Kishor Wasan - Academia.edu (original) (raw)

Papers by Kishor Wasan

Aberrant cholesterol metabolism is increasingly appreciated to be essential for prostate cancer i... more Aberrant cholesterol metabolism is increasingly appreciated to be essential for prostate cancer initiation and progression. Transcript expression of the high-density lipoprotein-cholesterol receptor scavenger receptor B1 (SR-B1) is elevated in primary prostate cancer. Hypothesizing that SR-B1 expression may help facilitate malignant transformation, we document increased SR-B1 protein and transcript expression in prostate cancer relative to normal prostate epithelium that persists in lethal castration-resistant prostate cancer (CRPC) metastasis. As intratumoral steroid synthesis from the precursor cholesterol can drive androgen receptor (AR) pathway activity in CRPC, we screened androgenic benign and cancer cell lines for sensitivity to SR-B1 antagonism. Benign cells were insensitive to SR-B1 antagonism, and cancer line sensitivity inversely correlated with expression levels of full-length and splice variant AR. In androgen-responsive CRPC cell model C4-2, SR-B1 antagonism suppressed cholesterol uptake, de novo steroidogenesis, and AR activity. SR-B1 antagonism also suppressed growth and viability and induced endoplasmic reticulum stress and autophagy. The inability of exogenous steroids to reverse these effects indicates that AR pathway activation is insufficient to overcome cytotoxic stress caused by a decrease in the availability of cholesterol. Furthermore, SR-B1 antagonism decreased cholesterol uptake, growth, and viability of the AR-null CRPC cell model PC-3, and the small-molecule SR-B1 antagonist block lipid transport-1 decreased xenograft growth rate despite poor pharmacologic properties. Overall, our findings show that SR-B1 is upregulated in primary and castration-resistant disease and is essential for cholesterol uptake needed to drive both steroidogenic and nonsteroidogenic biogenic pathways, thus implicating SR-B1 as a novel and potentially actionable target in CRPC.Significance:These findings highlight SR-B1 as a potential target in primary and castration-resistant prostate cancer that is essential for cholesterol uptake needed to drive steroidogenic and nonsteroidogenic biogenic pathways.

Supplementary information describing methods for cell culture, HDL assay,qPCR and western blottin... more Supplementary information describing methods for cell culture, HDL assay,qPCR and western blotting analysis, steroid analysis and BLT-1 pharmacokinetic analysis. Supplemental statistical analysis of IHC samples and results of in vivo profiling of BLT-1

Pharmaceutics, 2021

There have been several studies that have linked elevated scavenger receptor class b type 1 (SR-B... more There have been several studies that have linked elevated scavenger receptor class b type 1 (SR-B1) expression and activity to the development and progression of castration-resistant prostate cancer (CRPC). SR-B1 facilitates the influx of cholesterol to the cell from lipoproteins in systemic circulation. This influx of cholesterol may be important for many cellular functions, including the synthesis of androgens. Castration-resistant prostate cancer tumors can synthesize androgens de novo to supplement the loss of exogenous sources often induced by androgen deprivation therapy. Silencing of SR-B1 may impact the ability of prostate cancer cells, particularly those of the castration-resistant state, to maintain the intracellular supply of androgens by removing a supply of cholesterol. SR-B1 expression is elevated in CRPC models and has been linked to poor survival of patients. The overarching belief has been that cholesterol modulation, through either synthesis or uptake inhibition, w...

Pharmaceutics, 2019

Parenteral amphotericin B has been considered as first-line therapy in the treatment of systemic ... more Parenteral amphotericin B has been considered as first-line therapy in the treatment of systemic fungal and parasitic infections, however its use has been associated with a number of limitations including affordability, accessibility, and an array of systemic toxicities. Until very recently, it has been very challenging to develop a bioavailable formulation of amphotericin B due to its physical chemical properties, limited water and lipid solubility, and poor absorption. This perspective reviews several novel oral Amphotericin B formulations under development that are attempting to overcome these limitations.

