0 INSTITUT PASTEURIELSEVIER Res. Immunol. Paris 1995 1995, 146, 351-361 Bryostatins trigger human polymorphonuclear neutrophil and monocyte oxidative metabolism : association with in vitro antineoplastic activity A.H. Esa(l) (*), J.T. Warren c2) (**), A.D. Hess c3)and W.S. May t3) (I) Dkpartement d’Immunologie, Unite’ d’Immunophysiologie moie’culaire, (2) The Departme Znstitut Pasteur, 75724 Paris Cedex 1.5, nt of Environmental Health Sciences, The Johns Hopkins School of Hygiene and Public Health, Baltimore, MD 21205 (USA), and (jr)The Oncology Center, The Johns Hopkins School of Medicine, Baltimore, MD 21205 (USA) SUMMARY Bryostatin-1 - but not bryostatin-13 - a macrocyclic lactone isolated from the marine bryozoan Sugu/a neritina, triggered human polymorphonuclear neutrophil (PMN) and monocyte release of reactive oxygen radicals, as measured by the generation of lucige- nin chemiluminescence and by the ferricytochrome c reduction assay. The release of oxygen radicals by bryostatins was sensitive to inhibitors of protein kinases, but resist- ant to the inhibition of phospholipase A, activity and arachidonic acid metabolism (prior treatment with mepacrine or indomethacin). Comparison of the effect of protein kinase (PK) inhibitors H-8, H-7 and staurosporine on bryostatin-l-induced neutrophil oxygen radical release further suggested a requirement for activation of phospholipid-dependent PKC, but not for cGMP- or CAMP-dependent PK. In cytostatic assays, PMNs treated with bryostatin-1 inhibited the growth of the erythroleukaemic cell line K582 in a concentra- tion-dependent manner. These findings suggest that the reported antineoplastic effect of bryostatins may result, at least in part, from activation of PMNs and monocytes. Key-words : Bryostatin, Oxygen, Cytostasis, Polymorphonuclear leukocyte; Reactive oxygen radicals, Tumoricidal effect, Protein Kinase, Monocytes. INTRODUCTION In 1982, Petit et al. isolated bryostatin-1, an unsual macrocyclic lactone, from the marine bryozoan Bugula neritina. A number of bryos- tatin congeners were subsequently discovered and shown to have antineoplastic properties (Pettit et al., 1983, 1984). Studies also show that these agents stimulate in vivo colony for- mation in normal human bone marrow (May et al., 1987). Bryostatins share certain properties with haematopoietic growth factors that regu- late normal haematopoiesis. Like the multipo- tential human recombinant granulocyte-macro- Submitted May 9, 1995, accepted September 25, 1995. (*) Corresponding author. Address: 15, rue St. Pierre, 92200 Neuilly-sur-Seine, France. (**) Now at the National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD, USA.
Transcript
0 INSTITUT PASTEURIELSEVIER Res. Immunol. Paris 1995 1995, 146, 351-361
Bryostatins trigger human polymorphonuclear neutrophil and monocyte oxidative metabolism :
association with in vitro antineoplastic activity
A.H. Esa(l) (*), J.T. Warren c2) (**), A.D. Hess c3) and W.S. May t3)
(2) The Departme Znstitut Pasteur, 75724 Paris Cedex 1.5,
nt of Environmental Health Sciences, The Johns Hopkins School of Hygiene and Public Health, Baltimore, MD 21205 (USA), and
(jr) The Oncology Center, The Johns Hopkins School of Medicine, Baltimore, MD 21205 (USA)
SUMMARY
Bryostatin-1 - but not bryostatin-13 - a macrocyclic lactone isolated from the marine bryozoan Sugu/a neritina, triggered human polymorphonuclear neutrophil (PMN) and monocyte release of reactive oxygen radicals, as measured by the generation of lucige- nin chemiluminescence and by the ferricytochrome c reduction assay. The release of oxygen radicals by bryostatins was sensitive to inhibitors of protein kinases, but resist- ant to the inhibition of phospholipase A, activity and arachidonic acid metabolism (prior treatment with mepacrine or indomethacin). Comparison of the effect of protein kinase (PK) inhibitors H-8, H-7 and staurosporine on bryostatin-l-induced neutrophil oxygen radical release further suggested a requirement for activation of phospholipid-dependent PKC, but not for cGMP- or CAMP-dependent PK. In cytostatic assays, PMNs treated with bryostatin-1 inhibited the growth of the erythroleukaemic cell line K582 in a concentra- tion-dependent manner. These findings suggest that the reported antineoplastic effect of bryostatins may result, at least in part, from activation of PMNs and monocytes.
