The relationship between secretory leukocyte protease inhibitor expression and Epstein-Barr virus status among patients with nasopharyngeal carcinoma.
ABSTRACT The aim was to study the expression of Secretory Leukocyte Protease Inhibitor (SLPI) and to explore its correlation with the presence of Epstein-Barr Virus (EBV) among patients with nasopharyngeal carcinoma.
The expression levels of SLPI mRNA in NPC cell lines and in ten matched-pairs of NPC and adjacent normal tissue were examined by quantitative real-time Polymerase Chain Reaction (PCR). Furthermore, protein expression of SLPI in 71 paraffin-embedded NPC biopsies was assessed by immunohistochemistry. Finally, the serum level of SLPI in 177 NPC patients and 103 healthy controls was evaluated by enzyme-linked immunosorbent assay (ELISA).
The expression of SLPI mRNA in NPC cells was significantly lower than in the adjacent normal epithelium (p<0.001). When the expression of SLPI in EBV-positive and -negative NPC cell lines was compared, we found that both mRNA and protein expressions of SLPI were significantly higher in EBV-negative cells. Furthermore, the results of immunohistochemical analysis demonstrated that the frequency of reduced SLPI expression in EBV-positive biopsies was significantly higher than that in EBV-negative biopsies.
In this study, we have confirmed that SLPI is significantly down-regulated in NPC tissues. In addition, based on our preliminary results, we propose that the reduction of SLPI in NPC cells is associated with the presence of the EBV genome and/or the expression of EBV-encoded genes. SLPI may play an important role in EBV-mediated NPC tumorigenesis.
- SourceAvailable from: nih.gov[show abstract] [hide abstract]
ABSTRACT: Infection of adherent primary monocytes with HIV-1Ba-L is significantly suppressed in the presence of human saliva. By reverse transcriptase (RT) levels, saliva, although present for only 1 h during monocyte viral exposure, inhibited HIV-1 infectivity for 3 wk after infection, whereas human plasma and synovial fluid failed to inhibit HIV-1 infectivity. Antiviral activity was identified in the saliva soluble fraction, and to determine the factor(s) responsible, individual saliva proteins were examined. Of those proteins examined, only secretory leukocyte protease inhibitor (SLPI) was found to possess anti-HIV-1 activity at physiological concentrations. SLPI anti-HIV-1 activity was dose dependent, with maximal inhibition at 1-10 micrograms/ml (> 90% inhibition of RT activity). SLPI also partially inhibited HIV-1IIIB infection in proliferating human T cells. SLPI appears to target a host cell-associated molecule, since no interaction with viral proteins could be demonstrated. However, SLPI anti-HIV-1 activity was not due to direct interaction with or downregulation of the CD4 antigen. Partial depletion of SLPI in whole saliva resulted in decreased anti-HIV-1 activity of saliva. These data indicate that SLPI has antiretroviral activity and may contribute to the important antiviral activity of saliva associated with the infrequent oral transmission of HIV-1.Journal of Clinical Investigation 08/1995; 96(1):456-64. · 12.81 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Inflammatory lung injury is probably regulated by the balance between proteases and protease inhibitors together with oxidants and antioxidants, and proinflammatory and anti-inflammatory cytokines. Rat tissue inhibitor of metalloprotease-2 (TIMP-2) and secreted leukoprotease inhibitor (SLPI) were cloned, expressed, and shown to be up-regulated at the levels of mRNA and protein during lung inflammation in rats induced by deposition of IgG immune complexes. Using immunoaffinity techniques, endogenous TIMP-2 in the inflamed lung was shown to exist as a complex with 72- and 92-kDa metalloproteinases (MMP-2 and MMP-9). In inflamed lung both TIMP-2 and SLPI appeared to exist as enzyme inhibitor complexes. Lung expression of both TIMP-2 and SLPI appeared to involve endothelial and epithelial cells as well as macrophages. To assess how these endogenous inhibitors might affect the lung inflammatory response, animals were treated with polyclonal rabbit Abs to rat TIMP-2 or SLPI. This intervention resulted in significant intensification of