Profile of phospholipase A2 activity in subcellular fractions and lamellar bodies of developing, neonatal and adult rabbit lung. Correlation with intracellular levels of disaturated phosphatidylcholine

Phospholipase A2 activity was determined in subcellular fractions and lamellar bodies of fetal, neonatal and adult rabbit lungs. Specific activity in most fractions decreased from the 24th to the 28th day of gestation. All fractions except the mitochondrial and the nuclear fractions exhibited a sharp increase in activity in the newborn lung. Specific activity in the adult lung generally declined in comparison to neonatal values. During gestation total enzyme activity per gram of lung was concentrated in the cytosolic fraction. With the exception of the lamellar body fraction, the total content of phospholipase A2 activity increased dramatically in all fractions from the neonatal lung. The lamellar body fractions displayed both low specific activity and low total enzyme activity during gestation. Specific activity increased dramatically in the neonatal and adult lung but still accounted for only a small fraction of the activity in comparison to the other subcellular fractions. The subcellular content of disaturated phosphatidylcholine (PC) appeared to correlate well with the activity of phospholipase A2 in the neonatal mitochondrial, microsomal and cytosolic fractions. Since decreasing prenatal enzyme levels are associated with increasing disaturated PC content, the alkaline and calcium-dependent phospholipase A2 may not be directly involved in disaturated PC synthesis in the fetus. However, postnatally, the correlation between the pattern of production of disaturated PC and the activity of the phospholipase A2 indicates a role for this enzyme in surfactant-related disaturated PC synthesis.     

Ca2+.Calmodulin-dependent release of arachidonic acid for renal medullary prostaglandin synthesis. Evidence for involvement of phospholipases A2 and C

The present study examined (a) the source of arachidonic acid for Ca2+-stimulated renal inner medullary prostaglandin synthesis, (b) the Ca2+-dependence of enzymes of the phospholipase A2 and C pathways, and (c) the role of calmodulin in these Ca2+ actions. Ca2+ plus the ionophore A23187 stimulated (2-4-fold) release of labeled arachidonate, diglyceride, prostaglandin E2 or F2 alpha from inner medullary slices with a concomitant fall in labeled phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide hydrochloride (W-7) (10-100 microM) abolished or suppressed Ca++-stimulated immunoreactive prostaglandin E, labeled arachidonate and prostaglandin release, and the fall in labeled phospholipids but did not suppress labeled diglyceride or inositol accumulation. Studies in subcellular fractions demonstrated a particulate phospholipase A2 activity and a phosphatidylinositol-specific phospholipase C activity which was predominantly soluble (80%). W-7 or trifluoperazine (25 microM) abolished Ca2+-stimulated phospholipase A2 activity and particulate phospholipase C activity but were without effect on soluble phospholipase C. W-7 (100 microM) was without effect on Ca2+-stimulated diglyceride lipase and phosphatidic acid-specific phospholipase A2 activities. Hypertonic urea at concentrations that pertain in the inner medulla of hydropenic rats in vivo inhibited Ca2+-induced increases in labeled arachidonate release and immunoreactive prostaglandin E in slice incubates and Ca2+-responsive phospholipase C and A2. The results are consistent with the involvement of phospholipase A2, C, or both in the Ca2+ (+A23187)-stimulated release of free arachidonate for prostaglandin synthesis and support a role for calmodulin in Ca2+ activation of phospholipase A2 and particulate phospholipase C.     

Interleukin-1 beta and transforming growth factor-beta 2 enhance cytosolic high-molecular-mass phospholipase A2 activity and induce prostaglandin E2 formation in rat mesangial cells.

