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Advances in Brief |
B Activation1
Departments of Surgery [J. C. C.] and Biology [A. S. B.], Lineberger Comprehensive Cancer Center [J. C. C., R. L., A. S. B.], Curriculum in Genetics and Molecular Biology [A. S. B], University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7210
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ABSTRACT |
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B (NF-B), and that this
leads to suppression of the apoptotic potential of the chemotherapy. In
contrast, it was reported recently that stable inhibition of NF-B in
four different cancer cell lines did not lead to augmentation of the
chemotherapy-induced apoptosis. In this study, we have focused on
colorectal cancer, which is known to be highly resistant to genotoxic
chemotherapy and gamma irradiation. We show that the topoisomerase I
inhibitor
7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin
(CPT-11) activates NF-B in most colorectal cancer cell lines. We
then examine a therapeutic strategy that uses adenovirus-mediated
transfer of the super-repressor IB
to inhibit NF-B activation
as an adjuvant approach to promote chemosensitivity in colorectal tumor
cells to treatment with CPT-11. These data demonstrate that the
protection from apoptosis induced in response to CPT-11 treatment is
effectively inhibited by the transient inhibition of NF-B in a
variety of human colon cancer cell lines and in a tumor xenograft
model, resulting in a significantly enhanced tumoricidal response to
CPT-11 via increased induction of apoptosis. These findings indicate
that the activation of NF-B by chemotherapy is an important
underlying mechanism of inducible chemoresistance. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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B3
(1)
has been shown to regulate apoptosis in several
different settings (reviewed in Ref. 2
). Importantly,
several groups have shown that NF-B is activated by exposure of
cells to chemotherapy (reviewed in Ref. 3
). Recently, we
studied the human fibrosarcoma cell line HT1080 and showed that the
activation of NF-B by the chemotherapeutic agent daunorubicin
strongly suppressed the apoptotic potential of this chemotherapy
(4)
. Furthermore, tumors derived from HT1080 cells were
induced to undergo apoptosis and regression when animals were treated
with the topoisomerase I inhibitor CPT-11 in parallel with adenoviral
delivery of a modified form of IB, the natural inhibitor of
NF-B. These data argued that the activation of NF-B by
chemotherapy provides an important mechanism for inducible
chemoresistance. In apparent contrast with these studies, it was
reported recently (5)
that four different cell lines that
stably express the modified form of IB did not show enhanced
cytotoxicity in response to chemotherapy, although the chemotherapies
activated NF-B. We report here continued studies on the role of
NF-B in controlling inducible chemoresistance, and we report a
therapeutic regimen involving the inhibition of NF-B that leads to
dramatic antitumor responses. Additionally, we show that the stable
inhibition of NF-B via IB expression is not a consistent
experimental approach to test the role of NF-B in chemoresistance.
To extend our original studies, we have focused on colorectal cancers.
Like most solid tumors, colorectal cancers are frequently resistant to
chemotherapy and irradiation (6)
. Several of the cellular
mechanisms that determine sensitivity of cancer cells to genotoxic
therapies have been elucidated recently (7, 8, 9, 10)
. For
example, one mechanism for chemoresistance is the up-regulation of the
multidrug resistance gene product (MDR1), which is responsible for
pumping chemotherapy agents from the cells (11)
. Other
mechanisms of resistance appear to affect the ability of a cancer cell
to undergo apoptosis, the major mechanism by which chemotherapy and
radiation induce the killing of tumor cells (12, 13, 14)
.
Thus, mutation in the p53 tumor suppressor gene leads
to acquired resistance that impairs p53-dependent responses to
apoptotic stimuli and may promote cancer cell survival and
proliferation in some cancer cells (15)
. Another important
mechanism for resistance to chemotherapy is inducible chemoresistance,
a process whereby exposure of tumor cells to cancer therapy leads to
their resistance to apoptosis (11)
. As described above, we
have proposed that a major mechanism for inducible chemoresistance is
the up-regulation of the transcription factor NF-B.
The objectives of this report are to determine whether the principle of
reversing inducible chemotherapy resistance, as a means of enhancing
the apoptotic response to chemotherapy treatment, can be applied
broadly to the treatment of colorectal cancer in preclinical models.
