Arsenic and Cancer

1. “Long-term low-dose exposure of human
urothelial cells to sodium arsenite
activates lipocalin-2 via promoter
hypomethylation”
ARCHIVES OF TOXICOLOGY(2014) 88:1549–1559
TOXICOLOGY 9/87
Impact Factor- 5.078
Advisor: Speaker:
Yi-Wen Liu, PhD. Mezbahul Haque
Professor
Date: 2014.11.14
National Chiayi University
Dep. Of Microbiology, Immunology & Biopharmaceuticals.
1

2. Introduction
Both arsenic and its metabolites can have a variety of genotoxic effects,
which may be mediated by oxidants or free radical species. All of these
species also have effects on signaling pathways leading to proliferative
responses. Mutation Research 533 (2003): 37–65
DNA methylation is one of the most prominent mechanisms for epigenetic
control of gene expression. Nat Rev Genet 13 (2012):484–492
Recently, emerging evidence also has shown that LCN2 is overexpressed in
a variety of human cancers and facilitates tumorigenesis by promoting
survival, growth and metastasis. Cancer Lett 316 (2012):132–138
Lipocalin 2 (LCN2) was demonstrated to promote metastasis via several
different mechanisms, such as the formation the LCN2/MMP9 complex
and the enhancement of epithelial–mesenchymal transition (EMT)
signaling pathway. J Biol Chem 276 (2001):37258–37265
2

3. 3
DNA Methylation
DNA methylation is an epigenetic mechanism involving the transfer of a
methyl group onto the C5 position of the cytosine to form 5 -methylcytosine.
DNA methylation regulates gene expression by recruiting proteins involved in
gene repression or by inhibiting the binding of transcription factor to DNA.
Alcohol Res 35 (2013):6-16

4. 4
DNA Methylation and arsenic induced
Cancer
The long-term effects of acute exposure to arsenic and show that
the genetic instability was associated with structural chromosome
changes and concurrently with DNA hypomethylation.
Inorganic arsenic induced hypomethylation results in genomic
instability and oncogene upregulation.
Inorganic arsenic induced hypermethylation leads to the
downregulation of tumor suppressor genes.
Carcinogenesis 25 (2004):413-417
Toxicology and Applied Pharmacology 206 (2005) 288– 298
Toxicological Science 91(2006): 372–381

5. 5
Lipocalin-2 (LCN2)
LCN2 or Neutrophil Gelatinase Associated Lipocalin (NGAL), is a
dynamic 25 kDa protein with roles in innate immunity and in a
variety of pathologies.
LCN2 is a versatile molecule with an apparent dual role: a
beneficial one in innate immunity that protects against bacterial
pathogens, and a detrimental one when co-opted by cancer cells
into a tumor-promoting role.
Recently, emerging evidence also has shown that LCN2 is overexp-
ressed in a variety of human cancers and facilitates tumorigenesis
by promoting survival, growth and metastasis.
Cancer Letters 316 (2012) 132–138
Cancer Letters 316 (2012) 132–138
Cancer Letters 316 (2012) 132–138

6. 6
Lipocalin-2 in cancer
Cancer Letters 316 (2012) 132–138

7. 7
Aims
To further evaluation the involvement of DNA methylation
alterations in the regulation of gene expression in iAs exposed
human urothelial cells (iAs-HUCs).
Also wants to elucidate the oncogenic role of LCN2 and
demonstrate that its expression is enhanced in arsenic-exposed
cells via promoter hypomethylation or not.

8. Materials
8
Cell lines and cell culture:
The SV40-immortalized HUC line was exposed to 0.5 μM sodium arsenite
(NaAsO2) for more than 25 passages.
Tissue samples:
Twenty pairs of bladder cancer tissues and their adjacent normal tissues.
Tissue samples were from NTUH.

9. Methods
1. Treatment with 5-aza-2′-deoxycytidine (5-aza-dC)
2. Quantitative real-time PCR
3. Western blotting
4. Genome-wide methylation analysis
5. Bisulfite sequencing PCR (BSP)
6. Anchorage-independent cell growth
7. Cell growth in serum-free culture medium
8. Cell proliferation assay
9. Site-directed mutagenesis
10. Chromatin immunoprecipitation (ChIP) assay
11. Intracellular iron determination
12. Intracellular reactive oxygen species (ROS) determination
9

