J Cancer Res Clin Oncol (2002) 128: 91–95 DOI 10.1007/s00432-001-0300-7

O R I GI N A L P A P E R

S. Dog˘ru-Abbasog˘lu Æ U¨. Mutlu-Tu¨rkog˘lu S. Tu¨rkog˘lu Æ Y. Erbil Æ U. Barbaros M. Uysal Æ G. Aykac¸-Toker

Glutathione S-transferase-pi in malignant tissues and plasma of human colorectal and gastric cancers Received: 26 June 2000 / Accepted: 24 September 2001 / Published online: 13 December 2001 Ó Springer-Verlag 2001

Abstract Purpose: In this study, 16 paired samples of colorectal and gastric cancers and adjacent non-neoplastic tissues were analysed for the determination of glutathione S-transferase (GST) activities and the expression of GST-pi. Methods: Western blotting procedure as well as plasma GST-pi levels were used. Results: GST activities were found to be increased in malignant tissues of patient compared with adjacent normal tissues. A significant correlation was detected between GST activity and GST-pi expression in malignant tissues of patients. Plasma GST-pi levels increased in patients compared to aged-matched control subjects. When the patients were grouped according to TNM stage, GST-pi expression in malignant tissues as well as plasma GST-pi levels were higher in patients with more advanced tumor stages. Conclusions: Our results indicate that GST-pi expression in malignant tissues and plasma GST-pi levels in human colorectal and gastric cancers increased depending on the stages of tumor. Keywords Glutathione S-transferase-pi Æ Colorectal cancer Æ Gastric cancer Æ Tissue Æ Plasma

S. Dog˘ru-Abbasog˘lu (&) Æ U¨. Mutlu-Tu¨rkog˘lu M. Uysal Æ G. Aykac¸-Toker Department of Biochemistry, Istanbul Medical Faculty, University of Istanbul, C¸apa, 34390, Istanbul, Turkey E-mail: [email protected] S. Tu¨rkog˘lu Department of Microbiology, Istanbul Medical Faculty, University of Istanbul, C¸apa, Istanbul, Turkey Y. Erbil Æ U. Barbaros Department of General Surgery, Istanbul Medical Faculty, University of Istanbul, C¸apa, Istanbul, Turkey

Introduction Glutathione S-transferase (GST) isoenzymes which are classified as alpha, mu, pi, and theta in man, show different tissue characteristics and distribution (Tsuchida and Sato 1992; Schipper et al. 1997). They bind glutathione (GSH) to a variety of electrophiles including carcinogens and cytotoxic drugs for detoxification (Tsuchida and Sato 1992; Schipper et al. 1997). Previous studies have shown that GST activities (Howie et al. 1990; Moorghen et al. 1991; Peters et al. 1992; Butler et al. 1994; O¨zdemirler et al. 1998; Kanbag˘li et al. 2000) and GST-pi expression (Howie et al. 1990; Moorghen et al. 1991; Peters et al. 1992; Kantor et al. 1991; Mulder et al. 1995; Monden et al. 1997; Hengstler et al. 1998; Sutoh et al. 2000; De Bruin et al. 2000) increased in the tumoral tissues of patients with colorectal and gastric cancers and that these increases may play an important role in the prognosis and the resistance to chemotherapeutic agents of patients (Tsuchida and Sato 1992; Schipper et al. 1997; Peters et al. 1992; Mulder et al. 1995; Monden et al. 1997; Sutoh et al. 2000). Some investigators have found that the increase in GST activity in malignant tissues of patients with colorectal and gastric cancer is mainly related to increased GST-pi expression in these tissues (Howie et al. 1990; Moorghen et al. 1991; Peters et al. 1992). Being inspired by these studies, we thought that it would be interesting to compare the induction of GST-pi expression in tissues with monitoring the GST-pi levels in plasma. For this purpose, total GST activity and expression of GST-pi in colorectal and gastric tumor samples as well as GST-pi levels in plasma from patients undergoing primary surgical treatment were determined. The relationships between biochemical and histopathological results obtained from patients were also considered.

Patients and methods The experiments were approved by the human ethics committee of our university, and informed consent for participation was

92 obtained from each subject. Sixteen gastrointestinal cancer patients (range 44–96 years) operated on at the Department of General Surgery of Istanbul Medical Faculty were included in this study. Malignant and normal tissue specimens were taken from each patient at the operating room and immediately transported to the laboratory in ice. The specimens were cleaned by washing with icecold phosphate-buffered saline (pH 7.4) and stored –80 °C until analysis. Tumor-free tissues were taken from areas at least 10-cm away from the tumor site and used as normal tissues. The removal of malignant and benign tissues was done and sixteen paired samples were analyzed and histopathologically confirmed as adenocarcinoma at the Department of Pathology.