lung injury (as revealed by extravascular leak of albumin) and substantially increased neutrophil accumulation, as determined by cell content in bronchoalveolar lavage (BAL) fluids. These events were correlated with increased levels of C5a-related chemotactic activity in BAL fluids, while BAL levels of TNF-alpha and chemokines were not affected by treatment with anti-TIMP-2 or anti-SLPI. The data suggest that endogenous TIMP-2 and SLPI dynamically regulate the intensity of lung inflammatory injury, doing so at least in part by affecting the generation of the inflammatory mediator, C5a.The Journal of Immunology 04/1999; 162(6):3653-62. · 5.52 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Oropharyngeal candidiasis, typically caused by Candida albicans, is the most common oral disease associated with human immunodeficiency virus type 1 (HIV-1) infection. Secretory leukocyte protease inhibitor (SLPI), a 12-kDa antiprotease, suppresses the growth of C. albicans in vitro. To determine whether the mucosal protein plays a role in protecting oral tissues against fungal infection, we conducted a cross-sectional study investigating the oral and systemic health and salivary SLPI levels in 91 dentate HIV-1-infected adults receiving medical care in the southeastern United States. Participants with a self-reported history of clinical oropharyngeal candidiasis during the previous 2 years constituted the test group (n = 52), while the comparison group (n = 39) had no oropharyngeal candidiasis during that period. Data collected from medical records, oral examination, and SLPI enzyme-linked immunosorbent assay quantitation of whole saliva were analyzed by t test, analysis of variance, linear regression, and unconditional logistic regression. The test group had a significantly higher mean salivary SLPI level than the comparison group (1.9 microg/ml versus 1.1 microg/ml, P < 0.05). Linear regression modeling identified CD4 cell count and history of oropharyngeal candidiasis as key predictors of salivary SLPI and revealed a significant interaction (P < 0.05) between immunosuppression (CD4 cell count below 200 cells/ microl) and positive history of oropharyngeal candidiasis in predicting salivary SLPI level. By logistic regression modeling, a salivary SLPI level exceeding 2.1 microg/ml, low CD4 count, antiretroviral monotherapy, and smoking were key predictors of oropharyngeal candidiasis. These data support a key role for SLPI in the oral mucosal defense against C. albicans. The antimicrobial mucosal protein may serve as an indicator of previous oropharyngeal candidiasis infection among immunosuppressed persons.Infection and Immunity 04/2004; 72(4):1956-63. · 4.07 Impact Factor
Abstract. Aim: The aim was to study the expression of
Secretory Leukocyte Protease Inhibitor (SLPI) and to explore
its correlation with the presence of Epstein-Barr Virus (EBV)
among patients with nasopharyngeal carcinoma. Materials
and Methods: The expression levels of SLPI mRNA in NPC
cell lines and in ten matched-pairs of NPC and adjacent
normal tissue were examined by quantitative real-time
Polymerase Chain Reaction (PCR). Furthermore, protein
expression of SLPI in 71 paraffin-embedded NPC biopsies
was assessed by immunohistochemistry. Finally, the serum
level of SLPI in 177 NPC patients and 103 healthy controls
was evaluated by enzyme-linked immunosorbent assay
(ELISA). Results: The expression of SLPI mRNA in NPC
cells was significantly lower than in the adjacent normal
epithelium (p<0.001). When the expression of SLPI in EBV-
positive and -negative NPC cell lines was compared, we
found that both mRNA and protein expressions of SLPI were
significantly higher in EBV-negative cells. Furthermore, the
results of immunohistochemical analysis demonstrated that
the frequency of reduced SLPI expression in EBV-positive
biopsies was significantly higher than that in EBV-negative
biopsies. Conclusion: In this study, we have confirmed that
SLPI is significantly down-regulated in NPC tissues. In
addition, based on our preliminary results, we propose that
the reduction of SLPI in NPC cells is associated with the
presence of the EBV genome and/or the expression of EBV-
encoded genes. SLPI may play an important role in EBV-
mediated NPC tumorigenesis.
