Insoluble Cell Research Articles

Retention of Proanthocyanidin in Wine-like Solution Is Conferred by a Dynamic Interaction between Soluble and Insoluble Grape Cell Wall Components.

For better understanding of the factors that impact proanthocyanidin (PA) adsorption by insoluble cell walls or interaction with soluble cell wall-derived components, application of a commercial polygalacturonase enzyme preparation was investigated to modify grape cell wall structure. Soluble and insoluble cell wall material was isolated from the skin and mesocarp components of Vitis vinifera Shiraz grapes. It was observed that significant depolymerization of the insoluble grape cell wall occurred following enzyme application to both grape cell wall fractions, with increased solubilization of rhamnogalacturonan-enriched, low molecular weight polysaccharides. However, in the case of grape mesocarp, the solubilization of protein from cell walls (in buffer) was significant and increased only slightly by the enzyme treatment. Enzyme treatment significantly reduced the adsorption of PA by insoluble cell walls, but this effect was observed only when material solubilized from grape cell walls had been removed. The loss of PA through interaction with the soluble cell wall fraction was observed to be greater for mesocarp than skin cell walls. Subsequent experiments on the soluble mesocarp cell wall fraction confirmed a role for protein in the precipitation of PA. This identified a potential mechanism by which extracted grape PA may be lost from wine during vinification, as a precipitate with solubilized grape mesocarp proteins. Although protein was a minor component in terms of total concentration, losses of PA via precipitation with proteins were in the order of 50% of available PA. PA-induced precipitation could proceed until all protein was removed from solution and may account for the very low levels of residual protein observed in red wines. The results point to a dynamic interaction of grape insoluble and soluble components in modulating PA retention in wine.

Dispersion stability of non-refined turnip rapeseed (Brassica rapa) protein concentrate: Impact of thermal, mechanical and enzymatic treatments

Aqueous dispersion properties of a protein concentrate obtained from turnip rapeseed (Brassica rapa) press cake were investigated. Protein content of the press cake was enriched from 35.9% to 45.8% by sequential supercritical-CO2 extraction, milling and air classification. The protein denaturation degree and the solubility in the prepared protein concentrate were compared with a commercial canola protein isolate. Partial loss of the protein native structure was observed in the protein concentrate. The ζ-potential in the soluble fraction of the protein concentrate dispersion was ca. 20mV lower (more negative) compared with the commercial isolate at pHs 4–10, which was attributed to the presence of charged carbohydrates such as pectin in the protein concentrate. To improve the dispersion stability of the protein concentrate, enzymatic crosslinking with transglutaminase and microfluidization at 1850 bars were applied. Microfluidization resulted in the formation of rod-like aggregates formed mainly of the insoluble cell wall polysaccharides glued together with proteins as imaged by confocal laser scanning microscopy. Improvement in colloidal stability was observed in all samples upon microfluidization. Pre-treatment with transglutaminase resulted in different distributions of the components in the rods and further improved dispersion stability against sedimentation. Heat treatment caused compaction of the structures.

Hydrogel arrays formed via differential wettability patterning enable combinatorial screening of stem cell behavior

Here, we have developed a novel method for forming hydrogel arrays using surfaces patterned with differential wettability. Our method for benchtop array formation is suitable for enhanced-throughput, combinatorial screening of biochemical and biophysical cues from chemically defined cell culture substrates. We demonstrated the ability to generate these arrays without the need for liquid handling systems and screened the combinatorial effects of substrate stiffness and immobilized cell adhesion peptide concentration on human mesenchymal stem cell (hMSC) behavior during short-term 2-dimensional cell culture. Regardless of substrate stiffness, hMSC initial cell attachment, spreading, and proliferation were linearly correlated with immobilized CRGDS peptide concentration. Increasing substrate stiffness also resulted in increased hMSC initial cell attachment, spreading, and proliferation; however, examination of the combinatorial effects of CRGDS peptide concentration and substrate stiffness revealed potential interplay between these distinct substrate signals. Maximal hMSC proliferation seen on substrates with either high stiffness or high CRGDS peptide concentration suggests that some baseline level of cytoskeletal tension was required for hMSC proliferation on hydrogel substrates and that multiple substrate signals could be engineered to work in synergy to promote mechanosensing and regulate cell behavior. Statement of significanceOur novel array formation method using surfaces patterned with differential wettability offers the advantages of benchtop array formation for 2-dimensional cell cultures and enhanced-throughput screening without the need for liquid handling systems. Hydrogel arrays formed via our method are suitable for screening the influence of chemical (e.g. cell adhesive ligands) and physical (stiffness, size, shape, and thickness) substrate properties on stem cell behavior. The arrays are also fully compatible with commercially available micro-array add-on systems, which allows for simultaneous control of the insoluble and soluble cell culture environment. This study used hydrogel arrays to demonstrate that synergy between cell adhesion and mechanosensing can be used to regulate hMSC behavior.

