Anticancer Activity of the Choline Kinase Inhibitor PL48 Is Due to Selective Disruption of Choline Metabolism and Transport Systems in Cancer Cell Lines

Abstract

A large number of different types of cancer have been shown to be associated with an abnormal metabolism of phosphatidylcholine (PC), the main component of eukaryotic cell membranes. Indeed, the overexpression of choline kinase α1 (ChoKα1), the enzyme that catalyses the bioconversion of choline to phosphocholine (PCho), has been found to associate with cell proliferation, oncogenic transformation and carcinogenesis. Hence, ChoKα1 has been described as a possible cancer therapeutic target. Moreover, the choline transporter CTL1 has been shown to be highly expressed in several tumour cell lines. In the present work, we evaluate the antiproliferative effect of PL48, a rationally designed inhibitor of ChoKα1, in MCF7 and HepG2 cell lines. In addition, we illustrate that the predominant mechanism of cellular choline uptake in these cells is mediated by the CTL1 choline transporter. A possible correlation between the inhibition of both choline uptake and ChoKα1 activity and cell proliferation in cancer cell lines is also highlighted. We conclude that the efficacy of this inhibitor on cell proliferation in both cell lines is closely correlated with its capability to block choline uptake and ChoKα1 activity, making both proteins potential targets in cancer therapy.

Keywords: cancer; choline kinase inhibitors; choline uptake; lipid metabolism.

Figure 1

Chemical structure of synthetic ChoKα1 inhibitor PL48.

Figure 2

(A) [Methyl-¹⁴C]choline uptake by HepG2 and MCF7 cells was assayed for 10 min over a concentration range from 1 to 1000 μM choline. Representative Eadie–Hofstee plots for [methyl-¹⁴C]choline uptake in HepG2 (B) and MCF7 (C). The kinetic constants (K_M and V_max) for transporter proteins were determined using equations from the Eadie–Hofstee plots obtained with low (1–36 μM) and high (60–1000 μM) choline substrate concentrations. Data are representative of two independent experiments performed in triplicate.

Figure 3

Effects of hemicholinium-3 (HC-3) (A,B) or tetraethylammonium (TEA) (C,D) on choline uptake in HepG2 and MCF7 cell lines. Choline uptake was assayed in cells treated for 10 min with increasing concentrations of inhibitors dissolved in sodium-free buffer (SFB). Data represent the mean ± SEM of two independent experiments conducted in triplicate. Results are normalised to their respective controls. * p < 0.05, ** p < 0.001.

Figure 4

Effects of PL48 inhibitor on (A) HepG2 and (B) MCF7 cell proliferation. Cells growing in the log phase were incubated with MEM (HepG2) or RPMI-1640 (MCF7) in the presence or absence of PL48 at concentrations of up to 10 μM for 24 or 48 h. Cell number was determined by crystal violet staining and expressed as a percentage of the control cells. These experiments were performed twice in triplicate. * p < 0.05, *** p < 0.0001.

Figure 5

Effects of PL48 on choline kinase (ChoK)α1 activity. ChoKα1 activity was determined by the incorporation rate of ¹⁴C from [methyl-¹⁴C]choline into phosphocholine (PCho) in the absence or presence of different PL48 concentrations. The results are expressed as the percentage of enzymatic activity compared with the control. * p < 0.05, ** p < 0.001, *** p < 0.0001.

Figure 6

Effect of the linker bioisosteric change in the inhibitory activity towards the ChoKα1. The IC₅₀ values of compounds (a–d) are taken from [21,41,42], (e) corresponds to PL48.

Figure 7

Effects of PL48 on choline uptake in HepG2 (A) and MCF7 (B) cancer cell lines. Choline uptake was assayed in cells treated for 10 min with increasing concentrations of inhibitor. Data represent the mean ± SEM of two independent experiments conducted in triplicate. Results are normalised to their respective controls. * p < 0.05, ** p < 0.001, *** p < 0.0001.

Figure 8

Effect of PL48 on CTL1 and ChoK protein levels in HepG2 and MCF7 cells. Cells were incubated without PL48 (control) or with 1 μM PL48 for 48 h. (A) Western blots show representative results of experiments repeated three times. (B) Protein levels in the samples were normalised to their respective β-actin levels and expressed as x-fold change compared with the corresponding control ratio (1.0). Data represent the mean ± SEM of three independent experiments. * p < 0.05.

Figure 9

Effects of PL48 treatment on choline uptake (A) and on total cellular activity of ChoK (B) in HepG2 and MCF7 cancer cell lines. Cells were incubated with the inhibitor for 48 h, and after its withdrawal, choline uptake and ChoKα activity were determined. Data represent the mean ± SEM of two independent experiments conducted in triplicate. Results are normalised to their respective controls. * p < 0.05, *** p < 0.0001.

Figure 10

Scheme showing the action of PL48.