(CANCER RESEARCH 51, 682-687, January 15, 1991]
Successful Treatment of Murine Melanoma with Bryostatin Il
Lynn M. Schlichter,2 Ahmed H. Esa, W. Stratford May, Mary K. Laulis, George R. Pettit, and Allan D. Hess
The Johns Hopkins Oncology Center, Baltimore. Maryland 21205
marrow progenitor cells (3-5). Bryostatins bind to PKC' (6),
ABSTRACT
Bryostatins are a novel class of protein kinase C activators which
were isolated from the marine bryozoan Bugula neritina and found to
possess both antineoplastic and immunoenhancing properties. In this
report, we examined the relationship between the in vivo and in vitro
antineoplastic effects of bryostatin 1. The in vivo antitumor activity of
bryostatin 1 was demonstrated in a B16 melanoma pulmonary métastases
model, in which treatment of tumor-bearing C57BL/6 mice with 5 days
of bryostatin 1 resulted in a significant reduction in the number of lung
nodules (control, 87; bryostatin, 7). There was a clear dose-response
effect, with the optimal antimelanoma dose being 100 Mg/kg/day, but
even low doses of bryostatin 1 of 1 /ig/kg/day resulted in a 53% reduction
in the number of métastases.Although bryostatin 1 shares many biolog
ical properties with the phorbol esters, parallel treatment with 12-0tetradecanoyl 13-phorbol acetate was ineffective against B16 melanoma
in vivo. Using a clonogenic assay, bryostatin 1 was found to have a direct
antiproliferative effect against B16 melanoma. This inhibition occurred
at relatively high bryostatin 1 concentrations ( 10 " M), in comparison
with a sensitive cell line REH (IO '" M). Treatment of mice with bryo
the cellular receptor for phorbol esters, but unlike the phorbol
esters, which are tumor promoters, bryostatins lack tumorpromoting properties and under certain conditions can inhibit
tumor promotion mediated by phorbol esters (7).
In view of the unique activities of bryostatins, including their
potent immunomodulatory effects and antineoplastic proper
ties, we have evaluated the effects of bryostatin 1 against B16
melanoma, a tumor for which immune mechanisms play an
important role in its eradication. Our results described here
show that bryostatin 1 is effective against B16 melanoma in
vivo.
MATERIALS
AND
METHODS
statin 1 or preincubation of normal spleen cells with bryostatin 1 failed
to enhance nonspecific cell-mediated cytotoxicity against B16 melanoma
in vitro. Moreover, bryostatin 1 was found to inhibit both natural killer
cell activity and interleukin 2 generation of lymphokine-activated killer
cells. Thus, a role for an in vivo immune enhancement mechanism as the
basis for the antimelanoma activity observed with bryostatin 1 cannot be
invoked from these experiments. These findings indicate that bryostatin
1 may act directly on the B16 melanoma pulmonary métastases.The
precise mechanism whereby bryostatin exerts its antimelanoma effects
remains unclear.
Animals. Female C57BL/6 mice were purchased from The Jackson
Laboratory (Bar Harbor, ME) and were housed under sterile conditions.
Mice were 8-10 weeks of age when used for experiments.
Tumor Cell Lines. The B16 murine melanoma cell line was obtained
from the Frederick Cancer Research Facility and grown as an adherent
monolayer. The NK cell-sensitive Moloney virus-induced YAC-1 lymphoma cell line and REH, a human leukemia cell line, were grown as
suspensions (8). Cells were maintained in culture using Eagle's minimal
essential medium, supplemented with 10% heat-inactivated fetal calf
serum, nonessential amino acids, vitamins, I HIMsodium pyruvate, and
1 miviL-glutamine.
Treatment. Bryostatin 1 was isolated from the marine bryozoan
Bugula neritina as previously described (1). Stock bryostatin 1 was
maintained at —20°C
and, when needed, was resuspended in PBS with
INTRODUCTION
DMSO (<0.1%) just before use. TPA was purchased from Sigma
Chemical Co. (St. Louis, MO), dissolved in DMSO, and diluted in
PBS. The final concentration of DMSO was <0.1%. rIL-2 produced in
Escherichia coli was kindly supplied by Cetus Corporation (Emeryville,
CA). Each vial contained 1.5 x IO6 units of rIL-2 as a lyophilized
powder. Sterile water was used to reconstitute the product.