Specifically, these experiments explore whether the topoisomerase I
inhibitor CPT-11, a chemotherapy showing promise for cancers of
different tissue origin (6
, 16, 17, 18, 19, 20)
, induces activation of
NF-B in a variety of human colorectal cancer cells. We also examine
whether transient inhibition of NF-B activation concurrent with
CPT-11 exposure enhances the anticancer effect of CPT-11 among
different colorectal cancer cell lines, and several of the cell lines
used by Bentires-Alj et al. (5)
. Finally, in an
effort to determine the optimal dosing schedule necessary to maximize
tumoricidal response in a colon cancer xenograft model, we examine a
variety of treatment schedules that combine inhibition of NF-B
activation with CPT-11 administration. It is hoped that the results of
these studies will contribute to the design and implementation of a
novel therapeutic approach that improves patients’ responses to
systemic treatment for metastatic colorectal cancer as well as other
forms of cancer.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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B
expresses the super-repressor form of IB that is mutated at
serine residues 32 and 36 and functions as a potent and specific
repressor of NF-B-mediated events (4
, 22)
. The control
vector Ad.CMV3, generously provided by J. A. Roth (University of Texas
M. D. Anderson Cancer Center, Houston, TX), contains a CMV promoter
similar to Ad.CMV.IB but lacks a transgene insert. Studies of
transduction efficiency of the cell lines used in these experiments
indicate that moderate levels of transgene expression may be
accomplished using a MOI ranging from 20 to 100 (data not shown).
Chemotherapy Agents.
Camptothecin is a specific inhibitor of mammalian DNA topoisomerase I.
The camptothecin analogue CPT-11 and its active metabolite SN38 were
generously provided by J. Malczyn (Pharmacia and Upjohn Co., Kalamazoo,
MI).
Cell Culture.
The human colon cancer cell line LOVO was obtained from ATCC
(Rockville, MD). The LOVO cells were grown in F-12 (Ham) with 20% FBS.
The colon cancer cell lines SW1463, SW837, SW620, and SW480 were
obtained from ATCC and grown in L-15 with 10% FBS. The colon cancer
cell lines KM12-L4 and KM12-SM (generous gifts of J. Fidler, The
University of Texas M. D. Anderson Cancer Center, Houston, TX) and
HT-29, WiDR, and CL188 (obtained from ATCC) were grown in MEM with 10%
FBS. The colon cancer cell line CCD841 (ATCC) was grown in DMEM with
10% FBS, and NCI H508 was grown in RPMI with 10% fetal bovine serum.
All media were obtained from Life Technologies, Inc. (Gaithersburg, MD)
and supplemented with 100 µg/ml penicillin G and 100 µg/ml
streptomycin. MCF-7 and HCT116 were obtained from ATCC and grown in
Eagle’s MEM with 10% FBS and McCoy’s 5A medium with 10% FBS,
respectively. Cell cultures were maintained at 37°C.
Cell Growth Inhibition.
Human cancer cells (5–8 x 104)
were seeded in six-well plates and infected with Ad.CMV.IB at a
MOI of 20 per target cell when cells reached 20% confluence. The
control adenovirus vector Ad.CMV3 was used to infect the control group.
Drug treatment with SN38, the active metabolite of CPT-11, was
administered 24 h after virus infection at a final concentration
of 1000, 500, or 100 ng/ml. Daily cell counts were performed for 4
days. Experiments were performed in triplicate.
NF-B Activation Assay.
Activation of NF-B in response to treatment with chemotherapy was
determined by the EMSA as described previously (23)
. For
in vitro experiments, cancer cells were cultured in 100-mm
dishes until 50–70% confluence was achieved. Cells were infected with
Ad.CMV.IB (MOI, 100) for 1 h and then washed with PBS and
refed medium. Cells were treated with SN38 (1 µg/ml) 24 h after
adenovirus infection. Cells were then harvested at times 0, 1, 2, and
6 h after treatment with SN38. Nuclear extracts were prepared by
collecting cells and then washing and suspending them in hypotonic
buffer. The nuclear pellet was separated by centrifugation, and the
cytoplasmic supernatant was discarded. The nuclei were then resuspended
in a low-salt buffer to high-salt buffer, and the soluble protein was
released by centrifugation, collected, and stored at -70°C. The DNA
probe used contains an NF-B site (underlined) from the
H-2b gene
(5'-CAGGGCTGGGGATTCCCATCTCCACAGTTTCACTTC-3'; Ref.
