Dichloroacetate attenuates hypoxia-induced resistance to 5-fluorouracil in gastric cancer through the regulation of glucose metabolism

In this study, we investigated whether gastric cancer with hypoxia-induced resistance to 5-fluorouracil (5-FU) could be re-sensitized following treatment with low-dose dichloroacetate (DCA), an inhibitor of the glycolytic pathway. The expression profiles of hypoxia-inducible factor-1α (HIF-1α) and pyruvate dehydrogenase kinase-1 (PDK-1) were analyzed in tissues from 10 patients with gastric cancer who had different responses to adjuvant 5-FU treatment. For the in vitro assays, cell viability and apoptosis were evaluated with and without treatment with 20 mM DCA in the AGS and MKN45 cell lines, as well as in PDK1 knockdown cell lines. The expression levels of HIF-1α and PDK-1 were both elevated in the tumor tissues relative to the normal gastric tissues of most patients who showed recurrence after adjuvant 5-FU treatment. Cellular viability tests showed that these cell lines had a lower sensitivity to 5-FU under hypoxic conditions compared to normoxic conditions. Moreover, the addition of 20 mM DCA only increased the sensitivity of these cells to 5-FU under hypoxic conditions, and the resistance to 5-FU under hypoxia was also attenuated in PDK1 knockdown cell lines. In conclusion, DCA treatment was able to re-sensitize gastric cancer cells with hypoxia-induced resistance to 5-FU through the alteration of glucose metabolism.     

Controlled release of d-glucose from starch granules containing 29% free d-glucose and Eudragit L100-55 as a binding and coating agent

Waxy maize starch (100% amylopectin) granules were modified by reaction of the granules with glucoamylase in a minimum amount of water to give 29% (w/w) d-glucose inside the granules [Kim, Y.-K.; Robyt, J. F. Carbohydr. Res.1999, 318, 129−134]. These granules were made into beads by dropping an ethanol slurry of starch and different amounts of Eudragit L100-55 in a constant ratio of 100:1 from a pipette onto Whatman 3MM filter paper. The starch beads were air dried and then repeatedly sprayed 0-12 times with 2.0% (w/v) Eudragit L100-55 in ethanol, with drying between each spraying, to coat the surface of the starch beads, giving different amounts of Eudragit L100-55 coating. Seven different kinds of beads, with different amounts of Eudragit L100-55 binding and coating agent, were obtained. The rates of release of d-glucose into water from the seven kinds of beads were inversely proportional to the amount of binding and coating agent. Bead type I, which was without any binding and coating gave a fast 100% release of d-glucose in 30 min. Beads II and III also gave a fast 100% release in 60 min and 90 min, respectively. Bead IV gave a near linear release of 97% d-glucose in 150 min; Bead V gave a 50% release in 120 min followed by the remaining 50% in 60 min; and Beads VI and VII gave a slow release of 10% and 4%, respectively, from 0 to 120 min, followed by a rapid 100% release from 120 to 180 min.     

Abrus abrin derived peptides induce apoptosis by targeting mitochondria in HeLa cells

In our previous study, Abrus abrin derived peptide fraction (ABP) with molecular weight in range of 600-1500 Da was shown to have potent antitumor activity in Dalton's lymphoma (DL) tumor bearing mice. The purpose of this study was to elucidate the mechanism of mitochondrial apoptosis induced by the peptide fraction. ABP was found to have selective antiproliferative activity (10 ng-100 ng/ml) on several tumor cell lines in vitro without having any cytotoxic effect on normal cell lines with a dose of 1000 ng/ml. Analysis of the growth inhibitory mechanism in HeLa cells revealed DNA fragmentation with appearance of the sub G₀/G₁ peak indicative of apoptosis. Further investigation results showed that the apoptotic machinery of HeLa induced by ABP was associated with the release of reactive oxygen species, a drop in mitochondrial transmembrane potential, upregulation of Bax, downregulation of Bcl-2, and activation of caspase-3. The peptide fraction was found to target mitochondria of HeLa cells as observed by confocal microscopy. This peptide fraction offers a source of mitochondria penetrating peptides which might have therapeutic induction of apoptosis in cancer cells.     

