Wan-Li Cheng and Chia-Siu Wang contributed equally to this work.
Cancer Cell Biology
Overexpression of a secretory leukocyte protease inhibitor in human gastric cancer
Article first published online: 7 AUG 2008
Copyright © 2008 Wiley-Liss, Inc.
International Journal of Cancer
Volume 123, Issue 8, pages 1787–1796, 15 October 2008
How to Cite
Cheng, W.-L., Wang, C.-S., Huang, Y.-H., Liang, Y., Lin, P. Y., Hsueh, C., Wu, Y.-C., Chen, W.-J., Yu, C.-J., Lin, S.-R. and Lin, K.-H. (2008), Overexpression of a secretory leukocyte protease inhibitor in human gastric cancer. Int. J. Cancer, 123: 1787–1796. doi: 10.1002/ijc.23746
- Issue published online: 13 AUG 2008
- Article first published online: 7 AUG 2008
- Manuscript Accepted: 19 MAY 2008
- Manuscript Received: 14 DEC 2007
- Chang-Gung University (NMRPD, CMRPG). Grant Numbers: 150311, 640042
- National Science Council of the Republic of China. Grant Number: 95-2314-B-182-027
- gastrointestinal neoplasms;
- SLPI protein, human;
- reverse transcriptase polymerase chain reaction;
- tumor markers
Complementary DNA microarrays have identified aberrantly expressed genes in patients with gastric cancer. One that encodes secretory leukocyte protease inhibitor (SLPI) is among the aberrantly expressed genes and is associated with metastasis in gastric cancers. We evaluated the potential of SLPI expression as a helpful biomarker for detection of gastric cancer. Tumor tissue and matching noncancerous mucosa were obtained from 60 patients immediately after gastric resection. SLPI expression levels were determined by Northern and Western blot tests and quantitative-reverse transcriptase-polymerase chain reaction (Q-RT-PCR). Paraffin-fixed tumor tissues were used for immunohistochemistry study in 119 patients. A consistent result was obtained between all examinations except plasma SLPI. SLPI mRNA transcripts and protein were overexpressed in gastric cancer cells, and the depth of wall invasion was significantly greater in serosa-invading (T3 and T4) cancers compared to the serosa-free (T1 and T2) cancers. These enhanced expressions were significantly associated with lymph node metastasis, and were significantly higher in stages III and IV, and higher than those in stages I and II. Five-year survival of patients with lower expression of the SLPI gene was significantly better than among patients with a higher expression. To better understand the function of SLPI in human gastric cancer cells, isogenic SLPI overexpressing cell lines (AZ521) were prepared. The migratory and invasive abilities were increased 4.4-fold to 6.9-fold, or 3.0-fold to 4.1-fold, respectively, in SLPI-overexpressing cell lines. The results point to SLPI as a potential prognostic marker for gastric cancer and its function in cell invasion. © 2008 Wiley-Liss, Inc.
Gastric cancer is the second most common cancer in the world. It is the fifth leading cause of cancer-related deaths in Taiwan1 and, despite a declining incidence, it is similarly serious in Western countries.2 Surgery is the only cure for gastric cancer; however, curative resection is not feasible for the many patients whose cancer is too far advanced when detected.3 To improve the poor survival outcome and permit earlier diagnosis, there is a need for new and more sensitive prognostic indicators or tumor markers than those currently available, such as carcinoembryonic antigen and CA19-9.4
Gastric cancer is divided into 2 histomorphologic types, “intestinal-differentiated” and “diffuse-undifferentiated.”5, 6 However, similar lesions may differ in biological aggressiveness and response to therapy.7 The molecular events involved in development and progression of gastric cancer are complex, and involve multiple genes and steps that operate sequentially or in concert.6 Risk factors for gastric cancer include genetic alterations, chromosomal instability and infection caused by the bacterium Helicobacter pylori.8, 9
Numerous biomarkers have contributed to our knowledge of the molecular or cellular mechanisms of gastric carcinogenesis and progression.8 Most biomarkers are prognostic factors used to indicate the groups of patients at risk of relapse or metastasis.10 Biomarkers for the early detection of gastric cancer and monitoring of therapeutic efficacy are still lacking.