Therapeutic Experiments. B16 tumor cell suspensions were prepared
by trypsinization. The cells were washed 3 times with Hanks' balanced
Although major advances have been made in the treatment
of many malignancies, malignant melanoma remains refractory
to current therapies. New approaches are needed to treat mel
anoma and other resistant cancers. Bryostatin 1 belongs to a
newly discovered family of macrocyclic lactones isolated from
the marine bryozoan Bugula neritina which possess diverse
biological properties. Bryostatins have documented in vitro
antineoplastic properties against a number of cancer cell lines,
including melanoma, renal cell, and leukemia (1). In addition,
prolongation of survival in bryostatin 1-treated animals has
been reported in a murine P388 lymphocytic leukemia system
and in an ovarian sarcoma model (2). Other biological proper
ties of bryostatins include potent immunomodulatory effects,
including activation of T-cells and augmentation of neutrophil
and monocyte tumor cytotoxicity, and stimulation of bone
salt solution. Cell count and viability were determined by trypan blue
dye exclusion. On day 0, mice were given injections of 5 x IO4 cells/
0.2 ml PBS i.v. using the tail vein. This number of tumor cells estab
lishes approximately 100 microscopic melanoma pulmonary modules
by day 3 and macroscopic melanoma pulmonary foci after 12-15 days
in untreated mice. Three days after tumor injection, animals were
randomized into treatment groups, including bryostatin 1, TPA, and
rIL-2. All treatments were given as i.p. injections in 0.5 ml. The dose
of rIL-2 administered was 25,000 units every 8 h (9). Control animals
received PBS with 0.1% DMSO (diluent) in the same manner as the
treated animals. All treatments began on day 3 following tumor injec
tion and continued for 5 days. The mice were monitored daily for
morbidity and mortality. On day 14 after tumor inoculation the mice
were sacrificed. The melanoma pulmonary métastasesappeared as
discrete black nodules formed on the surface of the lungs. The lungs
were removed and melanoma nodules were counted in a blinded fashion
with the aid of dissecting microscope.
Preparation of Murine Splenocytes. C57BL/6 mice were sacrificed
and their spleens were aseptically removed, pooled, and passed through
Received 8/14/89; accepted 10/29/90.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by Grants CA-15396, ST32CA-09071, and ROI CA47993 from
the National Cancer Institute and IN-11-28 from the American Cancer Society.
W. S. M. and A. D. H. are Scholars of the Leukemia Society of America.
1 Present address: Hospital of the University of Pennsylvania, 3400 Spruce
Street, Division of Hematology/Oncology, 6 Penn Tower, Philadelphia, PA
19104. To whom requests for reprints should be addressed.
3The abbreviations used are: PKC, protein kinase C; NK, natural killer; LAK,
lymphokine-activated killer; DMSO, dimethyl sulfoxidc; PBS, phosphate-buff
ered saline; rIL-2, recombinant interleukin 2; IL-2, interleukin 2; TPA, 12-Otetradecanoyl-13-phorbol acetate.
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ANTIMELANOMA
EFFECTS OF BRYOSTATINS
Table 1 Treatment of melanoma pulmonary métastases
C57BL/6 mice were given i.v. injection of 5 x IO4 tumor cells; therapy was
initiated 3 days later and continued for 5 days.
of
métastases87
13.1°7±
±
DoseDiluentBryostatin
Agent
12.2*°
1
75 ßg/kgNo.
Mean ±SD.
" P value < 0.0001.
a mesh screen with the blunt end of a 10-ml syringe plunger. The cell
pellet was obtained and the RBC were lysed with Tris-NH4Cl. The cell
suspension was then centrifuged over fetal calf serum and washed 3
times in PBS. These effector cells were incubated for various time
periods with bryostatin 1 (1 x 10~8 M) and/or rIL-2 (1000 units/ml)
prior to cytotoxicity assays.
Induction of LAK Cells. C57BL/6 murine splenocytes (5 x IO6cells/
ml) were cultured in 25 ml of medium (as above) in 75-cm2 flasks in
the presence of rIL-2 (1000 lU/ml) (9), with or without bryostatin 1 (1
x 10~" M), for 3 days at 37°Cin a 5% CO2 atmosphere. LAK cell
cytotoxicity was measured against B16 melanoma cells using a chro
mium release assay.