Cancer Research, 2019

Molecular Pharmaceutics, 2019

Drug Delivery and Translational Research, 2017

The targeting and delivery of therapeutic and diagnostic agents to bone tissue presents both a ch... more The targeting and delivery of therapeutic and diagnostic agents to bone tissue presents both a challenge and opportunity. Osteoporosis, Paget's disease, cancer, and bone metastases are all skeletal diseases whose treatment would benefit from new targeted therapeutic strategies. Osteoporosis, in particular, is a very prevalent disease, affecting over one in three women and one in five men in Canada alone with the cost to the healthcare system estimated at over $2.3 billion in 2010. Bone tissue is often considered a rigid structure when in reality there is a process of continuous remodeling that takes place via complex endocrine-regulated cell signaling pathways in addition to the signaling pathways unique to bone tissue. It is these specific boneremodeling processes that provide unique targeting opportunities but also present a number of challenges.

Drugs in Context, 2015

Because guidelines for treatment dosing and timing vary quite widely, it is perhaps not apparent ... more Because guidelines for treatment dosing and timing vary quite widely, it is perhaps not apparent to the physician what the rationale and the BZD drug levels will be at any given time after the initiation of treatment. In addition, BZD drugs that are used for AWS are metabolized differently (Phase I oxidative

Prostate Cancer and Prostatic Diseases, 2015

The Prostate, 2011

BACKGROUNDScavenger Receptor Class B Type I (SR‐BI) facilitates influx of cholesterol to the cell... more BACKGROUNDScavenger Receptor Class B Type I (SR‐BI) facilitates influx of cholesterol to the cell from lipoproteins in the circulation. This influx of cholesterol may be important for many cellular functions, including synthesis of androgens. Castration‐resistant prostate cancer tumors are able to synthesize androgens de novo in order to supplement the loss of exogenous sources often induced by androgen deprivation therapy. Silencing of SR‐BI may impact the ability of prostate cancer cells, particularly those of castration‐resistant state, to maintain the intracellular supply of androgens by removing a supply of cholesterol.METHODSSR‐BI expression was knocked down using small interfering RNA in LNCaP and C4‐2 cells. The effect of down‐regulation of SR‐BI on PSA production, cell toxicity, and cell viability was measured in both cell types. In addition, compensatory cholesterol synthesis activity was measured using the radiolabeled precursor, 14C‐acetate.RESULTSSR‐BI protein expressio...

The Prostate, 2009

BACKGROUND. De novo androgen synthesis and subsequent androgen receptor (AR) activation has recen... more BACKGROUND. De novo androgen synthesis and subsequent androgen receptor (AR) activation has recently been shown to contribute to castration-resistant prostate cancer (CRPC) progression. Herein we provide evidence that fatty acids (FA) can trigger androgen synthesis within steroid starved prostate cancer (CaP) tumor cells. METHODS. Tumoral FA and steroid levels were assessed by GC-MS and LC-MS, respectively. Profiles of genes and proteins involved in FA activation of steroidogenesis were assessed by fluorescence microscopy, immunohistochemistry, microarray expression profiling and Western blot analysis. RESULTS. In human CaP tissues the levels of proteins responsible for FA activation of steroid synthesis were observed to be altered during progression to CRPC. Further investigating this mechanism in LNCaP cells, we demonstrate that specific FA, arachidonic acid, is synthesized in an androgen-dependent and AR-mediated manner. Arachidonic acid is known to induce steroidogenic acute regulatory protein (StAR) in steroidogenic cells. When bound to hormone sensitive lipase (HSL), StAR shuttles free cholesterol into the mitochondria for downstream conversion into androgens. We show that arachidonic acid induces androgen production in steroid starved LNCaP cells coincidently in the same conditions that HSL and StAR are predominantly localized in the mitochondria. Furthermore, their activities are verified by a functional increase in mitochondrial uptake of cholesterol in this steroid starved environment. CONCLUSIONS. We propose that this characterized arachidonic acid induced steroidogenesis mechanism significantly contributes to the activation of AR in CRPC progression and therefore recommend that fatty acid pathways be targeted therapeutically in progressing CaP.

