Download Synergistic Cytotoxicity and Apoptosis by Apo

Vol. 5, 2605–2612, September 1999
Clinical Cancer Research 2605
Synergistic Cytotoxicity and Apoptosis by Apo-2 Ligand and
Adriamycin against Bladder Cancer Cells1
Youichi Mizutani,2 Osamu Yoshida,
Tsuneharu Miki, and Benjamin Bonavida
Department of Urology, Faculty of Medicine, Kyoto University,
Kyoto 606-8507, Japan [Y. M., O. Y.]; Department of Urology,
Kyoto Prefectural University of Medicine, Kyoto 606-0841, Japan
[T. M.]; and Department of Microbiology and Immunology, UCLA
School of Medicine, University of California at Los Angeles,
California 90095 [B. B.]
ABSTRACT
Resistance to conventional anticancer chemotherapeutic agents remains one of the major problems in the treatment of bladder cancer. Hence, new therapeutic modalities
are necessary to treat the drug-resistant cancers. Apo-2
ligand (Apo-2L) is member of the tumor necrosis factor
ligand family, and it induces apoptosis in cancer cells. Several cytotoxic anticancer drugs, including Adriamycin
(ADR), also mediate apoptosis and may share the common
intracellular pathways leading to cell death. We reasoned
that combination treatment of the drug-resistant cancer
cells with Apo-2L and drugs might overcome their resistance. Here, we examined whether bladder cancer cells are
sensitive to Apo-2L-mediated cytotoxicity and whether
Apo-2L can synergize with ADR in cytotoxicity and apoptosis against bladder cancer cells.
Recombinant human soluble Apo-2L (sApo-2L), which
carries the extracellular domain of Apo-2L, was used as a
ligand. Cytotoxicity was determined by a 1-day microculture
tetrazolium dye assay. Synergy was assessed by isobolographic analysis.
Human T24 bladder cancer line was relatively resistant
to sApo-2L. Treatment of T24 line with combination of
sApo-2L and ADR resulted in a synergistic cytotoxic effect.
Synergy was also achieved in the ADR-resistant T24 line
(T24/ADR), two other bladder cancer lines, and three
freshly derived human bladder cancer cell samples. In addition, T24 cells were sensitive to treatment with sApo-2L
combined with epirubicin or pirarubicin. The synergy
achieved in cytotoxicity with sApo-2L and ADR was also
Received 3/23/99; revised 6/1/99; accepted 6/14/99.
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.
1
This work was supported by a Grant-in-Aid from Setsuro Fujii Memorial, The Osaka Foundation for Promotion of Fundamental Medical
Research.
2
To whom requests for reprints should be addressed, at Department of
Urology, Faculty of Medicine, Kyoto University, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. Phone: (075) 751-3337;
Fax: (075) 751-3740.
achieved in apoptosis. Intracellular accumulation of ADR
was not affected by sApo-2L. Incubation of T24 cells with
sApo-2L down-regulated the expression of glutathione Stransferase-p mRNA.
This study demonstrates that combination treatment of
bladder cancer cells with sApo-2L and ADR overcomes their
resistance. The sensitization obtained with established ADRresistant bladder cancer cells and freshly isolated bladder
cancer cells required low subtoxic concentrations of ADR,
thus supporting the in vivo potential application of combination of sApo-2L and ADR in the treatment of ADRresistant bladder cancer.
INTRODUCTION
Previous studies have demonstrated that a variety of cancer
cells have different degrees of sensitivity and resistance to
various kinds of anticancer cytotoxic agents (1). When anticancer chemotherapeutic agents are administered, only the drugsensitive cancer cells are deleted, and cancer cells with acquired
resistance develop (2). Consequently, drug resistance (inherent
or acquired) is the major cause of failure in cancer chemotherapy. Therefore, new effective therapeutic modalities are necessary to treat the drug-resistant cancers.
Apo-2L3 (also known as TRAIL) is a member of the TNF
ligand family, and it induces apoptosis in various transformed
cell lines, such as Fas ligand (3, 4). Unlike Fas ligand, the
transcripts of which are predominantly restricted to stimulated T
cells and sites of immune privilege, expression of Apo-2L is
detected in a lot of normal human tissues, most predominantly
in spleen, lung, and prostate (3). The receptors for Apo-2L,
DR4, and DR5 are also expressed in multiple human normal
tissues (5, 6). Interestingly, Apo-2L is not cytotoxic to most
normal tissues in vivo; however, it has marked apoptotic potential for cancer cells (3, 4). In contrast to injection of Fas ligand
and anti-Fas mAb, which is lethal to mice, injection of Apo-2L
is well tolerated in mice (7, 8). Thus, Apo-2L may have potential utility as an anticancer agent.
