In Vivo Evidence for Serine Biosynthesis-Defined Sensitivity of Lung Metastasis,but Not of Primary Breast Tumors,to mTORC1 Inhibition

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Diazepam inhibits cell respiration and induces fragmentation of mitochondrial reticulum

Diazepam (70-150 micrograms/ml) significantly inhibits oxygen consumption by pig kidney embryo cells and causes the cellular ATP level to fall. The maximum inhibitory effect develops after 1.5-2.5 h of diazepam treatment. In isolated mitochondria diazepam inhibits respiration in state 2 and 3u with glutamate and in state 3u with succinate. Ethylrhodamine staining and electron microscopic study reveal fragmentation of mitochondria in living cells.     

α-Tocospiro C,a novel cytotoxic α-tocopheroid from Cirsium setosum

α-Tocospiro C (1), the first example of nor-α-tocopheroid possessing unusual 7,8-dimethyl-1-oxaspiro[4.4]non-7-ene-6,9-dione carbon skeleton, was isolated and characterized from Cirsium setosum. Its structure and absolute configuration were determined by extensive spectroscopic methods, especially 2D NMR and ECD data analysis. The biosynthetic pathway was postulated. α-Tocospiro C (1) exhibited significant selective cytotoxic activities against human colon cancer (HCT8) cells, with IC₅₀ values of 0.03 μM.     

The sesquiterpene α-copaene is induced in tomato leaves infected by Botrytis cinerea

Abstract

Explorative experiments were done to investigate the possibility that tomato plants infected by Botrytis cinerea have a different emission of volatile organic compounds (VOC) than healthy plants. This was done by headspace analysis of volatiles emitted by detached leaves of infected and healthy plants. Principal component analysis (PCA) of GC-FID chromatograms revealed clearly separated clusters between infected and control leaves and identification of an interesting compound. In further analysis by GC-MS, the significantly distinctive component (p≤0.05) was identified as the sesquiterpene α-copaene. In previous work on herbivore damage, α-copaene was not distinctive, which may suggest that α-copaene may be specifically associated to fungal infections in tomato.     

Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD+

A new, acyclic NAD-analog, acycloNAD⁺ has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD⁺ with a redox potential of −324 mV and a 341 nm λ_max for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD⁺ by primary alcohols. With HLADH 1-butanol has the highest V_max at 49% that of β-NAD⁺. The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon–hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD⁺. In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD⁺ by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD⁺ has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases.     

NLS-RARα contributes to differentiation block and increased leukemogenic potential in vivo

The fusion oncogene, promyelocytic leukemia (PML)-retinoic acid receptor-α (RARα), is crucial for acute promyelocytic leukemia (APL) pathogenesis. Previous studies have reported that PML-RARα is cleaved by neutrophil elastase (NE), an early myeloid-specific serine protease, leading to translocation of the nuclear localization signal (NLS) of the PML protein to the N-terminal of RARα. This study was designed to evaluate the value of NLS-RARα in the early diagnosis of APL. To investigate the potential functional role of NLS-RARα in leukemogenesis, HL-60 and U937 cell lines were transfected with NLS-RARα lentivirus and negative control (LVNC). The results showed that the induced expression of NLS-RARα down-regulated expressions of CD11b, CD11c, and CD14 compared to the LVNC group induced by 1α, 25-dihydroxyvitamin D₃(1,25(OH)₂D₃). This suggested that NLS-RARα overexpression inhibited granulocytic and monocytic differentiation of myeloid leukemia cells. In addition, Wright-Giemsa staining, flow cytometry, respiratory burst assay, and NBT reduction assay all confirmed the importance of NLS-RARα in differentiation. The mechanistic investigations revealed that induced NLS-RARα expression inhibited 1,25(OH)₂D₃-induced granulocytic differentiation by regulating the cell cycle regulators p19^INK4D, p21^WAF1/CIP1, cyclinD1, cyclin E1, and pRB. Furthermore, the cleaved protein NLS-RARα enhanced the oncogenicity of U937 cells in NOD/SCID mice. These findings collectively demonstrated that NLS-RARα blocked granulocytic and monocytic differentiation of myeloid leukemia cells by inhibiting the downstream targets of the RARα signal pathway and the cell cycle. This may provide a promising new target and method for diagnosing and treating APL.     