Lipids in Health and Disease, 2014

Background: In castration-resistant prostate cancer (CRPC), recent evidence has demonstrated the ... more Background: In castration-resistant prostate cancer (CRPC), recent evidence has demonstrated the persistence of the intratumoral androgens. The multi-step androgen synthesis pathway originates from cholesterol, which can be obtained by cells from several major sources including intracellular synthesis through an enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR). The inhibition of this enzyme by the use of statins has been investigated in prostate cancer as a possible therapeutic target for blocking the de novo androgen synthesis resulting in decreased tumor growth. However, the effectiveness of statins in CRPC has not been investigated. Methods: Castration-resistant C4-2 and androgen-sensitive LNCaP cells were treated with Simvastatin for 48 hours. Dose-dependent responses to Simvastatin were analyzed using cell proliferation and cytotoxicity assays. Cellular growth curve was generated using haemocytometer. HMGCR activity was assessed using 14 C-acetic acid detected by thin layer chromatography, and the protein expression was quantified using western blot analysis. Intracellular cholesterol and prostate specific antigen (PSA) levels were quantified using enzyme-linked immunosorbent assays (ELISA). Results: Significant decrease in cell viability and growth curve observed at 75 μM of Simvastatin compared to no treatment group in the castration-resistant C4-2 cells. HMGCR activity was significantly decreased up to 50% and 70% at 50 μM and 75 μM of Simvastatin respectively compared to the vehicle control in C4-2 cells. Simvastatin did not affect the protein expression. 80% decrease in the amount of total intracellular cholesterol levels was observed in 75 μM Simvastatin treatment group compared to vehicle control. PSA secretion levels were significantly reduced in the C4-2 cell line at 50 μM and 75 μM of Simvastatin compared to vehicle control. Conclusion: The inhibition of HMGCR via Simvastatin lowered the viability of castration-resistant C4-2 cells. Simvastatin's ability to limit the endogenous supply of cholesterol contributes to the effects seen in cell viability.

Journal of Pharmacological and Toxicological Methods, 2005

The purpose of this study was to identify and characterize the presence of low-density lipoprotei... more The purpose of this study was to identify and characterize the presence of low-density lipoprotein receptors (LDLr) in LLC-PK(1) cells. LLC-PK(1) cells were assessed for the presence of LDLr by conducting dose-response, LDL specific binding and competitive studies with DiI-LDL, and Western blot and RT-polymerase chain reaction (PCR) analyses. Assay conditions with IgG-C7, a monoclonal antibody (mAb) to the LDLr, were optimized, including temperature, preincubation time, and concentration in LLC-PK(1) cells. LLC-PK(1) cells express LDL receptors as determined by LDL specific and competitive binding studies and Western blot and RT-PCR analysis (specific binding 0.5 ng DiI-LDL/mug of cellular protein). Taken together, these findings confirm the presence of LDL receptors on LLC-PK1 cells and support the appropriateness of using these cells in studies involving renal cell cholesterol uptake and metabolism.