Antibiotic-killed Staphylococcus aureus induces destructive arthritis in mice.

Permanent reduction in joint function is a severe postinfectious complication in patients with Staphylococcus aureus septic arthritis. We undertook this study to determine whether this reduction in joint function might be caused by persistent joint inflammation after the adequate eradication of bacteria by antibiotics. After intraarticular injection of cloxacillin-killed S aureus into mouse knee joints, we investigated whether antibiotic-killed S aureus induced joint inflammation and elucidated the molecular and cellular mechanisms of this type of arthritis. Intraarticular injection of antibiotic-killed S aureus induced mild-to-moderate synovitis and bone erosions that lasted for a minimum of 14 days. Compared with wild-type animals, mice deficient in tumor necrosis factor receptor type I (TNFRI), receptor for advanced glycation end products (RAGE), or Toll-like receptor 2 (TLR-2) had a significantly reduced frequency and severity of synovitis. Combined depletion of monocytes and neutrophils also resulted in a significantly lower frequency of synovitis. Among bacterial factors, insoluble cell debris played a more important role than bacterial DNA or soluble components in inducing joint inflammation. Importantly, anti-TNF therapy abrogated joint inflammation induced by antibiotic-killed S aureus. Antibiotic-killed S aureus induced and maintained joint inflammation mediated through TLR-2, TNFRI, and RAGE. The cross-talk between neutrophils and monocytes is responsible for this type of arthritis. Anti-TNF therapy might be used as a novel strategy, in combination with antibiotics, to treat staphylococcal septic arthritis.

UV radiation biomonitoring using cell compartmentation of UV-absorbing compounds in herbarium samples of a liverwort

Ultraviolet-absorbing compounds (UVACs) were analyzed in 90 herbarium samples of the liverwort Jungermannia exsertifolia subsp. cordifolia collected throughout Spain in the period 1913–2006, in order to evaluate their usefulness in UV radiation biomonitoring. This was assessed, for the first time in bryophytes, differentiating the soluble (mainly vacuolar) and insoluble (cell wall-bound) UVACs (respectively, SUVACs and IUVACs), which may represent different mechanisms to cope with UV radiation. The bulk levels of SUVACs and IUVACs, and the concentrations of seven individual compounds (five SUVACs and two IUVACs) were analyzed, and several environmental variables of the sampling points were obtained: longitude, latitude, altitude, ozone column, and modelled UV levels.Diverse positive relationships of UVAC variables with UV radiation levels were identified in both the temporal and spatial scales. The soluble compound p-coumaroylmalic acid showed significantly higher concentrations in the years after the onset of stratospheric ozone degradation (around 1975) with respect to the previous years. The bulk level and the concentrations of three individual SUVACs were higher in the Spanish samples analyzed in the present study than in the northern European samples analyzed in a previous study in which IUVACs were not considered. The bulk level of IUVACs was the biological variable best spatially correlated with the UV exposure of the samples and with important factors determining it, such as altitude and latitude. Furthermore, the bulk level of IUVACs was the biological variable selected by a stepwise multiple linear regression analysis to reconstruct the past levels of UV-B radiation. The reconstructed UV-B did not show a clear trend in the period 1913–2006, which was coincident with other climatic and biological UV reconstructions. Both SUVACs and IUVACs were significant loading factors differentiating summer and autumn samples in the Principal Components Analysis conducted using both UVACs and the environmental variables, but again the bulk level of IUVACs was the main loading factor differentiating the samples collected from the highest and most UV-exposed localities (Sierra Nevada). In general, these findings suggest that UVACs were stimulated by increased UV levels in time or space, and that IUVACs would be better UV protectants than SUVACs, probably due to their more efficient screening capacity.Overall, UVACs of J. exsertifolia subsp. cordifolia were good UV biomarkers, and their location in the soluble or cell wall-bound fractions determined their usefulness in the different aspects of UV biomonitoring, including the assessment of the UV-B increase associated to anthropogenic ozone degradation.

