[CANCER RESEARCH 49, 6285-6289. November 15, 1989|
Cytotoxic Interactions of Heat and an Ether Lipid Analogue in Human Ovarian
Carcinoma Cells1
Keiichi Fujiwara, Edward J. Modest, Charles E. Welander, and C. Anne Wallen2
Departments of Radiology [K. F., C. A. W.], Biochemistry [E. J. M.], and Obstetrics and Gynecology ¡C.E. W.], Bowman Gray School of Medicine of Wake Forest
University, Winston-Salem, North Carolina 27103
anticancer agents. These compounds are cytotoxic and, al
though their mechanism of action is not fully understood, the
Ether lipid (EL) analogues of platelet activating factor are known to
plasma membrane is their primary target (11,12). EL analogues
have a cell membrane-mediated antitumor activity. Although previous
interact in an additive mode with conventional DNA interactive
studies demonstrated additive interactions with EL and conventional
chemotherapeutic agents (13, 14). However, little is known
DNA-interacting chemotherapeutic agents, little is known about the
interaction of EL with heat. In this study, the cytotoxic interaction of one about the interaction of EL analogues with heat or ionizing
EL analogue, ET-18-OMe, with heat was measured at two different
radiation. Two reports have been published (15, 16) which
temperatures, 42 and 44°C,using BG-1 human ovarian carcinoma cells.
indicate that temperature strongly influences the cytotoxic ac
When the number of colonies, >40 /¿min diameter, was counted as a tivity of the EL analogue, ET-18-OMe. For example, incubation
of cells at 25°Celiminated the cytotoxicity of ET-18-OMe (15),
function of incubation time, the rate of colony formation was suppressed
by treatment with ET-18-OMe alone at doses >2.0 MMor with heat
while increasing the temperature from 37-42°C significantly
alone. The combination of ET-18-OMe with heat inhibited the colony enhanced the cytotoxicity of ET-18-OMe (16). This enhanced
formation of the slowest growing fraction of the heated cells. The dosecytotoxicity may be related both to absorption of the EL ana
response curve for BG-1 cells after continuous exposure to ET-18-OMe
alone was exponential with a small shoulder (/>„
= 0.25 MM).The 7",, logue by the tumor cells and to the increased turnover rate of
value (the time to reduce survival on the exponential portion of the curve the membrane phospholipids (16).
In this study, human ovarian cancer cells (BG-1) were assayed
by a factor of 1/<•)
of the -14( dose-response curve (30 min) was reduced
to half (15 min) by the addition of 0.25 to 1.0 MMET-18-OMe, but
for cell survival in double soft agarose after treatment with
either ET-18-OMe, heat (42°Cor 44°C),or various combina
increased again to 24 min when heat was combined with ET-18-OMe
concentrations >2.0 MM.The thermotolerant tail seen in the dose-re
tions of ET-18-OMe and heat. The interaction of this EL with
sponse curve after continuous heating at 42°Cwas removed by adding as
heat was investigated to determine if their interaction was
little as 0.25 MMET-18-OMe. Isobologram analysis for the combined
dependent on temperature or the concentration of the EL and
treatments with 44°Cheat and ET-18-OMe at surviving fractions of 0.5,
if their combined cytotoxicity was more than additive.
ABSTRACT
0.3, 0.1, and 0.01 showed that the treatments were supraadditive at low
concentrations (<0.5 MM)of ET-18-OMe and additive at moderate con
centrations (0.5 to 1.0 MM)of ET-18-OMe. Similarly, the interaction of
ET-18-OMe with 42°Cheat at surviving fractions of 0.3 and 0.1 was
supraadditive at low concentrations (<0.5 MM)of the ET-18-OMe and
additive with moderate concentrations (0.5 to 1.5 MM)of ET-18-OMe.
Because the greatest interaction of ET-18-OMe and heat occurred at
clinically achievable doses of both agents, this combination of agents
should be considered for use in clinical trials.
