Cannabidiol Overcomes Oxaliplatin Resistance by Enhancing NOS3- and SOD2-Induced Autophagy in Human Colorectal Cancer Cells.

Although oxaliplatin is an effective chemotherapeutic drug for colorectal cancer (CRC) treatment, patients often develop resistance to it. Therefore, a new strategy for CRC treatment is needed. The purpose of this study was to determine the effect of cannabidiol (CBD), one of the components of the cannabis plant, in overcoming oxaliplatin resistance in CRC cells. We established oxaliplatin-resistant cell lines, DLD-1 R and colo205 R, in CRC DLD-1 and colo205 cells. Autophagic cell death was induced when oxaliplatin-resistant cells were treated with both oxaliplatin and CBD. Additionally, phosphorylation of nitric oxide synthase 3 (NOS3) was increased in oxaliplatin-resistant cells compared to that in parent cells. Combined treatment with oxaliplatin and CBD reduced phospho-NOS3 levels and nitric oxide (NO) production and resulted in the production of reactive oxygen species (ROS) by reducing the levels of superoxide dismutase 2, an antioxidant present in the mitochondria, causing mitochondrial dysfunction. Taken together, these results suggest that elevated phosphorylation of NOS3 is essential for oxaliplatin resistance. The combination of oxaliplatin and CBD decreased NOS3 phosphorylation, which resulted in autophagy, by inducing the overproduction of ROS through mitochondrial dysfunction, thus overcoming oxaliplatin resistance.

Cannabidiol Enhances the Therapeutic Effects of TRAIL by Upregulating DR5 in Colorectal Cancer.

Cannabidiol, a major non-psychotomimetic compound derived from Cannabis sativa, is a potential therapeutic agent for a variety of diseases such as inflammatory diseases, chronic neurodegenerative diseases, and cancers. Here, we found that the combination of cannabidiol and TNF-related apoptosis-inducing ligand (TRAIL) produces synergistic antitumor effects in vitro. However, this synergistic effect was not observed in normal colonic cells. The levels of ER stress-related proteins, including C/EBP homologous protein (CHOP) and phosphorylated protein kinase RNA-like ER kinase (PERK) were increased in treatment of cannabidiol. Cannabidiol enhanced significantly DR5 expression by ER stress. Knockdown of DR5 decreased the combined effect of cannabidiol and TRAIL. Additionally, the combination of TRAIL and cannabidiol decreased tumor growth in xenograft models. Our studies demonstrate that cannabidiol enhances TRAIL-induced apoptosis by upregulating DR5 and suggests that cannabidiol is a novel agent for increasing sensitivity to TRAIL.

Cannabidiol-induced apoptosis is mediated by activation of Noxa in human colorectal cancer cells.

Cannabidiol (CBD), one of the compounds present in the marijuana plant, has anti-tumor properties, but its mechanism is not well known. This study aimed to evaluate the apoptotic action of CBD in colorectal cancer (CRC) cells, and focused on its effects on the novel pro-apoptotic Noxa-reactive oxygen species (ROS) signaling pathway. CBD experiments were performed using the CRC cell lines HCT116 and DLD-1. CBD induced apoptosis by regulating many pro- and anti-apoptotic proteins, of which Noxa showed significantly higher expression. To understand the relationship between Noxa and CBD-induced apoptosis, Noxa levels were downregulated using siRNA, and the expression of apoptosis markers decreased. After ROS production was blocked, the level of Noxa also decreased, suggesting that ROS is involved in the regulation of Noxa, which along with ROS is a well-known pro-apoptotic signaling agents. As a result, CBD induced apoptosis in a Noxa-and-ROS-dependent manner. Taken together, the results obtained in this study re-demonstrated the effects of CBD treatment in vivo, thus confirming its role as a novel, reliable anticancer drug.

Hepatoprotective Effect of Gallotannin-enriched Extract Isolated from Gall on Hydrogen Peroxide-induced Cytotoxicity in HepG2 Cells.