Gene expression profiling offers a new approach to cancer diagnosis, and may contribute to our understanding and lead to a cure in the future.11, 12 Complementary DNA (cDNA) microarrays have identified several highly expressed genes for gastric cancer. Our previous results showed that more than 2.9% (224/7,597) of aberrantly expressed genes were observed in these experiments. Among the numbers of highly upregulated genes were 86 of the intestinal type and 138 of the diffuse type. SPARC was the most highly expressed gene in the diffuse type, and thus was selected for previous study.13 Further, the gene-encoding secretory leukocyte protease inhibitor (SLPI, also called antileukoproteinase)14, 15 is associated with tumor metastasis. SLPI was selected for the present study because its overexpression is associated with aggressive, high-risk, or metastatic cancers, including pancreatic, papillary thyroid, uterine cervix, endometrial and ovarian cancers.14, 15 However, SLPI is underexpressed in nasopharyngeal carcinoma, bladder tumors and some breast carcinomas. To our knowledge, this is the first report of expression of SLPI in human gastric carcinoma.
The aim of this study was to identify helpful biomarkers for gastric cancer using cDNA microarrays, and to investigate their clinical significance. Our finding that SLPI expression is significantly higher in the advanced stages of gastric cancer in comparison to the early stages implicates the gene as a potential marker for the more advanced forms of gastric cancer.
Material and methods
After giving informed consent, 119 patients (65 males, 54 females; median age: 66 years, range 31–86 years) diagnosed with gastric cancer at Chang-Gung Memorial Hospital from 2000 to 2005 were enrolled in this study. Each patient had undergone gastric resection (31 had total gastrectomies and 88 had partial gastrectomies). The study protocol was approved by the Medical Ethics and Human Clinical Trial Committee at Chang-Gung Memorial Hospital.
Resected specimens were examined pathologically using the criteria in the Japanese General Rules for Gastric Cancer Study16 and the International Union Against Cancer (pTNM) classification system.17 The data included patient age and gender; tumor location and size; gross (Borrmann) type; wall invasion; resection margin; histologic type; lymph node metastasis; vascular invasion; lymphatic invasion; and perineural invasion. After discharge, all patients had periodic follow-up visits at the outpatient department at Chang-Gung Memorial Hospital until their deaths or the beginning of the preparation of this article.
Fresh samples of tumor tissue and adjacent noncancerous mucosa were harvested immediately after gastric resection. Samples dissected from resected specimens were immediately snap-frozen in individual vials using liquid nitrogen. Frozen specimens were stored at −70°C in a tumor bank until use.
Total RNA was extracted from cells using TRIzol® Reagent (Life Technologies, Rockville, MD), as described previously.18
Northern blot analysis
Northern blot analysis was performed, as described previously.19 Briefly, total RNA was extracted from cells with TRIzol reagent, and equal amounts of total RNA (20 μg) were analyzed on a 1.2% agarose-formaldehyde gel. The separated RNA molecules were then transferred to a nitrocellulose membrane and subjected to Northern blot analysis with a full-length SLPI cDNA fragment (∼0.6 kb) that was PCR-amplified and labeled with [α-32P]dCTP (3000 Ci/mmol; Amersham Inc., Piscataway, NJ). The membrane was subsequently reprobed with a 32P-labeled 18S rRNA fragment to verify equal application of RNA to each lane.
Real-time quantitative reverse-transcription polymerase chain reaction
Total RNA was extracted from cells using TRIzol as described earlier. Subsequently, the first strand of cDNA was synthesized using the SuperScript™ III One-Step RT-PCR System (Life Technologies; Invitrogen, Carlsbad, CA). Briefly, total RNA was denatured at 65°C for 5 min in the presence of 0.5 μg random hexamers and 1 mM deoxyribonucleotide triphosphate. After chilling on ice for at least 1 min, reverse transcription was allowed to proceed at 42°C for 5 min in the presence of 1× first-strand buffer, 5 mM dithiothreitol, and 40 U of RNase inhibitor. The reaction was then allowed to proceed at 42°C for another 60 min. The reaction was terminated by heat inactivation at 70°C for 10 min.
