J. Fred Nagelkerke

Effects of N-trimethyl chitosan chloride, a novel absorption enhancer, on caco-2 intestinal epithelia and the ciliary beat frequency of chicken embryo trachea.

N-trimethyl chitosan (TMC) polymers are quaternized chitosans in different degrees of trimethylation. These polymers enhance the absorption of macromolecules through mucosal epithelia by triggering the reversible opening of tight junctions and only allow for paracellular transport. To investigate the safety of these novel absorption enhancers cytotoxicity and ciliotoxicity studies have been performed. Intestinal Caco-2 cell monolayers were chosen to study possible membrane damaging effects of these polymers, using confocal laser scanning microscopy visualization of nuclear staining by a membrane impermeable fluorescent probe during transport of the paracellular marker Texas red dextran (MW 10 000). Ciliated chicken embryo trachea tissue was used to study the effect of the polymers on the ciliary beat frequency (CBF) in vitro. In both studies the TMC polymers of different degrees of substitution (20, 40 and 60%) were tested at a concentration of 1.0% (w/v). No substantial cell membrane damage could be detected on the Caco-2 cells treated with TMCs, while the effect on the CBF in vitro was found to be marginal. TMC60 and TMC40 enhance paracellular transport of Texas red dextran in Caco-2 cell monolayers, whereas TMC20 is ineffective. In conclusion, TMCs of high degrees of substitution may be effective and safe absorption enhancers for peptide and protein drug delivery.

Diffusion Rates and Transport Pathways of Fluorescein Isothiocyanate (FITC)-Labeled Model Compounds Through Buccal Epithelium

The aim of this study was to characterize transport of FITC-labeled dextrans of different molecular weights as model compounds for peptides and proteins through buccal mucosa. The penetration of these dextrans through porcine buccal mucosa (a nonkeratinized epithelium, comparable to human buccal mucosa) was investigated by measuring transbuccal fluxes and by analyzing the distribution of the fluorescent probe in the epithelium, using confocal laser scanning microscopy for visualizing permeation pathways. The results revealed that passage of porcine buccal epithelium by hydrophilic compounds such as the FITC-dextrans is restricted to permeants with a molecular weight lower than 20 kDa. The permeabilities of buccal mucosa for the 4- and 10-kDa FITC-dextran (of the order of 10−8 cm/sec) were not significantly different from each other or from the much smaller compound FITC. The confocal images of the distribution pattern of FITC-dextrans showed that the paracellular route is the major pathway through buccal epithelium.

Caspase-3 activity as a prognostic factor in colorectal carcinoma

Several techniques to determine apoptotic frequencies in tumors have been described. In this study, we report that biochemical detection of enzymatic caspase-3 activity is a simple and quantitative technique to measure apoptosis in colorectal tumor cells. The relevance of the level of apoptosis in colorectal cancer for the clinical course remains unclear. Therefore, we studied the correlation between caspase-3 activity and prognosis of the disease in relation to different factors known to be involved in apoptosis induction. High caspase-3 activity significantly correlated with a higher risk of recurrence and was preferentially found in tumors of the right side of the colon. No correlation was detected between high caspase-3 activity and altered protein expression of p53, β-catenin, or proteins of mismatched repair genes. This indicates that high caspase-3 activity has no evident correlation with the genetic Wnt-signaling or the mismatch repair mutational pathways. The caspase-3 activity significantly correlated with CD57+ tumor infiltrating cells. Therefore, high caspase-3 activity in right-sided tumors might be induced by a specific lymphocytic reaction.

Confocal laser scanning microscopic visualization of the transport of dextrans after nasal administration to rats: effects of absorption enhancers.

AbstractPurpose. To visualize the transport pathway(s) of high molecular weight model compounds across rat nasal epithelium in vivousing confocal laser scanning microscopy. Furthermore, the influence of nasal absorption enhancers (randomly methylated β-cyclodextrin and sodium taurodihydrofusidate) on this transport was studied. Methods. Fluorescein isothiocyanate (FITC)-labelled dextrans with a molecular weight of 3,000 or 10,000 Da were administered intranasally to rats. Fifteen minutes after administration the tissue was fixed with Bouin. The nasal septum was surgically removed and stained with Evans Blue protein stain or DiIC18(5) lipid stain prior to visualization with the confocal laser scanning microscope. Results. Transport of FITC-dextran 3,000 across nasal epithelium occurred via the paracellular pathway. Endocytosis of FITC-dextran 3,000 was also shown. In the presence of randomly methylated β-cyclodextrin 2% (w/v) similar transport pathways for FITC-dextran 3,000 were observed. With sodium taurodihydrofusidate 1% (w/v) the transport route was also paracellular with endocytosis, but cells were swollen and mucus was extruded into the nasal cavity. For FITC-dextran 10,000 hardly any transport was observed without enhancer, or after co-administration with randomly methylated β-cyclodextrin 2% (w/v). Co-administration with sodium taurodihydrofusidate 1% (w/v) resulted in paracellular transport of FITC-dextran 10,000, but morphological changes, i.e. swelling of cells and mucus extrusion, were observed. Conclusions. Confocal laser scanning microscopy is a suitable approach to visualize the transport pathways of high molecular weight hydrophilic compounds across nasal epithelium, and to study the effects of absorption enhancers on drug transport and cell morphology.

