Methyl-β-cyclodextrin potentiates the BITC-induced anti-cancer effect through modulation of the Akt phosphorylation in human colorectal cancer cells

ABSTRACT

Methyl-β-cyclodextrin (MβCD) is an effective agent for the removal of plasma membrane cholesterol. In this study, we investigated the modulating effects of MβCD on the antiproliferation induced by benzyl isothiocyanate (BITC), an ITC compound mainly derived from papaya seeds. We confirmed that MβCD dose-dependently increased the cholesterol level in the medium, possibly through its removal from the plasma membrane of human colorectal cancer cells. The pretreatment with a non-toxic concentration (2.5 mM) of MβCD significantly enhanced the BITC-induced cytotoxicity and apoptosis induction, which was counteracted by the cholesterol supplementation. Although BITC activated the phosphoinositide 3-kinase (PI3K)/Akt pathway, MβCD dose-dependently inhibited the phosphorylation level of Akt. On the contrary, the treatment of MβCD enhanced the phosphorylation of mitogen activated protein kinases, but did not potentiate their BITC-induced phosphorylation. These results suggested that MβCD might potentiate the BITC-induced anti-cancer by cholesterol depletion and thus inhibition of the PI3K/Akt-dependent survival pathway.

Abbreviations: CDs: cyclodextrins; MβCD: methyl-β-cyclodextrin; ITCs: isothiocyanates; BITC: benzyl isothiocyanate; PI3K: phosphoinositide 3-kinase; PDK1: phosphoinositide-dependent kinase-1; MAPK: mitogen activated protein kinase; ERK1/2: extracellular signal-regulated kinase1/2; JNK: c-Jun N-terminal kinase; PI: propidium iodide; FBS: fatal bovine serum; TLC: thin-layer chromatography; PBS(-): phosphate-buffered saline without calcium and magnesium; MEK: MAPK/ERK kinase; PIP2: phosphatidylinositol-4,5-bisphosphate; PIP3: phosphatidylinositol-3,4,5-trisphosphate     

An antitumor promoter from Moringa oleifera Lam.

In the course of studies on the isolation of bioactive compounds from Philippine plants, the seeds of Moringa oleifera Lam. were examined and from the ethanol extract were isolated the new O-ethyl-4-(α- -rhamnosyloxy)benzyl carbamate (1) together with seven known compounds, 4(α- -rhamnosyloxy)-benzyl isothiocyanate (2), niazimicin (3), niazirin (4), β-sitosterol (5), glycerol-1-(9-octadecanoate) (6), 3-O-(6′-O-oleoyl-β- -glucopyranosyl)-β-sitosterol (7), and β-sitosterol-3-O-β- -glucopyranoside (8). Four of the isolates (2, 3, 7, and 8), which were obtained in relatively good yields, were tested for their potential antitumor promoting activity using an in vitro assay which tested their inhibitory effects on Epstein–Barr virus-early antigen (EBV-EA) activation in Raji cells induced by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). All the tested compounds showed inhibitory activity against EBV-EA activation, with compounds 2, 3 and 8 having shown very significant activities. Based on the in vitro results, niazimicin (3) was further subjected to in vivo test and found to have potent antitumor promoting activity in the two-stage carcinogenesis in mouse skin using 7,12-dimethylbenz(a)anthracene (DMBA) as initiator and TPA as tumor promoter. From these results, niazimicin (3) is proposed to be a potent chemo-preventive agent in chemical carcinogenesis.     

Discrimination between lumenal and cytosolic sites of deglycosylation in endoplasmic reticulum-associated degradation of glycoproteins by using benzyl mannose in CHO cell lines

Recent studies demonstrated that deglycosylation step is a prerequisite for endoplasmic reticulum (ER)-associated degradation of misfolded glycoproteins. Here, we report the advantages of using benzyl mannose during pulse-chase experiments to study the subcellular location of the deglycosylation step in Chinese hamster ovary (CHO) cell lines. Benzyl mannose inhibited both the ER-to-cytosol transport of oligomannosides and the trimming of cytosolic-labeled oligomannosides by the cytosolic mannosidase in vivo. We pointed out the occurrence of two subcellular sites of deglycosylation. The first one is located in the ER lumen, and led to the formation of Man8GlcNAc2 (isomer B) in wild-type CHO cell line and Man4GlcNAc2 in Man-P-Dol-deficient cell line. The second one was revealed in CHO mutant cell lines for which a high rate of glycoprotein degradation was required. It occurred in the cytosol and led to the liberation of oligosaccharides species with one GlcNAc residue and with a pattern similar to the one bound onto glycoproteins. The cytosolic deglycosylation site was not specific for CHO mutant cell lines, since we demonstrated the occurrence of cytosolic pathway when the formation of truncated glycans was induced in wild-type cells.     

