Cholesterol Homeostasis in Two Commonly Used Human Prostate Cancer Cell-Lines, LNCaP and PC-3

BACKGROUND:Recently, there has been renewed interest in the link between cholesterol and prostate cancer. It has been previously reported that in vitro, prostate cancer cells lack sterol-mediated feedback regulation of the major transcription factor in cholesterol homeostasis, sterol-regulatory element binding protein 2 (SREBP-2). This could explain the accumulation of cholesterol observed in clinical prostate cancers. Consequently, perturbed feedback regulation to increased sterol levels has become a pervasive concept in the prostate cancer setting. Here, we aimed to explore this in greater depth. METHODOLOGY/PRINCIPAL FINDINGS:After altering the cellular cholesterol status in LNCaP and PC-3 prostate cancer cells, we examined SREBP-2 processing, downstream effects on promoter activity and expression of SREBP-2 target genes, and functional activity (low-density lipoprotein uptake, cholesterol synthesis). In doing so, we observed that LNCaP and PC-3 cells were sensitive to increased sterol levels. In contrast, lowering cholesterol levels via statin treatment generated a greater response in LNCaP cells than PC-3 cells. This highlighted an important difference between these cell-lines: basal SREBP-2 activity appeared to be higher in PC-3 cells, reducing sensitivity to decreased cholesterol levels. CONCLUSION/SIGNIFICANCE:Thus, prostate cancer cells are sensitive to changing sterol levels in vitro, but the extent of this regulation differs between prostate cancer cell-lines. These results shed new light on the regulation of cholesterol metabolism in two commonly used prostate cancer cell-lines, and emphasize the importance of establishing whether or not cholesterol homeostasis is perturbed in prostate cancer in vivo

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Does Changing Androgen Receptor Status during Prostate Cancer Development Impact upon Cholesterol Homeostasis?

<div><h3>Background</h3><p>Recent evidence associates prostate cancer with high cholesterol levels, with cholesterol being an important raw material for cell-growth. Within the cell, cholesterol homeostasis is maintained by two master transcription factors: sterol-regulatory element-binding protein 2 (SREBP-2) and liver X receptor (LXR). We previously showed that the androgen receptor, a major player in prostate cell physiology, toggles these transcription factors to promote cholesterol accumulation. Given that prostate cancer therapy targets the androgen receptor, selecting for cells with altered androgen receptor activity, how would this affect SREBP-2 and LXR activity? Using a novel prostate cancer progression model, we explored how this crosstalk between the androgen receptor and cholesterol homeostasis changes during prostate cancer development.</p> <h3>Methodology/Principal Findings</h3><p>Firstly, we characterised our progression model, which involved 1) culturing LNCaP cells at physiological testosterone levels to generate androgen-tolerant LNCaP-305 cells, and 2) culturing LNCaP-305 with the anti-androgen casodex to generate castration-resistant LNCaP-364 cells. This progression was accompanied by upregulated androgen receptor expression, typically seen clinically, and a reduction in androgen receptor activity. Although this influenced how SREBP-2 and LXR target genes responded to androgen treatment, cellular cholesterol levels and their response to changing sterol status was similar in all LNCaP sub-lines.</p> <h3>Conclusion/Significance</h3><p>Overall cholesterol homeostasis is unaffected by changing androgen receptor activity in prostate cancer cells. This does not negate the relationship between androgens and cholesterol homeostasis, but rather suggests that other factors compensate for altered androgen receptor activity. Given that cholesterol regulation is maintained during progression, this supports the growing idea that cholesterol metabolism is a suitable target for prostate cancer.</p> </div

Summary of studies that generated castration-resistant PCa cells, by long-term treatment of LNCaP cells with casodex (CDX).