Fas ligand and TNF-a as well as Apo-2L induce apoptosis
(9, 10). Several anticancer drugs, including ADR, also mediate
apoptosis and may share common intracellular signaling pathways leading to cell death. Indeed, we have reported that treatment with ADR in combination with anti-Fas mAb or TNF-a
resulted in significant potentiation of cytotoxicity and synergy
against a variety of sensitive and resistant human cancer cells
(11, 12). This study investigated whether the resistance of
3
The abbreviations used are: Apo-2L, Apo-2 ligand; TNF, tumor necrosis factor; mAb, monoclonal antibody; ADR, Adriamycin; sApo-2L,
soluble Apo-2L; EPI, epirubicin; THP, pirarubicin; GST-p, glutathione
S-transferase-p; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Topo II, topoisomerase II.
Downloaded from clincancerres.aacrjournals.org on June 18, 2017. © 1999 American Association for Cancer
Research.
2606 Chemoimmunosensitization of Bladder Cancer
Fig. 1 The synergistic cytotoxic effect of
sApo-2L and ADR used in combination against
T24 cells. The cytotoxic effect of sApo-2L and
ADR used in combination on T24 cells was assessed in a 1-day MTT assay (A) and estimated by
isobolographic analysis (B). Columns, means of
three different experiments; bars, SD. p, significantly higher than those achieved by treatment with
ADR alone at P , 0.05.
bladder cancer cells to ADR, one of anticancer agents used
clinically against bladder cancer, could be overcome by combination treatment with Apo-2L and ADR.
MATERIALS AND METHODS
Tumor Cells. The T24, J82, and HT1197 human bladder
cancer cell lines were maintained in monolayers on plastic
dishes in RPMI 1640 (Life Technologies, Inc., Bio-cult, Glasgow, Scotland), supplemented with 25 mM HEPES, 2 mM Lglutamine, 1% nonessential amino acids, 100 units/ml penicillin, 100 mg/ml streptomycin, and 10% heat-inactivated fetal
bovine serum (all from Life Technologies, Inc.), hereafter referred to as complete medium (13, 14). The T24/ADR line is an
ADR-resistant subline of the T24 cell line (11).
Fresh bladder cancer cells derived from three patients were
separated from surgical specimens as described previously (15, 16).
The histological diagnosis revealed that all patients had transitional
cell carcinoma of the bladder. Their histological classification and
staging according to the tumor-node-metastasis classification were:
patient 1, T2N0M0, grade 3; patient 2, T1N0M0, grade 2; and patient
3, T1N0M0, grade 2. Briefly, cell suspensions were prepared by
treating finely minced tumor tissues with collagenase (3 mg/ml;
Sigma Chemical Co., St. Louis, MO). After washing three times in
RPMI 1640, the cell suspensions were layered on discontinuous
gradients consisting of 2 ml of 100% Ficoll-Hypaque, 2 ml of 80%
Ficoll-Hypaque, and 2 ml of 50% Ficoll-Hypaque in 15-ml plastic
tubes and were centrifuged at 400 3 g for 30 min. Lymphocyterich mononuclear cells were collected from the 100% interface, and
tumor and mesothelial cells were collected from the 80% interface.
Cell suspensions that were enriched with tumor cells were sometimes contaminated by monocyte-macrophages, mesothelial cells,
or lymphocytes. To eliminate further contamination of host cells,
we layered the cell suspensions on a discontinuous gradient containing 2 ml each of 25, 15, and 10% Percoll in complete medium
Downloaded from clincancerres.aacrjournals.org on June 18, 2017. © 1999 American Association for Cancer
Research.
Clinical Cancer Research 2607
Fig. 2 The synergistic cytotoxic effect of
sApo-2L and ADR used in combination
against fresh bladder cancer cells. The cytotoxic effect of sApo-2L and ADR used in
combination on fresh bladder cancer cells
derived from three patients (data from patient
1 are shown as a representative sample) was
assessed in a 1-day MTT assay (A), and synergy was assessed by isobolographic analysis
(B). Columns, means of triplicate samples;
bars, SD. Similar data were found for the two
other patients. p, significantly higher than
those achieved by treatment with ADR alone
at P , 0.05.
in 15-ml plastic tubes and centrifuged them for 7 min at 25 3 g at
room temperature. Tumor cells that were depleted of lymphoid
cells were collected from the bottom, washed, and suspended in
complete medium. To remove further contamination from mesothelial cells and monocyte-macrophages, we incubated the cell
suspension in plastic dishes for 30 – 60 min at 37°C in a humidified
5% CO2 atmosphere. After incubation, nonadherent cells were
recovered, washed, and suspended in complete medium. Usually,
the nonadherent cells contained mainly tumor cells with ,5%
contaminating nonmalignant cells, as judged by morphological
examination of Wright-Giemsa-stained smears, and were .93%
viable, according to the trypan blue dye exclusion test. Cells having
,5% contamination with nonmalignant cells were accepted for use
as tumor cells.
Reagents. sApo-2L was kindly supplied by Pepro Tech
(Rocky Hill, NJ). ADR (lot no. 705ACB) and EPI (lot no.
