Synergistic Cytotoxicity and Apoptosis by Apo-2 Ligand and Adriamycin against Bladder Cancer Cells1
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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, achieved in apoptosis. Intracellular accumulation of ADR
Tsuneharu Miki, and Benjamin Bonavida was not affected by sApo-2L. Incubation of T24 cells with
sApo-2L down-regulated the expression of glutathione S-
Department of Urology, Faculty of Medicine, Kyoto University,
Kyoto 606-8507, Japan [Y. M., O. Y.]; Department of Urology, transferase-p mRNA.
Kyoto Prefectural University of Medicine, Kyoto 606-0841, Japan This study demonstrates that combination treatment of
[T. M.]; and Department of Microbiology and Immunology, UCLA bladder cancer cells with sApo-2L and ADR overcomes their
School of Medicine, University of California at Los Angeles, resistance. The sensitization obtained with established ADR-
California 90095 [B. B.]
resistant bladder cancer cells and freshly isolated bladder
cancer cells required low subtoxic concentrations of ADR,
ABSTRACT thus supporting the in vivo potential application of combi-
Resistance to conventional anticancer chemotherapeu- nation of sApo-2L and ADR in the treatment of ADR-
tic agents remains one of the major problems in the treat- resistant bladder cancer.
ment of bladder cancer. Hence, new therapeutic modalities
are necessary to treat the drug-resistant cancers. Apo-2 INTRODUCTION
ligand (Apo-2L) is member of the tumor necrosis factor Previous studies have demonstrated that a variety of cancer
ligand family, and it induces apoptosis in cancer cells. Sev- cells have different degrees of sensitivity and resistance to
eral cytotoxic anticancer drugs, including Adriamycin various kinds of anticancer cytotoxic agents (1). When antican-
(ADR), also mediate apoptosis and may share the common cer chemotherapeutic agents are administered, only the drug-
intracellular pathways leading to cell death. We reasoned sensitive cancer cells are deleted, and cancer cells with acquired
that combination treatment of the drug-resistant cancer resistance develop (2). Consequently, drug resistance (inherent
cells with Apo-2L and drugs might overcome their resist- or acquired) is the major cause of failure in cancer chemother-
ance. Here, we examined whether bladder cancer cells are apy. Therefore, new effective therapeutic modalities are neces-
sensitive to Apo-2L-mediated cytotoxicity and whether sary to treat the drug-resistant cancers.
Apo-2L can synergize with ADR in cytotoxicity and apop- Apo-2L3 (also known as TRAIL) is a member of the TNF
tosis against bladder cancer cells. ligand family, and it induces apoptosis in various transformed
Recombinant human soluble Apo-2L (sApo-2L), which cell lines, such as Fas ligand (3, 4). Unlike Fas ligand, the
carries the extracellular domain of Apo-2L, was used as a transcripts of which are predominantly restricted to stimulated T
ligand. Cytotoxicity was determined by a 1-day microculture cells and sites of immune privilege, expression of Apo-2L is
tetrazolium dye assay. Synergy was assessed by isobolo- detected in a lot of normal human tissues, most predominantly
graphic analysis. in spleen, lung, and prostate (3). The receptors for Apo-2L,
Human T24 bladder cancer line was relatively resistant DR4, and DR5 are also expressed in multiple human normal
to sApo-2L. Treatment of T24 line with combination of tissues (5, 6). Interestingly, Apo-2L is not cytotoxic to most
sApo-2L and ADR resulted in a synergistic cytotoxic effect. normal tissues in vivo; however, it has marked apoptotic poten-
Synergy was also achieved in the ADR-resistant T24 line tial for cancer cells (3, 4). In contrast to injection of Fas ligand
(T24/ADR), two other bladder cancer lines, and three and anti-Fas mAb, which is lethal to mice, injection of Apo-2L
freshly derived human bladder cancer cell samples. In ad- is well tolerated in mice (7, 8). Thus, Apo-2L may have poten-
dition, T24 cells were sensitive to treatment with sApo-2L tial utility as an anticancer agent.
