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Molecular Pathways with Human Sporadic Colorectal Tumors
Non-Decolorized Whole Leaf Extract-Induced Large Intestinal Tumors in F344 Rats Share Similar
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Aloe vera Non-Decolorized Whole Leaf Extract-Induced Large
Intestinal Tumors in F344 Rats Share Similar Molecular
Pathways with Human Sporadic Colorectal Tumors
ARUN R. PANDIRI1,2, ROBERT C. SILLS1, MARK J. HOENERHOFF1, SHYAMAL D. PEDDADA3, THAI-VU T. TON1,
HUE-HUA L. HONG1, GORDON P. FLAKE1, DAVID E. MALARKEY1, GREG R. OLSON4, IGOR P. POGRIBNY5,
NIGEL J. WALKER6, AND MARY D. BOUDREAU5
1Cellular and Molecular Pathology Branch, National Toxicology Program (NTP), National Institute of Environmental
Health Sciences (NIEHS), Research Triangle Park, North Carolina, USA
2Experimental Pathology Laboratories, Inc., Research Triangle Park, North Carolina, USA
3Biostatistics Branch, NIEHS, Research Triangle Park, North Carolina, USA
4Toxicologic Pathology Associates, Jefferson, Arkansas, USA
5National Center for Toxicological Research (NCTR), Jefferson, Arkansas, USA
6NTP/NIEHS, Research Triangle Park, North Carolina, USA
Aloe vera is one of the most commonly used botanicals for various prophylactic and therapeutic purposes. Recently, NTP/NCTR has
demonstrated a dose-dependent increase in large intestinal tumors in F344 rats chronically exposed to Aloe barbadensis Miller (Aloe vera)
non-decolorized whole leaf extract (AVNWLE) in drinking water. The morphological and molecular pathways of AVNWLE-induced large intestinal
tumors in the F344 rats were compared to human colorectal cancer (hCRC) literature. Defined histological criteria were used to compare AVNWLE-
induced large intestinal tumors with hCRC. The commonly mutated genes (Kras, Ctnnb1, and Tp53) and altered signaling pathways (MAPK, WNT,
and TGF-b) important in hCRC were evaluated within AVNWLE-induced large intestinal tumors. Histological evaluation of the large intestinal
tumors indicated eight of twelve adenomas (Ads) and four of twelve carcinomas (Cas). Mutation analysis of eight Ads and four Cas identified point
mutations in exons 1 and 2 of the Kras gene (two of eight Ads, two of four Cas), and in exon 2 of the Ctnnb1 gene (three of eight Ads, one of four
Cas). No Tp53 (exons 5–8) mutations were found in Ads or Cas. Molecular pathways important in hCRC such as MAPK, WNT, and TGF-b signaling
were also altered in AVNWLE-induced Ads and Cas. In conclusion, the AVNWLE-induced large intestinal tumors in F344 rats share several simi-
larities with hCRC at the morphological and molecular levels.
Keywords:Aloe vera; colon; F344 rat; human; colorectal tumors.
There has been an increased popularity in using tradition-
based botanicals as medicines and supplements. Increasingly,
these medicinal herbs and their respective extracts are being
used for newer arbitrary prophylactic and therapeutic purposes
at various dosages and duration. There is insufficient scientific
evidence on the indications and contraindications of many of
these herbal supplements and medicines. Also, the importance
of interactions of these herbal supplements and drugs cannot be
overstated (Hu et al. 2005; Izzo and Ernst 2009). These herbal
products are marketed as dietary supplements (rather than as
drugs), thereby obviating the burden of providing safety and
efficacy data to the United States Food and Drug Administra-
tion (USFDA), under the Dietary Supplement Health and Edu-
cational Act of 1994. The United States National Toxicology
Program (NTP) is testing several commonly used medicinal
herbs for their safety, for example, goldenseal, comfrey, pule-
gone, ginkgo, echinacea, Aloe vera, ginseng, kava kava, milk
thistle, and thujone (Anonymous 1999).
Aloe barbadensis (Miller), Aloe vera (AV), is one of the
most commonly used botanicals for a wide variety of ailments.
The composition of Aloe products depends on several factors
such as location, time of growth and harvest of Aloe crop, spe-
cies of Aloe, as well as the extraction and purification process.
Hence, aloe products may contain variable amounts of aceman-
nans along with other polysaccharides, as well as mixtures of
anthrones and anthraquinones (Boudreau and Beland 2006;
Elsohly et al. 2007). Aloe is used as aloe gel, decolorized whole
leaf extract, and non-decolorized whole leaf extract. The deco-
lorization process involves treatment with activated carbon to
Address correspondence to: Arun R. Pandiri, BVSc&AH, MS, PhD,
DACVP, BG 101 RM B358 MSC B3-06, 111 T. W. Alexander Dr., Research
Triangle Park, NC 27709, USA; e-mail: firstname.lastname@example.org.
This article may be the work product of an employee or group of employees
of the National Institute of Environmental Health Sciences (NIEHS), National
Institutes of Health (NIH); however, the statements, opinions or conclusions
contained therein do not necessarily represent the statements, opinions, or
conclusions of NIEHS, NIH, or the United States government. The authors
received no financial support for the research and/or authorship of this article.