Assay for Cytotoxicity. A 4-h chromium release assay was performed
using standard techniques (10). Targets included Yac-1 tumor cells and
B16 melanoma cells. Cells (1 x IO4) labeled with 100 Mlof 51Cr were
placed into each well, along with effector cells. Effectors consisted of
splenocytes from control or tumor-bearing C57BL/6 mice that were
incubated for 2 or 72 h with bryostatin 1, rIL-2 (LAK cells), or
bryostatin 1 and rIL-2. Effectors were plated in triplicate at three
different effector to target cell concentrations, 100:1, 40:1, and 10:1.
Maximum and spontaneous "Cr release were obtained by adding 0.1
ml of l N HC1 or 0.1 ml of medium, respectively, to wells with target
cells. Percentage of specific 5'Cr release was calculated according to the
formula:
% specific 51Cr release = cpm experimental —cpm spontaneous
cpm maximal - cpm spontaneous
Clonogenic Assay. The tumor cell clonogenic assays were performed
by culturing 500 to 1000 B16 melanoma cells or REH cells/ml in 0.3%
agar, bryostatin 1 (1 x 10~'°to 1 x 10~6 M), 20% fetal calf serum,
McCoys's medium 5A. One ml of cells with agar mixture was plated in
35-mm Petri dishes. The assays were performed in quadrupicate. The
plates were scored after 7 days of incubation for B16 melanoma cells
and after 18 days for REH cells, using an inverted microscope. A colony
was considered an aggregate of 50 or more cells.
Histológica! Procedures. For histológica! examination of in vivo tu
mors and tissues, animals were sacrificed and the relevant tissues were
removed aseptically and placed in formalin. The tissues were then
sectioned and stained with hematoxylin and eosin and reviewed in
consultation with a pathologist.
Statistical Analysis. For the in vivo experiments, the significance of
differences in the number of pulmonary métastasesbetween groups was
determined by a Wilcoxon rank test. In vitro results were analyzed for
statistical significance by analysis of variance and Student's t test.
0.0001). Histológica! examination of the lungs revealed the
presence of tumor cell foci in sections. Both experimental and
control lungs exhibited modest mononuclear cell infiltration;
however, the degree of cellular infiltration was not different
between the two groups. The size of the remaining nodules in
bryostatin 1-treated mice was significantly smaller. There was
no evidence of necrosis and the residual nodules were pigmented
with melanin.
The data in Fig. 1 indicate the inhibitory dose-response effect
of bryostatin 1. The maximal reduction in the number of
pulmonary nodules was achieved at a bryostatin 1 dose of 100
¿ig/kg/day(95% reduction). At 50 ¿¿g/kg/day,
bryostatin 1 re
sulted in a 76% reduction in nodules, and even at 1 ¿tg/kg/day,
the lowest dose evaluated, bryostatin 1 had a significant antimelanoma effect when compared to the control treatment (P <
0.01). Not only was the number of nodules in bryostatin 1treated animals effectively reduced, but the size of the residual
nodules also was smaller in the bryostatin 1-treated animals,
when compared to the nodules in the control animals receiving
diluent (Fig. 2). These results suggest that an optimal antimelanoma dose of bryostatin 1 is approximately 100 /ug/kg/
day. Of note, animals treated with doses greater than 50 Mg/kg
bryostatin 1 developed a transient clinical syndrome consisting
of diarrhea, weight loss, and oliguria. These toxicities were not
encountered at the lower dose levels. Bryostatin 1 doses greater
than 200 Mg/kg were uniformly lethal within 24 h of adminis
tration. Autopsy studies in these animals failed to reveal a
definite cause of death.
Effect of Bryostatin 1 and TPA on Established B16 Melanoma
Pulmonary Métastases.Because bryostatin 1 shares many bio
logical effects with the phorbol esters, we were interested in
comparing the antimelanoma effects of bryostatin 1 to those of
phorbol esters. C57BL/6 mice were inoculated with B16 mel
anoma cells on day 0. On days 3-7 the mice were treated with
either 100 /¿g/kg/day bryostatin 1, 100 ¿ig/kg/day TPA (a
phorbol ester), the combination, or diluent. As shown in Fig.
3, bryostatin 1 significantly reduced the number of pulmonary
nodules. In contrast, there was no antimelanoma effect observed
following TPA treatment. The combination of both agents
administered simultaneously was highly toxic, since all animals
120
o
o
100
o
2
0.