combined with epirubicin or pirarubicin. The synergy Fas ligand and TNF-a as well as Apo-2L induce apoptosis
achieved in cytotoxicity with sApo-2L and ADR was also (9, 10). Several anticancer drugs, including ADR, also mediate
apoptosis and may share common intracellular signaling path-
ways leading to cell death. Indeed, we have reported that treat-
ment with ADR in combination with anti-Fas mAb or TNF-a
resulted in significant potentiation of cytotoxicity and synergy
Received 3/23/99; revised 6/1/99; accepted 6/14/99. against a variety of sensitive and resistant human cancer cells
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
(11, 12). This study investigated whether the resistance of
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 Me-
morial, The Osaka Foundation for Promotion of Fundamental Medical
3
Research. The abbreviations used are: Apo-2L, Apo-2 ligand; TNF, tumor ne-
2
To whom requests for reprints should be addressed, at Department of crosis factor; mAb, monoclonal antibody; ADR, Adriamycin; sApo-2L,
Urology, Faculty of Medicine, Kyoto University, 54 Shogoin-Kawa- soluble Apo-2L; EPI, epirubicin; THP, pirarubicin; GST-p, glutathione
hara-cho, Sakyo-ku, Kyoto 606-8507, Japan. Phone: (075) 751-3337; S-transferase-p; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
Fax: (075) 751-3740. zolium bromide; Topo II, topoisomerase II.
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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 as-
sessed in a 1-day MTT assay (A) and estimated by
isobolographic analysis (B). Columns, means of
three different experiments; bars, SD. p, signifi-
cantly higher than those achieved by treatment with
ADR alone at P , 0.05.
bladder cancer cells to ADR, one of anticancer agents used cell carcinoma of the bladder. Their histological classification and
clinically against bladder cancer, could be overcome by combi- staging according to the tumor-node-metastasis classification were:
nation treatment with Apo-2L and ADR. patient 1, T2N0M0, grade 3; patient 2, T1N0M0, grade 2; and patient
3, T1N0M0, grade 2. Briefly, cell suspensions were prepared by
MATERIALS AND METHODS treating finely minced tumor tissues with collagenase (3 mg/ml;
Tumor Cells. The T24, J82, and HT1197 human bladder Sigma Chemical Co., St. Louis, MO). After washing three times in
cancer cell lines were maintained in monolayers on plastic RPMI 1640, the cell suspensions were layered on discontinuous
dishes in RPMI 1640 (Life Technologies, Inc., Bio-cult, Glas- gradients consisting of 2 ml of 100% Ficoll-Hypaque, 2 ml of 80%
gow, Scotland), supplemented with 25 mM HEPES, 2 mM L- Ficoll-Hypaque, and 2 ml of 50% Ficoll-Hypaque in 15-ml plastic
glutamine, 1% nonessential amino acids, 100 units/ml penicil- tubes and were centrifuged at 400 3 g for 30 min. Lymphocyte-
lin, 100 mg/ml streptomycin, and 10% heat-inactivated fetal rich mononuclear cells were collected from the 100% interface, and
bovine serum (all from Life Technologies, Inc.), hereafter re- tumor and mesothelial cells were collected from the 80% interface.
ferred to as complete medium (13, 14). The T24/ADR line is an Cell suspensions that were enriched with tumor cells were some-
ADR-resistant subline of the T24 cell line (11). times contaminated by monocyte-macrophages, mesothelial cells,
Fresh bladder cancer cells derived from three patients were or lymphocytes. To eliminate further contamination of host cells,
separated from surgical specimens as described previously (15, 16). we layered the cell suspensions on a discontinuous gradient con-
The histological diagnosis revealed that all patients had transitional taining 2 ml each of 25, 15, and 10% Percoll in complete medium
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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 cyto-
toxic 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 syn-
ergy 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 Pharmaceutical Co. Ltd. (Tokyo, Japan). GST-p cDNA, used in
room temperature. Tumor cells that were depleted of lymphoid making probes for Northern blot analysis, was a gift from
cells were collected from the bottom, washed, and suspended in Otsuka Pharmaceutical Co. Ltd. (Tokushima, Japan).