INTRODUCTION
Extensive studies have been performed on the use of heat as
a cancer treatment. However, because heat has had limited
efficacy as a single agent, recent clinical studies have primarily
focused on combining heat with ionizing radiation (1) or chem
otherapeutic agents (2, 3). The interaction of chemotherapy
and hyperthermia is highly complex, and the mechanisms in
volved in this interaction are dependent on the particular chem
otherapeutic agent (4). Membrane fluidizing agents such as the
alcohols, local anesthetics, and amphotericin B (4-10) interact
strongly with heat. These agents are generally noncytotoxic at
normothermic temperatures, but they are extremely toxic at
higher temperatures (4-10). Thus, the combination of heat with
a cytotoxic agent that acts primarily at the plasma membrane
might be extremely toxic to tumor cells.
EL3 analogues of platelet activating factor are a new class of
Received 5/16/89; revised 8/15/89; accepted 8/22/89.
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.
1Supported by NIH Grant CA 44105 from the National Cancer Institute.
2To whom requests for reprints should be addressed, at Department of
Radiology, Bowman Gray School of Medicine. 300 South Hawthorne Road,
Winston-Salem, NC 27103.
3 The abbreviations used are: EL. ether lipid; ET-18-OMe, l-octadecyl-2methyl-rac-glycero-3-phosphocholine; CFE, colony-forming efficiency.
MATERIALS
AND METHODS
Cell Line. BG-1 cells, derived from a human ovarian carcinoma (17),
were maintained in exponential growth in McCoy's Medium 5A sup
plemented with 10% fetal bovine serum, 0.05% L-glutamine, 1% basal
medium Eagle nonessential amino acids, 100 units/ml of penicillin G,
100 mg/ml of streptomycin sulfate, and 0.1 unit/ml of semilente
insulin. They were routinely tested and found to be Mycoplasma free.
The cells were rejuvenated from frozen stock every 6 mo.
Colony Assay. A double soft agarose clonogenic assay was used to
determine cell survival (17, 18). Exponentially growing (Day 2) BG-1
cells were trypsinized with 0.1% trypsin and 0.04% EDTA for 5 to 6
min and resuspended in McCoy's Medium 5A. After centrifugation,
cells (3 x 104/ml) were resuspended in McCoy's Medium 5A with 0.3%
molten agarose (SeaPlaque; FMC, Rockland, ME). A 1.0-ml aliquot of
cells was then placed onto the underlayer (1.0 ml of the same medium
with 0.5% agarose) in each of six 35-mm wells in Linbro culture plates
(Flow Laboratories, Inc., Mclean, VA). Following 20 min in the refrig
erator for gel formation, the cells were incubated at 37°Cfor 12 to 16
h prior to treatment. The initial multiplicity of the cells seeded in the
agarose was <1.1. After treatment, the plates were incubated at 37"C
in a 7.0% CO2-humidified atmosphere.
To determine the optimal time to score for CFE after the various
treatments, the colonies were sized and counted daily on an Omnicon
FAS-III tumor colony counter (Bausch & Lomb, Rochester, NY). Cell
clusters >30, 40, 50, and 60 ^m in diameter were counted each day.
From these data, it was determined that clusters >40 urn in diameter
were colonies and represented the cells able to undergo continuous
division. Based on the results of these experiments, colonies >40 ¿/m
in diameter were counted on the appropriate day after treatment to
determine clonogenic cell survival. The surviving fraction was calcu
lated as (CFE of treated cells)/(CFE of untreated cells). The average
CFE of untreated BG-1 cells in these experiments was 12.2 ±2.0%.
Ether Lipid Treatments. The ether lipid analogue used in this study
was ET-18-OMe (Fig. 1). ET-18-OMe was kindly provided by Dr.
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ETHER LIPIDS AND HEAT
H2C-0-(CH^17-CH3
HC-OCH3
0
H2C-0-P-0-(CH2)2-N*-(CH3)3
0e
Fig. 1. Chemical structure of 1-octadecyl^-methyl-rac-glycero-.Vphosphocholine(ET-18-OMe).
Wolfgang Berdel, Technical University of Munich, Federal Republic of
Germany. The stock solution was made by dissolving ET-18-OMe in
ethanol at a concentration of 10 mg/ml after which it was stored at
—¿20°C.
