668 research outputs found
The role of docosahexaenoic acid in mediating mitochondrial membrane lipid peroxidation and apoptosis in colonocytes.
No full textColon cancer is the second leading cause of cancer death in the United States. Epidemiological data indicate that the consumption of dietary fiber and fish/marine products favorably modulate colon tumorigenesis. Docosahexaenoic acid (DHA, 22:6n-3) from fish oil, and butyrate, a fiber fermentation product generated in colon, protect against colon tumorigenesis in part by inducing apoptosis. We have shown that DHA is incorporated into mitochondrial membrane phospholipids, which enhances oxidative stress and mitochondrial membrane potential (MP) dissipation. To elucidate the subcellular origin of oxidation induced by DHA and butyrate exposure, young adult mouse colonocytes (YAMC) were treated with 0200 ??M DHA, linoleic acid (LA, 18:2n-6) or no fatty acid (control) for 72 h with or without 5 mM butyrate for the final 6-24 h. Real time analysis of cellular membrane lipid oxidation, as indicated by oxidation of a lipophilic vital dye, mitochondrial permeability transition (MPT), as characterized by MP dissipation, and cytosolic ROS production, as depicted by hydrophilic ROS reactive fluorophore accumulation, were measured by living cell fluorescence microscopy. After 24 h of butyrate treatment, DHA primed cells showed a 29% increase in lipid oxidation (p<0.01), compared to no butyrate treatment, which could be blocked by a mitochondria targeted antioxidant, MitoQ (p <0.05), whereas LA treatment did not show an effect. In the absence of butyrate, DHA treatment, compared to LA, increased resting MP by 14% (p <0.01). In addition, butyrate-induced MP dissipation was greater (20%) in DHA primed cells as compared to LA (10%). This effect was blocked by pre-incubation with MPT inhibitors, cyclosporin A or bongkrekic acid at 1 ??M. These data suggest an increase in mitochondrial lipid oxidation and the resultant change in MP may contribute to the induction of apoptosis by DHA with butyrate as shown previously
Dietary fish oil and butyrate increase apoptosis and decrease aberrant crypt foci in colon cancer by enhancing histone acetylation and p21waf1/cip1 expression
No full textWe have previously shown that dietary fish oil and fiber, particularly the highly-fermentable pectin, are protective against colon cancer in a rat model of carcinogenesis. Therefore, based upon the current body of literature and our previous experimental findings, we hypothesized that one mechanism by which dietary fish oil+pectin suppress the promotion stage of colon cancer is through butyrate, the fermentation product of fiber, targeting (in particular) the p21Waf1/Cip1 gene and, via targeted histone hyperacetylation, inducing its expression. We found that dietary butyrate supplementation increased the concentration of fecal butyrate (mole %) in the distal colon, and that this increase corresponded to an increase in histone H4 acetylation. Similarly, diets supplemented with butyrate increased p21Waf1/Cip1 expression despite azoxymethane (AOM) treatment, which was not seen in non-butyrate supplemented diets. Furthermore, fish oil+butyrate diets resulted in the highest levels of apoptosis and the lowest levels of ACF, while corn oil+butyrate diets resulted in the lowest levels of apoptosis and the highest levels of ACF. Thus, it appears that the protective effect of fish oil+butyrate is due to the unique properties of fish oil, providing an environment in which butyrate��s enhancement of histone acetylation and p21 expression are pro-apoptotic, thereby diminishing pre-neoplastic ACF development
Docosahexaenoic acid differentially modulates plasma membrane targeting and subcellular localization of lipidated proteins in colonocytes
No full textCorrect localization of lipidated cytosolic proteins to the plasma membrane (PM) is mediated by interactions between lipid anchors of proteins and cell membranes. Previously, dietary fish oil and its major n-3 polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA), have been shown to decrease Ras membrane association, concomitantly reducing rat colon tumor incidence and Ras signaling, compared with corn oil and linoleic acid (LA), a highly prevalent vegetable fat and dietary PUFA in the U.S. diet. In order to explore the potential regulatory role of the cellular lipid environment in PM targeting of lipidated proteins, young adult mouse colon (YAMC) cells were treated with 50 ����M DHA, LA, or oleic acid (OA) 24 h prior to and 36-48 h after transfection with green fluorescent protein (GFP) fusion constructs of various lipidated cytosolic proteins. Relative expression of each GFP fusion protein at the PM and the Golgi in living cells was quantified using z-serial confocal microscopy and digital image processing. DHA differentially altered the subcellular localization of Ras isoforms and Src-related tyrosine kinases in a reversible manner. DHA significantly decreased the PM localization and increased the endomembrane association of H-Ras,
