Docosahexaenoic acid differentially modulates the cell cycle and metabolism- related genes in tumor and pre-malignant prostate cells

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Highlights

  • Cell cycle and metabolism-related genes are regulated by DHA in prostate cells.

  • DHA decreases proliferation of prostate cells with different AR and PTEN contexts.

  • DHA leads to cell cycle arrest in both prostate pre-malignant and tumor cells.

  • DHA modulates metabolism-related genes via nuclear receptors regulation.

  • The omega-3 differently induces ROS and lipid accumulation in AR+ and AR− cells.

Abstract

Prostate cancer (PCa) has different molecular features along progression, including androgen profile, which is associated to therapy inefficiency leading to more aggressive phenotype. Docosahexaenoic acid (DHA) has antiproliferative and pro-apoptotic properties in different cancers associated to cell metabolism modulation. The latter is of particular interest since metabolic reprogramming is one of PCa hallmarks, but is not clear how this occurs among disease progression. Therefore, we evaluated DHA antiproliferative potential in distinct androgenic backgrounds associated to metabolism modulation and androgen-regulated genes. For this purpose, pre-malignant PNT1A and tumor AR-positive 22rv1, and AR-negative PC3 cells were incubated with DHA at 100 μM–48 h. DHA reduced at least 26% cell number for all lineages due to S-phase decrease in AR-positive and G2/M arrest in AR-negative. Mitochondrial metabolic rate decreased in PNT1A (~38%) and increased in tumor cells (at least 40%). This was associated with ROS overproduction (1.6-fold PNT1A; 2.1 22rv1; 2.2 PC3), lipid accumulation (3-fold PNT1A; 1.8 22rv1; 3.6 PC3) and mitochondria damage in all cell lines. AKT, AMPK and PTEN were not activated in any cell line, but p-ERK1/2 increased (1.5-fold) in PNT1A. Expression of androgen-regulated and nuclear receptors genes showed that DHA affected them in a distinct pattern in each cell line, but most converged to metabolism regulation, response to hormones, lipids and stress. In conclusion, regardless of androgenic or PTEN background DHA exerted antiproliferative effect associated to cell cycle impairment, lipid deregulation and oxidative stress, but differentially regulated gene expression probably due to distinct molecular features of each pathologic stage.

Introduction

Most of available therapies for prostate cancer (PCa) are primary focused on ablation of tumor-stimulating hormones, as androgens. These therapies may drive to castrated-resistant phenotypes which are related to reoccurrence with increased aggressiveness and poor prognosis [1,2]. This lack of inefficient therapeutic strategies urges for new approaches as PCa still remains among the most frequent malignancy in men worldwide [3]. Metabolic reprogramming is a well-known event described in cancer and supports increased energy demand and anabolic processes required for high proliferative rates [4]. In PCa, deregulation of lipid metabolism is an early hallmark of this metabolic switch and each pathological stage of disease shows distinct patterns of overexpression for several enzymes [5] [6], such as fatty acid synthase (FASN) [[6], [7], [8], [9]] that is linked to castrated-resistant phenotype [7,10,11]. Also, lipolysis is the main catabolic pathway in PCa [12] which according to androgenic background exhibit different fatty acid transporters [13]. This evidence suggests that tumor metabolism is differently regulated during disease progression and that external lipids may play a distinct role among androgenic profiles found in the pathology spectrum.

This is of particular interest with regard to PCa etiology since dietary factors have been correlated with demographic differences in its incidence, particularly the amount and type of lipids [[14], [15], [16], [17], [18]]. On one hand, saturated fatty acids (SFAs) have been associated to prostate malignancy as well as polyunsatured fatty acids (PUFAs) omega-6 (ω-6) [19,20]. On the other, PUFAs of omega-3 (ω-3) class were reported to decrease PCa risk serving as preventive, therapeutic or even coadjuvant agents [21,22]. This is supported by evidence that lower ω-6/ω-3 ratio decreased tumor growth in xenograft models [23,24], delayed PCa progression [25] and impaired cell migration [26] whereas higher ratio increased the risk of PCa development [27]. The mechanism related to the protective property of lower ω-6/ω-3 ratio is not fully elucidated, but has been linked to the omega-3 fatty acids due to down-modulation of Akt/mTOR/NFκB axis, cell cycle arrest and anti-inflammatory mediators [23,25]. Moreover, preclinical studies suggest that oil fish-derived omega-3 have good outcome in androgen sensitive and castrated-resistant PCa [28,29], including in the absence of PTEN [30]. However, PUFAs' role in the gland carcinogenesis still remains under debate [18] and have been shown their increased levels in the blood [31].