3
). In brief, 7 µg of nuclear extracts were preincubated
with 1 µg of poly(deoxyinosinic-deoxycytidylic acid) in binding
buffer (10 mM Tris, 50 mM
NaCl, 20% glycerol, 0.5 mM EDTA, and 1
mM DTT) for 10 min at room temperature.
Approximately 20,000 cpm of 32P-labeled DNA probe
was then added and allowed to bind for 15 min. The complexes were then
separated on a 5% polyacrylamide gel and autoradiographed.
The procedure for obtaining nuclear extracts from cancer cells was modified for tumor samples as follows. s.c. tumors were harvested after treatment and snap-frozen in liquid nitrogen. Frozen tumor tissues were morselized over liquid nitrogen and scraped into 10-ml conical tubes containing 5 ml of solution A [0.3 M sucrose, 60 mM KCl, 15 mM NaCl, 15 mM HEPES (pH 7.5), 2 mM EDTA, 0.5 mM EGTA, 14 mM 2-ME, and 0.1% NP40]. The mixture was ground into a slurry, and the nuclear solution was layered. On the top of the slurry, 2.5 ml of solution B [0.88 M sucrose, 60 mM KCl, 15 mM NaCl, 15 mM HEPES (pH 7.5), 2 mM EDTA, 0.5 mM EGTA, and 14 mM 2-ME] were added, and the mixture was spun down for 10 min at 3000 rpm. The pellet was brought up to 1 ml of solution D [1 M sucrose, 15 mM KCl, 15 mM NaCl, 15 mM HEPES (pH 7.5), 0.1 mM EDTA, 0.1 mM EGTA, and 14 mM 2-ME] and layered on top of 2.5 ml of solution D and then spun down at 3000 rpm for 10 min. The supernatant was aspirated, and the pellet was resuspended in NE buffer [20 mM Tris (pH 8.0), 120 mM NaCl, 1.5 mM MgCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 25% glycerol] with proteinase inhibitor (10 µg/ml leupeptin, 25 µg/ml aprotinin, and 5 µg/ml pepstatin). The mixture was incubated on ice for 10 min and then spun down at full speed for 15 min at 4°C. The nuclear protein remaining in solution was then collected.
In Vivo Evaluation of Tumor Growth.
The ability of the super-repressor IB to enhance sensitivity to
CPT-11 was assessed in a LOVO xenograft model. The tumors were
established by injecting 5 x 106
LOVO cells s.c. into the flank of nude mice (NCR nu/nu
athymic nude mice, 5–6 weeks of age, female, and weight of 18–20 g;
Taconic Germantown, NY). Once tumors reached a mean diameter of 8–10
mm, treatment was initiated. Animals were treated on days 0, 7, and 14
with a single-pass intratumoral injection of adenovirus expressing the
super-repressor IB (Ad.CMV.IB at a concentration of
1 x 1010 pfu/200 µl), control
adenovirus Ad.CMV3 (1 x 1010
pfu/200 µl), or vehicle alone using a technique described previously
(21)
. On the basis of previous published reports of
tumoricidal response to CPT-11 in xenograft models (24
, 25)
, a dosage of 33 mg/kg every 4 days was selected and
administered i.v. via tail-vein injection during the 20-day treatment
period. A total of 5 drug treatments was administered. PBS was
administered i.v. as a control vehicle. Tumor diameters along two
orthogonal axes were recorded every other day until tumors approached
20 mm of mean diameter, at which point animals were sacrificed
according to protocol. Tumor volume was calculated by assuming a
spherical shape of the tumor, using the formula: volume = 4/3 
r3, where r is
1/2 (mean diameter of the tumor).
The effect of differing schedules of administration of Ad.CMV.IB
on tumoricidal response was assessed in a xenograft model. Nude mice
bearing s.c. LOVO tumors (mean diameter, 8–10 mm) were treated over a
50-day period with i.v. CPT-11 (33 mg/kg every 4 days) and differing
schedules (every 5, 10, 15, and 28 days) of intratumoral injection of
the adenovirus expressing the super-repressor IB (Ad.CMV.IB
at a dosage of 1 x 1010 pfu/200
µl).