BACKGROUND: Gall (Galla Rhois [GR]) is known to have antibacterial, anti-inflammatory, antimetastatic, and anti-invasion activities and exert hepatoprotective effects. However, the hepatoprotective effects of gallotannin-enriched GR (GEGR) and their mechanisms have not yet been investigated. OBJECTIVE: The potential protective effect of GEGR against hepatotoxicity induced by hydrogen peroxide (H2O2) was investigated. MATERIALS AND METHODS: Changes in cell viability, apoptosis protein expression, and reactive oxygen species (ROS) generation were determined in HepG2 cells that were pretreated with four different concentrations of GEGR (6.25-50 µg/ml) for 24 h before H2O2 exposure. RESULTS: GEGR consisted of gallotannin (69.2%), gallic acid (26.6%), and methyl gallate (4.2%) and showed remarkable 2,2-diphenyl-1-picrylhydrazyl scavenging activity (inhibitory concentration 50% = 0.212 µg/ml). The lethal dose 50% and effective dose 50% values for the response of HepG2 cells to GEGR were determined to be 178 and 6.85 µg/ml, respectively. Significant reductions in the immunofluorescence intensity indicating apoptosis were also detected in the nuclei of HepG2 cells stained with 4',6-diamidino-2-phenylindole and Annexin V after GEGR treatment. The Bax/Bcl-2 ratio and active caspase-3 level were higher in H2O2 + vehicle-treated cells. However, these levels gradually decreased to those of the No-treated group in the GEGR pretreated group even though little or no decrease was observed in response to low GEGR concentrations. Furthermore, the GEGR pretreated group showed a reduced level of 2',7'-dichlorofluorescein diacetate stained cells, indicating ROS generation relative to the H2O2 + vehicle-treated group. CONCLUSION: The results of this study provide strong evidence that GEGR can prevent cell death induced by H2O2 in HepG2 cells through the induction of antioxidant conditions. SUMMARY: The gallotannin (69.2%), gallic acid (26.6%), and methyl gallate (4.2%) are the main constituents of water extracts of GRGEGR was more potent in DPPH scavenging, and gallotannin contributes to this extract activityGEGR significantly reduced the increase of apoptosis, Bax/Bcl-2 ratio, and active caspase-3 level after H2O2 treatmentGEGR pretreatment showed protection against H2O2-induced ROS production in DCFH-DA staining analysis. Abbreviations used: COX: Cyclooxygenase; DAPI: 4',6-diamidino-2-phenylindole; DMSO: Dimethyl sulfoxide; DPPH: 2,2-diphenyl-1-picrylhydrazyl; GEGR: Gallotannin-enriched Galla Rhois; GR: Galla Rhois; HPLC: High-performance liquid chromatography; H2O2: Hydrogen peroxide; MMP: Metallopeptidase; MTT: 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; ROS: Reactive oxygen species; UV-Vis: Ultraviolet-visible.

Synthesis and Properties of Waterborne Polyurethane (WBPU)/Modified Lignin Amine (MLA) Adhesive: A Promising Adhesive Material.

A series of waterborne polyurethane (WBPU)/modified lignin amine (MLA) adhesives was prepared using MLA as a chain extender by a prepolymer mixing process. A successful Mannich reaction was achieved during the synthesis of MLA by reacting lignin with bis(3-aminopropyl)amine. Higher tensile strength, Young's modulus, and thermal stability were recorded for WBPU/MLA adhesives with higher MLA contents. The WBPU/MLA adhesive materials were used to coat polyvinyl chloride (PVC) substrates. The adhesive strength increased with increasing MLA content. More importantly, the MLA also enhanced the WBPU/MLA coating in terms of adhesive strength at moderately high temperatures as well as under natural weather exposed conditions. The adhesive strength was essentially unaffected with 6.48 mol % MLA in the WBPU/MLA coating after exposure to natural weather conditions for 180 days.

Effect of blend ratio of PP/kapok blend nonwoven fabrics on oil sorption capacity.

More research and development on novel oil sorbent materials is needed to protect the environmental pollution. New nonwoven fabrics (pads) of polypropylene (PP)/kapok blends (blend ratio: 100/0, 75/25, 50/50, 25/75 and 10/90) were prepared by needle punching process at a fixed (optimized) condition (punch density: 50 punches/cm2 and depth: 4mm). This study focused on the effect of blend ratio of PP/kapok nonwoven fabrics on oil sorption capacities to find the best blend ratio having the highest synergy effect. The PP/kapok blend (50/50) sample has the lowest bulk density and showed the best oil absorption capacity. The oil sorption capacity of PP/kapok blend (50/50) nonwoven fabric for kerosene/soybean oil [21.09/27.01 (g oil/g sorbent)] was 1.5-2 times higher than those of commercial PP pad oil sorbents. The highest synergy effect of PP/kapok blend (50/50) was ascribed to the lowest bulk density of PP/kapok blend (50/50), which might be due to the highest morphologically incompatibility between PP fibre and kapok. These results suggest that the PP/kapok blend (50/50) having the highest synergy effect has a high potential as a new high-performance oil sorbent material.