RESULTS
Effect of Bryostatin 1 on B16 Melanoma Pulmonary Métas
tases. To establish whether bryostatin 1 could mediate regres
sion of established B16 melanoma pulmonary métastases,
C57BL/6 mice were given i.v. injections of tumor cells. Therapy
with bryostatin 1 was initiated on day 3 following tumor injec
tion and continued for 5 days. On day 14 animals were sacri
ficed, lungs were harvested, and pulmonary nodules were enu
merated. As shown in Table 1, bryostatin 1 significantly reduced
the number of pulmonary nodules, compared to the control
animals that received diluent (PBS with 0.1% DMSO) (P <
u.
o
Diluent
1
10
50
100
BRYOSTATIN l (UG/KG/DAY)
Fig. 1. Antimelanoma activity of bryostatin 1. Dose-dependent effect of daily
administration of bryostatin 1 to animals with B16 melanoma nodules. C57BL/
6 mice were given injections of 5 x 10" B16 melanoma cells. On days 3 through
7, mice were treated with the indicated doses of bryostatin 1. The number of
macroscopic nodules was counted and the mean values are reported (n = 10).
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ANTIMELANOMA
EFFECTS OF BRYOSTATINS
Fig. 2. Photograph of the lungs from melanoma-bearing animals. C57BL/6 mice were treated as in Fig. 1. Left, control animal receiving diluent injections on days
3-7. Righi, animal receiving bryostatin 1 (100 pg/kg/day) on days 3-7.
TPA
TREATPuENT
DILUENT
BRYO
BRYO * L-2
Fig. 3. Effects of experimental therapies on pulmonary nodules. C57BL/6 mice were given injections of 5 x l O4B16 melanoma tumor cells. On days 3 through 7,
mice were treated as follows. Left, bryostatin 1 (BRYO) (100 /jg/kg/day), TPA (100 ng/kg/day). or diluent (n = 8 for each treatment group). Right, bryostatin 1 (50
/jg/kg/day), rIL-2 (25,000 units every 8 h), bryostatin 1 and rIL-2 (same doses), or diluent (n = 6 for each treatment group). On day 14, animals were sacrificed and
pulmonary métastaseswere counted. Values shown are the mean number of nodules.
expired within 24 h of the treatment. Autopsy studies did not
reveal a definite cause of death.
Effect of Bryostatin 1 and rIL-2 on B16 Melanoma Pulmonary
Métastases.Early reports have demonstrated synergy between
bryostatin 1 and IL-2 in terms of T-cell activation and cytotoxicity (11). Therefore, we evaluated whether IL-2 could also
enhance the antimelanoma effect of bryostatin 1. Animals were
given injections of tumor, as described. Tumor-bearing mice
were treated with bryostatin 1 (50 ¿/g/kg),bryostatin 1 (50 /zg/
kg) plus rIL-2 concurrently, rIL-2 alone, or diluent, beginning
on day 3, for 5 days as per protocol. Bryostatin 1 was given
once a day while the rIL-2 (25,000 units) was given 3 times a
day. As shown in Fig. 3, animals treated with bryostatin 1 had
an 86% reduction of pulmonary métastases,while rIL-2 therapy
alone caused only a 27% reduction. Combination of bryostatin
1 and rIL-2 resulted in a 27% reduction of pulmonary nodules,
indicating no enhanced antitumor effect for this combination.
In fact, the data suggest that rIL-2 interfered with or antago
nized the antimelanoma effect of bryostatin 1, since equivalent
doses of bryostatin 1 resulted in a decreased therapeutic effect
when combined with rIL-2. Additional experiments were con
ducted to evaluate synergy between rIL-2 and bryostatin 1. For
those experiments, tumor-bearing mice were sequentially
treated with bryostatin 1 followed by rIL-2 (i.e., 5 days of rlL2 followed by 5 days of bryostatin 1 or vise versa). No enhanced
antitumor effect for the combination was observed (data not
shown).