In 1982, Petit et al. isolated bryostatin-1, an unsual macrocyclic lactone, from the marine bryozoan Bugula neritina. A number of bryos- tatin congeners were subsequently discovered and shown to have antineoplastic properties
(Pettit et al., 1983, 1984). Studies also show that these agents stimulate in vivo colony for- mation in normal human bone marrow (May et
al., 1987). Bryostatins share certain properties with haematopoietic growth factors that regu- late normal haematopoiesis. Like the multipo- tential human recombinant granulocyte-macro-
Submitted May 9, 1995, accepted September 25, 1995.
(*) Corresponding author. Address: 15, rue St. Pierre, 92200 Neuilly-sur-Seine, France. (**) Now at the National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD, USA.
352 A.H. ESA ET AL.
phage colony-stimulating factor (GM-CSF), bryostatins can promote the growth of normal non-erythroid progenitor cells, as well as eryth- roid colony development, in vitro (May et al., 1987). The combination of antineoplastic activ- ity and the capability to promote normal hae- matopoietic progenitor cell growth makes the bryostatins potentially useful chemotherapeutic agents.
Bryostatins mimic the structurally unrelated tumour-promoting phorbol esters. Like phorbol esters, bryostatins bind to and activate protein kinase C (PKC) in normal neutrophils, stimu- lating the release of specific granules and superoxide (Berkow and Kraft, 1985; Kraft et al., 1986). The release of reactive oxygen radi- cals by neutrophils has been implicated in the putative tumoricidal and cytotoxic function of these cells (Clark and Klebanoff 1975, 1979; Clark and Szot, 1981; Celada and Schreiber, 1986 ; English and Lukens, 1983 ; Sacks et al., 1978 ; Simchowitz and Spilburg, 1979; Weiss and Slivka, 1982). This property of bryostatins may thus conceivably underlie the observed antineoplastic activity of these agents. In a pre- vious study, we reported that neutrophils exposed to bryostatin-1 can lyse the erythrol- eukaemic cell line K562 in short-term cyto- toxic assays (May et al., 1987). We now describe subsequent studies related to the role of PKC in the tumoricidal activity of bryosta- tin-activated PMNs, using specific PKC inhibi- tors and an inactive bryostatin congener. Our studies demonstrate a linkage between bryosta- tin-induced neutrophil tumoricidal activity and PKC-dependent release of free oxygen radi- cals.
MATERIALS AND METHODS
Isolation of polymorphonuclear leukocytes and monocytes
Human PMNs were isolated from heparinized blood by centrifugation through “Mono-Poly Resolving Medium” (density 1.114, Flow Labora- tories, McLean, VA). Briefly, 6 ml of fresh hepa- rinized normal blood were layered over 3.5 ml of medium in 15-ml conical tubes, and centrifuged at 300 g for 30 min. Two layers of cells are resolved with this procedure with the layer closest to the red cell pellet contaning 90-95 % PMNs. Cells in this layer were harvested and washed 3x in RPMI- 1640 supplemented with 10% foetal calf serum (FCS). Monocytes were isolated by countercurrent centrifugation elutriation (CCE) as described by Esa et aE., 1986. Approximately 500 x lo6 mono- nuclear cells from leukophoresed blood were introduced into a “Beckman JE-6B” elutriator. After lymphocyte depletion, two monocyte-rich fractions were isolated at predetermined flow rates.
Preparations of agents
Bryostatins (a kind gift from Dr. George Pettit of Arizona University) and TPA (Sigma) were dis- solved in DMSO at 10-2M and diluted in Dulbecco’s phosphate-buffered saline (DPBS) with Mg++ and Ca++. H-7, H-8 and staurosporine (Seikagaku Kogyo Co., Ltd., St. Petersburg, FL) were dissolved in distilled water and diluted in PBS. Opsonized zymosan was prepared as described by Roos et al. (198 1) and used at a final concentration of 0.1 mg/ml. Mepacrine indometha- tin and SOD (Sigma) were dissolved in DMSO and diluted in PBS. The final DMSO concentration in all preparations was less than 0.1 %, and PBS controls with 0.1% DMSO were included in all experiments.