3015AG) were supplied by Kyowa Hakkou Co. Ltd. (Tokyo,
Japan). THP (lot no. THPMR100) was obtained from Meiji
Pharmaceutical Co. Ltd. (Tokyo, Japan). GST-p cDNA, used in
making probes for Northern blot analysis, was a gift from
Otsuka Pharmaceutical Co. Ltd. (Tokushima, Japan).
Cytotoxicity Assay. The MTT assay was used to determine tumor cell lysis, as described previously (17, 18). Briefly,
100 ml of target cell suspension (2 3 104 cells) were added to
each well of 96-well flat-bottomed microtiter plates (Corning
Glass Works, Corning, NY), and each plate was incubated for
24 h at 37°C in a humidified 5% CO2 atmosphere. After
incubation, 100 ml of drug solution or complete medium for
control were distributed in the 96-well plates, and each plate
was incubated for 24 h at 37°C. Following incubation, 20 ml
of MTT working solution (5 mg/ml; Sigma) were added to
each culture well, and the cultures were incubated for 4 h at
37°C in a humidified 5% CO2 atmosphere. The culture medium was removed from the wells and replaced with 100 ml
of isopropanol (Sigma) supplemented with 0.05 N HCl. The
absorbance of each well was measured with a microculture
Downloaded from clincancerres.aacrjournals.org on June 18, 2017. © 1999 American Association for Cancer
Research.
2608 Chemoimmunosensitization of Bladder Cancer
plate reader (Immunoreader; Japan Intermed Co. Ltd., Tokyo,
Japan) at 540 nm. The percentage cytotoxicity was calculated
using the following formula: percentage cytotoxicity 5 [1 2
(absorbance of experimental wells/absorbance of control
wells) ] 3 100.
Chromatin Staining with Hoechst 33258. Apoptosis
was observed by chromatin staining with Hoechst 33258 as
described previously (19). T24 cells in a chamber/slide (Miles
Scientific, IL) were incubated with sApo-2L at 100 ng/ml in the
absence or presence of ADR at 1 mg/ml for 12 h at 37°C in a
humidified 5% CO2 atmosphere. After incubation, the supernatant was discarded, and T24 cells were fixed with 1% glutaraldehyde in PBS for 30 min at room temperature, washed four
times with PBS, and exposed to Hoechst 33258 at 10 mM for 30
min at room temperature. The cell preparations were examined
under UV illumination with an Olympus fluorescence microscope. Apoptosis was defined when apoptotic bodies, chromatin
condensation, or fragmented nuclei were observed.
ADR Determination. The ADR content in T24 cells was
determined by high-performance liquid chromatography using a
Hitachi model 635A (Hitachi Co. Ltd., Tokyo, Japan), as described in detail elsewhere (20).
Northern Blotting. Cytoplasmic RNA from tumor cells
was prepared as described in detail elsewhere (21, 22). Briefly,
tumor cell RNA (10 mg/lane) was electrophoresed in 1.2%
agarose-2.2 M HCHO gels in 13 4-morpholinepropanesulfonic
acid buffer (200 mM 4-morpholinepropanesulfonic acid, 50 mM
sodium acetate, and 10 mM sodium EDTA). The RNA was
transferred to Biodyne A membranes (Poll, CA) in 203 SSC [3
M NaCl and 0.3 M sodium citrate (pH 7.0)]. Fifty to 100 ng of
cDNA probe were labeled with [a-32P ]dCTP (NEN, Boston,
MA) by random oligoprimer extension. The nylon membranes
were UV cross-linked and hybridized.
Statistical Analysis. All determinations were made in
triplicate, and the results were expressed as the mean 6 SD.
Statistical significance was determined by Student’s t test. A P
of #0.05 was considered significant.
Calculations of synergistic cytotoxicity were determined
by isobolographic analysis, as described by Berenbaum (23, 24).
The nature of the effect of a particular dose combination was
determined by isobolographic analysis, as follows: the point
representing that combination would lay on, below, or above the
straight line joining the doses of the two drugs that, when given
alone, produce the same effect as that combination, representing
additive, synergistic, or antagonistic effects, respectively.
RESULTS
Synergistic Cytotoxicity against Bladder Cancer Cells
after Their Treatment with a Combination of sApo-2L
and ADR
We investigated the cytotoxic effect of a combination of
sApo-2L and ADR against the T24 bladder cancer cell line.