Real-time quantitative-reverse transcriptase-polymerase chain reaction (Q-RT-PCR) was performed in a 25-μL reaction mixture containing 50 nM forward and reverse primers, 1× SYBR® Green Master Mix (Applied Biosystems, Werrington, United Kingdom), and various amounts of template. The reaction was performed with preliminary denaturation for 10 min at 95°C, to activate Taq DNA polymerase, followed by 40 cycles of denaturation at 95°C for 15 sec, and annealing/extension at 60°C for 1 min. Fluorescence emitted by SYBR Green was detected online by the ABI PRISM 7000 Sequence Detection System (Applied Biosystems). Studies have shown that the initial copy number can be quantitated during real-time PCR analysis based on threshold cycle (Ct). The Ct is defined as the cycle at which fluorescence is determined to be statistically significant above the background. All PCR reactions were done in duplicate. For quantification of gene expression changes, the ΔCt method was used to calculate relative-fold changes normalized against the Human 18S rRNA. Human SLPI Q RT-PCR primer forward primer: 5′-TCCTGCCTTCACCATGAAGTC-3′; reverse primer 5′-AGCCCAAGGTGCCAGAGTT-3′. Human 18S rRNA Q RT-PCR forward primer: 5′-CGAGCCGCCTGGATACC-3′; reverse primer 5′-CCTCAGTTCCGAA AACCAACAA-3′.
Total cell lysates from tumors and adjacent noncancerous mucosa were prepared and the protein concentration determined with the method described by Bradford.20 Equal amounts of protein per lane were fractioned with sodium dodecyl sulfate polyacrylamide gel electrophoresis on a 15% gel. Separated proteins were transferred to a nitrocellulose membrane. Subsequently, the membrane was blocked for 2 hr at room temperature in 5% (w/v) nonfat dried milk in Tris-buffered saline (TBS). Next, the membrane was washed 3 times with TBS, and then incubated for 18 hr with rabbit polyclonal antibodies to SLPI (1:500 dilution in TBS; Abcam, Cambridge, MA). After further washing, the membrane was incubated for 1 hr with horseradish peroxidase-conjugated, affinity-purified antibodies to rabbit (1:1,000 dilution in TBS) (Santa Cruz Biotechnology Inc., Santa Cruz, CA). Immune complexes were then visualized by chemiluminescence with an ECL detection kit (Amersham Inc.).
Plasma SLPI determination
The assay employed the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for SLPI had been precoated onto a microplate. Standards, normal and patients' plasma (1:30 diluted) were pipetted into the wells. After washing, an enzyme-linked polyclonal antibody specific for SLPI was added to the wells. Following a wash, a substrate solution was added to the wells and color developed in proportion to the amount of SLPI bound in the initial step. The color development was stopped, and the intensity of the color was measured according to the manufacturer's (R&D Systems) instructions.
Formalin-fixed and paraffin-embedded tissues were examined by immunohistochemistry using monoclonal antibody to human SLPI (clone 31, HyCult biotechnology, Uden, The Netherlands; dilution 1:50) following the avidin-biotin complex method as described previously.21 Comparisons were made between the intensity of the staining of the carcinoma cells and benign superficial epithelium on the same slide. The negative group consisted of cancer cells with no detectable (−) or only trace staining of SLPI immunoreactivity (+1). The positive group consisted of cancer cells with moderate (+2) or high levels (+3) of SLPI immunoreactivity. The numerical scoring (Q) was confirmed by a second independent examination, blinded to the initial score. Results were scored by multiplying the percentage of positive cells (P) by the intensity (I).The formula was: Q = P × I. For example, a tissue section stained with 10% + 1, 60% + 2, 30% + 3, Q = 10 × 1 + 60 × 2 + 30 × 3 = 220. Normal bronchial gland was used as a positive control described by Ameshima et al.22 (data not shown).
Establishing stable SLPI overexpressing AZ521 cell lines
SLPI cDNA was amplified by RT-PCR and cloned into the pcDNA3 vector. Transfection of pcDNA3-SLPI gene was performed using Lipofectamine™ reagent (Invitrogen). After 24 hr of incubation, cells were transferred to G418 medium for selection. Overexpression of the SLPI gene was confirmed by detection of SLPI from culture medium via Western blot analysis. The pcDNA3 vector served as the control.
In vitro assay of invasive activity
The effect of SLPI overexpression on the invasive activity of the AZ521 cell line was assessed with a rapid in vitro assay (Transwell technique), as described previously.23 Briefly, cell density was adjusted to 1 × 105/mL, and 200 μL of this suspension was added to each well coating with Matrigel® (Becton-Dickinson, Franklin Lakes, NJ) in duplicate. The medium in the upper chamber was serum-free Roswell Park Memorial Institute medium 1640 and that in the lower chamber was supplemented with 10% fetal bovine serum. After incubation for 20 hr at 37°C, the number of viable cells that had traversed the filter to the lower chamber was determined.
