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Experimental Therapeutics, Preclinical Pharmacology |
Division of Hematology and Oncology, Karmanos Cancer Institute, Wayne State University School of Medicine, Department of Internal Medicine, Detroit, Michigan 48201
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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8%) after
96 h of bryostatin 1 exposure. The in vitro and
in vivo results emphasize the superiority of combining
bryostatin 1 with the CHOP regimen against the WSU-DLCL2
model. One possible mechanism may be the modulatory effects of
bryostatin 1 on the Bax:Bcl-2 family of apoptosis-regulatory proteins.
The use of this combination should be further explored clinically in
the treatment of lymphoma. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One approach to maximize the antitumor effect of certain regimens and possibly improve cure rates is to integrate new agents that have unique modes of action. The National Cancer Institute natural products program has identified a number of novel marine animal products with significant antilymphoid activity. The bryostatins represent one such group. Bryostatin 1, isolated from the marine bryozoan Bugula neritina (11) , exhibited both in vitro and in vivo antitumor activity in a number of model systems (12 , 13) . In addition, bryostatin 1 has biological effects on both T and B lymphocytes as well as on the hematopoietic system (13) . This agent has undergone preclinical evaluation against a variety of human lymphoid tumors and was found to have antitumor, immune modulating, and differentiating effects on a number of B-cell tumors including acute lymphoblastic leukemia (14) , chronic lymphocytic leukemia (15) , NHL (16) , and Waldenstrom’s macroglobulinemia (17) . In this study, we investigated the antitumor effect of the CHOP regimen alone and in combination with bryostatin 1 in a SCID mouse xenograft model bearing the WSU-DLCL2 cell line.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Growth.
WSU-DLCL2 cells were plated in 24-well culture
clusters (Costar, Cambridge, MA) at a density of 2 x
105 viable cells/ml/well. Triplicate wells were
treated with bryostatin 1 (10 nM), cyclophosphamide
monophosphate (C; 5.84 pM), doxorubicin (H; 1.5
pM), oncovin (O; 260 nM), or diluent (control).
Prednisone (P) was used only in the in vivo studies. Plates
were incubated at 37°C in a humidified incubator with 5%
CO2. All cultures were monitored throughout the
experiment by cell count and viability every 24 h for 4 days using
0.4% trypan blue stain (Life Technologies, Inc., Grand Island, NY) and
a hemocytometer.
Apoptosis.
For light microscopic examination, WSU-DLCL2
cells were seeded in 24-well culture plates as described above.
Briefly, untreated (control) and cells treated with bryostatin 1, CHO,
and B+CHO were set in three replications. Aliquots from cell cultures
were cytocentrifuged using a Cytospin II centrifuge (Shandon Southern
Instruments, Sewickley, PA). Cell smears were air dried, stained with
tetrachrome at full concentration for 5 min, and then stained at 50%
dilution with distilled H2O for another 5
min. Slides were analyzed under light microscopy (Nikon, Garden City,
WY). Three hundred cells were counted and analyzed under high power for
viability, mitosis, apoptosis and death. Features of apoptosis looked
for included nuclear chromatin condensation, formation of membrane
blebs, and apoptotic bodies.
WSU-DLCL2-Xenografts.
Four-week-old female ICR-SCID mice were obtained from Taconic
Laboratory (Germantown, NY). The mice were adapted, and
WSU-DLCL2 xenografts were developed as described
previously (10)
. Each mouse received
107 WSU-DLCL2 cells (in
serum-free RPMI 1640) s.c. in each flank area. When s.c. tumors
developed to approximately 1500 mg, mice were sacrificed, and tumors
were dissected and mechanically dissociated into single-cell
suspensions. Mononuclear cells were separated by Ficoll-Hypaque density
centrifugation and washed twice with RPMI 1640. These cells were
subjected to phenotypic analysis for comparison with the established
tumor cell line to insure the human origin and its stability. After
formation of s.c. tumors, serial propagation was accomplished by
excising the tumors, trimming extraneous material, and cutting the
tumors into fragments of 20–30 mg that were transplanted s.c. using a
12-gauge trocar into the flanks of a new group of mice.
Efficacy Trial Design.