complete medium. To remove further contamination from me- Cytotoxicity Assay. The MTT assay was used to deter-
sothelial cells and monocyte-macrophages, we incubated the cell mine tumor cell lysis, as described previously (17, 18). Briefly,
suspension in plastic dishes for 30 – 60 min at 37°C in a humidified 100 ml of target cell suspension (2 3 104 cells) were added to
5% CO2 atmosphere. After incubation, nonadherent cells were each well of 96-well flat-bottomed microtiter plates (Corning
recovered, washed, and suspended in complete medium. Usually, Glass Works, Corning, NY), and each plate was incubated for
the nonadherent cells contained mainly tumor cells with ,5% 24 h at 37°C in a humidified 5% CO2 atmosphere. After
contaminating nonmalignant cells, as judged by morphological incubation, 100 ml of drug solution or complete medium for
examination of Wright-Giemsa-stained smears, and were .93% control were distributed in the 96-well plates, and each plate
viable, according to the trypan blue dye exclusion test. Cells having was incubated for 24 h at 37°C. Following incubation, 20 ml
,5% contamination with nonmalignant cells were accepted for use of MTT working solution (5 mg/ml; Sigma) were added to
as tumor cells. each culture well, and the cultures were incubated for 4 h at
Reagents. sApo-2L was kindly supplied by Pepro Tech 37°C in a humidified 5% CO2 atmosphere. The culture me-
(Rocky Hill, NJ). ADR (lot no. 705ACB) and EPI (lot no. dium was removed from the wells and replaced with 100 ml
3015AG) were supplied by Kyowa Hakkou Co. Ltd. (Tokyo, of isopropanol (Sigma) supplemented with 0.05 N HCl. The
Japan). THP (lot no. THPMR100) was obtained from Meiji absorbance of each well was measured with a microculture
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Research.2608 Chemoimmunosensitization of Bladder Cancer
plate reader (Immunoreader; Japan Intermed Co. Ltd., Tokyo, Table 1 Effect of sequence of treatment with sApo-2L and ADR on
Japan) at 540 nm. The percentage cytotoxicity was calculated their cytotoxic activity against T24 cells
using the following formula: percentage cytotoxicity 5 [1 2 First treatmenta Second treatmenta % cytotoxicity
(absorbance of experimental wells/absorbance of control (6 h) (18 h) (mean 6 SD)b
wells) ] 3 100. sApo-2L Medium 5.8 6 0.9
Chromatin Staining with Hoechst 33258. Apoptosis ADR Medium 10.8 6 1.5
was observed by chromatin staining with Hoechst 33258 as Medium sApo-2L 11.8 6 1.0
Medium ADR 28.7 6 2.9
described previously (19). T24 cells in a chamber/slide (Miles Medium sApo-2L 1 ADR 74.8 6 6.0c,d
Scientific, IL) were incubated with sApo-2L at 100 ng/ml in the sApo-2L ADR 68.9 6 5.3c,d
absence or presence of ADR at 1 mg/ml for 12 h at 37°C in a ADR sApo-2L 49.4 6 1.1c
humidified 5% CO2 atmosphere. After incubation, the superna- a
T24 cells were pretreated with medium only, sApo-2L (100
tant was discarded, and T24 cells were fixed with 1% glutaral- ng/ml), or ADR (10 mg/ml) for 6 h (first treatment). The medium was
dehyde in PBS for 30 min at room temperature, washed four aspirated, and T24 cells were washed twice with RPMI 1640. The cells
times with PBS, and exposed to Hoechst 33258 at 10 mM for 30 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
min at room temperature. The cell preparations were examined assay.
under UV illumination with an Olympus fluorescence micro- b
Results are expressed as the mean 6 SD of three separate exper-
scope. Apoptosis was defined when apoptotic bodies, chromatin iments.
c
condensation, or fragmented nuclei were observed. Values in the combination treatment are significantly higher than
those achieved by treatment with ADR alone at P , 0.05.
ADR Determination. The ADR content in T24 cells was d
Values in the combination treatment are significantly higher than
determined by high-performance liquid chromatography using a those achieved when ADR was given first at P , 0.05.
Hitachi model 635A (Hitachi Co. Ltd., Tokyo, Japan), as de-
scribed in detail elsewhere (20).
