sphingosine-phosphorylcholine and Pancreatic-Neoplasms

We investigate connections between single-cell mechanical properties and subcellular structural reorganization from biochemical factors in the context of two distinctly different human diseases: gastrointestinal tumor and malaria. Although the cell lineages and the biochemical links to pathogenesis are vastly different in these two cases, we compare and contrast chemomechanical pathways whereby intracellular structural rearrangements lead to global changes in mechanical deformability of the cell. This single-cell biomechanical response, in turn, seems to mediate cell mobility and thereby facilitates disease progression in situations where the elastic modulus increases or decreases due to membrane or cytoskeleton reorganization. We first present new experiments on elastic response and energy dissipation under repeated tensile loading of epithelial pancreatic cancer cells in force- or displacement-control. Energy dissipation from repeated stretching significantly increases and the cell's elastic modulus decreases after treatment of Panc-1 pancreatic cancer cells with sphingosylphosphorylcholine (SPC), a bioactive lipid that influences cancer metastasis. When the cell is treated instead with lysophosphatidic acid, which facilitates actin stress fiber formation, neither energy dissipation nor modulus is noticeably affected. Integrating recent studies with our new observations, we ascribe these trends to possible SPC-induced reorganization primarily of keratin network to perinuclear region of cell; the intermediate filament fraction of the cytoskeleton thus appears to dominate deformability of the epithelial cell. Possible consequences of these results to cell mobility and cancer metastasis are postulated. We then turn attention to progressive changes in mechanical properties of the human red blood cell (RBC) infected with the malaria parasite Plasmodium falciparum. We present, for the first time, continuous force-displacement curves obtained from in-vitro deformation of RBC with optical tweezers for different intracellular developmental stages of parasite. The shear modulus of RBC is found to increase up to 10-fold during parasite development, which is a noticeably greater effect than that from prior estimates. By integrating our new experimental results with published literature on deformability of Plasmodium-harbouring RBC, we examine the biochemical conditions mediating increases or decreases in modulus, and their implications for disease progression. Some

Topics: Animals; Biomechanical Phenomena; Cell Line, Tumor; Elasticity; Erythrocyte Deformability; Erythrocytes; Gastrointestinal Neoplasms; Humans; In Vitro Techniques; Lysophospholipids; Malaria; Pancreatic Neoplasms; Phosphorylcholine; Plasmodium falciparum; Sphingosine

Cell migration and invasion are largely dependent on the complex organization of the various cytoskeletal components. Whereas the role of actin filaments and microtubules in cell motility is well established, the role of intermediate filaments in this process is incompletely understood. Organization and structure of the keratin cytoskeleton, which consists of heteropolymers of at least one type 1 and one type 2 intermediate filament, are in part regulated by post-translational modifications. In particular, phosphorylation events influence the properties of the keratin network. Sphingosylphosphorylcholine (SPC) is a bioactive lipid with the exceptional ability to change the organization of the keratin cytoskeleton, leading to reorganization of keratin filaments, increased elasticity, and subsequently increased migration of epithelial tumor cells. Here we investigate the signaling pathways that mediate SPC-induced keratin reorganization and the role of keratin phosphorylation in this process. We establish that the MEK-ERK signaling cascade regulates both SPC-induced keratin phosphorylation and reorganization in human pancreatic and gastric cancer cells and identify Ser431 in keratin 8 as the crucial residue whose phosphorylation is required and sufficient to induce keratin reorganization and consequently enhanced migration of human epithelial tumor cells.

Topics: Cell Line, Tumor; Cell Movement; Cytoskeleton; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Humans; Intermediate Filaments; Keratin-8; MAP Kinase Kinase Kinases; Pancreatic Neoplasms; Phosphorylation; Phosphorylcholine; Protein Kinase Inhibitors; Serine; Signal Transduction; Sphingosine; Stomach Neoplasms