Assays to Detect Apoptosis and Transgene Expression in
Vivo.
LOVO tumor xenografts (1-cm diameter) were treated with a single
intratumoral injection of Ad.CMV.IB (1 x 1010 pfu/200 µl). Twenty-four h after virus
administration, CPT-11 (33 mg/kg) was given i.v. by tail-vein
injection. Tumors were harvested at times 0, 1, 2, and 6 h after
drug treatment, embedded in OCT mounting medium (VWR, Sakura Finetec
U.S.A., Inc., Torrance, CA), snap-frozen in 2-methylbutane (Aldrich
Chemical Co., Milwaukee, WI) over liquid nitrogen, and then stored at
-70°C. Four-µm-thick sections were cut and collected on charged
and precleaned microscope slides (Fisher Scientific, Pittsburgh, PA).
Cryosections were stained using a double immunohistological staining
technique to detect the presence of exogenous IB in tumor cells
and to determine the level of apoptosis, determined by the TUNEL assay
in tumors. Exogenous IB was detected using a murine antibody
(anti-HA monoclonal antibody at the dilution of 1:200) to the HA tag
(fluorescent red) present on the exogenous IB expressed by the
Ad.CMV.IB. The reaction mixture was fixed with 10% neutral
buffered formalin and blocked with 2% BSA, followed by incubation for
1 h. Binding was detected with a 1:100 dilution of
rhodamine-conjugated goat antimouse secondary antibody. Apoptosis
was detected using the In Situ Cell Death kit (Boehringer
Mannheim Corp., Indianapolis, IN) and a fluorescent green antibody to
detect TUNEL-positive cells. Analysis of tumor sections was performed
using a two-color fluorescent microscope at x100.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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B Activation Is Induced by SN38 in Most Colorectal Cancer
Cell Lines.B in
fibrosarcoma cells (3
, 4)
. Although others have reported
that different chemotherapy agents activate NF-B in different cell
types, a recent report suggested that stable inhibition of NF-B did
not lead to enhanced cytotoxicity in several cancer cell lines,
including the colorectal cancer cell line HCT116 and the breast cancer
cell line MCF-7 (5)
. We explored the ability of SN38, the
active metabolite of CPT-11, to activate NF-B in a panel of
colorectal cancer cell lines plus MCF-7 cells. All of the cell lines
tested were relatively resistant to treatment with SN38 (data not
shown). To inhibit NF-B, we used transient inhibition via the
adenoviral expression of the super-repressor IB. Our data from
EMSAs demonstrated that NF-B activation, as measured by nuclear
translocation of NF-B, is induced at 1 h after SN38 treatment
and persisted up to 6 h in 10 of 11 cancer cell lines tested and
in MCF-7 cells (Fig. 1A)
. One of the cell lines, HT-29, did not show activation of
NF-B in response to SN38 treatment. The serine-to-alanine mutations
at serine residues 32 and 36 of the super-repressor form of IB
inhibit signal-induced phosphorylation and subsequent
proteasome-mediated degradation of IB. Cytoplasmic binding of the
mutant IB protein to the nuclear localization sequences of
NF-B thereby blocks nuclear translocation and subsequent binding of
NF-B to B binding sites on DNA, effectively blocking
NF-B-mediated transcription (4)
. We have shown
previously in vitro that pretreatment of LOVO cells with
Ad.CMV.IB prior to SN38 administration markedly decreased nuclear
translocation of NF-B in response to SN38 (26)
. We show
here that adenoviral delivery of the super-repressor IB, but not
control adenoviral infection, suppressed NF-B activation (Fig. 1A)
.