When appropriate, the Mann–Whitney U test or Fisher's exact test was used for comparisons between 2 groups, while Kruskal Wallis test or Pearson's chi-square test was used if more than 2 groups. The relationship between the results of 2 different examinations was analyzed by Spearman's correlation test. The patients were followed until the time of manuscript preparation or patient death. The cancer-specific survival outcome was expressed by applying the Kaplan-Meier method for all patients, except those who died from surgical complications. The log-rank test was used to compare the prognostic significance of individual variables on survival. Cox's proportional hazards model was used in a multivariate analysis to identify the independent predictors of survival. A p value of < 0.05 was considered statistically significant.
Clinical characteristics of the patients
The characteristics of all patients included in this study are listed in Tables I and II. Among the 119 patients with immunohistochemical (IHC) study, the mean tumor size (maximal diameter) was 5.1 ± 3.3 cm (median: 5.0 cm; range 0.3–15.0 cm). The tumors were located in the proximal third of the stomach in 21 patients, the middle third in 29, the distal third in 64, and throughout the stomach in 5. The histological types consisted of intestinal type in 37 patients and diffuse type in 82 others. As defined by the depth of wall invasion, early gastric cancer (T1) was noted in 26 cases (21.9%; comprising mucosa in 14 and submucosa in 12), while advanced cancer included T2 (muscle proper and subserosa) in 18 cases, T3 (serosa) in 58 cases, and T4 (invasion to adjacent organs) in 17 patients. Lymph node metastasis was found in 82 patients (68.9%). During operation, peritoneal seeding was found in 23 patients (19.3%), and liver metastasis was evident in 2 patients (1.7%). The occurrences of the various pathologic stages were IA (n = 22), IB (n = 11), II (n = 9), IIIA (n = 26), IIIB (n = 15) and IV (n = 36).
|Parameters||No.||IHC SLPI1||p value2||5-yr S.R.3||logrank p4|
|<65||58||61.8 ± 9.3||0.415||45.0||0.6421|
|≥65||61||67.3 ± 9.1||50.0|
|Male||65||79.3 ± 9.5||0.013||45.2||0.2737|
|Female||54||46.9 ± 7.9||51.7|
|Upper third||21||72.9 ± 17.9||0.551||58.3||0.0052|
|Middle third||29||57.4 ± 11.7||46.3|
|Lower third||64||68.4 ± 8.9||49.3|
|Whole||5||24.0 ± 14.3||0.0|
|Localized||49||45.6 ± 7.5||0.026||74.4||<0.0001|
|Infiltrative||70||77.9 ± 9.4||27.0|
|Size (maximal diameter)|
|<5 cm||65||53.7 ± 7.4||0.107||70.9||<0.0001|
|≥5 cm||54||77.8 ± 10.9||12.8|
|Intestinal||37||63.8 ± 11.3||0.970||67.6||0.0014|
|Diffuse||82||65.0 ± 7.9||39.0|
|Depth of invasion (pT)|
|T1||26||26.9 ± 6.6||0.017||90.4||<0.0001|
|T2||18||58.3 ± 11.7||77.5|
|T3||58||77.8 ± .10.0||26.3|
|T4||17||83.9 ± 20.1||0.0|
|No (T1, T2)||44||38.8 ± 6.5||0.008||85.4||<0.0001|
|Yes (T3, T4)||75||79.8 ± 9.1||22.5|
|Lymph node status (pN)|
|N0||37||41.2 ± 8.2||0.092||92.9||<0.0001|
|N1||38||74.7 ± 12.1||32.1|
|N2||21||91.0 ± 18.7||32.3|
