Download Soluble Fas and Fas ligand provide new information on metastasis

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Cancer Detection and Prevention xxx (2004) xxx–xxx
www.elsevier.com/locate/cdp
Soluble Fas and Fas ligand provide new information on metastasis
and response to chemotherapy in SCLC patients
Makoto Shimizu MDa, Masashi Kondo MDa, Yasushi Ito MDa, Hiroaki Kume MDa,
Ryujiro Suzuki MDa, Kenichi Yamaki MDb,*
a
Department of Respiratory Medicine, Nagoya University School of Medicine 65, Tsurumai-cho,
Showa-ku, Nagoya, Aichi 466-8560, Japan
b
Kariya Nursing College, Kariya Medical Corporation, 1, 1, 72, Araihata, Hajodo-cho, Kariya, Aichi 448-0804, Japan
Accepted 15 September 2004
Abstract
Background : The Fas/Fas ligand (FasL) system is a major regulator of apoptosis. Chemotherapeutic drugs have been shown to induce Fas
expression on the surface of lung cancer cells, and cancer cell apoptosis. However, this mechanism is not considered to be associated with Fas
expressed on lung cancer cells. Soluble Fas and FasL concentrations are reportedly elevated in the peripheral blood of patients with lung
cancer, but the roles of circulating soluble Fas and FasL in that disease have not been clarified.
Materials and methods : We measured the circulating soluble Fas and FasL levels in 21 patients with small cell lung cancer (SCLC), and 12
healthy matched controls, in order to examine whether such ligands could provide any important information and/or reveal any new clinical
features of SCLC.
Results : In the CR patients, the neuronal specific enolase (NSE), soluble Fas and soluble FasL concentrations were 21.26 3.65 ng/ml,
3.58 0.19 ng/ml and 0.50 0.15 ng/ml, while in the partial response (PR)/no change (NC)/progressive disease (PD) group of patients they
were 33.96 7.86 ng/ml, 5.29 0.29 ng/ml and 0.59 0.07 ng/ml, respectively. The NSE, soluble Fas and soluble FasL concentrations
were all elevated in the PR/NC/PD patients, however, significant differences were only seen in Fas concentration between CR and PR/NC/PD
patients and CR patients and the controls (p < 0.001).
Conclusions : Serum soluble Fas and FasL play important roles in the proliferation and metastasis of SCLC, as well as in the cytotoxic
reaction and apoptosis induced by anticancer drugs in SCLC. Further study of the mechanisms and participation of circulating soluble Fas and
FasL is necessary to develop treatment strategies for SCLC.
# 2004 International Society for Preventive Oncology. Published by Elsevier Ltd. All rights reserved.
Keywords: Fas; Fas ligand; SCLC; Chemotherapy
1. Introduction
The Fas/Fas ligand (FasL) system is a major regulator of
apoptosis. Fas is expressed on the surface of cell membranes
in a variety of normal tissue cells and malignant cells
including lung cancer cells [1,2], and FasL is expressed on
the cell membrane surface of activated T lymphocytes and
lung cancer cells [3,4]. Since FasL expressed on T
lymphocytes can induce apoptosis in Fas-expressing lung
* Corresponding author. Tel.: +81 566 27 5404; fax: +81 566 27 5407.
E-mail address: [email protected] (K. Yamaki).
cancer cells, the Fas/FasL system plays an important role in
the cell-mediated cytotoxic reaction against lung cancer. On
the other hand, malignant cells can escape the immune
system by the downregulation of the FasL and the killing of
lymphocytes through the expression of FasL [5,6]. Thus, the
balance of Fas/FasL interaction between the host immune
system and malignant cells may play an important role in
cancer progression, lymph node involvement, metastasis and
cancer cell death. Commonly used chemotherapeutic drugs
have been shown to induce Fas expression on lung cancer
cells and apoptosis of lung cancer cells [7]. The mechanism
of apoptosis induced by drugs has not been clarified, but is
0361-090X/$30.00 # 2004 International Society for Preventive Oncology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.cdp.2004.09.001
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M. Shimizu et al. / Cancer Detection and Prevention xxx (2004) xxx–xxx
not considered to be associated with the Fas expressed on
lung cancer cells [8,9]. Unlike membrane-bound Fas and
FasL in lung cancer cell lines, the role of soluble Fas and
FasL in the proliferation and death of lung cancer cells has
yet to be clarified. Soluble Fas and FasL levels rise in the
peripheral blood of lung cancer patients, yet the significance
of circulating soluble Fas and FasL has not been examined.
