Hesperidin-CAMKIV interaction and its impact on cell proliferation and apoptosis in the human hepatic carcinoma and neuroblastoma cells

Calcium/calmodulin-dependent protein kinase IV (CAMKIV) is a key regulatory molecule of cell signaling, and thereby controls its growth and proliferation, including expression of certain genes. The overexpression of CAMKIV is directly associated with the development of different types of cancers. Hesperidin is abundantly found in citrus fruits and exhibits wide range of pharmacological activities including anti-inflammatory, antibacterial and anticancerous effects. We have investigated binding mechanism of hesperidin with the CAMKIV using molecular docking methods followed by fluorescence quenching and isothermal titration calorimetric assays. An appreciable binding affinity of hesperidin was observed with CAMKIV during fluorescence quenching and isothermal titration calorimetric studies. Efficacy of hesperidin to inhibit the growth of human hepatic carcinoma (HepG2) and neuroblastoma (SH-SY5Y) cancer cell lines were investigated. Hesperidin has significantly reduced the proliferation of HepG2 and SH-SY5Y cells and induces apoptosis by activating the caspase-3-dependent intrinsic pathway through the upregulation of proapoptotic Bax protein. Hesperidin treatment reduces the mitochondrial membrane potential of HepG2 and SH-SY5Y cells. All these observations clearly anticipated hesperidin a potent inhibitor of CAMKIV which may be further exploited a newer therapeutic approach for the management of different cancer types.     

Defined concatenated α6α1β3γ2 GABAA receptor constructs reveal dual action of pyrazoloquinolinone allosteric modulators

Pyrazoloquinolinones (PQs) have been extensively studied as modulators of GABA_A receptors with different subunit composition, exerting modulatory effects by binding at α+/β- interfaces of GABA_A receptors. PQs with a substituent in position R7 have been reported to preferentially modulate α6- subunit containing GABA_A receptors which are mostly expressed in the cerebellum but were also found in the olfactory bulb, in the cochlear nucleus, in the hippocampus and in the trigeminal sensory pathway. They are considered potentially interesting in the context of sensori-motor gating deficits, depressive-like behavior, migraine and orofacial pain. Here we explored the option to modify the lead ligands’ R7 position. In the compound series we observed two different patterns of allosteric modulation in recombinantly expressed α6β3γ2 receptors, namely monophasic and biphasic positive modulation. In the latter case the additional phase occurred in the nanomolar range, while all compounds displayed robust modulation in the micromolar range. Nanomolar, near silent binding has been reported to occur at benzodiazepine binding sites, but was not investigated at the diazepam insensitive α6+/γ2- interface. To clarify the mechanism underlying the biphasic effect we tested one of the compounds in concatenated receptors. In these constructs the subunits are covalently linked, allowing to form either the α6+/γ2- interface, or the α6+/β3- interface, to study the resulting modulation. With this approach we were able to ascribe the nanomolar modulation to the α6+/γ2- interface. While not all compounds display the nanomolar phase, the strong modulation at the α6+/β3 interface proved to be tolerant for all tested R7 groups. This provides the future option to introduce e.g. isotope labelled or fluorescent moieties or substituents that enhance solubility and bioavailability.     

Inhibition of human leukocyte elastase and cathepsin G by extended peptides and subunits derived from human C-reactive protein

Extended peptides that derive from the primary sequence of the acute phase reactant C-reactive protein (CRP) are shown to inhibit in vitro the enzymatic activities of human leukocyte elastase (hLE) and human leukocyte cathepsin G (hCG), which are associated with the tissue damage that occurs during the course of several chronic inflammatory conditions. Major inhibitory activity was observed in the peptides CRP_70-98 and CRP_50-98 towards hLE (K_i = 4.0 µM) and hCG (K_i = 1.4 µM), respectively. In contrast to the inability of intact CRP pentamers to inhibit both enzymes, CRP subunits (monomers) inhibited hLE (3.0 µM) and hCG (3.6 µM) activity.     

Lysine 194 Is Functional in Isocitrate Lyase from Escherichia coli

Lysine 194 in conserved stretch 1 of tetrameric isocitrate lyase from Escherichia coli has been replaced by using the restriction-enzyme-site elimination method of directed mutagenesis. Expression of subunits of each variant and of wild-type (wt) enzyme was equivalent and all variants assembled into tetrameric proteins. The variants K194R and K194H had k_cat values relative to that of wt enzyme taken as 100 of 11 and 7, respectively. K _m values for Mg²⁺-D_s-isocitrate (in mM units) were: 0.13 for wt-enzyme; 0.12 for the K194R variant; and 0.55 for the K194H variant. Substitution at position 194 of Leu or Glu resulted in zero catalytic activity. These results establish that Lys 194 is another functional residue in conserved stretch one of isocitrate lyase from E. coli besides H184, K193, C195, and H197. Because K194 can be specifically replaced by the basic residues His and Arg with resultant lowered activity and by His with an increased K _m value, it may contribute to a cation center and facilitate substrate binding as well as orientation of the developing transition state. Received: 3 December 1996 / Accepted: 18 December 1996     

Potentiating vanadium-evoked glucose metabolism by novel hydroxamate derivatives

Summary L-glutamic acid (γ) monohydroxamate (L-Glu(γ)HXM) enhances the insulinomimetic activity of vanadium ions bothin vitro andin vivo. Based on this ligand as a lead compound, and in order to delineate molecular features relevant to its anti-diabetic potential, 14 related derivatives, including short peptides, were synthesized by solution as well as by solid phase methodologies. In addition, hydroxamate derivatives of (+) pantothenic acid and D-biotin were prepared. The vanadium binding, capacity of the hydroxamates synthesized was apparent, yet each had a different ligand-ions stoichiometry. Thein vitro lipogenic potency of several compounds toward rat adipocytes was demonstrated. Thus, vanadium complexes of L-Gln(α)HXM, L-Glu(γ)HXM-Gly, L-Aad(δ)HXM, di-Glu-γ,γ-HXM and of (+) pantothenic acid hydroxamate exhibited 82, 79, 76, 39 and 39% of maximal insulin activity, respectively. L-Aad (δ)HXM, L-Glu(γ)HXM-Gly and (+) pantothenic acid hydroxamate-by themselves —were found to possess 24, 14 and 10% of maximal insulin activity, respectively.In vivo potency, however, of L-Gln(α)HXM vanadium complex in streptozocin-treated rat diabetic model was less apparent.     