protein kinase. phospholipase A,. polymorphonuclear neutrophil. Roswell Park Memorial Institute tissue medium- 1640. superoxide dismutase. opsonized symosan. 12-O-tetradecanoyl- 13-phorbol acetate.
culture
BRYOSTATIN ACTIVATION OF POLYMORPHONUCLEAR NEUTROPHILS 353
Determination of superoxide release by ferricy- tochrome c reduction
The SOD-inhibitable reduction of ferricyto- chrome c was used as an indicator of the release of superoxide anion by activated PMNs as described by Babior et al., 1970. Briefly, PMNs (3.5 x lo6 cells) were incubated with stimulants and ferricy- tochrome c (Sigma) in the absence or presence of SOD. After 30 min of incubation at 37°C the absor- bance at 550 nm was measured using a Gilford “Response UV-VIS” spectrophotometer. In this assay, the amount of cytochrome c reduced by superoxide, determined by taking the difference between absorbance in the presence and absence of SOD, correlates quantitatively with the amount of superoxide anion.
Neutrophil chemiluminescence (CL)
Temporal generation of CL from 5~10~ cells was measured in 1 ml of DPBS supplemented with Mg++ and CA++ and stimulants or vehicle. Bryostatin-1 and tetradecanoyl-phorbol acetate (TPA) were dis- solved in DMSO and diluted in DPBS. One hundred pl of each stimulant were added to the assay buffer to give the desired final concentrations (100 nM). Control samples received 0.1% DMSO. All tubes then received 100 pM DBA (lucigenin). In this system, DBA is oxidized to electronically excited intermediates by way of dioxygenation (Allen, 1981; Allen and Loose, 1976). These intermediates revert to ground state after photon emission. The generation of CL from bryostatin-activated or con- trol neutrophils and monocytes was measured at the same time at 1.25-min intervals for up to 20 min, using an “LKB” luminometer (LKB, Turku, Fin- land). Each sample was monitored for up to 25 min and the generated CL recorded as relative CL inten- sity (mV/s).
Cytostatic activity of bryostatin
The ability of bryostatin-l-treated neutrophils to inhibit the growth of K562 cells was examined as follows. Cells (2~10~) were seeded in 96-well microtitre plates with bryostatin-1 and graded num- bers of neutrophils. The cells were incubated for 18 h at 37°C in 5 % CO and then pulsed with 0.1 pCi of 3H-thymidine or another 6 h and then t harvested for liquid scintillation counting.
Assessment of neutrophil cytotoxicity
The erythroleukaemic cell line, K-562, the human natural killer-cell-resistant cell line DAUDI,
the human T-cell leukaemia Jurkat and the murine YAC-1 tumour cell were all maintained in RPMI- 1640 supplemented with 10% FCS. These cells were harvested and washed with PBS and then labelled with 51Cr-NaCr,04 for 1 h at 37°C. Excess 51Cr was removed by washing cells three times in PBS, and the cells were resuspended in 10% FCS RPMI-1640 and adjusted to 1~10~ cells/ml. One hundred ~1 of the labelled cells were then seeded in each well of a 96-well round bottom microtitre plate. Effector cells, 3~10~ cells/ml, were incubated with bryostatin 1 (10e7 to lo-l2 M) or control vehicle alone for 2 h at 37°C. The cells were then washed and resuspended in 10 % FCS RPMI-1640. Effector cells were added to target cells in triplicate at the specified effector-to-target ratios in a final volume of 200 pl. The plates were then centrifuged at 250 g for 2 min and incubated 37°C in 5 % CO, for 4 h. To control for bryostatin toxicity for K-562 cells, bryos- tatin 1 at lo-’ and 10m8 M was added to control K- 562 wells. After incubation, the plates were again centrifuged and 100 pl of supernatant medium from each well were harvested. Radioactivity was counted with a gamma counter and expressed as mean counts per minute (cpm) of triplicate determi- nations. Spontaneous release was obtained in wells that received 100 pl of medium only, and maximal release in wells where labelled target cells were lysed with 6 M Hcl. Percentage specific lysis was calculated as follows :
mean cpm test - mean cpm spontaneous x 100 __-. mean cpm maximal - mean cpm spontaneous
RESULTS
Bryostatin stimulation of PMN superoxide release
We previously demonstrated the release of superoxide and other oxidants by bryostatin- activated PMN with the chemiluminogenic
probes, luminol and DBA (Warren et al., 1988). Those studies indicated that bryostatin- l-activ-
ated PMNs generated DBA-dependent CL. The kinetics of bryostatin-1 activation were similar
to those for the phorbol ester, TPA. Bryostatin-1 or TPA-activated PMNs generate CL within 2- 3 min and peak responses were attained after
lo-12 min. DBA CL is considered a relatively specific measure of superoxide anion generation (Allen, 1981). Therefore, induction of DBA CL by bryostatin indicates that bryostatin- 1 induces