Determination of GST-pi levels in plasma

Preparation of cytosolic fractions Tissue samples were homogenized in a ice-cold buffer containing 20 mM Tris/HCl pH 7.4, 1.4 mM dithiothreitol, and 250 mM sucrose using Teflon-homogenizer (20% w/v). The homogenates were centrifuged for 10 min at 10,000 g at +4 °C and the supernatant fractions were collected. Solid CaCl2 was added to obtain 8.0 mM final concentration and centrifuged again at 25,000 g for 15 min. The supernatant fraction was used for the determination of the enzyme activity and Western blotting (Schenkman and Cinti 1978). Determination of GST activity

Before the surgery and 7 days after surgery, blood samples of the patients were collected in tubes with EDTA. They were centrifuged at 2,500 g for 10 min at +4 °C within 6 h of collection. The supernatants were carefully decanted into the new tubes and recentrifuged at 6,000 g for 10 min at +4 °C to remove platelets completely. The supernatants were collected without giving any damage to pellet and kept at –35 °C until analysis. In these samples, the GST-pi levels were determined with ELISA method (Biotrin HEPKIT-pi).

Statistical analysis

The total GST activity was assayed by the spectrophotometric method using 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate. Results were calculated by using the absorbtion coefficient (9.6 mM–1 cm–1) of the product formed by the conjugation of GSH and CDNB. The enzyme activity was expressed as nmol/min/mg protein (Habig et al. 1974). Protein concentrations were measured by the method of Lowry et al. (Lowry et al. 1951).

Data were expressed means±SD and the statistical analyses were performed by using the Wilcoxon matched-pairs signed-ranks and Mann Whitney U-Wilcoxon rank sum W tests. Correlation coefficients were determined by Spearman’s method.

Results

SDS-PAGE and Western blotting Electrophoresis was performed in 12% polyacrylamide gels using the buffer system in the presence of sodium dodecyl sulfate (SDS) by the method described Laemmli (Laemmli 1970). The protein samples were solubilized in 60 mM Tris-HCl (pH 6.8) containing 14.4 mM 2-mercaptoethanol, 2% (w/v) SDS and 25% (v/v) glycerol. After they were boiled for 2–5 min, equal concentrations of each protein sample (50 lg per well) were subjected to electrophoresis. The separated proteins were transferred onto Immobilon-PVDF membrane (Millipore) in 25 mM Tris/192 mM glycine (pH 8.3) containing 10% methanol according to the method of Towbin et al. (Towbin et al. 1979) The membrane was left to dry overnight and then nonspecific binding sites were

Table 1 The properties of patients and histopathological evaluation of the specimens with regard to histopathological type and stage. (W well differentiated, M moderately differentiated, P poorly differentiated)

blocked with 5% low-fat dried milk in Tris-buffered saline (TBS) for 2 h at room temperature. After washing with TBS containing 0.01% Tween 20 (TBS-T), the membrane was probed with antiGST-pi monoclonal antibody (DAKO) (1:1,000 dilution) for 1 h. Membrane was rinsed with TBS-T, then exposed to peroxidaseconjugated goat immunoglobulin-G as a second antibody (DAKO). Immunodetection was carried out using an ECL Western blot detection kit (Amersham) according to the manufacturer’s instructions. The intensity of each band was semiquantified densitometrically by using the Bio-Rad Discovery Series Quantity One Software.

A total 16 patients were enrolled in this study. The mean age was 63.7±11.6 years. Ages and sex of the patients, localization of the tumor, the degree of their differentiation, and stage of TNM classification are shown in Table 1. GST activities were found to increase in malignant tissues in patients with colorectal and gastric cancer as compared with adjacent normal tissues (Table 2). The results of western blot analysis of GST-pi in normal and tumor tissues are shown in Fig. 1. GST activity and

Patients

Age at diagnosis

Sex

Localization of the tumor Histologic type TNM stage

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

64 55 70 96 65 65 50 70 62 60 70 73 55 60 60 44

F M F F M M F M F M M F F M M F

Stomach Rectum Colon Colon Stomach Stomach Sigmoid Rectosigmoid Stomach Rectum Rectum Sigmoid Colon Stomach Rectum Rectum

M W M M P M P W W P M M M M M M

III I II II II III III III II III III III II II II III

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Fig. 1 Western blot analysis of GST-pi content in normal (N) and tumor (T) tissue from colorectal and gastric cancers

Table 2 Tissue GST activity and plasma GST-pi levels in patients with colorectal and gastric cancers (mean±SD). Statistical analyses were performed by using the Wilcoxon matched-pairs signed-ranks and Mann-Whitney U–Wilcoxon rank sum W tests Tissue GST activity (nmol/min/mg protein) Normal tissue (n=16) Malignant tissue (n=16) Plasma GST-pi levels (lg/l) Controls (n=16) Patients (n=16) Preoperative Postoperative

144.1±34.5 191.1±64.3** 32.7±9.07 52.6±14.6* 47.8±15.2**,***

*P<0.001 **P<0.01 as compared to normals ***P<0.05 as compared to preoperative levels

did not correlate with malignant tissue GST activities (r=0.284, ns) and with GST-pi expression (r=0.412, ns).

