Electronic Theses and Dissertations

Date of Award

1-1-2015

Document Type

Dissertation

Degree Name

Ph.D. in Pharmaceutical Sciences

Department

Biomolecular Sciences

First Advisor

Babu L Tekwani

Second Advisor

John C. Matthews

Third Advisor

Stephen J. Cutler

Abstract

The inhibitors of histone deacetylases (HDACs) have shown promising neuroprotective and neuroactive properties. A set of pan- and isoform-specific HDAC inhibitors were evaluated for neurotrophic activity on Neuroscreen-1 (NS-1) cells, a subclone of PC-12 (rat pheochromocytoma) cells.

The HDAC inhibitors were tested alone and in combination with nerve growth factor (NGF). An in vitro method has been standardized that measures neurite outgrowth along with cytotoxicity of test compounds in a single assay. The neurotrophin signaling pathways were interrogated with selective inhibitors of MEK1/2 (PD98059/U0126) PI3K (LY294002) and TrkA (GW441756) and phosphorylation of target kinases. Associated factors namely, acetylation of histones (H3/H4) and α-tubulin, role of cAMP, and cell- cycle analysis with flowcytometry were also studied.

Vorinostat, a pan HDAC inhibitor, independently induced significant neurite outgrowth in NS-1 cells, similar to that of NGF. The inhibitors of MEK1/2 & PI3K attenuated the NGF and vorinostat mediated neurite outgrowth. Vorinostat induced phosphorylation of ERK1/2 was abolished in presence of U0126. Both, vorinostat-mediated neurite outgrowth and activation of ERK were attenuated in presence of the TrkA inhibitor, GW441756, indicating the role of activation of upstream neurotrophin receptor. Vorinostat produced hyperacetylation of α-tubulin and histones H3/H4. However, SQ22536 (the adenylate cyclase inhibitor) further stimulated iii neurotrophic action of vorinostat. Trichostatin A (TSA), belinostat and additional pan-HDAC inhibitors, showed variable effects. TSA was ineffective, while belinostat attenuated neurotrophic action of NGF. Belinostat though induced hyperacetylation of H3 & H4 but failed to produce acetylation of α-tubulin. PCI-34051, the HDAC 8 inhibitor, produced marginal neurotrophic action but did not produce hyperacetylation of H3/H4 and α-tubulin. Tubastatin-A, the HDAC 6 inhibitor, although produced hyperacetylation of α-tubulin but did not show any neurotrophic action. Tenovin-1, a sirtuin inhibitor, attenuated neurotrophic action of NGF and did not produce hyperacetylation of H3/H4.

These results suggest promising neurotrophic action of vorinostat via activation of MEK1/2 & PI3K pathways involving activation of upstream TrkA, a neurotrophin receptor. Bioactive small molecules with neurotrophic and neuritogenic actions, like vorinostat identified in present studies, hold great promise as therapeutic agents for treatment of neurodegenerative diseases and neuronal injuries by virtue of their ability to stimulate neuritic outgrowth.

Concentration/Emphasis

Emphasis: Pharmacology

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