Homocysteine Induces Iron-Catalyzed Lipid Peroxidation of Low-Density Lipoprotein that is Prevented by Alpha-Tocopherol

Homocystinuria is an inborn error of methionine metabolism that is characterized by the premature development of arteriosclerosis. As one of the major factors in the pathogenesis of arteriosclerosis, modification of low-density lipoprotein (LDL) has received widespread attention by many investigators. In this study, to elucidate the relationship between elevated homocysteine levels and premature arteriosclerosis, we investigated the role of homocysteine in the iron-catalyzed oxidative modification of LDL. When LDL isolated from a healthy subject was incubated with homocysteine and ferric ion, a gradual decrease of polyunsaturated fatty acids (PUFA), formation of thiobarbituric acid-reactive substances (TBARS) and fluorescent substances, and the fragmentation of apoprotein B (apoB) were observed. The extent of oxidative modification was dependent on the concentration of homocysteine. Modification of LDL was suppressed until the remaining α-tocopherol concentration reached a critical level. When the α-tocopherol content of LDL was increased by 2.6-fold, both the formation of TBARS and the fragmentation of apoB were suppressed. These results suggest that homocysteine might promote iron-catalyzed oxidation of LDL and imply its role for the development of premature arteriosclerosis.     

Post-transcriptional inhibition of ornithine decarboxylase induction by zinc in a difluoromethylornithine resistant cell line

Addition of Zn^2+_ to cell medium inhibited the induction of ornithine decarboxylase (ODC) activity in ODC overproducing L1210-DFMO^r cells. A significant effect was observed at a concentration as low as 0.01 mM, however a more marked inhibition was caused by the addition of 0.1 mM Zn²⁺. The inhibition of the induction of ODC activity was accompanied by a proportional decrease in the content of immunoreactive ODC protein, whereas the level of ODC mRNA, detemined by a solution hybridization RNase protection assay, was not affected signigicantly. Instead, some acceleration of ODC turnover was observed. the addition of 0.1 mM Co²⁺ or Mn²⁺, but not of other divalent metal ions, also inhibited ODC induction; differently from Zn²⁺ however, these metals affected cell viability and/or cell growth. Removal of endogenous Zn²⁺ by a chelator also provoked a strong decrease of ODC induction, which was reversed by Zn²⁺. However, addition of Zn²⁺ in excess of the chelator proved to be markedly inhibitory. These results indicate that both a restricted Zn²⁺ availability and an enhanced presence of the metal can inhibit the induction of ODC in L1210-DFMO^r cells.     

Synthesis and antitumor activities of novel rhein α-aminophosphonates conjugates

Several rhein α-aminophosphonates conjugates (5a–5q) were synthesized and evaluated for in vitro cytotoxicity against HepG-2, CNE, Spca-2, Hela and Hct-116 cell lines. Some compounds showed relatively high cytotoxicity. Especially, compound 5i exhibited the strongest cytotoxicity against Hct-116 cells (IC₅₀ was 5.32 μM). All the synthesized compounds exhibited low cytotoxicity against HUVEC cells. The mechanism of compound 5i was preliminarily investigated by Hoechst 33258 staining, JC-1 mitochondrial membrane potential staining and flow cytometry, which indicated that the compound 5i induced apoptosis in Hct-116 cancer cells. Cell cycle analysis showed that these compound 5i mainly arrested Hct-116 cells in G1 stage. The effects of 5i on the activation of caspases expression indicated that 5i might induce apoptosis via the membrane death receptor pathways. In addition, the binding properties of a model analog 5i to DNA were investigated by methods (UV–vis, fluorescence, CD spectroscopy and FRET-melting) in compare with that of rhein. Results indicated that 5i showed moderate ability to interact ct-DNA.     

Trypanosoma cruzi: Insights into naphthoquinone effects on growth and proteinase activity

In this study we compared the effects of naphthoquinones (α-lapachone, β-lapachone, nor-β-lapachone and Epoxy-α-lap) on growth of Trypanosoma cruzi epimastigotes forms, and on viability of VERO cells. In addition we also experimentally analyzed the most active compounds inhibitory profile against T. cruzi serine- and cysteine-proteinases activity and theoretically evaluated them against cruzain, the major T. cruzi cysteine proteinase by using a molecular docking approach. Our results confirmed β-lapachone and Epoxy-α-lap with a high trypanocidal activity in contrast to α-lapachone and nor-β-lapachone whereas Epoxy-α-lap presented the safest toxicity profile against VERO cells. Interestingly the evaluation of the active compounds effects against T. cruzi cysteine- and serine-proteinases activities revealed different targets for these molecules. β-Lapachone is able to inhibit the cysteine-proteinase activity of T. cruzi proteic whole extract and of cruzain, similar to E-64, a classical cysteine-proteinase inhibitor. Differently, Epoxy-α-lap inhibited the T. cruzi serine-proteinase activity, similar to PMSF, a classical serine-proteinase inhibitor. In agreement to these biological profiles in the enzymatic assays, our theoretical analysis showed that E-64 and β-lapachone interact with the cruzain specific S2 pocket and active site whereas Epoxy-α-lap showed no important interactions. Overall, our results infer that β-lapachone and Epoxy-α-lap compounds may inhibit T. cruzi epimastigotes growth by affecting T. cruzi different proteinases. Thus the present data shows the potential of these compounds as prototype of protease inhibitors on drug design studies for developing new antichagasic compounds.