Toxicological effects of inorganic nanoparticles on human lung cancer A549 cells

Many researches have shown that anionic clays can be used as delivery carriers for drug or gene molecules due to their efficient cellular uptake in vitro, and enhanced permeability and retention effect in vivo. It is, therefore, highly required to establish a guideline on their potential toxicity for practical applications. The toxicity of anionic clay, layered metal hydroxide nanoparticle, was evaluated in two human lung epithelial cells, carcinoma A549 cells and normal L-132 cells, and compared with that in other human cancer cell lines such as cervical adenocarcinoma cells (HeLa) and osteosarcoma cells (HOS). The present nanoparticles showed little cytotoxic effects on the proliferation and viability of four cell lines tested at the concentrations used (<250 μg/ml) within 48 h. However, exposing cancer cells to high concentrations (250-500 μg/ml) for 72 h resulted in an inflammatory response with oxidative stress and membrane damage, which varied with the cell type (A549 > HOS > HeLa). On the other hand, the toxicity mechanism seems to be different from that of other inorganic nanoparticles frequently studied for biological and medicinal applications such as iron oxide, silica, and single walled carbon nanotubes. Iron oxide caused cell death associated with membrane damage, while single walled carbon nanotube induced oxidative stress followed by apoptosis. Silica triggered an inflammation response without causing considerable cell death for both cancer cells and normal cells, whereas layered metal hydroxide nanoparticle did not show any cytotoxic effects on normal L-132 cells in terms of inflammation response, oxidative stress, and membrane damage at the concentration of less than 250 μg/ml. It is , therefore, highly expected that the present nanoparticle can be used as a efficient vehicle for drug delivery and cancer cell targeting as well.     

Curcumin and wikstroflavone B,a new biflavonoid isolated from Wikstroemia indica,synergistically suppress the proliferation and metastasis of nasopharyngeal carcinoma cells via blocking FAK/STAT3 signaling pathway

BackgroundCurcumin (CUR) is a natural diarylheptanoid with marked anti-tumor activities. Recent investigations demonstrate that CUR combines with some other phytochemicals exerts advantages over its single application manifested as lower toxicity, higher efficacy or more significant reversal of multidrug resistance.PurposeThis study aimed to elucidate a new biflavonoid (wikstroflavone B, WFB) isolated from Wikstroemia indica and to assess the synergistic inhibition of combined CUR and WFB (CUR/WFB) on human nasopharyngeal carcinoma (NPC) cell lines proliferation and metastasis.MethodsWFB was obtained through sequential chromatographic methods including silica gel, Sephadex LH-20 and preparative HPLC. Its structure was determined by HRESIMS, 1D and 2D NMR spectroscopic analysis. The absolute configuration of WFB was assigned through comparison of experimental and calculated optical rotation (OR) values. Changes in cellular viability, migration and invasion were assessed by MTT, colony formation, wound healing and Transwell assays. The nature of synergistic interaction of CUR/WFB was determined through the combination index (CI) method under the median-effect analysis. Expression levels of indicated mRNAs and proteins were measured by qRT-PCR and Western blotting assays, respectively.ResultsWFB was isolated and structural elucidated. Compared with CUR or WFB used alone, CUR/WFB treatment inhibited more effectively on the cell viability, colony formation, cell migration and invasion. Both CI and dose reduction index (DRI) values indicated the significant synergistic effects existed between CUR and WFB. Besides, CUR/WFB showed the marked modulation on the genes involved in cell proliferation (survivin, cyclin D1, p53 and p21) and metastasis (MMP-2, MMP-9 and FAK). CUR/WFB treatment was also found to restrain the phosphorylation of FAK and STAT3 proteins. When pretreatment with a FAK inhibitor, the cell viability and metastasis were significantly attenuated.ConclusionThe results indicate that WFB can synergistically increase the inhibitory effects of CUR on NPC cells proliferation and metastasis, and these findings may afford a rational approach for developing the antitumor medications.     

