Journal of Kidney Cancer and VHL 2015; 2(1): 25-29. Doi: http://dx.doi.org/10.15586/jkcvhl.2015.22
Short Communication
Glutathione-S-transferase-pi (GST-pi) expression in renal cell carcinoma
Christina Kaprilian1*, Maria Horti1, Kosmas Kandilaris1, Andreas Skolarikos3, Nikolaos Trakas2, Ioannis Kastriotis3, Charalambos Deliveliotis3
1Department of Pathology, Sismanoglio General Hospital, Sismanogliou 1, Marousi 15126, Athens, Greece; 2Department of Clinical Chemistry Sismanoglio General Hospital of Attica; 32nd Department of Urology Athens Medical School, Sismanoglio Hospital, Sismanogliou 1, Marousi 15126, Athens, Greece.
Abstract
Multidrug
resistance correlates with unfavourable treatment outcomes in numerous cancers
including renal cell carcinoma. The expression and clinical relevance of
Glutathione-S-transferase-pi (GST-pi), a multidrug resistance factor, in kidney
tumors remain controversial. We analyzed the expression of GST-pi in 60
formalin-fixed, paraffin-embedded renal cell carcinoma samples by
immunohistochemistry and compared them with matched normal regions of the
kidney. A significantly higher expression of GST-pi was observed in 87% of
clear cell carcinoma and 50% of papillary subtypes. GST-pi expression did not
correlate with tumor grade or patient survival. GST-pi is unlikely to be a
prognostic factor for renal cell carcinoma. However, further studies with large
number of samples are warranted to establish the role of GST-pi, if any, in intrinsic
or acquired resistance of renal cell carcinoma to conventional treatments. Copyright: The
Authors.
Received: 06 November 2014; Accepted after revision: 18 February 2015; Published: 22 February 2015
Author
for correspondence: Christina Kaprilian, Department
of Pathology, Sismanoglio General Hospital, Sismanogliou 1, Marousi 15126,
Athens, Greece. E-mail: [email protected]
How
to cite: Kaprilian C, Horti, M, Kandilaris K, Skolarikos
A, Trakas N, Kastriotis I, Deliveliotis C. Glutathione-S-transferase-pi
(GST-pi) expression in renal cell carcinoma. Journal of Kidney Cancer and VHL 2015;2(1):25-29.
Doi: http://dx.doi.org/10.15586/jkcvhl.2015.22
Introduction
Despite the introduction of targeted therapies,
metastatic renal cell carcinoma (RCC) continues to maintain its reputation as a
treatment-resistant cancer. About 30% of the patients display intrinsic
resistance to targeted therapy while the remaining 70% who initially respond
will eventually acquire resistance between 6 and 11 months (1-4). A better understanding
of molecules that influence drug resistance may shed light into the mechanisms
of resistance of this deadly disease. In this regard,
Glutathione-S-transferase-pi (GST-pi), has attracted some attention in the
past. GSTs are a family of detoxification enzymes that protect cells from
exogenous and endogenous electrophiles (5). There are eight mammalian GST
isoforms (6). RCC cell lines that are resistant to adriamycin (7) and cisplatin
(8) have a higher expression of GST-pi when compared with parental cell lines.
Ferric nitrilotriacetate-induced renal tumors in rats are associated with a
higher expression of GST-pi (9). However,
some studies reported no significant correlation between GST activity and drug
resistance (10). Baseline expression of GST-pi in human RCC samples has also
been controversial. Some studies show that GST-pi is decreased in RCC (11, 12) while others show no
difference (13, 14). On the contrary, some studies report a higher expression
of GST-pi in RCC (15-17). A recent evidence-based meta-analysis concluded no
association between GST-pi polymorphisms and the development of RCC (18). In
this study, we examined the expression of GST-pi on primary renal cancer
specimens to assess the value of this protein in RCC, any possible association
of this marker to prognosis or other clinicopathologic parameters, and finally
the importance of its expression in tumor aggressiveness.
Material and Methods
Ethics approval
Approval for this study was obtained from the
Scientific Committee of our hospital (Sismanoglio General Hospital, Athens,
Greece), Directors of the Departments, and the University of Athens, Greece.
Informed consent was obtained from patients before the collection of the
samples.
Patient samples
Formalin-fixed, paraffin-embedded archival renal
tumors from 60 patients along with matched morphologically normal regions of
kidneys were used in this study. The tissue samples were obtained from patients
who underwent nephrectomy for kidney cancer at the Department of Pathology
(Sismanoglio General Hospital, Athens, Greece) between 1997 and 2001. Medical
records showed that none of the patients received any pre-operative therapy.
Tumor grade was established according to the World Health Organization (WHO)
criteria and the clinical staging was conducted according to the TNM
classification scheme.