354 A.H. ESA ET AL.
the generation of superoxide anion in neutro- phils. The release of superoxide anion by bryo- statin activated-neutrophils is further demonstra- ted by the data shown in figure 1, derived from experiments using SOD-inhibitable reduction of ferricytochrome c as an indicator of superoxide anion induction (Babior et al., 1970). Both bryo- statin-l and TPA induced SOD-relative reduc- tion of ferricytochrome c. In contrast to TPA and bryostatin- 1, bryostatin- 13 was incapable of activating neutrophils to reduce ferricyto- chrome c.
The data summarized in figure 2 show that bryostatin-1 also stimulated monocytes to gen- erate CL, but that the response of monocytes was less vigorous in magnitude than that observed for PMNs. Two subsets of peripheral blood monocytes, separable on the basis of size differences by countercurrent centrifugation elu- triation (CCE), were tested. The results indicate that the larger monocyte subpopulation was more active in the generation of CL. These CCE-isolated large monocytes were previously shown to be more active in phagocytosis and the generation of superoxide anions than small
monocytes (Yasaka et al., 1981), and appear to be more responsive to bryostatin activation.
Mechanism of bryostatin-induced CL
The activation of the neutrophil respiratory burst enzyme, NADPH oxidase, results from a complex cascade of metabolic activities. PKC- activating phorbol esters stimulate this key enzyme directly. On the other hand, particulate stimuli, such as opsonized zymosan (STZ), act- ivate the PMN respiratory burst by a pathway apparently linked to phospholipase A, (PLA,) activation and may involve the liberation and metabolism of membrane-bound arachidonic acid (Cheung et al., 1983, Mareidonneau-Parini et al., 1986). To investigate the biochemical mechanism of reactive oxygen radical genera- tion induced by bryostatin, PMNs were prein- cubated for 10 min with inhibitors of PLA,, amino acid metabolism (Kelly et al. 1979 ; Schrezenmeir et al., 1986 ; Vargaftig and Dao Hui, 1972) or protein kinases (Kawamoto and Hidaka, 1984 ; Hidaka et al., 1984 ; Smolen and
TPA + SOD
Bryo 13 + SOD
Control + SOD
0 0.5 1 1.5
Abs 550 nm/3.6E6 PMN/30 min/37C
Fig. 1. Reduction of ferricytochrome c by TPA- or bryostatins-l- or -13-activated PMNs.
The ceils, 3 x 106, were incubated with the agents and ferricytochrome c (50 nmoles) in the absence or presence of 20 pg/ml of SOD. Absorbance at 550 nm was then measured after 30 min.
BRYOSTATIN ACTIVATION OF POLYMORPHONUCLEAR NEUTROPHILS 355
900
600
300
0 0 2 4 6 8
Time (mid
Fig. 2. Temporal generation of DBA CL by bryostatin-activated monocytes.
Human monocytes were separated into two fractions by centrifugation elutriation. Post-Ficoll peripheral blood mononuclear cells were loaded at a flow rate of 16 ml/min into a “Beckman” elu- triator at rotor speed of 3,000 rpm (900 g) and a temperature of 10°C. After lymphocyte removal at a flow rate of 24 ml/min, two monocyte fractions were collected: small monocytes, eluted at a flow rate of 29 ml/mm, and large monocytes eluted after the rotor has been turned off (R/O). The mono- cyte subpopulations, 5~ 106, were then stimulated with 10m7 M bryostatin and DBA CL measured as described in “Materials and Methods”.