Selective cytotoxicity of intense nanosecond-duration electric pulses in mammalian cells

Background

Nanosecond electric pulses (EP) disrupt cell membrane and organelles and cause cell death in a manner different from the conventional irreversible electroporation. We explored the cytotoxic effect of 10-ns EP (quantitation, mechanisms, efficiency, and specificity) in comparison with 300-ns, 1.8- and 9-μs EP.

Methods

Effects in Jurkat and U937 cells were characterized by survival assays, DNA electrophoresis and flow cytometry.

Results

10-ns EP caused apoptotic or necrotic death within 2–20 h. Survival (S, %) followed the absorbed dose (D, J/g) as: S = αD^(−K), where coefficients K and α determined the slope and the “shoulder” of the survival curve. K was similar in all groups, whereas α was cell type- and pulse duration-dependent. Long pulses caused immediate propidium uptake and phosphatidylserine (PS) externalization, whereas 10-ns pulses caused PS externalization only.

Conclusions

1.8- and 9-μs EP cause cell death efficiently and indiscriminately (LD₅₀ 1–3 J/g in both cell lines); 10-ns EP are less efficient, but very selective (LD₅₀ 50–80 J/g for Jurkat and 400–500 J/g for U937); 300-ns EP show intermediate effects. Shorter EP open propidium-impermeable, small membrane pores (”nanopores”), triggering different cell death mechanisms.

General significance

Nanosecond EP can selectively target certain cells in medical applications like tumor ablation.     

A truncated minimal-E1a gene with potency to support adenoviral replication mediates antitumor activity by down-regulating Neu expression and preserving Rb function

Oncolytic adenovirus is capable of infecting, replicating in and lysing cancer cells. In adenovirus infection and replication, the wild type E1a gene (wE1a) mediates various genetic events to facilitate viral replication and exert antitumor effect. To enhance its antitumor efficacy and optimize its safety, we manipulated the wE1a gene and designed a 720-bp truncated minimal-E1a (mE1a) by deletions and mutations of amino acid residues. The mE1a gene was incorporated in an adenovirus under the control of hTERT promoter, giving the vector AdDC315-mE1a. A variety of cancer cell lines infected with the virus expressed the mE1a protein and showed considerable down-regulation in Neu protein expression as compared to normal cell lines. mE1a also had a lower binding affinity to the Rb protein, preserving the Rb tumor suppressive function. The mE1a expression allowed efficient adenovirus replication with high and stable replication ratios in cancer cells (about 125- to 8500-fold higher at 48 h and 180- to 10,900-fold higher at 96 h post-infection). Further, the mE1a-supported oncolytic adenovirus induced higher cancer cell apoptosis, stronger cell cycle arrest and more effective antitumor efficacy in hepatocarcinoma xenografts in nude mice. In conclusion, the truncated minimal mE1a can act as a tumor inhibitor gene, and may be used to construct oncolytic adenovirus vectors for use in gene therapy of a variety of cancers.     

Use of TILLING and robotised enzyme assays to generate an allelic series of Arabidopsis thaliana mutants with altered ADP-glucose pyrophosphorylase activity

ADP-glucose pyrophosphorylase (AGPase) catalyses the synthesis of ADP-glucose, and is a highly regulated enzyme in the pathway of starch synthesis. In Arabidopsis thaliana, the enzyme is a heterotetramer, containing two small subunits encoded by the APS1 gene and two large subunits encoded by the APL1-4 genes. TILLING (Targeting Induced Local Lesions IN Genomes) of a chemically mutagenised population of A. thaliana plants identified 33 novel mutations in the APS1 gene, including 21 missense mutations in the protein coding region. High throughput measurements using a robotised cycling assay showed that maximal AGPase activity in the aps1 mutants varied from <15 to 117% of wild type (WT), and that the kinetic properties of the enzyme were altered in several lines, indicating a role for the substituted amino acid residues in catalysis or substrate binding. These results validate the concept of using such a platform for efficient high-throughput screening of very large populations of mutants, natural accessions or introgression lines. AGPase was estimated to have a flux control coefficient of 0.20, indicating that the enzyme exerted only modest control over the rate of starch synthesis in plants grown under short day conditions (8 h light/16 h dark) with an irradiance of 150 μmol quanta m⁻² s⁻¹. Redox activation of the enzyme, via reduction of the intermolecular disulphide bridge between the two small subunits, was increased in several lines. This was sometimes, but not always, associated with a decrease in the abundance of the APS1 protein. In conclusion, the TILLING technique was used to generate an allelic series of aps1 mutants in A. thaliana that revealed new insights into the multi-layered regulation of AGPase. These mutants offer some advantages over the available loss-of-function mutants, e.g. adg1, for investigating the effects of subtle changes in the enzyme's activity on the rate of starch synthesis.     

