Phospholipase A2, Eicosanoids, and Apoptosis
Phospholipase A2, Eicosanoids, and Apoptosis
By David P. Schuster, and Thomas Nicotera, PhD
Products formed during the metabolism of arachidonic acid (AA) play a deterministic role in regulating cell growth, function, and survival. In order for proper cellular functioning to occur, release of arachidonate from membrane phospholipids, as well as the constellation of enzymes directed toward eicosanoid metabolism must remain intact. Phospholipids acted upon by phospholipase A2 (PLA2) release free fatty acid from the sn-2 position. The sn-2 position is frequently occupied by unsaturated fatty acids, such as AA. Arachidonate can be processed by cyclooxygenase pathways (COX) or lipoxygenase pathways (LOX) to produce prostaglandins and leukotrienes, respectively. Arachidonate metabolites are potent mediators of a broad range of physiologic phenomena.
Perturbations in eicosanoid metabolism have been observed in a number of tumors and tumor-derived cell lines, and an increased ability to metabolize AA may contribute to the aggressiveness of tumor cells. Several enzymes have been identified that can significantly affect the concentrations of eicosanoids in cells. The mitogen inducible form of cyclooxygenase (COX-2) is elevated in the highly invasive MDA-MB-231 breast cancer cell line, as compared to the less invasive MCF-7 cell line.1 Specific inhibition of COX-2 can result in a decreased tumor growth rate and an increase in tumor cell apoptosis.2,3 Furthermore, the expression of COX-2 is elevated in gastric carcinomas and in neoplastic colon tissue as compared to adjacent, noncancerous tissue. Elevated COX activity should result in the generation of prostaglandins, and prostaglandins (particularly prostaglandin E) have been implicated as mediators in angiogenesis.3 Exogenous AA has been demonstrated to be mitogenic in human prostatic cancer cells,4 and there is evidence that leukotrienes can prevent programmed cell death in prostate tumor cell lines.4,13 These data suggest that altered regulation of arachidonate metabolism can provide multiple mitogenic stimuli.
Metabolism of AA in prostate tumors has been of interest in part because of epidemiological studies suggesting a role for dietary fats in the etiology of prostate cancer. Arachidonate is less abundant in the membrane of malignant prostate cells as compared to benign tissue.12 Addition of exogenous AA to prostate tumor cells results in an increase in production of leukotrienes4,5 and prostaglandins.15 Also, malignant tissue samples obtained during radical prostatectomy were shown to have elevated activity of PLA2 when compared to benign prostate tissue.12 No evidence of de novo synthesis of unsaturated fatty acids in prostate was found,12 and enzymes dedicated to the allocation of unsaturated fatty acids, AA-CoA hydrolase and lysophospholipid acyl transferase, were unchanged in malignant tissue as compared to benign tissue. This suggests that malignant cells have constitutively high PLA2 activity, and strip AA from membranes in malignant tissue.
Ability of Prostate Tumors to Establish Metastases
The availability of substrate for cyclooxygenase and lipoxygenase may be a critical factor in the ability of prostate tumors to establish metastases. In our lab, inhibition of PLA2 resulted in apoptosis in two well characterized prostate tumor cell lines, LNCaP, and PC3 cells. The more aggressive PC3 cells had a higher PLA2 activity and were more sensitive to inhibitor-induced apoptosis than the LNCaP cells. Exogenous AA generates an abundance of 5-HETE in these cell lines.4,5 LNCaP and PC3 cells are constitutive producers of the leukotriene precursor 5-HETE,5 and leukotrienes are suggested to impair programmed cell death in these cells.6 Other investigators report that blocking the lipoxygenase (LOX) pathway induced rapid apoptosis in both cell lines.5,6 It was also observed that apoptosis induced in human protaste cells by LOX inhibition was reversed by the addition of 5-HETE.6 This would suggest that AA liberated from membranes and metabolized by the lipoxygenase pathway has a cytoprotective effect in these tumor cell lines. In this case, constitutive elevation of PLA2 activity creates a proliferative advantage. Some sublines of these tumor cells selected for metastatic potential,7 also have high PLA2 activities, as determined in our laboratory. We are currently exploring the sensitivity of these sublines to inhibitor-induced apoptosis.
Treatment of Eicosanoid Metabolism
Three major enzymes have been considered so far in this treatment of eicosanoid metabolism, PLA2, COX, and LOX. As is the case with COX-1 and COX-2, LOX and PLA2 have a variety of isoforms. LOX isoforms differ in their specificity for particular ethylene moieties in the eicosanoid backbone, and characteristics attributed to 5-HETE have also been attributed to products of 15-LOX.13 Similarly, PLA2 activity is a composite of a variety of isoforms with different substrate specificity. The cPLA2s have an affinity for phospholipids with AA in the sn-2 position and require calcium for the translocation of active enzyme to the membrane. The sPLA2s require calcium to facilitate vessicular release, but do not have a preference for AA containing phospholipids. Furthermore, cPLA2 has been reported to be constitutively activated in PC3 cells,14 which may account for the relatively high PLA2 activity in these cells.
Conclusion
Recently, messenger RNA from a new class of PLA2s, the calcium-independent iPLA2, has been sequenced.8-10 Several splice variants of this species of RNA have been identified, and at least two of the splice variants code for truncated proteins with no consensus sequence for PLA2 activity.8 Active forms of this enzyme show specificity for phospholipids with AA in the sn-2 position, and one active form requires ATP.10 Truncated, inactive proteins predicted to exist are proposed to play a regulatory role in a multimeric iPLA2 complex. It has been documented that cells originating from different tissues maintain different ratios of these splice variants.9,10 We have also obtained preliminary data that suggest aberrant expression of this iPLA2 occurs in prostate tumor cells. Regulation-deficient iPLA2 isoforms in prostate tumors could result in the over production of cytoprotective eicosanoids, angiogenic catalysts, or mitogenic stimulation. Detailed information about AA metabolism in these cells may reveal markers of tumorigenicity, or new therapeutic approaches to this disease. (David P. Schuster is a graduate student in Molecular and Cellular Biophysics at Roswell Park Cancer Institute, Buffalo, NY; Dr. Nicotera is a Cancer Research Scientist in Molecular and Cellular Biophysics at Roswell Park Cancer Institute, Buffalo, NY.)
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