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B Inhibits SN38-induced Cytotoxicity.B activation by SN38 would
augment the cytotoxic response of this chemotherapy. We analyzed cell
numbers for six of the colorectal cancer cell lines and for MCF-7
breast cancer cells after SN38 treatment in the presence or absence of
adenovirus encoding the super-repressor form of IB. Treatment
with the adenovirus expressing the super-repressor IB inhibited
the growth of some cell lines compared with treatment with the control
vector (Fig. 1B)
. However, in all cell lines tested, the
addition of super-repressor IB to SN38 treatment resulted in a
significant increase in the level of growth inhibition compared with
treatment with chemotherapy alone, super-repressor IB alone,
control vector alone, and control vector combined with SN38
(P < 0.001, analyzed by ANOVA). Furthermore,
the reduced cell counts resulting from combined treatment of the
super-repressor IB combined with SN38 was associated with a
markedly increased level of apoptosis relative to controls receiving
either chemotherapy alone or Ad.CMV.IB alone (data not shown).
Previously, Bentires-Alj et al. (5)
established
several cancer cell lines, including HCT116 and MCF-7, stably
expressing super-repressor IB and found that cytotoxicity was not
enhanced when NF-B was inhibited. Our data contradict those
conclusions, at least in two of these cell lines, because we show that
transient inhibition of NF-B activation by adenovirus expression of
the super-repressor IB strongly augments the cytotoxic response
to SN38 (see "Discussion"). Furthermore, because the response is
seen in both p53 mutated (KM12-L4, KM12-SM, and WiDR) and p53 wild-type
(CCD841, MCF-7, HCT116, and LOVO) cell lines, our data suggest that the
enhancement of cytotoxicity induced by inhibition of NF-B is
independent of p53 function (see "Discussion").
Sensitivity of Colorectal Tumors to CPT-11 Is Markedly Enhanced by
NF-B Inhibition.
Experiments were performed to determine whether the super-repressor
IB could promote enhanced sensitivity of LOVO xenograft tumors to
CPT-11 (Fig. 2A)
. As described previously (3)
, tumor growth
after combined treatment with super-repressor IB and CPT-11 was
significantly less than treatment with the super-repressor IB or
CPT-11 alone (P < 0.0001, analyzed by
ANOVA). Delivery of IB once a week for 3 weeks combined with CPT-11
treatment twice a week for 3 weeks led to dramatic inhibition of tumor
growth (Fig. 2A)
. Interestingly, tumor size increased after
50 days and ultimately achieved a rapid growth rate. However,
modification of the treatment schedule to treat tumors every 5 or 10
days in combination with CPT-11 administration over a 50-day period
resulted in a persistent tumoricidal response (Fig. 2B)
and
ultimately long-term cures after cessation of treatment at day 50.
Tumor growth in groups receiving treatment with Ad.CMV.IB every 5
or 10 days was significantly less compared with groups receiving viral
treatment every 15, 21, or 28 days (P < 0.0001, analyzed by ANOVA). Tumor regrowth in the groups treated with
virus every 15, 21, or 28 days suggests that IB played an
important role in the enhancement of the tumoricidal response to
CPT-11. Long-term follow-up of treatment groups after cessation of both
viral and CPT-11 treatment on day 50 demonstrated that between 50 and
66% of animals receiving viral treatment every 5 or 10 days remained
tumor-free 5 months after discontinuation of all treatments (Fig. 2C)
.
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B Inhibits Nuclear Translocation of NF-B
induced by CPT-11 in Vivo.B in tumors
and that the adenoviral delivery of IB into tumors successfully
inhibited NF-B activation. EMSA assay for nuclear protein obtained
from tumor tissue extracts demonstrated that NF-B activation was
induced by CPT-11 at 1 h after systemic drug administration with a
peak activation of NF-B observed at 2 h (Fig. 3)
. NF-B activation in tumors was blocked by pretreatment with the
adenovirus expressing the super-repressor IB. The results
demonstrate that in vivo activation of NF-B after
treatment with CPT-11 was inhibited by the expression of the
super-repressor IB but not the control vector.
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B Super-Repressor IB.B expression along with the
induction of apoptosis in LOVO tumors after systemic CPT-11 treatment.