For the subsequent drug efficacy trials, small fragments of the
WSU-DLCL2 xenograft were implanted s.c. and
bilaterally into naive, similarly conditioned mice, as described
previously. Mice were checked three times per week for tumor
development. Once transplanted WSU-DLCL2
fragments developed into palpable tumors (100–200 mg), groups of five
animals were removed randomly and assigned to different treatment
groups.
To further extend our work in using bryostatin 1 as an antilymphoma and biological modulating agent, we combined it with CHOP using our WSU-DLCL2-SCID model. Bryostatin 1 (NSC 339555) was provided to us by the National Cancer Institute Division of Cancer Treatment and Diagnosis. In this model, the efficacy of bryostatin 1 given at 75 µg/kg, i.p., alone for 1 or 2 days was compared with that of CHOP alone, B+CHOP given concurrently, bryostatin 1 for 1 day followed by CHOP on day 2, and bryostatin 1 for 2 days followed by CHOP on day 3. CHOP doses were as follows: CHOP was given at MTD times one injection (i.e., cyclophosphamide, 40 mg/kg, i.v.; doxorubicin, 3.3 mg/kg, i.v.; vincristine, 0.5 mg/kg, i.v.; and prednisone, 0.2 mg/kg, p.o.] every day for 5 days. Mice were observed for measurement of s.c. tumors, changes in weight, and side effects of the drugs. The s.c. tumors were measured three times per week. Animals were euthanized when their total tumor burden reached 1500 mg to avoid discomfort. All studies involving mice were performed under Institutional Review Board-approved protocols. Tumor weights in SCID mice were plotted against time on a semilog sheet with the growth pattern resembling an S shape. Td is the time (in days) required for the tumor to double its weight during the exponential growth phase.
Assessment of Tumor Response.
The end points for assessing antitumor activity were established
according to standard procedures used in our laboratory (10
, 15
, 17)
and are as follows: (a) tumor weight (mg) =
(A x B2)/2, where
A and B are the tumor length and width (in mm),
respectively; (b) tumor growth inhibition (T/C) is
calculated by using the median tumor weight in the treated group (T)
when the median tumor weight in the control group (C) reached
approximately 900 mg. Tumor growth delay (T - C) is the
difference between the median time (in days) required for the treatment
group tumors (T) to reach 700 mg and the median time (in days) for the
control group tumors (C) to reach the same weight; and (c)
tumor cell kill net (log10) = (T -
C) - (duration of treatment in days)/(3.32)(Td). In this study,
the antitumor activity is considered highly active (++++) when the
log10 kill (net) is >2.0. Activity rating scores
of ++++ or +++ are needed for translation to clinical activity and
equate with complete and partial tumor regression, respectively. A
score of either + or ++ is not considered active by usual clinical
criteria (18)
.
Western Blot Analysis.
WSU-DLCL2 cells from bryostatin 1-treated SCID
mice (75 µg/kg, i.p., for days 1, 2, and 3) and cultures (10
nM, for days 1, 2, 3, and 4) or controls were washed twice
with 1x PBS, resuspended in Triton X-100 lysis buffer [300
mM sodium chloride, 50 mM Tris-HCl (pH 7.6),
5% Triton X-100, and protease inhibitors], and kept at 4°C for at
least 45 min. Cells were centrifuged at 14,000 x g for
10 min, and the supernatant containing the cytosolic extract was saved.
The protein concentration was determined using the Micro BCA protein
estimation kit (Pierce, Rockford, IL). For Western analysis, 20 µg
from each sample were separated on a 12% SDS-polyacrylamide gel and
transferred to nitrocellulose membranes. The membranes were blocked for
1 h in PBST (5% nonfat dry milk solution in PBS with 0.1% Tween
20). Membranes were then incubated in a 1:1000 concentration of Bax,
Bcl-2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and/or PARP
(Travigen, Gaithersburg, MD). The membranes were washed in PBST twice
and exposed to horseradish peroxidase-conjugated secondary antibodies
(Santa Cruz Biotechnology, Inc.). The proteins were visualized using
the enhanced chemiluminescence reagents (Amersham International Ltd.,
Buckinghamshire, United Kingdom).
7AAD Staining and Flow Cytometry.
