May 10, 1987 - production in TNF-treated cells (MDA is a free radical-induced lipid peroxidation .... support to free-radical involvement in TNF cytolysis.
Immunology 1987 62
BRIEF COMMUNICATION
153-155
Tumour cell killing by tumour necrosis factor: inhibition by anaerobic conditions, free-radical scavengers and inhibitors of arachidonate metabolism N. MATTHEWS, M. L. NEALE, S. K. JACKSON & J. M. STARK Department of Medical Microbiology, University of Wales College of Medicine, Cardif
Acceptedfor publication 10 May 1987
SUMMARY Previous work on the mechanism of tumour-cell killing by the macrophage product tumour necrosis factor (TNF) is consistent with a free radical-induced process. In this study, free-radical involvement was sought by (i) investigating the effects on TNF cytolysis of anaerobic conditions, free-radical scavengers and inhibitors of two potential pathways of free-radical generation (oxidative phosphorylation and arachidonate metabolism) and (ii) looking for increased malonyldialdehyde (MDA) production in TNF-treated cells (MDA is a free radical-induced lipid peroxidation product). Although TNF cytolysis of L929 cells was inhibited by anaerobic conditions, only limited effects were seen with free-radical scavengers. Suppression of arachidonate metabolism by steroids effectively inhibited TNF cytolysis but the mitochondrial poison rotenone did not. There was a marked, but late, increase in MDA production in TNF-treated cells. Overall, these results indicate that if free radicals are involved it is at a late stage in the cytolytic process. However the most striking observation in this study is that arachidonate metabolism is an essential link in the cytolytic process.
Tumour necrosis factor (TNF), as its name implies, has antitumour activity both in vitro and in vivo (Carswell et al., 1975). In vitro TNF does not usually kill untransformed cells but it is cytolytic to certain tumour cells by an undefined mechanism. In studying cytolysis of L929 tumour cells by TNF in vitro, we noted that there was a lag period of several hr before the cells succumbed, and that the first organelles to appear damaged were the mitochondria (Matthews, 1983). We concluded that these and other observations of the interaction of TNF with tumour cell lines were consistent with a free radical-induced cytolytic process (Matthews & Neale, 1987b). If free radicals are involved in the cytolytic process then cytolysis ought to be reduced by (a) anaerobic conditions (if oxygen-based radicals are involved), (b) free-radical scavengers and (c) inhibitors of pathways which generate free radicals. Also, it should be possible to find increased concentrations of the lipid peroxidation product malonyldialdehyde (MDA) in TNF-treated cells. To test the effects of oxygen deprivation, L929 cells were incubated with or without TNF for 3 days in a conventional gassed incubator (aerobic) or in a bacterial anaerobic jar (Gaspak system, Becton-Dickinson, Cockeysville, MD); cytolysis was determined as in Table 1. Under aerobic conditions, Correspondence: Dr N. Matthews, Dept. of Medical Microbiology, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, U.K.
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>95% cytolysis was noted with as little as 10 U/ml TNF. Anaerobically, the L929 cells survived surprisingly well in the absence of TNF, but also in its presence-even with TNF at 640 U/ml there was < 20% cytolysis. These data are consistent with an oxidative process but give no indication of whether oxygen-derived radicals are involved. Free-radical involvement would be indicated if cytolysis could be inhibited by free-radical scavengers. A number of freeradical scavengers were tested at a range of concentrations up to the toxic limit. No significant inhibition of TNF cytolysis to L929 cells was seen with dimethyl sulphoxide, vitamin E, thiourea, mercaptoethanol or methimazole. However, inhibition was seen with promethazine and the chelator desferal (Table 1) and also with butylated hydroxytoluene (23% inhibition at 20 pM). The ethanol diluent for these reagents was not itself inhibitory and inhibition was not simply due to reduction of growth rate: other scavengers reduced cell growth more effectively without inhibiting TNF cytolysis. Potential sites of free-radical production include mitochondria and membranes (during arachidonate metabolism). Treatment of cells with rotenone should prevent generation of H202derived radicals in the mitochondria, but rotenone concentrations up to 200 ng/ml did not inhibit cytolysis of L929 cells by TNF. In contrast, steroid inhibition of arachidonate metabolism markedly reduced cytolysis (Table 1). Cyclo-oxygenase inhibitors (indomethacin and acetyl salicylic acid) also reduced cytolysis but at concentrations much higher than normally