A combined microfluidic-microstencil method for patterning biomolecules and cells.

Despite the myriad of soft lithography based micropatterning methods available to researchers, it is still challenging to define small features (10-100 μm) that are spaced far apart (1-10 mm). In this report, we describe a combined microfluidic-microstencil patterning method that can produce multifunctional substrates of small features, O(10 μm), with a large pitch, O(1 mm). In that, we fabricate microstencils using an UV curable polyurethane (Norland Optical Adhesive 81) with dense arrays of 10-100 μm holes. Overlaying arrays of microfluidic channels over these microstencils allow for the control of the spacing between features and the ability to pattern multiple substrates. We show that this method is capable of patterning soluble proteins, fibrillar insoluble collagen, liposomes, cells, and nanoparticles. We demonstrate the utility of the method by measuring platelet adhesion under flow to three adhesive proteins (insoluble fibrillar collagen, laminin, and reconstituted acid solubilized collagen fibers) in a single assay.

Human stefin B role in cell's response to misfolded proteins and autophagy.

Alternative functions, apart from cathepsins inhibition, are being discovered for stefin B. Here, we investigate its role in vesicular trafficking and autophagy. Astrocytes isolated from stefin B knock-out (KO) mice exhibited an increased level of protein aggregates scattered throughout the cytoplasm. Addition of stefin B monomers or small oligomers to the cell medium reverted this phenotype, as imaged by confocal microscopy. To monitor the identity of proteins embedded within aggregates in wild type (wt) and KO cells, the insoluble cell lysate fractions were isolated and analyzed by mass spectrometry. Chaperones, tubulins, dyneins, and proteosomal components were detected in the insoluble fraction of wt cells but not in KO aggregates. In contrast, the insoluble fraction of KO cells exhibited increased levels of apolipoprotein E, fibronectin, clusterin, major prion protein, and serpins H1 and I2 and some proteins of lysosomal origin, such as cathepsin D and CD63, relative to wt astrocytes. Analysis of autophagy activity demonstrated that this pathway was less functional in KO astrocytes. In addition, synthetic dosage lethality (SDL) gene interactions analysis in Saccharomyces cerevisiae expressing human stefin B suggests a role in transport of vesicles and vacuoles These activities would contribute, directly or indirectly to completion of autophagy in wt astrocytes and would account for the accumulation of protein aggregates in KO cells, since autophagy is a key pathway for the clearance of intracellular protein aggregates.

Host-induced bacterial cell wall decomposition mediates pattern-triggered immunity in Arabidopsis.

Peptidoglycans (PGNs) are immunogenic bacterial surface patterns that trigger immune activation in metazoans and plants. It is generally unknown how complex bacterial structures such as PGNs are perceived by plant pattern recognition receptors (PRRs) and whether host hydrolytic activities facilitate decomposition of bacterial matrices and generation of soluble PRR ligands. Here we show that Arabidopsis thaliana, upon bacterial infection or exposure to microbial patterns, produces a metazoan lysozyme-like hydrolase (lysozyme 1, LYS1). LYS1 activity releases soluble PGN fragments from insoluble bacterial cell walls and cleavage products are able to trigger responses typically associated with plant immunity. Importantly, LYS1 mutant genotypes exhibit super-susceptibility to bacterial infections similar to that observed on PGN receptor mutants. We propose that plants employ hydrolytic activities for the decomposition of complex bacterial structures, and that soluble pattern generation might aid PRR-mediated immune activation in cell layers adjacent to infection sites.