|N3||23||61.5 ± 14.3||7.3|
|Lymph node metastasis|
|No||37||41.2 ± 8.2||0.021||92.9||<0.0001|
|Yes||82||75.2 ± 8.4||24.7|
|Distant metastasis (pM)|
|No||87||62.9 ± 7.4||0.596||63.0||<0.0001|
|Yes||32||69.4 ± 13.2||0.0|
|Pathological stage (pStage)|
|Stage I||33||33.3 ± 7.0||0.033||91.0||<0.0001|
|Stage II||9||73.3 ± 22.4||87.5|
|Stage III||41||83.8 ± 12.3||36.5|
|Stage IV||36||69.3 ± 12.6||0.0|
|Stage I,II||42||41.9 ± 7.6||0.015||89.8||<0.0001|
|Stage III,IV||77||77.0 ± 8.8||20.3|
|No||117||63.3 ± 6.5||0.126||49.0||0.0370|
|Yes||2||140.0 ± 40.0||0.0|
|No||96||63.1 ± 7.2||0.352||56.6||<0.0001|
|Yes||23||71.1 ± 14.4||0.0|
|No||91||65.0 ± 7.4||0.940||55.7||<0.0001|
|Yes||28||63.4 ± 13.3||10.9|
|No||50||53.9 ± 9.0||0.158||76.8||<0.0001|
|Yes||69||72.4 ± 9.0||21.7|
|No||73||56.2 ± 6.7||0.337||61.0||0.0005|
|Yes||46||78.0 ± 12.7||22.7|
|SLPI (IHC score)|
|<90||84||25.5 ± 2.8||<0.001||54.9||0.0422|
|≥90||35||158.6 ± 8.8||31.4|
|Parameters||No.||SLPI fold1||P value2|
|<65||29||6.23 ± 2.24||0.348|
|≥65||31||10.57 ± 4.21|
|Male||34||11.56 ± 4.14||0.371|
|Female||26||4.44 ± 1.19|
|Upper third||12||9.10 ± 4.38||0.207|
|Middle third||17||10.91 ± 7.21|
|Lower third||29||7.22 ± 2.21|
|Whole||2||2.17 ± 0.47|
|Localized||16||5.88 ± 3.15||0.025|
|Infiltrative||44||9.50 ± 3.11|
|Size (maximal diameter)|
|<5 cm||29||3.72 ± 1.66||0.002|
|≥5 cm||31||12.93 ± 4.32|
|Intestinal||21||5.63 ± 2.40||0.448|
|Diffuse||39||10.01 ± 3.50|
|Depth of invasion (pT)|
|T1||13||1.16 ± 0.28||<0.001|
|T2||5||1.17 ± 0.38|
|T3||33||13.52 ± 4.20|
|T4||9||4.60 ± 2.21|
|No (T1, T2)||18||1.27 ± 0.23||<0.001|
|Yes (T3, T4)||42||11.56 ± 3.37|
|Lymph node status (pN)|
|N0||16||1.30 ± 0.30||0.001|
|N1||16||12.08 ± 7.62|
|N2||12||15.41 ± 4.84|
|N3||16||6.85 ± 3.01|
|Lymph node metastasis|
|No||16||1.30 ± 0.30||<0.001|
|Yes||44||11.08 ± 3.23|
|Distant metastasis (pM)|
|No||44||6.50 ± 1.64||0.118|
|Yes||16||13.93 ± 7.95|
|Pathological stage (pStage)|
|Stage I||15||1.19 ± 0.26||<0.001|
|Stage II||2||1.25 ± 0.65|
|Stage III||23||10.53 ± 2.86|
|Stage IV||20||12.30 ± 6.38|
|Stage I,II||17||1.20 ± 0.23||<0.001|
|Stage III,IV||43||11.35 ± 3.30|
|No||58||8.29 ± 2.50||0.149|
|Yes||2||13.87 ± 9.80|
|No||46||6.18 ± 1.56||0.112|
|Yes||14||16.03 ± 9.01|
|No||49||8.11 ± 2.82||0.111|
|Yes||11||10.11 ± 4.40|
|No||20||1.47 ± 031||<0.001|
|Yes||40||11.98 ± 3.52|
|No||39||8.03 ± 3.36||0.046|
|Yes||21||9.30 ± 3.15|
|<median (2.47-fold)||30||1.21 ± 0.14||<0.001|
|≥median||30||15.74 ± 4.51|
Expression of SLPI mRNA and protein in gastric cancer tissues
In 60 patients, real-time Q-RT-PCR data for the expression of SLPI was measured by the ratio of SLPI signal in tumor tissue to that in noncancerous adjacent tissues. The median SLPI expression in tumor tissue was 2.47-fold (range: 0.01–124.5) greater than in noncancerous tissues. SLPI expression was significantly increased in the tumor tissues, compared to normal tissue (p < 0.001, by 1-sample t-test). Real-time Q-RT-PCR yielded very reproducible results that supported the microarray data.