Two-color immunohistochemical staining of tumors injected with the
adenovirus expressing the super-repressor IB and treated with
CPT-11 demonstrated HA-positive cells diffusely throughout the tumor at
all time points sampled, indicating successful adenovirus-mediated
transfer of the super-repressor IB gene to tumor cells
(Fig. 4)
. Staining for TUNEL-positive cells demonstrated that only a few tumor
cells were undergoing apoptosis prior to CPT-11 treatment and at 1 h after treatment. In comparison, increasing levels of apoptosis were
observed at both 2 and 6 h after drug treatment. Importantly,
treatment with the control virus Ad.CMV3 and CPT-11 or with
Ad.CMV.IB and PBS induced apoptosis in <1% of tumor cells at
all time points sampled (data not shown). These data demonstrate that
the activation of NF-B in colorectal tumors suppresses the apoptotic
potential of the chemotherapeutic response.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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B Activation Induced by Cancer Therapies Blocks the Induction
of Apoptosis.B by TNF-, ionizing
radiation, and the cancer chemotherapeutic compound daunorubicin leads
to an inhibition of the apoptotic response induced by these stimuli in
fibrosarcoma cells (4)
. Similar results were obtained by
others (27, 28, 29)
relative to TNF-. Thus, we and others
have proposed that potential apoptotic stimuli initiate two distinct
signaling pathways, one that leads to activation of apoptosis and one
that leads to NF-B activation, which induces a cell survival
response through the inhibition of apoptosis (2
, 4
, 30, 31, 32, 33, 34)
. Contrasting findings such as those reported by
Kasibhatla et al. (35)
, in which stress-induced
expression of Fas ligand (leading to Fas-mediated apoptosis) in human
leukemic Jurkat cells required NF-B activation, have led some
authors to conclude that NF-B serves multiple functional roles under
different conditions (36)
. The mechanism by which
chemotherapy activates NF-B is presently unknown and is the focus of
ongoing investigation in our laboratory. However, the mechanism whereby
NF-B suppresses apoptosis is better understood and involves the
induction of expression of genes that block the caspase cascade
(2)
. We have shown previously that inhibition of inducible
NF-B activation by transient expression of the super-repressor
IB leads to a dramatic improvement in the killing response of
tumor cells when exposed to apoptotic stimuli (3)
. On the
basis of these studies, we conclude that the apoptotic response to
conventional chemotherapy and irradiation may be augmented by the
inhibition of NF-B activation in resistant cancer cells. The
objective of this report was to evaluate the role of transient
inhibition of NF-B as an adjunct to the topoisomerase I inhibitor
CPT-11 for the treatment of colorectal cancer cells.
Use of CPT-11 in the Treatment of Colorectal Cancer.
Colorectal cancer is the second most common cause of mortality from
malignancy in the United States, accounting for 
57,000 deaths in the
United States in 1997 (37)
. Approximately 50% of these
patients will eventually die of metastatic disease (38)
.
The relatively high mortality rate of patients who are not cured by
surgical treatment results from the resistance that most cancers have
to conventional chemo- and radiation therapies (39, 40, 41)
.
Recently, efforts to overcome the 70–80% rate of resistance of
colorectal cancers to conventional therapies have directed the use of
new compounds with reported higher levels of sensitivity in clinical
trials. CPT-11 is one such promising agent used to treat a variety of
solid tumors including colorectal cancer and lung cancer (6
, 17, 18, 19, 20
, 42)
. In vitro studies have demonstrated
CPT-11 to have sustained activity against chemotherapy-resistant colon
cancer cell lines, including those having the multi-drug resistance
(MDR) phenotype (6)
. In addition, results from clinical
trials indicate CPT-11 to be a promising anticancer agent, used as a
single agent or in combination with other agents, with a duration of
response from several months to 1 year (17, 18, 19, 20
, 43)
. Because of its lack of cross-resistance with 5-FU,
promising clinical responses suggest that CPT-11 may be an effective
second-line agent in the treatment of patients who have failed
first-line treatment with 5-FU-based regimens (6)
. A
recently completed multicenter randomized clinical trial found that
CPT-11 increased the 1-year overall survival rate 2.6 times greater
than supportive care in patients who had failed conventional treatment
with 5-FU (16)
.
SN38 Induces Activation of NF-B in a Variety of Human Colorectal
Cancer Cell Lines.
