Determining the role of scavenger receptor class B type I as a source of cholesterol for de novo androgen synthesis in castration-resistant prostate cancer
department or school: Pharmaceutical Sciences, Faculty of
data provider: DSpace
degree campus: UBCV
creator: Midha, Ankur
issued: 2015-08-12T15:01:56Z
issued: 2015
related degree: Master of Science - MSc
degree grantor: University of British Columbia
description: Existing therapies for castration-resistant prostate cancer (CRPC) extend life and provide clinical benefit; however, patients develop therapeutic resistance. Persistent androgen signaling in CRPC is maintained in part by intratumoral steroidogenesis from the precursor cholesterol. The high density lipoprotein-cholesterol (HDL) receptor, scavenger receptor class B type I (SR-BI), is upregulated in CRPC models in vitro and in vivo. This thesis tests the hypothesis that depriving CRPC cells of HDL as a cholesterol source by silencing SR-BI will diminish de novo steroidogenesis and resultant androgen receptor-mediated signaling necessary for CRPC viability. The effects of SR-BI silencing were studied using CRPC C4-2 cells transfected with either Stealth RNAi duplexes targeting SR-BI (SRBI-KD) or Lo GC Negative Control (NC) duplexes. Cells cultured in androgen-depleted conditions for varying times post-transfection were assessed for SR-BI expression, prostate specific antigen (PSA) expression by chemiluminescence, cholesterol levels by fluorometry, and steroid levels by liquid-chromatography-mass spectrometry. HDL-uptake was measured by flow cytometry of the fluorescent cholesterol mimetic 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI-HDL). Cell cycle state was assessed by flow cytometry of propidium iodide DNA staining, while cell cycle markers were assessed by immunoblotting. Adaptive stress responses were assessed by immunoblotting for autophagy markers, as well by flow cytometry for senescence associated beta-galactosidase (SA-β-gal) using a fluorogenic substrate, 5-dodecanoylaminofluorescein di-β-D-galactopyranoside. SRBI-KD treatment reduced SR-BI levels by ~57% by 2 days and ~86% by 4 days post-transfection. This correlated with reduced DiI-HDL-uptake by 22%, reduced cellular testosterone levels two-fold, and reduced PSA secretion by 39% compared to NC cells. These changes were accompanied by reduced proliferation, G₁S cell cycle arrest,…
aggregated CHO: https://circle.library.ubc.ca/rest/handle/2429/54366
note: Determining the Role of Scavenger Receptor Class B Type I as a Source of Cholesterol for de novo Androgen Synthesis in Castration-resistant Prostate Cancer by Ankur Midha B.Sc. (Pharm), The University of British Columbia, 2010 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pharmaceutical Sciences) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2015 © Ankur Midha, 2015 ii Abstract Existing therapies for castration-resistant prostate cancer (CRPC) extend life and provide clinical benefit; however, patients develop therapeutic resistance. Persistent androgen signaling in CRPC is maintained in part by intratumoral steroidogenesis from the precursor cholesterol. The high density lipoprotein-cholesterol (HDL) receptor, scavenger receptor class B type I (SR-BI), is upregulated in CRPC models in vitro and in vivo. This thesis tests the hypothesis that depriving CRPC cells of HDL as a cholesterol source by silencing SR-BI will diminish de novo steroidogenesis and resultant androgen receptor-mediated signaling necessary for CRPC viability. The effects of SR-BI silencing were studied using CRPC C4-2 cells transfected with either Stealth RNAi duplexes targeting SR-BI (SRBI-KD) or Lo GC Negative Control (NC) duplexes. Cells cultured in androgen-depleted conditions for varying times post-transfection were assessed for SR-BI expression, prostate specific antigen (PSA) expression by chemiluminescence, cholesterol levels by fluorometry, and steroid levels by liquid-chromatography-mass spectrometry. HDL-uptake was measured by flow cytometry of the fluorescent cholesterol mimetic 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI-HDL). Cell cycle state was assessed by flow cytometry of propidium iodide DNA staining, while cell cycle markers were assessed by immunoblotting. Adaptive stress responses were assessed by immunoblotting for autophagy markers, as…
has type: Thesis/Dissertation
date issued: 2015-09
is shown at: 10.14288/1.0166529
language: eng
degree discipline: Pharmaceutical Sciences
provider: Vancouver : University of British Columbia Library
publisher: University of British Columbia
rights: Attribution-NonCommercial-NoDerivs 2.5 Canada
rights URI: http://creativecommons.org/licenses/by-nc-nd/2.5/ca/
scholar level: Graduate
type: Text
identifier URI: http://hdl.handle.net/2429/54366