Expression of the serine protease kallikrein 7 and its inhibitor antileukoprotease is decreased in prostate cancer.
Abstract: * Context.--Kallikreins are a subgroup of serine proteases with diverse physiologic functions. It has been confirmed that kallikrein 7 (KLK7) is differentially expressed in ovarian and breast cancer. Antileukoprotease (ALP) has been shown to be a specific inhibitor of human kallikrein 7 (hK7). Antileukoprotease overexpression is commonly associated with aggressive, high-risk, or metastatic cancer originating from various organs.

Objective.--To investigate the expression and potential role of hK7 and its inhibitor ALP in prostate cancer.

Design.--The mRNA expression of KLK7 and ALP transcript in benign prostate epithelial cells and prostate cancers was evaluated by semiquantitative reverse transcription-polymerase chain reaction. We examined hK7 and ALP protein expression by immunohistochemistry in 20 normal prostate tissues, 50 benign prostatic hyperplasia tissues, and 103 prostate cancers. Western blot examination showed protein expression of hK7 and ALP in benign prostate epithelial cells and prostate cancer cell lines.

Results.--Semiquantitative polymerase chain reaction examination revealed that the mRNA level of KLK7 and ALP was significantly decreased in prostate cancers compared with that in benign prostate epithelial cells (P < .001). Immunohistochemical expression of hK7 was observed in prostate epithelial cells, whereas little or no staining was observed in prostate cancer. Western blot analysis revealed that hK7 and ALP were decreased in malignant prostate epithelium.

Conclusions.--Like hK7, ALP is down-regulated in prostate cancers, which begs the question of whether it remains an effective inhibitor of hK7 or whether it is discordant in time or space and is ineffective as an inhibitor of hK7. The function of KLK7 and ALP in prostate cancer should be further studied.
Article Type: Report
Subject: Prostate cancer (Development and progression)
Kallikrein (Properties)
Serine (Properties)
Immunohistochemistry (Methods)
Gene expression (Physiological aspects)
Authors: Xuan, Qiang
Yang, Xiaoli
Mo, Linjian
Huang, Fengyu
Pang, Youhong
Qin, Min
Chen, Zhiqiang
He, Min
Wang, Qi
Mo, Zeng-Nan
Pub Date: 11/01/2008
Publication: Name: Archives of Pathology & Laboratory Medicine Publisher: College of American Pathologists Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2008 College of American Pathologists ISSN: 1543-2165
Issue: Date: Nov, 2008 Source Volume: 132 Source Issue: 11
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 230246869
Full Text: The extracellular matrix proteolysis accompanying tumor invasion and metastasis is a highly complicated process and probably involves a cascade of events requiring a variety of proteases. (1) In this process, protease plays a central role in paving the way for spreading tumor cells. The human kallikrein gene family is a subfamily of serine proteases and is located at chromosome locus 19q13.3-q13.4. In recent years, 15 of the tissue kallikrein family genes have been identified and cloned. Human kallikrein 7 (hK7) was first found in the human stratum corneum and with a possible involvement in desquamation. (2) There is now increasing evidence that many kallikrein family genes are related to human malignancies. (3, 4) We have found kallikrein 7 (KLK7) was upregulated in prostate epithelial cells cocultured with prostate fibroblasts compared with epithelial cells cultured separately. It has been suggested that hK7 may degrade adhesive interactions between individual corneocytes in the stratum corneum that are the primary substrate for cellular desquamation or skin shedding.5 The fact that inhibition of hK7 prevents normal desquamation of skin cells suggested that hK7 may represent a potential therapeutic target to inhibit the spread or metastasis of carcinoma cells. Whether hK7 has a similar function in prostate is worth studying.