For the treatments, the stock solution was diluted to 10 times
the desired concentration with phosphate-buffered saline. The ET-18OMe was added to each well and remained there throughout the colony
assay. The drug was added shortly (5 to 10 min) before heating when a
combination treatment was given. The maximum concentration of
ethanol in the medium during treatment was 0.02%. No killing or
enhanced heat sensitivity was observed at this ethanol concentration.
No adjustment was necessary for degradation of ET-18-OMe during
the incubation, since no loss of cytotoxic activity was observed when
ET-18-OMe was incubated in medium at 37°Cfor at least 4 days before
being used to treat BG-1 cells (data not shown).
Heat Treatment. The Linbro culture plates were sealed in freezer
bags and submersed in a temperature-controlled (±0.02°C)
water bath
at either 42°Cor 44°C.The transient time to the desired temperature
was 5 to 6 min. The equilibrium temperature was that of the water
bath. Once gelled the agarose did not melt when heated at 42°Cfor 24
h or 44'C for 3 h.
Isobologram Analysis. The interaction of the ET-18-OMe and heat
was analyzed by the isobologram method of Steel and Peckham (19).
The analysis was performed by surviving fractions of 0.5, 0.3, 0.1, and
0.01.
RESULTS
In previous studies using BG-1 cells to assess the effects of
chemotherapeutic anticancer agents with the double soft agarose colony formation system, the colonies were counted on
Days 7 to 9 (13, 14, 17, 18). However, because nothing was
known about the rate of colony growth of BG-1 cells treated by
heat or ET-18-OMe and because timing of colony counting is
known to be an important variable (20), the number of colonies
was counted daily after those treatments. For untreated BG-1
cells, the number of colonies (> 40 ^m) increased exponentially
as a function of incubation time, reached a maximum by Day
7, and remained there through Day 12 (Fig. 2). The cloning
efficiency was reduced in a dose-dependent manner by exposing
the BG-1 cells to ET-18-OMe. However, the rate of colony
formation was the same as for untreated cells when exposed to
1.0 or 2.0 Õ¿M
concentrations of ET-18-OMe (Fig. 2A). The
rate of colony formation was slowed slightly when the cells
were exposed to 4.0 /¿M
ET-18-OMe, but still reached a plateau
by Day 7 (Fig. 2A).
The increase in the number of colonies >40 firn in diameter
from BG-1 cells exposed to heat at 42°Cwas delayed for
exposures >60 min, but the rate of colony formation was the
same as that of untreated BG-1 cells. The time required for the
maximum number of colonies to form was lengthened in a
dose-dependent manner after 44°Cheat treatment (Fig. IB).
For example, when BG-1 cells were heated for >60 min at
44°C,the maximum number of colonies was not scored until
after Day 9. Furthermore, the rate of colony formation in the
44°C-heated cells was slowed compared with untreated cells.
However, when the cells were treated with both ET-18-OMe
and heat, the number of colonies >40 ^m in diameter reached
a plateau value by Day 7 (Fig. 2C). Therefore, ET-18-OMe
inhibited the colony formation of the slowest growing fraction
of the heated cells (i.e., those cells that form >40-fim colonies
between Days 7 and 12). Based on these results, the colonies
were counted for the survival studies on Days 7 to 9 for
untreated cells and cells treated with either ET-18-OMe alone
or in combination with heat. However, the colonies were
counted on Days 9 to 12 for cells treated with heat alone.
The dose-response curve for BG-1 cells continuously exposed
to ET-18-OMe (Fig. 3) was exponential with a small shoulder
(Z)q= 0.25 UM). The concentration of ET-18-OMe to reduce
the survival to 0.5, 0.1, and 0.01 was 0.8 /¿M,
2.2 UM, and 4.3
MM, respectively. These data are similar to those previously
obtained for this cell line (13, 14).