Interaction of 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate with mimetic membranes and cytotoxic effect on leukemic cells

10-(Octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC) is an alkylphospholipid that can interact with cell membranes because of its amphiphilic character. We describe here the interaction of ODPC with liposomes and its toxicity to leukemic cells with an ED-50 of 5.4, 5.6 and 2.9 μM for 72 h of treatment for inhibition of proliferation of NB4, U937 and K562 cell lines, respectively, and lack of toxicity to normal hematopoietic progenitor cells at concentrations up to 25 μM. The ED-50 for the non-malignant HEK-293 and primary human umbilical vein endothelial cells (HUVEC) was 63.4 and 60.7 μM, respectively. The critical micellar concentration (CMC) of ODPC was 200 μM. Dynamic light scattering indicated that dipalmitoylphosphatidylcholine (DPPC) liposome size was affected only above the CMC of ODPC. Differential calorimetric scanning (DCS) of liposomes indicated a critical transition temperature (T_c) of 41.5 °C and an enthalpy (?H) variation of 7.3 kcal mol⁻¹. The presence of 25 μM ODPC decreased T_c and ?H to 39.3 °C and 4.7 kcal mol⁻¹, respectively. ODPC at 250 μM destabilized the liposomes (36.3 °C, 0.46 kcal mol⁻¹). Kinetics of 5(6)-carboxyfluorescein (CF) leakage from different liposome systems indicated that the rate and extent of CF release depended on liposome composition and ODPC concentration and that above the CMC it was instantaneous. Overall, the data indicate that ODPC acts on in vitro membrane systems and leukemia cell lines at concentrations below its CMC, suggesting that it does not act as a detergent and that this effect is dependent on membrane composition.     

Up-regulation of annexin A2 in cholangiocarcinoma caused by Opisthorchis viverrini and its implication as a prognostic marker

Cholangiocarcinoma (CCA), or cancer of the bile ducts, is primarily associated with infection with the liver fluke Opisthorchis viverrini in northeast Thailand. The disease is associated with late presentation, poses challenges for diagnosis and has a high mortality rate - features that highlight the need for tumor markers. At present, there are no specific tumor markers that can indicate the early stages and status of CCA. Proteomic analysis of the proteins expressed on the surface of tumor cells is particularly difficult since proteome-wide analysis of surface membrane proteins has thus far been hampered by the lack of effective strategies to profile hydrophobic membrane proteins. In this study, a sequential protein extraction was utilized to overcome this problem. Membrane protein was extracted from four CCA cell lines with different tumor forming capabilities. The non-tumor H69 biliary cell line was used as a control. Two-dimensional-PAGE followed by MALDI-TOF-MS was used to identify differentially expressed proteins. Among 20 up-regulated membrane proteins identified in the CCA cell lines was ANXA2, a participant in tumor invasion and metastasis in other cancers. Accordingly, ANXA2 was verified in human subjects by probing, using a commercial anti-mouse monoclonal antibody and a tissue microarray of CCA (301 diagnosed cases), where it was found to associate with one of several tumor progression stages as reflected by lymphatic invasion (P = 0.014) and metastasis (P = 0.026). Patients with high expression of ANXA2 had a significantly shorter survival time (P = 0.011). ANXA2 expression in tumors may be useful for predicting the poor outcome of CCA patients.     

