Accumulation of Palmitoylcarnitine and Its Effect on Pro-Inflammatory Pathways and Calcium Influx in Prostate Cancer

Abstract

Background: Acylcarnitines are intermediates of fatty acid oxidation and accumulate as a consequence of the metabolic dysfunction resulting from the insufficient integration between β-oxidation and the tricarboxylic acid (TCA) cycle. The aim of this study was to investigate whether acylcarnitines accumulate in prostate cancer tissue, and whether their biological actions could be similar to those of dihydrotestosterone (DHT), a structurally related compound associated with cancer development.

Methods: Levels of palmitoylcarnitine (palcar), a C16:00 acylcarnitine, were measured in prostate tissue using LC-MS/MS. The effect of palcar on inflammatory cytokines and calcium (Ca(2+) ) influx was investigated in in vitro models of prostate cancer.

Results: We observed a significantly higher level of palcar in prostate cancerous tissue compared to benign tissue. High levels of palcar have been associated with increased gene expression and secretion of the pro-inflammatory cytokine IL-6 in cancerous PC3 cells, compared to normal PNT1A cells. Furthermore, we found that high levels of palcar induced a rapid Ca(2+) influx in PC3 cells, but not in DU145, BPH-1, or PNT1A cells. This pattern of Ca(2+) influx was also observed in response to DHT. Through the use of whole genome arrays we demonstrated that PNT1A cells exposed to palcar or DHT have a similar biological response.

Conclusions: This study suggests that palcar might act as a potential mediator for prostate cancer progression through its effect on (i) pro-inflammatory pathways, (ii) Ca(2+) influx, and (iii) DHT-like effects. Further studies need to be undertaken to explore whether this class of compounds has different biological functions at physiological and pathological levels. Prostate 76:1326-1337, 2016. © 2016 The Authors. The Prostate published by Wiley Periodicals, Inc.

Keywords: acylcarnitines; dihydrotestosterone; interleukine-6 pathway; intracellular calcium signaling; metabolic disruption.

Figure 1

Concentration of palcar in human prostate tissue. Palcar concentration (μM) was measured by LC/MS‐MS in benign (n = 10) and cancerous (n = 10) tissue samples. Mean values are shown as “+.” Statistical analysis was performed with unpaired t‐test (P = 0.015).

Figure 2

Effect of palcar on the pro‐inflammatory cytokine IL‐6. (A) IL‐6 secretion in PNT1A and PC3 cells in response to palcar as measured in culture medium using human IL‐6 ELISA kit. (B) IL‐6 gene expression in PNT1A and PC3 cells in response to palcar. IL‐6 gene expression was quantified using real time RT‐PCR and housekeeping 18 S gene was used for normalisation. (C) IL‐6 secretion in response to palcar and LPC in PC3 cells. Statistical analysis was performed using a one way ANOVA followed by Bonferroni's multiple comparison post‐test (***P < 0.001, **P ≤ 0.01, *P < 0.05 vs. untreated cells).

Figure 3

Effect of palcar on cell growth. Viability assay in PNT1A and PC3 cells in response to palcar treatment (0–100 μM) for 24 hr. Results represent means ± SD of six biological replicates (*P ≤ 0.05, **P ≤ 0.01; one way ANOVA followed by Tukey's multiple comparison post‐test).

Figure 4

Effect of palcar on Ca²⁺ influx. FURA‐loaded PNT1A (A), BPH‐1 (B), DU145 (C), and PC3 (D) cells were injected with DMSO (control), histamine (10–20 μM) or palcar (5–50 μM) 50 sec after starting Ca²⁺ measurement. The ratio of fluorescence emission (510 nm), excited at both 340 nm and 380 nm, indicated the [Ca²⁺]i. Data represent mean values of three independent experiments. The statistical analysis was performed with mixed effect model test using the Genstat software. Ratio of fluorescence after histamine injection was significantly different from control in all four cell lines (P ≤ 0.01). Ratio of fluorescence after palcar injection was not significantly different from the control in PNT1A (P = 0.110), BPH‐1 (P = 0.521), and DU145 cells (P = 0.110). In PC3 cells, palcar‐induced Ca²⁺ influx was significantly different from the control (P = 0.011), but there was no significant difference within the different concentrations of palcar (P = 0.839).

Figure 5

Effect of DHT on Ca²⁺ influx. FURA‐loaded PNT1A (A), BPH‐1 (B), DU145 (C), and PC3 (D) cells were injected with DMSO (control), 20 μM histamine or DHT (0.1‐1μM) 50 sec after starting Ca²⁺ measurement. The ratio of fluorescence emission (510 nm), excited at both 340 nm and 380 nm, indicated the [Ca²⁺]i. (E,F) The maximum fluorescence ratio was normalized to the baseline level at 50 sec of each sample injection. Data represent means ± SD of three independent experiments. Statistical analysis was performed using two ways ANOVA followed by Bonferroni's post‐test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 vs. untreated cells).

Figure 6

Venn diagram of genes common across palcar and DHT treatment in PNT1A cells. Cells were treated with palcar (0–5 μM) and 10 nM DHT for 8 hr. A, B, and C represent the genes common across 10 nM DHT and 50 nM, 500 nM, and 5 μM palcar, respectively.