VORINOSTAT INDUCES P-GLYCOPROTEIN ACTIVITY IN BLOOD-BRAIN BARRIER ENDOTHELIUM Taylor Liles

OUTLINE• 1. Introduction

• a. Blood brain barrier: Structure and Function 

• b. ABC Efflux transporters and their role in BBB function

• c. Vornistat • 2. Hypothesis• 3. Experiments and Data• 4. Conclusion and Outlook• 5. Acknowledgements

THE BLOOD BRAIN BARRIER

• Physical and chemical barrier

• 3D culture• Tight

Junctions• Efflux

Transporters

https://loonylabs.files.wordpress.com/2015/03/blood-brain-barrier.jpg?w=590https://www.google.com/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRxqFQoTCJLS25zV58YCFQY6kgod1ycM0w&url=http%3A%2F%2Fwww.pubfacts.com%2Ffulltext_frame.php%3FPMID%3D23678437%26title%3DImpacts%2520of%2520blood-brain%2520barrier%2520in%2520drug%2520delivery%2520and%2520targeting%2520of%2520brain%2520tumors.&ei=JNerVZKwN4b0yATXz7CYDQ&bvm=bv.98197061,d.cGU&psig=AFQjCNHtqILV8_qvW9ud2gAHlLbiUjz-Yw&ust=1437411150229860

P-GP (MDR1)

• ATP-binding cassette Transporter• Broad range of substrate

• Lipids• Xenobiotics• Peptides• Uncharged or positive charge• Hydrophobic

• Intestines, liver, kidneys, and brain micro-vasular endothelium

https://upload.wikimedia.org/wikipedia/commons/thumb/f/fe/MDR3_3g5u.png/500px-MDR3_3g5u.png

VORNISTAT (SAHA)• Non-Specific Histone Deacetylase (HDAC)

Inhibitor• FDA approval in 2006 for persistent or

recurrent Cutaneous T-Cell Lymphoma

VORNISTAT (SAHA)• Causes death and differentiation in

cancer cells ¹• Cellular changes through non-histone

proteins ²• Increase synaptic activity ³• Low brain penetration ³• P-GP substrate ³

1. Marks PA. Discovery and development of SAHA as an anticancer agent. Oncogene. 2007 Feb 26;26(9):1351-6. Review. PubMed PMID: 17322921. 2. Spange S, Wagner T, Heinzel T, Krämer OH. Acetylation of non-histone proteins modulates cellular signalling at multiple levels. Int J Biochem Cell Biol. 2009 Jan;41(1):185-98. doi: 10.1016/j.biocel.2008.08.027. Epub 2008 Sep 2. Review. PubMed PMID: 18804549.3. Hanson, J. E., La, H., Plise, E., Chen, Y.-H., Ding, X., Hanania, T., … Zhou, Q. (2013). SAHA Enhances Synaptic Function and Plasticity In Vitro but Has Limited Brain Availability In Vivo and Does Not Impact Cognition. PLoS ONE, 8(7), e69964. doi:10.1371/journal.pone.0069964

HYPOTHESIS• Can SAHA modulate

activity and/or expression of P-gp transporters in Brain microvascular endothelial cells?

METHODS• Mouse Primary and Human D3

cell line BMECs • Increasing concentrations of

SAHA over 1-6 hour time points• ATP-ase assay• P-GP GLO assay• Rhodamine efflux activity assay

1 hour

Crtl SAHA 10 nm

SAHA 100 nm

SAHA 1000 nm

3 hours

Crtl SAHA 10 nm SAHA 100 nm

SAHA 1000 nm

6 Hours

Crtl SAHA 10 nm SAHA 100 nm

SAHA 1000 nm

VIABILITY ASSAYS: D3 CELLSATP ASSAYLDH ASSAY

PGP ATP-ASE ASSAY• Assesses consumption of ATP• Metabolic activity

RHODAMINE ASSAYS• Rhodamine-123 is a lipid-soluble,

fluorescent dye• Cells undergo experimental conditions

and then are incubated in a R123 solution

• The solution is removed, the cells washed, and a fluorescent reading is taken

RHODAMINE ASSAYS: D3 CELLS

RHODAMINE ASSAYS: MOUSE PRIMARY CELLS

CONCLUSION•Vornistat does seem to exert an enhancing effect on P-gp activity.

•This could disqualify Vornistat as a potential therapeutic in Brain Cancers.

•Further tests need to be performed assessing other efflux transporters.

ACKNOWLEDGEMENTS• Ravi Sajja

Research Scientist• Shikha Prasad

Graduate Student• Mohammad Kaisar

Graduate Student• Luca Cucullo

Assistant Professor

•Teressa Carlisle GPPS, GSBS, ABRI Coordinator

All involved in organizing, funding, and speaking for the ABRI Program

Thank you for investing in my success as a scientist!

QUESTIONS?