Serine319 and 321 Are Functional in Isocitrate Lyase from Escherichia coli

With site-directed mutagenesis, Ser319 and Ser321 in conserved stretch 3 of tetrameric isocitrate lyase from Escherichia coli were each substituted with alanine, cysteine, asparagine, or threonine in addition to simultaneous alanine/alanine substitutions. Besides their absolute conservation in all aligned isocitrate lyase sequences, the location of these serine residues, which flank a completely conserved proline, had been suggested in the active site in previous research by studies of photoinactivation of the enzyme by vanadate [Ko et al. (1992) J Biol Chem 267:91]. All substitutions for Ser321 and 319 except by threonine appreciably reduced the k_cat of E. coli isocitrate lyase relative to that for wild-type (100) as follows: S319A, 0.4; S319C, 0.05; S319N, 0.01; S319T, 32.3; S321A, 2.9; S321C, 0.3; S321N, 0.1; S321T, 0.3; and S319A/S321A, 0, with little or no effect on the K _m for the substrate Mg²⁺-D_s-isocitrate. The most active variant S319T exhibited threefold less activity than the wild-type enzyme; all variants assembled into tetramers. The S319T mutant isocitrate lyase was 100-fold more active than the S321T variant. This observation suggests that the requirement for a β-hydroxymethyl group of serine in catalysis is less important at position 319 than at position 321. Although most singly substituted variants had very low isocitrate lyase activity, all variants harboring mutant isocitrate lyase of very low activity did grow on acetate as a sole carbon source albeit with longer doubling times and lag phases. Substitution of Pro320 by Ala, Asp, Gly, or His was highly detrimental to activity and increased the K _m for substrate 3.5- to 8-fold; this suggests that Pro fixes the location of adjacent Ser OH groups and facilitates substrate binding and catalysis. From these collective results, it is proposed that Ser319 and Ser321 are involved in E. coli isocitrate lyase catalysis, perhaps by stabilizing the postulated reaction intermediate succinate trianion in the aci-carboxylate form and the related transition state via hydrogen bonding. Received: 3 September 1996 / Accepted: 20 September 1996     

A new subclade of mtDNA haplogroup C1 found in icelanders: Evidence of pre‐columbian contact?

Although most mtDNA lineages observed in contemporary Icelanders can be traced to neighboring populations in the British Isles and Scandinavia, one may have a more distant origin. This lineage belongs to haplogroup C1, one of a handful that was involved in the settlement of the Americas around 14,000 years ago. Contrary to an initial assumption that this lineage was a recent arrival, preliminary genealogical analyses revealed that the C1 lineage was present in the Icelandic mtDNA pool at least 300 years ago. This raised the intriguing possibility that the Icelandic C1 lineage could be traced to Viking voyages to the Americas that commenced in the 10th century. In an attempt to shed further light on the entry date of the C1 lineage into the Icelandic mtDNA pool and its geographical origin, we used the deCODE Genetics genealogical database to identify additional matrilineal ancestors that carry the C1 lineage and then sequenced the complete mtDNA genome of 11 contemporary C1 carriers from four different matrilines. Our results indicate a latest possible arrival date in Iceland of just prior to 1700 and a likely arrival date centuries earlier. Most surprisingly, we demonstrate that the Icelandic C1 lineage does not belong to any of the four known Native American (C1b, C1c, and C1d) or Asian (C1a) subclades of haplogroup C1. Rather, it is presently the only known member of a new subclade, C1e. While a Native American origin seems most likely for C1e, an Asian or European origin cannot be ruled out.     

Selective cytotoxicity of Aniba rosaeodora essential oil towards epidermoid cancer cells through induction of apoptosis

Essential oils are complex mixtures of odorous and volatile compounds derived from secondary plant metabolism. They can be isolated from many plants by mechanical pressing or hydro- and steam-distillation and are known to induce a wide range of biological effects through their antibacterial, antifungal, cytotoxic, antioxidant and antimutagenic activities. In order to explore their beneficial properties on human skin cells, we investigated the effects of an essential oil from rosewood Aniba rosaeodora (REO) on the human epidermoid carcinoma cell line A431, on immortal HaCaT cells thought to represent an early stage of skin carcinogenesis, on transformed normal HEK001 keratinocytes and on primary normal NHEK keratinocytes. In a defined range of concentrations, REO selectively killed A431 and HaCaT cells. The same treatments had only a minor cytotoxic effect on HEK001 and NHEK cells. Preferentially in A431 and HaCaT cells, REO triggered the production of reactive oxygen species, induced depolarization of the mitochondrial membrane and caused caspase-dependent cell death characterized by phosphatidylserine externalization, an early marker of apoptosis. Both intrinsic and extrinsic apoptotic pathways were implicated in REO-induced cell death. The identification of selective induction of apoptosis in precancerous and cancerous skin cells by REO highlights the potential anticancer activity of this essential oil.