Antileukoprotease (ALP), also known as secretory leukocyte proteinase inhibitor, has been identified as a potent inhibitor of leukocyte chymotrypsin, trypsin, elastase, and cathepsin G (6) and plays a significant role in protection against neutrophil proteases during inflammatory responses. (7, 8) The hK7-dependent desquamation of skin cells can be inhibited by ALP. (9) This serine protease inhibitor is produced and released into mucus by secretory cells in the prostate, parotid, bronchus, cervix, and testis. (6) Antileukoprotease has a local protective function against proteolytic degradation of the male reproductive tract tissues (10) and cervical gland. Several studies have reported increased ALP expression in ovarian cancer tissues and in the serum of patients with non-small cell lung cancer. (11-13) Shigemasa et al (14) also have identified that ALP, a peptide inhibitor of hK7, is highly overexpressed in ovarian tumor cells, similar to hK7. These findings suggest that hK7 and ALP expression might play a role in human carcinogenesis and cancer metastases. However, at the current time little is known about the expression of hK7 and ALP in the development and progression of human prostate cancer.

The aim of this study was to investigate the expression of KLK7 and ALP in benign prostate tissue and prostate cancer development.

MATERIALS AND METHODS Cell Culture

Benign prostatic hyperplasia (BPH) tissue was obtained from men undergoing suprapubic prostatectomy. Normal prostate tissue was obtained from bladder cancer patients who underwent radical cystoprostatectomy. The prostate cancer tissue came from prostate cancer patients who underwent radical prostatectomy. The use of patient materials was approved by the ethical committee of our hospital, and informed consent was signed by each patient. The histologic status of the tissue was checked by an independent pathologist. Primary cultures of separated epithelial cells were established as described in Tsugaya et al (15) and Habib. (16) Briefly, the prostate tissue was diced into approximately 1-[mm.sup.3] pieces using forceps and scissors. The diced tissue was then incubated for 16 hours at 37[degrees]C in a collagenase solution (33 IU/ mL; Worthington, Lakewood, NJ) with shaking. After digestion with collagenase, the epithelial cells were separated by sedimentation and resuspended in WAJC 404 (Invitrogen Corporation, Grand Island, NY) supplemented with 6.7 g/L hydroxyethyl piperazine ethanesulfonic acid (Sigma-Aldrich, St Louis, Mo), 1.2 g/L NaHC[O.sub.3] (Sigma-Aldrich), 10 U/L epidermal growth factor (Sigma-Aldrich), 392 [micro]g/L dexamethasone (Sigma-Aldrich), 0.5 mg/L insulin (Sigma-Aldrich) (called epithelial medium), 0.5% fetal calf serum (FCS) (Invitrogen), 100 000 IU/mL penicillin, and 100000 [micro]g/mL streptomycin (Sigma-Aldrich). The separated cells were then incubated at 37[degrees]C in a 95% air-5% C[O.sub.2] humidified atmosphere. The identity and purity of the separated cultures were confirmed by immunohistochemistry and phase contrast microscopy as Habib previously described.

The prostate cancer cell lines PC-3, DU-145,22RV1, and LNCaP were purchased from American Type Culture Collection (Manassas, Va) and cultured according to the recommended protocol.

mRNA Extraction and cDNA Synthesis

Total RNA was extracted from the prostate tissue using TRIzol reagent (Invitrogen) following the manufacturer's instructions. Two micrograms of total RNA was reverse transcribed into firststrand cDNA using the RevertAid First Strand cDNA Synthesis Kit (Fermentas, Glen Burnie, Md) according to the manufacturer's instructions. Reverse transcription was performed with total RNA without reverse transcriptase (a mock reverse transcription sample) to detect possible contamination by genomic DNA in RNA samples.

