Expression of WWOX and FHIT is down regulated Expression of WWOX and FHIT is down regulated by exposure to arsenite in human uroepithelial cellsby exposure to arsenite in human uroepithelial cells

Advisor:Advisor:Yi-Wen Liu, PhD.Yi-Wen Liu, PhD.

Speaker: Speaker: Mezbahul HaqueMezbahul Haque

Date: 2015.04.18Date: 2015.04.18

Toxicology Letters (2013) : 220, 118-125Toxicology Letters (2013) : 220, 118-125TOXICOLOGY 20/87TOXICOLOGY 20/87

Impact Factor- 3.35Impact Factor- 3.35

Well water and arsenic

http://flipper.diff.org/app./items/info/5095

Ecological studies in Taiwan, Chile, Argentina, Bangladesh, and Mexico have confirmed significant dose dependent associations between ingestion of arsenic-contaminated drinking water and the risk of various human malignancies. Toxicology Letters (2013) 220:118– 125

Evidence for arsenic as a human carcinogen comes from studies of skin, lung, and bladder cancers in people exposed to drinking water containing arsenic or exposed therapeutically to arsenic.

Environ Health Perspect. (1991) 95:205-10

Blackfoot disease and Arsenic

Exposure to arsenic via drinking-water has been shown to cause a severe disease of the blood vessels, which leads to gangrene, known as ‘black foot disease.

http://www.who.int/water_sanitation_health/diseases/arsenicosis/en/

According to GreenFacts, "Blackfoot disease (BFD) is a severe form of peripheral vascular disease (PVD), in which the blood vessels in the lower limbs are severely damaged, resulting eventually in progressive gangrene. It has been observed in Taiwan."

http://www.academyofwater.org/public-health-and-rehabilitation/

Substances including arsenic, organic cholorides and ergot alkaloids have been identified in artesian well water; however, arsenic has been suggested as the most important determinants of BFD. (WHO,1981)

Previous studies have shown that crude cancer mortality rate was higher in the BFD endemic area than in the general population in Taiwan. Taiwan Yi Xue Hui Za Zhi. (1965) 64:470-84.

The BFD is a unique peripheral artery disease associated with chronic arsenic exposure in several southwestern townships of Taiwan. The residents of the BFD area had significantly dose-response relationship between concentration of arsenic in water and high mortality of bladder cancer.

Blackfoot disease and Arsenic and Cancer

Biomed Environ Sci. (2003) 16:355-68

The fragile histidine triad (FHIT) gene

Bis(5'-adenosyl)-triphosphatase also known as fragile histidine triad protein (FHIT) is an enzyme that in humans is encoded by the FHIT gene.

Various human cancers, particularly those resulting from environmentalcarcinogen exposure, exhibit aberrant mRNA and protein expression and inactivation of the FHIT gene resulting from allelic losses, homozygous deletions and epigenetic modifications.

J Pathol (2002) 196: 300–306

FHIT gene functions as tumor suppressor in many epithelial cell types and has important roles of FHIT in bladder cancer development.

Asian Pac J Cancer Prev. (2011) 12:2915-20.

The FHIT gene deletions could be important in the development and/or progression of urinary bladder cancers and may be used as an independent prognostic indicator for the clinical outcome in patients with these tumors.

Pak J Biol Sci. (2011) 14:212-8

WW domain-containing oxidoreductase (WWOX) gene

WW domain-containing oxidoreductase is an enzyme that in humans is encoded by the WWOX gene, located at chromosome 16q23.

The multiple functions of WWOX in physiological and pathological processes include cell growth, differentiation, tumor suppressor and apoptosis. The WWOX gene inactivation and alterations in human tumors may result from genomic, post-translational and epigenetic modification.

Virchows Arch (2010) 457:423–432

Recent evidence showed that WWOX gene could be silenced by methylation in lung, breast and bladder cancer.

Oncogene (2005) 24:1625-33

Partial or complete loss of WWOX expression has been reported in human cancers. Virchows Arch (2010) 457:423–432

Cancer Res. (2000) 60:2140-5.

AimsAims

The aims of this study were to observe the expression of ATR, WWOX and FHIT proteins in urothelial carcinoma (UC) between endemic and non-endemic

areas of blackfoot disease (BFD) by immunohistochemical analyses.

To compare expression of these genes between arsenite-treated SV-HUC-1 human epithelial cells and rat uroepithelial cells.

To determine the role of DNMT and MEK inhibitors on expressions of WWOX and FHIT in response to arsenite in SV-HUC-1.

Materials and Methods Materials and Methods

1. Western blotting2. Reverse transcription-polymerase chain reaction3. Immunocyto/histochemical staining4. Scoring of IHC antibody staining5. Statistical analysis

SV-HUC-1 1.with/without 5-aza-deoxycytidine2.with/without U01263.Sodium arsenite 1, 4 and 10 μM.

F344/N rats, Male1. sodium arsenite (0.13, 0.52 and 1.3 ppm)2. D.D. water as control

Animal experimentCell culture

Methods:

Flow charts Flow charts

Comparison of immunohistochemical staining results for ATR, WWOX andComparison of immunohistochemical staining results for ATR, WWOX andFHIT in urothelial carcinoma (UC) patients from blackfoot disease (BFD) areas andFHIT in urothelial carcinoma (UC) patients from blackfoot disease (BFD) areas and

non-blackfoot disease (non-BFD) areas.non-blackfoot disease (non-BFD) areas.