We further confirmed the SLPI expression levels in several patients with Northern blot analysis. Figure 1 illustrates SLPI expression in 10 representative patients. A 0.7-kb SLPI transcript was detected in all cancer tissues examined. The cancer tissues from both intestinal (G25, G27, G44, G46) and diffuse (G4, G21, G28, G36, G41, G49) types all highly expressed SLPI (except G36), compared to the matched noncancerous adjacent mucosa (Fig. 1). Northern blot analysis determined the mean expression of 0.7-kb SLPI mRNA in cancer tissues was 2.1-fold (range: 0.2–4.06) greater than in their noncancerous counterparts, confirming the trend observed in the real-time Q-RT-PCR.
Furthermore, the expression of SLPI protein was determined by Western blot analysis. Figure 2 shows SLPI expression in four representative patients. A 12-kDa SLPI protein was detected in all cancer tissues. An equal amount (100 μg) of protein was loaded for each specimen, and actin was used as the control. All cancer tissues from diffuse (G21, G18) and intestinal (G15, G48) types displayed upregulated SLPI expression compared with those in matched noncancerous adjacent mucosa (Fig. 2).
Overexpression of SLPI protein in gastric cancerous tissues demonstrated by immunostaining
To investigate the expression levels and location of SLPI in tissues, IHC staining was performed on gastric cancer tissues and matched noncancerous muscosa in 119 patients. Figure 3 shows four pairs of representative cases (a/b, c/d, e/f and g/h; a/b, c/d and e/f represented intestinal types, while g/h represented a diffuse type tumor). The noncancerous muscosa counterparts (a, c, e, g) and cancer tissues (b, d, f, h) were used. The dark brown immunostaining was most prevalent in the cancer cells, and low levels were observed in the stromal cells or fibroblasts in gastric cancer tissues. No or weak staining was observed for SLPI in the normal gastric epithelial cells (Figs. 3a, 3c, 3e and 3g). The staining was more intensive in advanced stages (stage III is depicted in Fig. 3f, and stage IV is depicted in Fig. 3h), compared to stages IB (Fig. 3b) and II (Fig. 3d). Among the 119 patients studied by IHC, the median score of IHC in tumor tissue was 40 (range: 0–270). These staining scores of tumor tissues were significantly greater than those in matching adjacent mucosa (p < 0.001, by Wilcoxon-signed rank test). Furthermore, the immmunoreactivity in the cancerous tissues was stronger than that in the nontumorous counterpart in 78 (65.5%) patients, equal in 24 patients, and weaker in 17 patients. SLPI expression levels in surgical specimens determined by IHC were significantly correlated with that ascertained by real-time Q-RT-PCR (n = 45; Spearman's rank correlation coefficient = 0.366, p = 0.013).
Plasma SLPI expression in patients vs. healthy controls
The plasma levels (mean ± SD) of SLPI in 19 healthy controls and 44 patients with gastric cancer were 28,954.0 ± 2,545.7 and 25,107.4 ± 1,064.8 pg/mL, respectively, which was not a significant difference (p = 0.621). However, extremely high levels of plasma SLPI were found only in the patient group. When a plasma SLPI level equal to the 95 percentile value (33,778.0 pg/mL) of healthy controls was set as the upper cutoff value, abnormally high levels of plasma SLPI were observed in 8 (8.6%) of the patients, compared to 1 (5.3%) of the healthy controls (p = 0.256, Pearson's chi-square test). When the 95th percentile of plasma SLPI values in healthy controls was used as the cutoff level, the sensitivity, specificity, positive predictive value and negative predictive value for plasma SLPI as a diagnostic test for gastric cancer were 8.6% (8/44), 94.7% (18/19), 88.9% (8/9) and 34.5% (19/55), respectively.
SLPI expression and clinicopathological correlation
SLPI expression in tumor tissue was not significantly associated with age, tumor location or histological type (Tables I and II). There was a higher expression of SLPI in females than in males (p = 0.013 for IHC, but p = 0.371 for Q-RT-PCR). Higher levels of SLPI were noted in the T3 and T4 groups, where the serosal surface of the gastric wall was invaded by the cancer, than in T1 and T2 groups, where no invasion was evident (p < 0.001 for Q-RT-PCR and p = 0.008 for IHC; Figs. 4a and 4e; Tables I and II). Expression of SLPI was significantly increased (p < 0.001) with metastasis to the lymph nodes (p < 0.001 for Q-RT-PR and p = 0.021 for IHC; Figs. 4b and 4f; Tables I and II). A higher expression was noted in patients with lymphatic invasion (p < 0.001) or perineural invasion (p = 0.046) on Q-RT-PCR analysis. Increased SLPI expression was not associated with vascular invasion or with distant metastasis, including peritoneal seeding or liver metastasis, in either Q-RT-PCR or IHC studies. Expression of SLPI was significantly higher (p < 0.001 for Q-RT-PCR and p = 0.015 for IHC) in patients with more advanced pathologic stages (stages III and IV) than for those in an earlier pathologic stage (stages I and II) (Figs. 4c and 4g; Tables I and II).
