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725
Involvement of the Mitochondrion Respiratory Chain
in the Synergy Achieved by Treatment of Human
Ovarian Carcinoma Cell Lines with both Tumor
Necrosis Factor-a and Cis-Diamminedichloroplatinum
Ruchan Uslu, M.D.’
Benjamin Bonavida, Ph.D.’
Address for reprints: Benjamin Bonavida, Ph.D.,
Department of Microbiology and Immunology,
UCLA School of Medicine, Center for Health Sciences, 10833 Le Conte Avenue, Los Angeles,
CA 90095-1747.
BACKGROUND. Previous studies have demonstrated that treatment of human tumor
cell lines with a combination of cis-diamminedichloroplatinum (CDDP) and tumor
necrosis factor-a (TNF-a) results in additivelsynergistic cytotoxic effects and reverses tumor cell resistance to TNF drugs. Free radical intermediates are induced
by both TNF-a and CDDP; however, the role of free radicals in synergy is not
known. This study investigated the effect of two inhibitors on synergy, phenoxan
(Phe) and butylated hydroxyanisole (BHA), which inhibit Complex I and Complex
1 and 11 of the mitochondrion respiratory chain, respectively.
METHODS. Three human ovarian carcinoma cell lines of different sensitivity to
TNF-a andlor CDDP were selected for the study and consisted of 222, a TNFl
CDDP-sensitive line, 222TR (TNF-resistant), a TNF-resistant, CDDP-sensitive line,
and AD10, a TNF-sensitive, CDDP-resistant line. Cytotoxicity was determined by
the microculture tetrazoliuni dye assay.
RESULTS. Synergy in cytotoxicity was achieved in all three lines treated with a
combination of TNF-a and CDDP. Cytotoxicity by either TNF-u or CDDP or by
both TNF-a and CDDP was inhibited in the presence of either Phe or BHA. Pretreatment of tumor cells with either Phe or BHA for up to 4 hours, washed and followed
by the addition of the cytotoxic agents (alone or combined), resulted in no inhibitory effect. Pretreatment of the cells with the cytotoxic agent for up to 2 hours,
washed and then followed by the addition of Phe, resulted in significant inhibition
of cytotoxicity. In contrast to Phe, the addition of BHA as late as 12 hours post
pretreatment of the cells with the cytotoxic agent(s) still inhibited cytotoxicity.
These results demonstrated that free radicals are involved in cytotoxicity mediated
by a single agent, and in synergy with both agents. Further, the results demonstrated that Phe acts at an early stage of the cytotoxic pathway and that BHA acts
at both an early and a late stage of the cytotoxic pathway.
CONCLUSIONS. These results demonstrated that both TNF-a and CDDP rapidly
stimulate the induction of free radicals but a lag of several hours was necessary
to initiate the irreversible program of cell death. Further, the studies demonstrated
that synergy and reversal of drug resistance in ovarian tumor cells by TNF-CY
and
CDDP, used in combination, share the same pathway of cytotoxicity as that mediated by TNF-a or CDDP used as a single agent. Cancer 1996; 7Z725-32.
0 1996 Ainericun Cancer Sociefy.
Received February 16, 1995; revisions received
August 14, 1995, and October 16, 1995; accepted October 16, 1995.
KEYWORDS: cytotoxicity, ovarian carcinoma, CDDP, TNF-a, synergy, resistance, free
radicals.
Department of Microbiology and Immunology,
UCLA Sc:hool of Medicine, University of California at Los Angeles, Los Angeles, California.
Supported in part by a grant from the Boiron
Research Foundation and a grant from Yuksek
Ogretim Kurumu, lurkey (RU).