Abbreviations: Ads, adenomas; AVNWLE, Aloe barbadensis Miller
(Aloe vera) non-decolorized whole leaf extract; Cas, carcinomas; CTNNB1,
b-catenin; KRAS, Kirsten rat sarcoma viral oncogene homolog; MAPK,
reverse-transcriptase polymerase chain reaction arrays; NTP, National Toxi-
cology Program; NCTR, National Center for Toxicological Research;
USFDA, United States Food and Drug Administration; WNT, Wg (wingless)
arrays, quantitative real-time
Toxicologic Pathology, 39: 1065-1074, 2011
Copyright # 2011 by The Author(s)
ISSN: 0192-6233 print / 1533-1601 online
remove the latex anthraquinones from the whole leaf extract.
Aloe gel is used as a skin balm for minor burns and is also taken
internally to treat constipation, colitis, peptic ulcers, diabetes,
and for some other therapeutic purposes. In addition, aloe juice
is marketed as a health tonic and as an ingredient of other fruit
juice mixtures. As with some other herbal products, there is a
paucity of safety data on the acute and chronic effects of aloe
ingestion. Recently, the NTP and the National Center for Tox-
icological Research have conducted a subchronic (ninety-day)
bioassay and a chronic (two-year) bioassay in F344 rats and
B6C3F1 mice of both sexes by administering Aloe barbadensis
Miller (Aloe vera) non-decolorized whole leaf extract
(AVNWLE) in drinking water (NTP 2011). In the chronic stud-
ies using B6C3F1 mice, there was no significant tumor inci-
dence. In contrast, there was a marked dose-related increase
in the incidence of large intestinal adenomas (Ads) and carci-
nomas (Cas) in F344 rats of both sexes, with higher incidence
in males than in females (NTP 2011).
Colorectal cancer (hCRC) is the third most frequently diag-
nosed cancer in men and women and the second leading cause
of cancer-related deaths in the United States (Edwards et al.
2010). About 15% of CRC are hereditary, whereas the other
85% are considered to be sporadic (Markowitz and Bertagnolli
2009). Vogelstein and colleagues proposed a multistep genetic
model involving accumulation of multiple, and in some cases
sequential, genetic mutations and aberrant gene expression that
lead to colorectal tumorigenesis (Fearon and Vogelstein 1990).
The common signal transduction pathways and genes involved
in CRC are WNT/CTNNB1, KRAS/BRAF, SMAD4/TGF-b,
PI3K/AKT, and TP53/BAX (Markowitz and Bertagnolli
2009). In addition to these canonical pathways, novel interac-
tions between these pathways as well as novel genes and path-
ways are being characterized in the pathogenesis of hCRC.
We hypothesizedthat the
AVNWLE-induced large intestinal tumors in F344 rats would
reflect major signaling pathways altered in hCRC. Hence, the
objective of the current study was to evaluate MAPK, WNT/
CTNNB1, and TGF-b signaling pathways in the large intestinal
Ads and Cas from AVNWLE-exposed F344 rats, and compare
them to the hCRC literature.
MATERIALS AND METHODS
Samples used in this study were collected during the two-
year NTP/NCTR bioassay. All of the animals in the study were
cared for and humanely handled according to the institutional
guidelines (NTP 2011). Malic Acid and aloin-A were used as
markers for evaluating the stability of the NTP test article
AVNWLE and to confirm the effective dosages. In this study,
drinking water solutions of 0.5%, 1.0%, and 1.5% AVNWLE
had an average malic acid content of 975, 1945, and 2920
mg/g water, respectively, and an average aloin A content of
32.3, 65.6, and 98.3 mg/g water, respectively. The mean percen-
tages of targeted values and standard deviations for malic
acid and aloin A in dosed waters were 95% + 7% and
100% + 12%, respectively. The F344 rats that were exposed
to AVNWLE in drinking water, ad libitum for two years, were
necropsied and examined for treatment-related lesions. The
twelve tumor samples larger than 0.5 cm in diameter were
sectioned in half; one half was fixed in 10% neutral buffered
formalin, and the other half was flash frozen in liquid nitrogen.
The negative controls (four samples) for mutation analysis
consisted of fresh frozen colons (transmural sections) from
concurrent untreated control two-year-old F344 rats. The neg-
ative controls (four samples) for the quantitative real-time
reverse-transcriptase polymerase chain reaction (RT-PCR)
arrays consisted of fresh, gently scraped colonic ‘‘mucosa
only,’’ without the outer muscular tunics, from adult untreated
six-month-old F344 rats to accurately calculate the differential
fold changes since the ‘‘tumor only’’ tissue (without the corre-
sponding outer muscular tunics) was collected for molecular
analysis. DNA was isolated from frozen tumor tissue (eight
Ads and four Cas) as well as the frozen normal colonic tissue
(four samples) from untreated rats for mutation analysis. RNA
was isolated from frozen tumor tissue (four Ads and four Cas)
as well as the normal fresh colonic tissue mucosa (four sam-
ples) from untreated rats for PCR arrays. The formalin-fixed
tissue was routinely processed and stained with hematoxylin
and eosin (H&E) for microscopic analysis.