Cytotoxicity was determined by a 1-day MTT assay, and synergy was assessed by isobolographic analysis, as described in
“Materials and Methods.” The T24 line was relatively resistant
to sApo-2L- and ADR-mediated cytotoxicity. However, when
T24 cells were treated with a combination of sApo-2L and
ADR, significant potentiation of cytotoxicity and synergy was
Table 1
Effect of sequence of treatment with sApo-2L and ADR on
their cytotoxic activity against T24 cells
First treatmenta
(6 h)
sApo-2L
ADR
Medium
Medium
Medium
sApo-2L
ADR
Second treatmenta
(18 h)
% cytotoxicity
(mean 6 SD)b
Medium
Medium
sApo-2L
ADR
sApo-2L 1 ADR
ADR
sApo-2L
5.8 6 0.9
10.8 6 1.5
11.8 6 1.0
28.7 6 2.9
74.8 6 6.0c,d
68.9 6 5.3c,d
49.4 6 1.1c
a
T24 cells were pretreated with medium only, sApo-2L (100
ng/ml), or ADR (10 mg/ml) for 6 h (first treatment). The medium was
aspirated, and T24 cells were washed twice with RPMI 1640. The cells
were then incubated with sApo-2L (100 ng/ml) and/or ADR (10 mg/ml)
for 18 h (second treatment). Cytotoxicity was assessed in a 1-day MTT
assay.
b
Results are expressed as the mean 6 SD of three separate experiments.
c
Values in the combination treatment are significantly higher than
those achieved by treatment with ADR alone at P , 0.05.
d
Values in the combination treatment are significantly higher than
those achieved when ADR was given first at P , 0.05.
achieved (Fig. 1). Preliminary experiments demonstrated that
the synergy was not observed when normal bladder cells were
used as targets.
To confirm the synergy, we then examined the sensitivity of
the T24 ADR-resistant variant cell line, T24/ADR, to treatment
with sApo-2L in combination with ADR. Significant synergy was
achieved by the combination treatment with sApo-2L and ADR
(data not shown). Furthermore, synergy was also obtained in two
other bladder cancer lines, J82 and HT1197 (data not shown).
On the basis of the above findings with established bladder
cancer cell lines, we examined for synergy on three freshly
isolated bladder cancer cells. In all three cases, significant
synergy was achieved, irrespective of the baseline sensitivity of
the cancer cells to either ADR or sApo-2L used alone (Fig. 2).
These findings demonstrate that treatment of established
and freshly derived bladder cancer cells with a combination
of sApo-2L and ADR resulted in potentiation of cytotoxicity.
Furthermore, the synergy was observed with both sensitive
and resistant cancer cells. In all of the above findings, synergy was achieved with subtoxic concentrations of ADR. The
synergy obtained with low concentrations of the agents is of
clinical relevance because high concentrations of drugs are
toxic in vivo.
Effect of the Sequence of Treatment with sApo-2L and
ADR on Synergy
The findings above demonstrate that simultaneous treatment of T24 cells with the sApo-2L and ADR resulted in
synergy. The effect of sequential treatment with sApo-2L and
ADR was examined and compared with treatment with both
agents together. The T24 bladder cancer cells were treated for
6 h with one agent, the medium was removed, the second agent
was subsequently added for another 18 h, and the cells were
tested for viability. The results show that the highest percentage
cytotoxicity was obtained when sApo-2L was given first or
together with ADR (Table 1). Similar results were obtained
Downloaded from clincancerres.aacrjournals.org on June 18, 2017. © 1999 American Association for Cancer
Research.
Clinical Cancer Research 2609
Fig. 3 The synergistic cytotoxic effect of sApo-2L in combination with EPI or THP against T24 cells. The cytotoxic effect of sApo-2L in
combination with EPI (A and B) or THP (C and D) on T24 cells was assessed in a 1-day MTT assay (A and C) and estimated by isobolographic analysis
(B and D). Columns, means of three different experiments; bars, SD. p, significantly higher than those achieved by treatment with EPI or THP alone
at P , 0.05.
when sApo-2L and ADR were used at different concentrations
(data not shown). These findings demonstrate that the sequence
of treatment with sApo-2L and ADR may not be critical to
obtain significant synergy but may determine the extent of
synergy.
Effect of the ADR-related Cytotoxic Drugs, EPI and THP,
on Synergy with sApo-2L
Two closely related compounds of ADR, EPI and THP, are
currently used clinically. The cytotoxic effect of these two drugs
used each in combination with sApo-2L was tested against T24
cells. Synergy was achieved with both EPI and THP in combination with sApo-2L (Fig. 3).
Mechanism of Synergy Achieved by sApo-2L and ADR
Induction of Apoptosis. Because both sApo-2L and ADR
can mediate apoptosis, we examined by Hoechst 33258 staining
whether the synergy achieved in cytotoxicity with sApo-2L and
ADR was also achieved in apoptosis. Apoptosis was defined when
apoptotic bodies, chromatin condensation, or fragmented nuclei
were observed. No apoptosis was seen in T24 cells cultured in
medium. ADR at a concentration of 1 mg/ml was cytotoxic against
T24 cells and mediated modest apoptosis (Fig. 4B). Swelling of
T24 cells was also observed. Likewise, sApo-2L, at a concentration
of 100 ng/ml, was slightly cytotoxic against T24 cells and also
mediated some apoptosis (Fig. 4C). When sApo-2L and ADR were
used in combination, fragmented nuclei of T24 cells were observed. We considered almost all T24 cells apoptotic, and synergy
Downloaded from clincancerres.aacrjournals.org on June 18, 2017. © 1999 American Association for Cancer
Research.