Secretory leukocyte protease inhibitor (SLPI MIM 107285),
also named antileukoprotease, is a member of the whey
acidic protein four-disulfide core family. It is an 11.7 kDa
non-glycosylated secretory protein that is produced by
neutrophils, macrophages and the epithelial cells lining the
respiratory, digestive, and reproductive tracts (1-4). SLPI can
be found abundantly in mucosal secretions, including breast
milk, seminal fluid, saliva and the secretions of the female
genital tract. In addition to the fact that it possesses
antiprotease activity which protects tissue from the damaging
effects of inflammation (3, 5-6), SLPI has been found to
have antibacterial (2, 7), antifungal (8) and antiviral activity
Dysregulation of SLPI has been reported in cancer;
however, depending on cancer type, both up-regulation and
down-regulation have been noted. For example, SLPI has
been showed to be overexpressed in ovarian (11-12), gastric
(13), papillary thyroid cancer (14) and non-small cell lung
carcinoma (15). In these cases, SLPI has been suggested to
promote tumorigenesis and invasiveness of cancer cells (16);
furthermore, the presence of elevated SLPI expression in
these types of cancer often seems to be associated with a
poor prognosis among these cancer patients (13, 17). In
contrast to this, down-regulation of SLPI has been reported
in prostate cancer (18), cervical adenocarcinoma (19) and
oral squamous cell carcinoma (20). In addition, down-
regulation of SLPI expression has also been reported to be
associated with infection-related diseases such as
Helicobacter pylori-related gastroduodenal diseases (21-22)
and exposure of human cervical epithelial cells to Herpes
Correspondence to: Ngan-Ming Tsang, MD, D.Sc., Department of
Radiation Oncology, Chang Gung Memorial Hospital at Lin-Kou,
Taoyuan, Taiwan, R.O.C. Tel: +886 975365772, Fax: +886
33280797, e-mail: firstname.lastname@example.org
Key Words: Nasopharyngeal carcinoma, Epstein-Barr virus,
Secretory leukocyte protease inhibitor.
ANTICANCER RESEARCH 32: 1299-1308 (2012)
The Relationship between Secretory Leukocyte Protease
Inhibitor Expression and Epstein-Barr Virus Status
among Patients with Nasopharyngeal Carcinoma
KA-PO TSE1, CHI-SHENG WU1, CHUEN HSUEH2, KAI-PING CHANG3,
SHENG-PO HAO4, YU-SUN CHANG1and NGAN-MING TSANG5,6
1Molecular Medicine Research Center and 6School of Traditional
Chinese Medicine, Chang Gung University, Taoyuan, Taiwan, R.O.C.;
Departments of 2Pathology, 3Otolaryngology-Head Neck Surgery and
5Radiation Oncology, Chang Gung Memorial Hospital at Lin-Kou, Taoyuan, Taiwan, R.O.C.;
4Department of Otolaryngology, Head and Neck Surgery,
Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan, R.O.C.
Simplex virus (HSV)-1 or HSV-2 (23). However, the
biological significance of SLPI reduction as part of these
diseases is still unknown.
Nasopharyngeal carcinoma (NPC) is the most common
type of cancer originating in the nasopharynx, and has a
remarkable geographical and racial distribution. It is
uncommon in Western countries, but is extremely common
in south-eastern regions of Asia, such as the southern parts of
China, Hong Kong, and Taiwan. Epstein-Barr virus (EBV)
has been suggested to be an important co-factor in the
etiology of NPC. Elevated levels of EBV-specific IgA can be
found in NPC patients compared to normal individuals and
patients with other cancer types (24, 25). Furthermore, the
detection of EBV nuclear antigen and viral DNA in NPC
cells has revealed that EBV is able to infect epithelial cells
and that this event is associated with their transformation
(26). All these finding strongly suggest a probable oncogenic
role for the virus in the genesis of this type of tumor (27).
