The Toxicologist - Society of Toxicology

creased level in the 46 kDa preproenzyme, under same conditions, there was no detectable
amount of the 50 kDa proenzyme and the 32 kDa mature enzyme suggesting
NNK perturbation of the LO processing to its mature form. Moreover, NNK
also induced a dose-dependent inhibition of LO activities in conditioned media of
treated cells, consistent with its inhibitory effects on the mRNA and protein levels.
At the promoter level, NNK suppressed LO promoter activities, enhanced methylation
of CpG and inhibited acetylation of histone 3 (H3) at the core promoter region
of the LO gene. These results support the hypothesis that transcriptional and
translational processes of LO are major targets for NNK. Thus, inactivation of the
tumor suppressor gene LO may play a critical role in NNK carcinogenesis (supported
by NIEHS 011340 and Philip Morris ERP)
220 GT-094, A NO-NSAID, INHIBITS COLON CANCER
CELL GROWTH BY ACTIVATION OF A REACTIVE
OXYGEN SPECIES (ROS)-MICRORNA-27A:ZBTB10-
SPECIFICITY PROTEIN (SP) PATHWAY.
S. Pathi 1 and S. Safe 1, 2 . 1 Veterinary Physiology and Pharmacology, Texas A&M
University, College Station, TX and 2 Institute of Biosciences and Technology, Texas
A&M Health Science Center, Houston, TX.
Ethyl 2-((2,3-bis(nitrooxy)propyl)disulfanyl)benzoate (GT-094) is a novel NO
chimera containing an NSAID and NO moieties and also a disulfide pharmacophore
that in itself exhibits cancer chemopreventive activity. In this study, the effects
and mechanism of action of GT-094 were investigated in RKO and SW480
colon cancer cells. GT-094 inhibited cell proliferation and induced apoptosis in
both cell lines and this was accompanied by decreased mitochondrial membrane
potential (MMP) and induction of reactive oxygen species (ROS), and these responses
were reversed after cotreatment with the antioxidant glutathione. GT-094
also downregulated genes associated with cell growth [cyclin D1, hepatocyte
growth factor receptor (c-Met), epidermal growth factor receptor (EGFR)], survival
(bcl-2, survivin), and angiogenesis [vascular endothelial growth factor (VEGF) and
its receptors (VEGFR1 and VEGFR2)]. Results of previous RNA interference studies
in this laboratory has shown that these genes are regulated, in part, by specificity
protein (Sp) transcription factors Sp1, Sp3 and Sp4 that are overexpressed in colon
and other cancer cell lines and not surprisingly, GT-094 also decreased Sp1, Sp3
and Sp4 in colon cancer cells. GT-094-mediated repression of Sp and Sp-regulated
gene products was due to downregulation of microRNA-27a (miR-27a) and induction
of ZBTB10, an Sp repressor that is regulated by miR-27a in colon cancer cells.
Moreover, the effects of GT-094 on Sp1, Sp3, Sp4, miR-27a and ZBTB10 were
also inhibited by glutathione suggesting that the anticancer activity of GT-094 in
colon cancer cells is due, in part, to activation of an ROS-miR-27a:ZBTB10-Sp
transcription factor pathway.
221 MOLECULAR MECHANISM OF ACTION OF INSULIN
SENSITIZING DRUG, METFORMIN, AS ANTICANCER
AGENT IN PANCREATIC CANCER.
V. Vasanthakumari 1 and S. Safe 1, 2 . 1 Veterinary Physiology & Pharmacology, Texas
A&M University, College Station, TX and 2 Institute of Biosciences and Technology,
Houston, TX.
The biguanide metformin is one of the most widely used insulin-sensitizing drugs
(ISDs) for treatment of type 2 diabetes mellitus. In addition to its antidiabetic effect
there is increasing evidence that metformin inhibits cancer cell proliferation
and induces cell cycle arrest. However the molecular mechanisms of the anticancer
activities of metformin are poorly understood. In the present study we evaluated
the antineoplastic effects of metformin in pancreatic cancer cells. The effects of
metformin on inhibition of cancer cell growth was demonstrated by cell proliferation
assays; western blot analysis and Real Time PCR were performed to determine
changes in expression of genes associated with the tumorigenicity of pancreatic cancer.
In Panc28 cells, metformin treatment resulted in a dose dependent inhibition
of cell proliferation. The specificity protein (Sp) transcription factors Sp1, Sp3 and
Sp4 that are usually overexpressed in pancreatic and other cancer cells were downregulated
by metformin and this response was dose dependent. Cotreatement of
pancreatic cancer cells with lactacystin (a compound which inhibits proteosome activity)
and metformin, reversed the downregulation of Sp proteins. Furthermore,
metformin-induced repression of Sp proteins was also reversed in Panc28 cells after
cotreatment with sodium orthovanadate - a phosphatase inhibitor which specifically
blocks tyrosine phosphatases. Treatment of Panc28 cells with metformin also
decreased expression of Sp-regulated genes involved in tumorigenesis and these include
cyclin D1( cancer cell growth), VEGF, VEGFR1( angiogenesis), survivin and
bcl2(survival). The mechanisms of metformin induced phosphatase/ proteosomemediated
downregulation of Sp proteins and Sp-regulated genes are currently being
evaluated.
