SelSA Compounds as HDAC Inhibitors

Katherine TaiMentor: Mohaiza Dashwood

Advisor: Rod DashwoodDepartment of Environmental & Molecular Toxicology

Linus Pauling Institute

Purpose:

To determine the anticancer effects of compounds SelSA-1 and SelSA-2 in cancer cells HCT 116 (colon cancer) and A431 (skin cancer) in vitro.

Histone post translational modifications

Histone Acetylation and Deacetylation

http://missinglink.ucsf.edu/lm/genes_and_genomes/acetylation.html

Histone Acetylation

Acetylated histones are usually associated with transcriptionally active chromatin

Histones are acetylated by Histone Acetyltransferases (HATs)

Deacetylated histones are usually associated with inactive chromatin

Histones are deacetylated by Histone Deacetylases (HDACs)

Classes of HDACs

4 classes of HDACs:• Class I: HDAC1, 2, 3, 8• Class II: HDAC4, 5, 6, 7, 9, 10• Class III: Sir2(yeast), SirT1, 2, 3, 4,

5, 6, 7• Class IV: HDAC11

Cancer Therapy with HDAC Inhibitors

Histone Deacetylase (HDAC) inhibition has been shown to elicit anticancer effects in several tumor cells by inhibition of cell growth (Desai et al, 2009)

HDAC inhibitors can induce p21 (WAF1) expression, a regulator of p53's tumor suppressor activity. (Richon etal, 2000)

HDAC inhibitors are currently used for anti-cancer chemotherapy (Desai et al, 2009)

Classes of HDAC Inhibitors

Hydroxamic Acids

Short-Chain Fatty Acids

Cyclic Tetrapeptides/epoxides

Aminobenzamides

Electrophilic ketones

Vorinostat (SAHA)(FDA Approved HDAC Inhibitor)

• Vorinostat or suberoylanilide hydroxamic acid (SAHA) is a member of a larger class of compounds that inhibit histone deacetylases (HDAC).

• Histone deacetylase inhibitors (HDI) have a broad spectrum of epigenetic activities.

• Vorinostat is marketed under the name Zolinza for the treatment of cutaneous T cell lymphoma (CTCL) when the disease persists, gets worse, or comes back during or after treatment with other medicines. (Merck & Co., 2006)

NH

O

O

NH

OH

SelSA Compounds Organoselenium compounds have been shown to be HDAC

inhibitors and reduce growth of colon and prostate cancer cells (Nian et al, 2009)

Two selenium analogs of SAHA have been reported as potent HDAC inhibitors (Desai et al, 2009)

SeNH

O

N

O

H

Se

NH

O

O

NH

OH

SAHA

NH

O

SeCN

Selenium Dimer (SelSA-1)

Selenocyanide (SelSA-2)

Hypothesis

Test SAHA derivatives SelSA-1 and SelSA-2 for their anti-cancer activity on cancer cell lines in vitro:

HCT116 (colon carcinoma) A431 (skin carcinoma)

Test SelSA-1 and -2 for their effect on HDAC activity and histone acetylation.

Test for cellular effects i.e. morphology, growth, cell cycle and cell death on cancer cells.

HDAC activity assay

The method requires two steps, both performed on the same microtiter plate.

First, the HDAC fluorogenic substrate, which comprises an acetylated lysine side chain, is incubated with a sample containing HDAC activity (e.g., HeLa nuclear extract).

Deacetylation of the substrate sensitizes the substrate, so that, in the second step, treatment with the Lysine Developer produces a fluorophore.

The fluorophore can be analyzed using a fluorescence plate reader (Ex 360 nm/Em 460 nm).

A standard curve of deactylated substrate is run in parallel.

Effect on HDAC Activity

0.2 2 20 2000

200400600800

1000120014001600

HDAC activity in Hela Nuclear extract

SAHA

Concentration (nM)

Ex 3

60/E

m 4

60

0.2 2 20 2000

500

1000

1500

2000

HDAC activity in Hela Nuclear extract

SELSA-1

Concentration (nM)

Ex 3

60/E

m 4

60

0.2 2 20 2000

500

1000

1500

2000

HDAC activity in Hela Nuclear extract

SELSA-2

Concentration (nM)

Ex 3

60/E

m 4

60

Western Blotting for HDAcs and Histone Modifications

IC50 concentrations were used.

Cancer cells were treated with SelSA-1, SelSA-2, and SAHA at 3, 6 and 24 hrs.

Cells were lysed and lysates collected.

Protein concentration in lysates was determined by BCA

Western blotting of equal amounts of protein was done on 4-12% Tris-Glycine pre-cast gels.

HisH3(9-10-10)

HisH3Acetylated

K9(9-10-10

HisH3Acetylated(9-10-10)

Western Blots of Colon Cancer:HisH3 Acetylation

No

Trea

tmen

t

SelS

A-1

10μM

SelS

A-1

0.1μ

MSe

lSA-

1 1μ

M SelS

A-1

5μMSe

lSA-

2 10

μM

SelS

A-2

0.1μ

MSe

lSA-

2 1μ

M SelS

A-2

5μMSA

HA

10μMSAH

A-1

0.1μ

MSA

HA-

1 1μ

M SAH

A-1

5μMDM

SO

Western Blots of Colon Cancer:HisH4 Acetylation

No

Trea

tmen

t

SelS

A-1

10μMSelS

A-1

0.1μ

MSe

lSA-

1 1μ

M SelS

A-1

5μMSelS

A-2

10μMSelS

A-2

0.1μ

MSe

lSA-

2 1μ

M SelS

A-2

5μMSA

HA

10μMSAH

A-1

0.1μ

MSA

HA-

1 1μ

M SAH

A-1

5μMDM

SO

HisH4(9-10-10)