Following defined histological criteria (Boivin et al. 2003;
Elwell and McConnell 1990), three board-certified patholo-
gists (ARP, MJH, and GPF) independently conducted micro-
DNA was extracted using a DNeasy Tissue Kit (Qiagen,
Valencia, CA) from snap-frozen AVNWLE-exposed tumors
(eight Ads and four Cas) as well as the normal colonic mucosa
(four samples) from untreated rats. The PCR primers used to
amplify the hot spot exons of Kras (1 and 2), Ctnnb1 (2), and
Tp53 (5-8) genes are presented in supplementary Table 1.
(A supplemental appendix to this article is published electroni-
cally only at http://tpx.sagepub.com/supplemental.) Gene ampli-
fication reactions were performed by semi-nested PCR; controls
lacking template DNA were run with all sets of reactions. Poly-
merase chain reaction–amplified products were purified using a
QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). The puri-
fied PCR products were cycled with Terminal Ready Reaction
Mix-Big Dye (Perkin Elmer,Foster City, CA), and the extension
products were purified with DyeEx 2.0 Spin Kit (Qiagen). The
lyophilized PCR products were sequenced with an automatic
sequencer (Perkin-Elmer ABI Model 3100).
Quantitative Real-Time RT-PCR Arrays
Two catalogued PCR arrays (MAP Kinase signaling
[PARN-061] and WNT signaling [PARN-043]) containing
eighty-four genes each (on a ninety-six–well plate) and one
custom PCR array containing ninety-two genes were obtained
from SABiosciences (Frederick, MD). The custom PCR array
was designed to include the canonical TGF-b signaling
1066 PANDIRI ET AL.TOXICOLOGIC PATHOLOGY
pathway (thirty-two genes), as well as sixty other genes
obtained from meta-analysis of ten human CRC microarray
GSE10972, GSE6988, GSE13471, GSE5364, GSE3294, and
GSE4107) using NextBio software (www.nextbio.com) and
data from the hCRC literature. Quantitative differential gene
expression levels were detected using arrays containing corre-
sponding PCR primers and SABiosciences SYBR Green qPCR
master mix, and the reactions were run on an ABI PRISM
7900HT Sequence Detection System (Foster City, CA) using
the manufacturer’s protocols.
Statistics and Data Analysis
Gene expression within each PCR array was normalized to
the housekeeping gene Actb, and the fold changes were calcu-
lated based on the DDCt method (Pfaffl 2001). The data were
analyzed on the basis of the progressive colorectal tumorigen-
esis paradigm proposed by Vogelstein and colleagues (Fearon
and Vogelstein 1990). According to this paradigm, a trend
analysis was conducted to evaluate an increasing or decreasing
trend in gene expression from normal colon tissue to adenoma
to carcinoma. In addition to trend analysis, pairwise compari-
sons (controlling for direction errors) were also performed, for
each gene, between Ads and normal, Cas and normal, and Cas
and Ads. The gene expression data are not necessarily normally
distributed, so the residual bootstrap-based methodology
(Efron and Tibshirani 1994) was employed to compute the p
values using 10,000 bootstrap samples. Since multiple stati-
stical tests were performed, the statistical significance was
determined by controlling the false discovery rate (FDR) at a
nominal 5% level. The trend tests and pairwise comparisons
were performed using the publicly available software ORIO-
GEN, version 4.01 (Peddada et al. 2005). The principal com-
ponent analysis (PCA) and
unsupervised cluster analysis (HCA) were done using the Par-
tek Genomics Suite, version 6.3 (St. Louis, MO). The venn dia-
grams were created using the bioconductor R/limma program
(Smyth 2004). Ingenuity Pathway Analysis (IPA) software was
used to create maps of canonical pathways and to decipher
interactions between various molecular pathways (Ingenuity
Rat Large Intestinal Ads and Cas are Morphologically
Similar to hCRC
The results of the microscopic evaluation of the tumors used
in this study were in concordance with the NTP pathology data
(NTP TR 577). The Ads were characterized by exophytic poly-
poid masses with distorted glands often lined by dysplastic
epithelium. Occasional glandular lumina and lamina propria
had mild to moderate mixed inflammatory cell infiltration.
There was no evidence of stromal invasion of the stalk of the
polyp or invasion into the muscularis mucosa (Figure 1A). The
Cas were characterized by neoplastic polypoid lesions with
anaplastic glandular epithelium invading the stalk stroma/mus-
cularis mucosa (Figure 1B). Mitoses, desmoplasia, and mixed
inflammatory cell infiltrates were often noted in the Cas.
Large Intestinal Tumors from AVNWLE-Exposed Rats
Have Point Mutations within Kras or Ctnnb1, but Not in
Summary of the mutation analysis is presented in Table 1,
and examples of the electropherogram indicating mutations
tumor sections from F344 rats exposed to Aloe vera non-decolorized
whole leaf extract in drinking water, ad libitum, for two years. (A) Ade-
noma (50?). The exophytic mass with dysplastic hyperchromatic cells
(arrows and inset box) was limited to the mucosa without evidence of
invasion into the muscularis mucosa. (B) Carcinoma (60?). Note the
neoplastic cells invading past the muscularis mucosa (arrows) and the
fibroplasia surrounding the neoplastic cells (inset box).