The role of cotyledon cell structure during in vitro digestion of starch in navy beans

Studies on the physico-chemical, microstructural characteristics and in vitro (under simulated gastric and small intestine conditions) starch digestibility of navy beans were carried out. The microstructure of raw and cooked beans observed through scanning electron microscopy (SEM) showed the presence of hexagonal or angular shaped cotyledon cells (50-100 μm size) containing starch granules with a size ranging between 10 and 50 μm. The extent of starch hydrolysis (%) after 120 min of in vitro gastro-intestinal digestion differed between whole navy beans (∼60%) and milled bean flour and bean starch (85-90%) after they were cooked under similar conditions. Starch hydrolysis (%) increased significantly when the cotyledon cells in the cooked whole navy beans were disrupted using high pressure treatment (French press). The storage of freshly cooked whole beans resulted in a lower (40-45%) starch hydrolysis whereas re-heating increased the same to 70-80% during in vitro small intestinal digestion. The SEM pictures of cooked navy bean digesta after different intervals of in vitro gastric and small intestinal digestion showed that the cotyledon cell structure is maintained well throughout the digestion period. However cotyledon cells appear shrunken and developed wrinkles during in vitro digestion. Particle size analysis of cooked bean paste taken before and after the in vitro gastro-intestinal digestion showed similar particle size distributions.     

Development and use of quantitative competitive PCR assays for relative quantifying rumen anaerobic fungal populations in both in vitro and in vivo systems

This paper describes the use of a quantitative competitive polymerase chain reaction (QC-PCR) assay; using PCR primers to the rRNA locus of rumen fungi and a standard-control DNA including design and validation. In order to test the efficiency of this method for quantifying anaerobic rumen fungi, it has been attempted to evaluate this method in in vitro conditions by comparing with an assay based on measuring cell wall chitin. The changes in fungal growth have been studied when they are grown in in vitro on either untreated (US) or sodium hydroxide treated wheat straw (TS). Results showed that rumen fungi growth was significantly higher in treated samples compared with untreated during the 12 d incubation (P < 0.05) and plotting the chitin assay's results against the competitive PCR's showed high positive correlation (R² ≥ 0.87). The low mean values of the coefficients of variance in repeatability in the QC-PCR method against the chitin assay demonstrated more reliability of this new approach. And finally, the efficiency of this method was investigated in in vivo conditions. Samples of rumen fluid were collected from four fistulated Holstein steers which were fed four different diets (basal diet, high starch, high sucrose and starch plus sucrose) in rotation. The results of QC-PCR showed that addition of these non-structural carbohydrates to the basal diets caused a significant decrease in rumen anaerobic fungi biomass. The QC-PCR method appears to be a reliable and can be used for rumen samples.     

Extraction and chemical characterization of starch from S. lycocarpum fruits

In this study the pulp from Solanum lycocarpum fruits was used as raw material for extraction of starch, resulting in a yield of 51%. The starch granules were heterogeneous in size, presenting a conical appearance, very similar to a high-amylose cassava starch. The elemental analysis (CHNS) revealed 64.33% carbon, 7.16% hydrogen and 0.80% nitrogen. FT-IR spectroscopy showed characteristic peaks of polysaccharides and NMR analysis confirmed the presence of the α-anomer of d-glucose. The S. lycocarpum starch was characterized by high value of intrinsic viscosity (3515 mPa s) and estimated molecular weight around 645.69 kDa. Furthermore, this starch was classified as a B-type and high amylose content starch, presenting 34.66% of amylose and 38% crystallinity. Endothermic transition temperatures (T_o = 61.25 °C, T_p = 64.5 °C, T_c = 67.5 °C), gelatinization temperature (ΔT = 6.3 °C) ranges and enthalpy changes (ΔH = 13.21 J g⁻¹) were accessed by DCS analysis. These results make the S. lycocarpum fruit a very promising source of starch for biotechnological applications.     