Discussion

GST-pi expression in malignant tissues were expressed as % of control tissue and a significant correlation (r=0.554, P<0.05) was detected between GST activity and GST-pi expression in malignant tissues of patients (Fig. 2). When the patients were grouped according to TNM stage, we did not observe any difference in malignant tissue GST activity. However, GST-pi expression in patients with TNM stage III were higher (36%) than in patients with stage I and II, but this difference was not significant (Table 3). Plasma GST-pi levels increased in patients with colorectal and gastric cancer as compared to aged-matched control subjects (60.3±6.9 years; ten men, six women). These levels were found to decrease 7 days after operation (Table 2). Plasma GST-pi levels in patients with TNM stage III were higher than in patients with TNM stage I and II (Table 3). However, plasma GST-pi levels Table 3 GST activity and GST-pi expression in malignant tissues as well as plasma GST-pi levels of patients with colorectal and gastric cancers grouped according to TNM stage (mean±SD).

TNM stage I and II (n=8) III (n=8)

Fig. 2 The relationship between GST activity and GST-pi expression in malignant tissues of patients with colorectal and gastric cancers (regression line and correlation coefficient)

GST-pi, an isoenzyme of the cytosolic glutathione S-transferase, is expressed in a number of tissues including placenta, breast, colon, and urinary bladder indicating that it is involved in normal physiological function such as detoxification (Tsuchida and Sato 1992; Schipper et al. 1997). The higher frequencies of overexpression of GST-pi have been reported by several research groups, although their biological importance has not been clarified yet (Tsuchida and Sato 1992; Schipper et al. 1997). However, it has been suggested that the induction of GST-pi expression may be used as a marker of malign transformation (Tsuchida and Sato 1992; Schipper et al. 1997). In fact, increases in GST activities have been proven in the malign tissues and the cell cultures of the patients, and overexpression of GST-pi has been held responsible from the increases observed in GST activity (Howie et al. 1990; Moorghen et al. 1991; Peters et al. 1992). On the other hand, previous studies have shown the importance of Statistical evaluation was made by using the Mann-Whitney U–Wilcoxon rank sum W tests. (ns nonsignificant)

Malignant tissue GST activity (nmol/min/mg protein)

Malignant tissue GST activity (% of control tissue)

Malignant tissue GST-pi expression (% of control tissue)

Plasma GST-pi level (lg/l)

193.9±85.9 188.2±38.2 U=26.5; ns

135.7±54.2 148.9±82.1 U=31.0; ns

109.7±28.8 149.2±65.2 U=18.5; ns

45.0±15.0 60.2±10.1 U=12; P<0.05

94

determining plasma GST-pi levels in the assessment of cancer status of malignant tissue (Tsuchida and Sato 1992; Hirata et al. 1992; Toffoli et al. 1992; Niitsu et al. 1989; Fan et al. 1995). Plasma GST-pi levels have been observed to increase in patients with some cancers (Hirata et al. 1992; Toffoli et al. 1992) including colorectal and gastric cancers (Tsuchida and Sato 1992; Niitsu et al. 1989; Fan et al. 1995). These high levels returned to normal after the surgical removal of tumors (Niitsu et al. 1989; Fan et al. 1995). In addition, plasma GST-pi levels of the patients with colorectal and gastric cancer with stage III+IV characterized by histopathological examination, without determining tissue GST-pi levels have been shown to be higher compared to stage I and II (Niitsu et al. 1989; Fan et al. 1995). Therefore, the determination of plasma GST-pi levels has been claimed to be promising also for monitorization of patients with cancer after surgery. In our study, total GST activities were found to increase in malignant tissues of patients. Western blot analysis of the same tissue samples also yielded increases in GST-pi expression. In addition, a significant correlation was found between total GST activity and GST-pi expression in malignant tissues. These results are in accordance with those of previous studies (Howie et al. 1990; Moorghen et al. 1991; Peters et al. 1992). In this study, when the patients were grouped according to their TNM stage, in malignant tissue of patients, GST activities were not found to be changed. However, GST-pi expression in malignant tissues was observed to be higher (36%) in patients with more advanced tumor stage, but this difference was not significant. There is inadequate knowledge in the literature about the relationships between tissue and plasma GST and/or GST-pi, but elevated plasma GST-pi in patients with malignancies may originate from the tumor cells themselves (Niitsu et al. 1989; Fan et al. 1995), although some controversial findings are available in the literature (Mulder et al. 1997). In this study, plasma GST-pi levels were also found to increase in patients with colorectal and gastric cancer and these levels were higher as the tumor progressed. However, we could not detect any correlation between GST-pi expression in malignant tissues and GST-pi levels in plasma. In conclusion, our results indicate that GST-pi expression in malignant tissues as well as plasma GST-pi levels in human colorectal and gastric cancers increased depending on the stages of tumor. Acknowledgement This work was supported by the Research Fund of the University of Istanbul. Project number: 1055 / 031297.

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