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Antimicrobial Agents and Chemotherapy, November 1999, p. 2787-2789, Vol. 43, No. 11
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Differential Modulatory Effects of
Clarithromycin on the Production of Cytokines by a
Tumor
Kazuhiko
Sassa,1
Yutaka
Mizushima,1,2,* and
Masashi
Kobayashi1
First Department of Internal Medicine, Toyama
Medical and Pharmaceutical University, Toyama
930-0152,1 and Department of Geriatrics,
Hirosaki University School of Medicine, Hirosaki
036-8562,2 Japan
Received 5 April 1999/Returned for modification 2 June
1999/Accepted 20 August 1999
 |
ABSTRACT |
In vitro treatment with clarithromycin inhibited the expression of
the matrix metalloproteinase-9, transforming growth factor 
, and
tumor necrosis factor alpha genes in 13762NF rat mammary adenocarcinoma
cells. Transient enhancement, rather than inhibition, was observed for
the interleukin-6 gene, and no significant change was observed for the
tissue inhibitor of metalloproteinase-2 gene. Such an effect was not
observed for cefotiam or gentamicin.
| |
TEXT |
A variety of macrolide activities as
biological response modifiers have already been reported on (1, 3,
6, 7, 9, 11, 14). We (13) also recently reported
finding that in a rat tumor model, clarithromycin (CAM) has an activity
which induces a beneficial therapeutic outcome, possibly by inhibiting the production of tumor-derived factors which induce cachexia (2,
5) or immunosuppression (15). In this study, we
examined in more detail the effects of CAM on the production of
cytokines by a tumor to define a characteristic property of CAM.
The tumor we used was the 13762NF (subclone MTLn3) mammary
adenocarcinoma (12) originating from an F-344 rat, and it
was maintained in vitro in RPMI 1640 medium containing 10% fetal calf serum (FCS). CAM (Abbott Co. Ltd.) was dissolved with 100% methanol (1 mg/ml) and then further diluted in RPMI culture medium to reach final
concentrations. Gentamicin sulfate (GM) and cefotiam dehydrochloride (CTM) were also diluted in RPMI medium. Expression of genes was measured by the reverse transcription-PCR method as described previously (13). The primers used for the amplification of
genes were as follows: matrix metalloproteinase-9 (MMP-9) gene,
5'-GGTCCCCCCACTGCTGGCCCTTCTACGGCC-3' and
5'-GTCCTCAGGGCACTGGAGGATGTCATAGGT-3'; transforming growth factor (TGF-) gene, 5'-GCCCTGGACACCTATTGC-3' and
5'-GCTGCACTTGCAGGAGCGCAC-3'; tumor necrosis factor alpha
(TNF-
) gene, 5'-CAAGGAGGAGAAGTTCCCAA-3' and
5'-CGGACTCCGTGATGTCTAAG-3'; interleukin-6 (IL-6) gene,
5'-ATGTAGCCGCCCCACACAGA-3' and
5'-CATCCATCTTTTTCAGCCAT-3'; inhibitor of metalloproteinase (TIMP-2) gene, 5'-TGCAGCTGCTCCCCGGTGCAC-3' and
5'-TTATGGGTCCTCGATGTCGAG-3'. As an internal control, a set
of primers for the glyceraldehyde-3-phosphate dehydrogenase gene
(5'-CAAAAGGGTCATCTCTG-3' and
5'-CCTGCTTCACCACCTTCTTG-3') was added to each sample. The
degree of gene expression, determined with a densitometer, was
expressed as a ratio to glyceraldehyde-3-phosphate dehydrogenase.
Gelatinolytic activity in the culture medium was assayed by the method
of Heussen and Dowdle (4), but in this assay, tumor cells
were cultured in the presence of 2%, instead of 10%, FCS because
significant gelatinolytic activity was found in the FCS. Data are
shown as means ± standard errors, and statistical significance was evaluated by Student's t test. A
P value of less than 0.05 was judged to be significant.