Interleukin-1 beta induces gene expression and secretion of group-II phospholipase A2 and release of prostaglandin E2 from rat mesangial cells. The interleukin-1 beta-induced synthesis of group-II phospholipase A2 is prevented by transforming growth factor-beta 2, whereas transforming growth factor-beta 2 potentiated the interleukin-1 beta-evoked prostaglandin E2 production. Transforming growth factor-beta 2 itself did not induce synthesis of group-II phospholipase A2, although it stimulated prostaglandin E2 formation. Here we describe the effect of interleukin-1 beta and transforming growth factor-beta 2 on a cytosolic phospholipase A2 activity and prostaglandin E2 formation in rat mesangial cells. Based on the resistance to dithiothreitol and migration profiles on a Mono-Q anion-exchange column and a Superose 12 gel-filtration column, the cytosolic phospholipase A2 activity was assigned to a high-molecular-mass phospholipase A2. Measured with 1-stearoyl-2-[1-14C]arachidonoylglycero-phosphocholine as substrate, both interleukin-1 beta and transforming growth factor-beta 2 enhanced the high-molecular-mass phospholipase A2 activity. The stimulation of rat mesangial cells with interleukin-1 beta and transforming growth factor-beta 2 was time- and dose-dependent with maximal cytosolic phospholipase A2 activities at 10 nM and at 10 ng/ml respectively, after 24 h of stimulation. Under these conditions, interleukin-1 beta and transforming growth factor-beta 2 enhanced the cytosolic phospholipase A2 activity 2.2 +/- 0.6-fold and 2.5 +/- 0.6-fold, respectively. These results strongly suggest that an enhanced cytosolic high-molecular-mass phospholipase A2 activity is involved in the formation of prostaglandin E2 mediated by transforming growth factor-beta 2. Whether interleukin-1 beta induced group-II phospholipase A2 and/or interleukin-1 beta-enhanced cytosolic phospholipase A2 activity is involved in prostaglandin E2 formation in rat mesangial cells is discussed.     

Phospholipase A activity in the skin. Modulators of arachidonic acid release from phosphatidylcholine.

The distribution of the hydrolysis of 1-acyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine and the simultaneous biosynthesis of prostaglandins by subcellular fractions from human and rat skin membrane preparations were determined. The phospholipase A2 activity was distributed among the subcellular particulate preparations with the highest specific activity in the 105000g particulate fraction. The activity was optimal at pH 7.5 in the presence of 1.0 mM-CaCl2 and was inhibited by EDTA. The hydrolysis of phosphatidylcholine by the skin 105000g particulate fraction was inhibited by cortisol and triamcinolone acetonide and it was stimulated by histamine, bradykinin, retinoic acid and cholera enterotoxin (freeze-dried Vibrio cholerae). Furthermore hydrolysis of phosphatidylcholine by the skin phospholipase A was also enhanced by low concentrations of prostaglandin E2 and prostaglandin F2 alpha. These last results suggest that the amplication of the hydrolysis of phosphatidylcholine by prostaglandin E2 and prostaglandin F2 alpha, with the consequent release of arachidonic acid (the substrate of prostaglandin synthesis) is likely a positive-feedback regulation of the arachidonic acid-prostaglandin cascade.     

Arachidonic acid metabolism by adult human osteoblast-like cells exhibits sexually dimorphic characteristics

The eicosanoids, including prostaglandin E₂ (PGE₂) and other bioactive arachidonic acid metabolites, are important local mediators of bone remodeling. Presumably, the limited or excessive synthesis of the eicosanoids could compromise bone homeostasis. We have noted that the stimulated release of arachidonic acid by adult male donor derived human osteoblast-like (hOB) cells exceeded the stimulated release measured for female-derived hOB cells by 1.5-fold. Assays of PGE₂ biosynthesis by cytokine-stimulated hOB cells also demonstrated a sex-linked difference, such that male hOB cell PGE₂ production exceeded female cell production by 1.6–2.2-fold. The calcium-dependent cytoplasmic phospholipase A₂ activity in subcellular fractions prepared from hOB cell homogenates was higher in both the cytosolic (1.6-fold) and particulate (1.5-fold) fractions from the male cells than in those prepared from female hOB cells, suggesting a molecular basis for the observed sexually dimorphic characteristics related to arachidonic acid metabolism by hOB cells. The relatively limited capacity of the female cells may limit needed intracellular and intercellular signaling during bone remodeling, thereby contributing to the development of bone pathology. J. Cell. Biochem. 71:74–81, 1998. © 1998 Wiley-Liss, Inc.     

Regulation of phosphatidylcholine biosynthesis in mammalian cells. II. Effects of phospholipase C treatment on the activity and subcellular distribution of CTP:phosphocholine cytidylyltransferase in Chinese hamster ovary and LM cell lines

