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Experimental Therapeutics |
Takasago Research Laboratories, Research Institute, Kaneka Corporation, Takasago 676-8688, Japan [S. Y., M. K.], and Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan [K. N.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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HSPs are a unique group of proteins induced by heat or other stressors such as sodium arsenite, heavy metals, or amino acid analogues in prokaryotic and eukaryotic cells. In mammalian cells, the expression of a number of HSPs, including HSP40, HSP47, HSP70, HSP90, and HSP100, is enhanced by heat shock and regulated at the transcriptional level (5) . HSPs bind to denatured proteins caused by various stresses including heat shock and serve to renature those proteins or to bring them to the degradation pathway. Some of the HSPs also have important roles as molecular chaperones for the maintenance of the intracellular environment, regulating the protein folding and translocation of proteins into endoplasmic reticulum, mitochondria, and so on (6) .
On the other hand, the induction of HSPs by hyperthermic treatment contributes to the acquisition of thermotolerance in tumor cells. Various studies have shown a close relationship between the level of thermotolerance and the cellular content of HSP72, the inducible form of HSP70 family proteins. For example, microinjection of antibodies against HSP70 makes cells more sensitive to thermal stress (7) , and HSP72 is associated with ribosomal subunits in thermotolerant cells, but not in normal cells (8) . HSP synthesis induced by heat shock is mainly regulated by HSF1 and HSE in the promoter region (9, 10, 11) . HSF1 exists in unstressed cells in an inactive form and is rapidly activated after heat shock.
The effectiveness of hyperthermia will definitely improve if the induction of HSPs including HSP70 in tumor cells is inhibited during heat treatment. For that reason, it is desirable to inhibit activation of HSF1 during heating for specific inhibition of inducible HSPs without affecting the expression and function of constitutive forms of HSPs. We reported previously that the bioflavonoid quercetin inhibited the synthesis of various HSPs, including inducible HSP70, as induced by heat shock or treatment with sodium arsenite in COLO 320DM human colon carcinoma cell lines (12 , 13) . Quercetin inhibited the induction of HSP70 at the mRNA level through inhibition of HSF1 activation (13) or was reported to cause retardation of the induction of HSP70 mRNA (14) . Quercetin was also reported to inhibit the acquisition of thermotolerance in COLO 320DM cells (15) . Thus far, quercetin is the only compound that has been proven to be involved in the inhibition of induction of HSPs as well as in the acquisition of thermotolerance.
In this study, we found a benzylidene lactam compound, KNK437, to be an inhibitor of the acquisition of thermotolerance. It was more effective than quercetin in inhibiting acquired thermotolerance and in inhibiting various HSPs at the mRNA level. These results demonstrate the usefulness of this compound as an effective sensitizer for cancer hyperthermic therapy and also suggest the strong relationship between thermotolerance and HSPs.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Hyperthermia and Drug Treatments.
Heat treatments at 42°C for 90 min were performed in a
CO2 incubator using 25-cm2
plastic flasks. Cells (1 x 105)
were seeded in the flasks, incubated at 37°C for 48 h, and then
heated by immersing the flasks in a water bath (45°C ± 0.05°C). KNK437
(N-formyl-3,4-methylenedioxy-benzylidene-
-butyrolactam;
synthesized by Kaneka Corp., Osaka, Japan) and quercetin (Nacalai
Tesque, Kyoto, Japan) were dissolved in DMSO before being added at the
indicated concentrations. The final concentration of DMSO in each
culture medium was 0.25% (v/v), irrespective of the concentrations of
the drugs. The same concentration of DMSO was used as a control. Sodium
arsenite was dissolved in PBS at a concentration of 80
mM and added to the medium. Cells were treated
with 300 µM sodium arsenite at 37°C for
1.5 h, rinsed, and then incubated at 37°C for 5 h before
45°C heat challenge.
Colony-forming Assay.
Cells were trypsinized, counted, and replated at the appropriate
dilutions. Surviving colonies were counted after 10–12 days of growth
at 37°C. Plating efficiencies were routinely about 80–90%. A set of
at least six dishes was used for each experimental condition. The
surviving fraction was calculated as the plating efficiency of the
treated cells divided by the plating efficiency of untreated control
cells.
Metabolic Labeling and Gel Electrophoresis.