N. Matthews et al.
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Table 1. Effect of promethazine, desferal and inhibitors of arachidonic acid metabolism on TNF cytotolysis of L929 cells
Mean absorbance (540 nm) x 1000+SD Exp. I 2 3
Inhibitor Medium Promethazine Medium Desferal Medium NDGA
Indomethacin 4
Medium Hydrocortisone Dexamethasone
Acetylsalicylic acid
Conc. -
20 pM 10 gM 0-07 mM 0-04 mM 6 pM
3 pM 1[5puM 100 pM 50 gM 25 PM
20OuM 10 PM 5 pM 100 PM 25SuM 6 pM 0 5 mM 0-25 mM
MDA production (pM/2 x 106 cells) mean + SD % % cyto- inhibi-
-TNF
+TNF
lysis
742+6 330 + 20 780+40 230+ 10 130+ 10 193 +19 468+20 354+30 359+24 419+27 256+ 16 419+21 416+22 397 + 14 232 +20 254+37 252 + 35 186+ 12 227+8 229+ 14
300+21 228 + 12 430+20 10+4 77+3 63 + 8 107+8 107+ 14 115+ 11 142+ 16 176+4 234+2 170+5 133 + 17 171 + 10 182+ 17 162 + 8 167+ 18 196+5 191 + 15
60 31 45 96 41 67 77 70 68 66 31 44 59 66 26 28 36 10 14 17
293 + 14 142+ 15 347 + 7 188 + 11
52 46
tion 48 25 57 30
9 12 14 60 43 23
61 56 45 85 79 74
Cell line
-TNF
+TNF
L929 L929/R U937 U937/R RK13 RK13/R
24+ 1 20+ 1 56+6 56 + 10 25+5 20+2
354+ 64 42+2 290+38 56 + 12 174+2 36+6
L929 (mouse fibroblastoid), RK 13 (rabbit kidney) and a U937 variant (human myelomonocytic) are TNF-susceptible, plastic-adherent cell lines from which TNF-resistant (-/R) sublines have been isolated (Matthews & Neale, 1987b). Triplicate cell cultures (2 x 106) in 5 ml growth medium were incubated with 1000 U/ml TNF. After 40 hr the cells were washed with isotonic saline, solubilized in 0-5 ml 1 % SDS, 50 gM butylated hydroxytoluene and heated at 950 for 30 min with 0-5 ml 1% aqueous thiobarbituric acid. The reaction product was extracted in to 5% HCl in butanol. MDA was quantified fluorimetrically (excitation 528 nm, emission 548 nm, assuming 50 fluorescence units/nM) and expressed relative to the 2 x 106 cells at the start of the
experiment.
21 30
TNF was purified from the serum of rabbits with endotoxic shock (Taverne et al., 1984) and the same batch (105 U/ml) was used throughout. A unit is defined as the minimal amount of TNF to kill 50% of the L929 cells in a 3-day assay. To measure inhibition of TNF cytolysis, L929 cells were plated in 96-well microtitre trays with the appropriate concentrations of the test agent plus enough TNF to give 70-90% cytolysis; the final culture volume was 175 p1. After 3 days at 37° the remaining adherent viable cells were stained with crystal violet. Dye uptake is proportional to the number of remaining cells and was quantified photometrically by an ELISA reader. Percentage cytolysis was calculated from the formula 100(a-b)/a, where a and b are respectively the mean absorbances of triplicate wells without or with TNF. This photometric assay is well established (Matthews, 1983), has been described in detail (Matthews & Neale, 1987a) and correlates well with other cytolytic assays (Flick & Gifford, 1984). Inhibition of cytolysis was calculated from the formula 100(c-d)/c where c and d are the percentage of cytolysis with medium or drug, respectively.