7AAD (Calbiochem-Novabiochem, La Jolla, CA) was dissolved in acetone,
diluted in PBS to a concentration of 200 mg/ml, and kept at -20°C
protected from light as described previously (15)
.
Briefly, 100 µl of 7AAD solution were added to 1 x
106 cells, suspended in 1 ml of PBS, and mixed
well. WSU-DLCL2 cells protected from light were
stained for 20 min on ice. Cells were pelleted, the supernatant was
removed, and the pellet was washed twice with PBS. Samples from
bryostatin 1-treated cells or untreated (control) cells were
analyzed by flow cytometry (FACScan; Becton Dickinson, Mountain View,
CA). Data were acquired on 20,000 cells and processed using Lysys II
software (Becton Dickinson). Scattergrams were generated by combining
forward light scatter with 7AAD fluorescence.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). However, the effect of adding bryostatin 1 to CHO proved
to inhibit the cell growth by greater than 100% (Fig. 1
A).
These results highlight the superiority of B+CHO over bryostatin 1 or
CHO alone. It is noteworthy that prednisone (P) was used only in the
in vivo studies.
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B). Again, these results confirm the advantage of adding
bryostatin 1 to CHO chemotherapy.
In Vivo Efficacy of Bryostatin 1.
When SCID mice received s.c. injection in each flank of equal numbers
of WSU-DLCL2 cells (1 x
107), 80% of the animals developed tumors. The
tumors in each flank were palpable by the third week. Dt in SCID mice
was 2.0 days. When WSU-DLCL2 was passaged in SCID
mice, the take rate was 100%. Drug efficacy trials were conducted on
animals with palpable tumors (tumors of approximately 200 mg).
Table 1
shows the antitumor activity of bryostatin 1, when given at 75 µg/kg
i.p., as one and two injections, CHOP alone, or B+CHOP given at MTDs,
against WSU-DLCL2-bearing SCID mice. Tumor growth
inhibition (T/C), tumor growth delay (T - C), and
log10 kill were 49%, 39%, 25.8%, 15.1%,
14.6%, and 12%; 6, 7, 16, 25, 12, and 15 days; and 0.6, 0.5, 2.2,
3.6, 1.7, and 2.0, respectively. T/C values are used to determine tumor
response. Bryostatin 1 is considered inactive when given as one
injection (T/C = 49%), all other treatments are considered active
against this type of human tumor (T/C values of 
42% are indicative
of antitumor activity). However, if log10 kill
net values are added as a criterion, only B+CHOP is clinically
considered highly active. All others were considered active by usual
clinical criteria. It should be noted that an activity rating score of
+++ (active) or ++++ (highly active) was needed to effect partial or
complete tumor regression. Thus, a score of + or ++ is not considered
active by the usual clinical criteria (18)
. The mean and
range of tumor weights varied dramatically among various treatments.
Animals treated with CHOP or B+CHOP showed the best tumor weight
and range. The smallest tumor (54 mg) was seen in the B+CHOP treatment
group, with a range of 0.0–171 mg. Tumor growth patterns of control,
bryostatin 1, CHOP, and B+CHOP are shown in Fig. 2
. B+CHOP showed a significant (P = 0.028) tumor
growth delay compared with all other treatments and demonstrated a much
better survival rate.
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A shows Bcl-2, Bax, and PARP expression in
WSU-DLCL2 before and after treatment with
bryostatin 1. Bryostatin 1 alone was unable to induce changes in the
expression of Bcl-2 protein, which is highly expressed in
WSU-DLCL2 cells. The proapoptotic Bax protein,
which is not highly expressed in control cells, increased in a
time-dependent manner after exposure of WSU-DLCL2
cells to bryostatin 1 (10 nM) for 24, 48, and
72 h. This modulation in the Bax:Bcl-2 protein ratio is important
for potentiating cells to undergo apoptosis. Cleavage of the PARP is
also an important indicator of apoptosis induction. PARP, which is
found as a Mr 116,000 band, was
dramatically cleaved 24 h after bryostatin 1 treatment to its
Mr 85,000 catalytic form. The parent
Mr 116,000 form is conserved over the
course of this study, as is the Mr
85,000 form. These data indicate that exposure to bryostatin 1 induced
PARP cleavage, the molecular marker for apoptosis.
