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Advances in Brief |
Departments of Gastrointestinal Medical Oncology [B. W., Q. X., Q. S., X. L., J. L. A., K. X.], and Cancer Biology [K. X.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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,
-ß, and -
. They are produced by many types of cells in response to
various stimuli and have antiproliferative and differentiation-inducing
effects and an immunomodulatory capacity, which are important for their
potential applications as therapeutic agents for treating tumors. These
effects have been demonstrated in many tumor cell lines in
vitro and in animal models (1, 2, 3, 4, 5)
. In clinical
trials, the benefits of IFN therapy are strongly correlated with
the intensity of the treatment. However, systemic administration of low
doses of IFNs is ineffective, whereas high doses of IFNs, which produce
beneficial antitumor effects, may inevitably produce dose-dependent
systemic side effects, which are intolerable for the patients and cause
the IFN therapy to fail (6
, 7)
. Recent studies
indicated that a local delivery of IFN-ß produced strong antitumor
activity without significant side effects, and the localized antitumor
activity correlates with the production of NO
(8)
. NO is a pleiotropic potent molecule that mediates diverse activities (9, 10, 11, 12) . Its influence on tumor growth and metastasis is highly circumstantial because of its nature as a double-edged sword (9, 10, 11, 12) . Intensive activation of NOS3 II and overproduction of NO in tumor cells and/or tumor stromal cells may suppress tumor growth and metastasis (11 , 12) , whereas low levels of NO may do the opposite (10 , 11) . Furthermore, tumor-associated NO is a result of the activities of NOS from both tumor and host-infiltrating cells (12) . The functional NOS II status of tumor-associated macrophages or other infiltration cells and the availability of NOS II stimuli may differ from tumor to tumor, which apparently correlate with different expression levels of NOS II in different tumors (12) . Therefore, the ultimate effect of tumor-associated NOS II activity on tumor growth and metastasis may be dictated by multiple sources and levels of NOS II expression. In general, macrophages produce much higher levels of NO than do tumor cells or other host cells and thus are the main source of NO production (9 , 12 , 13) . Our recent study indicated that genetic disruption of the host NOS II gene differentially affects tumor growth and metastasis. In B16 melanoma cells, expression of host NOS II apparently promotes growth and metastasis, whereas growth and metastasis of M5076 cells are inhibited by expression of host NOS II (14) . The sensitivity to NO-mediated cytotoxicity correlates with the outcome. However, it remains unclear whether a further up-regulation of NOS II expression produces pronounced protumor or antitumor activity.
In this study, we demonstrated that local production of IFN-ß suppressed the growth and metastasis of a highly metastatic murine pancreatic adenocarcinoma, and the antitumor activity was at least in part dependent on the persistent induction of NOS II gene by IFN-ß, thus providing the first evidence that host-derived NO was necessary for IFN-ß-mediated antitumor activity.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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(specific
activity, 1 x 107 units/mg
protein) was purchased from Genzyme (Cambridge, MA). All reagents used
in tissue culture were free of endotoxins as determined using the
Limulus amebocyte lysate assay (sensitivity limit of 0.125
ng/ml) purchased from Associates of Cape Cod (Woods Hole, MA).
PANC02-H7 Cells and in Vitro Culture Conditions.
PANC02 murine pancreatic adenocarcinoma cell line was originally
established by Corbett et al. (15)
by
implanting cotton thread-carrying 3-methyl-cholanthrene into the
pancreas of C57BL/6 mice followed by serial s.c. transplantation
and was generously provided by Dr. James A. Nelson (The University of
Texas M. D. Anderson Cancer Center). The highly metastatic PANC02-H7
cell line was established using an in vivo selection method
(16)
. All tumor cell lines were cultured in RPMI 1640
supplemented with 10% fetal bovine serum, sodium pyruvate,
nonessential amino acids, L-glutamine, and a
2-fold vitamin solution (Flow Laboratories, Rockville, MD). The cell
cultures were maintained in plastic flasks and incubated in 5%
CO2-95% air at 37°C. Cultures were free of
Mycoplasma.
Tumor Growth and Metastasis.