The cytotoxic interaction of ET-18-OMe (0 to 2 UM) and
44°C(0 to 180 min) was determined by measuring clonogenic
cell survival after the combined treatment (Fig. 4). The doseresponse curve for BG-1 cells after 44°Cheat alone was expo
nential with a shoulder width of 30 min. The Ta value (the time
to reduce survival on the exponential portion of the curve by a
factor of l/e) for the 44°Cdose-response curve was 30 min
(Fig. 4). By adding 0.25 to 1.0 UM ET-18-OMe, the T0 value
was reduced to 15 min (Fig. 4). However, the addition of 2.0
1000Fig. 2. Number of BG-1 colonies ^40 ^m
in diameter as a function of incubation time.
Treatments were A. •¿.
untreated; O, 1.0 tiM
ET-18-OMe; A. 2.0 nM ET-18-OMe; D, 4.0 M
ET-18-OMe; B, •¿.
untreated; O, 42'C for 60
min; A. 42'C for 360 min; D. 44'C for 60 min;
and O, 44'C for 90 min; C, 9, untreated; O.
2.0 MMET-18-OMe-H 42'C for 60 min;n, 2.0
MM+ 44°Cfor 60 min. Points, average of 3 to
5 independent experiments; bars, SE; if not
shown, bars lie within the point.
o
o
ü_
100
o
fr
Lü
CD
3
6
9
12
0
INCUBATION DAYS
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12
ETHER LIPIDS AND HEAT
1.000
1.00
00
0.001
0.01
01234
ET-18-OMe
3
(¿¿M)
Fig. 3. The dose-response curve of BG-1 cells continuously exposed to ET18-OMe. Points, average of 9 to 16 independent experiments; ears. SE; if not
shown, bars lie within the point. The exponential portion of the curve was fitted
by a linear least-squares regression analysis.
6
9
12 15
TIME at 42°C (hr)
18
Fig. 5. The dose-response curves for BG-1 cells as a function of time at 42°C
w¡lhor without ET-18-OMe. •¿.
cells heated at 42°C.Open symbols, cells treated
w¡,hcombinations of42°Cheat and 0.25 (A). 0.5 (D), or 1.0 (V) MMET-18-OMe.
Points, average of 3 to 5 independent experiments; oars, SE. The exponential
portion of the curves was fitted by linear least-squares regression analysis.
100
1.000
80
60
§
20
0.0
0.5
1.0
1.5
2.0
ET-18-OMe
Fig. 6. Isobologram analysis of the ET-18-OMe and 44°Cheat treatment at
the 0. 1 survival level. The lines represent the envelope of additivity. A. isoeffect
points from the dose-response curves of the various combination treatments.
0.001
0
30
60
90
120 150 180
TIME at 44°C (min)
Fig. 4. The dose-response curves for BG-1 cells as a function of time at 44'C
with or without ET-18-OMe. •¿.
cells treated at 44°C;open symbols, cells treated
with the combinations of 44'C heat and 0.25 (A), 1.0 (V), or 2.0 (O) MMET-18OMe. Points, average of 3 to 5 independent experiments; Aars, SE; if not shown,
bars lie within the point. The exponential portion of the curves was fitted by a
linear least-squares regression analysis.
fiM ET-18-OMe
reduced the T0 of the 44°Csurvival curve to
only 24 min (Fig. 4).
The dose-response curve to 42°Cheat alone was similar to
subadditive at higher concentrations. Analysis at the surviving
fractions of 0.5, 0.3, and 0.01 yielded similar results (data not
shown). The interaction of ET-18-OMe and 42°Cheat was
supraadditive at low concentrations of ET-18-OMe (<0.5 ^M)
and additive at concentrations between 0.5 and 1.5 /¿M
ET- 18OMe (Fig. 1A) when analyzed at a surviving fraction of 0.1.
Similar results were obtained at a survival fraction of 0.3.