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B shows Bcl-2, Bax, and PARP expression in
WSU-DLCL2 tumors excised from
WSU-DLCL2-SCID xenograft mice. High levels of
Bcl-2 and Bax protein expression are found in tumors from control mice,
and PARP is significantly cleaved from its
Mr 116,000 form to the catalytic
Mr 85,000 form. Tumors taken from mice
after 1, 2, and 3 days of bryostatin 1 show reduced Bax protein at days
1, 2, and 3, whereas the Bcl-2 protein level decreases at days 1 and 2
but increases to control level on day 3. A significant increase in the
Mr 116,000 PARP protein is recorded
over days 1, 2, and 3 with a concomitant decrease in the
Mr 85,000 form (Fig. 3
B).
Induction of Apoptosis in WSU-DLCL2 Cells by Bryostatin
1.
Typical fluorescence-activated cell-sorting scattergrams of
control and bryostatin 1-treated WSU-DLCL2 cells
are shown in Fig. 4
. The figure shows three regions defined by 7AAD staining:
(a) 7AAD-negative (viable cells, bottom);
(b) 7AAD-dim (early to mid-apoptotic cells,
middle); and (c) 7AAD-bright (late-apoptotic or
dead cells, top). The percentage of apoptotic cells in the
bryostatin 1-treated cells was insignificant compared with the control.
However, extending the incubation period of the
WSU-DLCL2 cells to 96 h did result in an 8%
increase in the percentage of apoptotic cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Two decades ago, using the CHOP combination of agents, 30–40% of
advanced-stage DLCL was found to be curable (19)
. Although
numerous attempts were made to improve the cure rate, no further
progress has been made (3
, 6)
. The
WSU-DLCL2 cell line was established from a
relapsed DLCL patient who was clinically resistant to therapy
(10)
. The disease relapsed after high-dose chemotherapy
and radiation, followed by bone marrow transplantation. In the in
vitro study, the WSU-DLCL2 cell line was
exposed to bryostatin 1 alone, CHO alone, and B+CHO (Fig. 1
A). The results highlighted the obvious superiority of
B+CHO over bryostatin 1 or CHO alone. B+CHO induced more in
vitro cell death compared with bryostatin 1 or CHO alone (Fig. 1
B).
The ability of these cells to grow as xenografts in SCID mice makes it a useful preclinical model to search for effective drugs against resistant lymphoma and to explore not only mechanisms of cell killing but mechanisms of resistance. Among the large number of marine invertebrate-derived anticancer agents, bryostatin 1 was selected for clinical development against hematopoietic disease. To further extend our work using bryostatin 1 as an antilymphoma and chemotherapy-modulating agent, we combine it with CHOP in the WSU-DLCL2-SCID model. In this study, we used the model of advanced disease, i.e., therapy was started when palpable tumors have developed (2–3 weeks after transplantation). CHOP was given at its one-time injection MTD as described previously, and all animals survived treatment without death or undue toxicity (<10% weight loss).
Tumor growth patterns of control, bryostatin 1, CHOP, and B+CHOP are
shown in Fig. 2
. B+CHOP showed significant (P = 0.028)
tumor growth delay compared with all other treatments and demonstrated
a much better survival rate. We concluded from this study that adding
one dose of bryostatin 1 (at MTD) to the CHOP regimen (at the MTD for
SCID mice) is well tolerated and improved the single-agent antitumor
activity. Animals were observed for toxicity, and their s.c. tumors
were measured. Animals were euthanized when their total tumor burden
reached 1500 mg (10% of body weight) to avoid discomfort. The end
points of study were tumor growth inhibition (T/C), tumor growth delay
(T - C), and log10kill. Table 1
shows the
antitumor activity of CHOP or B+CHOP given at the MTD against
WSU-DLCL2-bearing SCID mice.
When tumor responses are determined by the T/C value, bryostatin 1 is
considered inactive when given as one injection (T/C = 49%). All
other treatments are considered active against this type of human tumor
(a T/C value of 42% is indicative of antitumor activity). However,
when log10 kill net values are added as
criterion, only B+CHOP is clinically considered highly active, whereas
the others were considered active by usual clinical criteria. It should
be noted that an activity rating score of +++ (active) or ++++ (highly
active) was needed to effect partial or complete tumor regressions.
