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Katherine Tai Mentor: Mohaiza Dashwood Advisor: Rod Dashwood Department of Environmental & Molecular Toxicology Linus Pauling Institute.

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Presentation on theme: "Katherine Tai Mentor: Mohaiza Dashwood Advisor: Rod Dashwood Department of Environmental & Molecular Toxicology Linus Pauling Institute."— Presentation transcript:

1 Katherine Tai Mentor: Mohaiza Dashwood Advisor: Rod Dashwood Department of Environmental & Molecular Toxicology Linus Pauling Institute

2 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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4 http://missinglink.ucsf.edu/lm/genes_and_genomes/acetylation.html

5  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)

6  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

7  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)

8  Hydroxamic Acids  Short-Chain Fatty Acids  Cyclic Tetrapeptides/epoxides  Aminobenzamides  Electrophilic ketones

9 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) N H O O NH OH

10  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) Se N H O N O H N H O O NH OH SAHA N H O SeCN Selenium Dimer (SelSA-1) Selenocyanide (SelSA-2)

11  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.

12  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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14  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.

15 HisH3 (9-10-10) HisH3 Acetylated K9 (9-10-10 HisH3 Acetylated (9-10-10) No Treatment SelSA-1 10μM SelSA-1 0.1μM SelSA-1 1μM SelSA-1 5μM SelSA-2 10μM SelSA-2 0.1μM SelSA-2 1μMSelSA-2 5μM SAHA 10μM SAHA-1 0.1μM SAHA-1 1μM SAHA-1 5μM DMSO

16 No Treatment SelSA-1 10μM SelSA-1 0.1μM SelSA-1 1μM SelSA-1 5μM SelSA-2 10μM SelSA-2 0.1μM SelSA-2 1μMSelSA-2 5μM SAHA 10μM SAHA-1 0.1μM SAHA-1 1μM SAHA-1 5μM DMSO HisH4 (9-10-10) HisH4 Acetylated (9-10-10) HisH4 Acetylated K12 (9-10-10 1.071.101.171.251.301.021.061.301.321.071.281.371.261.00 Relative Densitometry 0.950.801.181.601.580.971.311.541.291.251.451.481.551.00 Relative Densitometry

17 No Treatment SelSA-1 10μM SelSA-1 0.1μM SelSA-1 1μM SelSA-1 5μM SelSA-2 10μM SelSA-2 0.1μM SelSA-2 1μMSelSA-2 5μM SAHA 10μM SAHA-1 0.1μM SAHA-1 1μM SAHA-1 5μM DMSO α-Tubulin Acetylated α-Tubulin

18 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) 3H6H24H

19 No Treatment SelSA-1 10μM SelSA-1 0.1μM SelSA-1 1μM SelSA-1 5μM SelSA-2 10μM SelSA-2 0.1μM SelSA-2 1μM SelSA-2 5μM SAHA 10μM SAHA-1 0.1μM SAHA-1 1μM SAHA-1 5μM DMSO β-Actin (9-16-10) HDAC3 (9-16-10)

20 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 3H6H24H

21 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 3H6H24H

22 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) 3H6H24H

23 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) 3H6H24H

24 All Compounds tested at 1μM for 72 hours DMSO SAHA

25 All Compounds tested at 1μM for 72 hours SELSA-1 SELSA-2

26  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.

27  Compound IC 50 (uM)  SAHA0.8  SELSA-10.6  SELSA-20.9

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

29 Treatment increases apoptotic sub-G1 phase

30  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

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

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