Plasma SLPI was not associated with most of clinicopathological parameters (Table III). However, it was significantly increased in the elderly (p = 0.018) and abnormally higher plasma levels were detected in 7 of 18 patients with stage III disease.
|Parameters||No.||Normal/high SLPI level1||p value2|
|Size (maximal diameter)|
|Depth of invasion (pT)|
|No (T1, T2)||17||15/2||0.455|
|Yes (T3, T4)||27||21/6|
|Lymph node status (pN)|
|Lymph node metastasis|
|Distant metastasis (pM)|
|Pathological stage (pStage)|
The mean duration of the follow-up period for 47 survivors was 47.5 months (range: 16–86 months). Three patients died of postoperative complications, and 6 died of other diseases. A total of 53 patients died as a result of the progression of their gastric cancer. The overall cumulative 5-year survival rate of the 119 patients with gastric resection was 48.07%. Figure 4d and 4h illustrate the cumulative survival curves of patients in the lower-expression and higher-expression SLPI groups. The lower-expression group was defined as those at or below the cutoff value (2.47-fold or the median for Q-RT-PCR, and 90 for IHC scores) of SLPI expression, whereas the higher-expression group consisted of patients expressing levels above the cutoff value of SLPI measured in the tumor. The 5-year survival rate of the lower-expression group was significantly better than that of the higher-expression groups (63.9% vs. 22.2%; log rank p = 0.0084 for Q-RT-PCR; 54.9% vs. 31.4%, log rank p = 0.0422 for IHC) (Figs. 4d and 4h). No significant survival difference was found in the patients examined by plasma SLPI study.
Univariate analysis showed significant prognostic factors, including the status of lymph node metastasis, serosal invasion, distant metastasis, lymphatic invasion, peritoneal seeding, vascular invasion, perineural invasion, liver metastasis and pathological stage, in addition to SLPI expression (Table I). Other significant parameters were histological type, tumor size, gross type and tumor location. Multivariate analysis revealed distant metastasis, and lymph node metastasis were independent prognostic factors, and that SLPI expression was not a significant one for survival.
Influence of ectopic overexpression of SLPI on AZ521 cell proliferation and migration
AZ521 cells expressing low levels of endogenous SLPI were transfected with pcDNA3-SLPI. After 2 weeks of transfection, the stable expression of secreted SLPI protein was determined from culture medium. Figure 5a shows that the expression of SLPI in 2 AZ521-SLPI sublines (nos. 5 and 7) was 6.5-fold and 3.0-fold higher, respectively, than in control cells that were transfected with control vector (V1 and V2).
To determine the effect of overexpression of SLPI in AZ521 cells, cell proliferation, migration and invasion were assayed. SLPI overexpressing cells exhibited significantly (p < 0.01) higher migration rates (6.9-fold or 4.4-fold) and invasive ability (4.1-fold or 3.0-fold) than did the controls (Figs. 5b and 5c). Additionally, cell proliferation was determined by cell counting and indicated by percentage of control for up to 6 days. The SLPI-overexpressing cells exhibited significantly (p < 0.05) higher proliferation rates than those that were transfected with control vector on days 4–6 (Fig. 5d).
The results of this study verified that SLPI is frequently overexpressed in gastric cancer cells. This was shown by using Northern blot analysis, real-time Q-RT-PCR and IHC examinations. Among the patients examined, higher SLPI expression was significantly associated with tumor progression and more advanced stages of gastric cancer. In addition, patients with lower SLPI expression had a better prognosis. SLPI inhibits the activities of neutrophil elastase, trypsin, cathepsin G and chymotrypsin.24 Theoretically, these biological activities may allow SLPI to function as a potential antiinvasive agent for mouse mammary tumor cells.25 However, SLPI overexpression has been reported in several other malignancies (see later section). Higher expression of SLPI has been found in human ovarian carcinoma specimens.26 In human epidermal tumors, SLPI expression is correlated with the degree of differentiation.27 The use of cDNA microarrays has identified genes, including SLPI, associated with the invasive or tumorigenesis phenotypes in breast carcinoma cell lines.28 Overexpression of mouse or human SLPI in 3LL-S cells is sufficient to enhance their malignant behavior.15 In contrast, decreased expression of SLPI has been described in all mouse tumors and over 80% of human breast carcinomas examined.29 However, its role during gastric tumorigenesis is not yet clarified.
