The Toxicologist - Society of Toxicology

1780 ANTIANDROGENIC AND ANTIPROLIFERATIVE
EFFECTS OF 3, 3’-DIINDOLYLMETHANE (DIM) AND
RING-SUBSTITUTED ANALOGS (RING-DIMS) IN
LNCAP HUMAN PROSTATE CANCER CELLS.
K. Abdelbaqi 1 , S. Stephen 2 and J. Sanderson 1 . 1 Institut Armand-Frappier, INRS,
Laval, QC, Canada and 2 Veterinary Physiology and Pharmacology, Texas A&M
University, College Station, TX.
Cruciferous vegetables have been found to protect against prostate cancer. Indole-
3-carbinol (13C) and its dimeric product 3,3’-diindolylmethane (DIM) exhibit
anti-tumor activities both in vitro and in vivo. Previous studies have shown that
DIM inhibits androgen receptor (AR) nuclear translocation, leading to down-regulation
of target genes. In this study, we observed structure-dependent differences for
the effects of the synthetic 4,4’-, and 7,7’-dihaloDIMs on AR and prostate specific
antigen (PSA) expression in LNCaP cells. We also report that DIM and ring-substituted
analogs (ring-DIMs) 4,4’-dibromo-, 4,4’-dichloro-, 7,7’-dibromo- and
7,7’-dichloro-DIM, inhibit the proliferation of androgen-sensitive LNCaP prostate
cancer cells at 10 and 30 μM. Western blot analysis showed that 4,4’- and 7,7’-dibromo-DIMs
reduced AR protein levels, whereas 4,4’- and 7,7’-dichloro-DIMs
had little effect. RT-PCR analysis clearly indicated that the 4,4’-dihaloDIMs and
7,7’-dihalo-DIMs (24 h exposure) inhibited the expression of AR at the mRNA
level. The 4,4’- and 7,7’-dihalo-DIMs also significantly decreased PSA mRNA expression
and cellular protein secretion levels at 10 and 30 μM. These anti-androgenic
effects of the dihalo-ring-DIMs suggest they may be interesting as (or form
the basis for) novel agents for the treatment of hormone-sensitive prostate cancer,
either alone or in combination with other therapeutics. This work will be continued
with the study of apoptotic effects of ring-DIMs in LNCaP as well as hormone-insensitive
PC-3 human prostate cancer cells.
1781 ACTIVATION OF THE TUMOR SUPPRESSOR P53 IS
NOT ESSENTIAL FOR GROWTH INHIBITION OF
HUMAN HEPATOCELLULAR CARCINOMA CELLS BY
ISOTHIOCYANATES.
V. Mersch-Sundermann, E. Lamy, H. Weimann and M. Wagner. Department of
Environmental Health Sciences, University Medical Center Freiburg, Freiburg,
Germany.
In a number of studies, Brassica-derived isothiocyanates (ITCs) decreased or inhibited
cell growth in cancer cell culture as well as in animal xenograft models. The induction
of programmed cell death (apoptosis) is a well-documented mechanism in
the anticancer activity of ITCs. A number of cell lines have been studied for apoptosis
induction by ITCs, and signal transduction pathways have been investigated.
It has been shown that apoptosis induction is generally mediated via the intrinsic
mitochondrial death pathway; however, the receptor death pathway may also be involved.
Even cancer cells, which are normally resistant to chemotherapeutic drugs
due to their high expression levels of anti-apoptotic Bcl-2 family proteins, have
been rendered to cell death by ITC treatment. The studies conducted by our group
showed proliferation inhibition of liver cancer cells by 4-methylthiobutyl ITC
(MTBITC), which arrested cells at the G2/M phase and depolarized mitochondria
at concentrations exceeding 10 μM. Although the tumor suppressor p53 was
markedly expressed in treated cells compared to control, RNAi experiments
demonstrated the independency of growth inhibition from p53. This detail could
be very valuable for the treatment of tumors with existing p53 mutation, e.g. 30 to
50 % of hepatocellular carcinomas (HCC), one of the most common malignant tumors
worldwide, are affected by this mutation. The therapeutic application of ITCs
for treatment of malignant tumors is promising; however, further research has to be
done to characterize the concentration-dependent ambivalent character of ITCs
and their specificity of tissue targeting.
1782 MODULATION OF HUMAN LYMPHOMA VIABILITY
AND PROLIFERATION BY RICE BRAN FROM
GENETICALLY DIVERSE VARIETIES.
E. P. Ryan 1 , A. Heuberger 2 , M. Lewis 3 and J. Leach 4 . 1 Clinical Sciences, Colorado
State University, Fort Collins, Co., 2 Soil and Crop Sciences, Colorado State
University, Fort Collins, Co., 3 Metabolomics Core-Biochemistry, Colorado State
University, Fort Collins, CO and 4 Bioagricultural Sciences, Colorado State University,
Fort Collins, CO.
Rice provides the majority of daily caloric intake for half of humanity and is a rich
source of bioactive food components (BFC) with disease prevention properties.