Q 1996 American Cancer Society
726
CANCER February 15,1996 / Volume 77 / Number 4
R
ecent studies from our laboratory have reported that
resistance of human tumor cells to drugsltumor necrosis factor (TNF)' can be reversed by the synergistic
cytotoxic activity of TNF-a and drugs.' ' In some studies,
we reported that synergy by TNF-a and adriamycin
(ADR)/cis-diamminedichloroplatinum(CDDP) is not influenced by the expression of gp170 in a multidrug resistance phenotype (MDR)' cell line but correlated with
the expression of TNF messenger ribonucleic acid
(mRNA).'--!TNF-a is a cytokine with a wide range of biologic activities. It was first described in serum of endotoxin-treated mice that can induce tumor necrosis in vivo
and selectively kills transformed and neoplastic cell lines
in vitro.' TNF-a has two types of receptors and its multiple activities are mediated by these two receptors. The
surface expression of TNF-a receptors is necessary but
not sufficient for the biologic activities of TNF-CX.~
Among
the well-known effects of TNF-a are its oxidative damageG
and DNA fragmentation.' Moreover, anaerobic conditions," glucocorticoids," and antioxidants' can block TNFa-mediated killing. These studies suggested that the generation of reactive oxygen intermediates by TNF-a may
be responsible for damaging cellular macromolecules
such as unsaturated lipids, DNA, RNA, and proteins and
further, free radicals may enhance enzymes that are involved in TNF-cu-mediated cytotoxicity pathway."'
One of the most important sources of reactive-oxygen intermediates is the mitochondria." Recently, it has
been shown that a functional mitochondrial respiratory
chain is crucial to the events leading to TNF-a-cytotoxicity in the classical TNF-a-sensitive fibrosarcoma cell line
L929 and in the WEHI164 clone 13." Also, mitochondrial
intermediates control both the cytotoxic and gene regulatory effects of TNF-a.' ' Other investigators have shown
that a high level of manganese-superoxide dismutase
(Mn-SOD), an important mitochondrial antioxidant enzyme, is up-regulated in TNF resistant lines and/or it
can be induced by TNF-cy in several tumor cell lines.'4
Conversely, inhibition of Mn-SOD and reduction of glutathione levels lead to augmentation of TNF-a cytotoxicitY.''.l4
Cisplatin is a widely used chemotherapeutic agent in
the treatment of several tumors such as testicular, ovarian, head and neck, and small cell lung cancer. However,
drug resistance is a major obstacle in cancer treatmentt5
Oxidative damage has been implicated in cytotoxicity by
cisplatin."' It has been shown that cellular resistance to
oxidative stress is accompanied by resistance to cisplatin." It has also been shown that the enzymatic activities of catalase, glutathione peroxidase and glutathioneS-transferase were significantly decreased whereas
Mn-SOD activity increased gradually after CDDP adniinistration." Studies on the subcellular distribution of cis-
FIGURE 1. Schematic diagram of the mitochondrion electron transport
system.
platin revealed that the mitochondrion and the cytosol
accuniulate the highest concentration of platinum in the
cell.'" Also, cisplatin inhibits enzymes of the mitochondrial respiratory chain." CDDP increases the accumulation of mitochondrial calcium,'' and can cause damage
to the mitochondrial DNA."
Since cytotoxicity by both TNF-cy a n d cisplatin involve the mitochondria, we hypothesized that both synergy a n d reversal of drug resistance of ovarian carcinoma cell lines by TNF-a a n d CDDP might b e sharing
a common pathway(s) of reactive-oxygen-mediated cytotoxicity. This hypothesis was tested by examining the
role of mitochondrial dysfunction in the generation of
reactive-oxygen intermediates by two inhibitors,
namely, phenoxan (Phe)'"''
a n d butylated hydroxyanisole (BHA), antioxidant'5 a n d potent mitochondrial
electron transport inhibitors at site of Complex I and
11, respectively (Fig. 1 ) .
MATERIALS AN0 METHOOS
Tumor Cells
Human tumor cells were obtained from malignant ascites
of a patient with a n ovarian tumor and were established
as a cell line named 222. Then, a TNF-resistant subline
of 222, named 222TR. was established by culturing it in
increasing concentrations of TNF-a.' The 222TR cell line
is TNF-resistant but remains sensitive to cisplatin. The
ADlO ovarian carcinoma cell line was obtained from Dr.
Robert Ozols, Philadelphia, PA. The A D l O cell line is an
adriamycin resistant variant developed from the ovarian
line A2780, and is an MDR' line, and sensitive to TNF-a
cytotoxicity. The three tumor cell lines are adherent in
culture and are maintained in 10% heat inactivated bovine calf serum (Gibco, Grand Island, NY) added to RPMI1640 medium (Gibco) with 1% pyruvate (Gibco), 1%
nonessential amino acids (Gibco) and 1% Fungi-bact solution (Irvine Scientific, Santa Ana, CA) which contains
10,000 Ulml penicillin G , 10 ing/ml streptomycin and 25
Role of the Mitochondrion in Resistance/Uslu et al.
pglml Fungizone. When the tumor cell lines were used
as target cells, they were first detached with TrypsinEDTA (ICN Biomedicals, Costa Mesa, CA), washed, and
resuspended in complete medium.
