The non-proteinogenic amino acids L-methionine sulfoximine and DL-phosphinothricin activate mTOR.

L-Methionine sulfoximine (MSO) and DL-Phosphinothricin (PPT), two non-proteinogenic amino acids known as inhibitors of Glutamine Synthetase, cause a dose-dependent increase in the phosphorylation of the mTOR substrate S6 kinase 1. The effect is particularly evident in glutamine-depleted cells, where mTOR activity is very low, but is detectable for PPT also in the presence of glutamine. The stimulation of mTOR activity by either MSO or PPT is strongly synergized by essential amino acids. Thus, the non-proteinogenic amino acids MSO and PPT are mTOR activators.

Towards a metabolic therapy of cancer?

It is increasingly appreciated that cancer cells must be endowed with specific metabolic adaptations to support enhanced growth and to ensure survival under stressful conditions. On the other hand, many oncogenic mutations of protooncogenes and tumor suppressor genes directly cause metabolic derangements and, conversely, mutations of enzymes have been found to underlie several forms of cancer. Thus, cancer-specific metabolic alterations are now considered among the hallmarks of malignant tumors. Most commonly, cancer cells exhibit enhanced glycolysis under aerobic conditions (the Warburg effect) but alterations in the metabolism of amino acids, such as glutamine, serine and proline are increasingly described as important metabolic features of selected tumor types. In theory, all these deranged cancer-specific metabolic pathways may constitute novel therapeutic targets, although the only "metabolic" drug in clinical use is still represented by the enzyme L-asparaginase. However, the increasing amount of experimental evidence, as well as the number of trials in progress, suggests that metabolic drugs will soon complement standard anti-cancer chemotherapy and modern biological drugs.

Copper binding agents acting as copper ionophores lead to caspase inhibition and paraptotic cell death in human cancer cells.

We report a quantitative structure-activity relationship study of a new class of pyrazole-pyridine copper complexes that establishes a clear correlation between the ability to promote copper accumulation and cytotoxicity. Intracellular metal accumulation is maximized when ligand lipophilicity allows the complex to rapidly cross the membrane. Copper and ligand follow different uptake kinetics and reach different intracellular equilibrium concentrations. These results support a model in which the ligand acts as an ionophore for the metal ion, cycling between intra- and extracellular compartments as dissociated or complexed entities. When treating cancer cells with structurally unrelated disulfiram and pyrazole-pyridine copper complexes, as well as with inorganic copper, the same morphological and molecular changes were reproduced, indicating that copper overload is responsible for the cytotoxic effects. Copper-based treatments drive sensitive cancer cells toward paraptotic cell death, a process hallmarked by endoplasmic reticulum stress and massive vacuolization in the absence of apoptotic features. A lack of caspase activation, as observed in copper-treated dying cells, is a consequence of metal-mediated inhibition of caspase-3. Thus, copper acts simultaneously as an endoplasmic reticulum (ER) stress inducer and a caspase-3 inhibitor, forcing the cell into caspase-independent paraptotic death. The establishment of a mechanism of action common to different copper binding agents provides a rationale for the exploitation of copper toxicity as an anticancer tool.

Thioamido coordination in a thioxo-1,2,4-triazole copper(II) complex enhances nonapoptotic programmed cell death associated with copper accumulation and oxidative stress in human cancer cells.

The thioamido function of [CuCl2(1H)]Cl (2) (1=4-amino-1,4-dihydro-3-(2-pyridyl)-5-thioxo-1,2,4-triazole), a cytotoxic copper complex, was converted into thioether moieties, leading to the synthesis of [CuCl2(3)]2 (4) and [CuCl2(5)] (6) (3=6-methyl-3-pyridin-2-yl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine; 5=4-amino-5-ethylthio-3-(2-pyridyl)-1,2,4-triazole). These complexes were structurally characterized, and their stability constants, along with their biological activity, were determined. 4 and 6 were slightly less stable and significantly less active than 2. However, as 2, both complexes induced nonapoptotic vacuolar cell death. Copper uptake, investigated in both 2-sensitive and -insensitive cell types, was markedly higher in sensitive cells where it was associated with an increase in oxidized glutathione. These data suggest that the thioamido function enhances the cytotoxicity of copper complexes in cancer cells promoting the accumulation of the metal and its interaction with cell thiols.

