{"paper_id":"fff4c7d0-47ec-45c6-933b-cb7cc6fe5b8d","body_text":"INTRODUCTION\nGamma-glutamyltransferase (GGT) is a transmembrane\nprotein that plays an important role in glutathione (GSH) salvage\nand homeostasis, particularly at low cysteine concentrations (1,\n2). GSH acts as an antioxidant and protects cells against\noxidative stress, especially by detoxifying peroxides and free\nradicals. It has an important role in maintaining the intracellular\nredox balance.\nGGT is upregulated after acute oxidative stress and during\npro-oxidant periods. The significant determinants of GGT\nexpression and activity are reactive oxygen species (ROS)\ngenerated from the nicotinamide adenine dinucleotide phosphate\n(NADPH) oxidase system (3). GGTis expressed in many human\ntissues, predominantly on the luminal surface of secretory\nepithelial cells, particularly of the hepatobiliary tract, pancreas\nand kidneys (4). The uterus contains a mixture of GGT -positive\nand -negative glands in both the secretory and proliferative\nphase of the menstrual cycle. The fluid within the GGT-positive\nsecretory glands is also GGT -positive (4). Germ cells, surface\nepithelium, and most stromal cells in the ovary are negative for\nGGT, while cilia on the epithelium of the Fallopian tubes stain\npositive for GGT.\nCancer cells are characterised by higher endogenous reactive\noxygen species production than normal, untransformed cells.\nThey can compensate and benefit from such increased oxidative\nstress situations. Many human tumours express high levels of\nGGT. However, the distribution and concentration of GGT in\nJOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2016, 67, 3, 395-402\nwww.jpp.krakow.pl\nK. PITYNSKI1, T. OZIMEK1, N. GALUSZKA1, T. BANAS1, K. MILIAN-CIESIELSKA2, M. PIETRUS1, \nK. OKON2, M. MIKOS3, G. JUSZCZYK4, A. SINCZAK-KUTA2, A. STOJ2\nASSOCIATION OF THE IMMUNOHISTOCHEMICAL DETECTION \nOF GAMMA-GLUTAMYL TRANSFERASE EXPRESSION \nWITH CLINICOPATHOLOGICAL FINDINGS IN POSTMENOPAUSAL WOMEN\nWITH ENDOMETRIOID ADENOCARCINOMA OF THE UTERUS\n1Department of Gynecology and Oncology, Jagiellonian University Medical College, Cracow, Poland; \n2Department of Pathomorphology, Jagiellonian University Medical College, Cracow, Poland; 3Dietl Specialistic Hospital, \nCracow, Poland; 4Department of Public Health, Medical University of Warsaw, Warsaw, Poland\nGamma-glutamyl transferase (GGT) is a membrane enzyme present not only in the liver but also in healthy endometrial\nepithelium. Its overexpression has been demonstrated in numerous malignancies, where it exerts an anti-apoptotic effect\nand causes drug resistance in response to oxidation stress. Aim of the study was investigation of GGT expression in\npostmenopausal patients with endometrioid adenocarcinoma of the uterus (EAC). The material comprised 98 paraf fin-\nembedded post-operative tumour samples of EAC from postmenopausal patients and a control group of 60 normal\nhuman postmenopausal endometrium samples. For immunohistochemical specimen staining, polyclonal IgG anti-GGT\nwas used; for GGT expression measurement, a semi-quantitative method was applied. In EAC patients, 16 (16.33%)\nwere diagnosed as stage IA, 46 (46.93%) as stage IB, 14 (14.29%) as stage II, and 22 (22.45%) as stage IIIA-C,\naccording to the International Federation of Gynaecology and Obstetrics (FIGO) classification. Fifty-six (57.14%)\npatients were diagnosed with low- or moderate-grade (G1-2) disease, and 42 (42.86%) were diagnosed with high-grade\n(G3) disease. Cytoplasmic GGT staining was confirmed in all samples, while apical membrane GGT staining was\nobserved only in G1-2 EAC specimens and the control group. In G3 EAC specimens, GGT cytoplasmic staining and\nhigh nuclear polymorphism areas were predominantly shown. Comparable high GGT median apical expression was\nconfirmed in healthy endometrium (2.0, S.E.M. = 0.28) and in G1-2 EAC (2.0, S.E.M. = 0.27); however, in G3 tumours,\nGGT expression was significantly lower (0.0, S.E.M. = 0.07) than in healthy endometrium (P < 0.001 and P < 0.001,\nrespectively). After stratification of the cancer cases according to FIGO staging, the lowest median apical GGT\nexpression levels were in II EAC (0.0, S.E.M. = 0.64) tumours compared with IA (4.0, S.E.M. = 0.47) tumours,\nspecimen and normal endometrium (2.0, S.E.M. = 2.8) (P < 0001). Stage IB EAC and IIIA-C EAC (1.0, S.E.M. = 0.16)\ncases showed only moderate median apical expression of GGT (1.0, S.E.M. = 0.24). We concluded that impaired GGT\nexpression has the potential to become a valuable tool for stratifying EEC patients’ prognosis and treatment planning.\nKey words: endometrioid adenocar cinoma, uter us, gamma-glutamyl transferase, immunohistochemistr y, postmenopausal\nwomen, cancer grade\n\nhuman tumours present several dif ferences from what is\nobserved in normal tissues (5, 6). Moreover , the heterogeneous\nexpression of GGT in different tumour types, and even different\ntumours of the same type, is observed (6). The enzyme has been\nsuggested to protect against apoptosis and is upregulated after\nacute oxidative stress via Ras and several downstream signalling\npathways (7, 8).\nThere is widespread interest in the role of this enzyme in\ntumour formation, progression, invasion, and drug resistance.\nHigh serum levels of GGT were shown to be associated with\ninferior prognosis in many human cancers. Epidemiologic studies\nhave indicated that elevated levels of γ-glutamyltransferase\n(GGT), a key enzyme of glutathione metabolism, might be\nassociated with increased cancer risk. Recent experimental\nmodels have further elucidated the ability of cellular GGT to\nmodulate crucial redox-sensitive functions, such as\nantioxidant/antitoxic defences and cellular proliferative/apoptotic\nbalance, and its role in tumour progression, invasion, and drug\nresistance has been proposed (6, 9-11).\nThe actions of GGT favouring tumour growth may be\ntwofold: it acts as a source of essential amino acids both for\nprotein synthesis and for the maintenance of intracellular GSH\nlevels. Under specific conditions, the metabolism of GSH by\nGGT can exert pro-oxidant ef fects, with modulatory ef fects on\nseveral redox-sensitive processes at the membrane surface and in\nthe extracellular microenvironment (6, 9). This suggests that the\npro-oxidant reactions stimulated by GGT serve as an additional\nsource of endogenous ROS in cancer cells, possibly contributing\nto the ‘persistent oxidative stress’ that has been described as a\nfactor in genomic instability and carcinogenesis (12).\nA recent interesting observation showed that GGT -rich\nexosomes are released from human cancer cells. In the resistant\nand invasive phenotype of malignant cells, secreted GGT may\nplay roles similar to those described for Helicobacter infection,\nleading to the establishment of cancer metastases (13).\nEndometrial cancer is presently the most common cause of\nmorbidity related to gynaecological cancers both in well-\ndeveloped European countries and the USA (14). Many women\nwho enter menopause face mood and sleep disorders that can\nincrease appetite and lead to obesity , which is consider to be a\nmajor risk factor of endometrial cancer . Fortunately recently\ndeveloped combined fluoxetine with melatonin therapy was\nproved to be ef fective in treatment of mood sleep and appetite\ndisorders in postmenopausal patients indirectly reducing the risk\nof endometrial cancer development (15). Endometrioid\nadenocarcinoma of the uterus (EAC) accounts for approximately\n80% of all endometrial cancer cases (16). Although the\nprognosis is favourable compared with other female\nmalignancies (e.g. ovarian cancer), doubts regarding the extent\nof treatment of EAC patients are still present. The discussion has\nfocused on finding the proper balance that would avoid both\nunder treatment resulting in an increased risk of recurrence and\novertreatment that could potentially lead to unnecessary