COLO 320DM cells (2 x 105) were
injected into each well of 12-well plastic plates 2 days before
incubation in the presence of KNK437 for 1 h before heat
shock. The cells were then heat-shocked at 42°C for 90 min or
kept at 37°C for the same length of time and incubated at 37°C for
2 h. KNK437 or 0.25% DMSO, was present in the medium from 1 h before heat shock until the end of the 2-h recovery period. For
metabolic labeling, cells were washed with PBS without
Ca2+ or Mg2+ and incubated
for 1 h with 1.22 MBq of [35S]methionine
in 250 µl of methionine-free DMEM supplemented with 10% dialyzed
fetal bovine serum. After metabolic labeling, cells were washed twice
with PBS and lysed in a buffer containing 1% NP40, 0.15 M
NaCl, 50 mM Tris-HCl (pH 8.0), 5 mM EDTA, and
protease inhibitors [0.2 mM
4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 2
mM N-ethylmaleimide, 1 µg/ml pepstatin, and 1
µg/ml leupeptin]. After centrifugation at 12,000 x g for 20 min, cell extracts containing equal amounts
of trichloroacetic acid-insoluble radioactivity were analyzed by
two-dimensional gel electrophoresis [the one-dimensional gel
electrophoresis was a nonequilibrium pH gradient gel electrophoresis,
and the two-dimensional gel electrophoresis was 10% SDS-PAGE (17
, 18)
].
Immunoblot Analysis.
About 4 x 105 cells were injected
in each well of 6-well plastic plates 2 days before a 1-h incubation
with or without KNK437 before heat shock. The cells were
heat-shocked and recovered as described above without labeling with
[35S]methionine. After washing, the cells were
lysed in Laemmli’s SDS sample buffer (18)
and sonicated.
Cell extracts containing equal amounts of protein were separated by
one-dimensional SDS-PAGE according to the methods of Laemmli
(18)
and blotted on nylon membrane (19)
.
After transfer, the membrane was incubated with monoclonal anti-HSP72
antibody (SPA-810; Stressgen Biotechnologies Co., Victoria, British
Columbia, Canada) or anti-ß-actin antibody (MAB1501; Chemicon,
Temecula, CA) and then incubated with peroxidase-conjugated goat
antibody against mouse IgG (Cappel; Organo Teknika Co., West Chester,
PA), followed by detection with enhanced chemiluminescence Western
blotting detection reagents (Amersham, Little Chalfont,
Buckinghamshire, England).
Northern Blot Analysis.
Cells (2 x 106) were plated in
25-cm2
plastic flasks and treated on the second
day with or without KNK437 for 1 h. The cells were then
heat-shocked at 42°C for 90 min or kept at 37°C. Cells were
harvested immediately after heat shock, and total RNA was purified,
with minor modifications, according to methods described previously
(13)
. Equal amounts of total RNA were electrophoresed in
formaldehyde-containing agarose gels, transferred to a nylon membrane,
and hybridized with [
-32P]dCTP-labeled
probes for HSP70 (pH 2.3 H-B; 2.3 kb; kindly provided by Dr. R. I.
Morimoto, Northwestern University, Chicago, IL) or ß-actin as
an internal control.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) on the acquisition of thermotolerance was examined in COLO 320DM
cells by a protocol of fractionated heat treatment. As shown in Fig. 2
, the thermotolerance was induced in COLO 320DM cells after the first
heat treatment at 45°C for 10 min in the presence of DMSO (control).
However, the acquisition of thermotolerance was almost completely
inhibited by the presence of 100 µM KNK437 when this
compound was added to the culture medium 1 h before the first heat
treatment and remained present until the end of the second heat
treatment (Fig. 2
).
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). A similar dose-dependent inhibitory effect of KNK437 on
the acquisition of thermotolerance was also observed for a different
cell line, HeLa S3 (Fig. 3B
). The inhibitory effect was less
prominent in HeLa S3 cells than in COLO 320DM cells, which may be
attributed to the less effective inhibition of induction of HSPs by
KNK437 in HeLa S3 cells (as shown later in Fig. 8
). Here, we performed
the preheat treatment at 42°C instead of 45°C, and these two
different preheating conditions provided essentially the same effects
on acquisition of thermotolerance (data not shown). KNK437 itself, even
at a concentration of 200 µM, did not show any
toxic effect on cell survival when cells were incubated at 37°C for
6 h (data not shown) or heated at 42°C for 90 min (Fig. 3A
).
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, KNK437 inhibited the acquisition of thermotolerance much more
effectively than quercetin when both compounds were examined at the
same concentrations.
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). The acquisition of thermotolerance induced by sodium arsenite was
also inhibited by the presence of KNK437 (Fig. 5
).
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, cells were incubated with this compound: (a) for the first
3.5 h, including the first heat treatment period (group 1);
(b) for the last hour and the second heat treatment period
(group 2); or (c) for the entire first and second heat
treatment periods (group 3). The inhibitory effect was most
prominent when KNK437 was present during the entire period (group 3).