HisH4Acetylated(9-10-10)

HisH4Acetylated

K12(9-10-10

1.07 1.10 1.17 1.25 1.30 1.02 1.06 1.30 1.32 1.07 1.28 1.37 1.26 1.00RelativeDensitometr

y

0.95 0.80 1.18 1.60 1.58 0.97 1.31 1.54 1.29 1.25 1.45 1.48 1.55 1.00RelativeDensitometr

y

Western Blots of Colon Cancer:α-Tubulin acetylation

No

Trea

tmen

t

SelS

A-1

10μM

SelS

A-1

0.1μ

MSe

lSA-

1 1μ

M SelS

A-1

5μMSe

lSA-

2 10

μM

SelS

A-2

0.1μ

MSe

lSA-

2 1μ

M SelS

A-2

5μMSA

HA

10μMSAH

A-1

0.1μ

MSA

HA-

1 1μ

M SAH

A-1

5μMDM

SO

α-Tubulin

Acetylatedα-Tubulin

Western Blots of Colon Cancer:Class I HDACs

Treatments:

β-Actin(8-3-10)

HDAC1(8-9-10)

HDAC2(8-3-10)

None SelsA-1 SelsA-1 SelsA-2 SelsA-2 SAHA

2.5μM 5μM 2.5μM 5μM 5μM

β-Actin(8-9-10)

HDAC8(8-3-10)

3H 6H 24H

Western Blots of Colon Cancer:Class I HDACs

No

Trea

tmen

t

SelS

A-1

10μM

SelS

A-1

0.1μ

MSe

lSA-

1 1μ

M SelS

A-1

5μMSe

lSA-

2 10

μM

SelS

A-2

0.1μ

MSe

lSA-

2 1μ

M SelS

A-2

5μMSA

HA

10μMSAH

A-1

0.1μ

MSA

HA-

1 1μ

M SAH

A-1

5μMDM

SO

β-Actin(9-16-10)

HDAC3(9-16-10)

Western Blots of Colon Cancer:Class II HDACs

Treatments:

β-Actin(8-3-10)

HDAC10(8-3-10)

None SelsA-1 SelsA-1 SelsA-2 SelsA-2 SAHA

2.5μM 5μM 2.5μM 5μM 5μM

3H 6H 24H

Western Blots of Colon Cancer:Class IV HDACs

Treatments:

β-Actin(8-3-10)

HDAC11(8-3-10)

None SelsA-1 SelsA-1 SelsA-2 SelsA-2 SAHA

2.5μM 5μM 2.5μM 5μM 5μM

3H 6H 24H

Western Blots of Skin Cancer:Class I HDACs

Treatments:

β-Actin(4-21-10)

HDAC1(4-21-10)

HDAC2(4-21-10)

None SelsA-1 SelsA-1 SelsA-2 SelsA-2 SAHA

1μM 5μM 1μM 5μM 5μM

HDAC3(6-21-10)

HDAC8(5-25-10)

3H 6H 24H

β-Actin(5-25-10)

β-Actin(6-21-10)

HDAC10(6-21-10)

Treatments:None SelsA-1 SelsA-1 SelsA-2 SelsA-2 SAHA

1μM 5μM 1μM 5μM 5μM

HDAC7(5-25-10)

3H 6H 24H

Western Blots of Skin Cancer:Class II HDACs

Effect on Colon Cancer Cell Morphology:

All Compounds tested at 1μM for 72 hours

DMSO SAHA

Effect on Colon Cancer Cell Morphology:

All Compounds tested at 1μM for 72 hours

SELSA-1 SELSA-2

Cell Counting Kit-8 (CCK8)

Cell Counting Kit-8 is a nonradioactive, sensitive colorimetric assay for the determination of the number of viable cells in cell proliferation and cytotoxicity assays.

Half maximal inhibitory concentration (IC50): the half maximal (50%) inhibitory concentration (IC) of a substance measuring the effectiveness of a compound in inhibiting biological or biochemical function.

CCK8: WST-8 is reduced by dehydrogenases to give a formazan product. The amount of formazan dye generated, which is soluble in the cell culture medium, is proportional to number of living cells.

CCK8 Assay-Dose Response 0f

HCT116 Colon Cancer Cells (72 hrs)

0.01 0.1 1 10 1000.00

0.50

1.00

1.50

2.00

2.50

SAHASELSA-1SELSA-2VEHICLE (DMSO)

Concentration (uM)

Absorb

ance (

A 4

50 n

m)

Compound IC50 (uM) SAHA

0.8 SELSA-1 0.6 SELSA-2 0.9

Ic50 of Organoselenium Compounds for 10,000 Skin

(A431) Cancer Cells

Treatment

% V

iab

le C

ell

s

0

20

40

60

80

100

120

140

160

180

SelsA-1SelsA-2SAHANo TreatmentDMSO (0.1%)

No Treatm

ent0.1

M

0.25 M

0.5 M

1.0 M

2.5 M

5.0 M

7.5 M

10 M

DM

SO

IC50• SelSA-1: 1.5 μM• SelSA-2: 1.75 μM• SAHA: 5 μM

Cell Cycle Analysis

Treatment increases apoptotic sub-G1 phase

Conclusions

SelSA-1 and SelSA-2 inhibit HDAC activity and induce histone acetylation

These compounds were found to be moderately more potent than SAHA in the activity assay

These compounds inhibit cell growth and cause cell death in colon and skin cancer cells

SelSA-1 and SelSA-2 are important SAHA derivatives which need to be further tested in animal models

Acknowledgements

HHMI Program Kevin Ahern Dashwood Lab Dr. Roderick Dashwood Mohaiza Dashwood Praveen Rajendran Rong Wang Hui Nian Pennsylvania State Hershey College of

Medicine