Semiquantitative Polymerase Chain Reaction

The ALP and KLK7 target sequences were amplified in parallel with the G3PD gene. G3PD was used as an internal control. The following specific oligonucleotide primers were used: for ALP amplification by polymerase chain reaction (PCR), forward 5'-GAGATGGATGGCCAGTGCAAGC-3' and the reverse 5'-GC TGTGTGCCAAGCCTTTCCC-3'; for KLK7 amplification by PCR, forward 5'-ATGGCAAGATCCCTTCTCC-3' and the reverse 5'-TTAGCGATGCTTTTTCATGGT-3'; and for G3PD internal control amplification by PCR, forward 5' -ACCACAGTCCATGCCAT CAC-3' and the reverse 5'-TCCACCACCCTGTTGCTGTA-3'. Polymerase chain reaction was carried out in a reaction mixture containing 0.5 [micro]g of the diluted reverse transcript product, 2 [micro]L of 2.5mM dNTPs, 2.5 [micro]L of 10 X EXTaq buffer, 0.2 [micro]L of EXTaq (Takara Biotechnology, Dalian, China), 1 [micro]L each forward and reverse intron-spanning primers (10[micro]M), respectively. The final volume was 25 [micro]L. Each cycle of PCR included 60 seconds of denaturation at 95[degrees]C, 60 seconds of primer annealing at 53[degrees]C, and 90 seconds of extension at 72[degrees]C. Polymerase chain reaction products were separated on 1.5% agarose containing ethidium bromide and visualized by UV transillumination (Gel Doc 2000, BioRad Laboratories, Hercules, Calif). All the bands were quantitated by Quantity One v4.1.1 (Bio-Rad). To correct for minor differences in using of cDNA on reverse transcription-PCR, a ratio of the relative density of each specific band to the relative density of G3PD band was calculated. The PCR products were sequenced by using ABI Prism Genetic Analyzer 3100 (Applied Biosystem, Foster, Calif). All the semiquantitative reverse transcription-PCR works were repeated.

Immunohistochemistry

Tissue microarray sections were purchased from Chaoying Biotechnology (Shanxi, China). Immunohistochemical staining was performed manually using the avidin-biotin peroxidase complex technique (Maixin Bio, Fujian, China) according to a previously described method with some modifications. (17) A goat antihK7 polyclonal antibody (R&D Systems, Minneapolis, Minn) was used as a 1:40 dilution (5 [micro]g/mL) and the tissue microarray sections were incubated with primary antibody at 4[degrees]C overnight. Endogenous peroxidase and nonspecific background staining were blocked by incubating slides with methanol with 0.3% hydrogen peroxide for 30 minutes. After washing with phosphatebuffered saline for 10 minutes, sections were incubated with biotinylated anti-goat immunoglobulin G (IgG) for 10 minutes. After washing with phosphate-buffered saline for 10 minutes, slides were incubated with avidin-biotin complex reagent for 30 minutes. The final products were visualized by using a 3,3-diaminobenzidine substrate system (Maixin Bio) and sections were counterstained with Mayer hematoxylin for 5 minutes before mounting. Positive controls included ovarian cancer tissue for hK7 and ALP immunostaining according to the published literature.18 Normal goat IgG (5 [micro]g/mL; R&D Systems) served as a negative control to replace the primary antibody. Digital images of each tissue microarray core were manually scored and displayed according to staining intensity and morphology. The staining was graded as - , +, ++,and + + + if less than 25%, 25% to 49%, 50% to 75%, and more than 75% of cells were stained, respectively; similarly, the staining intensity was graded as - , + , ++ ,and+++ if there was no yellow, light yellow, brownish yellow, and brown-yellow, respectively. The grades were given values of 0, 1, 2, and 3, respectively, for statistical analysis.19 The final staining result of a specific specimen was judged by adding the staining scope and degree scores, that is, negative as 0, low positive as 1, moderate positive as 2, and strongly positive as 3.

All cases were scored in an independent and blinded manner by 2 of the authors and 1 independent pathologist without knowledge of patient information. To promote the specificity, we consider staining grade 2 or greater as positive. The tumor grading was provided by a pathologist at the Shanxi Chaoying Biotechnology Company, the company from which the tissue microarray was purchased. The data of Gleason grade was confirmed by the pathologist at the First Affiliated Hospital, Guangxi Medical University, according to the modified Gleason grading system. (20) Statistical analysis of data was performed using the SPSS package (version 11.5 for Windows, SPSS, Chicago, Ill). The significance of associations was determined by means of the Kruskal-Wallis H tests and Mann-Whitney U test.