Numerous microarray studies have been done to identify genes such as Oct2, cadherin,30 thrombospondin 2,31 phospholipase A2 group IIA,32 dolichyl-diphosphooligosaccharide-protein glycosyltransferase,33 maspin34 and matrix metalloproteinase.35 Researchers have reported that claudin 18, trefoil factor 2, cadherin 11, sulfatase 1 and collagen,36 and endothelin A receptor37 are differentially expressed in gastric cancer. Better understanding of these genes has helped clarify the molecular or cellular mechanisms of gastric carcinogenesis associated with therapeutic targets, prognostic score, metastasis, tumor progression and diagnostic markers for gastric cancer. Haraguchi et al. also reported that cDNA microarray might be a useful tool to discover a prognostic indicator.38 However, all the genes are insufficiently sensitive for early detection of gastric cancer and subsequent therapy.
SLPI exerts pleiotropic activities in several biological systems. We and others39, 40 have reported that SLPI promotes in vitro cell proliferation. Furthermore, a positive correlation has been demonstrated between cellular SLPI production and proliferation, with SLPI selectively increasing cyclin D1 gene expression.39 A significantly lower SLPI detection rate is observed in cervical adenocarcinomas compared to normal endocervical glands, and the expression is tissue-specific.41 Thus, decreased expression of SLPI might play an important role in cervical adenocarcinoma development.41
We have demonstrated the positive correlation of increased SLPI expression with local tumor progression and lymph node invasion determined by Q-RT-PCR or IHC. These observations are entirely consistent with the previous observation that the expression of SLPI in tumors is often associated with poor prognosis.14 Additionally, elevated levels of SLPI expression increase both the tumorigenicity and lung-colonizing potential of low-malignant Lewis lung carcinoma cells.14 Additionally, SLPI can be downregulated by H pylori infection in gastric epithelial cell lines.42 Similar to our results, Wex et al. also reported that gastric tumor tissue had 2-fold higher SLPI levels than surrounding tumor-free gastric mucosa.43 However, no further physiological significance was discussed. Additionally, it is not surprising that plasma SLPI was not associated with most of the clinicopathologic parameters, since SLPI was also secreted from other tissues or organs, especially normal bronchial epithelium. Hence, serum SLPI is not a useful systemic marker for gastric cancer.
Invasion and metastasis are the inherent characteristics of malignant diseases involved in both intercellular and cell–matrix interactions. Genes associated with metastasis include SLPI in prostate cancer cells.44 However, SLPI can decrease the liver-metastasizing potential of carcinoma cells, and this protective effect correlates with the decreased production of hepatic TNF-alpha and E-selectin.45 These observations differ from our experience and that of others.26, 28, 46 Interestingly, overexpression of SLPI can promote in vivo growth and spontaneous metastasis to the lung, whereas it suppresses invasive activity in vitro,25 perhaps indicating that SLPI=overexpressing cells are capable of inducing a sinusoidal vasculature and subsequently produce endothelial-coated tumor emboli, which are morphologic indices of the invasion-independent pathway.25 Additionally, SLPI can suppress the production of matrix metalloproteinases 1 and 9, which are important for cancer invasion, independent of its antiprotease activity in monocytes47 Therefore, the role of SLPI in tumor metastasis is still controversial. It may be due to cell type or assay system used. In the present system, overexpression of SLPI by transfection or the addition of exogenous SLPI promoted cancer cell migration and invasion in vitro.
In summary, this study defined the misregulation of SLPI in gastric carcinoma and its strong association with the more advanced stages of this disease. The overexpression of SLPI in a variety of malignancies excludes it as a suitable diagnostic biomarker for any specific tumor, although it does display promise as a prognostic marker of tumor progression and advanced cancers. Finally, this study demonstrates that further investigation of SLPI is warranted, because of its potential as a prognostic and therapeutic agent.
The authors thank Dr. Ting-Chang Chang, Director of the Biostatistics Consulting Unit, Chang Gung Memorial Hospital, for contributing to the statistical analysis.
- 3Resectable gastric cancer: operative mortality and survival analysis. Chang Gung Med J 2002; 25: 216–27., , , , , , , , , .