CTP:phosphocholine cytidylyltransferase was located in both the cytosolic and particulate fractions from Chinese hamster ovary cells. The activity of the cytosolic form of the enzyme was greatly enhanced by incubation with sonicated preparations of several different lipids, although incubations with either phosphatidylcholine or 1,2-sn-diolein did not increase activity. The activation of the cytidylyltransferase in Chinese hamster ovary cells treated with phospholipase C from Clostridium perfringens occurred with a concomitant shift in the subcellular distribution of the enzyme from cytosolic to particulate fractions. This shift was rapid and did not require protein synthesis. Removal of phospholipase C from the cell cultures resulted in a return to basal levels of incorporation of [3H]choline into phosphatidylcholine, a decrease in the activity of cytidylyltransferase, and a loss of the membrane-bound form of the enzyme. Similar experiments with LM cells, which are resistant to exogenous phospholipase C, showed no change in subcellular distribution of cytidylyltransferase, suggesting that the activation of CTP:phosphocholine cytidylyltransferase required a change in membrane phospholipid composition. The results presented are discussed in terms of a mechanism of regulation of phosphatidylcholine production involving monitoring of membrane phospholipid composition.     

Intracellular actions of group IIA secreted phospholipase A2 and group IVA cytosolic phospholipase A2 contribute to arachidonic acid release and prostaglandin production in rat gastric mucosal cells and transfected human embryonic kidney cells

Gastric epithelial cells liberate prostaglandin E(2) in response to cytokines as part of the process of healing of gastric lesions. Treatment of the rat gastric epithelial cell line RGM1 with transforming growth factor-alpha and interleukin-1beta leads to synergistic release of arachidonate and production of prostaglandin E(2). Results with highly specific and potent phospholipase A(2) inhibitors and with small interfering RNA show that cytosolic phospholipase A(2)-alpha and group IIA secreted phospholipase A(2) contribute to arachidonate release from cytokine-stimulated RGM1 cells. In the late phase of arachidonate release, group IIA secreted phospholipase A(2) is induced (detected at the mRNA and protein levels), and the action of cytosolic phospholipase A(2)-alpha is required for this induction. Results with RGM1 cells and group IIA secreted phospholipase A(2)-transfected HEK293 cells show that the group IIA phospholipase acts prior to externalization from the cells. RGM1 cells also express group XIIA secreted phospholipase A(2), but this enzyme is not regulated by cytokines nor does it contribute to arachidonate release. The other eight secreted phospholipases A(2) were not detected in RGM1 cells at the mRNA level. These results clearly show that cytosolic and group IIA secreted phospholipases A(2) work together to liberate arachidonate from RGM1 cell phospholipids in response to cytokines.     

Luteal phospholipase A2 activity increases during functional and structural luteolysis in pregnant rats.

We determined cytosolic phospholipase A2 activity of the corpus luteum during luteolysis in pregnant and post-partum rats. Phospholipase A2 activity and its metabolite prostaglandin F2alpha in the corpus luteum remarkably increased just before parturition and further rose transiently during post-partum structural luteolysis. The absence of a pups' suckling stimulus delayed corpus luteum involution, being associated with an altered fluctuation in phospholipase A2 activity and depressed prostaglandin F2alpha levels. Exogenous prolactin had a reversal effect. Pharmacological and immunochemical characterization suggests multiple isoforms of phospholipase A2 in a pregnant corpus luteum. These results show the increased phospholipase A2 activity and its possible implication in luteolysis in pregnant rats.     

Characterization of the major phosphoinositide-specific phospholipase C of human amnion

Most of the phosphoinositide-specific phospholipase C activity in human amnion at term was found to be attributable to a single isoform (Mr 85,000). Phospholipase C purified from amnion catalyzed the calcium-dependent hydrolysis of both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate. The high phospholipase C activity of amnion cells isolated at 38-41 weeks of gestation declined greater than 80% during the initial 2-5 days of culture to values characteristic of amnion tissue in early gestation. Activities of phospholipase A2 and phosphatidylinositol synthase remained essentially unaltered during this period of culture. Loss of phospholipase C activity was apparently due neither to the appearance of an inhibitor nor to the loss of an activator and most likely reflected a decrease in the amount of enzyme in amnion cells. Basal production of prostaglandin E2 (PGE2) by amnion cells also declined greatly during the period of loss of phospholipase C activity. Involvement of phospholipase C in the regulation of amnion prostaglandin production was also supported by the finding that the phospholipase C inhibitor, U-73122, potently inhibited amnion cell PGE2 production. In contrast, vasopressin, which appears to stimulate prostaglandin production in amnion cells by a phospholipase C-dependent mechanism, was equipotent in stimulating PGE2 production by amnion cells on Day 2 and Day 5 of culture, even though phospholipase C activity had declined by more than 75%. Furthermore, epidermal growth factor stimulation of PGE2 production by amnion cells appeared to be largely attributable to an increase in prostaglandin H synthase activity and did not involve an increase in phospholipase C activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subcellular localization of the phospholipases A of rat heart: evidence for a cytosolic phospholipase A1