Synthesis and properties of waterborne polyurethane hydrogels for wound healing dressings.

To accomplish ideal wound healing dressing, a series of waterborne polyurethane (WBPU) hydrogels based on polyethylene glycol (PEG) were synthesized by polyaddition reaction in an emulsion system. The stable WBPU hydrogels which have remaining weight of above 85% were obtained. The effect of the soft segment (PEG) content on water absorbability of WBPU hydrogels was investigated. Water absorption % and equilibrium water content (%) of the WBPU hydrogel significantly increased in proportion to PEG content and the time of water-immersion. The maximum water absorption % and equilibrium water content (%) of WBPU hydrogels containing various PEG contents were in the range of 409-810% and 85-96%, respectively. The water vapor transmission rate of the WBPU hydrogels was found to be in the range of 1490-3118 g/m(2)/day. These results suggest that the WBPU hydrogels prepared in this study may have high potential as new wound dressing materials, which provide and maintain the adequate moist environment required to prevent scab formation and dehydration of the wound bed. By the wound healing evaluation using full-thickness rat model experiment, it was found that the wound covered with a typical WBPU hydrogel (HG-78 sample) was completely filled with new epithelium without any significant adverse reactions.

Characteristics of crosslinked blends of Pellethene and multiblock polyurethanes containing phospholipid.

A series of segmented multiblock polyurethanes (MPUs) were synthesized by polyaddition reaction using hexamethylene diisocyanate (HDI)/poly(ethylene oxide) (PEO, as a hydrophilic component)/ poly(tetramethylene oxide) (PTMO)/ poly(butadiene diol)(PBD)/1,4-butanediol(BD)/(2-[bis(2-hydroxyethyl) methyl ammonio]ethyl stearyl phosphate)[BESP, as a phospholipids component: 0-42 mol% (0-9 wt%)]. To improve the blood compatibility of biomedical grade polyurethane (Pellethene), the Pellethene was blended with MPUs and then crosslinked using dicumyl peroxide as a crosslinking agent. Effects of BESP content [0-42 mol% (0-9 wt%)] in MPUs on the properties of MPUs and blend (Pellethene/MPUs) films were investigated. The X-ray photoelectron spectra indicated that the BESP moieties were located at the surface of the crosslinked blend (Pellethene/MPUs) films. As the BESP content in MPUs increased, the water contact angle on the surfaces of crosslinked blend film was decreased but the water absorption and mechanical properties were markedly increased. By the test of platelet adhesion on the surfaces of crosslinked blend film, it was found that the platelet adhesion on the surface was significantly decreased from 70% to 6% by increasing BESP content from 0 to 42 mol% (0-9 wt%) in MPUs. These results suggest that crosslinked blend films may have more potential as a new material for biomedical applications, which are directly in contact with blood.

Inhibitory effect of Spirodela polyrhixa on the secretion of NO in LPS-stimulated macrophages.

Spirodela polyrhixa Schleid has been used in folk medicine to treat inflammatory diseases. Since nitric oxide (NO) is one of the major inflammatory parameters, we studied the effect of aqueous extracts of Spirodela polyrhixa (AESP) on NO production in lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages. AESP inhibited the secretion of NO in macrophages, without affecting cell viability. The protein level of inducible nitric oxide synthase (iNOS) in peritoneal macrophages was also decreased by AESP. Transient transfection assay of reporter plasmid and gel shift assay indicated that AESP blocked the activation of nuclear factor-kappa B (NF-kappa B), which was considered to be a potential transcription factor for iNOS expression. AESP also blocked the phosphorylation and degradation of inhibitory protein I kappa B-alpha (I kappa B-alpha). These results suggest that AESP could exert its anti-inflammatory actions by suppressing the synthesis of NO through inhibition of NF-kappa B activity.

Coactivator ASC-2 mediates heat shock factor 1-mediated transactivation dependent on heat shock.