and\nbothersome complications (17, 18). Thus far, there have been\nsome established histopathological risk factors, such as FIGO\n(International Federation of Gynaecology and Obstetrics) stage,\ndepth of myometrial invasion, histological grading, or presence\nof lymphovascular invasion that help predict the group of\npatients at high risk for nodal disease that would require\nlymphadenectomy or adjuvant treatment (19, 20). Additionally\nhigher intratumoral micro-vessels density was confirmed in\nendometrial cancer compared to benign endometrial changes\nand normal human endometrium and it is considered as an\nindependent, poor prognostic factor of overall survival and\nrecurrence-free survival (21). Recent studies have focused on the\nidentification of novel biomarkers (e.g., stem cell-associated\nnuclear transcription factors) that could be helpful in the\nidentification of patients diagnosed with low-stage EAC who\nmight be at high risk of metastases and disease recurrence (22).\nNew histological markers would be helpful to gain a better\nunderstanding of the disease, stratify patient risk and plan\ntailored treatments.\nThe connection between GGT expression and endometrial\ncancer pathogenesis has not been explored extensively thus far .\nSeebacher et al. proved that the elevated pre-therapeutic GGT\nserum level in endometrial cancer patients correlates with\nshorter progression-free survival and can be used as an\nindependent prognostic factor (23). Since only Hanigan and\nFrierson have investigated GGT expression in EC specimens\npreviously (their study was limited to only four EAC cases) (4),\nour study sought to assess GGT activity in EAC tissue as a\npossible novel histopathological prognostic factor in the future.\nMATERIALS AND METHODS\nPatients and materials\nThe material included in the study came from 158\npostmenopausal patients: 98 cases of EAC and 60 women with\npelvic or gan prolapse (control). Before sur gery, every patient\nwas weighed, and their heights were ensured. Body mass index\n(BMI) was calculated as the body mass divided by the square of\nthe body height and is expressed as kg/m 2 units. Formalin-fixed\nparaffin-embedded (FFPE) tissue sections were taken from the\nfiles of our hospitals’ tissue archives. Histopathological\nclassification and grading were performed on haematoxylin and\neosin-stained slides according to standard histopathological\nprocedures.\nDetection of gamma-glutamyl transferase (GGT)\nImmunohistochemistry was performed on 4- to 5-µm-thick\nFFPE tissue sections. Briefly , slides were deparaf finised and\ndehydrated in 100% ethanol, washed in distilled water and\nmicrowaved (600 W for 10 min and 5 min) in antigen retrieval\nsolution (EDTA, pH 8.0). Next, they were washed in distilled\nwater, cooled at room temperature (R T) for 20 – 30 min and\nimmersed in 3% H 2O2 to block endogenous peroxidase. After\nwashing in distilled water (5 min) and in Wash Buffer (Tris-HCl;\nDakoCytomation, S3006) twice for 5 min, 100 µl of the gamma-\nglutamyltransferase 1 precursor antibody (GGT1; polyclonal;\nThermo Scientific P A5-21344) was applied to each tissue\nsection. The antibody was diluted 1:400 in Dako Antibody\nDiluent with Background Reducing Components (S3022). Next,\nthe slides were incubated for 30 min at R T. After washing in\nWash Buffer, the secondary antibody was applied and incubated\nfor 30 min at R T (Dako REAL EnVision HRP Rabbit/Mouse,\nK5007). The enzymatic reaction was performed with DAB\n(incubation for 10 – 30 s). Tissue sections were counterstained\nwith haematoxylin and coverslipped.