During myocardial ischemia increased levels of lysoglycerophospholipids have been reported which may be deleterious to myocardial function. Phospholipases are presumed to be important in the regulation of this process. To further quantify and characterize the activity of heart phospholipases, we carried out a systematic analysis of phospholipase A activity in rat heart subcellular fractions isolated by the method of Palmer et al. (J. Biol. Chem. 1972. 262: 8731-8739). Neutral phospholipase A was recovered predominately in the cytosolic (soluble) fraction which represented 46% of recovered activity, while the microsomal and subsarcolemmal mitochondrial fractions represented 15% and 12% of the total recovered activity, respectively. Cytosolic phospholipase A differed from the two principal membrane-bound phospholipases A in its pH dependence and apparent Km for substrate. The cytosolic enzyme had a Km (apparent) for dioleoylphosphatidylcholine of 0.07 mM versus 0.28-0.33 mM for the membrane-associated phospholipases A. Acid phospholipase A activity had a subcellular distribution consistent with a lysosomal localization. Lysophospholipase was found principally in the cytosolic, microsomal, and the subsarcolemmal and interfibrillar mitochondrial fractions where it represented 46, 17, 6.3, and 6.9% of the recovered activity, respectively. The positional specificity of the respective phospholipases was assessed. This analysis was complicated by the fact that in heart, lysophospholipase has an observed Vmax 3.6- to 4.5-fold greater than that of phospholipase A in the various subcellular fractions. Equations were derived to obtain corrected values for the activity of phospholipases A1 and A2. Using this method we found that the cytosolic and lysosomal fractions contained phospholipase A1, while the mitochondrial fractions contained primarily phospholipase A2. In heart microsomes, the positional specificity of phospholipase A could not be determined because lysophospholipase activity was very high and lysophosphatidylcholine did not accumulate.     

Identification of type-2 phosphatidic acid phosphohydrolase (PAPH-2) in neutrophil plasma membranes

Plasma membrane phosphatidic acid phosphohydrolase (PAPH) plays an important role in signal transduction by converting phosphatidic acid to diacylglycerol. PAPH-2, a Mg²⁺-independent, detergent-dependent enzyme involved in cellular signal transduction, is reportedly absent from the plasma membranes of neutrophilic leukocytes, a cell that responds to metabolic stimulation with abundant phospholipase -dependent diacylglycerol generation. The present study was designed to resolve this discrepancy, focusing on the influence of cellular disruption techniques, detergenta availability and cation sensitivity on the apparent distribution of PAPH in neutrophil sub-cellular fractions. The results clearly indicate the presence of two distinct types of PAPH within the particulate and cytosolic fractions of disrupted cells. Unlike the cytosolic enzyme, the particulate enzyme was not potentiated by magnesium and was strongly detergent-dependent. The soluble and particulate enzymes displayed dissimilar pH profiles. Separation of neutrophil particulate material into fractions rich in plasma membranes, specific granules and azurophilic granules by high speed discontinuous density gradient centrifugation revealed that the majority of the particulate activity was confined to plasma membranes. This activity was not inhibited by pretreatment with n-ethyl-maleimide in concentrations as high as 25 mM. PAPH activity recovered in the cytosolic fraction of disrupted neutrophils was almost completely inhibited by 5.0 mM n-ethylmaleimide. We conclude that resting neutrophils possess n-ethylmaleimide-resistant PAPH (type 2) within their plasma membranes. This enzyme may markedly influence the kinetics of cell activation by metabolizing second messengers generated as a result of activation of plasma membrane phospholipase D.     

Multiple phospholipase A2 activities in canine vascular smooth muscle.