Inhibition of lipid peroxidation by disulfiram and diethyldithiocarbamate does not prevent hepatotoxin-induced cell death in isolated rat hepatocytes. A study with allyl alcohol, tert-butyl hydroperoxide, diethyl maleate, bromoisovalerylurea and carbon tetrachloride

The relationship between lipid peroxidation and cell death, induced by a number of hepatotoxins, was studied in isolated rat hepatocytes. Disulfiram (DSF) and diethyldithiocarbamate (DDC) completely prevented lipid peroxidation, induced by allyl alcohol, tert-butyl hydroperoxide (t-BHP), diethyl maleate (DEM), bromoisovalerylurea (BIU) and carbon tetrachloride (CCl4). Lipid peroxidation was measured by the formation of both thiobarbituric acid positive material and conjugated dienes. However, DSF and DDC did not protect against cell death, induced by these hepatotoxins. In the presence of DSF or DDC, cell death occurred even earlier in time. We conclude that cell death can occur in the absence of lipid peroxidation. Therefore, lipid peroxidation is not a requisite for the induction of cell death.

Lipid peroxidation-dependent and -independent protein thiol modifications in isolated rat hepatocytes: Differential effects of vitamin E and disulfiram

Exposure of isolated rat hepatocytes to allyl alcohol (AA), diethyl maleate (DEM) and bromoisovalerylurea (BIU) induced lipid peroxidation, depletion of free protein thiols to about 50% of the control value and cell death. Vitamin E completely prevented lipid peroxidation, protein thiol depletion and cell death. A low concentration (0.1 mM) of the lipophylic disulfide, disulfiram (DSF), also prevented the induction of lipid peroxidation by the hepatotoxins; however, in the presence of DSF, protein thiol depletion and cell death occurred more rapidly. Incubation of cells with a high concentration (10 mM) of DSF alone led to 100% depletion of protein thiols and cell death, which could not be prevented by vitamin E. The level of free protein thiols in cells, decreased to 50% by exposure to AA, DEM and BIU, could be reversed to 75% of the initial level by dithiothreitol (DTT) treatment, indicating that the protein thiols were partially modified into disulfides and partially into other, stable thiol adducts. The 100% depletion of protein thiols by DSF was completely reversed by DTT treatment. The involvement of lipid peroxidation in protein thiol depletion was studied by measuring the effect of a lipid peroxidation product, 4-hydroxynonenal (4-HNE), on protein thiols in a cell free liver fraction. 4-HNE did not induce lipid peroxidation in this system, but protein thiols were depleted to 30% of the initial value, irrespective of the presence of vitamin E. DTT treatment could reverse this for only 25%. Similar, DSF-induced protein thiol depletion could be reversed completely by DTT. We conclude that (at least) two types of protein thiol modifications can occur after exposure of hepatocytes to toxic compounds: one due to interaction of endogeneously generated lipid peroxidation products with protein thiols, which is not reversible by the action of DTT, and one due to a disulfide interchange between disulfides like DSF and protein thiols, which can be reversed by the action of DTT.

Prevention of cycloheximide-induced apoptosis in hepatocytes by adenosine and by caspase inhibitors.

The mechanism by which cycloheximide induces apoptosis in isolated rat hepatocytes was studied. Cycloheximide (1-300 microM) induced apoptosis within 3-4 hr in the hepatocytes. Specific apoptotic characteristics such as blebbing, phosphatidyl serine (PS) exposure, chromatin condensation, and nuclear fragmentation were induced. Cycloheximide (CHX) dose dependently activated the caspase-3-like proteases, but not the caspase-1-like proteases. Pretreatment of the hepatocytes with 100 microM of the caspase inhibitors z-Val-Ala-DL-Asp-fluoromethylketone or Ac-Asp-Glu-Val-Asp-aldehyde completely abrogated the caspase activation and the apoptosis. Addition of adenosine (100 microM) reduced phosphatidyl serine exposure and other morphological characteristics of apoptosis by 50%; however, it did not prevent the activation of the caspases, suggesting that adenosine inhibited downstream of caspase activation. The adenosine receptor antagonist 8-[4-[[[[(2-aminoethyl)amino]-carbonyl]methyl]oxy]phenyl]-1,3-dipropylxa nthine abolished the capacity of adenosine to prevent apoptosis, indicating that prevention was receptor-mediated. During apoptosis, the mitochondrial membrane potential in apoptotic cells (cells with PS exposition) was decreased to 50-60% of the control value; in the population viable cells, however, the mitochondrial membrane potential remained stable. Prevention of apoptosis by the caspase inhibitor z-Val-Ala-DL-Asp-fluoromethylketone or adenosine prevented the decrease in mitochondrial membrane potential. In conclusion, CHX rapidly induces apoptosis in isolated rat hepatocytes, which is inhibited by adenosine at a relatively late step.