10. Flow Chart
10
Gene expression in iAs-HUCs due to alterations in their DNA
methylation status
Evaluation of the relationship between the NF-κB and
hypomethylated promoter of Lipocqalin2 (LCN2)
LCN2 expression with iAs-induced cellular transformation and
anti-apoptotic activity
Observation of NF-κB activity and expression of inflammatory
cytokines regulated by the LCN2
Identification of intracellular iron and ROS levels induced by
Lipocqalin2 (LCN2)

11. 11
|Δ range|*
CpG sites (%)
Hypomethylated (Δ<0)
Hypermethylated
(Δ>0)
0~0.25 895 (46.3%) 475 (43.4%)
0.25~0.5 721 (37.3%) 445 (40.6%)
0.5~0.75 257 (13.3%) 141 (12.9%)
0.75~1.0 62 (3.2%) 34 (3.1%)
Total 1935 (63.9%) 1095 (36.1%)
Methylation levels between the iAs-HUCs
and HUCs

12. 12
Genes regulated by DNA methylation
Hypomethylated
(Δβ<−0.75)
Hypermethylated
(Δβ>0.75)
Total genes analyzed 40 21
No expression 3 (0) 2 (0)
Upregulated (p<0.05) 33 (20)a 7 (2)b
No significant change (p>0.05) 3 (0)a 5 (2)b
Downregulated (p<0.05) 1 (0)a 7 (5)b
a HUCs was significantly enhanced by treatment with 5-aza-dC (p<0.05).
b iAs-HUCs was significantly enhanced by treatment with 5-aza-dC (p<0.05).

13. 13
Summary
There are many genes has expressed typically in iAs-HUCs , the
genes whose expression levels were associated with their DNA
methylation status.
Larger number of gene expression level has shown due to
hypomethylation than hypermethylation.
Using 5-aza-dC treatment, it confirmed that the expression levels
of 20 out of 40 (50 %) hypomethylated and 5 out of 21 (23.8 %)
hypermethylated genes were likely associated with their promoter
methylation status.

14. Flow Chart
14
Gene expression in iAs-HUCs due to alterations in their DNA
methylation status
Evaluation of the relationship between the NF-κB and
hypomethylated promoter of LCN2
LCN2 expression with iAs-induced cellular transformation and
anti-apoptotic activity
Observation of NF-κB activity and expression of inflammatory
cytokines regulated by the LCN2
Identification of intracellular iron and ROS levels induced by
LCN2

15. 15
The methylation status of the LCN2 gene promoter and DNA
methylation ratios for individual CpG sites

16. 16
Identification of LCN2 promoter activity
1. Luciferase Reporter assay
2. ChIP assay
An inhibitor of NF-κB

17. 17
Summary
The promoter of the LCN2 gene in iAs-HUCs and bladder
tumors was apparently hypomethylated as compared with
their counterparts.
RelA and NF-κB1 are the major NF-κB members regulating
LCN2 expression in iAs-HUCs. However, C/EBP-α may also play
certain roles as a transcriptional activator of LCN2.

18. Flow Chart
18
Gene expression in iAs-HUCs due to alterations in their DNA
methylation status
Evaluation of the relationship between the NF-κB and
hypomethylated promoter of Lipocqalin2 (LCN2)
LCN2 expression with iAs-induced cellular transformation and
anti-apoptotic activity
Observation of NF-κB activity and expression of inflammatory
cytokines regulated by the LCN2
Identification of intracellular iron and ROS levels induced by
Lipocqalin2 (LCN2)

19. 19
Identification of protein level of LCN2 and
involvement of LCN2 in cellular transformation
Anchorage-independent growth assayWestern blot analysis

20. 20
Determination of cell viability and protein level with
or without serum in the cell culture medium
AlamarBlue assay Western blotting

21. 21
Determination of effect in cell growth and anti-apoptotic
activity of the iAs-HUC cells in serum-free medium

22. 22
Summary
The mRNA levels of BCLX (an anti-apoptotic gene), LC3 and
DAPK1 (autophagic genes) were remarkably enhanced in the iAs-
HUCs but was significantly suppressed upon the silencing of LCN2.
These results suggest that LCN2 upregulation is associated with
the enhanced anti-apoptotic activity of the iAs-HUC cells in serum-
free medium.