To prepare tumor cells for inoculation, cells in the exponential growth
phase were harvested by brief exposure to a 0.25% trypsin/0.02% EDTA
solution (w/v). Cell viability was determined by trypan blue exclusion,
and only single-cell suspensions >95% viable were used. To evaluate
the tumor growth and metastasis, 0.05 ml of tumor cell suspensions
(1 x 105 cells/mouse) was
orthotopically injected into the pancreas of anesthetized syngeneic
NOS II+/+ or NOS
II-/- C57BL/6 mice (Jackson Laboratory, Bar
Harbor, ME) as described previously (16
, 17)
. The animals
were sacrificed 30 days after tumor implantation or when they became
moribund. Primary tumors in the pancreas, metastases to liver or other
organs, and the amount of ascites were determined as described
previously (14)
.
NO-mediated Cytostasis.
Mouse peritoneal exudate macrophages were collected by peritoneal
lavage from mice given an i.p. injection of 1.5 ml of thioglycollate
broth (Baltimore Biological Laboratories, Cockeysville, MD) 4 days
before harvesting. Macrophages and PANC02-H7 cells (1 x 105 and 1 x 104 cells/well in 96-well plate, respectively)
were cocultured at 37°C in 0.2 ml of medium or medium containing 10
units/ml IFN- and 1 µg/ml LPS in the presence or absence of 1
mM aminoguanidine. Sixty h after cell seeding, 0.1
µCi/well of [3H]thymidine was added.
Free [3H]thymidine was removed 12 h
later, cells were lysed by 0.1 N NaOH, and
[3H]thymidine incorporation was monitored in a
beta counter (8)
. The cytostasis was calculated according
to the formula: cytostasis (%) = [1 - (B/A)] x 100, where A is
the cpm of the coculture in medium, and B is the cpm of the
coculture in medium containing IFN-, LPS, and/or AG.
Determination of NO Production.
NO production in vivo was determined by measuring
nitrate/nitrite in the serum and ascites after conversion of nitrate
into nitrite by nitrate reductase (8)
. NO production
in vitro was determined by measuring nitrite accumulation in
culture supernatants. Nitrite was measured using a microplate assay
with Griess reagent (1.0% sulfanilamide, 0.1% naphthylethylene
diamine dihydrochloride, and 2.5%
H3PO4) as described
previously (12)
. In brief, 50-µl samples were allowed to
react with an equal volume of Griess reagent at room temperature for 10
min. The absorbance at 540 nm was monitored with a microplate reader.
Nitrite concentration was determined by using sodium nitrite as a
standard.
Analysis of NOS II and IFN-ß Gene
Expression.
NOS II and IFN-ß mRNA expression was determined by
Northern blot analysis essentially as described previously
(8)
.
Statistical Analyses.
The in vitro data were analyzed for significance by using
Student’s t test (two-tailed), and the in vivo
data were analyzed for significance by using the Kruskal-Wallis test.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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, parental PANC02-H7 (H7-P) and control vector-transfected
H7-N1, -N2, -N3, and -N4 cells did not express IFN-ß mRNA, whereas
IFN-ß-transfected H7 (H7-ß1, -ß2, -ß3, and -ß4) cells
expressed IFN-ß mRNA. The activity of IFN-ß secreted from the tumor
cells was determined as described previously (8)
. The
IFN-ß-transfected H7-ß1, -ß2, -ß3, and -ß4 cells produced
active IFN-ß ranging from 3 x 103 to 7 x 103 units/106 cells/24 h
(Fig. 1B)
.
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, all control tumors grew progressively in the pancreas and produced
numerous liver metastases and large amount of ascites. In contrast,
IFN-ß-transfected cells grew poorly in the pancreas and rarely
produced liver metastasis and ascites. These data indicated that
enforced expression of IFN-ß strongly suppress growth, metastasis,
and ascites formation of the highly aggressive pancreatic
adenocarcinoma.
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, all control tumors grew progressively in the pancreas and produced
numerous liver metastases and ascites in both NOS
II+/+ and NOS II-/- mice,
whereas a slight increase in the growth of these cells was seen in NOS
II-/- mice (statistically not significant). In
contrast, IFN-ß-transfected cells grew poorly in the pancreas and
rarely produced liver metastasis and ascites in NOS
II+/+ mice, whereas the same tumor cells grew
much better in NOS II-/- mice (statistically
significant). These data indicated that disruption of NOS II
gene impaired the antitumor activity of enforced expression of IFN-ß.