Purification and biocompatibility of fermented hyaluronic acid for its applications to biomaterials.

BackgroundHyaluronic acid (HA) is of importance due to its diverse applications in pharmaceuticals and medical devices such as dermal filler, adhesion barriers, carrier for cells and bioactive molecules as well as scaffold biomaterials for tissue engineering. Evaluations of purification and biocompatibility of HA are required for its applications to biomaterials.ResultsAfter synthesizing HA by fermentation of streptococcus zooepidemicus for 25 hr, extensively purification of the fermented broth was performed to remove impurities using a filtration process for insoluble components and cells, and diverse adsorbents for soluble impurities. Its in vitro biocompatibility has been evaluated by measurement of cell counting and assay of cell live and dead. 60% yield of white HA powder was obtained, having 15–17 dL/g intrinsic viscosity with a molecular weight of approximately 1,000 kDa. While low molecular weight impurities and insoluble impurities were successfully removed using a ultrafiltration membrane with 50 KDa molecular weight cut, endotoxins, high molecular weight proteins and nucleic acids were removed from the broth by employing adsorbents such as alumina and activated carbons. Alumina showed the best results for the removal of endotoxins, all of the activated carbons were very effective in the removal of high molecular weight proteins and nucleic acids. The purified HA solution showed excellent cell compatibility with no cell damages as observed by both measurement of cell proliferation and observation of cell viability.ConclusionsWe obtained high molecular weight HA with excellent biocompatibility as judged by both measurement of cell proliferation and viability, indicating high possibility of its applications to biomaterials.

Characterisation of soluble and insoluble cell wall fractions from rye, wheat and hull-less barley endosperm flours

Within cereal endosperm flours, arabinoxylan and β-glucan molecules exist in either a soluble or an insoluble form. From a nutritional functionality viewpoint, soluble and insoluble forms offer different potential health advantages, so it is important to define both the features controlling solubilisation and the properties of each of the soluble and insoluble fractions. Factors known to affect the stability of arabinoxylan (AX) and β-glucan (BG) solutions include AX branching extent and type, and the ratio of cellotriose to cellotetraose units (DP3/DP4) in BG. Through studying the solubilisation of AX and BG from wheat, rye, and hull less barley endosperm under conditions that avoid the use of alkali or ethanol during the solubilisation process, we report (a) similar A/X ratios and fine structures for extracted soluble arabinoxylan and the corresponding insoluble AX within the cell walls for rye and wheat endosperm flours, (b) comparable DP3/DP4 ratios for soluble β-glucan, flour and insoluble β-glucan within the endosperm cell wall of hull less barley, and (c) evidence for enrichment of β-glucan at the exterior of residual insoluble cell walls. Therefore, the factors determining solubilisation of AX and BG from endosperm cell walls are different to those that determine the stability of aqueous solutions of the same polymers, and β-glucan may show limited solubilisation by being trapped within restraining cross-linked arabinoxylans in the cell wall.

Identification of Ligand Specificity Determinants in Lantibiotic Bovicin HJ50 and the Receptor BovK, a Multitransmembrane Histidine Kinase

Lantibiotic bovicin HJ50 is produced by Streptococcus bovis HJ50 and acts as the extracellular signal to autoregulate its own biosynthesis through BovK/R two-component system. Bovicin HJ50 shows a linear N-terminal and glubolar C-terminal structure, and the sensor histidine kinase BovK contains eight transmembrane segments lacking any extensive surface-exposed sensory domain. The signal recognition mechanism between bovicin HJ50 and BovK is still unknown. We performed saturated alanine scanning mutagenesis and other amino acid substitutions on bovicin HJ50 using a semi-in vitro biosynthesis. Results of the mutants inducing activities indicated that several charged and hydrophobic amino acids in ring B of bovicin HJ50, as well as two glycines were key residues to recognize BovK. Circular dichroism analyses indicated that both glycines contributed to bovicin HJ50 structural changes in the membrane. Biotin-labeled bovicin HJ50 could interact with the N-terminal sensor of BovK, and several charged residues and a conserved hydrophobic region in the N-terminal portion of BovK sensor domain were important for interacting with the signal bovicin HJ50. By combining the results, we suggested a mechanism of bovicin HJ50 recognizing and activating BovK mainly through electrostatic and hydrophobic interactions.