In our previous microarray data analysis of matched-pair
NPC tumor tissue and a pool of adjacent normal tissue, we
found that SLPI is one of the genes significantly down-
regulated in NPC cells compared to normal nasopharyngeal
epithelium (28). A similar observation was also found when
the expression profile of NPC was assessed by laser capture
microdissection (29). Given the potent anti-inflammatory and
antiviral activities of SLPI and the fact that silencing of SLPI
has been reported in infection-associated diseases, an
evaluation of the expression of SLPI in NPC may help to
unravel various tumorigenesis processes. In this study, we
compared the mRNA and protein expression levels of SLPI
using matched-pair specimens of NPC tumor and adjacent
normal nasopharyngeal epithelium. In addition, the
correlation between SLPI reduction and EBV status in
clinical biopsies was also examined.
Materials and Methods
Patients and samples. Paraffin-embedded tissues and serum samples
were collected from consecutively consenting patients suffering
from newly diagnosed and untreated NPC who attended oncology
clinics at the Department of Radiation Oncology and
Otolaryngology-Head Neck Surgery of Chang Gung Memorial
Hospital (CGMH), Lin-Kou, Taiwan, ROC, between September
2002 and February 2007. Samples of NPC tissues and adjacent
normal nasopharyngeal epithelium were obtained during surgery
and were frozen immediately after surgical resection. Tumor-node-
metastasis (TNM) stage was defined according to the 2002 revision
of the cancer staging systems promulgated by the American Joint
Committee on Cancer (AJCC) (30). Histopathological classification
of the tumor samples was based on the “Pathology and Genetics of
Head and Neck Tumors” published by the World Health
Organization in 2005 (31). All stages and histological types were
present among the samples obtained from our patients and therefore
the patients displayed a full range of NPC symptoms. One hundred
and three serum samples were collected from volunteers undergoing
routine health examinations as healthy controls; those presenting
with hypertension, cardiovascular disease, or diabetes mellitus were
excluded from the study.
Based on the typing of lymphomas and the detection of EBV
encoded small RNAs (EBER) by ISH given below, 16 cases of
characterized nasal Natural Killer/ T cell Lymphoma (NKTCL)
collected from 1999 to 2007 at CGMH, Linkou, Taiwan, ROC, were
studied. This project was reviewed and approved by the Institutional
Review Board and Ethnics Committee of CGMH (authorization no.
IRB 98-3215B). Informed consent was obtained from all participants.
Cell culture. NPC cell lines HK1 and HK1/EBV (kindly provided by
Dr. Sai-Wah Tsao, University of Hong Kong, Hong Kong SAR,
China) were maintained in RPMI-1640 medium supplemented with
10% fetal bovine serum, 2 mM sodium pyruvate and 2 mM L-
glutamate at 37˚C under 5% CO2. For HK1/EBV cells, 500 μg/ml of
Geneticin (G418) was added to the culture medium for EBV-positive
cell selection. For the ELISA assay, cells were cultured in 96-well
plates; after 48 h, the culture medium were harvested and centrifuged
at 4˚C and 1500 ×g for 5 min. The resulting pellets were discarded
and supernatants were collected and stored at –80˚C until use.
Quantitative real-time polymerase chain reaction (qRT-PCR). Total
RNA from NPC tissues and cell lines were isolated using TRIzol
reagent (Invitrogen, Carlsbad, CA, USA) according to the
manufacturer’s instruction. The concentration of RNA was quantified
using a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher
Scientific, Waltham, MA, USA). Reverse transcription of the RNA
samples (1 μg) was performed using ImProm-II (Promega, Madison,
WI, USA) and oligo (dT) 15 primers (Promega). The reverse
transcription products were diluted 20-fold and used as template in a
10 μl PCR reaction containing 1× SYBR Master Mix (Applied
Biosystems, Foster City, CA, USA). The primers used to amplify the
cDNA corresponding to SLPI and internal control 18S rRNA were
as follows: SLPI _F: 5’-TCCTGCCTTCACCATGAAGTC-3’; and
SLPI _R: 5’-AGCCCAAGGTGCCAGAGTT-3’; 18S rRNA _F: 5’-
CGAGCCGCCTGGATACC-3’and 18S rRNA _R: 5’-CCTCAGT
TCCGAAAACCAACAA-3’. The quantitative RT-PCR was carried
out on an ABI 7500 (Perkin-Elmer Applied Biosystems, Inc., Foster
City, CA, USA), running pre-denaturation at 95˚C for 10 min, then
40 cycles of the following program: 15 s at 95˚C, 1 min at 60˚C.