Effect of Bryostatin 1 on Growth of B16 Melanoma Cells in
Vitro. To examine whether the antineoplastic property of bryo
statin 1 in whole animals results from a direct antiproliferative
effect, a clonogenic assay was performed. B16 melanoma cells
and REH cells, a line sensitive to bryostatin 1 in culture, were
cultured in 0.3% agar in the presence of increasing concentra
tions of bryostatin 1 from 1 x 10~'°M to 1 x 10~" M. Fig. 4
250
200
150
MELANOMA
REH
100
50
Diluent
10-10
BRYOSTATIN
10-9
10-8
10-7
CONCENTRATION
10-6
(molar)
Fig. 4. Tumor colony inhibition by bryostatin 1. Effects of bryostatin 1 on the
growth of B16 melanoma (500 cells) and REH (1000 cells) in a clonogenic assay.
Colonies were incubated with bryostatin 1 or diluent, cultured for 7 days, and
then counted. The mean number of colonies/plate are reported.
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ANTIMELANOMA
EFFECTS OF BRYOSTATINS
summarizes the data. B16 melanoma cells were resistant to the
inhibitory effects of bryostatin 1 at concentrations ranging from
10~'°M to 10~7 M. At 10~6 M bryostatin 1, the highest concen
tration tested, there was a 97% reduction in melanoma colonies
(6 colonies in the bryostatin 1-treated plates versus 176 colonies
in the control plates, P< 0.001). A significant antiproliferative
effect for bryostatin 1 was observed against REH cells over the
concentration range tested, demonstrating the exquisite sensi
tivity of this pre-B-cell line. These data indicate that bryostatin
1 can have a potent direct inhibitory effect on B16 melanoma
clonogenic cell growth at 1 x 10~6 M. Thus, in a clonogenic
assay, there is evidence for a direct antiproliferative effect of
bryostatin 1 against B16 melanoma.
In Vitro Cytotoxic Activity of Mononuclear Cells against B16
Melanoma Cells. B16 melanoma has been shown previously to
be sensitive to cell-mediated immune responses and immune
interventions in vivo (12-14). We were, therefore, interested in
examining whether the suppression of melanoma pulmonary
nodules in vivo by bryostatin 1 could potentially result from
enhanced lymphocyte cytotoxicity. In the first set of experi
ments, we evaluated the capacity of C57BL/6 splenocytes in
cubated i/i vitro with bryostatin 1 to lyse melanoma or Yac-1,
an NK-sensitive cell line. The data, shown in Fig. 5, indicate
that bryostatin 1 did not enhance splenocyte cytotoxicity
against B16 melanoma. As expected, Yac-1 target cells were
sensitive to lysis by normal murine splenocytes. This lytic
activity, however, was inhibited when splenocytes were incu
bated with bryostatin 1.
Since splenocytes incubated with bryostatin I were unable to
mediate lysis of B16, we examined whether bryostatin 1 treat
ment i/i vivo could generate an antimelanoma effect in vitro.
Thus, spleen cells obtained from C57BL/6 mice which had been
treated with bryostatin 1 (75 jugAg) or control diluent daily for
5 days were tested for in vitro antimelanoma cytotoxicity. The
percentage of lysis at effector to target ratios of 100:1, 40:1,
and 10.1 was 8%, 6%, and 6%, respectively, which indicates
that bryostatin 1 treatment in vivo was unable to enhance
splenocyte-mediated antimelanoma activity in vitro. These re
sults were not different whether splenocytes were isolated from
tumor-bearing or non-tumor-bearing animals (data not shown).
Finally, we sought to determine the effect of bryostatin 1 on
the generation of LAK cell activity in vitro. Splenocytes were
cultured in the presence of rIL-2 (1000 units/ml) (positive
control) and/or bryostatin 1 (1 x 10~8 M) for 3 days, and
cytotoxicity against B16 melanoma was assessed. This concen
tration of bryostatin was utilized because previous studies have
shown this to be the optimal concentration for activation of
lymphocytes (3). The data are shown in Fig. 5. IL-2, but not
bryostatin 1, induced LAK cell activity from murine splenocytes
when tested against B16 melanoma targets. Nevertheless, the
addition of bryostatin 1 inhibited the ability of rIL-2 to generate
LAK cell activity, since splenocytes treated with bryostatin 1
plus rIL-2 displayed no lytic activity against B16 melanoma.