Upon exposure to elevated temperatures, mammalian cells increase the expression of the heat shock proteins (HSP) through activation of the heat shock factor 1 (HSF1). Since most transcription factors require coactivators for efficient transcriptional activity, we tried to identify the coactivator(s) that interacts with and modulates the activities of HSF1. In vitro glutathione S-transferase (GST) pull-down assay revealed that HSF1 strongly interacts with activating signal cointegrator (ASC)-2 and weakly with cyclic adenosine monophosphate responsive element binding protein (CBP). We also show that cotransfection of ASC-2, but not CBP, potentiates HSF1-mediated transactivation based on its cognate element (heat shock element, HSE) linked to luciferase reporter. The molecular interaction of HSF1 and ASC-2 was stimulated by heat shock in cells and the overexpression of HSF1-interacting domain of ASC-2 inhibited the specific induced protein association and HSF1-mediated transactivation. Taking these results together, we suggest that ASC-2 in a novel coactivator for HSF1 and heat shock stress may contribute the strong active transcription complex through sequential recruitment of HSF1 and ASC-2.

Ca2+/calmodulin-dependent protein phosphatase calcineurin mediates the expression of iNOS through IKK and NF-kappaB activity in LPS-stimulated mouse peritoneal macrophages and RAW 264.7 cells.

Ca(2+) and Ca(2+)/calmodulin-dependent protein phosphatase calcineurin (CN) have been known to play crucial roles in immune response and inflammation. Using mouse peritoneal macrophages and RAW 264.7 macrophage cells, we demonstrated that LPS mobilized intracellular free Ca(2+) and induced CN phosphatase activity. iNOS expression and NO secretion in response to LPS were suppressed by Ca(2+) antagonists (TMB-8, BAPTA/AM, and nifedipine) and CN inhibitor (cyclosporin A). Transient expression of constitutively active CN in mouse peritoneal macrophages and RAW 264.7 macrophages strongly activated NF-kappaB, a key mediator of iNOS expression. We also found that CN mediates NF-kappaB activation via IkappaB-alpha hyperphosphorylation and degradation. Overexpression of dominant negative mutant of IKKalpha and -beta demonstrates that only IKKbeta is the target for CN. These results indicate that CN is required for full iNOS expression and the effective activation of NF-kappaB in RAW 264.7 and peritoneal macrophages.

Redox regulation of DNA binding activity of DREF (DNA replication-related element binding factor) in Drosophila.

DREF [DRE (DNA replication-related element) binding factor] is an 80 kDa polypeptide homodimer which plays an important role in regulating cell proliferation-related genes. Both DNA binding and dimer formation activities are associated with residues 16-115 of the N-terminal region. However, the mechanisms by which DREF dimerization and DNA binding are regulated remain unknown. Here, we report that the DNA binding activity of DREF is regulated by a redox mechanism, and that the cysteine residues are involved in this regulation. Electrophoretic mobility shift analysis using Drosophila Kc cell extracts or recombinant DREF proteins indicated that the DNA binding domain is sufficient for redox regulation. Site-directed mutagenesis and transient transfection assays showed that Cys59 and/or Cys62 are critical both for DNA binding and for redox regulation, whereas Cys91 is dispensable. In addition, experiments using Kc cells indicated that the DNA binding activity and function of DREF are affected by the intracellular redox state. These findings give insight into the exact nature of DREF function in the regulation of target genes by the intracellular redox state.

Phosphorylation of NF-kappa B by calmodulin-dependent kinase IV activates anti-apoptotic gene expression.

We previously presented that calmodulin-dependent kinase IV (CaMKIV) mutually interacts with NF-kappa B and phosphorylates it directly, inducing the increased transcriptional regulation dependent on NF-kappa B target genes [J. Biol. Chem. 276 (2001) 20005]. Here, we show that Ser(535) residue is phosphorylated by CaMKIV. S535A mutant of p65 was specifically defective in transactivation of NF-kappa B target gene expression induced by CaMKIV. While coexpression of active CaMKIV with wild-type p65 led to a recovery from etoposide-induced apoptosis and an increase of Bcl-2 protein in cells, cells expressing S535A mutant did not. Taken together these results suggest that phosphorylated NF-kappa B p65 on Ser(535) by CaMKIV increases NF-kappa B target gene expression, including anti-apoptotic gene, hence leading to inhibition of apoptosis.

Anti-inflammatory effects of aqueous extract from Dichroa febrifuga root in rat liver.