In the present study, we measured the concentrations of
circulating soluble Fas and FasL in patients with small cell
lung cancer (SCLC), as they have higher sensitivity to
chemotherapeutic agents than non-small cell lung cancer
(NSCLC) patients, in order to examine whether soluble Fas
and FasL participate in any as yet unidentified clinical
features of SCLC.
2. Patients and methods
2.1. Patients
Twenty-one patients (15 males, 6 females; mean age,
65.7 years; range, 47–81 years; Table 1) diagnosed with
SCLC by transbronchial lung biopsy and treated at the
Second Department of Internal Medicine of Nagoya
University Hospital from 1998 to 2000 were included in
this study. Blood samples were obtained prior to
chemotherapy in order to measure soluble Fas and soluble
FasL concentrations. All patients were evaluated according to the simple two-stage system developed by the
Veteran’s Administration Lung Cancer Study Group [10].
Distant organ metastasis was found in 12 of the 21
patients. Limited disease (LD) stage SCLC describes a
tumor confined to the hemithorax of origin, the mediastium, and the supraclavicular lymph node, which can be
encompassed within tolerable radiation therapy. Extensive
disease (ED) stage SCLC indicates a tumor that is too
widespread to be included within the LD stage definition.
Blood samples were also obtained from 12 age-matched
healthy controls (eight males, four females; mean age,
63.8 years, range, 50–78 years). Blood serum was stored at
80 8C immediately after separation by centrifugation at
4 8C until the assay.
2.2. Therapy and response to therapy
The patients in the ED stage received intensive weekly
anticancer chemotherapy (vincristine, doxorubicin, etoposide and carboplatin), while patients in the LD stage were
treated with the same intensive weekly anticancer chemotherapy as those in the ED stage plus sandwich thoracic
irradiation. The response to therapy was documented at 4
weeks following the completion of therapy. Complete
response (CR) was defined as the absence of detectable
disease, partial response (PR) indicated a reduction in tumor
size of more than 50%, no change (NC) indicated a tumor
reduction of less than 50% or an enlargement of less than
25%, and progressive disease (PD) indicated a tumor
enlargement greater than 25% or additional tumor involvement. Two independent observers evaluated the radiological
findings based on X-ray, computed tomography or magnetic
resonance imaging films. Prophylactic cranial irradiation
was applied only to patients who achieved CR, as proved by
negative histology of a transbronchial biopsy after chemotherapy.
2.3. Measurement of soluble Fas and FasL
Table 1
Characteristics of the 21 small cell lung cancer patients
Patient
Age
Sex
Smoker
Stage
Distant
metastasis
Response to
chemotherapy
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
58
60
65
71
69
46
51
63
73
68
72
55
64
65
67
69
70
70
71
81
71
M
M
M
F
F
F
M
M
M
M
M
F
F
F
M
M
M
M
M
M
M
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
LD
LD
LD
LD
LD
ED
ED
LD
LD
ED
ED
ED
ED
ED
ED
ED
ED
ED
ED
LD
ED
No
No
No
No
Yes
Yes
Yes
No
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
CR
CR
CR
CR
CR
CR
CR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PD
PD
Soluble Fas and FasL in blood serum were measured
using a soluble Fas ELISA Kit and soluble FasL ELISA Kit
(Medical & Biological Laboratories Co. Ltd., Nagoya,
Japan) according to the manufacturer’s instructions. Blood
samples (0.1 ml) were poured into antibody-coated microwells using multichannel pipettes, then incubated for 60 min
at room temperature. After washing four times, 0.1 ml of
peroxidase-conjugated monoclonal antibody solution was
added to each well and incubated for 60 min at room
temperature with shaking. After washing, the peroxidase
substrate reagent (0.1 ml) was added to each well. Stop
solution (0.1 ml) was then added to the wells after 30 min
shaking at room temperature. The absorbance of each well
was measured at a wavelength of 450 nm and the reference
at 620 nm using a microplate reader MPQ A4i (Tosoh,
Tokyo, Japan).