222
I
CDDPluM)
222
losssl
TNF(pM)
727
1033
Reagents
TNF-a w,as purchased from Peprotech (Nutley, NJ). TNFa was diluted in PBS at 10 pg/ml and stored at 4" C. CDDP
(Lot. No. 103H3451) and butylated hydroxyanisole (BHA)
(Lot. No. 12H0147) were purchased from Sigma, St.
Louis, MO. The phenoxan (Phe) was a gift from Dr. Elena
Burlakova (Moscow, Russia). Stock solutions of CDDP,
BHA, and phenoxan were prepared in DMSO (Sigma, Lot.
No. 93H4650) and the DMSO concentration in the assay
did not exceed 0. I % and was not cytotoxic to the tumor
cells.
Cytotoxicity Assay
The microculture tetrazolium dye (MTT) assay was used
to determine cytotoxicity as described elsewhere.' Briefly,
target tumor cells were resuspended in medium at lo5
cells/ml after verifying the cell viability by trypan blue
dye (Sigma) exclusion. One hundred pl of cell suspension
was distributed into each well of a 96-well flat bottom
microtiter plate (Costar, Cambridge, MA) and each plate
was incubated for 24 hours at 37" C in a humidified 5%
CO, atmlosphere to allow cells to proliferate. The next
day, 100 ,ul of reagent solutions at the desired concentrations, or medium alone used for positive control, were
distributed into each well. 'Two hundred p1 of the medium
alone without cells and reagents were also distributed for
use as negative control. Then, the microtiter plate was
incubated for the desired periods of time. Thereafter, 20
p1 of MTT dye (5 mg/ml) (Sigma) was added into each
well. The unreactive supernatants in the wells were carefully aspirated and replaced with 100 p1 of isopropanol
(Sigma) supplemented with 0.05N HC1 to solubilize the
reactive dye. The OD values of each well at 540 nm were
read using an automatic multiwell spectrophotometer
(Titerek Multiscan MCC/340). The negative control well
was used for zeroing the OD. The percent cytotoxicity was
calculated using the background-corrected OD as follows:
Percent of Cytotoxicity
11
~
=
(OD of experimental wells/OD
of positive control wells)] x 100.
Statistical Analysis
All assays were set up in triplicates, and the results were
expressed as the mean plus or minus the standard deviation (SD:I (mean -C SD). Statistical analysis was determined b y the Student's t test. Synergy was calculated
0
I
4
180
400
0
I
222TR
4
160
400
222TR
80
70
-10
-10
0
4
1 0
400
AD10
0
4
180
400
AD10
.0
160
4
400
PHE
FIGURE 2. Inhibitory effect of Phe on TNF-a and CDDP-mediated cytotoxicity against 222, 222TR, and AD10 cell lines. Cytotoxicity was determined in a 24-hour MTT assay. The results are derived from three different
experiments (standard deviation < 5).
and illustrated by a modified isobole analysis based on
Berenbaum's methods.26
RESULTS
Inhibition by Phe and BHA of TNF-a and CDDP-Mediated
Cytotoxicity on Human Ovarian Carcinoma Tumor Cell
Lines
Three human ovarian carcinoma tumor cell lines were
selected for the study, namely 222 (TNF-a, CDDP-sensitive), 222TR (TNF-resistant, CDDP-sensitive), and AD10
(TNF-a-sensitive, CDDP-resistant). The addition of Phe
inhibited TNF-a cytotoxicity of all three lines tested, with
maximal inhibition obtained at concentrations of Phe 2
160 pM and TNF-a < 588 pM (Fig. 2). The addition of
Phe also inhibited CDDP-mediated cytotoxicity at concentrations 2 160 pM (Fig. 2). Optimal inhibition of TNFa and CDDP cytotoxicity was achieved at BHA concentrations > 100 pM for all three lines (Fig. 3 ) . These results
demonstrate that inhibition of free radicals by Phe and
BHA abrogates both TNF-a and CDDP cytotoxicity. Based
CANCER February 15,1996 / Volume 77 / Number 4
728
222
-10
222
TNF(pH
0
0
033
33
0
~.
