INTRODUCTION

Prostate cancer (CaP) is an irreversible scarring process, which changes the normal gland structure to the abnormal one. This also leads to changes in protein fractions.1 In men, 95% of prostate cancer is observed between 45 and 89 years of age. During the last decade significant research has been conducted using prostate-specific antigen (PSA) in the basic and clinical sciences and many advances have occurred in the clinical use of PSA for detecting and monitoring prostate cancer. Separation methods including polyacrylamide gel electrophoresis have made significant contributions to the discovery and identification of different molecular forms of PSA, which have improved its ability to detect early CaP. However, the sensitivity and specificity of PSA are still limited and unnecessary biopsies are required for men with slightly elevated levels of PSA.

It has been reported that the systematic analysis and cataloging of electrophoresed proteins from identified cancers and their comparison with the protein patterns from histologically indeterminate cancers should provide a general means of identifying the latter.2 The possible application of different electrophoretic methods in the basic science of prostate cancer may be associated with the identification of more cancer-specific forms of PSA and the discovery of other serum proteins useful not only for detecting but also for staging and prognosis of  CaP. Such novel markers might lead to a better understanding of CaP aggressiveness and to developments in the clinical field of treatment.3 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) is a versatile and powerful technique widely used for protein separation based on their molecular weights Laemmli (1970)4

In this study a qualitative analysis of Coomassie Brilliant Blue stained proteins separated by SDS-PAGE was undertaken to detect some novel proteins, which could possibly serve as markers for cancer detection.

SUBJECTS AND METHODS

The study was conducted from January to March 2003.

Inclusion Criteria

CaP cases were selected on the basis of lower urinary tract symptoms (urgency, frequency and dribbling) and digital rectal examination (DRE) results (hard consistency, adherent mucosa overlying prostate gland, etc.), which were further confirmed as malignant on biopsy. While the control subjects displayed none of these symptoms.

Exclusion Criteria: Patients that had undergone any surgical intervention were excluded. The cases confirmed as BPH on biopsy were also excluded.

Serum samples of 36 biopsy-confirmed CaP patients of different ages were collected from National Health Research Complex and the Urology Units of Jinnah Hospital and Mayo Hospital, Lahore. A control group of 36 matched healthy males were also included from the general community, ensuring similarities of their basic confounding factors. The samples were grouped according to age as follows: 50-59 (Group I), 60-69 (Group II), 70-79 (Group III) and >80 (Group IV) years. Sera were stored at -80°C till analysis.

Reagent Preparation

12% resolving gel:  It was prepared by dissolving 8 ml 30% acrylamide-bisacrylamide, 3.35ml of 3M Tris-HCl (pH 8.8), 0.2ml of 10%SDS, 8.45ml of distilled water, 4ml TEMED and 65ml of 10% ammonium persulfate.

Stacking gel: Prepared by displving 0.9ml of 30% acrylamide-bisacrylamide, 55ml 10% SDS, 0.35ml of 1M Tris-HCl (pH 6.8) in 4.90ml distilled water and 38ml bromophenol blue, 5ml TEMED and 55ml 10% ammonium persulfate was added.

Tris-gycine buffer: 15g of trizma base, 72g of glycine and 5g SDS were dissolved in distilled water and volume was made 1000ml. The solution was 5x diluted before electrophoresis.

Staining solution: 0.5g Coomassie Brilliant Blue R-250 in 450ml methanol, 90ml glacial acetic acid was dissolved in  450ml distilled water.

De-staining solution

300ml of  (30%) methanol, 100ml of (10%) acetic acid and   were added to 600ml water.     Serum samples of one control and 4-5 cases of each age group were separated by SDS-PAGE on a 12%  gel.(5 μg of protein was loaded in each well). The gel was then electrophoresed at constant supply of 12mA and voltage of 150V in a minicold lab, where the teperature was maintained at 40C. The gel was stained using Coomassie Brilliant Blue R-250 and then destained. Protein fractions appeared as dark bands on a light background. The gels were photographed using “GeneSnap” and their images were formatted and analyzed by “GeneTools”, which were part of the computer software program “GeneGenius Gel Documentation & Analysis System”.

The molecular weight of each protein fraction was determined by using molecular weight markers as standard. The raw volume, calculated by the software using the band height and intensity, was a measure of the relative quantity of protein in each sample.

RESULTS AND DISCUSSION

The appearance and relative raw volumes of 14 major protein fractions ranging in molecular weight from 0.23-157 kD were studied in each group.

Group I (Fig.1) included N1 (control), C1, C2, C3, C4 and C5 (cases).

            M      N1    C1      C2     C3        C4        C5

Fig 1. Photograph showing serum protein fractions of control (N1) and cases (C1- C5) in Group I

(50-59 years), resolved on 12% SDS-PAGE gel. 

M: Protein size markers (from top to bottom): 67, 45, 24, 18, 13 and 1.45 kD, respectively.

Group II (Fig.2) included N2 (control), C6, C7, C8, C9 and C10 (cases).

                         M       N2      C6      C7         C8       C9        C10

Fig 2. Photograph showing serum proteins fractions of control (N2) and cases (C6- C10) in Group II (60-69 years), resolved on 12% SDS-PAGE gel.

M: Protein size markers (from top to bottom): 67, 45, 24, 18, 13 and 1.45 kD, respectively.

Group III (Fig.3) included N3 (control), C11, C12, C13, C14 and C15 (cases).

     M       N3     C11     C12     C13     C14     C15

Fig 3. Photograph showing serum proteins fractions of control (N3) and cases (C11- C15) in Group III (70-79 years), resolved on 12% SDS-PAGE gel.