2610 Chemoimmunosensitization of Bladder Cancer
Table 2
Effect of treatment of T24 cells with sApo-2L on the
intracellular accumulation of ADR
Treatment
Intracellular accumulation of ADR
(ng/105 cells)a
Control
sApo-2L
35.8 6 4.2
33.1 6 5.0
a
T24 cells were treated with ADR (10 mg/ml) in combination with
medium or sApo-2L (100 ng/ml) for 12 h. The medium was aspirated,
and T24 cells were washed three times with RPMI 1640. The intracellular concentration of ADR was measured by high-performance liquid
chromatography, as described in “Materials and Methods.” The results
are expressed as the mean 6 SD of three separate experiments.
Fig. 5 Effect of treatment of T24 cells with sApo-2L on the level of
GST-p mRNA. T24 cells were treated with medium or sApo-2L (100
ng/ml) for 12 h. Total RNA was then separated, subjected to Northern blot
analysis, and probed for GST-p, as described in “Materials and Methods.”
Each lane is GST-p mRNA of T24 cells after the following treatments (A):
Lane 1, medium only; and Lane 2, sApo-2L. B, ethidium bromide staining
of the same filter as control of the amount of RNA present in each lane.
Fig. 4 Apoptosis in T24 cells treated with sApo-2L and/or ADR. Apoptosis in T24 cells was determined by chromatin staining with Hoechst
33258, as described in “Materials and Methods.” Shown are fluorescence
microscopic views of chromatin staining with Hoechst 33258 (3200) after
the following treatments: A, ADR at 1 mg/ml; B, sApo-2L at 100 ng/ml; C,
ADR at 1 mg/ml and sApo-2L at 100 ng/ml.
in apoptosis was also observed (Fig. 4D). The apoptosis was also
examined by DNA ladder assay. Treatment of T24 cells with
combination of sApo-2L and ADR resulted in strong DNA ladder
(data not shown). These results indicate that there was a good
correlation between cytotoxicity and apoptosis after treatment of
bladder cancer cells with a combination of sApo-2L and ADR.
ADR Intracellular Accumulation. A possible mechanism of augmentation of cytotoxicity may be the result of
increased accumulation of ADR in the cells treated with sApo2L. When T24 cells were treated with combination of ADR and
sApo-2L, ADR accumulation inside the cells did not change
significantly (Table 2).
Expression of GST-p mRNA. Previous findings indicated that ADR resistance may be, in part, due to up-regulation
of the antioxidant enzyme GST-p expression (25, 26). T24 cells
constitutively expressed mRNA for GST-p (Fig. 5). Treatment
of T24 cells with sApo-2L down-regulated GST-p mRNA expression.
Altogether, these findings suggest that the synergistic cytotoxicity achieved with sApo-2L and ADR is, in part, due to
up-regulation of apoptosis and down-regulation of GST-p
mRNA expression.
DISCUSSION
Here, we showed that the combined treatment of sApo-2L
and ADR resulted in a synergistic cytotoxicity and apoptosis
Downloaded from clincancerres.aacrjournals.org on June 18, 2017. © 1999 American Association for Cancer
Research.
Clinical Cancer Research 2611
against bladder cancer cells and reversed their resistance. It has
been reported that ADR augmented Apo-2-induced apoptosis in
breast cell lines (27). The synergy was achieved with low
concentrations of each agent, thus minimizing their toxicity in
vivo and maximizing their therapeutic application in vivo. Because Apo-2L is not toxic to most of normal tissues (3, 4), a
combination of sApo-2L and ADR may be a promising strategy
to eliminate bladder cancer cells.
The mechanisms responsible for cellular resistance to ADR
are believed to be multifactorial and include alterations in the
transmembrane transport of ADR, decreased formation of DNA
single- and double-strand breaks, earlier onset of DNA repair,
the cytosolic quenching of ADR due to increased levels of
glutathione and its related enzymes, and the decreased cellular
level of DNA Topo II. Alterations in the transmembrane transport of ADR in cancer cells by P-glycoprotein or multidrug
resistance-associated protein result in reduced intracellular accumulation of ADR and resistance to ADR (28, 29). The resistance to ADR has been attributed to reduced levels of DNA
single- and double-stranded breaks induced by the drug in
ADR-resistant P388 leukemia cells (30, 31). The ADR-resistant
P388 cells appeared to display an earlier onset of DNA repair
than did drug-sensitive cells (32). Some ADR-resistant cancer
cells have higher levels of intracellular glutathione or its related
enzymes (25, 26). Because ADR inhibits DNA Topo II, the
reduced cellular level of DNA Topo II has been proposed as a
possible mechanism of cancer cell resistance to ADR (32, 33).
