Lysophosphatidate Reverses G2/M Arrest by Taxol Figure 2. Lysophosphatidate releases the cells from G2/M phase. Cells were preincubated with 50 nM Taxol for 24 h and cells passing through S-phase were labeled for 2 h with BrdU. Cells were then incubated for 12 h with 10% delipidated serum in the presence or absence of 50 nM Taxol or 5 mM LPA (Protocol B). The progression of cells treated with Taxol alone or Taxol and LPA through S, G2/M and G1 phases of the cell cycle (Panels B and C, respectively) was quantified by FACS analysis (Panel A). DNA histograms were composed with CellQuest software. The histogram for time 0 for all treatments shows the same results, repeated for clarity of the figure. Results are means 6 SD for three independent experiments. doi:10.1371/journal.pone.0020608.g002 this effect (p,0.05). LPA also facilitated mitosis (p,0.05) and the production of G1 mononucleated cells regardless of whether Taxol was maintained or removed (Fig. 4C). In addition, LPA increased the percentage of multinucleated cells (Fig. 4D). This latter result indicates that some cells escaped Taxol-induced mitotic arrest in the absence of cytokinesis. After incubating with Taxol for 26 h, 1363% of the remaining cells were dead or dying as identified because of their apoptotic or necrotic morphology (Fig. 3B). Incubating the cells for a further 12 h in Taxol increased (p,0.05) the percentage of dead or dying cells to 2464% (p,0.01) and this value was similar when Taxol was removed. LPA decreased (p,0.05) the percentage of dead or dying cells regardless of whether Taxol was maintained or removed during the final 12 h of incubation (Fig. 4E). We also used MDA-MB-468 cells to establish if the effects of LPA on Taxol-induced mitotic arrest and cell death occur in other breast cancer cells. Treatment for 26 h with 50 nM Taxol caused about 4665% to accumulate in mitosis (Fig. 5A). We then chose to incubated these cells for 6 or 12 h with LPA since this was where the major effects of LPA were observed in MCF-7 cells (Fig. 4). Incubating MDA-MB-468 cells with LPA in the presence of Taxol for 12 h decreased (p,0.05) the percentage of cells that were in mitosis from 5366% with Taxol alone to about 3265% (Fig. 5A). The percentage of cells with abnormal spindles increased with time of incubation, whereas LPA reversed this effect (p,0.05) (Fig. 5B). LPA also increased (p,0.05) the percentage of mononucleated cells in G1 from 1464% to 2063% and 2666% after 6 and 12 h, respectively (Fig. 5C). LPA also significantly increased the percentage of multinucleated cells after 6 and 12 h (Fig. 5D). By contrast, continued treatment with Taxol in the absence of LPA decreased the proportion of mononucleated and multinucleated cells. Incubation with Taxol for the initial 26 h of the experiment induced significantly higher killing of MDA- MB-468 cells compared to MCF-7 cells (2767% compared to 1363%) (Figs 4E and 5E). An additional incubation of MDA-MB- 468 cells for 12 h with Taxol increased the percentage of dead and dying cells to 3864%. LPA prevented the increase in the percentage of dead and dying cells and this percentage stayed at 2465% after the 12 h incubation. LPA does not increase the expulsion of Taxol from MCF-7 cells or increased Taxol metabolism, but it does cause dissociation of Taxol from the tubulin polymer compartment. The next experiments were performed to investigate the mechanisms of how LPA decreases Taxol-induced cell death through the release of breast cancer cells from mitotic arrest. Chemo-resistance is already known to result partially through the activation of drug transporters [33], which can cause cross- resistance to other chemotherapeutic agents [34]. We, therefore, hypothesized that LPA decreases Taxol-induced accumulation of the cells in G2/M [7] by stimulating Taxol efflux through ABCB1 (multidrug resistance gene 1), p-glycoprotein (ABCC1) or ABCG2, since these transporters account for a major portion of drug resistance in human tumor cells [35]. MCF-7 cells express high levels of ABCG2, less ABCC1 and no ABCB1 [36]. To investigate whether LPA stimulates the export of Taxol, MCF-7 cells were pretreated for 26 h with 50 nM [3H]Taxol to PLoS ONE | www.plosone.org 4 May 2011 | Volume 6 | Issue 5 | e20608