Vol. 39, No. 7, 2011 AVNWLE-INDUCED LARGE INTESTINAL TUMORS IN RATS1067
in various codons of Kras and Ctnnb1 genes are presented
in Figure 2. Point mutations within the Kras were observed
in two of eight (25%) Ads and two of four (50%) Cas. Both
the Ads samples had Kras mutations within codon 13 (G to
C). The Kras mutations within the Cas samples were within
codon 12 (G to A) and codon 61 (A to G). Point mutations
within the regulatory domain (codons 29–50) of Ctnnb1
were observed in three of eight (38%) Ads and one of four
(25%) Cas. The Ctnnb1 mutations within the Ads samples
were observed within codon 32 (G to A), codon 41 (A to
C), and codon 45 (C to T). The Ctnnb1 mutation within the
Cas sample was in codon 34 (G to A). The mutations within
Kras and Ctnnb1 were mutually exclusive within the tumor
samples. No Tp53 (exons 5–8) mutations were observed
within either the Ads or Cas. No mutations were observed
within the Kras, Ctnnb1, or Tp53 in control colonic epithe-
Differential Gene Expression Indicates Up-Regulation of
Pathways Associated with hCRC Principal Component
Analysis (PCA) and Hierarchal Cluster Analysis (HCA)
Principal componentanalysis plots revealed a clear-cut clus-
tering between normal mucosal epithelial samples (negative
controls) and tumor samples. However, within the tumor sam-
ples, there was a slight overlap between Ads and Cas samples
(Figure 3A). Likewise, HCA of all of the 110 statistically sig-
nificant genes revealed a tight clustering of normal mucosal
epithelium (negative controls) and Ads and Cas segregated
reasonably well, with the exception of a single Cas that has
clustered with the Ads (Figure 3B).
The MAPK signaling pathway was highly represented
within both the Ads and Cas samples (Table 2, Figure 4A,
supplementary Figure 1). Literature citing the roles played
by each of the significantly altered genes within CRC is
presented in the references column of supplementary Table
2. Trend analysis of differential gene expression after nor-
malizing to the housekeeping gene Actb indicated twenty-
five genes and two genes with increasing and decreasing
differential gene expression patterns, respectively (supple-
mentary Table 3). Pairwise comparisons (controlling for
directional errors and FDR at a 5% nominal level) indicated
that twenty-two genes and thirteen genes were significantly
altered within the Ads and Cas samples, respectively (Figure
4A, supplementary Table 2). There was an excellent agree-
ment between the trend test and pairwise comparisons. There
were only five of twenty-seven unique genes within the trend
test and one of twenty-three unique genes within the pairwise
comparisons. There was up-regulation of transcription regula-
tors (Ccne1, Creb1, Ets1, and Tp53), kinases (Cdk4, Cdk6,
and Ksr1), and cell cycle genes (Ccnb2, Ccnd1, and Ccnd2)
in both Ads and Cas. In contrast, some transcription factors
(Cdnk2c and Myc), and kinases (Cdc42, Egfr, Mapk7,
Mapk2k1, Mapk2k4, Mapk12, and Mapk13) were signifi-
cantly up-regulated only in Ads (they did not meet the FDR
criteria for significance in Cas). Heat shock protein Hspb1
was up-regulated in Ads and Cas, whereas Hspa5 was
down-regulated only in Cas. Col1a1, a profibrogenic extra-
cellular matrix gene, was highly expressed both within Ads
The WNT signaling pathway was highly represented
within both the Ads and Cas samples (Table 2, Figure 4B,
supplementary Figure 2). Literature citing the roles played
by each of the significantly altered genes within CRC is pre-
sented in the references column of supplementary Table 4.
Trend analysis of differential gene expression after normaliz-
ing to the housekeeping genes Actb indicated thirty-four
genes and one gene with increasing and decreasing differen-
tial gene expression patterns, respectively (supplementary
Table 5). Pairwise comparisons (controlling for directional
TABLE 1.—Summary of gene mutations in F344 rat large intestinal tumors.a
Animal IDDx Codon 12 Codon 61Exons 5–8
Codon 32 GAT->AAT (Asp->Asn)
Codon 45 TCC->TTC (Ser->Phe)
Codon 41 ACC->CCC (Thr->Pro)
Codon 34 GGA->GAA (Gly->Glu)
Note: There were no Kras, Ctnnb1, and Tp53 point mutations within the four age-matched untreated colon tissue from F344 control rats.
aF344 rats were treated ad libitum with Aloe vera non-decolorized whole leaf extract in drinking water for 2 years.
Abbreviations: Ads, adenoma; Cas, carcinoma; Dx, diagnosis; N, normal colonic mucosal epithelium; wt, wild type.