In our previous studies, we have demonstrated that NF-B activation
may result from exposure of cancer cells to a variety of apoptotic
stimuli including TNF-, chemotherapy, and irradiation
(4)
. In this report, we have evaluated a wide variety of
human colorectal cancer cells to determine whether this inducible
response is widely observed or incidental. To evaluate this response,
we have selected a variety of resistant colorectal cancer cell lines
including those that are mutated for the p53 gene (WiDR,
KM12-L4, KM12-SM, SW480, and SW620), contain the K-ras
oncogene (LOVO, HCT116, SW480, and SW620), as well as those that
overexpress Bcl-2 (KM12-L4 and SW480). The enhanced cytotoxicity
responses attained through inhibition of NF-B were found to be
independent of the status of p53, K-ras, or
Bcl-2 expression. These results do not imply that expression
of these proteins is not relevant to cancer therapy, but that the
enhancement of cytotoxicity can be attained in their absence of
expression. In preliminary studies, anticancer agents that
traditionally have been used to treat patients with metastatic
colorectal cancer, including 5-FU and mitomycin C, were found to only
weakly induce NF-B activation in a variety of colorectal cancer cell
lines tested (data not shown). In some cell lines, no activation of
NF-B was observed after treatment with these agents. In contrast,
SN38, the active metabolite of CPT-11, was found to activate NF-B in
10 of 11 colorectal cancer cell lines tested. Importantly, in all cases
in which NF-B activation was induced by SN38, inhibition of
activation was facilitated by pretreatment with super-repressor
IB but not the control vector. These findings are consistent with
our observations in a variety of different cancer subtypes including
pancreatic cancer, sarcoma, and breast cancer in which SN38 was found
to be a very potent and consistent inducer of NF-B activation (data
not shown). In addition, relative to a variety of different anticancer
agents we have tested, the level of inducible NF-B activation after
treatment with SN38 is surpassed only by TNF- (data not shown).
Inhibition of SN38-induced NF-B Activation Enhances Sensitivity
to SN38 in a Variety of Resistant Colorectal Cancer Cell Lines.
To evaluate the role of SN38-induced NF-B activation on the
chemosensitivity of a variety of resistant colon cancer cell lines, we
pretreated these cells with the super-repressor IB. In all cell
lines tested, sensitivity to SN38 was markedly enhanced by transient
inhibition of NF-B activation. Furthermore, enhanced
chemosensitivity was observed at all concentrations of SN38 tested.
This has important clinical relevance because of the inability to
achieve the requisite therapeutic dosages of chemotherapy in patients
and dose-limiting toxicity. At the lower tolerated levels of
chemotherapy attained in patients, we would predict, based on these
findings, that enhanced chemosensitivity may be achieved by effective
NF-B inhibition in those cells. Although the ability to predict
sensitivity of a patient’s tumor to a specific chemotherapy agent is
limited, the observed ability to augment the sensitivity of the variety
of cell lines tested suggests that application of this combination
therapy approach may have a broad impact on colorectal cancer patients
receiving CPT-11.
Enhanced sensitivity to SN38 after inhibition of NF-B activation was
observed in cell lines that ranged in SN38 sensitivity from high (WiDR)
to low (HCT116). The response of the various cell lines to combination
therapy using the super-repressor IB and SN38 may reflect
differences in sensitivity to chemotherapy, small differences in
adenovirus infectivity, as well as variations in the level of
activation of NF-B after chemotherapy exposure. The mechanism
underlying chemotherapy resistance is likely multifactorial
(44)
, as demonstrated in the wide range of genetic errors
represented in the cell lines tested. Previous analysis of CPT
resistance in colorectal cancer cell lines from the NCI Anticancer
Screen reported by Goldwasser et al. (45)
suggested that CPT uptake and expression of DNA topoisomerase I did not
predict cytotoxicity in response to CPT, although the formation of
cleavable complexes did reasonably predict CPT sensitivity. Our results
suggest that inducible chemotherapy resistance, mediated by the
transcription factor NF-B, may also be a major determinant of
sensitivity to CPT-11 and appears to be a shared survival mechanism
among a wide variety of colon cancer cell lines. As described above, we
observed enhanced chemosensitivity in both p53 mutated and
wild-type cell lines, cells with and without oncogenic
K-ras, and independent of high or low levels of
Bcl-2 expression.
Our findings using transient inhibition of NF-B via
adenovirus-mediated delivery of the super-repressor IB are in
contrast to those reported recently by Bentires-Alj et al.