~
02
04
06
08
~~
1
~
222TR
-10
1w Zen
10
1
222
CDDP(uMJ
J
0
10
1w
200
222TR
222TR
80
70
0
40
E*
-10
0 33
AD10
30
70
33
0
02
04
06
08
1
-
t
AD10
-
20
10
0
-10 I
0
10
1w
200
AD10
0
10
100
200
AD10
0
0 33
CDDP ["MI
0
02
04
06
F I C of CDDP
08
I
FIGURE 4. Synergy of cytotoxicity of AD10 by combination of TNF and
CDDP. Synergy is demonstrated in the right side by isobolograph analysis.
0
10
100
2w
BHA
FIGURE 3. Inhibitory effect of BHA on TNF-cr and CDDP-mediated cytotoxicity against 222, 222TR, and AD10 cell lines. Cytotoxicity was determined in a 24-hour MTT assay. The results are derived from three different
experiments (standard deviation > 5).
on the optimal concentration of inhibition, a constant
concentration of Phe at 400 p,M and of BHA at 200 pM
were used in subsequent experiments.
All three lines tested showed synergy when TNF-a
and CDDP were used in combination (Fig. 4). Synergy
was achieved with low concentrations (58.8 pM TNF-a
and 0.33 pM CDDP) and higher concentrations (294 pM
TNF and 3.3 ph4 CDDP) (data not shown) of the agents.
The addition of Phe or BHA significantly inhibited the
synergistic activity of TNF-a and CDDP used in combination (Fig. 5). There were no noticeable differences in the
degree of inhibition obtained in all three lines tested.
Mechanism of Phe and BHA-Mediated Inhibition of
Cytotoxicity
Phe inhibits the mitochondria1 electron transport at the
site of Complex I and B H A acts at site of Complex I and I1
(Fig. 1 ) . We wished to examine how these two inhibitors,
which act at different steps in the metabolic pathway
involved in free radical formation and detoxification,
block cytotoxicity. Initially, experiments were performed
whereby sequential treatments were done for various periods of time. Tumor cells were treated with Phe for a
period of 0.5-4 hours, washed, and TNF-a, CDDP, or a
combination of TNF-a and CDDP were added for the
duration of the MTT cytotoxicity assay. There was no
detectable inhibitory effect of pretreatment of tumor cells
with Phe on cytotoxicity by TNF-a, CDDP, or a conibination on all three cell lines tested. Also, there were no
differences when different concentrations of the agents
were used. Similar findings to those obtained with Phe
were also observed with BHA.
The time kinetics of addition of inhibitors subsequent to pretreatment with the cytotoxic agent(s) was
examined. Pretreatment with TNF-a, CDDP, or a combination for up to two hours, and wash, and addition of
Phe subsequently for the remaining of the assay, resulted
in inhibition of cytotoxicity (Table 1). However, a fourhour pretreatment with TNF-a, or a CDDP four-hour pretreatment when Phe was added four hours after, the inhibitory effect was no longer detected (Table 1, Fig. 5).
These results demonstrate that Phe acts at an early step in
the pathway of cytotoxicity after initiation of the cytotoxic
pathway. After two hours, the cells are not inhibitable by
Phe and thus, the pathway for cytotoxicity could not be
reversed.
Unlike Phe, BHA reversed cytotoxicity up to 12 hours
following pretreatment of tumor cells with the cytotoxic
agents. Pretreatment of tumor cells for four hours with
Role of the Mitochondrion in Resistance/Uslu et al.
ADIO
AD10
ADIO
45
1
r--
0
45
EM(+)
5
-5
-5
0.5h
ih
2h
4h
4h
6h
8h
12h
CDDP+BHA
CDDP+PHE
AD10
AD10
50
H
y
--
-
.Phe(+)
10
0
CISh
ih
2h
4h
Time
_ _ _ ~
4h
6h
8h
Time
'TNF+CDOP+PHE
way by which synergy is achieved by TNF-a and CDDP
involves the mitochondrion as two anti-oxidant inhibitors
abrogated cytotoxicity. Furthermore, the data suggest
that induction of cytotoxicity is as late as 12 hours following treatment and, prior to that time, cytotoxicity can be
blocked.