However, analysis at a surviving fraction of 0.5 showed only
additive interactions for concentrations of ET-1 8-OMe between
0.125 and 0.5 ^M ET-18-OMe (Fig. IB).
that observed for other mammalian lines, with a resistant tail
developing after 2 h of heating (Fig. 5). The resistant tail
probably resulted from the development of thermotolerance (4). DISCUSSION
The addition of ET-18-OMe at either 0.25, 0.5, or 1.0 MM
The unique plasma membrane-mediated cytotoxicity ob
immediately prior to heating resulted in the complete disap
pearance of this thermotolerant tail (Fig. 5). The 42°Cdose- served for EL analogues of platelet activating factor makes
response curve after all of the combined treatments was expo
these compounds interesting for use as anticancer agents either
nential with a To of 2.8 h. Therefore, the sensitization of BG-1 alone or with other agents. This study has focused on the
cells to 42°Cheat-induced cytotoxicity was not dependent on interaction of one of the EL analogues, ET-18-OMe, with heat,
the concentration of ET-18-OMe.
because heat causes cell membrane damage (21), and the re
Isobologram analysis of the combined ET-18-OMe and 44°C sponse to heat is increased by membrane-acting agents such as
alcohol, local anesthetics, and amphotericin B (4-10).
heat treatment at the 0.1 surviving fraction showed a supraad
The survival data for BG-1 cells treated at 44°Cwith or
ditive interaction with concentrations less than 0.5 ¿IM
ET-18OMe (Fig. 6). The interaction was additive when ET-18-OMe
without ET-18-OMe showed that the T0value of the heat dosewas added at concentrations between 0.5 and 1.0 UM and response curve was reduced significantly by adding a low con6287
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ETHER LIPIDS AND HEAT
0.0
cells by changing the break point (4, 5, 8). Ethanol, the most
studied of these agents, acts as a heat mimic, and the combi
nation of ethanol and heat is essentially equivalent to exposure
at a higher temperature (4, 22). Furthermore, if ethanol is given
prior to heating, it induces both heat shock proteins and thermotolerance (22, 23). Maximal interaction of ET-18-OMe with
either 42"C or 44°Cheat was observed at concentrations <0.25
MMET-18-OMe (Figs. 4 and 5). Although the cell kill from
ET-18-OMe increased at higher concentrations, the slope of
the heat survival curve remained the same at these higher
concentrations. Only when ET-18-OMe killed >90% of the
BG-1 cells was any difference in heat sensitization observed
(Fig. 4). The increase in the slope of the 44°Cheat doseresponse curve after 2.0 MM ET-18-OMe exposure probably
indicates that, at these high doses, both agents kill some of the
same cells. Therefore, although the toxicities of ET-18-OMe
and heat may overlap at high concentrations (>2.0 MM),it does
not appear that ET-18-OMe is a heat mimic, since there is no
dose-dependent heat sensitization at doses <2.0 MM(Figs. 4
and 5). Furthermore, preliminary studies in our laboratory have
shown that pretreatment of BG-1 cells with ET-18-OMe 4 to
24 h prior to heating either for 60 min at 42°Cor 15 min at
44°Cdid not induce thermal resistance (data not shown). There
fore, it is more likely that ET-18-OMe eliminates the thermo
tolerant tail at 42°Cby inhibiting the development of thermo-
0.5
1.0
ET-18-OMe
3.0
B
.2.0
11.0
0.0
0.0
0.2
0.4
0.6
ET-1B-OM«(/iM)
0.8
Fig. 7. Isobologram analysis of the ET-18-OMe and 42°Cheat treatment at
the 0.1 survival level (A) and 0.5 survival level (B). The lines represent the
envelope of addilivity. A, isoeffect points from the dose-response curves of the
various combination treatments.
centratici! (0.25 MM)of ET-18-OMe (Fig. 4). Reduction of the
TO was not dependent on the concentration of ET-18-OMe
between 0.25 and 1.0 MM, but it was less when heat was
combined with 2.0 MMET-18-OMe (Fig. 4). Alterations in the
TOvalues were directly mirrored in the isobologram analysis
(Fig. 6), which showed that ET-18-OMe and 44°Cheat inter
acted supraadditively at low concentrations, additively at mod
erate concentrations, and subadditively at higher concentrations
of ET-18-OMe.
The interaction of ET-18-OMe and 42"C heat had two major
manifestations, (a) The thermotolerant tail seen in the doseresponse curve to 42°Cheat alone was eliminated by the addi
tion of concentrations as low as 0.25 MMET-18-OMe (Fig. 5).
(b) Increasing the concentration of ET-18-OMe did not reduce
the slope of the resulting 42°Cdose-response curve further (Fig.