1068PANDIRI ET AL.TOXICOLOGIC PATHOLOGY
errors and FDR at a 5% nominal level) indicated that thirty-
one genes and thirty-two genes were significantly altered
within the Ads and Cas samples, respectively (Figure 4B,
supplementary Table 4). There were only two of thirty-four
unique genes within the trend test and nine of thirty-nine
unique genes within the pairwise comparisons. There was
up-regulation of transcription regulators (Ctnnb1, Lef1, Tcf3,
Tcf4, and Tcf7), WNT genes (Wif1, Wnt3, Wnt4, and Wnt7b),
APC complex genes (Axin1, Dvl1, Frzb, Fzd2, Fzd5, and
Fzd6), cell cycle genes (Csnk2a1 Ccnd1, Ccnd2, and Ccnd3),
and other important WNT pathway genes (RhoA, Bcl9,
Dkk3, Nkd2, Sfrp1, and Sfrp4) in both Ads and Cas. Tran-
scription regulators Myc, Pitx2, and Tle2 and WNT pathway
genes Fbxw2, Axin2, Nkd1, RGD1561440, and Ppp2ca were
up-regulated only in Ads. Conversely, WNT genes Lrp5,
Porcn, Wnt2b, Wnt3a, and Wnt5b were up-regulated only
TGF-β Signaling and Other Genes Relevant for Human
Several important genes within the TGF-b signaling path-
way were altered in both Ads and Cas (Table 2, Figure 4C,
supplementary Figure 3). Literature citing the roles played by
each of the significantly altered genes within CRC is presented
in the references column of supplementary Table 6. Trend anal-
ysis of differential gene expression after normalizing to the
housekeeping genes Actb indicated thirty-eight genes and ten
genes with increasing and decreasing differential gene expres-
sion patterns, respectively (supplementary Table 7). Pairwise
comparisons (controlling for directional errors and FDR at a
5% nominal level) indicated that forty-four genes and thirty-
seven genes were significantly altered within the Ads and Cas
samples, respectively (Figure 4C, supplementary Table 6).
There were only seven of forty-eight unique genes within the
trend test and six of forty-seven unique genes within the pair-
wise comparisons. Relevant TGF-b pathway transcription reg-
ulators (Smad2), growth factors (Bmp1, Bmp4, Inhba, Tgfb1,
Tgfb2, and Tgfb3), and kinases (Tgfbr1, Tgfbr2, and Tgfbr3)
were altered in both Ads and Cas. Smad5 was altered only in
Ads, and Smad1 was altered only in Cas.
Among other relevant human CRC genes, there was differ-
ential expression of transcription regulators (Klf4, Sox9, Stat1,
Stat3, and Tcf7l2), kinases (Akt1, Akt2, Akt3, Sgk1, and Stk11),
phosphatases Cdc25a, Dusp4, Ptpro and Ptprs), and other col-
orectal cancer genes (Tnf, Ca2, Hpgd, Hsd17b2, Msh2, Nos2,
FIGURE 2.—Examples of electropherograms indicating point mutations within Kras and Ctnnb1 genes amplified from large intestinal adenomas
and carcinomas of F344 rats chronically exposed to Aloe vera non-decolorized whole leaf extract. (A) Identification of point mutations in codons
12 and 13 (exons 1 and 2) of the Kras gene. (a) Normal Kras codon 12 (GGT) and codon 13 (GGC); (b) adenoma with mutated codon 13
(GGC>CGC); (c) carcinoma with mutated codon 12 (GGT>GAT). (B) Identification of point mutations in codons 32 and 34 (exon 2) of Ctnnb1
gene. (a) Normal Ctnnb1 codon 32 (GAT) and codon 34 (GGA); (b) adenoma with mutated codon 32 (GAT>AAT); (c) carcinoma with mutated
codon 34 (GGA->GAA).
Vol. 39, No. 7, 2011 AVNWLE-INDUCED LARGE INTESTINAL TUMORS IN RATS1069
Psat1, Bax, and Sparc) in both Ads and Cas. Some kinases
(Fgfr1, Pik3cb, Pik3r1, and Pik3r1) and other colorectal cancer
genes (Hspd1, Top2a, Birc5, and Timp1) were up-regulated
only in Ads, and Casp3 was down-regulated only within Cas.
We have demonstrated that the AVNWLE-induced large
intestinal tumors in F344 rats share several similarities with
human CRC at the morphological and molecular level. There
was excellent correlation between the trend analysis and pair-
wise comparisons and the resulting genes from each pathway
yielded a very meaningful molecular insight into the possible
pathogenesis of AVNWLE-induced colorectal tumors in
F344 rats. There was no single molecular pathway that was
unique to Ads or Cas samples. Within the PCA and HCA, the
segregation between Ads and Cas samples was not very dis-
crete and there was a slight overlap. These findings were not
surprising since these plots were based on a limited set of genes
(not based on whole genome arrays) and may also be due to the
fact that the pathogenesis of the Ads and Cas is part of a con-
tinuum. Histological re-examination of the Cas sample that had
clustered with the Ads (in the HCA plot) unequivocally con-
firmed our previous diagnosis of Cas.
FIGURE 3.—Principal component analysis (A) and hierarchical cluster
analysis (B) of differentially expressed genes within untreated colon
tissues as well as large intestinal adenomas and carcinomas from
F344 rats exposed to Aloe vera non-decolorized whole leaf extract
in drinking water, ad libitum, for two years.
TABLE 2.—Summary of differentially expressed genes in the large
intestinal tumors of F344 rats chronically exposed to Aloe vera non-
decolorized whole leaf extract in drinking water.