Thus, a score of + or ++ is not considered active by usual clinical
criteria (18)
. The mean and range values of tumor weights
varied dramatically among various treatments. CHOP or its combination
with bryostatin 1 showed the best mean tumor weight and range.
The smallest tumor (54 mg) was seen in the B+CHOP treatment group
(range, 0.0–171 mg). Previously, we have shown that the highest
activity in this model was demonstrated by vincristine given
sequentially after bryostatin 1 (10)
. Bryostatin 1
potentiates the antitumor activity of vincristine in
WSU-DLCL2-SCID xenograft mice, with bryostatin 1
sensitizing WSU-DLCL2 cells to the effect of
vincristine because the reverse sequence of vincristine followed by
bryostatin 1 did not increase apoptosis (10)
. In a
separate experiment, our data showed that treatment giving bryostatin 1
two days after CHOP did not show better antitumor activity compared
with either CHOP alone or B+CHOP (data not shown).
It has been shown in some types of NHLs and in acute myeloid leukemia that Bcl-2 down-regulation augmented sensitivity to chemotherapeutic agents (20, 21, 22) . However, other studies have demonstrated that in pediatric acute lymphoblastic leukemia, high levels of Bcl-2 are not correlated with resistance to cytotoxic drugs (23 , 24) . Clearly, expression of other Bcl-2-related genes, presumably Bax, may modulate the effect of Bcl-2 itself and influence whether or not cells will undergo apoptosis.
The Bax gene encodes a Mr 21,000 protein that shares considerable amino acid homology with Bcl-2 and is capable of binding to the latter protein (25 , 26) . Thus, Bax can form Bcl-2:Bax heterodimers as well as Bax:Bax homodimers, the latter being preferentially formed when the level of Bax exceeds that of Bcl-2. Formation of Bcl-2:Bax heterodimers appears to inhibit the antiapoptotic function of Bcl-2, whereas the Bax:Bax homodimers contribute to initiation of apoptosis (27) . Thus, higher levels of Bax relative to Bcl-2 after a death signal may increase the cell susceptibility to apoptosis.
WSU-DLCL2 cells taken from in vitro
(Fig. 3
A) or from excised tumor (Fig. 3
B) express
high levels of Bcl-2 protein and a low level of Bax. Bryostatin 1
caused a gradual increase in Bax expression but did not change the
expression of Bcl-2. Bryostatin 1 induced marginal cleavage of PARP
from its Mr 116,000 form to its
Mr 85,000 form, which has been shown
to result in apoptosis (28)
. Marginal apoptosis induction
was also recorded (8%) using a flow cytometry method that takes
advantage of DNA strand breaks being recognized and bound by 7AAD (Fig. 4)
.
Collectively, the results obtained from this work highlight the obvious superiority of B+CHOP over CHOP or bryostatin 1 alone and begin to elucidate some of the mechanisms behind the potentiating effects of bryostatin 1. The results from this study should prove useful in guiding the clinical application of these novel agents in the treatment of lymphomas.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by NIH Grant CA79837 and
Leukemia Society of America Grant 6323-97. The Flow Cytometry
Facility is supported in part by the NIH Grant P30 CA22453-20. 
2 To whom requests for reprints should be
addressed, at Division of Hematology and Oncology, Wayne State
University School of Medicine, P. O. Box 02143, Detroit, MI 48201.
Phone: (313) 577-7919; Fax: (313) 577-7925; E-mail: Mohammad{at}karmanos.org
3 The abbreviations used are: NHL, non-Hodgkin’s
lymphoma; CHOP, cyclophosphamide, doxorubicin, vincristine, and
prednisone; DLCL, diffuse large cell lymphoma; SCID, severe combined
immunodeficient; PARP, poly(ADP-ribose) polymerase; CHO,
cyclophosphamide monophosphate, doxorubicin, and oncovin; MTD, maximum
tolerated dose; Td, tumor doubling time; 7AAD, 7 amino-actinomycin D;
B+CHO, bryostatin 1 + CHO; B+CHOP, bryostatin 1 + CHOP.
Received 4/21/00; revised 9/14/00; accepted 9/14/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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