<p>It should be noted that the assays for growth and AR activity differed between experiments, incuding pre-treatment, androgen used, and assay duration. N/D, not determined; ↑, increase; ↓ decrease; ↕, biphasic response; -, no effect.</p>a<p>Time during CDX treatment.</p>b<p>Or in comparison to parental LNCaP-104S cells for reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054007#pone.0054007-Kokontis2" target="_blank">[50]</a>. In our study, comparison to LNCaP or 305 cells yielded the same trends.</p>c<p>This includes luciferase assays or target gene (e.g., <i>PSA</i>) mRNA and protein levels. In cases where there was a discrepancy between assays, luciferase assay results were given priority, followed by target gene mRNA levels and finally target gene protein levels.</p>d<p>Parenthesised numbers below refer to the clone number (2, 11 or 12).</p>e<p>These cells were derived from LNCaP-104S cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054007#pone.0054007-Kokontis1" target="_blank">[17]</a> rather than LNCaP cells.</p>f<p>CDX treatment reduces androgen-induced proliferation, but above 1 µM CDX, growth is increased in a CDX-dose-dependent fashion independently of androgens.</p>g<p>CDX increased nuclear localisation of AR.</p>h<p>The study showed that CDX was not agonistic, but did not show it was antagonistic.</p

Overexpression of a key regulator of lipid homeostasis, Scap, promotes respiration in prostate cancer cells

AbstractProstate metabolism is unique, characterised by cholesterol accumulation and reduced respiration. Are these related? We modulated cholesterol levels and despite changes in mitochondrial cholesterol content, we saw no effects on lactate production or respiration. Instead, these features may be related via sterol regulatory element-binding protein 2 (SREBP-2), the master transcriptional regulator of cholesterol synthesis. SREBP-2 diverts acetyl-CoA into cholesterol synthesis and may thus reduce respiration. We examined LNCaP cells overexpressing the SREBP-2 regulator, Scap: although having higher SREBP-2 activity, these cells displayed higher respiration. This striking observation warrants further investigation. Given that SREBP-2 and Scap are regulated by factors driving prostate growth, exploring this observation further could shed light on prostate carcinogenesis

The effect of androgen receptor status on basal cholesterol homeostasis.

<p>Cells were grown in their basal media: Medium A (LNCaP), supplemented with 10 nM testosterone (305) or 10 μM casodex (364). RNA was harvested and (A) <i>LDLR</i> and <i>HMGCR</i>, and (B) <i>ABCG1</i> and <i>ABCA1</i>, mRNA levels were determined by qRT-PCR, normalised to the LNCaP cells. (A–B) Data presented as mean + S.E., from three separate experiments per cell-line, each performed with triplicate wells per condition.</p

The effect of androgen receptor status on androgen-regulated cholesterol homeostasis.

<p>(A) Schematic outlining the effects of the androgen receptor (AR) on key transcription factors in cholesterol homeostasis. Details in the text. (B–C) Cells were starved in Medium B for 24 h, before treatment with 1 nM dihydrotestosterone (DHT) and/or 10 μM CDX in Medium B for another 24 h. Following treatment, RNA was harvested and (B) <i>LDLR</i> and <i>HMGCR</i>, and (C) <i>ABCG1</i> and <i>ABCA1</i>, mRNA levels were determined by qRT-PCR, normalised to the vehicle condition in each cell-line. (B–C) Data presented as mean ± S.E., from three separate experiments per cell-line, each performed with triplicate wells per condition.</p

The effect of androgen receptor status on cholesterol levels and LDL uptake.

<p>(A) Cells were grown in their basal media: Medium A (LNCaP), supplemented with 10 nM testosterone (305) or 10 μM casodex (364). Cholesterol levels were determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054007#s4" target="_blank">Materials and Methods</a>. (B) Cells were treated in their basal media, after which LDL uptake was determined. (C) Cells were plated in their basal media, then starved overnight in Medium C. The next days, cells were treated for 6 h with or without 10 μM 25-hydroxycholesterol (25-HC) in Medium C, after which LDL uptake was determined. (A–C) Data presented as mean + S.E., from three separate experiments per cell-line, each performed with triplicate wells per condition.</p