Correlation of ATR, WWOX and FHIT proteins in patients with urothelial carcinoma(UC) from

BFD areas and non-BFD areas.

Correlation of WWOX, FHIT and ATR proteins and known clinicopathologic parameters in 33 patients with UC from BFD and non-BFD areas.

Summary Summary

The immunostaining results for ATR, WWOX and FHIT showedthat low expression of these proteins was more common in patientswith UC from BFD areas than in UC patients from non-BFD areas.

The WWOX and FHIT expressions had statistically significant correlation with tumor sites but expressions of ATR, WWOX and

FHIT expression showed no significant correlations with gender, age, tumor invasiveness, histological grade or recurrence.

Flow charts Flow charts

Reduction of ATR, WWOX and FHIT expression by arsenite inhuman uroepithelial cells

Effects of sodium arsenite on the ATR, WWOX and FHIT labeling index of immunostaining.

Western blot and RT-PCR results after treated with arsenic

Western blot RT-PCR

Summary Summary

Sodium arsenite treatment decreased the LI in a dose-dependent manner at 1–4 uM . But, the LI of WWOX and FHIT increased

with high concentration of arsenite (10 uM)

The ICC and Western blot results showed that arsenite reduced ATR, WWOX and FHIT proteins in SV-HUC-1 cells

RT-PCR showed that arsenite suppressed mRNA expression in WWOX and FHIT

Flow charts Flow charts

Effect of MEK and DNMT inhibitors on expressions of WWOX and FHIT in human uroepithelial cells treated with arsenite

Western blot RT-PCR

Summary Summary

The experiments showed that 24 h pretreatment with U0126 or 5-aza-CdR suppressed the effect of arsenite on expressions of

WWOX and FHIT proteins and mRNA

The ICC, western blot and RT-PCR results showed that arsenite reduced expressions of WWOX and FHIT proteins and mRNA

That is, U0126 and 5-aza-CdR treatment caused restoration of WWOX and FHIT expressions.

Flow charts Flow charts

Expressions of ATR, WWOX and FHIT proteins in raturoepithelial cells

Western blotting resultsIHC results

Summary Summary

In the rat bladder, urothelial cells of the arsenite-treated groupsshowed only low nuclear and cytoplasm immunostaining for ATR,

WWOX and FHIT compared to the non-exposed controls

Western blot results for expressions of ATR, WWOX and FHIT proteins between control groups and arsenitetreated groups

showed that expressions of ATR, WWOX and FHIT were lower in the arsenite treated groups than in the control groups

Discussion Discussion

Mutation of the ATR/ATM checkpoint causes cell proliferation, tumor progression, DNA replication stress, and genomic instability.

Journal of Cellular Biochemistry (2008) 104:1525–1533

Exposure to carcinogens increased chromosomal fragile site expression. The data also suggest that FHIT and WWOX genes are common targets of carcinogen-induced damage and have a central role in the early stage of carcinogenesis.

Cell Cycle (2007) ;6(9):1044-8

Inactivation of FHIT has an important role in the development of bladder cancer and abnormal WWOX expression has been demonstrated at the genomic, mRNA and protein levels in various human tumors, including prostate and bladder tumors.

Histopathology (2008) 52:831–839

ConclusionConclusion

Arsenite decreased expressions of ATR, WWOX and FHIT via ERK1/2 activation in SV-HUC-1 cells.

These findings confirm that dysregulations of these markers may contribute to arsenite-induced carcinogenesis.

This study provides the first evidence that frequence of ATR,WWOX and FHIT low expressions in UC patients from BFD areas was higher than the UC patients from non-BFD areas.

MEK inhibitorsMEK inhibitors

A MEK inhibitor is a chemical or drug that inhibits the mitogen-activated protein kinase kinase enzymes MEK1 and/or MEK2. They can be used to affect the MAPK/ERK pathway which is often overactive in some cancers.

Hence MEK inhibitors have potential for treatment of some cancers,[1] especially BRAF-mutated melanoma,[2] and KRAS/BRAF mutated colorectal cancer.[3]

Some MEK inhibitors:Trametinib (GSK1120212), FDA-approved to treat BRAF-mutated melanoma. Also studied in combination with BRAF inhibitor dabrafenib to treat BRAF-mutated melanoma.Selumetinib, had a phase 2 clinical trial for non-small cell lung cancer (NSCLC) which demonstrated an improvement in PFS,[4] and is now in phase III development in KRAS mutation positive NSCLC (SELECT-1,NCT01933932). Other clinical trials underway include uveal melanoma, and differentiated thyroid carcinoma.Binimetinib (MEK162), had phase 1 trial for biliary tract cancer and melanoma[5] and in Sept 2014 reported encouraging results from a phase II clinical trial for NRAS melanoma.[6]

PD-325901, for breast cancer, colon cancer, and melanoma[7]

Cobimetinib or XL518, in a Phase III trial, in combination with vemurafenib (Zelboraf(R)), for treatment of advanced melanoma.

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5‑aza‑2′‑deoxycytidine (5‑aza‑dC) 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 descriptionDecitabine 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.

ATRATR

Serine/threonine-protein kinase ATR also known as ataxia telangiectasia and Rad3-related protein (ATR) or FRAP-related protein 1 (FRP1) is an enzyme that, in humans, is encoded by the ATR gene.[1][2] ATR belongs to the phosphatidylinositol 3-kinase-related kinase protein family.

ATR is a serine/threonine-specific protein kinase that is involved in sensing DNA damage and activating the DNA damage checkpoint, leading to cell cycle arrest.[3]

FHIT

FHIT