Several possible mechanisms of resistance to the killing of
cells by Apo-2L in cancer cells have been reported, such as lack of
the expression of Apo-2L receptors, DR4 and DR5, and the enhanced expression of antagonistic Apo-2L receptors, DcR1 and
DcR2 (5, 6, 8). The existence of multiple receptors for Apo-2L
suggests an unexpected complexity in the regulation of signaling
by this cytokine. Protection against apoptosis via synthesis of an
intracellular protein is a well-established paradigm. The protein
product of bcl-2 has been shown to inhibit DNA fragmentation
induced by a variety of stimuli (34). Because Apo-2L induces
apoptosis in target cells in a caspase-dependent fashion, the resistance to Apo-2L might be dependent on the level of the expression
of caspases (35, 36). Further studies are required to elucidate the
mechanisms responsible for the acquisition of resistance of bladder
cancer cells to Apo-2L-mediated cytotoxicity.
Although the down-regulation of GST-p mRNA expression by sApo-2L is suggestive for synergistic cytotoxicity of
sApo-2L and ADR, the precise mechanism of the synergy by
combination treatment with sApo-2L and ADR is not fully
understood. The expression of GST-p protein might have been
increased by posttranslational stabilization of the protein, or the
activity of GST-p itself might have been increased. The mechanisms responsible for synergistic cytotoxicity by combination
treatment of sApo-2L and ADR await further investigation.
Both Apo-2L and Fas ligand are coexpressed on the cell
surface of immune cells (37, 38). Apo-2L as well as Fas ligand
may play an important role in cytotoxic T cell-mediated apoptosis in cancer cells (39). The cells that are resistant to Fasmediated apoptosis are sensitive to Apo-2L-mediated apoptosis
(39). In this study and in a previous report, it has been shown
that treatment with ADR in combination with sApo-2L or antiFas mAb resulted in significant potentiation of cytotoxicity and
synergy against bladder cancer cells (10). In addition, preliminary experiments showed that treatment of freshly isolated
bladder cancer cells with ADR enhanced their susceptibility to
lysis by autologous lymphocytes. These findings suggest that a
combination of ADR chemotherapy and immunotherapy might
be an alternative approach in the treatment of ADR-/immunotherapy-resistant bladder cancer.
The overall response rate of patients with bladder cancer
to current anticancer chemotherapeutic agents involving
ADR has improved. However, drug resistance and recurrence
of cancers remain major problems, and a more effective
therapy is necessary for these patients. This study shows that
combination treatment with sApo-2L and ADR resulted in a
synergistic cytotoxicity and apoptosis against both acquired
and natural ADR-resistant bladder cancer cells, and the synergistic effect is not restricted to established cell lines: it is
also observed in freshly derived cancers. These findings
suggest that the therapeutic use of ADR in combination with
sApo-2L might be useful in patients with ADR-resistant
bladder cancer.
ACKNOWLEDGMENTS
We are deeply indebted to Professor Osamu Ogawa at Department
of Urology, Faculty of Medicine, Kyoto University, for his kind advice
during this investigation.
REFERENCES
1. Safrit, J. T., and Bonavida, B. Hierarchy of sensitivity and resistance
of tumor cells to cytotoxic effector cells, cytokines, drugs, and toxins.
Cancer Immunol. Immunother., 34: 321–328, 1992.
2. Birkhead, B. G. Evaluating and designing cancer chemotherapy
treatment using mathematical models. Eur. J. Cancer Clin. Oncol., 22:
3– 8, 1986.
3. Willy, S. R., Schooley, K., Smolak, P. J., Din, W. S., Huang, C. P.,
Nicholl, J. K., Sutherland, G. R., Smith, T. D., Rauch, C., Smith, C. A., and
Goodwin, R. G. Identification and characterization of a new member of the
TNF family that induces apoptosis. Immunity, 3: 673–682, 1995.
4. Pitti, R. M., Marsters, S. A., Ruppert, S., Donahue, C. J., Moore, A.,
and Ashkenazi, A. Induction of apoptosis by Apo-2 ligand, a new
member of the tumor necrosis factor cytokine family. J. Biol. Chem.,
271: 12687–12690, 1996.
5. Pan, G., O’Rourke, K., Chinnaiyan, A. M., Gentz, R., Ebner, R., Ni,
J., and Dixit, V. M. The receptor for the cytotoxic ligand TRAIL.
Science (Washington DC), 276: 111–113, 1997.
6. Sheridan, J. P., Marsters, S. A., Pitti, R. M., Gurnay, A., Skubatch,
M., Baldwin, D., Ramakrishnan, L., Gray, C. L., Baker, K., Wood, W. I.,
Godard, A. D., Godowski, P., and Ashkenazi, A. Control of TRAILinduced apoptosis by a family of signaling and decoy receptors. Science
(Washington DC), 277: 818 – 821, 1997.