ELISA. Patient blood was obtained at time of diagnosis by
peripheral venous puncture and was immediately centrifuged at
3,000 ×g for 15 min. The serum was collected and frozen at - 80°C
until use. The SLPI concentration in the serum samples and culture
medium were determined by a commercially available ELISA kit
(Quantikine®; R&D Systems, Inc., Minneapolis, U.S.A.). All SLPI
analyses were performed in duplicate according to the
Immunohistochemistry (IHC). Immunohistochemical staining was
performed using an automatic IHC-staining device, Bond-max
Bannockburn, IL, USA). Paraffin-embedded tissue sections were
retrieved using Bond Epitope Retrieval Solution 1 and stained with
antibodies against SLPI (NCL-SLPI 1:50 dilution; Novocastra
Laboratories Ltd., Newcastle, UK). Tissue sections were treated
with 30-diaminobenzidine tetrahydrochloride (DAB) as the
chromogen and hematoxylin as the counterstaining reagent. Images
Immunostainer (Vision Biosystems, Leica,
ANTICANCER RESEARCH 32: 1299-1308 (2012)
of the slides were captured using a ScanScope CT automated slide
scanning system (Aperio Technologies, Vista, CA, USA) and
evaluated by experienced pathologists.
In situ hybridization (ISH). ISH to detect EBV-encoded RNA
transcripts (EBER) was performed using the EBV probe ISH kit
(Novocastra) in accordance with the manufacturer’s instruction.
Statistical analysis. Differences in SLPI mRNA between NPC tumor
tissue and the corresponding adjacent normal nasopharyngeal
epithelium were assessed using paired t-test. Descriptive statistics
(mean, SE, SD, median, minimum and maximum) were used to
summarize the distribution of serum SLPI levels. The one-sample
Kolmogorov-Smirnov test was used to test for the normal distribution
of serum SLPI levels within each group, and the results suggested
that the data was not normally distributed. Based on this, the
statistical difference in SLPI protein concentration between NPC
patients and healthy controls was assessed using the Mann-Whitney
U-test. The statistical difference in SLPI protein concentration
between the culture medium from HK1 versus HK1/EBV cells was
assessed using Student’s t-test. All statistical tests were two-sided
and significance was set as p<0.05 (except where a Bonferroni
correction was made to adjust for multiple testing during subgroup
analysis, where the significance was set at p<0.05/n, where
n=number of subgroups). All statistical analysis was performed using
SPSS version 13.0 for Windows (SPSS Inc., Chicago, IL, USA).
Down-regulation of SLPI in NPC biopsies. Firstly, to confirm
the expression status of SLPI in the NPC biopsies, qRT-PCR
was performed on 10 matched-pairs of NPC tumor and
adjacent normal nasopharyngeal epithelium specimens. As
shown in Figure 1A, expression of SLPI mRNA was
dramatically lower in all NPC tumor tissue samples when
compared to the corresponding adjacent normal tissue
(p<0.001, paired t-test). In addition, we further investigated
the protein expression levels of SLPI in paraffin-embedded
tissues from the same patients using IHC (Figure 1C). All
samples showed abundant SLPI signal in the adjacent normal
epithelium (N), but the IHC results were negative for the
tumor cells (T). These results demonstrate that the expression
of SLPI in NPC cells is significantly reduced compared to
the adjacent normal nasopharyngeal epithelium.
Serum level of SLPI in NPC patients and healthy controls.