\nScoring system\nGGT staining was evaluated in each tumour specimen and\nnormal postmenopausal endometrium. Every tumour was\nscored according to the intensity of staining and number of\nstained cells (0, expression in < 25%; 1, 26 – 50%; 2, 51 – 75%;\nand 3, > 76% of cells). Positive staining was observed in the cell\nmembrane, particularly in the apical part of glandular cells\n(staining scored as 1 - weak, 2 - moderate or 3 - strong). In cases\nwhere staining was classified as 3 (strong), the apical part of the\ncytoplasm also exhibited a positive reaction for GGT . The final\nimmunoreactivity score was determined by multiplying the\n396\n\nintensity scores by the extent of the positivity scores of stained\ncells, providing a score range of 0 – 9. Each sample was\nassessed blindly by three independent observers (K.M-C., N.G .,\nand T.O.). In the case of any discrepancy between the\nobservations, samples were verified once again together to\nachieve a consensus.\nStatistical analysis\nThe Shapiro-Wilk test was used to examine the distribution\nof variables in the patients. The clinical features of the study\ngroup and control group were compared using the parametric\nStudent’s t-test and non-parametric Mann-Whitney U-test or χ2\ntest where appropriate. One-way analysis of variance (ANOVA)\nand Kruskal-W illis test were used to evaluate more than two\ngroups of variables followed by Fisher ’s post-hoc test where\nappropriate. Multivariate analysis of variance (MANOV A) was\nused to identify factors that may influence GGT expression.\nGamma correlation or multivariate regression was used to\nevaluate the relationship between GTT expression and clinical\nfeatures expressed as continuous variables. Clinical features of\nthe study patients are presented as median values and the\nstandard error of the median (S.E.M.) or number of cases and\npercentage. GGT immunoreactivity is presented using an\narbitrary relative scale (points) as the median and interquartile\nrange (IQR). P = 0.05 was accepted as statistically significant.\nAll calculations were carried out using S tatistica software v. 10\n(StatSoft, USA, 2011).\nRESULTS\nThe average age of the patients with EAC was 59.31 ± 9.76\nyears, and they did not dif fer significantly from the average age\nof women in the control group (59.80 ± 11.56 years) (P = 0.779).\nPatients with EAC had a significantly higher mean BMI,\nsignificantly lower mean age of the first period, and a lower\nmedian number of pregnancies and deliveries. There were no\nsignificant differences between the mean age at menopause and\nmenstrual cycle characteristics; however , the women from the\nstudy group had a significantly lower median number of\ngestations and deliveries compared with the controls (1.5,\nS.E.M. = 0.28, vs. 3.0, S.E.M. = 2.45, P= 0.028, and 0.5, S.E.M.\n= 0.67, vs. 3.0, S.E.M. = 1.17, P = 0.016, respectively). Detailed\nclinical features are presented in Table 1. From the 98 patients\nwith diagnosed EAC, 16 (16.33%) were in stage IA, 46\n(46.93%) in stage IB, 14 (14.29%) in stage II, and 22 (22.45%)\nin stage III, according to FIGO. Fifty-six (57.14%) patients were\ndiagnosed with low- or moderate-grade (G1-2) disease, and 42\n(42.86%) were diagnosed with high-grade (G3) disease. High-\ngrade cases showed significantly more advanced clinical FIGO\nstaging than G1-2 samples (P < 0.001) (Table 2).\nExpression of gamma-glutamyl transferase in tumour tissues\nand control specimens\nIn all EAC samples and control group specimens, GGT\nmembrane expression was predominant compared with\n397\n Patients with endometrial \nadenocarcionoma \n(n=98)  \nHealthy controls \n \n(n=60) \nP \nMean age [years] (S.D.*)    59.31 (± 9.76)      59. 80 (± 11.56)     NS #\nMean BMI [kg/m 2] (±S.D.*)    28.72 (± 3.81)      23.41 (± 2.03)     NS #\nGrading  \n- Low/moderate grade (G 1–2 ) \n- High grade (G 3) \n   57 (58.16%) \n   42 (41.84%)      NA ###     NA ###  \nMean age of first menstrual period \n[years] (±SD*)    9.52 (± 1.52)      11.45 (± 1.81)     NS # \nMean age of menopause [years]    53.21 (± 2.21)      52.45 (± 2.31)    NS # \nMean duration of menstrual cycle \n[days] (± S.D. *)    27.50 (± 2.5)      28.00 (± 2.0)     NS # \nMenstrual cycles \n- regular  \n- irregular  \n \n   86 (87.80%) / \n   12 (12.20%) \n \n     46 (81.67) /  \n     14 (18.33%)  \n   NS # \nMenstrual cycles \n- painful  \n- painless  \n \n   38 (48.72 / \n   40 (51.28%) \n \n     32 (21.48) /  \n     117 (78.52%)  \n   NS # \nMean duration of menstruation \n[days]    4.25 (± 1.52)      4.32 (± 0.97)     NS # \nType of menstrual bleeding  \n- scant \n- normal  \n- heavy  \n \n   6 (6.12%) \n   75 (76.53%) \n   17 (17.35%) \n \n     22 (3.33%) \n     49 (81.67%) \n     9 (15.00%) \n    NS # \nMedian number of gestations \n(S.E.M.