Engineering Hydrogen Gas Production from Formate in a Hyperthermophile by Heterologous Production of an 18-Subunit Membrane-bound Complex

Biohydrogen gas has enormous potential as a source of reductant for the microbial production of biofuels, but its low solubility and poor gas mass transfer rates are limiting factors. These limitations could be circumvented by engineering biofuel production in microorganisms that are also capable of generating H2 from highly soluble chemicals such as formate, which can function as an electron donor. Herein, the model hyperthermophile, Pyrococcus furiosus, which grows optimally near 100 °C by fermenting sugars to produce H2, has been engineered to also efficiently convert formate to H2. Using a bacterial artificial chromosome vector, the 16.9-kb 18-gene cluster encoding the membrane-bound, respiratory formate hydrogen lyase complex of Thermococcus onnurineus was inserted into the P. furiosus chromosome and expressed as a functional unit. This enabled P. furiosus to utilize formate as well as sugars as an H2 source and to do so at both 80° and 95 °C, near the optimum growth temperature of the donor (T. onnurineus) and engineered host (P. furiosus), respectively. This accomplishment also demonstrates the versatility of P. furiosus for metabolic engineering applications.

Cell signalling molecules

All periodontal cells are capable of expressing a broad range of gene products, including matrix proteins, proteases involved in tissue remodeling (such as collagenases) and cytokines. Mesenchymal cells in the periodontal connective tissues encounter a complex array of stimuli that include soluble agents (such as polypeptide growth factors, cytokines, neuropeptide and chemotherapeutic agents), insoluble extracellular matrix proteins and other cells. The mechanisms that permit cells to respond to extracellular stimuli are essential for normal growth and function. Characterization of how these pathways are up-regulated or down-regulated in response to other agents may eventually allow the predictability of periodontal regeneration to be enhanced. The current review aims to provide a comprehensive overview to the various cell signaling pathways and molecules that mediate the periodontal disease process and can serve as a target for periodontal therapy.

Interactions between grape skin cell wall material and commercial enological tannins. Practical implications

Commercial enological tannins were used to investigate the role that cell wall material plays in proanthocyanidin adsorption. Insoluble cell wall material, prepared from the skin of Vitis vinifera L. cv. Monastrell berries, was combined with solutions containing six different commercial enological tannins (proanthocyanidin-type tannins). Analysis of the proanthocyanidins in the solution, after fining with cell wall material, using phloroglucinolysis and size exclusion chromatography, provided quantitative and qualitative information on the non-adsorbed compounds. Cell wall material showed strong affinity for the proanthocyanidins, one of the commercial tannins being bound up to 61% in the experiment. Comparison of the molecular mass distribution of the commercial enological tannins in solution, before and after fining, suggested that cell walls affinity for proanthocyanidins was more related with the proanthocyanidin molecular mass than with their percentage of galloylation. These interactions may have some enological implications, especially as regards the time of commercial tannins addition to the must/wine.

Rapid and Deep Human Proteome Analysis by Single-dimension Shotgun Proteomics

Multiparameter optimization of an LC-MS/MS shotgun proteomics experiment was performed without any hardware or software modification of the commercial instrument. Under the optimized experimental conditions, with a 50-cm-long separation column and a 4-h LC-MS run (including a 3-h optimized gradient), 4,825 protein groups and 37,550 peptides were identified in a single run and 5,354 protein groups and 56,390 peptides in a triplicate analysis of the A375 human cell line, for approximately 50% coverage of the expressed proteome. The major steps enabling such performance included optimization of the cell lysis and protein extraction, digestion of even insoluble cell debris, tailoring the LC gradient profile, and choosing the optimal dynamic exclusion window in data-dependent MS/MS, as well as the optimal m/z scan window.