The synthesis of SNAT2 transporters is required for the hypertonic stimulation of system A transport activity.

In cultured human fibroblasts incubated under hypertonic conditions, the stimulation of system A for neutral amino acid transport, associated to the increased expression of the mRNA for SNAT2 transporter, leads to an expanded intracellular amino acid pool and to the recovery of cell volume. A protein of nearly 60 kDa, recognized by an antiserum against SNAT2, is increased both in the pool of biotinylated membrane proteins and in the total cell lysate of hypertonically stressed cells. The increased level of SNAT2 transporters in hypertonically stressed cells is confirmed by immunocytochemistry. DRB, an inhibitor of transcription, substantially inhibits the increase of SNAT2 proteins on the plasma membrane, completely suppresses the stimulation of system A transport activity, and markedly delays the cell volume recovery observed during the hypertonic treatment. On the contrary, if the transport activity of system A is adaptively increased by amino acid starvation in the presence of DRB, the increase of SNAT2 transporters on the plasma membrane is still clearly detectable and the transport change only partially inhibited. It is concluded that the synthesis of new SNAT2 transporters is essential for the hypertonic stimulation of transport system A, but accounts only in part for the adaptive increase of the system.

SNAT2 silencing prevents the osmotic induction of transport system A and hinders cell recovery from hypertonic stress.

Under hypertonic conditions the induction of SLC38A2/SNAT2 leads to the stimulation of transport system A and to the increase in the cell content of amino acids. In hypertonically stressed human fibroblasts transfection with two siRNAs for SNAT2 suppressed the increase in SNAT2 mRNA and the stimulation of system A transport activity. Under the same condition, the expansion of the intracellular amino acid pool was significantly lowered and cell volume recovery markedly delayed. It is concluded that the up-regulation of SNAT2 is essential for the rapid restoration of cell volume after hypertonic stress.

Synthesis, solution equilibria and antiproliferative activity of copper(II) aminomethyltriazole and aminomethylthioxotriazoline complexes.

The aim of the present study was the synthesis, the determination of formation constants, and the evaluation of the antiproliferative activity of two copper(II) complexes formed with triazole-type ligands. The synthesis of the unsymmetrical triazole ligand 4-amino-3-aminomethyl-5-methyl-1,2,4-triazole (L1), and its copper(II) complex is reported. The ligand was prepared by functionalization of the carboxylate function of tert-butyloxycarbonyl (BOC) protected glycine O-methyl ester. All intermediates and final products were isolated and characterized with IR, 1H NMR, and elemental analysis. X-ray structures of the ligand as a sulfate salt ((H2L1)2SO4.H2O) and the copper(II) complex [CuCl2(L1)(2)] are described. The ligand forms a (N,N) bidentate chelate with the amino group and one triazole nitrogen atom. The tetragonally distorted octahedral coordination of Cu(II) results from two axially coordinated chloride ions. Protonation constants for L1 and speciation of the Cu(II)/L1 system were determined in 0.1 M aqueous KCl solution at 25 degrees C. Complexes formed in solution were also characterized by visible spectrophotometry. Ligand substitution competition between L1 and glycine has also been studied using potentiometric titrations. Antiproliferative activities of ([CuCl2(L1)2]) and [CuCl2(H2L2)]Cl, where HL2 is the 5-thioxo analog of L1, against human tumor cell lines HT1080 and HT29 as well as normal human fibroblasts (HF) are presented along with the antiproliferative activities of L1, CuCl2, and cisplatin. Activity of these two complexes are discussed and compared with the activity of analogous compounds reported in the literature which contain pyridyl groups in place of the aminomethyl group. In particular, it is suggested that a lypophilic residue such as a pyridyl group is important for antiproliferative activity of this class of compounds.