N-Ras, and Lck, which are targeted to the PM via the exocytic pathway, regardless of their functional state. In contrast, the subcellular distribution of K-Ras and Fyn, of which transport is independent of the vesicular transport pathway, was unaffected by DHA. Moreover, DHA selectively inhibited lipidated cytosolic protein targeting since the PM delivery of transmembrane protein cargo was unaffected, indicating that DHA does not alter the bulk flow of secretory vesicular traffic. Overall, the present study presents compelling evidence that select dietary constituents with membrane lipid-modifying properties can differentially modulate subcellular localization of important lipidated signaling proteins depending on their intracellular trafficking route to the PM
Noninvasive detection of colonic biomarkers using fecal messenger RNA
No full textA noninvasive method utilizing feces, containing sloughed colonocytes, as a sensitive technique for detecting diagnostic colonic biomarkers as well as a method for isolating poly A +RNA from feces. The method allows the isolation and quantitation of specific eukarotic mRNAs as candidate biomarkers for colon cancer isolated from feces.U
Summarizing FLARE assay images in colon carcinogenesis
No full textIntestinal tract cancer is one of the more common cancers in the United States. While in some individuals a genetic component causes the cancer, the rate of cancer in the remainder of the population is believed to be affected by diet. Since cancer usually develops slowly, the amount of oxidative damage to DNA can be used as a cancer biomarker. This dissertation examines effective ways of analyzing FLARE assay data, which quantifies oxidative damage. The statistical methods will be implemented on data from a FLARE assay experiment, which examines cells from the duodenum and the colon to see if there is a difference in the risk of cancer due to corn or fish oil diets. Treatments of the oxidizing agent dextran sodium sulfate (DSS), DSS with a recovery period, as well as a control will also be used.
Previous methods presented in the literature examined the FLARE data by summarizing the DNA damage of each cell with a single number, such as the relative tail moment (RTM). Variable skewness is proposed as an alternative measure, and shown to be as effective as the RTM in detecting diet and treatment differences in the standard analysis. The RTM and skewness data is then analyzed using a hierarchical model, with both the skewness and RTM showing diet/treatment differences. Simulated data for this model is also considered, and shows that a Bayes Factor (BF) for higher dimensional models does not follow guidelines presented by Kass and Raftery (1995).
It is hypothesized that more information is obtained by describing the DNA damage functions, instead of summarizing them with a single number. From each function, seven points are picked. First, they are modeled independently, and only diet effects are found. However, when the correlation between points at the cell and rat level is modeled, much stronger diet and treatment differences are shown both in the colon and the duodenum than for any of the previous methods. These results are also easier to interpret and represent graphically, showing that the latter is an effective method of analyzing the FLARE data
Dietary apigenin and naringenin protect against colon carcinogenesis by lowering high multiplicity aberrant crypt foci and enhancing apoptosis in azoxymethane-treated rats
No full textColon cancer is the third most common cancer in the United States. However,
evidence indicates that a proper diet abundant in fruits and vegetables may be protective
against colon cancer development. Bioactive compounds in fruits and vegetables, such
as flavonoids and limonoids, have been shown to possess anti-proliferative and antitumorigenic
effects in various in vitro and in vivo models of cancer. Since there are few
animal studies involving flavonoids and limonoids and colon cancer, this experiment
investigated the potentially protective effects of four citrus flavonoids and one limonoid
mixture against the promotion stage of chemically-induced colon cancer in rats. Male
SD rats (n =60; 10 rats/group) were assigned to receive diets containing 0.1% apigenin,
0.02% naringenin, 0.1% hesperidin, 0.01% nobiletin, 0.035% limonin
glucoside/obacunone glucoside mixture, or a control diet (0% flavonoid/limonoid). Rats
received the diets for 10 wk and were injected with azoxymethane (15 mg/kg) at wk 3
and 4. The excised colons were evaluated for aberrant crypt foci (ACF) formation, cell
proliferation (PCNA assay), apoptosis (TUNEL assay), and iNOS and COX-2
expression. When compared to the control diet, apigenin lowered the number of high