Differentially expressed genes in Aloe vera
non-decolorized whole leaf extract–induced tumors
Cell cycle genes
Map2k4, Mapk7, Mapk12, Mapk13, Mapk8ip3
Ets1, Tp53, Creb1, Ccne1, Myc, Cdkn2c
Ccnd1, Ccnd2, Ccnd3, Ccnb2, Cdc42, Cdk6, Cdk4,
Egfr, Ksr1, Col1a1, Hspa5a, Hspb1
WNT ligands and
Wnt 2b, Wnt3, Wnt3a, Wnt4, Wnt7b, Frzb, Fzd2,
Dvl1, Axin1, Axin2, Ctnnb1
Wnt5a, Fzd6, Dvl1, RhoA, Nkd1, Nkd2
Tcf3, Tcf4, Tle1, RhoA, Bcl9, Dkk3, Nkd2, Sfrp1,
Sfrp4, Fbxw2, Lef1, Lrp5, Pitx2, Porcn,
important in hCRC
Smad1, Smad2, Smad5
Tgfb1, Tgfb2, Tgfb3, Bmp1, Bmp4, Bmpr2, Inhba
Tgfbr1, Tgfbr2, Tgfbr3
Klf4a, Sox4, Sox9, Stat1, Stat3, Tcf7l2
Pik3cb, Pik3r1, Pik3r2, Akt1, Akt2, Akt3, Fgfr1,
Sgk1a, Stk11, Fgfr1
Cdc25a, Dusp4, Ptpro, Ptprs,
Birc5, Bax, Casp3a
Tnf-a, Nos2, Ca2a, Prdx6, Hpgda, Hsd17b2a, Msh2,
Psat1, Timp1a, Hspd1, Top2a, Sparc
aDown-regulated gene. All other genes are up-regulated compared to untreated
control colonic epithelium.
1070PANDIRI ET AL.TOXICOLOGIC PATHOLOGY
In this study, the ideal samples for differential gene expres-
sion would have been age-matched controls, since it is known
that there can be differences in gene expression between intest-
inal mucosa of control animals of different ages. However,
because of logistical issues (described in sample collection),
this method was not possible. Nevertheless, the differential
gene expression between tumor tissue (from two-year-old rats)
and control colonic epithelium (from six-month-old rats) is
highly suggestive of a tumor signature.
Kras mutations are an early event in chemical-induced col-
orectal carcinogenesis and are seen with increasing frequency
within aberrant crypt foci<adenoma<carcinoma. As illustrated
in Table 3, the incidence of Kras mutations in AVNWLE-
treated F344 rats was 33% and was comparable to human col-
orectal cancers (40%), 1,2-dimethylhydrazine (1,2-DMH)–
induced colon tumors in albino rats (66%), and azoxymethane
(AOM)-induced colon tumors in F344 rats (37%; Bos et al.
1987; Jacoby et al. 1991; Vivona et al. 1993). Point mutations
in exon 1 of Kras observed in this study were similar to those
reported in1,2-DMH and AOM-induced colon tumors in ratsas
well as in human CRC. Ctnnb1 mutations are also potential
early events in human CRC (Markowitz and Bertagnolli
2009). The incidence of Ctnnb1 mutations in this study was
33% (Table 3) and was comparable to human CRC (10–
26%), but significantly lower than that reported in rats exposed
to AOM (80%) and cooked meat–derived heterocyclic amines
(75%; Dashwood et al. 1998; Morin et al. 1997; Takahashi et
al. 1998). AVNWLE-induced Ctnnb1 mutations observed in
codons 32, 34, 41, and 45 are in the proximity of threonine and
serine sites that are important for phosphorylation. The muta-
tions within Kras and Ctnnb1 were mutually exclusive within
the tumor samples, that is, no single tumor sample had muta-
tions in both Kras and Ctnnb1. In addition, the Kras mutations
in adenomas (codon 13) and carcinomas (codons 12 and 61)
were also mutually exclusive. These seemingly mutually exclu-
sive mutations may be a result of the small sample size,
FIGURE 4.—The venn diagrams represent the number of differentially
expressed genes (*), within the Aloe vera non-decolorized whole leaf
extract exposed F344 large intestinal adenoma and carcinoma samples
compared to control colon tissue, by multiple pairwise comparisons
FIGURE 4 (continued). and the statistical significance was determined
by controlling the false discovery rate (FDR) at a nominal 5% level.
(A) MAPK signaling; (B) WNT signaling; (C) TGF-b signaling.
TABLE 3.—Comparison of incidence of point mutations within large
intestinal tumors of F344 rats chronically exposed to Aloe vera non-
decolorized whole leaf extract with human colorectal canceraand
rodent models of chemically induced colon cancer.b
Aloe vera non-decolorized
whole leaf extract
Human colon cancer
* Fisher exact test, p < .008.
aBos et al. 1987; Morin et al. 1997.
bJacoby et al. 1991; Vivona et al. 1993; Dashwood et al. 1998; Takahashi et al. 1998;
Takahashi and Wakabayashi 2004.
Vol. 39, No. 7, 2011 AVNWLE-INDUCED LARGE INTESTINAL TUMORS IN RATS 1071
especially considering the Vogelstein’s paradigm of adenoma–
carcinoma continuum, where the carcinoma samples should
include the mutations observed within the adenoma samples.