required to inhibit this pathway: the lipo-oxygenase inhibitor, nordihydroguaiaretic acid, had little effect on TNF killing (Table 1). Therefore arachidonate mobilization seems to be important in the cytolytic process but it may be metabolized by unconventional route. Peroxidation of lipids is a common consequence of tissue damage by free radicals and the presence of the reaction product MDA is an indication that such a process has taken place. TNF treatment of the susceptible lines L929, U937 and RKI 3, but not their TNF-resistant sublines, resulted in a marked increase in MDA production after 40 hr (Table 2). In time-course studies an
Table 2. MDA production by cells treated for 40 hr with TNF
with L929 cells, a significant increase in MDA was first noted about 8 hr after TNF treatment (16+2 pM/2 x 106 cells in control and 22 + 2 pM in TNF treated). Superficially, the above observations support the idea of a free radical-induced mechanism. However, some of the data can be interpreted differently. Firstly, the reduction of cytolysis under anaerobic conditions may be interpreted as a requirement not for oxygen but for a respiration-dependent process. This latter alternative is unlikely because rotenone-treated cells have impaired respiration yet are susceptible to TNF killing. Secondly, increased MDA was not detected until after 8 hr and reached high levels 40 hr after TNF addition when most of the cells were dead. Either lipid peroxidation is a slow primary event or it is purely secondary, triggered perhaps by organelle disruption initiated by some other process. Thirdly, although desferal, promethazine and butylated hydroxytoluene inhibited killing, other free-radical scavengers did not. It is debatable whether this can be explained by differences between scavengers in reactivity with different radicals or in ability to penetrate to the site of radical generation. Perhaps more likely, the inhibitory scavengers are acting by other mechanisms, for example promethazine stabilizes membranes and inhibits prostaglandin synthetase (Seeman, 1966; Collier, 1974). Similarly, it can be argued that steroids affect other cellular processes in addition to arachidonate metabolism. Nevertheless, recently we have shown that TNF releases arachidonate metabolites from TNF susceptible cells but not from their TNF-resistant sublines; dexamethasone and promethazine at concentrations that inhibit cell killing also prevent release of arachidonate metabolites from susceptible cells. On balance, the above observations give only limited
Tumour necrosis factor support to free-radical involvement in TNF cytolysis. However, the most important finding in this study is the involvement of arachidonate metabolism in the cytolytic process.
ACKNOWLEDGMENT We thank the Cancer Research Campaign for financial support.
REFERENCES CARSWELL E.A., OLD, L.J., KASSEL R.L., GREEN S., FIORE N. & WILLIAMSON B. (1975) An endotoxin-induced serum factor that causes necrosis of tumors. Proc. natin. Acad. Sci. U.S.A. 72, 3666. COLLIER H.O. (1974) In: Prostaglandin Synthetase Inhibitors (eds H.J. Robinson & J.R. Vane), p. 121. Raven Press, New York.
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FLICK D.A. & GIFFORD G.E (984) Comparison of in vitro cell cytotoxic assays for tumor necrosis factor. J. immunol. Meth. 68, 167. MATrHEWs N. (1983) Anti-tumour cytotoxine produced by human monocytes: studies on its mode of action. Br. J. Cancer, 48, 405. MArrHEws N. & NEALE M.L. (1987a) Cytotoxicity assays for tumour necrosis factor and lymphotoxin. In: Lymphokines and interferons: a practical approach (eds. A.J.M. Gearing, A.G. Morris and M.J. Clemens) IRL Press, Oxford (in press). MArrHEws N. & NEALE M.L. (1987b) Studies on the mode of action of tumour necrosis factor in vitro. In: Lymphokines (ed. E. Pick). Vol. 14, pp. 223-251. Academic, Orlando Press (in press). SEEMAN, P.M. (1966) Membrane stabilization by drugs: tranquilizers, steroids and anesthetics. Int. Rev. Neurobiol. 9, 145. TAVERNE J., MATTHEws N., DEPLEDGE P. & PLAYFAIR J.H.L. (1984) The same factor in tumour necrosis serum kills malarial parasites and tumour cells. Clin. exp Immunol. 57, 293.