Journal of Antimicrobial Chemotherapy, 2010

Journal of Antimicrobial Chemotherapy, 2009

To assess the impact of visceral leishmaniasis (VL) on the concentration of amphotericin B (AmB) ... more To assess the impact of visceral leishmaniasis (VL) on the concentration of amphotericin B (AmB) recovered in the liver and spleen following either intravenous (AmBisome w) or oral (iCo-009) AmB administration to mice. Methods: Livers and spleens previously obtained from VL-infected BALB/c mice (following intravenous AmBisome w or oral AmB treatments) were analysed for AmB concentrations. Then, non-infected BALB/c mice were divided into three treatment groups: a single dose of intravenous AmBisome w (2 mg/kg, n¼ 5); and oral AmB every 12 h for 5 days (10 mg/kg, n¼ 6 and 20 mg/kg, n¼ 6). The animals were sacrificed 7 days after the initiation of the treatment and the livers and spleens were harvested for drug analysis by HPLC. Results: The single intravenous injection of AmBisome w resulted in a 77-fold lower concentration of AmB in infected compared with non-infected liver tissue, while the difference in AmB concentration in the spleen was only 5-fold. The multiple dose oral administration of AmB resulted in a 3-fold lower concentration of AmB in infected compared with non-infected livers for both oral doses, while the differences in AmB concentrations in the spleen were not statistically different for the oral treatment groups. Conclusions: VL significantly lowered the concentration of AmB in the liver and the spleen when compared with uninfected animals. This effect seems to correlate with the degree of infection of the tissue. In the case of the intravenous liposomal formulation (AmBisome w), the differences between the infected and non-infected tissues are of a higher magnitude than in the case of orally administered AmB (iCo-009).

Dalton Transactions, 2007

(K.M.W.). † Electronic supplementary information (ESI) available: Table of IR data and 1 H NMR ex... more (K.M.W.). † Electronic supplementary information (ESI) available: Table of IR data and 1 H NMR example of metal binding (Hma and La(ma) 3 spectrum).

Cancer Research, 2016

Existing therapies for castration-resistant prostate cancer (CRPC) extend life and provide clinic... more Existing therapies for castration-resistant prostate cancer (CRPC) extend life and provide clinical benefit; however, patients continue to develop therapeutic resistance. Androgen pathway activity persists in CRPC even under maximal androgen blockade conditions. A proposed mechanism for continued androgen pathway signaling is de novo intratumoral synthesis of androgen receptor agonists from the precursor cholesterol. The high density lipoprotein(HDL)-cholesterol receptor, scavenger receptor class B type I (SR-BI), is upregulated in CRPC models in vitro and in vivo. Here, we test the hypothesis that SR-BI is a source of cholesterol for de novo steroidogenesis in CRPC cells using the castration-resistant LNCaP-derived cell line, C4-2. Cells were transfected with either non-targeted (NC) or stealth RNAi duplexes (Thermo Fisher) targeting SR-BI to silence protein expression (SR-B1 KD). Prostate specific antigen (PSA) levels in media from SR-B1 KD samples were reduced to 39% of that seen...

The Prostate, 2012

Scavenger Receptor Class B Type I (SR-BI) facilitates influx of cholesterol to the cell from lipo... more Scavenger Receptor Class B Type I (SR-BI) facilitates influx of cholesterol to the cell from lipoproteins in the circulation. This influx of cholesterol may be important for many cellular functions, including synthesis of androgens. Castration-resistant prostate cancer tumors are able to synthesize androgens de novo in order to supplement the loss of exogenous sources often induced by androgen deprivation therapy. Silencing of SR-BI may impact the ability of prostate cancer cells, particularly those of castration-resistant state, to maintain the intracellular supply of androgens by removing a supply of cholesterol. SR-BI expression was knocked down using small interfering RNA in LNCaP and C4-2 cells. The effect of down-regulation of SR-BI on PSA production, cell toxicity, and cell viability was measured in both cell types. In addition, compensatory cholesterol synthesis activity was measured using the radiolabeled precursor, (14) C-acetate. SR-BI protein expression is higher basally in C4-2 cells than LNCaP cells. Silencing of SR-BI expression to greater than 85% reduced PSA production in LNCaP and C4-2 SRBI-KD cells by 55% and 58% compared to negative control cells, respectively. SR-BI-KD C4-2 cells demonstrated significantly reduced cell viability (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;25%) compared the NC cells. The down-regulation of SR-BI significantly impacts PSA production of prostate cancer cells, as well as the viability of C4-2 cells in the presence and absence of HDL. This may indicate a deficiency in cholesterol availability to the androgen synthesis pathway or may implicate a role for SR-BI in prostate cancer signal transduction pathways.