Human melanoma TrkC: its association with a purine-analog-sensitive kinase activity

The various members of the Trk tyrosine kinase family and p75 neurotrophin receptor (p75(NTR)) have been identified as signaling receptors for the structurally related members of the neurotrophins (NT) family. We have previously reported that NT treatment of murine and human brain-metastatic melanoma cells affects their invasive capacities and increases the production of extracellular-matrix degradative enzymes. These cells express aberrant levels of functional p75(NTR) and TrkC, the putative high-affinity receptor for the neurotrophin NT-3. Here we demonstrate that, by using sensitive immune-complex kinase assays in human brain-metastatic (70W) melanoma cells, TrkC receptors associate with a kinase activity exhibiting a dose-dependent susceptibility to inhibition by the purine-analogs 6-thioguanine and 2-aminopurine. The activity of this purine-analog-sensitive kinase (PASK) was induced by NT-3 in a time-dependent fashion, phosphorylating exogenous myelin basic protein (MBP) but not denatured enolase. It is similar to the one reported to relate with p75(NTR) and TrkA receptors and stimulated by the prototypic NT, nerve growth factor. Thus, PASKs may represent unique signaling components common to NT receptors that could engage joint downstream signaling effectors in brain-metastatic melanoma.     

Benzyl adenine restores anthocyanin pigmentation in suspension cultures of wild Vaccinium pahalae

The overriding influence of cytokinin source on flavonoid production in vitro was explored using a suspension culture system for Vaccinium pahalae. The substitution of kinetin by 20 μM benzyl adenine (BA) in the suspension culture media resulted in a three-fold increase in total anthocyanin yield, and a more rapid production during the cell culture cycle. Anthocyanin production reached a maximum after a 16–20 day interval in cultures containing an optimal kinetin concentration, but pigment accumulation peaked at only 12–16 days when BA was used as the sole cytokinin source. Unlike some other production systems which increase secondary metabolite production at the expense of cell growth, BA-supplementation promoted both increased growth and increased anthocyanin productivity. In BA-supplemented medium, cultures were not susceptible to typical osmotically-induced cell growth suppression. When, after multiple subcultures in kinetin-containing media, anthocyanin production capability was lost or diminished, productivity could be restored within 3 days after transfer of cells to a BA-supplemented medium. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biosorption of tetradecyl benzyl dimethyl ammonium chloride on activated sludge: kinetic, thermodynamic and reaction mechanisms

The biosorption of tetradecyl benzyl dimethyl ammonium chloride (C₁₄BDMA) onto activated sludge was examined in aqueous solution with respect to the contact time, temperature and particle size. Equilibrium reached in about 2 h contact time. An decrease in the temperature increases of biosorption capacity of C₁₄BDMA onto activated sludge, which also increases with decreasing particle size. The experimental data fit the pseudo-second-order kinetics model well. The Langmuir and Freundlich models were applied to describe equilibrium isotherms, and the equilibrium partitioning data was described well by both models. Thermodynamic data showed that C₁₄BDMA biosorption onto activated sludge was feasible, spontaneous and exothermic. The Fourier transform infrared (FT-IR) spectrophotometry results show that both physisorption and chemisorption were involved. The measured zeta potential values and the enhanced cation concentration indicate the presence of electrostatic interactions, hydrophobic interactions and ion exchange.     

Design, synthesis and biological activity of novel peptidyl benzyl ketone FVIIa inhibitors

Herein is described the synthesis of a novel class of peptidyl FVIIa inhibitors having a C-terminal benzyl ketone group. This class is designed to be potentially suitable as stabilization agents of liquid formulations of rFVIIa, which is a serine protease used for the treatment of hemophilia A and B inhibitor patients. A library of compounds was synthesized with different tripeptide sequences, N-terminals and d-amino acids in the P3 position. Cbz-d-Phe-Phe-Arg-bk (33) was found to be the best candidate with a potency of K_i = 8 μM and no substantial inhibition of related blood coagulation factors (thrombin and FXa). Computational studies revealed that 33 has a very stable binding conformation due to intramolecular hydrogen bonds, which cannot be formed with l-Phe in the P3 position. Nonpolar amino acids were found to be superior, probably due to a minimization of the cost of desolvation upon binding to FVIIa.     