23. Flow Chart
23
Gene expression in iAs-HUCs due to alterations in their DNA
methylation status
Evaluation of the relationship between the NF-κB and
hypomethylated promoter of Lipocqalin2 (LCN2)
LCN2 expression with iAs-induced cellular transformation and
anti-apoptotic activity
Observation of NF-κB activity and expression of inflammatory
cytokines regulated by the LCN2
Identification of intracellular iron and ROS levels induced by
Lipocqalin2 (LCN2)

24. 24
NF-κB activity and expression LCN2 also activates NF-κB
in iAs-HUCs
Luciferase reporter assay Western blotting

25. 25
The mRNA levels of pro-inflammatory genes

26. 26
The mRNA levels of pro-inflammatory genes were examined
by qPCR after treated with the NF-κB inhibitor Bay 11- 7082

27. 27
Summary
The increase in NF-κB activity resulting from LCN2
overexpression promotes the increased expression of inflammatory
genes in iAs-HUCs.
NF-κB is a transcription factor of LCN2 in iAs-HUCs, the
enhanced expression of LCN2 may as well activate NF-κB activity.

28. Flow Chart
28
Gene expression in iAs-HUCs due to alterations in their DNA
methylation status
Evaluation of the relationship between the NF-κB and
hypomethylated promoter of Lipocqalin2 (LCN2)
LCN2 expression with iAs-induced cellular transformation and
anti-apoptotic activity
Observation of NF-κB activity and expression of inflammatory
cytokines regulated by the LCN2
Identification of intracellular iron and ROS levels induced by
Lipocqalin2 (LCN2)

29. 29
Measurement of the intracellular iron levels and the
ROS levels
Mean fluorescence intensities (MFI)

30. 30
The relative ROS levels, NF-κB activities, or mRNA
levels were calculated by comparing with control cells
Iron chelator-2,2′-bipyridyl (BPD), Antioxidant-N-acetyl-cysteine (NAC)

31. 31
Summary
Enhanced NF-κB activity and expression of pro-
inflammatory cytokines as a result of LCN2
overexpression in iAs-HUCs.
LCN2-enhanced NF-κB activation in iAs-HUC cells is
likely due to increased iron accumulation and ROS
production.

32. 32
Conclusion
LCN2 LCN2
LCN2LCN2
LCN2
Fe Fe
Fe
Fe
Micro environmental
StimuliER
NF-κB
ROS
Inflammatory
Cytokine
Autophagy &
Anti-apoptotic
activity
Tumor
growth
MMP9
Stablization
EMT
Promotion
Tumor cell
Fe
LCN2
Inorganic arsenic

33. 33
Thank you

34. 34
5-aza-2′-deoxycytidine (5-aza-dC)
Decitabine (trade name Dacogen), or 5-aza-2'-deoxycytidine, is a drug for the
treatment of myelodysplastic syndromes, a class of conditions where certain
blood cells are dysfunctional, and for acute myeloid leukemia (AML).
Chemically, it is a cytidine analog.
Mechanism:
Decitabine is a hypomethylating agent. It hypomethylates DNA by inhibiting
DNA methyltransferase. It functions in a similar manner to azacitidine, although
decitabine can only be incorporated into DNA strands while azacitidine can be
incorporated into both DNA and RNA chains.
General description
Decitabine is an epigenetic modifier that inhibits DNA methyltransferase activity
which results in DNA demethylation (hypomethylation) and gene activation by
remodeling "opening" chromatin, structure detectable as increased nuclease
sensitivity. This remodeling of chromatin structure allows transcription factors to
bind to the promoter regions, assembly of the transcription complex, and gene
expression. Genes are synergistically reactivated when demethylation is combined
with histone hyperacetylation.

35. 35
NF-κB
Mechanism of NF-κB action. In this figure, the NF-κB heterodimer between Rel and p50 proteins is
used as an example. While in an inactivated state, NF-κB is located in the cytosol complexed with the
inhibitory protein IκBα. Through the intermediacy of integral membrane receptors, a variety of
extracellular signals can activate the enzyme IκB kinase (IKK). IKK, in turn, phosphorylates the IκBα
protein, which results in ubiquitination, dissociation of IκBα from NF-κB, and eventual degradation of
IκBα by the proteosome. The activated NF-κB is then translocated into the nucleus where it binds to
specific sequences of DNA called response elements (RE). The DNA/NF-κB complex then recruits other
proteins such as coactivators and RNA polymerase, which transcribe downstream DNA into mRNA,
which, in turn, is translated into protein, which results in a change of cell function.
NF-κB (nuclear factor kappa-light-chain-enhancer
of activated B cells) is a protein complex that controls
transcription of DNA. NF-κB is found in almost all
animal cell types and is involved in cellular responses
to stimuli such as stress, cytokines, free radicals,
ultraviolet irradiation, oxidized LDL, and bacterial or
viral antigens.