Weissmann, 1980 ; Tamaoki et al., 1986) before from these experiments, illustrated in figure 4, exposure to CL inducing stimuli. The results show that H-7 at 60 or 100 l.tM significantly shown in figure 3 indicate that PLA, or AA inhibited both TPA- and bryostatin-induced metabolism inhibition with mepacrine or indo- DBA CL, but that H-8 (fig. 4 top right) could methacin did not affect bryostatin- l-induced not abrogate bryostatin-induced DBA CL when DBA CL (fig. 3A and B). As expected, mepa- used at a concentration as high as 10 PM (the crine, which blocks PLA,, inhibited STZ- inhibitory constants [Ki] of H-S for cGMP or induced PMN DBA CL. In contrast, the results CAMP are 0.48 and 1.2 FM, respectively, and depicted in figure 3C show that H-7, a selective its Ki for PKC is 15 l.tM). In addition, the data inhibitor of PK activation, depressed bryostatin- in figure 4 (bottom) show that staurosporine, a l-induced PMN CL at 60 and 100 l.tM. These more potent PK-C inhibitor with a reported Ki data suggest that PK activity, but not PLA, acti- for PKC lo-fold lower than its Ki for the other vation or arachidonic acid metabolism, was kinases, caused approximately 57 % inhibition necessary for bryostatin activation of PMNs. of DBA CL at a concentration of 1 nM, a dose Further, the data summarized in figure 4 dem- that approximates its reference Ki for PKC, but onstrate that PKC activation, in particular, plays well below its Ki for CAMP or cGMP PK inhi- a role in bryostatin stimulation of neutrophils. bition. These results indicate that PKC, but not In these experiments, the inhibitory effect of H- other protein kinases, plays a role in the gener- 8 and staurosporine on bryostatin-induced DBA ation of superoxide bryostatin and TPA-stimul- CL were compared to that of H-7. The data ated neutrophils.
356 A.H. ESA ET AL.
6.000 my0 only ’ -3
oT 0 5 10 15 20 25
f E6cc4 ii % Pm P z~
0 45 -1.5 1.5 4s I . ! ! 105 1.35 16.5 19s 22.5
rlme (mln)
Time (min)
Fig. 3. Effects of cyclooxygenase, PLA, or PKC inhibitors on DBA CL generation by bryostatin-l- activated PMN.
A) Neutrophils (5x 106) were preincubated for 10 min with 10 pM indomethacin or buffer and stimulated with 10e7 M bryostatin-1 or buffer.
B) Neutrophils were preincubated with 10 pM mepacrine or vehicle and then stimulated with 10m7 M bryostatin or 0.1 mg/ml S’IZ.
C) Neutrophils were preincubated with graded concentrations of H-7 followed by stimulation with bryostatin. DBA CL was measured as described.
Cytostatic effect of bryostatin-1 and of bryosta- tin and PMNs
The ability of bryostatin-1 to inhibit the growth of K562 cells was evaluated by a 24-h thymidine uptake assay. Neutrophils by them- selves did not inhibit the proliferation of these cells. Bryostatin- 1 at 0.1 and 0.01 pM caused sig- nificant reduction in the proliferation of these cells. At both of these concentrations, bryostatin- 1 caused 70-75 % reduction in the proliferation of
K562 cells. The data in figure 5 show the effect of neutrophils in the growth of bryostatin-l- treated cells. The inclusion of graded numbers of neutrophils ranging from 4x lo4 to 3 X 105, along with bryostatin- 1, increased the cytostatic activity of bryostatin. At a concentration of 0.1 pM, bryostatin-1 in the presence of 4x lo4 PMNs caused 90-95 % inhibition of K562 thymidine uptake, increasing the cytostatic effect of bryosta- tin-l by a further 25 %. At the lower bryostatin-1 concentration of 0.01 pM, the same degree of
BRYOSTATIN ACTIVATION OF POLYMORPHONUCLEAR NEUTROPHILS 357
,oo . H-7. Kl-5.8 (COIIIP)
3.0 (CAMP)
6.0 [PK-C) 80 -
,oo _ H-8. Kb 0.43 (&UP)
1.2 (CUP)
15 (PK-C) 80 .
Dose of lnhbito (uU) I
100 - S’SP. Kl-8.5 (COW)
7.0 (CNIIP)
0.7 (m-c)
a0 .
Fig. 4. Comparison of various PK inhibitors on their effects on bryostatin stimulation of neutrophils.
Top left: neutrophils were incubated for 10 min with graded doses of H-7 before exposure to bryostatin-1 or TPA. Top right and bottom: preincubation with H-8 or staurosporine (Stsp), respec- tively, and activation with bryostatin-1. Values shown are percent inhibition of control DBA CL.
inhibition could be achieved with a higher num- ber of PMNs (8x 104).