Western Blot Analysis of Prostate Epithelial Cells and Prostate Cancer Cell Lines

Prostate epithelial cells and cell lines (DU-145, LNCaP, PC3, 22RV1) were lysed by using M-PER Mammalian Protein Extraction Reagent (Pierce Biotechnology Inc, Rockford, Ill). The lysis was centrifuged at 4[degrees]C for 30 minutes at 14 000g to remove debris. Cytoplasmic extracts from different cells were mixed with loading buffer containing 10% [beta]-mercaptoethanol, respectively. Samples mixed with loading buffer were denatured by boiling for 5 minutes and separated on 12% denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gels, followed by transfer (TransBlot Semi-Dry, Bio-Rad) to nitrocellulose Hybond membrane (Amersham Biosciences, Piscataway, NJ). Membranes were blocked with 5% nonfat milk in Tris-buffered saline (TBS)-Tween 20 (50mM Tris-HCl, pH 7.5, 150mM NaCl) for 1 hour at room temperature and then incubated with anti-hK7 primary antibodies (1:150 dilution; R&D Systems) in diluent (5% nonfat milk in TBS-Tween 20) for 2 hours. [beta]-Actin (Lab Vision Corporation, Fremont, Calif) was used as an internal control. Primary antibody complexes were detected using horseradish peroxidase-conjugated secondary rabbit anti-goat antibodies (1:3000 dilution; KPL, Gaithersburg, Md). The signals were visualized on x-ray film by enhanced chemiluminescence (Super Signal West Pico System, Pierce Biotechnology). For detecting ALP, the same quantity of all the samples were separated on 15% denaturing Tricine-SDS-polyacrylamide gels, followed by transfer to nitrocellulose membrane.

[FIGURE 1 OMITTED]

We did the reprobing with antibody of [beta]-actin as follows. After the film exposure, we incubated the membrane for 30 minutes at 50[degrees]C in the stripping buffer (62.5mM Tris-HCl, pH 6.8, 2% SDS, 100mM 2-mercaptoethanol) followed b washing in TBS-Tween 20. The membrane was incubated for 1 minute in the Western Lightning Chemiluminescence Reagent and exposed to film for 1 hour to make sure that the original signal was removed. The membrane was washed again for four 5-minute sessions in TBSTween 20 and then started at the blocking step.

RESULTS

Semiquantitative PCR

Prostate cancer samples and prostate cancer cell lines, when compared with benign prostate epithelial cells, were shown to have decreased levels of KLK7 transcript and ALP transcript (Figure 1, A). G3PD was used as an internal control. By using 1-way analysis of variance, we found a significant decrease in KLK7 expression levels in prostate cancer tissue and cell lines, compared with benign prostate epithelial cells (P < .001). Also, ALP was down-regulated in prostate cancer and cell lines compared with benign prostate epithelial cells (P < .001) (Figure 1, B; Table 1). There was a significant positive correlation between ALP and KLK7 decreased expression status in pros tate cancer (P < .001). However, we did not find a difference of KLK7 and ALP expression between normal prostate and BPH epithelial cells.

Immunohistochemistry

Immunohistochemical expression of hK7 and ALP was examined in 20 normal prostate tissues, 50 BPH tissues, and 103 prostate cancer tissues. Figure 2 shows an example of the immunohistochemical colocalization of hK7 with ALP in the cytoplasm of normal prostate epithelium using adjacent tissue sections. We observed positive staining of hK7 and ALP in normal prostate (Figure 2, A and C) and BPH epithelium; however, little or no staining of hK7 and ALP was observed in prostate cancer (Figure 2, B and D). The positive ratio of hK7 and ALP expression in various tissues are described later and further summarized in Table 2. hK7 protein expression was detected in 13 (65%) of 20 normal prostate tissues with ALP protein expression evident in 10 (50%) of 20 samples of normal prostate. hK7 was detected in 38 (76%) of 50 BPH tissues with ALP protein expression evident in 26 (52%) of 50 samples of BPH tissues. In contrast, hK7 was detected in 18 (17.5%) of 103 prostate cancer tissues with ALP protein expression evident in 1 (1.0%) of 103 samples of prostate cancers tissues. Significant difference (Table 2; P < .001) was found between 3 groups by using Kruskal-Wallis test according to the staining score. hK7 and ALP expression were not significantly different, however, between the normal prostate and benign hyperplastic prostate. Using Spearman correlation, a significant negative association was found between Gleason grade and KLK7 expression ([r.sub.S] =--0.342, P < .001). Also, a significant negative association was found between Gleason grade and ALP expression (rS =-- 0.240, P = .02). A positive association was found between KLK7 and ALP expression ([r.sub.S] = 0.703, P < .001). These data indicate that hK7 and ALP expression is decreased in prostate cancer compared with benign prostate tissue.