Quantification and visualization of the transport of octreotide, a somatostatin analogue, across monolayers of cerebrovascular endothelial cells

Confocal laser scanning microscopy (CLSM) was used to quantify and visualize the transport of the octapeptide and somatostatin analogue, octreotide (SMS 201-995, Sandostatin), across monolayers of bovine cerebrovascular endothelial cells, an in vitro model of the blood–brain barrier. The concentrations of octreotide and its conjugates in the cell culture medium were determined by radioimmunoassay (RIA). Two fluorescent conjugates of octreotide (FITC- and NBD-octreotide) were used to obtain CLSM images. The peptides did not undergo significant degradation in the presence of brain endothelial cell monolayers. The transport rate of octreotide expressed as clearance (Cl) and endothelial permeability (Pe) did not depend on either the initial concentration (between 10 nM and 1 µM) or the site of administration (luminal or abluminal side of the mono-layer), indicating the absence of saturable and/or asymmetrical transport mechanisms. The Pe of octreotide and that of the paracellular permeability marker fluorescein correlated well. Although the conjugates are more lipophilic than octreotide itself, they exhibited lower Cl and Pe, values probably because of their larger molecular size. On the CLSM images, FITC-octreotide was present only in the intercellular space, while the cells did not exhibit detectable fluorescence. Transport studies and CLSM images suggest that octreotide passes the endothelial monolayer primarily via the paracellular route without significant contribution of carrier-mediated transport.

Regional loss of the mitochondrial membrane potential in the hepatocyte is rapidly followed by externalization of phosphatidylserines at that specific site during apoptosis.

The spatio-temporal relationship between a decrease in the mitochondrial membrane potential (MMP) and externalization of phosphatidylserines (PS) during induction of apoptosis was investigated in single freshly isolated hepatocytes. Apoptosis was induced in the hepatocytes in three different ways: attack by activated Natural Killer cells, exposure to ATP, or exposure to the inhibitor of protein synthesis cycloheximide. Fluorescence microscopy showed staining of externalized PS at those areas where the staining for MMP was lost whereas in other areas the mitochondria remained intact for longer periods of time, indicating coupling between local loss of MMP and local PS exposure. To discriminate whether the decrease in MMP itself or a decrease in ATP induced PS externalization, hepatocytes were treated with rotenone, which resulted in a rapid collapse of cellular ATP but left the MMP intact for a much longer period. Addition of fructose prevented the decrease of ATP to ∼30% and also delayed the collapse of the MMP. This indicates that ATP was needed for the maintenance of the MMP probably via reverse action of the ATP synthase. In a subsequent study hepatocytes were incubated with Natural Killer cells for induction of apoptosis followed by addition of rotenone to deplete ATP. Under these conditions the PS staining co-localized with mitochondrial MMP indicating that PS externalization does not require a collapse in MMP. Moreover, exposure of PS was evenly distributed over the whole plasma membrane. In conclusion, we propose that after an apoptotic stimulus some mitochondria start to loose their MMP, which results in cessation of ATP production and perhaps even consumption of ATP. This results in an overall decrease in cellular ATP. ATP-consuming enzyme reactions most distal from still intact mitochondria will be most sensitive to such a decrease. Apparently the translocase that keeps phosphatidylserines inward-oriented is such a sensitive enzyme.

Disrupted expression of CXCL5 in colorectal cancer is associated with rapid tumor formation in rats and poor prognosis in patients.

Purpose: We isolated a subline (CC531M) from the CC531S rat colon carcinoma cell line, which grows and metastasizes much more rapidly than CC531S. We found, using RNA expression profiling, that one of the major changes in the CC531M cell line was a 5.8-fold reduction of the chemokine CXCL5. The purpose of this study was to determine the effect of CXCL5 expression on colorectal tumor growth and metastasis. Experimental Design: CC531 clones were generated with either knockdown or restored expression of CXCL5. These clones were inoculated in the liver of rats. In addition, in two independent cohorts of colorectal cancer patients, the level of CXCL5 expression was determined and associated to clinical variables. Results: Knockdown of CXCL5 expression in CC531S resulted in rapid tumor growth and increased number of metastasis, whereas restored expression of CXCL5 in CC531M resulted in a return of the “mild” tumor growth pattern of the parental cell line CC531S. In vitro, no difference was found in proliferation rate between clones with either high or low expression of CXCL5, suggesting that environmental interactions directed by CXCL5 determine tumor outgrowth. Finally, the importance of our findings was established for patients with colorectal cancer. We found that low expression of CXCL5 was significantly associated with poor prognosis for colorectal cancer patients. CXCL5 showed a trend (P = 0.05) for a positive correlation with intratumoral CD8+ T-cell infiltration, suggesting a possible explanation for the observed poorer prognosis. Conclusions: Our results show that CXCL5 is important in growth and development of colorectal cancer, implicating a future role in both cancer therapy and diagnosis.