Structure, Activity, and Substrate Selectivity of the Orf6 Thioesterase from Photobacterium profundum

Thioesterase activity is typically required for the release of products from polyketide synthase enzymes, but no such enzyme has been characterized in deep-sea bacteria associated with the production of polyunsaturated fatty acids. In this work, we have expressed and purified the Orf6 thioesterase from Photobacterium profundum. Enzyme assays revealed that Orf6 has a higher specific activity toward long-chain fatty acyl-CoA substrates (palmitoyl-CoA and eicosapentaenoyl-CoA) than toward short-chain or aromatic acyl-CoA substrates. We determined a high resolution (1.05 Å) structure of Orf6 that reveals a hotdog hydrolase fold arranged as a dimer of dimers. The putative active site of this structure is occupied by additional electron density not accounted for by the protein sequence, consistent with the presence of an elongated compound. A second crystal structure (1.40 Å) was obtained from a crystal that was grown in the presence of Mg(2+), which reveals the presence of a binding site for divalent cations at a crystal contact. The Mg(2+)-bound structure shows localized conformational changes (root mean square deviation of 1.63 Å), and its active site is unoccupied, suggesting a mechanism to open the active site for substrate entry or product release. These findings reveal a new thioesterase enzyme with a preference for long-chain CoA substrates in a deep-sea bacterium whose potential range of applications includes bioremediation and the production of biofuels.

Effect of Cd(ii) and Se(iv) exposure on cellular distribution of both elements and concentration levels of glyoxal and methylglyoxal in Lepidium sativum

In this work, the effect of cadmium (0-5.0 mg L(-1) as cadmium chloride, Cd(II)) and selenium (0-2.0 mg L(-1) as sodium selenite, Se(IV)) was studied in Lepidium sativum with specific focus on glyoxal (GO) and methylglyoxal (MGO) and on the cellular distribution of both elements under different exposure conditions. The concentrations of two reactive α-ketoaldehydes present as natural metabolites and as by-products of lipid peroxidation, were increased in plants treated with Cd(II), providng complementary experimental evidence on element phytotoxicity in garden cress, in terms of oxidative damage. Even though for higher than 1.0 mg L(-1) Se in medium similar adverse effect was found, under simultaneous exposure to both elements the changes in GO and MGO concentrations were clearly attenuated as compared to a single stressor treatment. This effect was accompanied by lower uptake of the two elements, significant decrease of their relative distribution in the fraction containing polar compounds and their increase in fraction corresponding to insoluble cell fragments/components, suggesting that the direct in vivo interaction between two element forms might be involved in the favorable effects of simultaneous treatment with Cd(II) + Se(IV). The fluorescence spectra obtained for biomass extracts corresponding to different exposure conditions suggested possible in vivo formation of CdSe quantum dots; however further studies are needed for ultimate identification and characterization of such nanoparticulate species.

Proline and hydroxyproline containing cell wall proteins of red raspberry (Rubus idaeus L.) cane cortex

The formation of insoluble wall bound proline-rich proteins (PRPs) and hydroxyproline-rich glycoproteins (HRGPs) in relation to the growth stage and developmental changes was examined in red raspberry primocane cortex. Two cultivars - 'Latham' and 'Malling Promise' - differing in growth intensity and in sensitivity to biotic stress were used for the experiments. The level of insoluble cell wall bound PRPs of the cortical tissues was almost similar in the course of the plants growth period. HRGPs were significantly higher at the end of the vegetation season (August/September), especially in 'Malling Promise'. The plant sensitivity to biotic stress, based on the response to the fungus Didymella applanata changed in the period of vegetative growth. At the end of summer the both cultivars became more resistant. It has been inferred that the insolubilization of wall PRPs and HRGPs was responsible for the acquiring resistance to D.applanata by 'Malling Promise', which was previously very susceptible to this fungus.