Sphingosylphosphorylcholine (SPC) is found at increased levels in the malignant ascites of tumor patients and induces perinuclear reorganization of keratin 8 (K8) filaments that contribute to the viscoelasticity of metastatic cancer cells. In this study, we investigated the role and molecular mechanisms of Tgase-2 in SPC-induced K8 phosphorylation and perinuclear reorganization in PANC-1 cells (PAN(WT)), and in PANC-1 cells that stably expressed shTgase-2 or Tgase-2 (PAN(shTg2) and PAN(Tg2)). SPC induces the expression of Tgase-2 in a time- and dose-dependent manner. Gene silencing of Tgase-2 or cystamine suppressed the SPC-induced phosphorylation and perinuclear reorganization of K8 and suppressed the SPC-induced migration of PANC-1 cells. An inhibitor of c-Jun N-terminal kinase (JNK), SP600125, suppressed the SPC-induced phosphorylation of serine 431 in K8 and keratin reorganization. Next, we examined the effect of Tgase-2 on JNK activation of serine 431 phosphorylation in K8. Tgase-2 gene silencing suppressed the expression of active form JNK (pJNK). Constitutive or tetracyclin-induced conditional expression of Tgase-2 increased the levels of pJNK. Tgase-2 was coimmunoprecipitated with K8 and JNK. In addition, K8 was coimmunoprecipitated with Tgase-2 and JNK. JNK was also coimmunoprecipitated with K8 and Tgase-2. Overall, these results suggest that Tgase-2 is involved in SPC-induced phosphorylation and perinuclear reorganization of K8 by activating JNK and forming a triple complex with K8 and JNK. Therefore, SPC-induced Tgase-2 might be a new target for modulating keratin reorganization, metastasis of cancer cells and JNK activation.

Topics: Anthracenes; Cell Line, Tumor; Cell Movement; Cystamine; Cytoskeleton; Gene Expression Regulation, Neoplastic; Gene Silencing; GTP-Binding Proteins; Humans; Immunoprecipitation; JNK Mitogen-Activated Protein Kinases; Keratin-8; MAP Kinase Signaling System; Pancreatic Neoplasms; Phosphorylation; Phosphorylcholine; Protein Glutamine gamma Glutamyltransferase 2; Protein Kinase Inhibitors; RNA, Small Interfering; Serine; Sphingosine; Transglutaminases

Sphingosylphosphorylcholine (SPC) is a naturally occurring bioactive lipid that is present in high density lipoproteins (HDL) particles and found at increased levels in blood and malignant ascites of patients with ovarian cancer. Here, we show that incubation of human epithelial tumour cells with SPC induces a perinuclear reorganization of intact keratin 8-18 filaments. This effect is specific for SPC, largely independent of F-actin and microtubules, and is accompanied by keratin phosphorylation. In vivo visco-elastic probing of single cancer cells demonstrates that SPC increases cellular elasticity. Accordingly, SPC stimulates migration of cells through size-limited pores in a more potent manner than lysophosphatidic acid (LPA). LPA induces actin stress fibre formation, but does not reorganize keratins in cancer cells and hence increases cellular stiffness. We propose that reorganization of keratin by SPC may facilitate biological phenomena that require a high degree of elasticity, such as squeezing of cells through membranous pores during metastasis.

Topics: Actin Cytoskeleton; Carcinoma; Cell Movement; Cell Size; Cytoskeleton; Elasticity; Fluorescent Antibody Technique; Humans; Keratins; Microscopy, Electron; Neoplasm Metastasis; Pancreatic Neoplasms; Phosphorylcholine; Sphingosine; Stress, Mechanical; Tumor Cells, Cultured

Sphingosylphosphorylcholine (SPC) has been shown to be a potent mitogen for Swiss 3T3 fibroblasts and also to be an inhibitor of cell growth of some cancer cells, suggesting cell-selective action of the lipid. We examined the effects of SPC, and its structurally-related sphingosine (SP), sphingosine 1-phosphate (S1-P) and membrane-permeable derivatives of ceramides on cell growth of four strains of human pancreatic cancer cells, MLA PaCa-2, PANC-1, PK-1 and PK-9. Under the reported conditions for SPC-induced stimulation of 3T3 fibroblasts, where cells were grown to confluency in the presence of 10% fetal bovine serum (FBS) in culture prior to experiments and insulin was supplemented in experimental culture, none of the agents tested stimulated DNA synthesis in MIA PaCa-2 cells and ceramide at high concentration even inhibited it. On the other hand, in reduced FBS concentration in preculture and in the absence of insulin in experimental culture, SP, S1-P and ceramides suppressed cell growth of all the cells tested including Swiss 3T3 fibroblasts. However, under these conditions, SPC inhibited three out of four species of pancreatic cancer cells but stimulated Swiss 3T3 fibroblasts in terms of both DNA synthesis and cell proliferation. Cell cycle analysis showed that SPC stimulated cell cycle progress from the G1 to the S phase in Swiss 3T3 fibroblasts but inhibited it in PANC-1 cells in reduced FBS concentrations. We suggest that extracellular SPC can inhibit cell growth of human pancreatic cancer cells through regulation of the cell cycle process depending upon both the cell species and environmental conditions.

Topics: 3T3 Cells; Animals; Cattle; Cell Culture Techniques; Cell Cycle; Cell Division; Ceramides; DNA; Humans; Lysophospholipids; Mice; Nucleic Acid Synthesis Inhibitors; Pancreatic Neoplasms; Phosphorylcholine; Serum Albumin; Sphingosine; Tumor Cells, Cultured