**)    1.5 (0.28)      3 (2.45)  0.028 ##  \nMedian number of deliveries \n(S.E.M.**)    0.5 (0.67)      3 (1.17)  0.016 ##  \n \n*S.D., standard deviation; **S.E.M., standard error of median; #NS, statistically not significant; ## statistically significant value; ###NA,\ndata not available.\nTable 1. Clinical characteristics of the patients with endometrioid adenocarcinoma of the uterus compared with healthy controls.\n\ncytoplasmic staining in both glandular and stromal cells. In\nglandular areas, GGT-positive cells were mostly localised at the\nupper parts of glands, which was described as apical staining. No\narea-related pattern was observed in GGT-positive stromal cells.\nThe homogeneous cytoplasmic type of GGT staining was\npresent in all assessed samples from all groups and was not\nfurther analysed.\nApical staining was observed in both the control group and\nG1-2 EAC tumours ( Figs. 1 and 2); however , in G3 EAC\nspecimens, cytoplasmic staining and high nuclear polymorphism\nareas were predominant. The majority of G1-2 EAC tumours\n(52/56) and control specimens (55/60) showed apical staining\nfor GGT, while significantly fewer G3 EAC samples (10/42)\nwere GGT-positive (46/69) for apical expression (P < 0.001).\nComparable high GGT median apical expression was confirmed\nin normal postmenopausal endometrium (2.0, S.E.M. = 0.28)\nand in G1-2 EAC (2.0, S.E.M. = 0.26) tumours. In G3 tumours,\nGGT expression was significantly lower (0.0, S.E.M. = 0.07)\nthan in normal postmenopausal endometrium (P= 0.02) (Fig. 3).\nAfter the stratification of cancer cases according to FIGO\nstringing, the highest median apical GGT expression was\nobserved in IA EAC tumours (4.0, S.E.M. = 0.47), a finding that\nwas similar to that in normal postmenopausal endometrium\nderived from the control group (2.0, S.E.M. = 0.28). The lowest\nmedian apical GGT expression was in I EAC (0.0, S.E.M. =\n0.24) tumours. In IB EAC and IIIA-C EAC specimens, only\nmoderate median apical GGT expression (1.0, S.E.M. = 0.24,\nand 1.0, S.E.M. = 0.16) was observed. The median apical\nexpression of GGT in the control group and IA EAC tumours\nwere significantly higher than that in IB – IIIC EAC specimens\n(P < 0.001) (Fig. 4).\nWhile analysing sections, we also noticed that, in the\nmajority of G1-2 EAC cases, both the adjacent endometrial\nstromal cells and stroma between them were noticeably GGT -\npositive compared with the control group, where significantly\nfewer cells presented this staining pattern. Indeed, in 75% (21\nout of 28) of G1-2 specimens, all observers noticed stromal cell\nstaining; however , in the control group, it was observed in\n51.67% (31 out of 60) of cases (P = 0.038). No stromal staining\nwas noticed in G3 EAC specimens.\nMultivariate regression analysis confirmed a significant\nassociation between GGT apical expression and tumour grading\n(P < 0.001) and FIGO staging (P = 0.002), while there was no\nrelationship between GGT cytoplasmic expression and tumour\ngrading, staging, age of EAC diagnosis parity, or age of the first\nperiod and BMI. The age of EAC diagnosis, parity, or age of the\nfirst period and BMI also had no ef fect on the median apical\nGGT expression in EAC samples.\n398\nTumor grading FIGO staging \nG1-2  G 3\nP#\nIA 16 (16.33%) 0 (0.00%) \nIB 30 (30.61%) 16 (16.33%) \nII 6 (6.12%) 8  (8.16%) \nIII A–C 4 (4.08%) 18 (18.37%) \n< 0.001 \n# Statistically significant at the level P < 0.05.\nTable 2 . Association between the International Federation of\nGynaecology and Obstetrics staging and grade in patients with\nendometrioid adenocarcinoma of the uterus.