Northern Blotting. Cytoplasmic RNA from tumor cells
was prepared as described in detail elsewhere (21, 22). Briefly, achieved (Fig. 1). Preliminary experiments demonstrated that
tumor cell RNA (10 mg/lane) was electrophoresed in 1.2% the synergy was not observed when normal bladder cells were
agarose-2.2 M HCHO gels in 13 4-morpholinepropanesulfonic used as targets.
acid buffer (200 mM 4-morpholinepropanesulfonic acid, 50 mM To confirm the synergy, we then examined the sensitivity of
sodium acetate, and 10 mM sodium EDTA). The RNA was the T24 ADR-resistant variant cell line, T24/ADR, to treatment
transferred to Biodyne A membranes (Poll, CA) in 203 SSC [3 with sApo-2L in combination with ADR. Significant synergy was
M NaCl and 0.3 M sodium citrate (pH 7.0)]. Fifty to 100 ng of achieved by the combination treatment with sApo-2L and ADR
cDNA probe were labeled with [a-32P ]dCTP (NEN, Boston, (data not shown). Furthermore, synergy was also obtained in two
MA) by random oligoprimer extension. The nylon membranes other bladder cancer lines, J82 and HT1197 (data not shown).
were UV cross-linked and hybridized. On the basis of the above findings with established bladder
Statistical Analysis. All determinations were made in cancer cell lines, we examined for synergy on three freshly
triplicate, and the results were expressed as the mean 6 SD. isolated bladder cancer cells. In all three cases, significant
Statistical significance was determined by Student’s t test. A P synergy was achieved, irrespective of the baseline sensitivity of
of #0.05 was considered significant. the cancer cells to either ADR or sApo-2L used alone (Fig. 2).
Calculations of synergistic cytotoxicity were determined These findings demonstrate that treatment of established
by isobolographic analysis, as described by Berenbaum (23, 24). and freshly derived bladder cancer cells with a combination
The nature of the effect of a particular dose combination was of sApo-2L and ADR resulted in potentiation of cytotoxicity.
determined by isobolographic analysis, as follows: the point Furthermore, the synergy was observed with both sensitive
representing that combination would lay on, below, or above the and resistant cancer cells. In all of the above findings, syn-
straight line joining the doses of the two drugs that, when given ergy was achieved with subtoxic concentrations of ADR. The
alone, produce the same effect as that combination, representing synergy obtained with low concentrations of the agents is of
additive, synergistic, or antagonistic effects, respectively. clinical relevance because high concentrations of drugs are
toxic in vivo.
RESULTS Effect of the Sequence of Treatment with sApo-2L and
Synergistic Cytotoxicity against Bladder Cancer Cells ADR on Synergy
after Their Treatment with a Combination of sApo-2L The findings above demonstrate that simultaneous treat-
and ADR ment of T24 cells with the sApo-2L and ADR resulted in
We investigated the cytotoxic effect of a combination of synergy. The effect of sequential treatment with sApo-2L and
sApo-2L and ADR against the T24 bladder cancer cell line. ADR was examined and compared with treatment with both
Cytotoxicity was determined by a 1-day MTT assay, and syn- agents together. The T24 bladder cancer cells were treated for
ergy was assessed by isobolographic analysis, as described in 6 h with one agent, the medium was removed, the second agent
“Materials and Methods.” The T24 line was relatively resistant was subsequently added for another 18 h, and the cells were
to sApo-2L- and ADR-mediated cytotoxicity. However, when tested for viability. The results show that the highest percentage
T24 cells were treated with a combination of sApo-2L and cytotoxicity was obtained when sApo-2L was given first or
ADR, significant potentiation of cytotoxicity and synergy was together with ADR (Table 1). Similar results were obtained
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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 Mechanism of Synergy Achieved by sApo-2L and ADR
(data not shown). These findings demonstrate that the sequence Induction of Apoptosis. Because both sApo-2L and ADR
of treatment with sApo-2L and ADR may not be critical to can mediate apoptosis, we examined by Hoechst 33258 staining
obtain significant synergy but may determine the extent of whether the synergy achieved in cytotoxicity with sApo-2L and
synergy. 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
Effect of the ADR-related Cytotoxic Drugs, EPI and THP, medium. ADR at a concentration of 1 mg/ml was cytotoxic against
on Synergy with sApo-2L T24 cells and mediated modest apoptosis (Fig. 4B). Swelling of
Two closely related compounds of ADR, EPI and THP, are T24 cells was also observed. Likewise, sApo-2L, at a concentration
currently used clinically. The cytotoxic effect of these two drugs of 100 ng/ml, was slightly cytotoxic against T24 cells and also
used each in combination with sApo-2L was tested against T24 mediated some apoptosis (Fig. 4C). When sApo-2L and ADR were
cells. Synergy was achieved with both EPI and THP in combi- used in combination, fragmented nuclei of T24 cells were ob-
nation with sApo-2L (Fig. 3). served. We considered almost all T24 cells apoptotic, and synergy
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Research.2610 Chemoimmunosensitization of Bladder Cancer
Table 2 Effect of treatment of T24 cells with sApo-2L on the
intracellular accumulation of ADR
Intracellular accumulation of ADR
Treatment (ng/105 cells)a
Control 35.8 6 4.2
sApo-2L 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 intracel-
lular 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. Apo-
ptosis in T24 cells was determined by chromatin staining with Hoechst
33258, as described in “Materials and Methods.” Shown are fluorescence sApo-2L, ADR accumulation inside the cells did not change
microscopic views of chromatin staining with Hoechst 33258 (3200) after
significantly (Table 2).