13762NF tumor cells were treated with CAM at 5 µg/ml, and total RNAs
were extracted from the tumor cells at 6, 12, 24, 48, and 72 h.
Expression of the MMP-9, TGF-, and TNF- genes was shown to be
inhibited significantly by treatment with CAM (Fig. 1A to
C). Conversely, transient enhancement was
observed for the IL-6 gene (Fig. 1D). Next, tumor cells were treated
for 24 h with different concentrations of CAM (1 to 50 µg/ml).
Significant decreases in expression were observed for the MMP-9,
TGF-, and TNF- genes (data not shown), and an increase was
observed for the IL-6 gene (Fig. 1E). The gelatinolytic activity in the
culture medium was shown to be inhibited by treatment of tumor cells
with CAM (data not shown). We also examined the effects of two other
antimicrobial agents, CTM and GM, on the expression of the MMP-9 gene
(24 h), and no significant effect was observed for these two agents
(Fig. 2). We further examined the effect
of CAM on the expression of the TIMP-1 or TIMP-2 gene (8) in
13762NF tumor cells. In the tumor cells, the TIMP-2 gene was found to
be expressed highly but the TIMP-1 gene was not (data not shown). As
shown in Fig. 3, no significant change
due to CAM (5 µg/ml) treatment was observed.
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FIG. 1.
Effects of CAM (5 µg/ml) treatment time on expression
of the MMP-9 (A), TGF- (B), TNF- (C), and IL-6 (D) genes. 13762NF
tumor cells were treated with CAM at 5 µg/ml for different lengths of
time, and then total RNAs were extracted for analysis. (E) Effect of
CAM concentration on expression of the IL-6 gene. 13762NF tumor cells
were treated with different concentrations of CAM for 24 h, and
then total RNAs were extracted for analysis. GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
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FIG. 2.
Effect of treatment with CTM (A) or GM (B) on expression
of the MMP-9 gene in 13762NF tumor cells. 13762NF tumor cells were
treated with CTM (A) or GM (B) for 24 h, and then total RNAs were
extracted for analysis. GAPDH, glyceraldehyde-3-phosphate
dehydrogenase.
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FIG. 3.
Effect of CAM on expression of the TIMP-2 gene in
13762NF tumor cells. 13762NF tumor cells were treated with CAM (5 µg/ml) for different lengths of time (given in hours on the
x axis). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
|
|
As a consequence, three patterns were observed after CAM treatment:
suppression (MMP-9, TGF-, and TNF-), transient enhancement (IL-6), and no change (TIMP-2). It is difficult to explain why these
different patterns appeared following CAM treatment. Such a modulatory
effect was not observed for CTM or GM. We suppose that the effect
observed in this study is specific to the macrolides, probably specific
to the 14-membered ring macrolides. For example, Yatsunami et al.
(Kyushu University School of Medicine) showed, in the B16 tumor-C57BL6
mouse model, that CAM and roxithromycin (14-membered ring) could
inhibit angiogenesis but josamycin (16-membered ring) and azithromycin
(15-membered ring) could not (personal communication). We have no
explanation for the difference in action among the 14-, 15-, and
16-membered ring macrolides.
It is interesting that macrolide antibiotics cause a beneficial
therapeutic outcome in hosts bearing cancer (3, 10, 13). We
hope that CAM may become a beneficial tool for the treatment of certain cancers.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Geriatrics, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan. Phone: 81-172-33-5111, ext. 6424. Fax: 81-172-39-5346.
| |
REFERENCES |
| 1.
|
Bailly, S.,
J. J. Pocidalo,
M. Fay, and M. A. G. Pocidalo.
1991.
Differential modulation of cytokine production by macrolides: interleukin-6 production is increased by spiramycin and erythromycin.
Antimicrob. Agents Chemother.
35:2016-2019[Abstract/Free Full Text].
|
| 2.
|
Espat, N. J.,
E. M. Copeland, and L. L. Moldawer.
1994.
Tumor necrosis factor and cachexia: a current perspective.