In addition to measuring the expression level of SLPI in
tumor cells in situ, we also wanted to evaluate the clinical
relevance of SLPI. The level (mean±S.D.) of SLPI was
quantified by ELISA in serum samples collected from 177
NPC patients and 103 healthy individuals. As shown in
Figure 2A, the mean serum level of SLPI in NPC patients
was significantly higher than in the healthy control group
(46.89±17.22 vs. 41.06±10.22 pg/ml; p=0.003, Mann-
Whitney U-test). Moreover, to clarify if the serum level of
SLPI is associated with disease progression, we compared
the serum level of SLPI in patients at different stages (Figure
2B) with that of the healthy control group. The mean serum
SLPI level of the patients at stage I and II were found to be
39.66 and 42.75 pg/ml, respectively, which are not
significantly different from that of the healthy group
(p=0.704 and 0.86 for stage I and II vs. control, respectively;
Mann-Whitney U-test). However, the mean serum SLPI level
of the patients at stage III and IV were found to be 46.22 and
53.39 pg/ml, respectively, which are significantly higher than
that in the control group (p=0.006 and <0.001 for stage III
and IV vs. control, respectively; Mann-Whitney U-test). The
results demonstrated that the serum SLPI level is elevated in
NPC patients at an advanced stage of the disease, but not in
patients at an early stage of the disease.
Expression of SLPI in EBV-infected nasopharyngeal epithelial
cells. According to previous reports, down-regulation of SLPI
has been reported in various types of infectious diseases such
as Helicobacter pylori-related gastroduodenal disease (21-22)
and when human cervical epithelial cells are exposed to either
HSV-1 or HSV-2 (23). Given the fact that NPC is known to
be closely associated with EBV infection, we speculated that
down-regulation of SLPI in NPC tumor cells might be a
phenomenon associated with EBV infection. To verify the
association between EBV infection and reduced SLPI gene
expression in NPC cells, we examined the SLPI expression
status of various NPC cell lines. Specifically, HK1, an NPC
cell line that does not carry EBV (32) and HK1/EBV, to
which EBV had been introduced by co-culture with infected
Akata cells, were used (33). Firstly, the concentration of SLPI
protein in culture medium from cell lines was measured by
ELISA. As shown in Figure 3A, the level of SLPI in the HK1
culture medium (mean±S.D.=1036.8±197.8 pg/ml) was
significant higher than that in the HK1/EBV culture medium
(mean±S.D.=134.1±80.6 pg/ml; p<0.001, t-test). Similarly,
when SLPI mRNA levels for the two cell lines were
measured, the mRNA level of SLPI in HK1/EBV cells was
~21% that of the HK1 cells (Figure 3B). These results show
that both the mRNA and protein levels of SLPI in HK1/EBV
cells were significantly lower than that in parental HK1 cells.
Expression of SLPI in EBV-positive and -negative NPC
biopsies. In addition to the above cell lines, we also tested
the association between EBV status and expression of SLPI
using clinical samples. The expression of SLPI protein was
evaluated by IHC using EBV-positive and EBV-negative
paraffin-embedded NPC tissues (n=71); the presence of EBV
in these samples was demonstrated by EBV-encoded small
RNA (EBER; Figure 4A and B) staining.
The demographic and pathological data of the studied
cases are summarized in Table I. Among the 71 biopsies
samples investigated, 64 cases (90.1%) were EBV positive
and 7 cases (9.9%) were EBV negative. Overall, SLPI tumor
Tse et al: Down-regulation of SLPI in NPC
cell signal was only detected in 4 out of the 71 cases (5.6%),
with all the tumor cells from the remaining NPC samples (67
out of 71, 94.4%) being negative for SLPI staining. No
association was found between SLPI expression and the
various different clinicopathological characteristics, such as
gender, age, and disease stage. However, a significant
correlation between reduced SLPI expression and the
presence of EBER was observed (p=0.002, Fisher’s exact
test). Among the 64 EBER-positive biopsies, 63 (94%)
exhibited a loss of SLPI expression, which is significantly
higher than for the EBER-negative specimens (3 out of 7).
