Arsenic and Cancer

“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

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

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

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

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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

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Lipocalin-2 in cancer

Cancer Letters 316 (2012) 132–138

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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.

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.

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

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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)

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|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

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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).

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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.

Flow Chart

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methylation status


hypomethylated promoter of LCN2






LCN2

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The methylation status of the LCN2 gene promoter and DNA

methylation ratios for individual CpG sites

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Identification of LCN2 promoter activity

1. Luciferase Reporter assay

2. ChIP assay

An inhibitor of NF-κB

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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.

Flow Chart

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methylation status








Lipocqalin2 (LCN2)

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Identification of protein level of LCN2 and

involvement of LCN2 in cellular transformation

Anchorage-independent growth assayWestern blot analysis

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Determination of cell viability and protein level with

or without serum in the cell culture medium

AlamarBlue assay Western blotting

21

Determination of effect in cell growth and anti-apoptotic

activity of the iAs-HUC cells in serum-free medium

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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.

Flow Chart

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methylation status








Lipocqalin2 (LCN2)

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NF-κB activity and expression LCN2 also activates NF-κB

in iAs-HUCs

Luciferase reporter assay Western blotting

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The mRNA levels of pro-inflammatory genes

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The mRNA levels of pro-inflammatory genes were examined

by qPCR after treated with the NF-κB inhibitor Bay 11- 7082

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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.

Flow Chart

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methylation status








Lipocqalin2 (LCN2)

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Measurement of the intracellular iron levels and the

ROS levels

Mean fluorescence intensities (MFI)

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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)

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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.

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Conclusion

LCN2 LCN2

LCN2LCN2

LCN2

Fe Fe

Fe

Fe

Micro environmental

Stimuli ER

NF-κB

ROS

Inflammatory

Cytokine

Autophagy &

Anti-apoptotic

activity

Tumor

growth

MMP9

Stablization

EMT

Promotion

Tumor cell

Fe

LCN2

Inorganic arsenic

33

Thank you

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.

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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.

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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.

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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

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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

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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.

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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

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NF-κB activities and mRNA levels of pro-

inflammatory genes

2,2′-bipyridyl (BPD), N-acetyl-cysteine (NAC)

Arsenic and Cancer

Health & Medicine

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