2.4. Statistical analysis
Statistical analysis was performed using Fisher’s PLSD
and Scheffe method of Post Hoc Test (StatView 5.0).
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M. Shimizu et al. / Cancer Detection and Prevention xxx (2004) xxx–xxx
Differences with a p-value less than 0.05 were considered
significant. Data represent the mean S.E.
3. Results
The serum soluble Fas and FasL concentrations of the 21
SCLC patients were 4.72 0.27 ng/ml and 0.56 0.06 ng/
ml (mean S.E.), respectively, and were significantly
higher than those of the 12 controls, at 1.98 0.12 ng/ml
and 0.27 0.09 ng/ml, respectively (p < 0.001). The
neuronal specific enolase (NSE) concentration of the SCLC
patients (29.70 25.00 ng/ml) was significantly higher than
that of the controls (<10 ng/ml) (p < 0.001) (Table 2).
The NSE concentration of the LD and ED patients was
16.19 3.01 ng/ml (n = 8) and 38.05 7.87 ng/ml
(n = 13), respectively. The NSE concentration of the ED
patients, but not the LD patients, was significantly higher
than those of the LD patients and controls. The NSE
concentration of patients with distant metastasis
(38.02 8.62 ng/ml, n = 12) was significantly higher than
that of patients without distant metastasis (8.66 3.37 ng/
ml, n = 9). In SCLC patients without distant metastasis, the
NSE concentration was higher than that in the controls, but
the difference did not reach statistical significance. Thus,
elevation of NSE concentration in serum appears to indicate
the spread of SCLC.
The soluble Fas concentration of the LD and ED patients
was 4.05 0.33 ng/ml (n = 8) and 5.14 0.34 ng/ml
(n = 13), respectively. Each was significantly higher than
in the controls, and the soluble Fas concentration of ED
patients was significantly higher than that of the LD patients,
while the soluble Fas concentration of ED patients was the
highest of all groups. Therefore, elevation of soluble Fas in
blood may indicate progressive proliferation and metastasis
of SCLC. The soluble Fas concentration of patients with
distant metastasis (5.12 0.39 ng/ml, n = 12) was significantly higher than that of patients without distant metastasis
(4.19 0.29 mg/ml, n = 9), and the latter was significantly
higher than that of the controls value. Thus, elevation of
3
soluble Fas concentration in serum may indicate a
progressive invasion of SCLC, suggesting that soluble Fas
may be a valuable marker for SCLC, in addition to NSE, the
usual marker of SCLC (Fig. 1).
The soluble FasL concentration of the LD and ED
patients was 0.44 0.14 ng/ml (n = 8) and 0.64 0.05 ng/
ml (n = 13), respectively, and a significant difference was
recognized between the ED patients and controls value but
not between LD and ED patients. The soluble FasL
concentration of patients with and without distant metastasis
were 0.70 0.03 ng/ml (n = 13) and 0.38 0.12 mg/ml
(n = 8), respectively. The former was significantly higher
than both the latter and that of the controls value, but there
was no significant difference between the latter and the
controls. The soluble FasL concentration of patients with ED
or distant metastasis was higher than in the controls. These
data suggest that elevation of soluble FasL concentration in
SCLC may indicate an extensive invasion by cancer cells.
Elevation of NSE or soluble FasL concentration may
indicate the ED stage or distant metastasis of SCLC. In
addition, elevation of soluble Fas concentration was detected
even in the LD stage and in patients with no distant
metastasis of SCLC. These results suggest that serum
soluble Fas may be one of the most sensitive markers of
SCLC.