Transglucosylation reaction of amylomaltase for the synthesis of anticariogenic oligosaccharides

Abstract Synthesis of maltooligosylsucroses by the recombinant amylomaltase from Corynebacterium glutamicum as a N terminal (His)6 chimera is reported. From the analysis of the products, by TLC and HPLC analysis on a Rezex RSO-Oligosaccharide column, the suitable glucosyl donor was found to be raw tapioca starch. The optimal condition was 2.0% (w/v) sucrose, 2.5% (w/v) raw tapioca starch and 9 U/ml of amylomaltase at 30 °C for 48 h, giving an overall 82% yield of maltooligosylsucrose products. After purified by Bio-Gel P-2 size exclusion column chromatography, the main products were determined by MS and NMR analysis to be maltosyl-, maltotriosyl-, maltotretraosyl- and maltopentaosyl-fructosides (G2F, G3F, G4F and G5F, respectively, where G = glucosyl unit and F = fructose) with an α-1,4 linkage between the added glucosyl unit and the sucrose. The low cariogenic property of these maltooligosylsucrose products was confirmed by analyzing the effect on the synthesis of water insoluble glucan, acid fermentation, plaque formation and cell aggregation of Streptococcus mutans when compared to those exerted by sucrose. Moreover, by adding sucrose to maltooligosylsucrose products at ratios of 1:1, 1:2 and 1:4, the inhibitory effects on glucosyltransferase activity of S. mutans by 7, 33 and 50%, respectively, were observed. These results suggest that the obtained maltooligosylsucrose products have an anticariogenic property and could be used to substitute for sucrose in food or related products.

Induction of Tumor Cell Death through Targeting Tubulin and Evoking Dysregulation of Cell Cycle Regulatory Proteins by Multifunctional Cinnamaldehydes

Multifunctional trans-cinnamaldehyde (CA) and its analogs display anti-cancer properties, with 2-benzoyloxycinnamaldehyde (BCA) and 5-fluoro-2-hydroxycinnamaldehyde (FHCA) being identified as the ortho-substituted analogs that possess potent anti-tumor activities. In this study, BCA, FHCA and a novel analog 5-fluoro-2-benzoyloxycinnamaldehyde (FBCA), were demonstrated to decrease growth and colony formation of human colon-derived HCT 116 and mammary-derived MCF-7 carcinoma cells under non-adhesive conditions. The 2-benzoyloxy and 5-fluoro substituents rendered FBCA more potent than BCA and equipotent to FHCA. The cellular events by which these cinnamaldehydes caused G2/M phase arrest and halted proliferation of HCT 116 cells were thereby investigated. Lack of significant accumulation of mitosis marker phospho-histone H3 in cinnamaldehyde-treated cells indicated that the analogs arrested cells in G2 phase. G2 arrest was brought about partly by cinnamaldehyde-mediated depletion of cell cycle proteins involved in regulating G2 to M transition and spindle assembly, namely cdk1, cdc25C, mad2, cdc20 and survivin. Cyclin B1 levels were found to be increased, which in the absence of active cdk1, would fail to drive cells into M phase. Concentrations of cinnamaldehydes that brought about dysregulation of levels of cell cycle proteins also caused tubulin aggregation, as evident from immunodetection of dose-dependent tubulin accumulation in the insoluble cell lysate fractions. In a cell-free system, reduced biotin-conjugated iodoacetamide (BIAM) labeling of tubulin protein pretreated with cinnamaldehydes was indicative of drug interaction with the sulfhydryl groups in tubulin. In conclusion, cinnamaldehydes treatment at proapoptotic concentrations caused tubulin aggregation and dysegulation of cell cycle regulatory proteins cdk1 and cdc25C that contributed at least in part to arresting cells at G2 phase, resulting in apoptotic cell death characterized by emergence of cleaved forms of caspase 3 and poly (ADP-ribose) polymerase (PARP). Results presented in this study have thus provided further insights into the intricate network of cellular events by which cinnamaldehydes induce tumor cell death.