Two sites of the mitochondrial electron transport
chains have been identified as sources of superoxide radicals. The first is dependent on the auto-oxidation of the
flavin mononucleotide from reduced nicotinamide adenine dinucleotide (NADH)-dehydrogenase (Complex I),
and the second is dependent o n the autooxidation of the
unstable ubisemiquinone (Complex III)." The generation
of oxygen radicals at the ubiquinone site is supported by
NADH-linked substrates and is decreased by inhibitors
of Complex 120,29(Fig. 1). Schulze-Osthoff et al".'' have
shown that inhibition of the production of oxygen radicals at the site of Complex I inhibits TNF-@-mediated
cytotoxicity. Other investigators have shown that antioxidants and free radical scavengers protect cells against
TNF-a-induced cytolysis.R.'2.'"Likewise, oxidative damage
has also been implicated in cytotoxicity by CDDP.'"'"
Also, CDDP has been shown to have a n effect on the
mitochondrial membrane, the respiratory chain, and the
mitochondrial DNA."-"
The phenolic compound 2,6-di-tert-butyl+methoxy-phenol (butylated hydroxyanisole1BHA) is a widely
used antioxidant to prevent foods from oxidative deterioration and rancidity. In addition to its preventive effect
on lipids and proteins from peroxidation,25BHA has been
shown to inhibit the mitochondrial electron transport
system3"."' at the site of Complex I and Complex 11. Further, BHA has been shown to increase glutathione and
glutathione-s-transferase levels,'* to increase gammaglutamylcysteine synthetase activity,"3 to increase cytosolic free calcium levels,34 and to induce certain oncogenes.35 These studies demonstrated that BHA acts at
both the production of free radicals and at the detoxification of free radicals. In contrast, Phenoxan is a new oxazole-pyrone from myxobacteria and inhibits the mitochondria] electron transport system at the site of Complex
I and acts primarily on the production of free radicals.""?'
The present study demonstrates that both TNF-a and
CDDP-mediated cytotoxicity and synergy by TNF-a and
CDDP are inhibited by both Phenoxan and BHA, suggesting a common initial trigger of cytotoxicity. The results also demonstrate that induction by the cytotoxic
agents must precede the block by the inhibitors suggesting d e novo induction of free radicals. Thus, modulation of the mitochondria1 respiratory chain by TNF-a and
CDDP is a n early event in the cytotoxic pathway. Phenoxan acts at this early stage of the cytotoxic pathway,
and is effective only for 2 hours following which the cells
;VTri
AD10
i2h
TNF+CDDP+BH
FIGURE 5. Effect of sequence of treatment of AD10 tumor cells with
Phe or BHA and TNF-a. CDDP, or TNF and CDDP on cytotoxic activity.
The tumor cells were pretreated with TNF-a (58.8 pM), CDDP (0.33p M ) ,
or both for desired periods of time. The medium was aspirated and the
tumor cell:; were washed twice with RPMl medium. The cells were then
incubated with Phe (400 p M ) or BHA (200 pM) for desired periods of
time. Cytotoxicity was assessed in a 24-hour MTT assay. The results are
derived from three different experiments (SD < 5).
the cytotoxic agents and wash was sufficient to trigger
cytotoxicity to the same extent as cells pretreated for 12
hours or longer (Table 1 and Fig. 5). These results demonstrate that BHA acts at an early and at a late stage of the
pathway of cytotoxicity (i.e. both Phe and BHA block the
inductive phase while BHA also blocks a post inductive
phase of cytotoxicity).