\nFig. 1. Gamma-glutamyltransferase (GGT) apical staining in a low-grade (G1) endometrioid adenocarcinoma of the uterus (EAC) in\npostmenopausal patients: A, weak staining (+); B, moderate staining ( ++); C, strong staining ( +++).\nFig. 2. Gamma-glutamyltransferase (GGT) apical staining in a high-grade (G3) endometrioid adenocarcinoma of the uterus (EAC) in\npostmenopausal patients: A, weak staining (+); B, moderate staining (++); C, strong staining (+++). The arrow shows positive membrane\nstaining for GGT.\n\nDISCUSSION\nThis study is the first detailed investigation of GGTexpression\nin EAC tissue. Excluding the study of Hanigan et al. that was\nlimited to only four endometrial cancer cases, no one has\ninvestigated GGTexpression in EAC specimens (5). Based on the\nvariable clinicopathological parameters that low- and moderate-\ngrade EAC are regarded as type I endometrial cancer while high-\n399\nFig. 3. Expression of gamma-glutamyltransferase (GGT) according to tumour grade in patients with endometrioid adenocarcinoma of\nthe uterus.\n* GGT-IHC, gamma-glutamyltransferase immunohistochemistry staining.\nFig. 4. Expression of gamma-glutamyltransferase (GGT) according to tumour International Federation of Gynaecology and Obstetrics\nFIGO staging in patients with endometrioid adenocarcinoma of uterus.\n* GGT-IHC, gamma-glutamyltransferase immunohistochemistry staining.\n\ngrade tumours can be categorised as type I and type II disease, we\ndivided patients with EAC into G1-2 and G3 subgroups for proper\ndata analysis. Among the investigation concerning the relationship\nbetween GGTand endometrial cancer, Seebacher et al. proved that\nan elevated pre-therapeutic GGTserum level in endometrial cancer\npatients correlates with shorter progression-free survival and can\nbe used as independent prognostic factor (23). We carefully\nanalysed the results presented by Seebacher et al., who reported\nimpaired 5-year survival in women with elevated serum GGT\nlevels. However, they also found no stage- or grade-dependent\ndifferences in the GGT serum concentration. According to their\ndetailed findings, women diagnosed with G3 endometrial cancer\n(of both endometrioid and non-endometrioid origin) had lower\nGGT serum levels than those of patients with G1 tumours, but the\ndifferences were not significant (29.5 ± 31.4 ng/dl vs. 33.1 ± 52.9\nmg/dl; P = 0.600). These data are in contrast to the findings of\nPolterauer et al., who investigated pre-therapeutic serum GGT\nlevels in patients with cervical cancer and found significant\nassociations between serum GGT levels and FIGO stage (P <\n0.0001) and age (P < 0.0001), which were not reported by\nSeebacher et al . for endometrial cancer (23, 24). Similarly to\nwomen with endometrial cancer, patients with cervical cancer and\nelevated GGTserum levels were associated with poor disease-free\nand overall survival in univariate analyses, although these\nassociations were not confirmed in a multivariate Cox-regression\nmodel (24). As the role of GGTin gynaecological malignancies is\nnot clear due to non-unequivocal results, we evaluated GGT\nexpression in normal endometrium and in EAC specimens. At first\nglance, our results seem to contrast strongly with those of\nSeebacher et al ., as we showed significantly decreased GGT\nexpression in G3 EAC. However, we must emphasise that these\nGGT serum and tissue expression levels cannot be compared\ndirectly (23). First, the serum GGT level depends not only on\nmalignant tissue secretion but also on its involvement in metabolic\nreactions, including oxidative stress, kidney excretion, and liver\nmetabolism. Consequently, the GGT serum level may not be a\ndirect function of apical GGT expression in endometrial tissues.