Figure 6. LPA does not affect Taxol expulsion form MCF-7 cells. Panel A, approximately 26105 MCF-7 breast cancer cells were grown to confluence in 3.5 cm cell culture dishes. The medium was replaced with 1.5 ml RPMI 1640 medium containing 10% of charcoal-treated FBS with 50 nM Taxol containing 0.5 mCi [3H]Taxol. After incubation for 26 h, the medium was supplemented in some cases with 5 mM LPA or not as indicated and the incubation was continued for another 12 h. Media and cells were collected at the times indicated and Taxol was extracted. Results are expressed as the percentage of total radioactivity recovered in the cells and medium. Panel B, cells were treated and analyzed as above but after 26 h incubation, Taxol was removed and 5 mM LPA was added or not as indicated. The results are expressed as the percentage of the radioactivity added to the medium presented as mean 6 S.D. (where large enough to be shown) for three independent experiments. doi:10.1371/journal.pone.0020608.g006 from Taxol-induced arrest in G2/M and cell death, as we showed using our experimental protocols. In vivo, ATX expression increases the exposure of tumors to LPA and this is associated with increased tumor aggressiveness, angiogenesis and metastasis [22,23,24,44]. MCF-7 cells express mainly LPA2 [40] and, as expected, neither LPA1 and LPA3 antagonists or pertussis toxin blocked LPA antagonism towards Taxol in these cells [7]. LPA also enabled MDA-MB-468 cells to escape from Taxol-induced mitotic arrest and consequent cell death. These cells also have high LPA2 receptor expression [40], but whereas MCF-7 cells do not express caspase-3 [31], MDA- MB-468 cells express relatively high caspase-3 activity [32]. This probably explains why they displayed greater Taxol-induced cell death. LPA2 receptors are especially associated with increased development of breast and other cancers [25,45]. Activation of LPA2 receptors protects against adriamycin-induced caspase-3 cleavage and cell death [46]. This depends on the LPA2- dependent degradation of the pro-apoptotic protein, Siva-1 [46]. Our work describes another mechanism by which LPA can protect against Taxol-induced cell death, which involves displace- ment of Taxol from the polymerized tubulin fraction and escape from mitotic arrest. We also showed that LPA protects MDA-MB- 435 cells, which show relatively high LPA1 expression, from Lysophosphatidate Reverses G2/M Arrest by Taxol Taxol-induced death [40]. Consequently, the effects of LPA in causing Taxol resistance appear to occur in several cancer cell lines. We showed that LPA-induced PI3K activation decreases the Taxol-induced formation of ceramide, a proapoptotic lipid [7]. Now we showed that the role of LPA in displacement of Taxol from polymerized tubulin depends on the differential expression of LPA receptors (LPA 1/3 in MDA-MB-231 cells and LPA2 in MCF- 7 cells). In fact, Taxol treatment increases mRNA expression for LPA3 in MDA-MB-231 cells and decreased LPA2 mRNA expression in MCF-7 cells. We do not know the mechanisms by which Taxol effects these changes. Activation of several LPA receptors, including LPA1, LPA2 and LPA3 results in PI3K activation [47]. We propose that LPA-induced displacement of Taxol from polymerized tubulin is PI3K dependent based on studies with LY294002 and wortmannin where we see inhibition in the expected concentration ranges. We recognize that all inhibitors have off target effects, but these are different between LY294002 and wortmannin. Also, we used a casein kinase-2 inhibitor since LY294002 can inhibit this enzyme [38]. Casein kinase-2 can be activated in Wnt signaling, and it is also involved in cell cycle progression [48,49]. To investigate the possible role of casein kinase-2 in the LPA effect, we also used TBB (a selective inhibitor of casein kinase-2) and observed no effect on LPA-induced Taxol displacement from polymerized tubulin even at 100 mM TBB. Tumor cells are particularly responsive to the effects of ATX and LPA compared to non-transformed cells since they often express low LPP activities [6,50,51,52,53]. In addition, MCF-7 and MDA-MB- 435 cells exhibit very low expression of LPP1 and LPP3, and relatively high LPP2 compared non-transformed MCF10A breast epithelial cells (G. Venkatraman and D.N. Brindley, unpublished observations based on real-time RT-PCR). This distribution is opposite to that in non-transformed fibroblasts [54,55]. The LPPs have a dual function: the ecto-activities balance the action of ATX by decreasing extracellular LPA concentrations and thus receptor activation [20,21]. Increased LPP1 expression enhances LPA degradation by ovarian cancer cells, which inhibits cell proliferation and colony-forming activity, and produces a marked increase in apoptosis [52]. The intracellular activities of the LPPs decrease signaling downstream of the activation of LPA and tyrosine kinase receptors including the platelet-derived growth factor receptor [54,56]. Importantly, high LPP1 expression also decreases the ability of LPA to activate downstream signaling targets including ERK, Rac and Rho [54]. This appears to depend upon the expression of LPP1 within the cell and its ability to degrade a wide variety of bioactive lipid phosphates downstream of receptor activation [56]. Increased expression of LPP3 also decreases the growth, survival, and tumorigenesis of ovarian cancer cells [53]. By contrast, LPP2 has effects on cell signaling that are different from those of LPP1 and LPP3 since LPP2 expression specifically stimulates the progression of cells through the cell cycle [55]. We, therefore, conclude that many cancer cells may be particularly sensitive to the effects of LPA [6]. The LPA effects that we observe on mitosis in MCF-7 cells could, therefore, be higher than those in non-transformed cells. The present work establishes that the effects of ATX and LPA in antagonizing Taxol-induced death of breast cancer cells results from the PI3K-dependent displacement of Taxol from polymer- ized tubulin and escape from mitotic arrest. This is an unexpected observation that adds to our knowledge of how LPA signaling downstream of PI3K antagonizes Taxol action. Our work also provides further evidence that ATX and LPA could be an important source of chemo-resistance to the therapeutic use of Taxol in treating breast and other cancers. The roles of ATX and LPA in aggravating resistance to chemotherapy have received little PLoS ONE | www.plosone.org 8 May 2011 | Volume 6 | Issue 5 | e20608