DISCUSSION
Studies have reported that TNF-a and CDDP used in
combination can exert a synergistic cytotoxic activity
against 1°F-a andlor CDDP-resistant human tumor cell
lines and can also reverse tumor cell resistance to TNFN andlor CDDP. The mechanisms of synergy and overriding tumor cell resistance, however, have not been elucidated. The present study provides evidence that the path-
729
730
CANCER February 15, 1996 / Volume 77 / Number 4
TABLE 1
Effect of Sequential Treatment of TNF-a or CDDP with Wash on the Inhibitory Effect of PHE and BHA
Firstlsecond treatment
Tumor
222
Time of 1st
treatment (h)
0.5
1
7
222TK
AD10
4
0.5
1
2
4
0.5
Tie of 1st
TNFl
medium
TNFlPhe
Phe'lTNF
CDDPl
medium
CDDPlPhe
Phe"l
CDDP
TNF t CDDPl
Med
TNF t CDDPl
Phe
Phea TNF t
CDDP
4.1 i 2.1
5.2 ? 2.5
9.2 2 3.5
11.0 2 1.8
1.1 t 2.8
1.2 ? 1.7
1.8 2.9
3.1 i 1.3
5.8 i 3.2
6.2 t 2.3
8.8 i 2.7
12.1 ? 2.7
0.3 i 2.1
0.9 ? 2.1
5.5 i 1.7
11.1 i 2.4
1.0 2 3.1
0.8 1 2.1
0.2 i- 1.1
3.2 i- 2.0
-1.2 2 3.1
2.3 2 1.2
4.2 i 0.8
12.3 i 2.4
13.5 ? 2.7
13.7 t- 1.9
12.5 2 1.7
12.7 2 1.3
4.5 i 2.5
4.2 t 2.4
3.7 2 1.8
4.3 i 0.7
14.3 i 1.7
14.0 t- 3.3
13.5 2 2.2
13.3 i 1.2
6.1 1 1.3
7.8 i 2.9
10.9 i- 2.4
12.1 I 1.5
7.0 1 0.7
8.2 I1.9
9.5 i 1.9
13.2 i 1.0
O . i 5 1.2
1.0 i- 0.3
1.5 t 2.7
2.2 i- 1.8
1.8 t 1.0
3.2 ? 2.6
8.1 i 3.2
12.3 1' 2.1
0.8 ? 2.7
1.1 i 2.2
6.9 i 2.8
13.1 2 3.4
-0.7 2 1.4
-0.3 t 1.4
0.9 2 2.8
2.4 2 3.0
14.0 i- 1.4
13.9 ? 1.7
13.7 ? 1.9
13.0 i 2.6
14.9 i- 1.5
14.5 2 2.3
13.4 i 1.7
14.0 t 2.4
3.8 t- 0.4
3.7 2 1.8
2.9 i 1.0
2.9 i 3.4
26.9 i 3.1
28.1 ? 3.3
39.8 2 3.0
40.9 ? 2.7
18.2 2 3.0
20.9 2.1
26,8 t 0.8
36.1 ? 1.8
15.5 2 2.1
17.5 1.3
24.9 t- 1.3
34.8 2 2.1
6.8 I 2.4
10.5 2 2.7
2 0.5 2 3.2
41.1 i 1.9
3.2 2 1.0
4.1 2 3.0
15.9 i 2.7
36.5 i 1.8
0.6 5 1.9
3.5 2 2.8
13.7 i 1.8
34.5 i 2.3
45.0 i- 3.2
44.3 t- 1.2
43.3 2 1.1
42.4 t 1.9
38.9 t- 2.2
38.3 i- 1.7
36.3 ? 3.2
36.9 i 1.7
38.4 t 2.0
36.8 i- 2.0
35.1 i 2.4
36.2 i 2.4
+_
treatment (h)
TNFl
medium
TNF/BHAa
BHA/TNF
CDDPl
medium
CDDPl
BHA
BHA/
Tumor
CDDP
TNF t CDDPl
med
TNF t CDDPl
BHA
BHA/TNF t
CDDP
222
4
8
12
4
8
12
4
8
12
12.0 i- 2.1
12.3 2 2.3
11.9 2 3.7
2.9 i 1.7
3.1 1' 1.6
2.9 2 0.4
13.9 t 1.8
13.1 2 1.3
12.1 t 1.7
-2.8 2 1.7
-2.2 i 0.7
-0.9 t 0.8
-2.7 ? 1.1
-2.0 % 1.3
-1.0 2 3.0
-2.3 i- 0.7
-1.9i 1.8
-0.6 2 2.2
12.3 2 2.8
12.6 ? 2.7
11.5 t- 0.5
3.2 i 1.5
2.9 i- 0.9
2.9 i 2.1
14.3 t 2.2
13.8 2 1.8
13.2 i 1.1
12.7 i 1.0
11.6 i 0.7
11.7 t 2.6
12.5 i 2.0
12.9 i- 1.0
12.9 i 2.4
2.4 t 1.6
2.6 i- 0.6
2.2 i 1.6
-2.7
-2.1
-1.1
-2.4
-1.7
-0.5
-2.2
-1.2
-0.8
13.1 2 1.0
12.2 ? 1.1
11.2 5 0.9
14.0 i- 0.7
13.0 ? 1.7
11.8 t 1.7
3.1 t- 1.9
1.2 ? 0.7
2.8 1' 1.7
45.0 i 2.3
41.0 i 2.4
40.9 t 1.7
33.7 ? 0.9
14.8 ? 1.3
33.6 2 1.1
32.0 I 2.1
31.9 i 1.7
31.9 t 2.8
5.4 2 2.2
8.1 i 1.0
9.6 5 2.6
-1.9 i- 2.9
1.0 i 3.4
5.3 2 2.2
-1.4 2 0.8
-1.0 ? 2.3
6.9 2 1.2
46.0 i- 3.2
41.8 2 0.9
40.3 i 3.1
37.9 ? 3.5
35.9 i 3.0
32.7 2 2.8
33.1 2 3.4
33.8 2 1.4
32.3 2 1.8
222TR
AD10
2 2.1
t- 2.1
i 2.0
2 2.4
2 1.6
? 1.3
i 1.8
t- 1.9
? 1.1
-