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, and NO production was determined by measuring
nitrate/nitrite in the ascites and/or serum (Fig. 2B)
. A low
level of NOS II expression was observed in the control tumors growing
in the pancreas and ascites of NOS II+/+ mice,
but not in the control tumors of the NOS
II-/- mice. The increased level of NOS II
expression was obvious in the small tumor of H7-ßp cells in the
pancreas and ascites of NOS II+/+ mice; no NOS II
was detected in the tumors of either H7-Np or H7-ßp cells in NOS
II-/- mice. These data suggested that H7-Np
cells themselves expressed a very low level of NOS II gene,
whereas H7-ßp cells stimulated NOS II expression in NOS
II+/+ mice. NOS II expression in the tumor lesion
(tumor section) and ascites (smear) was further confirmed by
immunostaining using anti-NOS II and antimacrophage antibodies analysis
of NOS II protein. NOS II was clearly expressed in the infiltration
cells e.g., macrophages but not in the tumor cells (data not
shown).
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and 1 µg/ml LPS in the presence or absence of 1
mM AG. NO production was determined 24 h after
coculture, and cytotoxicity was determined 72 h after coculture.
As shown in Fig. 3A and B
, macrophages activated with IFN-/LPS
produced high levels of NO and cytostasis. H7-ßp cells
stimulated NO production in macrophages and cytotoxicity in the
presence of LPS, whereas control H7-Np did not. Furthermore, H7-ßp
cell-stimulated NO was totally inhibited by the addition of
IFN-ß-neutralizing antibody or specific NOS II inhibitor, AG (Fig. 3C)
. In contrast, IFN-ß-secreting H7-ßp cells did not
induce NO production and cytostasis in macrophages from NOS
II-/- mice (Fig. 3D)
. These data
suggested that IFN-ß was responsible for NO production, and NO was
responsible for macrophage-mediated cytotoxicity.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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IFNs can potentially influence tumor growth through various mechanisms. It has been shown in several in vitro systems that IFNs are antiproliferative through affecting different phases in the mitotic cycle (1, 2, 3, 4, 5, 6, 7) . IFNs may also act by antagonizing the function of various growth factors or by inhibiting the genes regulated by the growth factors (18 , 19) . In many of these in vitro studies, tumor cells die after exposure to IFNs. In the present study, the IFN-ß-producing tumor cells grew equally well as control tumor cells under in vitro conditions (data not shown), which suggests that they were resistant to autocrine or paracrine antitumor activity of IFN-ß, and the direct antiproliferative effect of IFN-ß on tumor cells was minimal. However, those resistant tumor cells did not grow well in vivo, which suggests that an indirect antitumor effect of IFN-ß occurred. This observation was consistent with several previous reports (18, 19, 20, 21, 22, 23) that show that IFNs have indirect effects on tumor growth through modulating host immune functions, the tumor stromal cells, or the vascularization of the tumor. For example, IFNs have been shown to regulate cell surface molecules, which may regulate cell growth by influencing the adhesion of malignant cells to stromal components or immune cells. IFNs may also inhibit tumor angiogenesis through the suppression of gene expression such as basic fibroblast growth factor (24) , matrix metalloproteinases (25) , and interleukin-8 (26 , 27) . Recent studies (8) have further suggested that IFN-ß can elicit a strong local nonspecific antitumor activity, which was presumably mediated by NO.
To provide direct evidence for the role of NO derived from host stromal cells in IFN-ß-mediated antitumor activity, highly metastatic pancreatic cancer cells, which did not express a significant level of NOS II (data not shown), were injected into the pancreas of syngeneic C57BL/6J mice with or without intact NOS II gene. Although IFN-ß-secreting cells were poorly tumorigenic and metastatic in wild-type mice, they produced larger tumors and more metastasis in NOS II-/- mice. The increased tumor growth and metastasis were correlated with absent expression of NOS II gene in the infiltration macrophages. These data directly indicated that NOS II expression and NO production were involved in IFN-ß-mediated antitumor activity. Therefore, this finding provides a new mechanistic insight into how to maximize the therapeutic effect of IFN-ß. It is also predicted that IFN-ß-related systemic side effect is most likely caused by the systemic production of NO. It remains to determine whether NO-related systemic side effect can be minimized without affecting IFN-ß-elicited and NO-dependent therapeutic effect.
The effect of NO derived from host cells on tumor growth and metastasis
appears to depend on the levels of NO production and cellular status of
tumor cells. In general, the intensity of NO-mediated cytotoxicity
depends on the intrinsic sensitivity of tumor cells and level of NO
production (10, 11, 12, 13, 14)
. Our recent study has shown that the
disruption of host NOS II gene promotes metastasis of
NO-sensitive M5076 cells but inhibits metastasis of NO-resistant B16
melanoma (14)
. Therefore, sensitivity to NO-mediated
cytotoxicity may be responsible for the differential ability to grow
and metastasize by IFN-ß-secreting cells and control cells. To test
this possibility, IFN-ß-secreting cells and control cells were
cocultured with LPS/IFN--activated macrophages. There was no
significant difference in LPS/IFN--activated macrophage-mediated
NO-dependent cytotoxicity (Fig. 3)
under in vitro
conditions. Furthermore, the tumor cells were also treated with NO
donors, i.e., S-Nitroso-N-acetyl-DL-penicillamine.