When the compound was present only for the first 3.5 h (group 1),
the inhibition was smaller than that seen in group 3. The inhibitory
effect in group 2 was modest compared with that of the other groups,
but it was still significant compared with the control (group 4). These
data indicate that the presence of the compound during the first heat
treatment is necessary for inhibition of the acquisition of
thermotolerance. In light of these results, we next examined the effect
of KNK437 on the induction of HSPs in COLO 320DM cells.
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). Adding 100 µM KNK437 to the medium during heat shock
specifically inhibited the induction of these HSPs, whereas synthesis
of other proteins remained essentially the same. At this concentration
of KNK437, cell numbers neither increased nor decreased after 3 days of
incubation. KNK437 did not inhibit the expression of
constitutively expressed HSP family proteins such as HSC73 and HSP90
(Fig. 7 and B
, spots d and
e).
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). KNK437 dose-dependently suppressed the level of HSP72 after heat
shock in COLO 320DM cells (Fig. 8A
), HeLa S3 cells, and SCC
VII cells (Fig. 8B
). To evaluate the inhibitory effect of
KNK437 accurately, the amount of ß-actin was also examined by Western
blot analysis as an internal control. The inhibitory effect of
KNK437 at the same concentration was higher for COLO 320DM cells
than for HeLa S3 cells. This is consistent with the previous results
that KNK437 inhibited the acquisition of thermotolerance less
effectively for HeLa S3 cells than for COLO 320DM cells (Fig. 3 and B
). The inhibitory effect of KNK437 on the
acquisition of thermotolerance was approximately 4-fold higher in COLO
320DM cells than in HeLa S3 cells (see Fig. 3 and B
), and the inhibitory effect on the induction of HSP72 was
again about 4-fold higher for COLO 320DM cells than for HeLa S3 cells
(compare Fig. 8A
with Fig. 8B
).
When the compound was removed from the culture medium after 24 h
of incubation, cell proliferation started again, and the cell numbers
recovered to the control level (data not shown), suggesting that the
effect of KNK437 is cytostatic and reversible rather than cytotoxic.
Next we examined whether the inducibility of HSPs is restored when the
compound is removed from the medium after preincubation with the cells.
As shown in Fig. 8C
, the induction of HSP72 by heat shock
was clearly inhibited when COLO 320DM cells were preincubated with
KNK437 for 24 h (Lane 2). However, when the COLO 320DM
cells preincubated with KNK437 were further incubated for 24 h in
medium without KNK437, the inducibility of HSP72 (Lane 4)
was restored to the normal level without incubation with KNK437
(Lane 3).
To investigate the effect of KNK437 on the level of HSP70 mRNA, we
performed Northern blot analysis (Fig. 9
). HSP70 mRNA was markedly induced by heat treatment at 42°C for 90
min, and the accumulation of HSP70 mRNA by heat shock was almost
completely inhibited by the presence of 100 µM KNK437.
The level of ß-actin mRNA as an internal control remained virtually
unchanged under all of the conditions examined.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because it is critical for cancer therapies using hyperthermia to prevent the induction of HSPs, a specific inhibitor of induction of various HSPs in cancer cells after the first heat treatment would be useful for effective hyperthermic therapy. Quercetin, a bioflavonoid, was reported to inhibit the induction of HSPs including HSP70, HSP90, HSP47, and HSP27 at the mRNA levels (12 , 13) and to prevent the acquisition of thermotolerance in COLO 320DM human colon cancer cells (15) . Quercetin is the only specific inhibitor for the induction of HSPs reported thus far.
We report here that KNK437 dose-dependently inhibited the acquisition of thermotolerance after fractionated heat treatment. KNK437 is a novel compound that we first isolated from organic source libraries available in our laboratory. It does not show any anticancer effect by itself, nor does it inhibit the activities of protein kinases A and C and tyrosine kinase. A structurally similar compound shows antidepressant activity, but this pharmacological function has not yet been revealed.3
Inhibition of the acquisition of thermotolerance by this compound was
observed in COLO 320DM cells as well as in HeLa S3 cells (Fig. 3
). The
inhibitory effect was most prominent when this compound was added
before the first heat treatment and was present throughout the
experimental period, including the second heat treatment. This suggests
that the prevention of thermotolerance acquisition in the cells treated
with this compound may be due to the inhibition of HSP induction. As
shown in Fig. 7
, KNK437 inhibited the induction of various HSPs
including HSP70, HSP40, and HSP105. HSP105 has a homologous region for
HSP70 (21)
and has been reported to contribute to the
induction of thermotolerance in mouse cells (27)
. When
KNK437 was added to the medium only during the second heat treatment at
45°C, it still showed a weak inhibitory effect, suggesting that the
HSPs induced during the second heat treatment may also partially
contribute to the induction of thermoresistance. The inhibitory effect
on the acquisition of thermotolerance was also observed when the cells
were pretreated with sodium arsenite. Thus, KNK437, as noted here, is
the second reported specific inhibitor of the induction of
thermotolerance as well as the induction of HSPs. This compound was
more effective than quercetin in inhibiting the induction of
thermotolerance, and the toxic effect of this compound on the cells was
less than that of quercetin, suggesting that this compound might be a
more useful potentiator for hyperthermic cancer therapy.
