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SelSA Compounds as HDAC Inhibitors

Katherine Tai Mentor: Mohaiza Dashwood Advisor: Rod Dashwood Department of Environmental & Molecular Toxicology Linus Pauling Institute. SelSA Compounds as HDAC Inhibitors. Purpose:.

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SelSA Compounds as HDAC Inhibitors

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  1. Katherine Tai Mentor: MohaizaDashwood Advisor: Rod Dashwood Department of Environmental & Molecular Toxicology Linus Pauling Institute SelSA Compounds as HDAC Inhibitors

  2. 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.

  3. Histone post translational modifications

  4. Histone Acetylation and Deacetylation http://missinglink.ucsf.edu/lm/genes_and_genomes/acetylation.html

  5. Histone Acetylation • Acetylated histones are usually associated with transcriptionally active chromatin • Histones are acetylated by Histone Acetyltransferases (HATs) • Deacetylatedhistones are usually associated with inactive chromatin • Histones are deacetylated by Histone Deacetylases (HDACs)

  6. 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

  7. 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. (Richonetal, 2000) • HDAC inhibitors are currently used for anti-cancer chemotherapy (Desai et al, 2009)

  8. Classes of HDAC Inhibitors • Hydroxamic Acids • Short-Chain Fatty Acids • Cyclic Tetrapeptides/epoxides • Aminobenzamides • Electrophilicketones

  9. O H NH N OH O 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)

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

  11. 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.

  12. 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.

  13. Effect on HDAC Activity

  14. 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.

  15. Western Blots of Colon Cancer:HisH3 Acetylation SelSA-1 10μM SelSA-2 10μM SAHA-1 0.1μM SelSA-1 0.1μM SelSA-2 0.1μM SAHA-1 5μM SelSA-1 5μM SAHA 10μM DMSO SAHA-1 1μM SelSA-1 1μM No Treatment SelSA-2 5μM SelSA-2 1μM HisH3 (9-10-10) HisH3 Acetylated (9-10-10) HisH3 Acetylated K9 (9-10-10

  16. Western Blots of Colon Cancer:HisH4 Acetylation SAHA-1 0.1μM SelSA-1 0.1μM SelSA-2 10μM SelSA-2 0.1μM SelSA-1 10μM SAHA-1 5μM SelSA-1 5μM SAHA 10μM DMSO SAHA-1 1μM SelSA-1 1μM No Treatment SelSA-2 5μM SelSA-2 1μM HisH4 (9-10-10) HisH4 Acetylated (9-10-10) Relative Densitometry HisH4 Acetylated K12 (9-10-10 Relative Densitometry

  17. Western Blots of Colon Cancer:α-Tubulin acetylation SelSA-1 10μM SelSA-2 10μM SAHA-1 0.1μM SelSA-1 0.1μM SelSA-2 0.1μM SAHA-1 5μM SelSA-1 5μM SAHA 10μM DMSO SAHA-1 1μM SelSA-1 1μM No Treatment SelSA-2 5μM SelSA-2 1μM α-Tubulin Acetylated α-Tubulin

  18. Western Blots of Colon Cancer:Class I HDACs 3H 6H 24H β-Actin (8-9-10) HDAC1 (8-9-10) β-Actin (8-3-10) HDAC2 (8-3-10) HDAC8 (8-3-10) Treatments: None SelsA-1 SelsA-1 SelsA-2 SelsA-2 SAHA 2.5μM 5μM 2.5μM 5μM 5μM

  19. Western Blots of Colon Cancer:Class I HDACs SelSA-1 10μM SelSA-2 10μM SAHA-1 0.1μM SelSA-1 0.1μM SelSA-2 0.1μM SAHA-1 5μM SelSA-1 5μM SAHA 10μM DMSO SAHA-1 1μM SelSA-1 1μM No Treatment SelSA-2 5μM SelSA-2 1μM β-Actin (9-16-10) HDAC3 (9-16-10)

  20. Western Blots of Colon Cancer:Class II HDACs 3H 6H 24H β-Actin (8-3-10) HDAC10 (8-3-10) Treatments: None SelsA-1 SelsA-1 SelsA-2 SelsA-2 SAHA 2.5μM 5μM 2.5μM 5μM 5μM

  21. Western Blots of Colon Cancer:Class IV HDACs 3H 6H 24H β-Actin (8-3-10) HDAC11 (8-3-10) Treatments: None SelsA-1 SelsA-1 SelsA-2 SelsA-2 SAHA 2.5μM 5μM 2.5μM 5μM 5μM

  22. Western Blots of Skin Cancer:Class I HDACs 3H 6H 24H β-Actin (4-21-10) HDAC1 (4-21-10) HDAC2 (4-21-10) HDAC3 (6-21-10) HDAC8 (5-25-10) Treatments: None SelsA-1 SelsA-1 SelsA-2 SelsA-2 SAHA 1μM 5μM 1μM 5μM 5μM

  23. Western Blots of Skin Cancer:Class II HDACs 3H 6H 24H β-Actin (5-25-10) HDAC7 (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

  24. Effect on Colon Cancer Cell Morphology: DMSO SAHA All Compounds tested at 1μM for 72 hours

  25. Effect on Colon Cancer Cell Morphology: SELSA-1 SELSA-2 All Compounds tested at 1μM for 72 hours

  26. 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.

  27. CCK8 Assay-Dose Response 0fHCT116 Colon Cancer Cells (72 hrs) • CompoundIC50 (uM) • SAHA 0.8 • SELSA-1 0.6 • SELSA-2 0.9

  28. Ic50 of Organoselenium Compounds for 10,000 Skin (A431) Cancer Cells • IC50 • SelSA-1: 1.5 μM • SelSA-2: 1.75 μM • SAHA: 5 μM

  29. Cell Cycle Analysis Treatment increases apoptotic sub-G1 phase

  30. 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

  31. Acknowledgements • HHMI Program • Kevin Ahern • Dashwood Lab • Dr. Roderick Dashwood • MohaizaDashwood • Praveen Rajendran • Rong Wang • HuiNian • Pennsylvania State Hershey College of Medicine

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