[FIGURE 2 OMITTED]

Western Blot Analysis of hK7

To quantify hK7 and ALP protein in prostate epithelial cells and prostate cancer cell lines we performed Western blot analysis. As shown in Figure 3, hK7 was detected as a 28-kd band and ALP was detected as a 12-kd band in benign prostate epithelial cells. We found that hK7 and ALP were down-regulated in prostate cancer cell lines (DU145, LNCaP, PC3, 22RV1) compared with benign prostate epithelial cells (Figure 3). Antileukoprotease was also detected in prostate epithelial cells, whereas weak expression of ALP was detected in PC3 cell lines. In this study, expression of hK7 and ALP in the prostate epithelial cell is consistent with mRNA level of KLK7 and ALP. Western blot analysis of [beta]-actin expression showed the relatively equal loading of protein.

[FIGURE 3 OMITTED]

COMMENT

There is now growing evidence that the human kallikrein genes are involved in human malignancies. Prostatespecific antigen (hK3) has been used successfully as a circulating tumor marker for the early detection of prostate cancer. Recently, hK2, hK4, and hK11 have been shown to be potential serum biomarkers in prostate cancer. (21-25) It has been confirmed that kallikrein 4 (hK4) and prostate-specific antigen are associated with the loss of E-cadherin and an epithelial-mesenchymal transition-like effect in prostate cancer cells. (26) Under the newly established kallikrein gene nomenclature, the stratum corneum chymotrypsin enzyme gene is now known as KLK7 and the stratum corneum chymotrypsin enzyme protein is designated as hK7. Using in vitro assays, hK7 was shown to cleave E-cadherin and the soluble E-cadherin fragment produced significantly enhanced Panc-1 cell invasion through extracellular matrix proteins with a corresponding reduction in Panc-1 cell aggregation. These results suggest that aberrant expression of KLK7 plays an important role in pancreatic cancer and provides novel insight into the effects of elevated hK7 proteinase activity in this and perhaps other adenocarcinomas. (27)

Specific inhibitors for most of the proteolytic enzymes have been identified and it has been contemplated that these inhibitors inhibit extracellular degradation, which in turn prevents tumor cell invasion. Probably the proteolysis activity associated with tumors is a highly regulated cascade and the interplay between proteases and their inhibitors may play a specific role in tumor development and progression. The role of KLK7 in prostate cancer is still unknown.

In the present study, we demonstrated that ALP, a specific inhibitor of hK7, is frequently decreased in prostate cancer. Recently, Franzke et al (9) reported that ALP is the major inhibitor of stratum corneum chymotrypsin enzyme in the epidermis and that it seems to be involved in the regulation of desquamation under physiologic and pathologic conditions. hK7 inhibition by secretory leukocyte proteinase inhibitor was of the hyperbolic, mixed type with a strong competitive component. At saturating inhibitor concentration, a residual 7.1% of activity is still present in the presence of synthetic substrates. The inhibitory activity of secretory leukocyte proteinase inhibitor on hK7 was also associated with the concentration of secretory leukocyte proteinase inhibitor. However, hK7 may be inhibited by several modulating factors. Recently, the study by Debela et al (28) indicated that hK7 activity is inhibited by [Zn2.sup.+] and [Cu2.sup.+] at low micromolar concentrations. The concentration of [Zn2.sup.+] was found at high level in normal prostate while at low level in prostate cancer. Although the physiologic roles of kallikreins are generally still unknown, hK7 has been clearly implicated in pathologic situations such as cancer and psoriasis. We assume that hK7 may play a role in tumorigenesis of prostate. In normal prostate tissue, the function of hK7 may be inhibited by some modulating factors (including ALP, [Zn2.sup.+]). We think that the precise mechanism of hK7/ALP interaction and the modulating factors of hK7 are complicated in prostate. The fact that inhibition of hK7 causes the concomitant complete inhibition of cell shedding from plantar stratum corneum in vitro led to the hypothesis that hK7 may be involved in the process of physiologic desquamation. (29)

We confirmed positive correlation of KLK7 and ALP mRNA expression in benign prostate epithelial cells from different cases. It was also confirmed by using immunohistochemistry that coexpression of hK7 existed with ALP in the cytoplasm. This observation in one sense is paradoxical, because one would expect low ALP levels if hK7 activity plays an important role in normal prostate epithelium. These data are entirely consistent with the data observed for hK7/ALP expression in differentiated keratinocytes. In light of the fact that desquamation of skin cells is hK7 dependent and can be inhibited by ALP, it is suggested that some dyssynchrony in time or space allows hK7 activation in the presence of ALP.