Although quercetin has been reported to inhibit the induction of HSP70
synthesis at the mRNA level (12)
, the inhibitory mechanism
has not been clearly established. The binding of HSF1 to HSE is blocked
when quercetin is added before and during heat treatment
(13)
, and the phosphorylation of HSF1 is partially
inhibited (28)
. This is consistent with a recent study
(29)
reporting that the targeted disruption of murine HSF1
abolishes thermotolerance as well as protection against heat-inducible
apoptosis. On the other hand, quercetin is also reported as only
delaying the synthesis of HSP70 after heat shock (14)
.
Quercetin is also reported to have scavenger activity for reactive
oxygen species, and reactive oxygen is reported to induce HSPs. Like
quercetin, KNK437 also inhibits the induction of HSP70 at the mRNA
level. Because KNK437 did not inhibit the constitutive expression of
HSP family proteins as shown in Fig. 7
, the inhibitory mechanism of
this compound might be attributed to inhibition of the activation of
HSF1 or the interaction of HSF1 with HSE as reported for quercetin
(13)
. However, it should be more carefully studied,
considering the involvement of other factors including reactive oxygen
species (30
, 31)
. KNK437 had an inhibitory effect on cell
growth, which is reversible. However, there is no relationship between
growth inhibition and inhibition of the acquisition of thermotolerance
because we have already shown that cycloheximide inhibited cell growth
but did not inhibit the acquisition of thermotolerance
(15)
.
A number of studies (4 , 32 , 33) have demonstrated the possible involvement of apoptosis in killing tumor cells exposed to hyperthermia, and these studies suggest a strong relationship between the induction of stress proteins and the prevention of apoptosis. Quercetin was previously reported to induce apoptosis in tumor cells by inhibiting the induction of HSP70 synthesis after heat shock (34) . Furthermore, inhibition of HSP70 synthesis as well as induction of apoptosis by treatment with quercetin combined with hyperthermia is reported to be confined to leukemic cells, and not to normal hematopoietic progenitor cells (35) . The inhibition of Jun NH2-terminal kinase activity by elevated levels of HSP70 is suggested to be responsible for the protection against apoptosis caused by various stresses including heat shock (36) . The effect of KNK437 on the induction of apoptosis remains an area for future study.
In conclusion, we show here that KNK437 is a novel inhibitor for the acquisition of thermotolerance through inhibition of the induction of various HSPs. In addition, this compound has been found to enhance the efficiency of hyperthermic therapy against the growth of solid tumors transplanted into mice.4 We have also found that modified derivatives of KNK437 inhibited the induction of various HSPs after heat shock.3 Therefore, further screening of benzylidene lactam derivatives that inhibit the acquisition of thermotolerance more efficiently would contribute to the efficient application of hyperthermic therapy in human cancers.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Department of Molecular and Cellular Biology, Institute
for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho
Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. 
2 The abbreviations used are: HSP, heat shock
protein; HSF, heat shock factor; HSE, heat shock element.
3 S. Yokota, K. Yamamoto, M. Kitahara, and
K. Nagata, unpublished observations.
4 M. Koishi, S. Yokota, T. Mae, Y. Nishimura, S.
Kanamori, N. Horii, K. Shibuya, and M. Hiraoka. KNK437, an inhibitor of
HSP synthesis, inhibits the acquisition of thermotolerance in a murine
transplantable tumor in vivo, submitted for
publication.
Received 8/19/99. Accepted 3/30/00.
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REFERENCES |
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) or without (
) KNK437 for 5 h
before a challenging heat shock at 45°C. Cells preincubated with
(
) or without (•) KNK437 for 1 h before the first heat
treatment at 45°C for 10 min were recovered at 37°C for 4 h
and then treated with a challenging heat shock at 45°C for the
indicated periods. Surviving fractions of the treated cells were
examined as described in "Materials and Methods," and the results
are expressed as the means of triplicate samples. Bars,
SD.
), 25 (
), 50 (
) during the entire experimental
period from 1 h before the first heat treatment. B,
HeLa S3 cells were incubated with KNK437 at concentrations of 0 (DMSO;