DISCUSSION
The results from this study reveal that short term treatment
of animals with bryostatin 1 causes a dramatic reduction in the
number of established pulmonary melanoma métastases.Dose
escalation studies clearly demonstrate an antitumor dose-re
sponse effect for bryostatin 1, with 100 ^g/kg/day being the
most effective dose (Fig. 1). The sensitivity of this tumor in
vivo to bryostatin 1 is exquisite, apparent even at very low
nontoxic doses of 1 /ug/kg/day. This finding suggests that
combining bryostatin 1 with other anticancer therapies may be
easily accomplished without significant toxicity.
The primary question raised by our results is the mechanism
of bryostatin's therapeutic effect. Our results demonstrate that
there is significant direct inhibitory effect on the growth of B16
melanoma cells in culture (Fig. 4), but only at the highest
concentration tested. This is consistent with previously pub
lished data, in which direct antiproliferative effects of bryostatin
1 were shown against a number of cancer cell lines (1, 15). The
concentration of bryostatin 1 necessary to achieve in vitro
antiproliferative effects against B16 melanoma, however, is
substantially higher than that required to inhibit other sensitive
cell lines (1). This suggests that direct antiproliferative activity
of bryostatin 1 in vivo may not be entirely responsible for the
antineoplastic effect observed. The ability to monitor serum
and intracellular levels of bryostatin 1 would help to clarify the
differences in the concentrations of bryostatin 1 necessary for
the in vivo and in vitro antitumor effect, but many technical
limitations in performing these assays have not yet been re
solved.
Previous reports have confirmed the important role of im
mune mechanisms in the inhibition of B16 melanoma tumor
growth. Biological response-modifying agents such as interferon and rIL-2, as well as cell-mediated cytotoxicity by LAK
cells, have all been reported to inhibit B16 melanoma in vivo
and i/i vitro (12-14). Recent reports have confirmed that bryo
statin 1 has potent immunoenhancing properties (11). Data
from our laboratory demonstrate that bryostatin 1 causes acti
vation of T-cells and can induce the expression of functional
IL-2 receptors on human CD4+ and CD8+ lymphocytes (3).
Other investigators have reported that bryostatin 1 can stimu
late resting T-cells to proliferate and differentiate into cytotoxic
T-lymphocytes (11).
Based upon these data, we investigated whether such immune
mechanisms might be involved in the observed antimelanoma
100:1
10:1
effect of bryostatin 1. In a series of experiments, we tested
EFFECTOR
RATIO
whether bryostatin 1 could enhance cell-mediated cytotoxicity
Fig. 5. Cell-mediated cytotoxicity against B16 melanoma of splenocyte effector
cells incubated for 2 or 72 h with bryostatin 1 (I x 10~8 M) (Bryo), rIL-2, or
against B16 melanoma. However, our experiments indicate
diluent. Spleen cells were obtained from 8-10-week-old C57BL/6 mice, and
that, at least in the in vitro setting, bryostatin 1 does not
single-cell suspensions were treated as indicated. These cells were then washed 3
stimulate an antimelanoma cell-mediated immune response.
times and tested for cytotoxic activity against "Cr-labeled B16 melanoma, as
described in "Materials and Methods."
Thus, C57BL/6 splenocytes incubated with bryostatin 1 for 2
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ANTIMELANOMA
EFFECTS OF BRYOSTATINS
to 72 h fail to mediate cytotoxicity against B16 melanoma (Fig.
5). In addition, bryostatin 1 was also found to inhibit NK cell
cytotoxicity (Fig. 6), because normal splenocytes treated in vitro
with bryostatin 1 had lower NK activity than untreated control
cells. As has been previously reported, splenocytes which are
cultured in the presence of rIL-2 cause proliferation and gen
eration of LAK cells that can lyse a variety of tumor cells,
including B16 melanoma (9). This was confirmed in our studies,
as indicated in Fig. 5. As the data in Fig. 5 indicate, however,
bryostatin 1 interfered with the generation of LAK cells. Sple
nocytes incubated with IL-2 showed pronounced lytic activity
against B-16, but this was strongly inhibited by the inclusion of
bryostatin 1. Thus, bryostatin 1 has dual effects on immune
function, stimulating T-cell proliferation and cytotoxicity while
inhibiting LAK and NK cell activity. This is consistent with the
data recently reported by Nishimura et al. (16), indicating that
agents that activate PKC can interfere with the generation of
LAK cells by IL-2 but enhance IL-2-induced cell proliferation
and IL-2 receptor expression on lymphocytes. As suggested by
these studies, it is possible that bryostatin 1 treatment causes
PKC desensitization or down-regulation of the PKC receptor
on lymphocytes (17). This may impede any PKC-dependent
signal transduction necessary for bryostatin 1 effects.
