SelSA Compounds as HDAC Inhibitors
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Transcript of SelSA Compounds as HDAC Inhibitors
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SelSA Compounds as HDAC Inhibitors
Katherine TaiMentor: Mohaiza Dashwood
Advisor: Rod DashwoodDepartment of Environmental & Molecular Toxicology
Linus Pauling Institute
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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.
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Histone post translational modifications
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Histone Acetylation and Deacetylation
http://missinglink.ucsf.edu/lm/genes_and_genomes/acetylation.html
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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)
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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
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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)
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Classes of HDAC Inhibitors
Hydroxamic Acids
Short-Chain Fatty Acids
Cyclic Tetrapeptides/epoxides
Aminobenzamides
Electrophilic ketones
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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
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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)
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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.
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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.
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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
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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.
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HisH3(9-10-10)
HisH3Acetylated
K9(9-10-10
HisH3Acetylated(9-10-10)
Western Blots of Colon Cancer:HisH3 Acetylation
No
Treat
ment
SelSA
-1
10μM
SelSA
-1 0.1
μM SelSA
-1
1μM SelSA
-1
5μMSe
lSA-2
10μM
SelSA
-2
0.1μM SelSA
-2
1μM SelSA
-2 5μ
MSAHA
10
μMSAHA
-1 0.1
μM SAHA
-1 1μ
M SAHA
-1 5μ
MDMSO
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Western Blots of Colon Cancer:HisH4 Acetylation
No
Treat
ment
SelSA
-1
10μMSelSA
-1 0.1
μM SelSA
-1 1μ
M SelSA
-1 5μ
MSelSA
-2 10
μMSelSA
-2 0.1
μM SelSA
-2 1μ
M SelSA
-2 5μ
MSAHA
10
μMSAHA
-1 0.1
μM SAHA
-1 1μ
M SAHA
-1 5μ
MDMSO
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
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Western Blots of Colon Cancer:α-Tubulin acetylation
No
Treat
ment
SelSA
-1
10μM
SelSA
-1 0.1
μM SelSA
-1
1μM SelSA
-1
5μMSe
lSA-2
10μM
SelSA
-2
0.1μM SelSA
-2
1μM SelSA
-2 5μ
MSAHA
10
μMSAHA
-1 0.1
μM SAHA
-1 1μ
M SAHA
-1 5μ
MDMSO
α-Tubulin
Acetylatedα-Tubulin
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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
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Western Blots of Colon Cancer:Class I HDACs
No
Treat
ment
SelSA
-1
10μM
SelSA
-1 0.1
μM SelSA
-1 1μ
M SelSA
-1 5μ
MSelSA
-2
10μM
SelSA
-2 0.1
μM SelSA
-2 1μ
M SelSA
-2 5μ
MSAHA
10
μMSAHA
-1 0.1
μM SAHA
-1 1μ
M SAHA
-1 5μ
MDMSO
β-Actin(9-16-10)
HDAC3(9-16-10)
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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
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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
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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
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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
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Effect on Colon Cancer Cell Morphology:
All Compounds tested at 1μM for 72 hours
DMSO SAHA
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Effect on Colon Cancer Cell Morphology:
All Compounds tested at 1μM for 72 hours
SELSA-1 SELSA-2
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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.
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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)
Abso
rban
ce (
A 45
0 nm
)
Compound IC50 (uM) SAHA
0.8 SELSA-1 0.6 SELSA-2 0.9
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Ic50 of Organoselenium Compounds for 10,000 Skin
(A431) Cancer Cells
Treatment
% V
iabl
e C
ells
0
20
40
60
80
100
120
140
160
180
SelsA-1SelsA-2SAHANo TreatmentDMSO (0.1%)
No Treatment
0.1 M
0.25 M0.5 M1.0 M2.5 M5.0 M
7.5 M
10 M
DMSO
IC50• SelSA-1: 1.5 μM• SelSA-2: 1.75 μM• SAHA: 5 μM
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Cell Cycle Analysis
Treatment increases apoptotic sub-G1 phase
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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
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Acknowledgements HHMI Program Kevin Ahern Dashwood Lab Dr. Roderick Dashwood Mohaiza Dashwood Praveen Rajendran Rong Wang Hui Nian Pennsylvania State Hershey College of
Medicine