Synthesis, molecular structure, solution equilibrium, and antiproliferative activity of thioxotriazoline and thioxotriazole complexes of copper II and palladium II.

Preparations of copper(II) and palladium(II) complexes of 4-amino-5-methylthio-3-(2-pyridyl)-1,2,4-triazole (L(1)) and the copper(II) complex of 1,4-dihydro-4-amino-3-(2-pyridyl)-5-thioxo-1,2,4-triazole (HL) are described. These complexes have been characterized by means of spectroscopy and microanalysis. Molecular structures of HL (1), [CuCl(2)(H(2)L)]Cl.2H(2)O (2a), cis-[CuCl(2)(L(1))] (3), and cis-[PdCl(2)(L(1))] (4) have been determined by single-crystal X-ray diffraction. The HL ligand acts as a N,S bidentate through the thioxo moiety and the exo-amino group whilst the ligand L(1) forms N,N coordination complexes through the pyridine and triazole nitrogen atoms. Speciation in solution of the systems Cu/HL and Cu/L(1) have been determined by means of potentiometry and spectrophotometry as well as for the Cu/L(1)/A (HA=glycine) system in order to determine species present at physiological pH. Antiproliferative activity of these complexes and their ligands was evaluated, using the AlamarBlue Assay, on normal human fibroblasts (HF) and human fibrosarcoma tumor (HT1080) cells. The copper compounds cis-[CuCl(2)(H(2)L)]Cl and cis-[CuCl(2)(L(1))] exerted significant antiproliferative activity of both normal and neoplastic cells; although dose-response experiments revealed that the HT1080 cell line was more sensitive to the tested drugs than normal fibroblasts.

Copper-dependent cytotoxicity of 8-hydroxyquinoline derivatives correlates with their hydrophobicity and does not require caspase activation.

This study reports the structure-activity relationship of a series of 8-hydroxoquinoline derivatives (8-HQs) and focuses on the cytotoxic activity of 5-Cl-7-I-8-HQ (clioquinol, CQ) copper complex (Cu(CQ)). 8-HQs alone cause a dose-dependent loss of viability of the human tumor HeLa and PC3 cells, but the coadministration of copper increases the ligands effects, with extensive cell death occurring in both cell lines. Cytotoxic doses of Cu(CQ) promote intracellular copper accumulation and massive endoplasmic reticulum vacuolization that precede a nonapoptotic (paraptotic) cell death. The cytotoxic effect of Cu(CQ) is reproduced in normal human endothelial cells (HUVEC) at concentrations double those effective in tumor cells, pointing to a potential therapeutic window for Cu(CQ). Finally, the results show that the paraptotic cell death induced by Cu(CQ) does not require nor involve caspases, giving an indication for the current clinical assessment of clioquinol as an antineoplastic agent.

Synthesis, structural characterisation and solution chemistry of ruthenium(III) triazole-thiadiazine complexes.

Two ruthenium(III) complexes structurally similar to the anticancer compound NAMI were prepared: Na[RuCl4(DMSO)(L1)] ( 1) and Na[RuCl4(DMSO)(L2)] ( 2), where L1 and L2 are differently functionalised triazole-thiadiazine ligands. To facilitate the crystallisation of the complex anions, Na+ was substituted with the [bis(triphenylphosphoranylidene)ammonium] cation (PPN+), allowing the X-ray characterisation of PPN[RuCl4(DMSO)(L1)].2H2O ( 1a.2H2O) and PPN[RuCl4(DMSO)(L2)].3H2O ( 2a.3H2O), respectively. The two compounds undergo stepwise hydrolytic processes, as assessed by means of UV-vis and 1H NMR spectroscopy. The first hydrolytic step consists of the replacement of a chloride anion with a water molecule, with a half-life of 50 min ( 1) and 110 min ( 2), while the subsequent hydrolytic steps are more complicated to describe since more than one product is generated at the same time. The redox potential of the Ru(III)/Ru(II) couple (0.31 V for 1 and 0.28 V for 2) suggests that these complexes can be reduced in the intracellular environment, in agreement with the activation by reduction mechanism proposed for NAMI and NAMI-A. 1 and 2 were tested on a human cancer cell line derived from a fibrosarcoma (HT1080), and on non-cancerous primary human fibroblasts (HF), where they showed a modest inhibitory effect.