5). This lack of concentration-dependent
heat sensitization was
similar to the observations made in the experiments combining
ET-18-OMe with 44°Cheat. From the isobologram analysis,
the lower concentrations of ET-18-OMe again showed a strik
ing supraadditive interaction, while the interaction was additive
with higher concentrations of ET-18-OMe. Because the begin
ning point of the thermotolerant tail is so close to the surviving
fraction of 0.5 (Fig. 5), the isobologram analysis at the 0.5
surviving level showed only an additive interaction with all
concentrations of ET-18-OMe tested (Fig. IB).
The most interesting finding in this study is the elimination
of the thermotolerant tail induced by continuous heating at
42°Cusing concentrations of ET-18-OMe as low as 0.25 MM.
Elimination of the thermotolerant tail could occur either by
shifting the break point in the Arrhenius plot for cell inactivation by heat or by inhibiting the development of thermotolerance (4). The alcohols and local anesthetics appear to sensitize
tolérance,rather than by shifting the break point of the Arrhen
ius plot. Additional studies on (a) the expression of heat shock
proteins after ET-18-OMe treatment, (b) the ability of ET-18OMe to inhibit thermotolerance after acute heat shock, and (c)
the influence of ET-18-OMe on the expression of thermotoler
ance when it is given after heating are necessary before a clear
hypothesis on the mechanism for this inhibition of thermoto
lerance can be formulated.
Unlike many combinations of drugs and heat, the most strik
ing interaction between heat and ET-18-OMe occurred when
low concentrations of ET-18-OMe (<0.25 MM)and 42°Cheat
were used (Fig. "IA). These low levels of both agents make it
much more likely that this combination could be used in the
clinic. In a Phase I trial of the thioether lipid analogue, BM
41.440, the peak plasma concentration after a single 4-mg/kg
dose (p.o.) was 1.8 Me/ml or 3.4 MM(24). Although no drug
concentrations have been measured after i.v. administration of
ET-18-OMe in humans, the maximum tolerated daily dose was
20 mg/kg (25). In that study, the most severe side effect reported
was Grade 4 pulmonary edema observed in one of 16 patients.
All other side effects of ET-18-OMe were minor (25). There
fore, it is highly probable that a plasma concentration of 0.25
MMET-18-OMe can be achieved clinically. However, further
preclinical investigations are needed to determine (a) the opti
mal sequencing and timing of the administration of the two
agents, (b) the effect of repeated treatments, and (c) if ionizing
radiation or other chemotherapeutic agents should be used in
combination with these two agents.
In conclusion, the ether lipid analogue of platelet activating
factor, ET-18-OMe, has several characteristics that make it
different from the alcohols and local anesthetics, (a) It is
cytotoxic at 37°C(Fig. 3). (¿>)
Its sensitization of BG-1 cells to
heat is not dose dependent at doses <2.0 MM(Figs. 4 and 5).
(c) It does not appear to induce thermotolerance. The combi
nation of ET-18-OMe and heat treatment on BG-1 cells showed
supraadditive cytotoxicity when low concentrations of ET-18OMe were combined with either 42"C or 44"C heat. They also
exhibit an additive interaction when moderate to high concen-
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ETHER LIPIDS AND HEAT
trations of ET-18-OMe were used. The addition of as little as
0.25 ¿¿M
ET-18-OMe eradicated the thermotolerance induced
by continuous 42°Cheating. These findings suggest that com
binations of ET-18-OMe and heat may be an effective approach
in the treatment of cancer, although preclinical and mechanistic
studies on the interaction of ether lipid analogues and hyperthermia will be required to optimize their use.
ACKNOWLEDGMENTS
The authors thank Jessie Anderson for the technical assistance and
Pat Tomlinson and Pam Cregger for preparation of this manuscript.
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Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1989 American Association for Cancer Research.
Cytotoxic Interactions of Heat and an Ether Lipid Analogue in
Human Ovarian Carcinoma Cells
Keiichi Fujiwara, Edward J. Modest, Charles E. Welander, et al.
Cancer Res 1989;49:6285-6289.
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