In human CRC, the frequency of mutations in the Tp53 gene
is about 50% (Nigro et al. 1989). However, no Tp53 mutations
(in exons 5–8) were found within the AVNWLE-induced large
intestinal tumors. Interestingly, no Tp53 mutations were
observed in rodent models of colon cancer, such as 2-amino-
1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) and azoxy-
methane (Takahashi and Wakabayashi 2004). These data sug-
gest that AVNWLE-induced large intestinal tumors in the rat
develop independently of the TP53 signal transduction path-
way, but they involve other significant genetic pathways that
are relevant to human colon cancer.
The MAPK signal transduction pathway is very important in
colon cancer. It consists of four main pathways: extracellular
signal-related kinases (Ras/Raf1/MEK/ERK), ERK5 (BMK1
or MAPK7), c-Jun N-terminal kinases or stress activated pro-
tein kinases (JNK or SAPK), and p38 kinases (p38a, p38b,
p38g, p38d; Johnson and Lapadat 2002). These pathways may
be activated through consistent activation of Kras and by var-
ious stimuli such as growth factors, cytokines, G-protein–
coupled receptor ligands, stress, carcinogens, and transforming
agents (Johnson and Lapadat 2002). In this study, several mem-
bers of the MAPK pathway were significantly altered, such as
the Map2k1, Map2k4, Mapk1, Mapk12, Mapk13, Mapk7, and
Mapk8ip3. Map2k1 activates Mapk3 and Mapk1, and plays
an important role in the epithelial-to-mesenchymal transition
(Lemieux et al. 2009). Map2k4, an upstream kinase of the
p38 and JNK pathway, is primarily activated by environmental
stress. Mapk12 and Mapk13 are members of the p38 MAPK
signaling and Mapk7 is a member of the ERK5 signaling. The
up-regulation of Map2k1 and Mapk1 may be related to mito-
genic stimulation by the mutated Kras, and the up-regulation
of Mapk7, Map2k4, Mapk12, Mapk13, and Mapk8ip3 may be
caused by various cellular stresses. Thus, the overrepresenta-
tion of the MAPK pathway within these large intestinal tumors
is likely a result of a multi-pronged stimulation caused by the
mutated Kras as well as cellular stress.
In addition to activation of the MAPK pathway, there was
alteration of several downstream cell cycle genes (Cdc42,
Cdk4, Cdk6, Ccnb2, Ccnd1, and Ccnd2) that play an important
role in human CRC. Creb1, up-regulated in both Ads and Cas,
inhibits apoptosis in colon cancer cells by inducing cellular
inhibitor of apoptosis protein 2 (Nishihara et al. 2004). Ets1,
a proto-oncogene, was highly up-regulated in AVNWLE
tumors, and its expression was correlated with tumor invasion
in human CRC (Nakayama et al. 2001). Consistent with the
role of inflammation in hCRC, ETS1 can also regulate COX2
promoter activity (Zhang et al. 2007).
WNT/CTNNB1 signaling is altered in almost all cases of
CRC (Kinzler and Vogelstein 1996; Morin et al. 1997). Several
genes within the WNT/CTNNB1 pathway were also altered
within the AVNWLE-induced large intestinal tumors in F344
rats, including up-regulation of Ctnnb1. The catalog of differ-
entially expressed genes within AVNWLE-induced large
intestinal tumors in rats indicates that all stages of the canonical
WNT/CTNNB1 pathway were represented. However, several
negative regulators (Dkk3, Wif1, Sfrp1, Sfrp4, Nkd1, and Nkd2)
of the canonical WNT/CTNNB1 pathway were up-regulated
within our dataset, perhaps because of negative feedback up-
regulation of these genes (Cebrat et al. 2004; Katoh 2001). In
addition, some of these canonical WNT antagonists and ago-
nists may regulate CTNNB1-independent WNT signaling path-
ways, such as the WNT/Ca2þpathway or the WNT/planar cell
polarity (PCP) pathway (Katoh 2005; Katoh 2007). Several
non-canonical WNT signaling mediators that were up-
regulated within our dataset included Wnt5a, Fzd6, Dvl1,
RhoA, Nkd1, and Nkd2. Also, some of these genes may interact
with the MAPK signaling through non-canonical WNT signal-
ing pathways (Katoh 2005; 2007).
The TGF-b pathway is a critical pathway that is usually
altered during the later stages of human CRC pathogenesis
(Markowitz and Bertagnolli 2009). A potent pleotrophic cyto-
kine, TGF-b has myriad functions based on the cell type, stage,
and context of signaling. It acts as a tumor suppressor in normal
intestinal cells butacts as a potenttumor promoterin colon can-
cer (Wakefield and Roberts 2002). In tumor cells, TGF-b pro-
motes uncontrolled cell proliferation, epithelial-mesenchymal
transition (EMT), invasion, metastasis, angiogenesis, and dys-
regulated immunosurveillance (Tian et al. 2010).
cal TGF-b signaling pathwayinAVNWLE-inducedlarge intest-
Tgfb3) that bind and activate TGFb receptors (Tgfbr1 and
Tgfbr2). The activated Tgfr1 phosphorylates Smad2, but its
heteromericcomplexpartnersSmad3 andSmad4 were not repre-
sented within the AVNWLE-induced rat large intestinal tumors.