\nSecond, we investigated only the endometrioid type of endometrial\ncancer, while Seebacher et al . evaluated GGT serum\nconcentrations in women with endometrioid and non-endometrioid\nendometrial cancers. Our study focused on postmenopausal\npatients, while Seebacher et al . included every woman with\nendometrial cancer irrespective of menopausal status. Seebacher et\nal. found no association between endometrial cancer grade or stage\nand the GGT serum level; therefore, they considered the serum\nGGT concentration as an independent predictor of 5-year survival,\nwhile we found a strong negative association between GGTapical\nexpression and grade (23). Our results are seemingly opposite\nthose of Seebacher et al., and we believe that further studies should\nbe undertaken to elucidate the role of GGTin endometrial cancer\nand evaluate its clinical significance.\nWe found that GGTexpression in EAC tissue differs with that\nof healthy endometrium. The presented differences in the assessed\nfeatures correlated with both tumour biology (histological\ngrading) as well as the stage of EAC. In summary , the best\nexpression of membrane GGTwas present in the control group of\nhealthy endometrium and well- to moderate-differentiated (G1-2)\nEAC. In G3, GGT expression of the EAC samples was much\npoorer, and over 75% of samples were GGT -negative.\nSurprisingly, apical expression of GGT was more intense in\nspecimens with better cell dif ferentiation and more favourable\ngrading. In G1-2 EAC patients, it was highly expressed compared\nwith G3 patients, where most G3 cases showed no membrane\nstaining at all. Carcinomas arising from some GGT -positive\nepithelium retained their GGT -positive phenotype. The more\nstructural and functional dif ferences between normal and\nneoplastic cells, the more diverse the GGT expression. There is\nevidence that GGT is dysregulated in malignant cells by\nproducing reactive oxygen species, causing tumour progression\ntowards more aggressive phenotypes associated with a poorer\nprognosis (5, 25). Many human tumours express high levels of\nGGT. However, the distribution and concentration of GGT in\nhuman tumours present several differences from what is observed\nin normal tissues (5, 6). Moreover, the heterogeneous expression\nof GGT in different tumour types, and even different tumours of\nthe same type, was observed (26). In a study of human GGT -\ntransfected melanoma cells, higher levels of GGT activity were\nassociated with greater levels of background DNA damage and\noxidised bases (27); this activity was unrelated to dif ferences in\ncell cycle distribution and apoptotic rates.\nTo identify a significant determinant for GGTexpression and\nactivity in endometrial cancer cells, Ravuri et al . tested the\nIshikawa cell line and established that endogenous production of\nreactive oxygen species by the NADPH oxidase complexes is a\ndeterminant of γ-glutamyltransferase expression (3). Neoplastic\ncells in many tumours are not polarised and, therefore, express\nGGT on their entire cell surface (5). Unlike normal cells in which\nGGT only has access to substrates in ductal fluids, the GGT on\ntumour cells can cleave glutathione (GSH) in interstitial fluid and\nblood. The expression of GGT provides tumour cells with an\nadditional source of cysteine and cystine from the cleavage of\nextracellular GSH and oxidised glutathione (GSSG). In our\ninvestigation, in addition to its expression in tumour cells, GGT\nexpression was also found in the stromal cells between them. It is\ninteresting that GGT staining was present not only on the\nmembrane but also within the cytoplasm. Both membrane and\ncytoplasmic cancer was observed in human prostate carcinoma.\nThe cytoplasmic staining may reflect GGT protein that is being\nsynthesised and processed within the cell (28). Because differences\nin GGT expression between the controls and G1-2 EAC were not\nobserved, it would be interesting to understand the origin of the\nincreased G1-2 endometrial stromal staining and elevated serum\nGGT levels in some EAC patients (20). It is possible that cancer\ntissue could be the source for stromal and serum GGT elevation.