The IHC results further support our hypothesis that the
presence of EBV or the expression of EBV genes is
associated with a reduction in SLPI expression by NPC cells.
ANTICANCER RESEARCH 32: 1299-1308 (2012)
Figure 1. Comparison of Secretory Leukocyte Protease Inhibitor (SLPI) mRNA and protein expression levels between ten paired pericancerous
adjacent normal tissue and tumor tissue. A: SLPI mRNA levels in the ten paired samples assessed by qRT-PCR. 18S rRNA was used as an internal
control. B: The PCR products were confirmed by agarose gel electrophoresis. C: Immunohistochemical staining of SLPI in paired pericancerous
adjacent normal tissue (N) and tumor tissue (T) from two representative cases; bar, 100 μm.
Down-regulation of SLPI in EBV-associated nasal type,
natural killer (NK)/T-cell lymphoma. In addition to the
situation with NPC, a constant association of nasal type
NK/T-cell lymphoma (NKTCL) with EBV has also been
reported (34-37). To further investigate if down-regulation of
SLPI is a general phenomenon among EBV-associated
malignancies, the expression of SLPI was evaluated in a pilot
study of 16 NKTCL biopsies using IHC staining. All
Tse et al: Down-regulation of SLPI in NPC
Figure 2. Box and Whisker plots showing serum Secretory Leukocyte Protease Inhibitor (SLPI) levels in healthy control individuals (control) and
patients with nasopharyngeal carcinoma (NPC patients). A: Serum SLPI levels in healthy control and patients with NPC. B: Serum SLPI levels in
healthy controls and patients with NPC according to stage. Box, the range of the middle 50% of SLPI level; line inside box, median; whiskers, 5th
and 95th percentile; circles, outliers; stars or triangles, extreme cases. Significance was calculated by two-side Mann-Whitney U-test.
Figure 3. The expression of Secretory Leukocyte Protease Inhibitor (SLPI) in HK1 and HK1-EBV cells. A: The SLPI concentration in the culture
medium from the cell lines was measured by ELISA. B: The mRNA levels of SLPI were measured by quantitative RT-PCR using 18S rRNA as the
internal control. C: The PCR products were examined by agarose gel electrophoresis. The presence of EBV genomic DNA in the HK1-EBV cells was
confirmed by PCR using primers specific for circularized EBV genomic DNA.
samples examined were found to be EBV positive. As shown
in Figure 5, abundant expression of SLPI protein was
detected in normal nasopharyngeal mucosa (Figure 5A and
C), but all the NKTCL biopsies examined were negative for
SLPI signal by IHC (Figure 5B and D). This result suggests
that a reduction in SLPI expression might be a general
phenomenon in EBV-associated malignancies.
In this study, using real-time qPCR and IHC, we confirmed
that expression of SLPI at the mRNA and protein levels is
significantly lower in NPC tumor cells than in adjacent
normal nasopharyngeal epithelium. Expression of SLPI was
dramatically reduced in the NPC tumor mass, with more than
94% of biopsies examined being negative for the presence of
SLPI protein. Moreover, when the expression levels of SLPI
in EBV-positive and -negative NPC cell lines and paraffin-
embedded NPC tissues were compared, we found that a
reduction in SLPI expression was also associated with the
presence of EBV in tumor cells.
Local inflammation is suspected to play a major role in
NPC tumorigenesis, because of the consistent presence of
massive lymphoid infiltrate in primary tumors and the
intense local production of inflammatory cytokines (38). In
addition, it has been shown that SLPI is able to negatively
ANTICANCER RESEARCH 32: 1299-1308 (2012)
Figure 4. Representative examples of Secretory Leukocyte Protease Inhibitor (SLPI) expression in EBV-positive (EBER-positive) and EBV-negative
(EBER-negative) NPC specimens. A, B: In situ hybridization with EBER probe; C, D: Immunohistochemical staining with SLPI-specific antibody.
Figure 5. Secretory Leukocyte Protease Inhibitor (SLPI) protein expression in EBV-associated Natural Killer T-cell Lymphoma (NKTCL).