Surg. Oncol.
3:255-262[Medline].
|
| 3.
|
Hamada, K.,
E. Kita,
M. Sawaki,
K. Mikasa, and N. Narita.
1995.
Antitumor effect of erythromycin in mice.
Chemotherapy
41:59-69[Medline].
|
| 4.
|
Heussen, C., and E. B. Dowdle.
1980.
Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates.
Anal. Biochem.
102:196-202[Medline].
|
| 5.
|
Kishimoto, T.
1989.
The biology of interleukin 6.
Blood
74:1-10[Free Full Text].
|
| 6.
|
Kita, E.,
M. Sawaki,
F. Nishikawa,
K. Mikasa,
Y. Yagyu,
S. Takeuchi,
K. Yasui,
N. Narita, and S. Kashiba.
1990.
Enhanced interleukin production after long-term administration of erythromycin stearate.
Pharmacology
41:177-183[Medline].
|
| 7.
|
Kobayashi, H.
1995.
Airway biofilm disease: clinical manifestation and therapeutic possibilities using macrolides.
J. Infect. Chemother.
1:1-15.
|
| 8.
|
Kugler, A.,
B. Hemmerlein,
P. Thelen,
M. Kallerhoff,
H. J. Radzun, and R. H. Ringert.
1998.
Expression of metalloproteinase 2 and 9 and their inhibitors in renal cell carcinoma.
J. Urol.
160:1914-1918[Medline].
|
| 9.
|
Majima, T.,
K. Mikasa,
K. Hamada,
M. Konishi,
K. Maeda,
M. Sakamoto,
E. Yoshimoto,
K. Murakawa,
K. Ueda,
E. Kita, and N. Narita.
1999.
Changes of cytokine mRNA in peripheral blood mononuclear cells from unresectable non-small cell lung cancer patients before and after clarithromycin therapy.
Jpn. J. Chemother.
47:345-348. (In Japanese.)
|
| 10.
|
Mikasa, K.,
M. Sawaki,
E. Kita,
K. Hamada,
S. Teramoto,
M. Sakamoto,
K. Maeda,
M. Konishi, and N. Narita.
1997.
Significant survival benefit to patients with advanced non-small-cell lung cancer from treatment with clarithromycin.
Chemotherapy
43:288-296[Medline].
|
| 11.
|
Morikawa, K.,
F. Oseko,
S. Morikawa, and K. Iwamoto.
1994.
Immunomodulatory effects of three macrolides, midecamycin acetate, josamycin, and clarithromycin, on human T-lymphocyte function in vitro.
Antimicrob. Agents Chemother.
38:2643-2647[Abstract/Free Full Text].
|
| 12.
|
Nakajima, M.,
D. R. Welch,
D. M. Wynn,
T. Tsuruo, and G. L. Nicolson.
1993.
Serum and plasma Mr 92,000 progelatinase levels correlate with spontaneous metastasis of rat 13762NF mammary adenocarcinoma.
Cancer Res.
53:5802-5807[Abstract/Free Full Text].
|
| 13.
|
Sassa, K.,
Y. Mizushima,
T. Fujishita,
R. Oosaki, and M. Kobayashi.
1999.
Therapeutic effect of clarithromycin on a transplanted tumor in rats.
Antimicrob. Agent Chemother.
43:67-72[Abstract/Free Full Text].
|
| 14.
|
Sawaki, M.,
E. Kita, and N. Narita.
1995.
Clarithromycin as a potent anti-angiogenesis agent: possible application for antitumor agent.
Can. J. Infect. Dis.
6(Suppl. c):213.
|
| 15.
|
Sporn, M. B.,
A. B. Roberts,
L. M. Wakefield, and B. de Crombrugghe.
1986.
Some recent advances in the chemistry and biology of transforming growth factor-.
J. Cell Biol.
105:1039-1045[Free Full Text].
|
Antimicrobial Agents and Chemotherapy, November 1999, p. 2787-2789, Vol. 43, No. 11
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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