Induction of PMN tumoricidal activity with bryostatin or TPA
We also performed short-term chromium release assays to determine the cytolytic activ- ity of bryostatin- l-activated neutrophils. Pre-
liminary experiments determined that, although bryostatins caused a neutrophil oxidative burst that peaked within twenty mm, simultaneous incubation of bryostatin with PMNs and tumour cells did not result in enhanced lytic activity of the PMNs. However, neutrophils first incubated with bryostatin for 2 h, washed and then exposed to 5 1 Cr-labelled target cells in microtitre wells showed significant lytic activity against a number of cell lines in vitro.
358 A.H. ESA ET AL.
2XiOE4 K562
I I I 5 10 15
Number of Neutrophils Added (iOE4)
, 20
Fig. 5. Inhibitory effect of bryostatin-1 on the growth of K562 cells in vitro.
Cells, 2x 104, were cultured in diluent, 0.1 or 0.01 pM of bryostatin. Graded numbers were also added. The cells were incubated for 24 h at 37°C in 5% CO, and their proliferative response was measured with thymidine uptake. The inclusion of neutrophils in the absence of bryostatin- 1 did not affect the growth of the cells. The data are presented as mean cpm derived from six samples. Mean cpm of control=86975f9975; this was reduced to 25656k5658 by the addition of bryostatin and further reduced by the addition of neutrophils and bryostatin as depicted. PMNs by themselves did not cause a significant effect (mean cpm 75882+ 13612).
The results shown in figure 6 indicate that K562, Daudi, YAC and to a lesser extent Jurkat cells were sensitive to lysis by the bryostatin- l-activated neutrophils. In these experiments, bryostatin 1 was found to be most effective when used at a concentration of 10m7 M and had little or no activity at 1O-9 M, and the data presented are from experiments using 10e7 M. Neutrophils not treated with bryostatin-1 dis- played negligible lysis against K562 (n = 10, mean % lysis 0.53, range -5.9 to 4.9%) or against the other target cells. Bryostatin-l-activ- ated PMNs were capable of causing significant lysis at an E:T ratio of 15:l (p < .Ol, in com- parison with lysis obtained with unstimulated PMNs by Student’s t test). In all cases, bryosta- tin-l was unable to lyse target cells directly when added in the absence of neutrophils, sug- gesting that the bryostatin-mediated lytic activ- ity required neutrophil activation and was not due to a direct toxic effect of bryostatin on the target cells.
40
Fig. 6. Sensitivity of various tumour cells to bryostatin-l- activated PMNs.
The neutrophils were treated with bryostatin-1 for 2 h, washed and then exposed to SICr-labelled target cells, K562, Daudi, Yac 1 or Jurkat, in round-bottom 96-well plates. Percent specific K562 lysis with neutrophils incu- bated in control medium alone ranged from -5.9 to 4.9 with a mean of 0.53 (n=lO) E:T=15:1.
K562 DAWI YAC J-RKAl
BRYOSTATIN ACTIVATION OF POLYMORPHONUCLEAR NEUTROPHILS 3.59
DISCUSSION
The experiments reported here were designed to study the mechanism(s) by which bryostatins activate mature PMN and monocyte reactive oxygen metabolite release, and to assess whether such activation might contrib- ute to their observed antineoplastic activity by determining the capacity of bryostatin-stim- ulated neutrophils to inhibit the growth of tumour cell lines in vitro. Although bryostatins are structurally unrelated to phorbol esters, our experiments confirm and extend previous reports, which indicate that these agents can functionally activate PMN superoxide genera- tion in a manner similar to TPA (Berkow and Kraft, 1985; Kraft et al., 1986). The genera- tion of DBA CL, as well as the induction of SOD-relative reduction of ferricytochrome c, suggest that bryostatin-activated PMNs released superoxide anions. We have previ- ously determined the generation of luminol- enhanced azide-sensitive CL by bryostatin- activated T cells, indicating that other oxidant species, in particular, myeloperoxidase-cata- lysed HOCl-, are also generated in response to bryostatin (Warren et al., 1988).
