Activation of phosphatidylcholine cycle enzymes in human epithelial ovarian cancer cells

Abstract

Altered phosphatidylcholine (PC) metabolism in epithelial ovarian cancer (EOC) could provide choline-based imaging approaches as powerful tools to improve diagnosis and identify new therapeutic targets. The increase in the major choline-containing metabolite phosphocholine (PCho) in EOC compared with normal and nontumoral immortalized counterparts (EONT) may derive from (a) enhanced choline transport and choline kinase (ChoK)-mediated phosphorylation, (b) increased PC-specific phospholipase C (PC-plc) activity, and (c) increased intracellular choline production by PC deacylation plus glycerophosphocholine-phosphodiesterase (GPC-pd) or by phospholipase D (pld)-mediated PC catabolism followed by choline phosphorylation. Biochemical, protein, and mRNA expression analyses showed that the most relevant changes in EOC cells were (a) 12-fold to 25-fold ChoK activation, consistent with higher protein content and increased ChoKalpha (but not ChoKbeta) mRNA expression levels; and (b) 5-fold to 17-fold PC-plc activation, consistent with higher, previously reported, protein expression. PC-plc inhibition by tricyclodecan-9-yl-potassium xanthate (D609) in OVCAR3 and SKOV3 cancer cells induced a 30% to 40% reduction of PCho content and blocked cell proliferation. More limited and variable sources of PCho could derive, in some EOC cells, from 2-fold to 4-fold activation of pld or GPC-pd. Phospholipase A2 activity and isoform expression levels were lower or unchanged in EOC compared with EONT cells. Increased ChoKalpha mRNA, as well as ChoK and PC-plc protein expression, were also detected in surgical specimens isolated from patients with EOC. Overall, we showed that the elevated PCho pool detected in EOC cells primarily resulted from upregulation/activation of ChoK and PC-plc involved in PC biosynthesis and degradation, respectively.

Figure 1. Phosphatidylcholine metabolites

A, The phosphatidylcholine (PC) cycle. Metabolites: CDP-Cho, cytidine diphosphate choline; Cho, choline; DAG, diacylglycerol; FFA, free fatty acid; G3P, sn-glycerol-3-phosphate; GPC, glycerophosphocholine; LPC, lysophosphatidylcholine; PA, phosphatidate; PCho, phosphocholine. Enzymes: Kennedy pathway: Chok, choline kinase (EC 2.7.1.32); ct, cytidylyltransferase (EC 2.7.7.15); pct, phosphocholine transferase (EC 2.7.8.2). Headgroup hydrolysis pathways: plc, phospholipase C (EC 3.1.4.3); pld, phospholipase D (EC 3.1.4.4). Deacylation pathway: plA1, phospholipase A₁ (EC 3.1.1.32); plA2, phospholipase A₂ (EC 3.1.1.4); lpl, lysophospholipase (EC 3.1.1.5); pd, glycerophosphocholine phosphodiesterase (EC 3.1.4.2). B, Quantification of aqueous metabolites (mean values ± SEM) in EOC and EONT cells [OSE (n=2); IOSE (n=4); hTERT (n=15)]). C, Relative PC contents (± maximum deviation for n=2; ± SD for n≥3) in EOC normalized to hTERT cells. In parenthesis, number of independent experiments.

Figure 2. Choline transporters and Kennedy pathway enzymes in EOC and EONT cells

A, Microarray analysis of choline transporters (dotted line, sensitivity threshold). * Significance of differences: P ≤ 0.015; B, RT-qPCR analysis of enzymes’ expression in EOC (OSE cells used as internal calibrator; horizontal line at quantification level = 1). A representative experiment of three performed is shown. For each gene the mean value (± SD) of the analyzed EOC cell lines is reported.

Figure 3. ChoK protein expression and activity

A, ChoK protein expression by Western blotting, as detected by the custom-made rabbit polyclonal antibody. B, Chok densitometric evaluation referred to GAPDH (mean ± SEM, n≥4. * = P<0.05; ** = P<0.01, relative to EONT by one-way ANOVA. C, Chok activity(mean ± SD, n ≥3).

Figure 4. Enzymes of pld-mediated and deacylation pathways in EOC and EONT cells

A, pld* and GPC-pd activity(mean value ± SD, n ≥6). B, RT-qPCR analysis of PLD1 and PLD2 in EOC cells (OSE used as internal calibrator; horizontal line at quantification level = 1). A representative experiment of three performed is shown. For each gene mean value ± SD is reported. C, Relative 2-Lyso-C6PC formation (± SD, n ≥3) in EOC and hTERT cell lysates at 1h of exposure to C6PC. D, Gene expression of plA1 and of plA2 isoforms. The dotted line represents sensitivity threshold.

Figure 5. Activation of PC-plc in EOC cells

A, PC-plc activity (± SD, n ≥4). B, PC-plc activity and C, PCho content in OVCAR3 cells following incubation in absence (CTRL) or presence of D609 (53 μg/mL, 24 h). Inset: ¹H NMR tCho profile in aqueous extracts. D, OVCAR3 cell growth in complete medium in absence (CTRL) or presence of D609 and in serum-deprived medium (-FCS). Error bars, ± SD, n ≥ 5 (Student’s t-test * P<0.05; ***P< 0.001).

Figure 6. ChoK and PC-plc detection in EOC surgical specimens

A, Gene expression analysis of ChoKα in OSE cells, surgical EOC specimens and EOC cell lines. Horizontal bars represent relative median expression for each group. Western blot analyses of total lysates of EOC surgical specimens and hTERT cells are shown for ChoK protein expression, as detected by the commercially available goat polyclonal antibody (B) and PC-plc protein expression (C). Actin, internal loading control.