ylable form of Smad2 found in colorectal cancers, induces
inhibit TGFb-mediated growth arrest (Prunier et al. 1999). The
expression ofSmad4, a bonafide tumor suppressor, is lostin sev-
eralcoloncancers andwas not detectedinthis study.Inaddition,
it has been shown the SMAD4 silencing and Ras hyperstimula-
tion can overcome TGF-b’s tumor suppressor effects (Calonge
and Massague 1999). BMP-mediated SMAD signaling appears
to be down-regulated within our data set since Bmpr2 is
down-regulated with corresponding up-regulation of Bmp1 and
Bmp4, which is possibly a consequence of a feedback loop. In
human colorectal tumors, down-regulation of BMPRII and
ways such as MAPK (ERK1/ERK2, p38, and JNK), growth and
survival kinases (PI3K, AKT/PKB, and mTOR), and small
GTP-binding proteins (RAS, RHOA, RAC1, and CDC42; Tian
et al. 2010). Several of these mediators are up-regulated within
AVNWLE-induced intestinal tumors, suggesting an interplay
between multiple signal transduction pathways.
Several mediators that play important roles in inflammation
as well as cellular proliferation were altered in AVNWLE-
1072 PANDIRI ET AL.TOXICOLOGIC PATHOLOGY
induced intestinal tumors. It is well known that inflammation
plays a promoter role in carcinogenesis, and examples include
CRC within ulcerative colitis, dextran sodium sulfate rodent
colitis-tumor models, and genetically engineered mouse
models such as IL-2/b2M DKO, IL-10 KO, and Rag2 KO
(Itzkowitz and Yio 2004). Within these models, many of the
genes that were altered within colon tumors were also altered
within inflamed colonic mucosa without any histological evi-
dence of dysplasia (Itzkowitz and Yio 2004). Tnf, an important
pleotrophic ligand for activation of NF?B, stress related-
MAPK JNK, and p38 signaling, and apoptosis signaling was
markedly up-regulated within both AVNWLE-induced Ads
and Cas samples (Beissert et al. 1989; Yoshimi et al. 1994).
Nos2, an important inducer of TNF, was also markedly up-
regulated within all colon tumors examined in this study (Ambs
et al. 1998; Zhang et al. 1998). STAT1 and STAT3 are impor-
tant mediators within the JAK-STAT signaling pathway and
are activated by interferon g and IL-6 ligands, respectively.
Activated STAT1 and STAT3 regulate several target genes
important in inflammation, and innate and adaptive immune
responses. In addition, activation of STAT3 has been reported
in several tumors in which it has been associated with growth
promotion and anti-apoptosis (Aaronson and Horvath 2002).
Several mediators of the PI3K/Akt signaling pathway (Pik3cb,
Pik3r1, Pik3r2, Akt1, Akt2, Akt3, and Stk11) were up-regulated
in this study. This pathway is important in effecting inflamma-
tory response, anti-apoptosis, and cell proliferation (Paez and
Sellers 2003). Evaluation of IPA pathway interactions based
on the differentially expressed genes in this study indicated
altered canonical inflammatory pathways such as IL-8, NF?B,
Further prospective studies are warranted to examine the roles
played by these inflammatory pathways in colon carcinogen-
esis in greater detail.
Most anthranoid laxatives, and probably those from
AVNWLE as well, exert their laxative effects by inhibition of
Naþ-KþATPase, damaging colonic epithelial cells, and
increasing intestinal motility. In addition, the damaged epithe-
in recruitment of inflammatory cells that subsequently release
additional inflammatory mediators with myriad functions in
inflammation and cell proliferation (van Gorkom et al. 1999).
tration within the tumors (NTP 2011). However, it is uncertain
whether the inflammatory infiltrate preceded or is the result of
tumorigenesis. The genotoxicity of anthraquinone compounds
nature and structure of the anthraquinones (Mueller et al. 1996;
Westendorf et al. 1990). Though AVNWLE was found to be
non-genotoxic in the NTP’s Ames test, it does not necessarily
mean that the test article is indeed non-genotoxic at the tissue
level (colonic epithelial interface) because of the numerous
variables that can influence the genotoxic status of the test com-
pound. Thus, it is possible that genotoxicity along with chronic
inflammation may have contributed to AVNWLE-induced large
intestinal tumorigenesis in F334 rats.
In conclusion, we have demonstrated that the molecular
pathways important in the pathogenesis of hCRC such as
MAPK, WNT, and TGF-b signaling were also altered in large
intestinal tumors from F344 rats chronically exposed to
AVNWLE in drinking water. Further research, particularly ear-
lier pre-disease time points, may provide data on potential pre-
dictors or biomarkers of disease that can be useful in hazard
identification and protecting human health.
The authors would like to thank Dr. Darlene Dixon and
Dr. Michael Devito for their review of this manuscript; Dr.
Keith Shockley and Dr. Grace Kissling for help with statistics;
and Ms. Natasha Clayton and Ms. Beth Mahler for their excel-
lent technical assistance. This study was supported in part by
IAG #224-07-0007 between the FDA and the NTP as well as
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