(5)
, in which clones of HCT116 and the highly
CPT-resistant breast cancer cell line MCF-7 were selected for stable
expression of mutated (super-repressor) IB. Our data shown here
and published previously suggest that the resistance to
chemotherapy-induced apoptosis is mediated by the activation of
NF-B. However, in apparent contrast to our findings, in which
inhibition of NF-B activation in the parental cells led to
dramatically enhanced sensitization to SN38, the selected clones that
were stably transfected with mutated IB in the Bentires-Alj study
were not more sensitive to the various chemotherapy agents and TNF-,
despite activation of NF-B by these stimuli. This suggests that the
process of selecting clones that contain stable expression of mutated
IB leads to the acquisition of alternative survival mechanisms,
necessary to overcome the NF-B inhibition that occurs in the
presence of constitutively expressed, mutated IB. In contrast to
the conclusions of the Bentires-Alj report, our data suggest that, in
fact, NF-B does play a central role in inducible
chemoresistance. Furthermore, our findings indicate that transient
inhibition of NF-B activation is a potent adjuvant to the treatment
of colon cancer with CPT-11. Additional studies are indicated to
further evaluate the mechanisms of chemotherapy-induced NF-B
activation and the resulting antiapoptotic response.
Dose Intensification Using Serial Administration of Combined CPT-11
and NF-B Inhibition Leads to Complete Tumor Eradication in a
Colorectal Cancer Xenograft Model.
Application of combined NF-B inhibition with CPT-11 for the
treatment of human colorectal cancer appears to be most promising for
chemotherapy-resistant tumors. Cell lines such as WiDR, which were most
sensitive to SN38, were found to have the minimum degree of enhanced
response to NF-B inhibition. Similarly, a minimal amount of enhanced
sensitivity was observed in vivo in WiDR tumors that
received combined treatment (data not shown). In contrast, the moderate
resistance of LOVO to SN38 and CPT-11 in vitro and in
vivo, respectively, was dramatically overcome by inhibition of
NF-B activation. By inhibiting the inducible survival response that
occurs with each individual drug treatment, through the activation of
NF-B, the maximum apoptotic response to CPT-11 was obtained.
Although inhibition of constitutive NF-B activation as a single
therapeutic intervention may have potential in the treatment of some
types of malignancy, our findings suggest that inhibition of inducible
NF-B activation when combined with chemotherapy may improve the
response to conventional chemotherapies. Furthermore, the results from
these preclinical studies provide a rational basis for how to most
effectively apply this combination therapeutic strategy to optimize the
apoptotic response to CPT-11 in patients receiving treatment for
metastatic colorectal cancer. Currently, studies are under way to
evaluate the potential role of small molecule inhibitors of NF-B to
enhance the apoptotic response to chemotherapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the University of North
Carolina Lineberger Comprehensive Cancer Center (to J. C. C.); NCI
Grant CA75528 (to J. C. C.) and Grants CA72771, CA75080, and CA73756
(to A. S. B.); and the American Cancer Society Clinical Oncology
Career Development Award 96-21 (to J. C. C.). Additional support for
this project was provided by the University of North Carolina
Specialized Program of Research Excellence program in breast cancer NCI
Grant CA58223. 
2 To whom requests for reprints should be
addressed, at Department of Surgery, CB#7210, University of North
Carolina at Chapel Hill, Chapel Hill, NC 27599-7210. Phone:
(919) 966-8007; Fax: (919) 966-8806.
3 The abbreviations used are: NF-B, nuclear
factor-B; CPT-11,
7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin;
CMV, cytomegalovirus; MOI, multiplicity of infection; SN38,
7-ethyl-10-hydroxycamptothecin; ATCC, American Type Culture Collection;
FBS, fetal bovine serum; EMSA, electrophoretic mobility shift assay;
2-ME, 2-mercaptoethanol; pfu, plaque-forming unit(s); TUNEL, terminal
deoxynucleotidyl transferase-mediated dUTP nick end labeling; HA,
hemagglutinin; 5-FU, 5-fluorouracil; CPT, camptothecin; TNF, tumor
necrosis factor; NCI, National Cancer Institute.
Received 9/10/99. Accepted 3/15/00.
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