multiplicity ACF (> 4 AC/focus) by 57% (P<0.05) and tended to lower the proliferative
index (28%; P=0.07), while naringenin lowered both the number of high multiplicity
ACF by 51% (P<0.05) and the proliferative index by 32% (P<0.05). Both apigenin and
naringenin increased apoptosis of surface colon cells (78% and 97%, respectively;
P<0.05) when compared to control diet. Hesperidin, nobiletin, and the limoninglucoside/obacunone glucoside mixture did not have any effects on the above variables
measured in this model of colon carcinogenesis. The colonic mucosal protein levels of
iNOS or COX-2 were not different among the six diet groups. Evidence suggests that
high multiplicity ACF are indicative of future tumor development in both humans and
rats. Furthermore, dysregulated proliferation and apoptosis may also lead to
tumorigenesis. Therefore, the ability of dietary apigenin and naringenin to reduce high
multiplicity ACF, lower proliferation, and increase apoptosis may contribute toward
colon cancer prevention. However, their protection is not due to their influence on iNOS
and COX-2 protein levels
Ligand binding proteins: roles in ligand transfer and activation of nuclear receptors
No full textCholesterol and fatty acyl-coenzymeA thioesters are signalling molecules with role in regulation of genes involved in lipid and glucose transport and metabolism. The studies described herein focused on three proteins that bind lipids and have different cellular functions: steroidogenic acute regulatory protein (StAR), hepatocyte nuclear factor-4a (HNF-4a) and acyl-CoA binding protein (ACBP). First, StAR mediates delivery of cholesterol to inner mitochondrial membrane in steroidogenesis by a poorly understood mechanism. In our studies, fluorescent NBD-cholesterol binding assays demonstrate that StAR binds cholesterol at two binding sites with 32 nM Kds and circular dichroism spectra show that cholesterol binding results in changes of StAR secondary structure. Fluorescent sterol exchange assays between donor and acceptor mitochondrial membranes indicate that StAR significantly increased the formation of rapidly transferable cholesterol domains. Second, HNF-4a, a nuclear receptor, had been shown to bind fatty acyl-CoAs as natural ligands with apparent low affinities obtained with radiolabeled ligand binding assays. Our fluorescence spectroscopy studies demonstrate that HNF-4a ligand binding domain (HNF-4aLBD) binds acyl-CoAs at a single binding site with Kds of 1.6-4 nM. Fluorescence resonance energy transfer (FRET) between HNF-4aLBD tryptophan residues and cis-parinaroyl-CoA yielded an intermolecular distance of 42 �� thus pointing to direct molecular interaction. Third, although ACBP has been detected in the nucleus, it is not known whether ACBP may directly and/or functionally interact with a nuclear acyl-CoA binding protein such as HNF-4a to regulate transcription. Our present studies in vitro and in intact cultured cells, including circular dichroism of HNF-4a in the presence of ACBP, coimmunoprecipitation of HNF-4a/ACBP complexes, ACBP and HNF-4a colocalization in nuclei of cells by confocal microscopy demonstrate a physical association of ACBP and HNF-4a. FRET microscopy data indicated an intermolecular distance of 53 �� between ACBP and HNF-4a in rat hepatoma cells. Functional assays (transactivation of an HNF4a-dependent reporter gene) showed significant increase in the presence of ACBP in two different cell lines. Expression of ACBP anti-sense RNA decreased HNF-4a-mediated transactivation, pointing to a role of ACBP in co-regulating HNF-4a-dependent transcription
Reflectance and Fluorescence Confocal Microscope for Imaging of the Mouse Colon
No full textMany Americans are afflicted with inflammation of the colon. They are also at a
higher risk of developing colon cancer. Confocal microscopy of bulk epithelial tissue has
the potential to provide information on tissue structural properties that may be lost in the
fixation and slicing procedures required for histopathology. Optical sectioning provides
images in three dimensions capturing the organizational structure of cells and colon
crypts throughout the entire colon. I have constructed a custom built fluorescence and
reflectance confocal microscope for imaging molecular and morphological changes
associated with development of inflammation in a mouse model.
A confocal microscope is a point scanning system that removes out of focus
light by placing a pinhole aperture in the conjugate image plane located in front of the
detector. We have two sources, 488 nm and 811 nm, for fluorescence and reflectance
imaging, respectively. A polygon scanning mirror and a galvanometer scanning mirror
allow for a variable scan rate between 8 and 15 fps. The lateral resolution of the system
is approximately 3 ��m with an axial resolution of 6 ��m and 4 ��m for reflectance and
fluorescence mode, respectively.