State of water in starch-water systems in the gelatinization temperature range as investigated using dielectric relaxation spectroscopy

The dielectric response of native wheat starch-water slurries containing 5-60% starch (w/w) was measured in the frequency range of 0.2-20 GHz after heating the slurries to 7 different temperatures between 25 and 90 °C for 30 min. Three relaxations, with relaxation time range of 4-9 ps, 20-25 ps and 230-620 ps at 25 °C, were identified from the dielectric spectra of starch slurries. The fastest relaxation process (4-9 ps) was attributed to bulk water while the two slower relaxations were attributed to the confined water molecules present in the starch-water system. The amount of water exhibiting the slowest relaxation (230-620 ps) was calculated to be 0.08-0.16 g water/g starch, which was close to the monolayer water associated with wheat starch. Mobility of bulk water was significantly reduced (P < 0.001) upon gelatinization at low starch concentration (10% starch), but remained unaffected at higher starch concentrations. The mobility of two slower relaxing water species was not significantly influenced (P > 0.19) by gelatinization at all starch concentrations.     

Formulation optimization of chelerythrine loaded O-carboxymethylchitosan microspheres using response surface methodology

The aims of this investigation were to develop a procedure to prepare chelerythrine (CHE) loaded O-carboxymethylchitosan (O-CMCS) microspheres by emulsion cross-linking method and optimize the process and formulation variables using response surface methodology (RSM) with a three-level, three-factor Box-Behnken design (BBD). The independent variables studied were O-CMCS/CHE ratio, O/W phase ratio, and O-CMCS concentration, dependent variables (responses) were drug loading content and encapsulation efficiency. Mathematical equations and response surface plots were used to relate the dependent and independent variables. The process and formulation variables were optimized to achieve maximum drug loading content and entrapment efficiency by the desirability function. The optimized microsphere formulation was characterized for particle size, shape, morphology and in vitro drug release. Results for mean particle size, drug loading content, entrapment efficiency, and in vitro drug release of CHE-loaded O-CMCS microspheres were found to be of 12.18 μm, 4.16 ± 3.36%, 57.40 ± 2.30%, and 54.5% at pH 7.4 after 70 h, respectively. The combination use of RSM, BBD and desirability function could provide a promising application for O-CMCS as controlled drug delivery carrier and help to develop procedures for a lab-scale microemulsion process.     

Physicochemical properties and in vitro digestibility of flour and starch from pea (Pisum sativum L.) cultivars

Flours and isolated starches from three different cultivars (1544-8, 1658-11 and 1760-8) of pea grown under identical environmental conditions were evaluated for their physicochemical properties and in vitro digestibility. The protein content, total starch content and apparent amylose content of pea flour ranged from 24.4 to 26.3%, 48.8 to 50.2%, and 13.9 to 16.7%, respectively. In pea starches, the 1760-8 showed higher apparent amylose content and total starch content than the other cultivars. Pea starch granules were irregularly shaped, ranging from oval to round with a smooth surface. All pea starches showed C-type X-ray diffraction pattern with relative crystallinity ranging between 23.7 and 24.7%. Pea starch had only a single endothermic transition (12.1-14.2 J/g) in the DSC thermogram, whereas pea flour showed two separate endothermic transitions corresponding to starch gelatinization (4.54-4.71 J/g) and disruption of the amylose-lipid complex (0.36-0.78 J/g). In pea cultivars, the 1760-8 had significantly higher setback and final viscosity than the other cultivars in both pea flour (672 and 1170 cP, respectively) and isolated starch (2901 and 4811 cP). The average branch chain length of pea starches ranged from 20.1 to 20.3. The 1760-8 displayed a larger proportion of short branch chains, DP (degree of polymerization) 6-12 (21.1%), and a smaller proportion of long branch chains, DP ≥ 37 (8.4%). The RDS, SDS and RS contents of pea flour ranged from 23.7 to 24.1%, 11.3 to 12.8%, and 13.2 to 14.8%, respectively. In pea starches, the 1760-8 showed a lower RDS content but higher SDS and RS contents. The expected glycemic index (eGI), based on the hydrolysis index, ranged from 36.9 to 37.7 and 69.8 to 70.7 for pea flour and isolated pea starch, respectively.