7. Ogasawara, J., Watanabe-Fukunaga, R., Adachi, M., Matsuzawa, A.,
Kasugai, T., Kitamura, Y., Itoh, N., Suda, T., and Nagata, S. Lethal effect
of the anti-Fas antibody in mice. Nature (Lond.), 364: 806–809, 1993.
8. Rieger, J., Naumann, U., Glaser, T., Ashkenazi, A., and Weller, M.
APO2 ligand: a novel lethal weapon against malignant glioma. FEBS
Lett., 427: 124 –128, 1998.
9. Suda, T., Takahashi, T., Golstein, P., and Nagata, S. Molecular
cloning and expression of the Fas ligand, a novel member of the tumor
necrosis factor family. Cell, 75: 1169 –1178, 1993.
10. Smith, C. A., Farrah, T., and Goodwin, R. G. The TNF receptor
superfamily of cellular and viral proteins: activation, costimulation, and
death. Cell, 76: 959 –962, 1994.
11. Mizutani, Y., Okada, Y., Fukumoto, M., Bonavida, B., and Yoshida, O. Doxorubicin sensitizes human bladder carcinoma cells to
Fas-mediated cytotoxicity. Cancer (Phila.), 79: 1180 –1189, 1997.
Downloaded from clincancerres.aacrjournals.org on June 18, 2017. © 1999 American Association for Cancer
Research.
2612 Chemoimmunosensitization of Bladder Cancer
12. Safrit, J. T., and Bonavida, B. Sensitivity of resistant human tumor cell
lines to tumor necrosis factor and Adriamycin used in combination: correlation between down-regulation of tumor necrosis factor-messenger RNA
induction and overcoming resistance. Cancer Res., 52: 6630 –6637, 1992.
13. Mizutani, Y., Nio, Y., Fukumoto, M., and Yoshida, O. Effects of
Bacillus Calmette-Guérin on cytotoxic activities of peripheral blood
lymphocytes against human T24 lined and freshly isolated autologous
urinary bladder transitional carcinoma cells in patients with urinary
bladder cancer. Cancer (Phila.), 69: 537–545, 1992.
14. Mizutani, Y., Okada, Y., Terachi, T., Kakehi, Y., and Yoshida, O.
Serum granulocyte colony-stimulating factor levels in patients with
urinary bladder tumour and various urological malignancies. Br. J.
Urol., 76: 580 –586, 1995.
15. Mizutani, Y., Yoshida, O., and Bonavida, B. Sensitization of human
bladder cancer cells to Fas-mediated cytotoxicity by cis-diamminedichloroplatinum (II). J. Urol., 160: 561–570, 1998.
16. Mizutani, Y., Yoshida, O., and Bonavida, B. Prognostic significance of soluble Fas in the serum of patients with bladder cancer.
J. Urol., 160: 571–576, 1998.
17. Mizutani, Y., and Yoshida, O. Overcoming TNF-a resistance of human renal and ovarian carcinoma cells by combination treatment with
buthionine sulfoximine and TNF-a. Role of TNF-a mRNA down-regulation in tumor cell sensitization. Cancer (Phila.), 73: 730 –737, 1994.
18. Mizutani, Y., Fukumoto, M., Bonavida, B., and Yoshida, O. Enhancement of sensitivity of urinary bladder tumor cells to cisplatin by
c-myc antisense oligonucleotide. Cancer (Phila.), 74: 1546 –2554, 1994.
19. Boix, J., Llecha, N., Yuste, V. J. K, and Comella, J. X. Characterization of the cell death process induced by staurosporine in human
neuroblastoma cell lines. Neuropharmacology, 36: 811– 821, 1997.
20. Shinozawa, S., Mikami, Y., and Araki, Y. Determination of the
concentration of Adriamycin and its metabolites in the serum and tissues
of Ehrlich carcinoma-bearing mice by high-performance liquid chromatography. J. Chromatogr., 196: 463– 469, 1980.
21. Mizutani, Y., Bonavida, B., Koishihara, Y., Akamatsu, K., Ohsugi,
Y., and Yoshida, O. Sensitization of human renal cell carcinoma cells to
cis-diamminedichloroplatinum (II) by anti-interleukin-6 monoclonal antibody or anti-interleukin-6-receptor monoclonal antibody. Cancer Res.,
55: 590 –596, 1995.
22. Mizutani, Y., Bonavida, B., Nio, Y., and Yoshida, O. Overcoming
TNF-a and drug resistance of human renal cell carcinoma cells by
treatment with pentoxifylline in combination with TNF-a or drugs.
J. Urol., 151: 1697–1702, 1994.
23. Berenbaum, M. C. Synergy, additivism and antagonism in immunosuppression. Clin. Exp. Immunol., 28: 1–18, 1977.
24. Berenbaum, M. C. A method for testing for synergy with any
number of agents. J. Infect. Dis., 137: 122–130, 1978.