Controlled Expression of Recombinant Proteins in <Emphasis Type="Italic">Physcomitrella patens</Emphasis> by a Conditional Heat-shock Promoter: a Tool for Plant Research and Biotechnology

The ability to express tightly controlled amounts of endogenous and recombinant proteins in plant cells is an essential tool for research and biotechnology. Here, the inducibility of the soybean heat-shock Gmhsp17.3B promoter was addressed in the moss Physcomitrella patens, using β-glucuronidase (GUS) and an F-actin marker (GFP-talin) as reporter proteins. In stably transformed moss lines, Gmhsp17.3B-driven GUS expression was extremely low at 25 °C. In contrast, a short non-damaging heat-treatment at 38 °C rapidly induced reporter expression over three orders of magnitude, enabling GUS accumulation and the labelling of F-actin cytoskeleton in all cell types and tissues. Induction levels were tightly proportional to the temperature and duration of the heat treatment, allowing fine-tuning of protein expression. Repeated heating/cooling cycles led to the massive GUS accumulation, up to 2.3% of the total soluble proteins. The anti-inflammatory drug acetyl salicylic acid (ASA) and the membrane-fluidiser benzyl alcohol (BA) also induced GUS expression at 25 °C, allowing the production of recombinant proteins without heat-treatment. The Gmhsp17.3B promoter thus provides a reliable versatile conditional promoter for the controlled expression of recombinant proteins in the moss P. patens.     

丝氨酸内肽酶在黄瓜叶片衰老中的作用

采用丝氨酸内肽酶抑制剂和植物生长调节剂处理离体黄瓜叶片,研究了黄瓜叶片暗诱导衰老过程中丝氨酸内肽酶的作用。结果表明,6-BA50μmol／L与丝氨酸内肽酶抑制剂AEBSF能抑制叶片内肽酶活性的升高,延缓蛋白质降解,而ABA50μmol/L则促进了内肽酶活性的升高：其作用效果与AEBSF相反。活性电泳结果显示,黄瓜叶片中检测到6条内肽酶同工酶,其中4条（CEP2、3、4、6）为丝氨酸类型内肽酶,而ABA使丝氨酸内肽酶CEP2、3、4、6的活性明显增强,提示了丝氨酸类型内肽酶在黄瓜叶片衰老过程中具有重要作用。     

Effect of N-Guanyl Modifications on Early Steps in Catalysis of Polymerization by Sulfolobus solfataricus P2 DNA Polymerase Dpo4 T239W

Translesion DNA polymerases are more efficient at bypass of many DNA adducts than replicative polymerases. Previous work with the translesion polymerase Sulfolobus solfataricus Dpo4 showed a decrease in catalytic efficiency during bypass of bulky N²-alkyl guanine (G) adducts with N²-isobutylG showing the largest effect, decreasing ∼ 120-fold relative to unmodified deoxyguanosine (Zhang, H., Eoff, R. L., Egli, M., Guengerich, F. P. Versatility of Y-family Sulfolobus solfataricus DNA polymerase Dpo4 in translation synthesis past bulky N²-alkylguanine adducts. J. Biol. Chem. 2009; 284: 3563-3576). The effect of adduct size on individual catalytic steps has not been easy to decipher because of the difficulty of distinguishing early noncovalent steps from phosphodiester bond formation. We developed a mutant with a single Trp (T239W) to monitor fluorescence changes associated with a conformational change that occurs after binding a correct 2′-deoxyribonucleoside triphosphate (Beckman, J. W., Wang, Q., Guengerich, F. P. Kinetic analysis of nucleotide insertion by a Y-family DNA polymerase reveals conformational change both prior to and following phosphodiester bond formation as detected by tryptophan fluorescence. J. Biol. Chem. 2008; 283: 36711-36723) and, in the present work, utilized this approach to monitor insertion opposite N²-alkylG-modified oligonucleotides. We estimated maximal rates for the forward conformational step, which coupled with measured rates of product formation yielded rate constants for the conformational step (both directions) during insertion opposite several N²-alkylG adducts. With the smaller N²-alkylG adducts, the conformational rate constants were not changed dramatically (<  3-fold), indicating that the more sensitive steps are phosphodiester bond formation and partitioning into inactive complexes. With the larger adducts (≥  (2-naphthyl)methyl), the absence of fluorescence changes suggests impaired ability to undergo an appropriate conformational change, consistent with previous structural work.