Fingolimod interrupts the cross talk between estrogen metabolism and sphingolipid metabolism within prostate cancer cells☆
Graphical abstract
Introduction
Prostate cancer accounts for 29% of all cancers in men. Prostate cancer is the second most common cancer after non-melanoma skin cancer, and it is the second highest cause of cancer deaths after lung cancer among men of all races (Barqawi et al., 2012). It has long been believed that androgens are the major underlying hormonal cause of prostate cancer (PC) (Chua et al., 2005). However, current epidemiological, laboratory and clinical evidence suggests that estrogens may play a critical role in predisposing, or even causing, prostate cancer (Nelles et al., 2011). In this respect, it is noteworthy that local estrogen production by aromatase (CYP19) in prostate tumors, rather than circulating steroids, was a major player in driving prostate cancer initiation and progression in men (Stocco, 2012).
Indeed, studies demonstrated a clear link between estradiol (E2) metabolism and prostate carcinogenesis. The metabolic bio-activation of estrogens is mainly mediated by CYP1A1 and CYP1B1 enzymes to produce 2- and 4-hydroxylated estrogens and to a lesser extent 16α-hydroxylated estrogens. These hydroxylated metabolites (catechol estrogens) possess superior genotoxic and pro-malignant properties compared to their parent estrogens (Smith et al., 2008; Hevir et al., 2011). Catechol estrogens are detoxified by catechol-O-methyltransferase (COMT) enzyme to antiproliferative and anticancer methoxy estrogens, maintaining intracellular homeostasis (Ho et al., 2011; Mosli et al., 2013). Estrogen receptors, ERα and ERβ, are considered important risk factors for the initiation and progression of prostate cancer. ERα signaling mediates the proliferative effects of estrogens, whereas ERβ signaling directs their antiproliferative effects (Marino and Acconcia, 2011).
Sphingolipid-mediated cell death pathways are currently an emerging field of research for the treatment of autoimmune diseases and neoplasia (Gestaut et al., 2014). A major therapeutic intervention of sphingolipids in cancer focuses on inhibiting activity and/or expression of the enzyme SphK1 (Pyne et al., 2012). SphK1 is a tumor-associated enzyme; its expression level is increased in prostate cancer patients and has been associated with poor prognosis and survival (Pyne et al., 2014). Moreover, in prostate cancer, overexpression of SphK1 is associated with resistance to chemotherapy (Pchejetski et al., 2008) and radiotherapy (Pchejetski et al., 2010). In addition, SphK1 overexpression in prostate cancer influences progression toward a hormone-refractory state (Dayon et al., 2009), regulating Warburg effect (Watson et al., 2013), P53 signaling (Lim et al., 2012) and hypoxia (Cho et al., 2011). These findings highlighted the potential of SphK1 as a therapeutic target in prostate cancer. Accordingly, SphK1 inhibitors may have a potent role in treating prostate cancer patients.
Interestingly, there is emerging evidence establishing a link between estrogens and sphingolipids in a number of studies. These studies showed that E2 serves as a potent SphK1 activator in addition to upregulating its expression. SphK1 is an important mediator in the cytoplasmic signaling of estrogens and promotes mitogenic and oncogenic effects in estrogen-related cancers (Sukocheva et al., 2009; Takabe et al., 2010). Indeed, it has been suggested that SphK1 induces cancer progression via an ER-dependent pathway (Watson et al., 2010). Some selective SphK1 inhibitors possess therapeutic potential in treating estrogen-related cancers via both inhibiting SphK1 enzyme and interfering with ER signaling (Antoon et al., 2011).
FTY720 is the first clinically approved SphK1 inhibitor for the treatment of autoimmune diseases (Estrada-Bernal et al., 2012). FTY720 was approved by the U.S. Food and Drug Administration in 2010 as the first oral bioavailable drug for patients with multiple sclerosis (Chun and Hartung, 2010). Over the past ten years, FTY720 was found to be a potential player in cancer therapy. FTY720 showed antitumor efficacy both in vivo and in vitro in multiple types of cancer including breast, prostate, ovarian, lung, liver, bladder, glioblastoma and hematopoietic malignancies (Zhang et al., 2013b). In these malignancies, several mechanisms were proposed to explain its antitumor effects depending on the type of malignancy (Pereira et al., 2013). Collectively, the anticancer activity of FTY720 in prostate cancer was attributed to inhibiting proliferation, angiogenesis and metastasis and promoting apoptosis (Permpongkosol et al., 2002; Zhou et al., 2006; Chua et al., 2009). After that, studies shed further light on the mode of action of FTY720 by demonstrating its direct inhibition of the activity of SphK1 enzyme in vivo and in vitro (Pchejetski et al., 2010).
To date and to the best of our knowledge, there are no published studies on the cross talk between SK inhibitors and estrogens in prostate cancer. Yet, in this study we demonstrate the effect of FTY720 (SphK1 inhibitor) on estrogen synthesis, metabolism, ER expression and signaling in prostate cancer cells. Moreover, we show the reciprocal effect of estrogen and its metabolites on SphK1 expression in prostate cancer cells.
Section snippets
Chemicals and drugs
RPMI-1640 medium, penicillin-streptomycin (×100), 0.25% trypsin-EDTA, phosphate buffered saline (PBS) and fetal bovine serum (phenol red free) were purchased from Lonza Group Ltd. (Basel, Switzerland). Sulforhodamine-B (SRB) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Quantikine® human active caspase-3 kit was purchased from R&D Systems, Inc. (Minneapolis, MN, USA) and Protease Inhibitor tablets were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Antiproliferative and cytotoxic effects of FTY720 against prostate cancer cells
To assess the antiproliferative effects of FTY720 against prostate cancer cells, three different prostate cancer cell lines (LNCaP, DU-145 and PC-3) were exposed to increasing concentrations of FTY720 (0.01–100 μM) for 72 h. Cell viability was assessed using SRB assay as mentioned in the methods section. The dose response curve of FTY720 was fitted using the Emax model, and IC50 values were calculated as shown in Fig. 1A. FTY720 exerted gradual antiproliferative activity in LNCaP, DU-145 and
Discussion
FTY720, a SphK1 inhibitor interfering with the intracellular sphingolipid metabolism, has been involved in several studies concerning prostate cancer. Estrogenic microenvironments such as estrogen, estrogen metabolites and intracellular estrogen signaling are drawing great attention in the field of prostatic neoplasia. In the current study, we studied the anticancer properties of FTY720 and its role in disrupting the cross talk between estrogen metabolism/signaling and sphingolipid metabolism
Conflict of interest
None.
Author contributions
R. M. A. performed the experimental procedures, combined data, performed statistical analysis and manuscript preparation. A.M.A. generated the research idea, set the experimental design and manuscript preparation. A.K. shared the experimental procedures (LC/MS) and revised manuscript preparation. O.A.S. supervised the experimental performance, combining data and statistical analysis. S.M.N. supervised the experimental performance, combining data and statistical analysis. A.E.K. supervised the
Acknowledgements
Our project was supported by grants from the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under grant No. (260/140/1433). The authors, therefore, acknowledge with thanks DSR technical and financial support. All authors are extremely thankful to Ohoud Y. Alshehri, Tissue Culture Facility Unit, King Faisal Specialist Hospital & Research Center, Jeddah, Saudi Arabia for authenticating the cell lines used in the current study.
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This work was presented in part at The American Association of Cancer Research Annual Meetings 2014 (San Diego, CA, USA; Abstract # 3800) and 2017 (Washington-DC, USA; Abstract # 2115).