M: Protein size markers (from top to bottom): 67, 45, 24, 18, 13 and 1.45 kD, respectively.

Group IV (Fig.4) included N4 (control), C16, C17, C18 and C19  (cases).

              M        N4     C16    C17      C18       C19      

Fig 4. Photograph showing serum proteins fractions of control (N4) and cases (C16-C19) in Group IV (>80 years), resolved on 12% SDS-PAGE gel.

 M: Protein size markers (from top to bottom): 67, 45, 24, 18, 13 and 1.45 kD, respectively.

It was seen, on the whole, that the raw volume of most of the protein fractions decreased in the CaP cases as compared to the controls. These included protein fractions of 1.27, 14, 55, 100, 114, 122, 131, 140 and 155 kD. Similar findings were reported by Wada et al. (1985) who compared the subcellular proteins of normal, BPH and CaP subjects using SDS-PAGE. They found that the concentration of non-histone proteins of 42, 55 and 190 kD were significantly lower in CaP than in normal.5

It was, furthermore, observed that the protein fraction of 140 kD, prominent in normal samples of each group, was absent in 6 cases (C3, C6, C16, C17, C18 and C19), while its level was significantly decreased in other diseased samples of each group.

The protein fraction of molecular weight 122 kD showed a decreased raw volume in almost all the cases, but it was altogether absent in 5 cases (C3, C6, C9, C11 and C19). The 114 kD protein fraction showed a similar behavior such that it decreased by volume in most of the cases and was absent in 4 cases (C3, C9, C12 and C19). A 100 kD protein fraction, which is known as haptoglobin type 1-1, was also decreased in cases compared to the control subjects. Moreover, it was not absent in three of the cases (C3, C9 and C19). Another low molecular weight protein fraction of 1.27 kD was absent in 2 cases (C3 and C19), whereas a very significant decrease in its raw volume, as much as 99%, was observed in other cases of different age groups.

It was noteworthy that in samples of two cases (C3 and C19) protein fractions were lost. Almost similar findings were reported by Meehan et al. (2002) who compared the protein maps of normal and malignant prostate tissue and identified 20 different proteins that were lost in malignant transformation.6

On the contrary, the raw volume of major protein fraction of 66.3 kD increased in the cases. This was also reported by Mikolajczyk et al. (1999) who purified, sequenced and identified a novel hK2 complex in prostate tissue consisting of hK2 and a serine protease inhibitor known as protease inhibitor-6 (PI-6).7 This 64 kD complex is elevated in the tumor as compared to the normal and benign conditions. This complex may possibly have merged in the serum albumin fraction of 66.3 kD, and expressed as a single band. The apparently elevated level of this protein fraction might thus be due to the elevation of this complex. To confirm this, it is suggested that these samples be analyzed by 2D-PAGE followed by immunoblotting. 2D-PAGE has advantage over SDS-PAGE in that it separates proteins on the basis of isoelectric point (pI) as well as molecular weight.

It helps to resolve the proteins with same molecular weight but different pI. Regarding this protein fraction (66.3 kD) exceptional behavior was seen in 2 cases (C3 and C19), where it was significantly decreased by 47% and 37%, respectively.

Another abnormal protein fraction of 0.23 kD appeared in most of the cases but was absent in all the control subjects. It was not, however, considered as unique to their respective samples since amounts of proteins that do not exceed the minimum sensitivity of the dye may have been present in the other samples. But it was found that this fraction was also absent in 5 cases (C5, C10, C11, C14 and C16). Further investigation of these samples could yield more information. It is suggested that immunoblotting be undertaken in order to identify the abnormal protein. Silver staining, which is more sensitive than Coomassie Blue, would tell whether minute quantities of these protein fractions were present. 2D-electrophoresis would separate the proteins according to pI as well as molecular weight and thus achieve better resolution.

ACKNOWLEDGEMENT

Authors gratefully acknowledge Dr. Amanullah Khan from College of Physicians and Surgeons Pakistan for his guidance. The authors also appreciate cooperation and support of all the staff members of N.H.R.C., and Institute of Biochemistry and Biotechnology, University of the Punjab Lahore, for providing all the facilities. We are also thankful to Dr. Naveed Iqbal and Dr. Nadeem, Urology Department, Jinnah Hospital Lahore for their cooperation in sample collection. 

REFERENCES

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Sumi S, Arai K, Yoshida K, Separation methods applicable to prostate cancer diagnosis and monitoring therapy. J Chromatogr B Biomed Sci Appl. 2001 764(1-2):445-55.

Laemmli E K, Rozanski T A, Morey A.F, Rholl V, (1993). The effects of exercise activity on serum prostate specific antigen levels. Urology 150: 893-894.

Wada F, Nishi N, Tanaka Y, Muguruma Y, Tanaka K, Usami M, et al. Comparison of subcellular proteins of normal prostate, benign prostatic hypertrophy and prostatic cancer; presence of BPH associated non-histone proteins. Prostate 1985 7(1):107-15.

Meehan KL, Holland JW & Dawkins HJ Proteomic analysis of normal and malignant prostate tissue to identify novel proteins lost in cancer. Prostate (2002) 50(1): 54-63.

Mikolajczyk SD, Millar LS, Marker KM, Rittenhouse HG, Wolfert RL, Marks LS, et al. Identification of a novel complex between human kallikrein-2 and protease inhibitor-6 in prostate cancer tissue. Cancer Res 1999 59(16):3927-30.