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. Expression of GST-p mRNA. Previous findings indi-
cated 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
in apoptosis was also observed (Fig. 4D). The apoptosis was also of T24 cells with sApo-2L down-regulated GST-p mRNA ex-
examined by DNA ladder assay. Treatment of T24 cells with pression.
combination of sApo-2L and ADR resulted in strong DNA ladder Altogether, these findings suggest that the synergistic cy-
(data not shown). These results indicate that there was a good totoxicity achieved with sApo-2L and ADR is, in part, due to
correlation between cytotoxicity and apoptosis after treatment of up-regulation of apoptosis and down-regulation of GST-p
bladder cancer cells with a combination of sApo-2L and ADR. mRNA expression.
ADR Intracellular Accumulation. A possible mecha-
nism of augmentation of cytotoxicity may be the result of DISCUSSION
increased accumulation of ADR in the cells treated with sApo- Here, we showed that the combined treatment of sApo-2L
2L. When T24 cells were treated with combination of ADR and and ADR resulted in a synergistic cytotoxicity and apoptosis
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Research.Clinical Cancer Research 2611
against bladder cancer cells and reversed their resistance. It has synergy against bladder cancer cells (10). In addition, prelimi-
been reported that ADR augmented Apo-2-induced apoptosis in nary experiments showed that treatment of freshly isolated
breast cell lines (27). The synergy was achieved with low bladder cancer cells with ADR enhanced their susceptibility to
concentrations of each agent, thus minimizing their toxicity in lysis by autologous lymphocytes. These findings suggest that a
vivo and maximizing their therapeutic application in vivo. Be- combination of ADR chemotherapy and immunotherapy might
cause Apo-2L is not toxic to most of normal tissues (3, 4), a be an alternative approach in the treatment of ADR-/immuno-
combination of sApo-2L and ADR may be a promising strategy therapy-resistant bladder cancer.
to eliminate bladder cancer cells. The overall response rate of patients with bladder cancer
The mechanisms responsible for cellular resistance to ADR to current anticancer chemotherapeutic agents involving
are believed to be multifactorial and include alterations in the ADR has improved. However, drug resistance and recurrence
transmembrane transport of ADR, decreased formation of DNA of cancers remain major problems, and a more effective
single- and double-strand breaks, earlier onset of DNA repair, therapy is necessary for these patients. This study shows that
the cytosolic quenching of ADR due to increased levels of combination treatment with sApo-2L and ADR resulted in a
glutathione and its related enzymes, and the decreased cellular synergistic cytotoxicity and apoptosis against both acquired
level of DNA Topo II. Alterations in the transmembrane trans- and natural ADR-resistant bladder cancer cells, and the syn-
port of ADR in cancer cells by P-glycoprotein or multidrug ergistic effect is not restricted to established cell lines: it is
resistance-associated protein result in reduced intracellular ac- also observed in freshly derived cancers. These findings
cumulation of ADR and resistance to ADR (28, 29). The resist- suggest that the therapeutic use of ADR in combination with
ance to ADR has been attributed to reduced levels of DNA sApo-2L might be useful in patients with ADR-resistant
single- and double-stranded breaks induced by the drug in bladder cancer.
ADR-resistant P388 leukemia cells (30, 31). The ADR-resistant
P388 cells appeared to display an earlier onset of DNA repair ACKNOWLEDGMENTS
than did drug-sensitive cells (32). Some ADR-resistant cancer
We are deeply indebted to Professor Osamu Ogawa at Department
cells have higher levels of intracellular glutathione or its related of Urology, Faculty of Medicine, Kyoto University, for his kind advice
enzymes (25, 26). Because ADR inhibits DNA Topo II, the during this investigation.
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
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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.
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