Docosahexaenoic acid (DHA) is a PUFA omega-3 that shows much reduced synthesis in humans, especially in men, and is obtained mainly from nutritional supplementation or diet with higher levels found in marine cold fishes [32]. Along the past years, studies have reported DHA antitumor properties in several tissues which raised discussions about its role in PCa initiation and progression [32]. Compared to others omega-3 with biological activity, DHA shows higher effectiveness in decrease cell viability and induce apoptosis compared to others, as EPA (eicosapentanoic acid) and ALA (α-linoleic acid) in PCa cells [33]. The underlying mechanisms in prostate are partially known and involves the reduction in cell migration [34] and proliferation through ROS-mediated AKT/mTOR suppression [35], downregulation of lipid metabolism enzymes [36], modulation of cell death or survival pathways as AKT/PIP3 [37] and PDK1/AKT/Bad [38], which are most tightly related to cell metabolism. Recently, we reported the preventive potential of DHA by decreasing proliferation of PNT1A pre-malignant cells due to mitochondria bioenergetics modulation, decreasing their capacity to respond to stress stimuli and enhancing antiproliferative effect of melatonin [22]. Several studies have reported that such omega-3 regulated metabolic sensors as PPARγ, PPARδ and RXRs [[39], [40], [41]]. These nuclear receptors together with others were described to play a role in several cancers [42] and may respond to extracellular compounds, as hormones and factors modulating cell proliferation, bioenergetics [41], mitochondrial dynamics and lipid metabolism.

PCa metabolic vulnerabilities have been investigated as therapeutic strategy [7]. Taken together, these evidence supports that metabolism regulation may be a potential mechanism of DHA, but is not clear how this omega-3 affects different molecular contexts found among disease progression. In the present study, we tested DHA potential to decrease proliferation of pre-malignant (benign) and castrated-resistant tumor cell lines with different androgenic backgrounds associated to metabolism regulation and androgen signaling. For this purpose we used three different cell lines: PNT1A, which maintain androgen sensibility, PTEN expression, normal secretory phenotype but also exhibit non-malignant alterations [43], as observed in benign prostatic hyperplasia (BPH) and early stages of carcinogenesis; castrated-resistant tumor 22rv1 (express full-length AR and variants with constitutional activation similar to hormone refractory phenotype; PTEN-positive) and, more aggressive, PC3 (AR-negative, neuroendocrine-like adenocarcinoma; PTEN-negative) [44]. Our findings revealed that although DHA led to lipid accumulation, ROS overproduction and reduced cell number, it differently modulated cell cycle in each line by decreasing S-phase in AR-positive and arresting AR-negative cells in G2/M. In addition, this omega-3 differently regulated gene expression in distinct pattern in each pathological stage, despite most of them converged to metabolism regulation and response to stress and hormones.

Section snippets

Cell culture

PNT1A (#95012614—Health Protection Agency, England, UK), 22rv1 and PC3 (CRL1435) cells were cultured in RPMI 1640 medium (#R6504—Sigma-Aldrich, St. Louis, Missouri, EUA) enriched with 10% fetal bovine serum (FBS) (#S0011—Vitrocell, Campinas, São Paulo, Brazil), 1% of penicillin, streptomycin, and amphotericin B (#15240062—Life Technologies, Paisley, UK) and kept in a wet incubator with 5% of CO2 at 37 °C. For cell maintenance, the medium was replaced every 2–3 days and subculture was done when

DHA conversely modulates MMR from benign and malignant prostate cells

After 24 h, DHA did not affect MMR of PNT1A at any concentration used here, however, decreased of PC3 at 10 and 20 μM (22% and 37%, respectively) and increased about 40% of 22rv1 at 100 μM (Fig. 1A). Regarding the longer incubation (48 h), DHA stimulated PNT1A mitochondrial metabolism at 10 μM (35%) and 50 μM (40%) and reduced about 38% at 100 μM (Fig. 1B). The omega-3 increased mitochondrial metabolism of both PCa lines PC3 and 22rv1 at 100 μM when incubated for 48 h (Fig. 1B). Then, this

Discussion

Despite DHA antiproliferative potential has already been described in several cancers [[34], [35], [36], [37], [38]], its protective effects against PCa progression have been discussed and the underlying mechanisms remain to be elucidated. To the best of our knowledge, this is the first study to compare the ability of DHA to decrease cell proliferation in pre-malignant and PCa castrated-resistant cells with different androgenic backgrounds and the association with metabolism regulation. Our

Conclusion

The present study provided evidence that DHA regardless of PTEN status and androgenic background was able to decrease cell proliferation associated to metabolism regulation, but via different mechanisms. However, for most of genes, the omega-3 regulated them in an opposite manner that those observed in PCa samples which supports its antitumor property at least in vitro. Especially for castrated-resistant cells that lack clinical efficient treatment, DHA may be an alternative therapeutic

CRediT authorship contribution statement

Guilherme Henrique Tamarindo: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing - original draft. Rejane Maira Góes: Conceptualization, Methodology, Resources, Writing - review & editing, Supervision, Project administration, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors are grateful to Dr. Nishtman Dizeyi (Lund University, Malmo, Sweden) and Dr. Hernandes Faustino Carvalho (Institute of Biology, State University of Campinas, São Paulo, Brazil) for kindly providing PNT1A and PC3 cell lines; Dr. Joice Biselli and Dr. Ana Elizabete da Silva for collaboration in qRT-PCR arrays; Dr. Sebastião Roberto Taboga, Dr. Mary Massumi Itoyama and MSc. Luiz Roberto Falleiros Júnior (Institute of Biosciences, Humanities and Exact Sciences, São Paulo State

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