222 PHARMACOLOGICAL INHIBITION OF THE TGFβ1
TYPE I RECEPTOR INDUCES PREMALIGNANT
KERATINOCYTE TERMINAL DIFFERENTIATION.
K. E. Masiuk, L. Mordasky Markell, N. Blazanin and A. B. Glick. Center for
Molecular Toxicology and Carcinogenesis, Penn State University, University Park, PA.
The Transforming Growth Factor β1 (TGFβ1) pathway is important in the regulation
of tumor suppression and promotion in skin cancer. To target this pathway,
pharmaceutical companies have developed small molecule inhibitors of the TGFβ1
type 1 receptor (ALK5) kinase such as SB431542 (SB) that are potent in blocking
the TGFβ1 signaling pathway. Our recently published data show that SB inhibits
papilloma formation but enhances malignant conversion in a mouse multi-stage
carcinogenesis assay. To test if SB inhibits tumor development through altered terminal
differentiation (TD), we used a RAS-oncogene induced neoplastic keratinocyte
model. SB enhanced RAS-induced cornification which correlated with
the increased expression of TD genes transglutaminase 1 (TGM1) and 3 (TGM3)
and small proline-rich protein 1A (SPR1A) and 2H (SPR2H) which crosslink structural
proteins of the cornified envelope. There was a similar increase in cornified
layers and TGMs following SB treatment of mice expressing inducible epidermal
RAS. Alternatively, treatment of RAS-expressing keratinocytes with TGFβ1 or overexpression
of TGFβ1 and RAS in an inducible epidermal model resulted in reduced
expression of TGMs and cornification in vitro. Papilloma (SP1) and squamous cell
carcinoma (PAM2.12) cell lines were less responsive to TGFβ1 suppression of
markers and marker induction by SB. Interestingly, a subset of RAS-expressing, SBtreated
keratinocytes were resistant to TD and overcame senescence and rapidly immortalized,
suggesting that they represent a progressed phenotype. However, these
cells were unable to grow in elevated calcium or produce tumors after grafting nude
mice indicating that they were not fully malignant. Thus, selective responsiveness
to TD may represent a mechanism by which blocked TGFβ1 signaling can inhibit
the outgrowth of preneoplastic lesions but may cause a more progressed phenotype
in a separate keratinocyte population.
223 PHENOBARBITAL-LIKE MODE OF ACTION FOR
LIVER TUMORS IN CD1 MICE AND F344 RATS
EXPOSED TO A NEW DEVELOPMENTAL COMPOUND
(X11422208).
D. Geter 1 , L. H. Kan 1 , A. Wood 1 , M. J. LeBaron 1 , B. B. Gollapudi 1 , J.
Murray 1 , C. Terry 3 , R. Rasoulpour 1 , C. Elcombe 2 , A. Vardy 2 , J. Ross 2 and R.
Billington 3 . 1 The Dow Chemical Company, Midland, MI, 2 CXR BioSciences,
Dundee, United Kingdom and 3 Dow AgroSciences, Abingdon, Oxfordshire, United
Kingdom.
D. Geter1, H.L. Kan1, A. Wood1 M. LeBaron1, B. Gollapudi1, J. Murray1, C.
Terry3, R. Rasoulpour1, C. Elcombe2, A. Vardy2, J. Ross2, R. Billington3. 1. The
Dow Chemical Company, Midland, Michigan, USA, 48674. 2. CXR Biosciences,
Dundee, Scotland, UK. 3. Dow AgroSciences, Abingdon, Oxfordshire, UK. In repeat
dose rodent toxicity studies, a new developmental compound (X11422208)
induced liver hypertrophy in mice and rats during the first week of exposure. In
guideline carcinogenicity studies it produced liver tumors in CD1 mice at the high
dose of 79.6 or 176 mg/kg/day, and in F344 rats at the high dose of 21.3
mg/kg/day. A series of mode-of-action (MoA) studies was undertaken to determine
if the liver effects were mediated by nuclear receptor activation. Several receptors
known to be involved in liver enlargement were investigated: AhR, CAR, PXR, and
PPARα. In both the mouse and rat, transcriptional studies showed elevations in
CAR, and to a lesser extent PXR, but not AhR or PPARα mediated gene responses.
Activation of CAR and PXR were further confirmed by increases in PROD and
BROD enzyme activity. BrdU and Ki-67 investigations showed a large increase in
hepatocellular proliferation. Additional studies using a transgenic mouse that contained
human receptors for PXR and CAR (hPXR/hCAR) and a second mouse
model that was null for both receptors (PXRKO/CARKO) were conducted. In the
PXRKO/CARKO mice, X11422208 did not induce liver changes demonstrating
that activation of these receptors was required to elicit a liver response. X11422208
exposed hPXR/hCAR mice developed slight liver hypertrophy and CAR and PXR
related genes and enzyme activity were elevated; however, hepatocellular proliferation
was not observed in these humanized mice. It is concluded that X11422208
induces hepatocellular tumors in both the mouse and rat via a CAR-mediated MoA
similar to phenobarbital and thus, would not induce tumors in humans.
SOT 2011 ANNUAL MEETING 47