As colon tissue becomes inflamed, there is a distinct change in the structure and
architecture of the tissue. The colon crypts are no longer uniform in size or distribution
throughout the tissue. Having a large field of view of 1mm2 allows for many colon
crypts to be visualized within a single frame. Histology was performed on the same
tissue imaged for the inflammatory study confirming the constructed confocal
microscope���s ability to characterize inflamed tissue and the potential use for guided
biopsy.
Mosaicing, or image tiling, is an imaging technique that stitches single frames
together to produce a much larger field of view. An extended frame with 1 mm x 2 cm
field of view is achieved within seconds. This extended frame would allow mosaicing of
the entire mouse colon much faster than conventional methods without loss of
resolution.
The acquired confocal images of colon tissue demonstrate the microscope���s
ability to resolve cell nuclei lining the colon crypts within a relatively large field of
view
The evaluation of melamine dendrimers as potential macromolecular vehicles for anticancer drug delivery
No full textOften associated with chemotherapy are the dangerous and sometimes lifethreatening
side effects towards non-cancerous tissue that can occur while on such drug
regimens. The design and utilization of macromolecular drug delivery vehicles is
gaining much attention because the vascular system of tumor tissue possesses properties
that make it permeable to macromolecules. The attachment or encapsulation of
anticancer drugs to macromolecules can be used to selectively deliver these drugs to
tumor tissue thereby minimizing the toxic effects towards healthy tissue while
specifically targeting the tumor. Moreover, the association of poorly water soluble drugs
with soluble macromolecules can increase the water solubility of such hydrophobic
drugs. Finally, association of chemotherapeutic agents with macromolecules can also
increase the drugs?? circulation time by decreasing the rate of renal clearance thus leading
to improve pharmacokinetics.
A class of spherical, hyperbranched polymers known as dendrimers has received
much attention as potential vehicles for anticancer drug delivery. Dendrimers based on
melamine might afford such use as macromolecular carriers for drug delivery. Therefore
an evaluation of melamine dendrimers is reported. The goal of objective one was to
assess both the in vitro and in vivo biocompatibilities of a cationic dendrimer based on
melamine. The results reported herein indicate that this particular species of dendrimer is
not suitable for in vivo use and did not warrant further investigation.
The goals of objective two were to see what impact surface modification had on
the in vitro and in vivo toxicities of a melamine dendrimer. The results presented here
indicate that surface modification of a cationic dendrimer to anionic or neutral species
can extensively increase biocompatibility. Moreover, the introduction of neutral
poly(ethylene glycol) (PEG) grafts affords the most protection in vitro and in vivo.
Interaction with albumin, controlled drug release, cellular uptake along with
favorable biodistribution patterns are vital factors that must be evaluated when screening
a drug delivery system. Evaluating the PEGylated dendrimer as a vehicle for anticancer
drug delivery, the goal of objective three, provides initial evidence that the PEGylated
dendrimer displays favorable characteristics as a vehicle for drug delivery and justifies
additional studies utilizing in vivo models
The effects of diet and ionizing radiation on azoxymethane induced colon carcinogenesis
No full textThe ability of ionizing radiation to enhance colon carcinogenesis and the role of diet in this process has not been documented. We hypothesized that radiation would enhance the formation of aberrant crypt foci, ACF, known precursor lesions to colon cancer, by suppressing apoptosis and upregulating proliferation in colonocytes. Diets contained a combination of fish oil or corn oil and either pectin or cellulose. We exposed 40 male Sprague-Dawley rats to 1 Gy ionizing radiation (1 GeV Fe) 10 d prior to injection with AOM. Colons were resected at the promotion stage of carcinogenesis (7 wk post initial injection) and assayed for ACF and apoptosis. Radiation treatment increased (P=0.0327) the incidence of high multiplicity ACF (foci with four or more aberrant crypts) and decreased (P=0.0340) the apoptotic index compared to non-irradiated rats. Radiation also resulted in an increase (P<0.0001) in the proliferative index compared to the nonirradiated rats. The fish oil containing diets resulted in fewer (P=0.0002) high-multiplicity ACF compared to the corn oil treatment. Dietary pectin significantly increased (P=0.0204) the apoptotic index compared to cellulose treatment. These data suggest that ionizing radiation can work synergistically with AOM and increase the formation of high-multiplicity ACF, upregulate cellular proliferation and decrease apoptosis in colonocytes. The data also suggest that diets containing fish oil and pectin may protect against colon cancer by increasing apoptosis and reducing the formation of high multiplicity ACF
- …