KLK7 and ALP expression were found to be down-regulated, at the mRNA and protein level, in prostate cancer tissues compared with normal tissue. It should be noted, however, that the tissue expression levels might not reflect the serum levels of the protein, which at present cannot be measured due to lack of methodology. Although levels of prostate-specific antigen and hK2 are elevated in the serum of prostate cancer patients, their tissue concentration is lower in the prostate cancer tissues. (30) The elevation of serum concentration may be attributed to angiogenesis, destruction of the tissue architecture, and leakage of these proteins to the general circulation. (31) The inhibition of hK7 of skin cells points to the potential of hK7 as a target for inhibition or down-regulation in the spread or metastasis of prostate cancer. Because ALP is a specific inhibitor of hK7, it may also be useful in the abatement of tumor growth and progression in low-ALP-expressing cancers.

We have demonstrated that KLK7 and ALP were downregulated in malignant prostate epithelial cells. They may have a potential for being present in the circulation of tumor-bearing patients. The role of hK7 and ALP is still unknown. These proposals and hypotheses need experimental verification.

This study was performed at the Institute of Urology, the First Affiliated Hospital of Guangxi Medical University. This work was supported by a grant from the National Natural Science Foundation of China (30260110/C03030305) and the First Affiliated Hospital of Guangxi Medical University.

Accepted for publication April 15, 2008.

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Qiang Xuan, MD; Xiaoli Yang, MD; Linjian Mo, MD; Fengyu Huang, MD; Youhong Pang, MD; Min Qin, MD; Zhiqiang Chen, MD; Min He, MD; Qi Wang, MD; Zeng-Nan Mo, MD

From the Institute of Urology, the First Affiliated Hospital (DrsXuan, Yang, L. Mo, Huang, Pang, Qin, Chen, and Z.-N. Mo), the Laboratory Center For Medical Science (Dr He), and the Affiliated Tumor Hospital (Dr Wang), Guangxi Medical University, Nanning, China

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Zeng-Nan Mo, MD, Institute of Urology, The First Affiliated Hospital of Guangxi Medical University, Nanning, China (e-mail: mozengnan@263.net).
Table 1. Relative mRNA Expression Levels of
Kallikrein 7 (KLK7) and Antileukoprotease (ALP) in
Benign and Malignant Prostate Epithelia

                               Ratio of      Ratio of
                        n *    KLK7/G3PD     ALP/G3PD

                               Mean   SD     Mean   SD     P

Normal ([dagger])         5    0.59   0.41   0.42   0.16   <.001
BPH ([double dagger])    13    0.52   0.23   0.51   0.23
Prostate cancer           8    0.02   0.06   0.09   0.11

* The number of the cases includes 4 prostate cancer cell lines
(DU145, LNCaP, PC3, and 22RV).

([dagger]) Normal prostate.
([double dagger]) Benign prostatic hyperplasia.

Table 2. Immunohistochemical Expression of Antileukoprotease (ALP) and
Human Kallikrein 7 (hK7) in Benign Prostate and Prostate Cancer

Tissue Type         n *   hK7 Expression, No. (%)

                          Negative    Positive
                                      ([dagger])

Normal ([dagger])    20    7 (35.0)   13 (65.0)

BPH ([section])      50   12 (24.0)   38 (76.0)

Prostate cancer     103   85 (82.5)   18 (17.5)

Tissue Type         ALP Expression, No. (%)   P

                    Negative     Positive

Normal ([dagger])    10 (50.0)   10 (50.0)   <.001

BPH ([section])      24 (48.0)   26 (52.0)

Prostate cancer     102 (99.0)    1 (1.0)

* Number of the cases.

([dagger]) To promote the specificity, we consider
staining grade 2 or greater as positive.

([double dagger]) Normal prostate tissue.

([section]) BPH indicates benign prostatic hyperplasia.
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