Immunohistochemical staining of SLPI in normal nasal mucosa (A, C) and NKTCL (B, D). Bar-100 μm.
regulate the inflammatory response, firstly, by inhibiting
Nuclear Factor-KappaB (NF-κB) activation (39); secondly,
by inhibiting inflammatory infiltrate recruitment and
activation (40-41); thirdly, by inhibiting histamine release
from mast cells (42); and fourthly, by inhibiting C5a
production in the inflamed lung (5). Here, based on our IHC
staining results, we confirmed that SLPI down-regulation can
be found in more than 94% of NPC biopsies and that this is
associated with the presence of the EBV genome and/or the
expression of EBV-encoded genes. Given that EBV infection
is closely associated with NPC and SLPI is such an
important broad-spectrum innate immune molecule with anti-
inflammatory properties, which is generally silenced in NPC,
we can speculate that the decreased expression of SLPI may
help EBV-associated NPC tumorigenesis to occur (43).
Further studies are required to clarify the biological
significance of SLPI down-regulation in EBV-positive NPC
Although our results show that expression of SLPI mRNA
and protein in tumor cells is significantly reduced when
compared to adjacent normal nasopharyngeal epithelium, the
serum level of SLPI in the NPC patients at advanced stages
of the disease was significantly higher than among the
healthy control group. Since the source of SLPI found in
serum remains unclear, it is suggested that this contradiction
may be explained by the reduction in SLPI being a local
event associated with the NPC tumor mass, while the
elevated level of serum SLPI may reflect an activation of the
systematic antitumor immune response when a larger tumor
mass at advanced stage is present.
In addition to previous published expression profiling
information, a recent report in 2012 also demonstrated that
SLPI is one of the genes that is consistently down-regulated
in NPC tissues (44), which supports our findings. However,
although the biological significance and underlying
mechanism remain unknown, this report is the first showing
that SLPI down-regulation is associated with the presence of
EBV in NPC cells. It is just the beginning for understanding
the biological significance of SLPI in EBV-mediated
tumorigenesis of NPC. Functional assays of SLPI protein in
NPC cells should help us to further clarify its roles and its
interaction with EBV.
We thank our colleagues in the Department of Radiation Oncology for
their kind assistance, and we gratefully acknowledge the skill and
dedication of our laboratory technicians, Shu-Tsen Kao, Yu-Ling Hsieh
and Hsiao-Yun Cheng. We are grateful to Lai-Chu See (Department
of Public Health, Chang Gung University, Taoyuan, Taiwan) for
helpful discussions about our results. This study was supported by a
Chang Gung Memorial Hospital Research Grant, no. CMRPG360221.
1 Abe T, Kobayashi N, Yoshimura K, Trapnell BC, Kim H,
Hubbard RC, Brewer MT, Thompson RC and Crystal RG:
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epithelial cells. J Clin Invest 87(6): 2207-2215, 1991.
Fahey JV and Wira CR: Effect of menstrual status on
antibacterial activity and secretory leukocyte protease inhibitor
production by human uterine epithelial cells in culture. J Infect
Dis 185(11): 1606-1613, 2002.
Jin FY, Nathan C, Radzioch D and Ding A: Secretory leukocyte
protease inhibitor: a macrophage product induced by and
antagonistic to bacterial lipopolysaccharide. Cell 88(3): 417-426,
Shugars DC: Endogenous mucosal antiviral factors of the oral
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Gipson TS, Bless NM, Shanley TP, Crouch LD, Bleavins MR,
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Tse et al: Down-regulation of SLPI in NPC
Table I. Clinical characteristics and Secretory Leukocyte Protease
Inhibitor (SLPI) expression in the patients with Nasopharyngeal
Characteristic All SLPI-
(2002 AJCC criteria)
(2002 AJCC criteria)
(2002 AJCC criteria)
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Received February 15, 2012
Revised March 15, 2012
Accepted March 16, 2012
Tse et al: Down-regulation of SLPI in NPC