AIM: To study the anti-inflammatory effects of aqueous extract from Dichroa febrifuga root (AEDF) for suppression in the process of lipopolysaccharide (LPS)-induced sepsis in the rat liver. METHODS: The inhibitory effect of AEDF on the alteration of inflammatory proteins was investigated by Western blot and immunohistochemical analysis. RESULTS: Western blot analysis showed that the level of nuclear factor (NF)-kappaBp65 was markedly up-regulated and (I)-kappaBalpha was down-regulated by LPS (8 mg/kg) challenge. However, AEDF 100 mg/kg inhibited induction of NF-kappaBp65 and degradation of I-kappaBalpha in the liver of LPS-challenged rats. Immunohistochemical analysis showed that while the expression of the NF-kappaBp65, tumor necrosis factor (TNF)-alpha and inducible nitric oxide synthase (iNOS) tended to increase, that of I-kappaBalpha was decreased in the hepatocytes of rats challenged with LPS. A slight decline of NF-kappaBp65, TNF-alpha and iNOS, but an increase of I-kappaBalpha were observed in the hepatocytes of the rats pretreated with AEDF. CONCLUSION: AEDF may act as a therapeutic agent for inflammatory disease through a regulation of inflammation-related proteins.

Fluorescence and folding properties of Tyr mutant tryptophan synthase alpha-subunits from Escherichia coli.

The fluorescence of tyrosine has been used to monitor a folding process of tryptophan synthase alpha-subunit from Escherichia coli, because this protein has 7 tyrosines, but not tryptophan. Here to assess the contribution of each Tyr to fluorescence properties of this protein during folding, mutant proteins in which Tyr was replaced with Phe were analyzed. The result shows that a change of Tyr fluorescence occurring during folding of this protein is contributed to approximately 40% each by Tyr(4) and Tyr(115), and to the remaining approximately 20% by Tyr(173) and Tyr(175). Y173F and Y175F mutant proteins showed an increase in their fluorescence intensity by approximately 40% and approximately 10%, respectively. These increases appear to be due to multiple effects of increased hydrophobicity, quenching effect of nearby residue Glu(49), and/or energy transfer between Tyrs. Two data for Y173F alpha-subunit of urea-induced unfolding equilibrium monitored by UV and fluorescence were different. This result, together with ANS binding and far UV CD, shows that folding intermediate(s) of Y173F alpha-subunit, contrary to that of wild-type, may contain self-inconsistent properties such as more buried hydrophobicity, highly quenched fluorescence, and different dependencies on urea of UV absorbance, suggesting an ensemble of heterogeneous structures.

Syntenin is overexpressed and promotes cell migration in metastatic human breast and gastric cancer cell lines.

Two human breast cancer cell lines of differing invasive and metastatic potential, MDA-MB-435 and MCF7, were examined using subtractive suppression hybridization in a search for any genes associated with metastasis. Of the 17 cDNAs identified as being differentially expressed genes, it was determined that syntenin was overexpressed in metastatic MDA-MB-435 cells. Expression analysis showed that the expression level of syntenin was well correlated with invasive and metastatic potential in various human breast and gastric cancer cell lines. Moreover, gastric tumor tissues exhibited a much higher syntenin mRNA expression than their normal counterparts. Syntenin-transfected MCF7 cells migrated more actively, and showed an increased invasion rate relative to vector-transfectants or parental MCF7 in vitro, without evidencing any effect on the adhesion to fibronectin, type I collagen and laminin. Similarly, the forced expression of syntenin to human gastric cancer cell line Az521 increased its migratory and invasive potential in vitro. Syntenin-expressing MCF7 cells were associated with the appearance of numerous cell surface extensions and with pseudopodia formation on collagen I, suggesting that syntenin may be involved in the signaling cascade to actin-reorganization. Mutation study suggested that PDZ2 domain of syntenin could be an essential role in its stimulatory effect on the cell migration. This is the first demonstration that syntenin, a PDZ motif-containing protein, can be overexpressed during the metastatic progression of human breast and gastric cancer cells and that it can function as a metastasis-inducing gene.

Mild heat shock induces cyclin D1 synthesis through multiple Ras signal pathways.

Hyperthermia such as that occurring during fever may improve cell survival during infection, although its mechanism of action is largely unknown. Here we show that acute exposure to mild, but not severe, heat shock induces the synthesis of cyclin D1 that plays a critical role(s) in G1 progression of the cell cycle. This induction seemed to be regulated through multiple Ras signal pathways involving extracellular signal-regulated kinase, phosphatidylinositol 3-kinase, and Rac1/NADPH oxidase, all of which have well been documented to be responsible for growth factor-induced cyclin D1 expression. In a physiological sense, mild heat shock may regulate cell proliferation through inducing cyclin D1 along with growth factors.