Immunohistochemistry
The expression of GST-pi was examined by routine
immunohistochemistry technique. In brief, the blocks were cut into 5 micron
sections, mounted onto polylysine-coated slides, dewaxed in xylene, rehydrated
in ethanol, treated with Trilogy solution (Code S3307; DAKO, Denmark) and
heated in a microwave for 15 minutes. The sections were peroxidase-incubated
for 10 minutes using 3% hydrogen peroxide and
incubated for 20 minutes at room temperature with the GST-pi primary antibody
(Thermo Scientific; Cat. No. RB-050-A1) at 1: 100 dilution. Then Envision/HRP
(CodeK4010; DAKO, Denmark) was used for 20 minutes, followed by
diaminobenzidine (DAB) for 5 minutes. The slides were counterstained with
hematoxyline for 5 minutes, dehydrated in in ethanol, cleared in xylene and
mounted in DePex mounting medium. Two pathologists independently reviewed the
slides without the knowledge of former diagnosis. The intensity of GST-pi
immunoreactivity was scored as negative (<10% cells with intense positive
cytoplasmic and/or nuclear expression) or positive (>10% cells with intense
positive cytoplasmic and/or nuclear expression) when compared with the matched
morphologically normal regions. The distal tubular cells have been reported to
have elevated concentrations of GST-pi [19]. Therefore we used the distal renal
tubular cells as positive control. For negative controls, the primary antibody
was omitted.
Statistical analysis
The statistical program SPSS for Windows, version
13.0 (Statistical Package for the Social Sciences, Chicago, IL, USA) was used.
Correlation of staining with baseline characteristics was performed using chi-square
test and Fisher’s exact test. Life tables were estimated by Kaplan-Meier
statistics and survival curves were compared using the Log-rank test. Survival
was calculated from the day of surgery. All P values were two-sided and 5% was
chosen as the level of statistical significance.
The characteristics, tumor grade and immunoreactivity of all 60 sample are shown in the supplementary file. Of the 60 samples, 54 were clear cell carcinomas and the remaining 6 were papillary subtypes. Eighty-seven percent (47/54) of clear cell and 50% (3/6) of papillary subtypes showed a higher expression of GST-pi when compared with matched normal regions. A representative photomicrograph for each subtype is shown in Figure 1. No significant correlation between GST-pi expression and tumor grade was observed (Table 1). The median survival of GST-pi positive patients was 90.37 months, whereas for negative patients, it was 86.04 months. Given that only a small number of samples were negative, these results are insufficient to reach any reasonable conclusion on the effect of GST-pi on survival.
Figure1. GST-pi
expression in renal cell carcinoma. A, Normal distal tubular cells (negative
control, primary antibody was excluded). B, Normal distal tubular cells acting
as positive control for GST-pi expression. C, Normal cortical region showing
weak or no expression of GST-pi. D, Clear cell RCC showing high expression of
GST-pi. E, Papillary RCC showing weak expression of GST-pi. F, Papillary RCC showing high expression of
GST-pi. Of the six papillary subtypes, 3 were positive and 3 were negative for
GST-pi.
Discussion
RCC encompasses a set of tumors originating from
the epithelial cells of the renal tubules (20). The two predominant subtypes of
RCC are clear cell (60-70%) and papillary (10-15%). In our series, 54 (90%)
cases were clear cell and 6 (10%) were papillary RCCs. GST-pi was increased in
RCC, however, no correlation with tumor grade or stage was observed. Our
findings contrast with previous studies that showed a decrease (11,12) or no
change in GST-pi in RCC (13, 14).
It has been suggested that a decrease in GST expression may represent a
dedifferentiation program in RCC and that GST expression could be used as a
prognostic marker in RCC (11). Our observations deviate from this assumption.
Our findings are in agreement with studies that showed an increased expression
of GST-pi in RCC. In one study 76% of samples were positive for GST-pi (21),
and in another study, 100% of samples (12/12) were positive (17). The five-year
survival for patients with GST-pi positive tumors was 88% versus 50% for those
with GST-pi negative tumors (21), a pattern reflected in our findings. Both
these studies employed immunohistochemistry to detect GST-pi expression.
Toffoli and colleagues (16) analysed mRNA expression in 38 human RCC samples
and showed a significantly higher GST-pi mRNA expression in RCC. In conclusion,
an increase in expression of GST-pi was observed in our study. However it does
not appear to have a correlation with tumor aggressiveness. GST-pi is unlikely
to be a prognostic factor for RCC. Further studies, using a large number of
samples, are warranted to establish the role of GST-pi in intrinsic or acquired
resistance of RCC.
Acknowledgments
The authors wish to thank E. Anagnostopoulou, A. Zabarelou,
M. Moscho and G. Tzoy for their excellent technical assistance.
Conflicts of interest
The authors declare that they have no competing
interests.
References
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