The thioxotriazole copper(II) complex A0 induces endoplasmic reticulum stress and paraptotic death in human cancer cells.

The copper(II) complex A0 induces a type of non-apoptotic cell death also known as paraptosis. Paraptosis involves extensive endoplasmic reticulum vacuolization in the absence of caspase activation. A wide panel of human cancer cell lines was used to demonstrate differences in cytotoxicity by the paraptosis-inducing drug A0 and the metal-based pro-apoptotic drug cisplatin. Gene expression profiling of the human fibrosarcoma HT1080 cells showed that, while cisplatin induced p53 targets, A0 up-regulated genes involved in the unfolded protein response (UPR) and response to heavy metals. The cytotoxic effects of A0 were associated with inhibition of the ubiquitin-proteasome system and accumulation of ubiquitinylated proteins, in a manner dependent on protein synthesis. Cycloheximide inhibited the accumulation of ubiquitinylated proteins and hampered A0-induced cell death process. The occurrence of the UPR during A0-induced death process was shown by the increased abundance of spliced XBP1 mRNA, transient eIF2alpha phosphorylation, and a series of downstream events, including attenuation of global protein synthesis and increased expression of ATF4, CHOP, BIP, and GADD34. Mouse embryonic fibroblasts expressing a mutant eIF2alpha, which could not be phosphorylated, were more resistant to A0 than wild type cells, pointing to a pro-death role of eIF2alpha phosphorylation. A0 may thus represent the prototypical member of a new class of compounds that cause paraptotic cell death via mechanisms involving eIF2alpha phosphorylation and the UPR.

Amino acid starvation induces the SNAT2 neutral amino acid transporter by a mechanism that involves eukaryotic initiation factor 2alpha phosphorylation and cap-independent translation.

Nutritional stress caused by amino acid starvation involves a coordinated cellular response that includes the global decrease of protein synthesis and the increased production of cell defense proteins. Part of this response is the induction of transport system A for neutral amino acids that leads to the recovery of cell volume and amino acid levels once extracellular amino acid availability is restored. Hypertonic stress also increases system A activity as a mechanism to promote a rapid recovery of cell volume. Both a starvation-dependent and a hypertonic increase of system A transport activity are due to the induction of SNAT2, the ubiquitous member of SLC38 family. The molecular mechanisms underlying SNAT2 induction were investigated in tissue culture cells. We show that the increase in system A transport activity and SNAT2 mRNA levels upon amino acid starvation were blunted in cells with a mutant eIF2alpha that cannot be phosphorylated. In contrast, the induction of system A activity and SNAT2 mRNA levels by hypertonic stress were independent of eIF2alpha phosphorylation. The translational control of the SNAT2 mRNA during amino acid starvation was also investigated. It is shown that the 5'-untranslated region contains an internal ribosome entry site that is constitutively active in amino acid-fed and -deficient cells and in a cell-free system. We also show that amino acid starvation caused a 2.5-fold increase in mRNA and protein expression from a reporter construct containing both the SNAT2 intronic amino acid response element and the SNAT2-untranslated region. We conclude that the adaptive response of system A activity to amino acid starvation requires eukaryotic initiation factor 2alpha phosphorylation, increased gene transcription, and internal ribosome entry site-mediated translation. In contrast, the response to hypertonic stress does not involve eukaryotic initiation factor 2alpha phosphorylation, suggesting that SNAT2 expression can be modulated by specific signaling pathways in response to different stresses.