Complete response was achieved in 7 (33.3%), PR in 12
(57.2%), NC in 0 (0%), and PD in 2 (9.5%) of the 21 SCLC
patients. In CR patients, NSE, soluble Fas and soluble FasL
concentrations were 21.26 3.65 ng/ml, 3.58 0.19 ng/ml
and 0.50 0.15 ng/ml, respectively, while they were
33.96 7.86 ng/ml, 5.29 0.29 ng/ml and 0.59 0.07 ng/
ml, respectively, in the PR/NC/PD group of patients.
Elevation of NSE, soluble Fas and soluble FasL concentrations were detected in the PR/NC/PD patients, however,
only Fas concentration was significantly different between
the CR and PR/NC/PD patients. NSE, soluble Fas and
soluble FasL concentrations were elevated in both CR patients
and the controls, however only soluble Fas concentration significantly differed between the CR patients and
controls.
Table 2
Concentrations of soluble Fas, soluble Fas ligand and NSE in the 21 small cell lung cancer patients before chemotherapy
Soluble Fas
Small cell lung cancer
Limited disease
Extensive disease
Distant metastasis ( )
Distant metastasis (+)
CR
PR/NC/PD
Controls
n = 21
n=8
n = 13
n = 10
n = 11
n=7
n = 14
n = 12
Data represent mean S.E.
a
Significant increase compared with controls (p < 0.001).
b
Significant increase compared with the former (p < 0.001).
a
4.72 0.27
4.05 0.33a
5.14 0.34a,b
4.13 0.27a
5.26 0.40a,b
3.58 0.19a
5.29 0.29a,b
1.98 0.12
Soluble FasL
a
0.56 0.06
0.44 0.14
0.64 0.05a
0.42 0.12
0.70 0.03a
0.50 0.15a
0.59 0.07a
0.27 0.09
NSE
29.72 5.44a
16.19 3.01
38.05 7.87a,b
17.53 3.22
40.81 8.92a,b
21.26 3.65
33.96 7.86a
<10.00
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Fig. 1. Soluble Fas concentration of the ED patients was significantly higher than that of the LD patients and controls (p < 0.001) (a). The soluble Fas
concentration of patients with distant metastasis was higher than that of patients without distant metastasis (p < 0.001) (b). In addition, the soluble Fas levels in
patients achieving CR were significantly lower than in patients with PR/NC/PD (p < 0.001) (c). Data represent means S.D. Serum elevation of soluble Fas
may indicate lymph node involvement and/or distant metastasis of SCLC.
4. Discussion
The Fas/FasL system is a major regulator of apoptosis.
Fas is expressed on the surface of cell membranes in a
variety of normal lung cells and lung cancer cells [1,2], and
Fas mRNA is expressed in non-small cell lung cancer cells
[11]. Serum soluble Fas is produced from the cell surface of
malignant cells in a form lacking the 21 amino acid residues
containing the transmembrane domain by alternative
splicing. Soluble Fas is supposed to act as a FasL inhibitor
to bind Fas and prevent Fas-mediated apoptosis. Circulating
soluble Fas has been reported to increase in hepatocellular
carcinoma [12], hematopoietic malignancies [13], renal cell
carcinoma [14], breast cancer [15] and bladder cancer [16].
We demonstrated in the present study that serum soluble Fas
levels are higher in SCLC patients with distant metastasis or
in ED patients than in patients without distant metastasis or
LD patients. Although the concentrations of serum soluble
Fas or FasL did not parallel the levels of Fas or FasL
expression on the surface of cancer cells, their expression on
lung cancer cell membranes will reflect the circulating
soluble Fas and FasL. Thus, measurement of the serum
soluble Fas and FasL concentrations is important to
understand the tumorigenesis of SCLC. Higher expression
of Fas correlates with a lower incidence of lymph node
involvement in NSCLC [17]. The expression of Fas on the
membrane of cancer cells or mRNA in cancer cells was not
determined in the 21 SCLC samples examined in the present
study. However, most patients with a higher level of soluble
Fas were ED cases and/or distant metastasis cases. Although
the highly metastatic potential of lung cancer may not be
induced by an alteration in the expression of a single gene,
the soluble Fas level may be an important marker of
metastasis of SCLC.