Three phospholipase A2 activities from canine vascular smooth muscle were identified and characterized including: (1) a cytosolic calcium-independent phospholipase A2 which is activated by nucleotide di- and triphosphates; (2) a cytosolic calcium-dependent phospholipase A2 which is activated by physiologic increments in calcium ion concentration; and (3) a microsomal calcium-independent phospholipase A2 which was highly selective for plasmenylcholine substrate. Vascular smooth muscle cytosolic calcium-independent phospholipase A2 was activated 338% +/- 11 (X+S.E.; n = 15) by physiologic concentrations of ATP. Similar amounts of activation were also present utilizing other nucleotide di- and triphosphates (e.g., ADP, CTP, GDP and GTP) as well as non-hydrolyzable nucleotide triphosphate analogs (e.g., ATP-gamma-S, AMP-PNP and GTP-gamma-S). Vascular smooth muscle cytosolic calcium-dependent phospholipase A2 was purified 455-fold by sequential DEAE-Sephacel, Phenyl-Sepharose, Mono Q, hydroxyapatite and Superose 12 chromatographies. The partially purified calcium-dependent phospholipase A2 was activated by physiologic increments in calcium ion concentration (e.g., 1 microM) and possessed an apparent native molecular weight of 95 kDa, an acidic isoelectric point (pI = 4.8) and a neutral pH optimum (pH 7.0). Vascular smooth muscle microsomal phospholipase A2 activity was predominantly calcium-independent and was over six-fold selective for hydrolysis of plasmenylcholine substrate. Taken together, these results demonstrate the existence of three separate and distinct phospholipase A2 activities in vascular smooth muscle and identify ATP and calcium ion as independent modulators of discrete phospholipase A2 activities in vascular smooth muscle cells.     

Regulation of cytosolic phospholipase A2 in rat endometrial stromal cells: the role of epidermal growth factor

The effect of epidermal growth factor on the levels of cytosolic phospholipase A2 mRNA and protein in cultured rat endometrial stromal cells isolated from uteri sensitized for the decidual cell reaction was examined. Treatment with epidermal growth factor increased the steady-state cytosolic phospholipase A2 mRNA and protein levels as demonstrated by Northern and Western blot analyses, respectively. Immunocytochemical analysis demonstrated an increase of cytosolic phospholipase A2 protein in most cells, as opposed to a small subpopulation of cells in culture. These results show that epidermal growth factor causes an increase in steady-state cytosolic phospholipase A2 mRNA and protein levels in rat endometrial stromal cells from uteri sensitized for the decidual cell reaction. Epidermal growth factor receptor ligands may regulate cytosolic phospholipase A2 and thus prostaglandin production in the endometrial stromal cells during implantation.     

Evidence for multiple metabolic pools of phosphatidylinositol in stimulated platelets

Stimulation of platelets with ionophore A23187 or thrombin indicates the existence of three distinct metabolic fractions of phosphatidylinositol. Two of those pools of phosphatidylinositol are degraded by phosphatidylinositol-specific phospholipase C and the third one by a phospholipase A2 activity. Low concentrations of ionophore A23187 (100 nM) or thrombin (0.25 units/ml) induce the degradation by phospholipase C of a minor fraction of phosphatidylinositol which is involved in the phosphatidylinositol cycle. In addition, thrombin, but not ionophore A23187, leads to the degradation by phospholipase C of a larger fraction of phosphatidylinositol and the subsequent accumulation of phosphatidic acid. A third fraction of phosphatidylinositol, sensitive to thrombin (0.5-2 units/ml) or ionophore A23187 (0.5-2 microM), can be degraded by phospholipase A2 to lysophosphatidylinositol with the concomitant liberation of arachidonic acid. Degradation of phosphatidylinositol by the phospholipase C pathway precedes that of the phospholipase A2 pathway. The results also suggest that the phosphatidylinositol cycle is sensitive to a small rise in cytosolic Ca2+ concentration. A further mobilization of cytosolic Ca2+ interrupts the phosphatidylinositol cycle by inhibiting conversion of phosphatidic acid to phosphatidylinositol and also activates phospholipases of the A2 type.     

Calcium is sufficient but not necessary for activation of sheep platelet cytosolic phospholipase A2

In this study we demonstrate that: (1) although the major phospholipase A2 present in sheep platelets is activated by calcium ions, it can effectively catalyze hydrolysis of the sn-2 ester linkage in phospholipids in the absence of calcium; (2) expression of calcium-independent phospholipase A2 activity can be induced by NaCl utilizing purified (but not crude) cytosolic enzyme; and (3) calcium-independent phospholipase A2 activity is regulated by a reconstitutable cytosolic protein. Collectively, these results underscore the fundamental catalytic differences between extracellular and intracellular calcium-dependent phospholipases A2 and demonstrate that calcium is sufficient, but not necessary, for the activation of this class of intracellular phospholipases A2.     