Two pathways have been identified for the release of oxidants from PMNs. Particulate stimuli, such as STZ, primarily stimulate neu- trophil NADPH-oxidase through PLA, activa- tion. On the other hand, soluble stimulants, such as phorbol esters that activate PKC, can directly stimulate NADPH-oxidase. Our results show that inhibitors of PLA, or cyclooxyge- nase, mepacrine and indomethacin, respect- ively, did not affect bryostatin-induced neu- trophil CL, indicating that this pathway played only a minor role in the bryostatin activation process. In contrast, selective inhibition of PK activation was effective in blocking CL gener- ation by bryostatin and TPA. Furthermore, bryostatin stimulation of neutrophil oxidant generation results from the activation of PKC and is not dependent on other kinases, since concentrations of inhibitors at the Ki for PKC inhibition, but well below the Ki of other PKs, blocked CL. Even inhibitor concentrations which are greater than the Ki for inhibition of
other protein kinases, but too low for PKC inhibition (e.g. 6.0 pM of H-7 and 10 PM of H-8), were ineffective, further supporting the role of PKC activation in bryostatin-induced CL generation.
The release of reactive oxygen radicals and degranulation are commonly associated with neutrophil microbicidal activity (Baehner et al., 1975). The roles, if any, of these radicals in tumoricidal activity remain unclear. A number of studies demonstrate that a variety of human and murine tumours can be lysed in vitro by activated neutrophils (Clark and Szot, 198 1 ; English and Lukens, 1983). These studies have also suggested a role for the peroxidase-mye- loperoxidase system in the tumoricidal capac- ity of neutrophils. Our results indicate that neutrophils activated with bryostatin- 1 can enhance the growth-inhibiting capacity of bryostatin against K562 in vitro, and in short- term chromium assays are lytic for a number of cell lines.
The significance of bryostatin-induced in vitro tumoricidal activity of PMNs for any in vivo antineoplastic activity remains to be elu- cidated. Although non-specific in their activ- ity, ihese cells possess strong chemotactic action and are rapidly attracted to sites of immunological reactivity where, in concert with antibody, a specific anti-tumour antibody- dependent cell-mediated cytotoxic reaction may take place. Interestingly, PKC activation is also essential for neutrophil chemotaxis (Harvath et al., 1987). Further, bryostatins share some properties with GM-CSF which, after in vivo administration or extended in vitro incubation, primes neutrophils for a more potent superoxide release and antiparasitic effect (Reed et al., 1987; Vadas el nl., 1983). Trenn et al. (1988) report that administration of bryostatin is synergistic with IL4 for the development of non-specific cytotoxic T lym- phocytes and that in vitro incubation with bryostatin enhances the efficacy of IL2-primed CTLs. These properties suggest a useful thera- peutic role for these unique immunomodulat- ing substances and warrant further investiga- tions.
360 A.H. ESA ET AL.
Acknowledgements
The authors wish to thank Julie Megerian for her excellent technical assistance.
This work was supported in part by NIH Grants AI24682 and CA15396 and American Cancer Society grant IM398.
Les bryostatines d&clenchent le m&abolisme oxydatif des polynuclCaires neutrophiles et des monocytes humains : on note une relation avec
I’activit6 antitumorale in vitro
Les premibres mol&ules de bryostatine-1 (bryo- 1) ont CtC isoltes B partir du bryozan marin, Bugulu neritina, il y a plus de dix ans. D’autres formes de bryostatines, actives et inactives, ont tt& dCcouvertes par la suite. Bien que ces bryostatines ne soient pas structurellement apparentkes aux esters de phorbol, elles sont capables de lier la protCine-kinase C (PKC) via un site qui a Ctk dtcrit ulttrieurement. Nous avons mis en Cvidence, de meme que d’autres Cquipes, les propriCtCs antitumo- rales des bryostatines ainsi que leurs effets sur I’hCmatopoiZse. Dans la pr&ente Ctude, nous mon- trons que la production de radicaux oxygtnCs libres par les monocytes et leucocytes neutrophiles humains est induite par la bryo-1, mais non par la bryo-13. Nous avons Cgalement observC que le m&anisme g&&ant des radicaux oxygCnts libres est, dans ce modble, directement 1% & I’activation de la PKC et non aux autres kinases ou 5 la pho- pholipase A, comme c’est le cas au tours de l’opsonisation du zymosan. De plus, nos rCsultats montrent que la bryo-1 stimule les neutrophiles qui, ainsi activts, inhibent in vitro la croissance des cel- lules K562. Ces observations suggbrent que les propriCtCs antitumorales de la bryo-1 peuvent Ctre dues ZI la 1ibCration de superoxydes par les neutro- philes stimults.
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