four systemic effects differed in time course: tolerance to the hypothermia was induced even with daily injections, while tolerance to the salivation and rotarod effects could be detected only when the drug was given every 4 h. No tolerance developed to the tremor with any of the schedules and doses used. The maximal achievable tolerance degree and the pattern of changes of the duration were different for each systemic effect. The tolerance was found to be reversible, with different rates of recovery, for the different effects. The tolerance state could not be correlated with changes in the pattern of brain acetylcholinesterase (AcChE) inhibition by physostigmine in vivo or with changes in the rate of the enzymes spontaneous reactivation.Scopolamine. HBr given 10 min before physostigmine prevented tolerance development. In addition, cross-tolerance to various muscarinic agonists and cholinesterase inhibitors was found in the physostigmine-tolerant mice. The correlation between these results and our previous findings concerning possible biochemical adaptations is presented and discussed.

Muscarinic Receptors in the Preoptic Area Are Sensitive to 17β-EstradioI during the Critical Period

The relationship between the steroid hormone 17 beta-estradiol and the muscarinic cholinergic receptors present in the preoptic area (POA), median hypothalamus and posterior hypothalamus of female rats was examined in vitro at various stages of the estrous cycle. Muscarinic receptors varied in a cyclic manner, specifically in the POA, as shown by an increase in the proportion of high-affinity agonist binding sites (RH) to 60% during the proestrus, as compared to RH proportion observed during diestrus-2 and during the afternoon of proestrus (35%). Exposure of POA homogenates to 17 beta-estradiol resulted in conversion of RH to low-affinity agonist binding sites (RL). This effect of the hormone was also restricted to the POA taken from rats during the morning of proestrus. It was blocked by the antiestrogenic drug, clomiphene, and could be prevented by preoccupation of the muscarinic receptors by their own ligands prior to the addition of hormone. It follows that significant changes in POA muscarinic receptors in situ exactly coincided with the known critical time period characterized by high estrogen levels and high levels of estrogen receptors in the POA. These changes in muscarinic receptors might thus conceivably reflect variations in cholinergic activity in the POA during the estrous cycle.