We also evaluated whether rIL-2 and bryostatin 1 in combi
nation could enhance the antimelanoma effect of bryostatin 1.
Treatment of mice with rIL-2 alone caused only a marginal
reduction in melanoma pulmonary métastases.Moreover, com
bining rIL-2 and bryostatin 1 was apparently antagonistic,
because animals receiving this combined treatment had a
greater number of pulmonary nodules than mice treated with
equivalent doses of bryostatin 1 alone (Fig. 3). This antagonistic
effect cannot be explained by the available data, although a
similar inhibitory interaction has been previously reported by
Trenn et al. (11), who found that low concentrations of bryo
statin 1 enhanced IL-2 cytotoxicity but high concentrations of
bryostatin 1 either had no effect or inhibited IL-2 cytotoxicity.
Previous studies have reported that bryostatins share some
biological properties with phorbol esters. Both bryostatin 1 and
TPA are equipotent activators of PKC in mediating phosphorylation of cell surface protein receptors, including the transferrin receptor, and in activating T-cells and neutrophils (3, 6, 11,
18). These findings suggest that the biological effects of bryo
statin 1 are similar to those of phorbol esters and may be
mediated through activation of PKC, although marked differ
ences in the biological effects of these two agents have been
reported (19, 20). Bryostatin 1 blocks phorbol ester-induced
differentiation of certain cell lines (21), possess antineoplastic
effects (22), and can mimic certain stimulating effects of hematopoietic growth factors (5) and, whereas phorbol esters are
potent tumor promoters, bryostatins lack both first and second
stage tumor-promoting activity (7). Both types of agents can
inhibit cell growth, but the growth-inhibitory effects of TPA
and other phorbol esters against some cell lines appears to be
associated with their activation of PKC and induction of ter
minal differentiation (23). The mechanism whereby bryostatin
1 exerts its antiproliferative activity remains unknown.
Given these effects, we thought it necessary to compare
bryostatin 1 with TPA in terms of an antimelanoma effect. Our
data reveal that treatment of animals with bryostatin 1 for 5
days consistently reduced the number of established pulmonary
métastases,whereas treatment with TPA at equivalent doses
had no effect on tumor growth (Fig. 3). The data from these
experiments further support the divergent effects of TPA and
bryostatin 1 on tumor cell growth. Whether the antimelanoma
activity of bryostatin 1 is independent of PKC activation or
perhaps involves the differential activation of PKC isozymes
(24) is unknown at present but remains an intriguing topic for
further studies.
In conclusion, our results demonstrate that bryostatin 1 can
have direct antiproliferative effects in vitro against B16 mela
noma. Since bryostatin 1 can also inhibit B16 melanoma tumor
growth in vivo, it is likely that this is, at least in part, the result
of a direct effect. Furthermore, in our experimental setting we
could not find any evidence that bryostatin 1 can stimulate
nonspecific cell-mediated cytotoxicity against B16 melanoma
in vitro, despite the potent immunoenhancing properties of
bryostatin 1 (3, 11). Although bryostatins share many biological
properties with phorbol esters, our data demonstrate certain
divergent biological effects of bryostatin 1 and phorbol esters.
Whether the significant therapeutic effects of bryostatin 1 result
solely from a direct antiproliferative effect is unknown. It is
also possible that indirect immunomodulatory effects (i.e., re
lease of tumor necrosis factor or other cytokines), eytotoxic Tlymphocytes, or other factors may be involved in the antimelanoma effect observed in vivo. Further investigation into
the mechanism of action of these new agents will likely lead to
improvements in the treatment of human cancers such as mel
anoma.
REFERENCES
1. Pettil, Ci. R.. Herald, S. L.. Doubck, D. L.. Arnold. E., and Clardy. .1.
Isolation and structure of bryostatin 1. J. Am. C'hem. Soc., 104:6X46-6848,
Diluent
Bryo
o
at
100:1
40:1
EFFECTOR:TARGET
20:1
RATIO
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Successful Treatment of Murine Melanoma with Bryostatin 1
Lynn M. Schuchter, Ahmed H. Esa, W. Stratford May, et al.
Cancer Res 1991;51:682-687.
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