25. Volm, M., Mattern, J., and Samsel, B. Relationship of inherent
resistance to doxorubicin, proliferative activity and expression of Pglycoprotein 170, and glutathione S-transferase-p in human lung tumors. Cancer (Phila.), 70: 764 –769, 1992.
26. Ahn, H., Lee, E., Kim, K., and Lee, C. Effect of glutathione and its
related enzymes on chemosensitivity of renal cell carcinoma and bladder
carcinoma cell lines. J. Urol., 151: 263–267, 1994.
27. Keane, M. M., Ettenberg, S. A., Nau, M. N., Russell, E. K., and
Lipkowitz, S. Chemotherapy augments TRAIL-induced apoptosis in
breast cell lines. Cancer Res., 59: 734 –741, 1999.
28. Breuninger, L. M., Paul, S., Gaughan, K., Miki, T., Chan, A.,
Aaronson, S. A., and Kruh, G. Expression of multidrug resistanceassociated protein in NIH/3T3 cells confers multidrug resistance associated with increased drug efflux and altered intracellular drug distribution. Cancer Res., 55: 5342–5347, 1995.
29. Nooter, K., and Herweijer, H. Multidrug resistance (MDR) genes in
human cancer. Br. J. Cancer, 63: 663– 669, 1991.
30. Goldenberg, G. J., Wang, H., and Blair, G. W. Resistance to Adriamycin: relationship of cytotoxicity to drug uptake and DNA single- and
double-strand breakage in cloned cell lines of Adriamycin-sensitive and
-resistant P388 leukemia. Cancer Res., 46: 2978–2983, 1986.
31. Deffie, A. M., Alam, T., Senevirante, C., Beenken, S. W., Batra,
J. K., Shea, T. C., Henner, W. D., and Goldenberg, G. J. Multifactorial
resistance to Adriamycin: relationship of DNA repair, glutathione transferase activity, drug efflux, and P-glycoprotein in cloned cell lines of
Adriamycin-sensitive and -resistant P388 leukemia. Cancer Res., 48:
3595–3602, 1988.
32. De Jong, S., Zijlstra, J. G., De Vries, E. G. E., and Mulder, N. H.
Reduced DNA topoisomerase II activity and drug-induced cleavage
activity in an Adriamycin-resistant human small cell lung carcinoma cell
line. Cancer Res., 50: 304 –309, 1990.
33. Deffie, A. M., Batra, J. K., and Goldenberg, G. J. Direct correlation
between DNA topoisomerase II activity and cytotoxicity in Adriamycinsensitive and -resistant P388 leukemia cell lines. Cancer Res., 49:
58 – 62, 1989.
34. Sentman, C. L., Shutter, J. R., Hockenberry, D., Kanagawa, O., and
Korsmeyer, S. J. bcl-2 inhibits multiple forms of apoptosis but not
negative selection in thymocytes. Cell, 67: 879 – 888, 1991.
35. Marsters, S. A., Pitti, R. M., Donahue, C. J., Ruppert, S., Bauer, K. D.,
and Ashkenazi, A. Activation of apoptosis by APO-2 ligand is independent
of FADD but blocked by crmA. Curr. Biol., 6: 750–752, 1996.
36. Mariani, S. M., Matiba, B., Armandola, E. A., and Krammer, P. H.
ICE-like proteases/caspases are involved in TRAIL-induced apoptosis
of melanoma and leukemia cells. J. Cell Biol., 137: 21–29, 1997.
37. Mariani, S. M., and Krammer, P. H. Differential regulation of
TRAIL and CD95 ligand in transformed cells of the T and B lymphocyte
lineage. Eur. J. Immunol., 28: 973–982, 1998.
38. Mariani, S. M., and Krammer, P. H. Surface expression of TRAIL/
Apo-2 ligand in activated T and B cells. Eur. J. Immunol., 28: 1492–
1498, 1998.
39. Jeremias, I., Herr, I., Boehler, T., and Debatin, K. M. TRAIL/Apo-2
ligand-induced apoptosis in human T cells. Eur. J. Immunol., 28:
143–152, 1998.
Downloaded from clincancerres.aacrjournals.org on June 18, 2017. © 1999 American Association for Cancer
Research.
Synergistic Cytotoxicity and Apoptosis by Apo-2 Ligand and
Adriamycin against Bladder Cancer Cells
Youichi Mizutani, Osamu Yoshida, Tsuneharu Miki, et al.
Clin Cancer Res 1999;5:2605-2612.
Updated version
Cited articles
Citing articles
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://clincancerres.aacrjournals.org/content/5/9/2605
This article cites 39 articles, 12 of which you can access for free at:
http://clincancerres.aacrjournals.org/content/5/9/2605.full.html#ref-list-1
This article has been cited by 10 HighWire-hosted articles. Access the articles at:
/content/5/9/2605.full.html#related-urls
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from clincancerres.aacrjournals.org on June 18, 2017. © 1999 American Association for Cancer
Research.