"First treaunent with Ilhe or Btl4 showed the same findings as first treatment with medium alone i.e. Phe and BHA are not toxic to the cells. The bold nuinbers represent the inhibition mediated by the addition
of Phe.
The cells w e pretreated with medium only, TNF-n 158.8 pM), CDDP 10.33 pM),Phe 1400 pM1 or BHA 1200 phl) or TNF-a 158.8 pM) t CDDP 10.33 pMJ for desired periods of time (first treatment). The medium
was aspirated and the cells were washed twice with RPMl medium. I h e cells were then incubated with medium only, TNF-n (58.8 pMI, CDDP 10.33 !MI, Phe (400 phl] or BHA (200 pMi or TNF-a (58.8 pM1 t
CDDP 10.33 pMI for desired periods of time [second treatment). Cytoroxicity was assessed in one day MTT assay. The results are erpressed as the mean S.D.
are programmed for cell death and cannot be reversed.
However, in addition to what Phe does, BHA also has a
free radical scavenger effect which prevents peroxidation
of lipids and proteins. This preoxidation stage appears to
be a late stage of the cytotoxic pathway initiated by the
free radicals. Hence, BHA can block the synergistic cytotoxicity as late as 12 hours post treatment of the cells with
the cytotoxic agent(s).
Clement et a13' have shown that DNA fragmentation
by anti-fas antibody takes place in less than 2 hours, but
that TNF-mediated DNA fragmentation can occur as late
as 24 hours. It is generally accepted that DNA fragmentation is irreversible and cannot be b10cked.~'In our experiments, no significant DNA fragmentation was detected
up to 12 hours following treatment with a concentration
of CDDP or TNF-a (data not shown), and accordingly,
BHA could reverse CDDP and TNF-a-mediated cytotoxicity up to 12 hours prior to irreversible DNA damage.
Tumor cells develop resistance to chemotherapy.
Ovarian cancer is a model disease to investigate chemotherapeutic resistance because of both its intrinsic and
its acquired resistance to drugs. Approximately 75% of
patients have surgically-incurable disease at time of diagnosis, thus chemotherapy plays a major role in the management of ovarian cancer.38 Aggressive treatment of
these patients with platinum-based combination chemotherapy usually results in severe adverse effects and it is
rarely curative. Therefore, effective subtoxic therapeutic
modalities are needed. Such modalities include biological
response modifiers, cytokine based chemotherapy, passive administration of tumor infiltrating lymphocytes,
genes therapy, and targeting of tumor cells with immunotoxins. In addition, the development of drugs that inhibit
cellular anti-oxidant pathways should reverse tumor cell
sensitivity to cytotoxic agents.
Recent studies have shown that treatment of tumor
Role of the Mitochondrion in Resistance/Uslu et al.
cells with combinations of biologic response modifiers
such as IFN-a, IL-2, and TNF-a and drugs or toxins results
in reverse drug resistance and often,
These combination treatments were also effective at concentrations that were subtoxic. Such treatment modalities
may also boost the immune system for a more effective
and complimentary antitumor immune response.
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