Surprisingly, IFN-ß-secreting cells showed a slightly decreased
sensitivity to NO-mediated cytotoxicity as compared with that of
control tumor cells (data not shown). The relevance of NO donors to the
macrophage-derived NO remains unclear. However, IFN-ß-secreting
cells elicited a cytotoxicity by LPS-primed macrophages, whereas the
control cells did not. The cytotoxicity was abrogated by either NOS II
inhibitor, AG, or an IFN-ß-neutralizing antibody, which suggests that
IFN-ß released from tumor cells induce NOS II expression and NO
production in LPS-primed macrophages (Fig. 3)
. Therefore,
IFN-ß-secreting and control tumor cells have similar sensitivity to
NO-mediated cytotoxicity but a different capacity to induce NO
production in host cells.
It is known that the level of NO production by tumor cells or tumor
infiltration cells depends on the interaction between the tumor cells
and host cells. The source of NO can be tumor cells and/or host cells.
Tumor cells may not be the major source of NO because both control
cells and IFN-ß-secreting cells did not express detectable levels of
NOS II. NO may be produced from host cells, which is consistent
with our previous observations (12, 13, 14)
, showing that
tumor cells stimulate the NOS II expression in host cells. To test this
possibility, both control cells and IFN-ß-secreting cells were
cocultured with host macrophages, which have been shown to be the major
source of NO production (14)
. Control cells and
IFN-ß-secreting cells did not induce NO production in macrophages
under in vitro conditions, whereas IFN-ß-secreting cells
did in LPS-primed macrophages. However, elevated NOS II expression was
clearly observed in the tumor formed by IFN-ß-secreting cells as
compared with the control tumor (Fig. 2)
. These data suggested that
IFN-ß released from tumor cells induces NOS II expression and NO
production from host cells, e.g., macrophages in
vivo conditions. This may be attributable to the presence of other
cytokines, e.g., interleukin 1 and tumor necrosis factors,
which can synergize with IFN-ß for NO induction (8)
.
Elevated NO production may produce diverse effects on tumor growth and metastasis. Upon interaction between tumor cells and stromal cells, a well-controlled expression of NOS II may benefit the growth and metastasis of tumor cells (10, 11, 12) . In this report, a strong antitumor activity was produced by an elevated expression of NOS II in the host stromal cells, which may be attributable to sustained NO induction by persistent stimulation of IFN-ß secreted from tumor cells. Therefore, a switch from a beneficial level of tumor-associated NO to a destructive level of NO production can suppress tumor growth and metastasis.
In summary, we demonstrated that localized high concentration of IFN-ß suppressed tumor growth and metastasis, in part through induction of NO from host cells such as macrophages. Our data further suggest a hypothesis that tumor-associated NO production can be switched toward to a vigorous antitumor activity from a potentially beneficial status through manipulating NOS II expression.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Lustgarten Pancreatic Cancer
Research Foundation, Research Project Grant #RPG-00-054-01-CMS from the
American Cancer Society, and Cancer Center Support Core Grant CA 16672
from the National Cancer Institute, NIH (to K. X.). 
2 To whom requests for reprints should be
addressed, at the Department of Gastrointestinal Medical Oncology, Box
78, The University of Texas M. D. Anderson Cancer Center, 1515
Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-2013; Fax:
(713) 745-1163; E-mail: kepxie{at}mail.mdanderson.org
3 The abbreviations used are: NOS, NO synthase;
LPS, lipopolysaccharide; AG, aminoguanidine.
Received 8/28/00. Accepted 11/15/00.
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REFERENCES |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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. Proc. Natl. Acad. Sci. USA, 91: 1198-1205, 1994.-interferon on epidermal growth factor-mediated receptor-dependent events. Cancer Res., 53: 5148-5157, 1993. and ß down-regulate the expression of basic fibroblast growth factor in human carcinomas. Proc. Natl. Acad. Sci. USA, 92: 4562-4566, 1995., -ß, and -. Arch. Dermatol. Res., 281: 11-18, 1989.[Medline]
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