Enzymatic cross-linking of involucrin and other proteins by keratinocyte particulates in vitro

A transglutaminase-catalyzed cross-linking process characteristic of keratinocytes leads to the formation of the insoluble corneocyte envelope. The essentials of this process take place in vitro in a reconstituted system derived from subcellular fractions. A particulate fraction containing membrane-bound envelope precursor proteins and the enzyme transglutaminase is combined with cytosolic proteins; when the enzyme is activated by Ca++, cytosolic proteins are removed from solution and cross-linked to particulate proteins. This interaction is cell-type-specific, since particulates derived from fibroblasts and also containing transglutaminase activity cannot substitute for those of keratinocytes. Involucrin, a cytosolic protein known to be a precursor of the envelope, is more efficiently cross-linked than other cytosolic proteins. The cross-linking of proteins of the particulate fraction (membrane proteins) is promoted by the presence of involucrin.     

GTP and GDP will stimulate platelet cytosolic phospholipase C independently of Ca2+

The hydrolysis of [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) by cytosolic phospholipase C from human platelets was determined. Cytosolic fractions were prepared from platelets that had or had not been preactivated with thrombin. Thrombin pretreatment did not affect cytosolic phospholipase C activity. In both cytosolic fractions, phospholipase C was activated by GTP and GTP gamma S. This action is observed in the presence of 2 mM EGTA. GDP was as effective as GTP in stimulating cytosolic phospholipase C in the presence of Ca2+ or EGTA. Partially purified phospholipase C obtained from platelet cytosol is activated by GTP, but not by GTP gamma S, in the presence of 2 mM EGTA. However, in the presence of 6 microM Ca2+, both GTP and GTP gamma S stimulated the partially purified phospholipase C. Our present information indicates that GTP and GDP have a direct effect on the cytosolic phospholipase C.     

Phospholipase C in rabbit thymocytes: subcellular distribution and influences of calcium and GTP gamma S on the substrate dependence of cytosolic and plasma membrane-associated phospholipase C

The subcellular distribution of phospholipase C (PLC) activity in rabbit thymocytes was examined by measuring the enzyme's activity in different subcellular fractions. PLC activity was determined using exogenously added [3H]PIP2 as substrate. Approx. 80% of the activity of the cell homogenate was found in the cytosolic fraction. A minor portion of PLC activity was attached to the particulate fraction. This membrane-associated PLC activity was found to be predominantly bound to the plasma membrane. Both PIP2-cleaving PLCs (the PLC associated with the plasma membrane and the PLC in the cytosol) exhibited maximum activity at pH 5. GTP gamma S stimulated the cytosolic and the membrane-bound PLC. As revealed by computer analysis of the substrate dependence of both basal and GTP gamma S-stimulated PLC activity, GTP gamma S enhanced the Vmax of the enzymes. Calcium, at a concentration of 1 mM, decreased PLC activity, as compared to a calcium concentration of 100 nM. The characteristic increase in Vmax induced by GTP gamma S was observed at a concentration of 1 mM calcium and was similar to that at 100 nM. These data suggest that the stimulatory effect of GTP gamma S is not due to an increased affinity of PLCs to calcium.     

Inhibition of cytosolic phospholipase A2 mRNA expression: a novel mechanism for acetylsalicylic acid-mediated growth inhibition and apoptosis in colon cancer cells

Acetylsalicylic acid (ASA) has been confirmed to inhibit proliferation and to induce apoptosis in human colorectal cancer cells in vitro. However, the mechanism by which ASA exhibits antiproliferative and proapoptotic effects in cyclooxygenase 2 (COX-2)-negative cells remains to be further elucidated. In the present study, SW480, a COX-2-negative colon cancer cell line, was treated with various concentrations of ASA (0, 2.5, 5, and 10 mM). The antiproliferative and proapoptotic effects of ASA were confirmed by MTT assay, flow cytometry of propidium iodide (PI)-stained cells, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. After treatment with ASA, intracellular cyclic AMP (cAMP) levels were increased and the production of prostaglandin E2 (PGE2) was decreased. RT-PCR analysis revealed that treatment of ASA induced a concentration-dependent downregulation of cytosolic phospholipase A2 (cPLA2) mRNA expression in SW480 cells and also in two other colorectal cancer cell lines, Colo320 and HT-29 cells. Intracellular calcium levels were unaffected by ASA treatment. Our results indicate that the ASA-induced downregulation of cytosolic phospholipase A2 mRNA expression might be a novel mechanism for ASA-mediated growth inhibition and apoptosis in colon cancer cells.