Emerging evidence supports that BFC in rice bran may work synergistically and in
parallel to elicit protective host immunity and prevent cancer, and thus focusing on
nutritional magic bullets as in dietary supplements may be problematic over promoting
a general increase in whole grain consumption. Metabolomics was performed
using a standard operating procedure developed by our laboratory to investigate
the phytochemical diversity of rice bran isolated from 10 genetically diverse
rice cultivars with distinct grain characteristics. Methanol-soluble compounds were
detected by liquid chromatography-mass spectrometry and analyzed using principal
components analysis. Three rice varieties with distinguishable bran global
metabolite profiles (>200 metabolites) and that significantly differs in vitamin E
content were assessed for their ability to effect human lymphoma viability and proliferation
as measured by MTT assay and CFSE staining with flow cytometry.
Relative differences across rice varieties were also examined for phytate, ferulic acid,
phytosterols and salicylic acid content. A 30-50% difference in viability and proliferation
index was detected across rice varieties examined. Identifying the phytochemicals
and rice genic regions in rice bran that account for differential anticancer
activity can be used to inform future crop improvement strategies for disease prevention.
These findings support that cultivar information should be reported when
investigating BFCs as unique phytochemical contents across varieties demonstrate
not only differential anticancer activity as reported herein, but may also differentially
influence cellular stress responses to toxicants.
1783 SUPPRESSION OF LIPOPOLYSACCHARIDE-INDUCED
INFLAMMATION BY ELLAGITANNIN.
S. Lee 1 and S. Kim 2 . 1 Pharmacology, Kyungpook National University, Daegu,
Republic of Korea and 2 Toxicology Branch, National Toxicology Program/NIEHS,
Durham, NY.
Ellagitannin isolated and purified from Euphorbia species. Ellagitannin is known to
possess anti-cancer and anti-oxidative activity. Recently, ellagitannin has been reported
to be effective in producing anti-inflammatory activity. However, the mechanism
of effective action is still unknown. We investigated on anti-inflammatory
actions of ellagitannin and its possible mechanisms of action in lipopolysaccharide
(LPS)-stimulated macrophages. LPS activates inducible nitric oxide synthase
(iNOS) and produces nitric oxide (NO) in macrophages. However, ellagitannin inhibited
LPS-induced NO production dose-dependently through inhibition of
iNOS. LPS induces phosphorylation of mitogen activated protein kinase (MAPK)
including extracellular regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK),
p38 and activation of nuclear factor-κB (NF-κB) and Cyclooxygenase-2 (COX-2).
On the contrary, ellagitannin decreased LPS-induced activation of MAPK, NF-κB,
and COX-2. In addition, ellagitannin reduced LPS-induced inflammatory cytokines
and chemokines. Therefore, we suggest that ellagitannin inhibits LPS-induced
inflammation such as NO production, inflammatory cytokines and
chemokines by suppressing the activation of MAPK, NF-κB and COX-2. Our results
suggest that ellagitannin might be a candidate for developing anti-inflammatory
and cancer chemopreventive agents.
1784 CONSUMPTION OF DOCOSAHEXAENOIC ACID
ATTENUATES LUPUS NEPHRITIS-RELATED GENE
EXPRESSION AND DISEASE PROGRESSION IN
NZBWF1 MICE.
L. L. Vines 1, 2 , I. M. Langohr 3 and J. J. Pestka 1, 2, 4 . 1 Food Science & Human
Nutrition, Michigan State University, East Lansing, MI, 2 Center for Integrative
Toxicology, Michigan State University, East Lansing, MI, 3 Pathobiology & Diagnostic
Investigation, Michigan State University, East Lansing, MI and 4 Microbiology &
Molecular Genetics, Michigan State University, East Lansing, MI.
Systemic lupus erythematosus (SLE) is a debilitating autoimmune disease of unknown
etiology, where mortality is associated with the onset of lupus nephritis. Of
note, n-3 polyunsaturated fatty acids (n-3 PUFAs) have shown efficacy in preventing
and treating lupus nephritis in rodents and human clinical studies, however,
mechanisms are unknown. The purpose of this study was to validate the capacity of
n-3 fatty acid docosahexaenoic acid (DHA) to suppress murine lupus nephritis and
profile ensuing transcriptome changes in lupus-prone mice. Four week-old female
NZBWF1 mice were divided into three groups and fed daily an AIN-93G diet consisting
of 1% corn oil and 6% of either DHA-enriched fish oil (DHA [n-3]), higholeic
safflower oil (SAF [n-9]), or corn oil (CRN [n-6]). Plasma and urine were collected
at regular intervals, and mice terminated at 16 (pre-nephritic) or 36wks
(nephritic) for further histological, protein, and PCR analyses. At 36wk termination,
SAF and CRN alone exhibited marked nephritis (both class IV, blind ISN-
PRS grading scale) and proteinuria (75% and 63%, respectively). Also, plasma antidsDNA
IgG, an SLE biomarker, was significantly greater in SAF (216%) and CRN
(240%). For gene expression profiling, DHA suppressed numerous genes in blood,
kidney, and spleen samples. However, consistent suppression of Ccl7, Cxcr3, IL-
4Rα, and osteopontin (OPN) was observed. Notably, OPN is a pleiotropic cytokine
linked to autoantibody formation. When OPN was assessed in 36wk plasma
SOT 2010 ANNUAL MEETING 379