36. 36
DNA methylation levels
The Beta-value is a ratio between Illumina methylated probe intensity and total
probe intensities (sum of methylated and unmethylated probe intensities). It is in
the range of 0 and 1, which can be interpreted as the percentage of methylation.
Granada, 18-20 March, 2013
Beta-value for an ith interrogated CpG site is defined as:
Du et al. BMC Bioinformatics 2010, 11:587
where yi,menty and yi,unmenty are the intensities measured by the ith
methylated and unmethylated probes, respectively. To avoid negative values
after background adjustment, any negative values will be reset to 0. Illumina
recommends adding a constant offset a (by default, a = 100) to the
denominator to regularize Beta value when both methylated and unmethylated
probe intensities are low. The Beta-value statistic results in a number between
0 and 1, or 0 and 100%. Under ideal conditions, a value of zero indicates that
all copies of the CpG site in the sample were completely unmethylated (no
methylated molecules were measured) and a value of one indicates that every
copy of the site was methylated. If we assume the probe intensities are
Gamma distributed, then the Beta-value follows a Beta distribution. For this
reason, it has been named the Beta-value.

37. 37
LCN2 in Innate immunity
The binding of NGAL to bacterial siderophores is important in the innate immune
response to bacterial infection. Upon encountering invading bacteria the toll-like
receptors on immune cells stimulate the synthesis and secretion of NGAL.
Secreted NGAL then limits bacterial growth by sequestering iron-containing
siderophores.Lipocalin-2 also functions as a growth factor.
LCN-2 also known as neutrophil gelatinase associated lipocalin is a component
of the innate immune system with a key role in the acute-phase response to
infection.
Here, we show that this event is pivotal in the innate immune response to
bacterial infection. Upon encountering invading bacteria the Toll-like receptors
on immune cells stimulate the transcription, translation and secretion of lipocalin
2; secreted lipocalin 2 then limits bacterial growth by sequestrating the iron-laden
siderophore. Our finding represents a new component of the innate immune
system and the acute phase response to infection.
NATURE |VOL 432 | 16 DECEMBER 2004
Front. Physiol., 02 October 2013

38. 38
Mechanism of Bay-11-7082
NF-κB pathway inhibitors
parthenolide and Bay 11-7082 are
potent inhibitors of the
inflammasome independent of their
inhibitory effect on the NF-κB
pathway. We show that
parthenolide is a direct inhibitor of
caspase-1 and NLRP3 whereas Bay
11-7082 and several structurally
related vinyl sulfone compounds
are selective inhibitors of NLRP3.
This study identifies parthenolide
as the first natural product that
directly targets caspase-1 and the
NLRP3 inflammasome and vinyl
sulfones as the first small
molecules that selectively inhibit
activation of the NLRP3
inflammasome, possibly by
targeting its ATPase activity.
J Biol Chem. 2010 Mar 26;285(13):9792-802

39. 39
Principal of ROS detection and why H2DCFDH used?
In order to observe the formation of
reactive oxygen species a fluorescent
detector is employed. The acetate ester
form of 2',7' dichlorodihydrofluorescein
diacetate (H2DCFDA-AM) is a membrane
permanent molecule that passes through
the cell membrane. Once inside the cell,
cellular esterases act on the molecule to
form the non-fluorescent moiety
H2DCFDA, which is ionic in nature and
therefore trapped inside the cell. Oxidation
of H2DCFDA by ROS converts the
molecule to
2',7‘ dichlorodihydrofluorescein (DCF),
which is highly fluorescent. Upon
stimulation, the resultant production of
ROS causes an increase in fluorescence
signal over time.

40. 40
ChiP assay
Chromatin is composed of proteins, DNA, and RNA.
Found in the nucleus of eukaryotic cells, it mediates
several central biological processes, such as
regulating cell-specific or tissue-specific gene
expression and DNA replication and repair. The ChIP
assay has become one of the most practical and
useful techniques to study the mechanisms of gene
expression, histone modification, and transcription
regulation. In addition, ChIP assays are particularly
useful for the identification of transcription factors
and their target genes. This assay determines whether
a certain protein-DNA interaction is present at a
given location, condition, and time point. The use of
an appropriate antibody for the immunoprecipitation
step is considered the most critical factor for a
successful ChIP assay.
http://www.rndsystems.com/literature_chromatin_immunoprecipitation_chip.aspx

41. 41
NF-κB activities and mRNA levels of pro-
inflammatory genes
2,2′-bipyridyl (BPD), N-acetyl-cysteine (NAC)