Novel pleiotropic effects of bioactive phospholipids in human lung cancer metastasis

Abstract

We previously proposed that one of the unwanted side effects of chemotherapy and radiotherapy is the increase in several peptide- and non-peptide based chemoattractants in damaged tissues, leading to induction of a prometastatic microenvironment for remaining cancer cells. Herein, we turned out our attention to a potential role of bioactive phospholipids (BphsLs), such as sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), lysophosphatidylcholine (LPC), and lysophosphatidic acid (LPA) in lung cancer (LC) metastasis. We report that LC cells express several functional BphL receptors (for S1P, LPC, and LPA) as well as several enzymes involved in their metabolism and that BphsLs are potent chemokinetic and adhesion factors for these cells. We also demonstrate for the first time the novel role of C1P as a prometastatic factor in LC cells. In addition to their chemokinetic activities, BphsLs also sensitize or prime the chemotactic responsiveness of LC cells to known prometastatic factors such as hepatocyte growth factor/scatter factor (HGF/SF). Thus, for the first time we demonstrate a prometastatic effect that is based on the priming of a cell's responsiveness to chemotactic factors by chemokinetic factors. To our surprise, none of the bioactive lipids induced proliferation of LC cells or ameliorated toxic effects of vincristine treatment. Interestingly, BphsLs increase adhesion of LC cells to bone marrow-derived stromal cells and stimulate these cells to release ExNs, which additionally increase LC cell motility. In conclusion, our results show that BphsLs are important modulators of prometastatic environment. Therefore, their inhibitors could be considered as potential anti-metastatic drug candidates to be included as a part of post radio- and/or chemo- therapy treatment.

Keywords: HGF/SF; bioactive phospholipids; lung cancer; metastasis; priming.

Figure 1. Expression of receptors and enzymes involved in BphsL signaling and synthesis

RT-PCR analysis of the expression of S1P, LPA, and LPC receptors as well as enzymes involved in the synthesis and degradation of the tested BphsLs.

Figure 2. BsphL receptors in lung cancer cells are functional

Phosphorylation of p42/44 MAPK and AKT in human NSCLC (Panel A) and SCLC (Panel B) cell lines stimulated for 5 min with S1P (1 μM), C1P (0.5 μM), or LPA (0.1 μM) or stimulated for 2 h with LPC (20 μM). Representative WB (left) and densytometric analysis (right graphs) from three independent analysis are presented *p ≤ 0.05.

Figure 3. BphsLs induce migration of LC cells

(Panel A) Responsiveness of NSCLC and SCLC cells to gradients of S1P (1 μM), C1P (0.5 μM), LPA (0.1 μM), or LPC (20 μM) compared with the known chemoattractant HGF employed at supraphysiological (H; 10 ng/ml) and physiological (L; 0.3 ng/ml) concentrations. The experiment was performed at least twice in duplicate, *p ≤ 0.05. (Panel B) Chemotaxis versus chemokinesis analysis by comparing the migration of A549 cells in response to BphsL gradients (BphsL in the lower chamber) or no gradient (BphsL in both chambers). The experiment was performed twice in duplicate, *p ≤ 0.05. (Panel C) Adhesion of LC cells to fibronectin. The cells were unstimulated (control) or stimulated with S1P (1 μM), C1P (0.5 μM), LPA (0.1 μM), or LPC (20 μM) for 10 min. The number of adherent cells was measured by microscopic analysis. Data from two separate experiments are pooled together, and means ± SD are shown, *p ≤ 0.05.

Figure 4. Pretreatment of A549 cells with BphsLs regulates their response to HGF

The effect of pretreatment of A549 cells with S1P (Panels A and B), C1P (Panels C and D), LPA (Panels E and F), or LPC (Panels G and H) on the migration of cells in response to HGF employed at supraphysiological (Panel A, C, E, and G) or physiological (Panels B, D, F, and H) concentrations. The experiment was performed at least twice in duplicate, *p ≤ 0.05.

Figure 5. BphsLs regulate LC–stromal interactions

(Panel A) The adhesion of A549 cells to human stromal cells. LC cells stained with calcein as well as stromal cells were stimulated with S1P (1 μM), C1P (0.5 μM), LPA (0.1 μM ), or LPC (20 μM) for 30 min. After a 20-minute incubation, non-adherent cells were removed, and adherent cells counted under a fluorescence microscope. Data from two separate experiments are pooled together, and means ± SD are shown, *p ≤ 0.05. Lower panel includes representative pictures of calcein-labeld A549 cells that adhered to stroma cells after stimulation (or not) with different BphsLs. (Panel B) The level of ATP in conditioned medium from stromal cells stimulated for 1 min with S1P, C1P, LPA, or LPC. Data from two separate experiments are pooled together, and means ± SD are shown, *p ≤ 0.05.

Figure 6. BphsL levels create a prometastatic microenvironment in irradiated organs

(Panel A) Conditioned media (CM) from irradiated BM, liver, and lung cells enhance the migration of A549 cell lines across Transwell membranes. The results from two independent experiments are shown as means ± SD. *p ≤ 0.05 compared with the control (CM from cells from untreated animals). (Panel B) The effect of pertussis toxin (PTx, 1 μg/ml), VPC32183 (10 μM), VPC23019 (20 μM), and the autotaxin inhibitor S32826 (ATX inh, 10 μM) on the migration of A549 cells in response to CM from irradiated BM. The results from two independent experiments are shown as means ± SD. *p ≤ 0.05 compared with the control (CM from cells from untreated animals). Migration in response to RPMI is shown as reference.

Figure 7. Schematic representation of the role of BphsLs, ExNs, and chemotactic peptides in the regulation of the metastasis of lung cells as a side effect of radio- and/or chemo-therapy