In addition to Fas, FasL also exists in soluble form
released from cell surfaces after cleavage by metalloproteinases [18,19]. FasL is a type II transmembrane protein
that is a member of the tumor necrosis factor family of
cytokines [20,21], expressed in T-cells, NK cells and a wide
variety of tumors, many of which also express Fas [3,4], and
induce apoptosis in Fas-expressing cells. High levels of
soluble FasL have been observed in some hemopoietic
malignancies [22], hepatocellular malignancy [23], colon
cancer [24], esophageal cancer [25], and pancreatic cancers
[3,4]. The cytotoxic potential of soluble FasL is significantly
less than that of membrane-bound FasL. However, soluble
FasL retains the capacity to induce apoptosis [26,27]. The
soluble Fas and FasL system at the interface of tumor cells
and the immune system is more complex than expected. The
aberrant expression of FasL is a common feature of
malignant cells and can result in tissue invasion, metastatic
spread, and immune escape [28]. Invading and metastatic
tumor cells typically secrete matrix metalloproteinases and
induce matrix metalloproteinase production by surrounding
stromal cells that may overwhelm the local tissue capacity to
maintain their proteolytic activity in check. Synthetic
metalloproteinase inhibitors have been able to prevent
tumor cell-induced remodeling of extracellular matrix and
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angiogenesis and to provide potentially useful reagents for
the control of cancer spread. FasL highly expressed on
SCLC cells and circulating soluble Fas may counteract the
host’s anticancer immunity in their vicinity thereby
enhancing immune escape of cancer cells, cancer cell
growth and metastases in patients [5,6]. The expression of
FasL is downregulated in highly metastatic subpopulations
of NSCLC [29]. We demonstrated that the serum soluble
FasL concentration is elevated in SCLC patients, as is
soluble Fas, suggesting that soluble FasL plays an important
role in tumorigenesis and anticancer cytotoxic activity,
similar to soluble Fas.
A role for the Fas/FasL signaling system in apoptosis
induced by chemotherapy has been proposed for some cell
types of lung cancer [30]. The expression of Fas and FasL on
the surface of NSCLC cells is upregulated after exposure to
anticancer agents, and apoptosis is induced in some cell lines
upon exposure to the Fas agonistic monoclonal antibody.
However, the drug response to doxorubicin has been
demonstrated to be associated with Fas expression in 94
NSCLC cells lines [7], and sensitivity to chemotherapy has
not been shown to correlate with Fas-mediated apoptosis
[8,9]. Therefore, chemotherapy-induced apoptosis is
expected to occur in the absence of Fas and FasL interaction.
However, soluble Fas may play a key role in the
development of SCLC, and may act as an inhibitor and
blocker of the chemotherapy-induced apoptosis of SCLC
cells. In the present study, the concentrations of serum
soluble Fas were statistically lower in CR patients than in the
other patients, although the difference between them was not
particularly great. In six of the seven (85.7%) CR patients,
the soluble Fas concentration was lower than 4.20 ng/ml,
which was the mean minus one S.D. of the soluble Fas
concentration of the PR/NC/PD patients, and in only 1 of 14
(7.1%) of the other patients was the soluble Fas concentration lower than 4.20 ng/ml. Therefore, serum soluble Fas
may predict the responsiveness to chemotherapy in SCLC
patients. However, the concentration at which Fas becomes
serum soluble must be determined using a large SCLC
patient cohort and the standard assay. Furthermore, the
response will be predicted more accurately by a analysis of a
combination of soluble Fas and other tumor markers such as
NSE and pro GRP.
In conclusion, serum soluble Fas and FasL play important
roles in the proliferation and metastasis of SCLC, as well as
in the cytotoxic reaction and apoptosis induced by anticancer drugs in SCLC. The mechanisms and participation of
circulating soluble Fas and FasL require further study in
order to develop treatment strategies for SCLC.
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