\nGGT expression in ovarian cancer tissue is reflected in GGTserum\nlevels (2). The elevated serum level of GGT detected in several\ntypes of neoplasia may be due not only to its release from cancer\ncells, but might also be associated with systematic changes in the\ndisease, such as example, inflammation (29). A positive correlation\nbetween greater advancement of the tumour and serum GGTlevel\nwas observed in renal and cervical cancers (24, 30).\nTo our knowledge, this is the first comprehensive study\nevaluating GGT expression in EAC and its correlation with\nclinicopathological features. All laboratory evaluations were\nperformed in one setting by staf f highly experienced in\nimmunohistochemistry, and were conducted during a short period\nof time to reduce research bias. The numerous limitations of our\nstudy need to be acknowledged when interpreting and applying the\noutcomes. Foremost, we did not evaluate serum GGT levels and\noxidative stress markers in our study and are unable to draw\nreliable conclusions on the role of GGT in EAC. However ,\nexploring GTT mechanisms in EAC was not an aim of our study.\nAs the roles of GGT in physiological and pathological processes\nwere broadly investigated and described in detail, even if no\nunequivocal results were obtained, as discussed earlier, we aimed\nonly to evaluate the possible clinical utility of GTT. Therefore, IHC\nwas chosen as an evaluation method because it is an essential\npathological technique. A second limitation is that we failed to\nanalyse whether GGT expression is directly associated with\nprediction of the final therapeutic result in patients with EAC. As\nwomen with low-grade EAC have very favourable progression-\nfree and overall survival prognoses, patients need to be re-\nevaluated, at least at the 5-year follow-up, to evaluate the utility of\nGTT as a therapy predictor for women with EAC; such data will be\n400\n\navailable in the future. We also acknowledge that the number of\npatients with G3 EAC was low; however, the predominant EAC\ngrades are 1 and 2, accounting for 80% of cases (18). Based on the\nabove discussion, we conclude that our initial results regarding\nGTT expression in EAC need to be further validated in a lar ger\ncohort to gain more epidemiological and clinical impact. Only a\nprospective follow-up GGT expression analysis in the EAC\nspecimen conducted in a larger population and with age-matched\ncontrols over a longer period of time will fully allow us to elucidate\nthe clinical usefulness of GGTexpression. We believe that the most\nimportant unresolved issue to address for further investigation is\nthe low rate of membrane staging of GGT in less-differentiated\nendometrial cancer cases compared with better-differentiated ones.\nThe result conflicts with what was found in other cancer types,\nincluding ovarian and cervical cancer, and further investigation is\nessential to resolve this issue unequivocally.\nAlthough exploring GTTinvolvement in EAC cell metabolism\nwas not the aim of our study, the decreased apical GTTexpression\nin high-grade EAC should be discussed in the context of its\npossible role in tumour development. First, it is known that\nimmature high-grade tumours have significantly dif ferent\nmetabolism and protein expression patterns compared with well-\ndifferentiated low-grade neoplasms and healthy tissues. Therefore,\nit is not surprising that we observed lower GTTexpression in G3\nEAC. Altered GTT levels may render EAC cells more susceptible\nto oxidative stress. Normal cells and tissues have developed many\nmechanisms to reduce oxidative stress, as it directly impairs their\nfunction and may result in apoptotic death. Uterine endometrium is\ndirectly exposed to dif ferent infection agents that can lead to\nsubclinical or clinical inflammation. In a bovine model an elevated\nexpression of mRNAs of chemokines (CXCL1 and CXCL2),\ninterleukins, prostaglandins and metallopeptidase was confirmed\nduring the late puerperium preventing, according to authors, cows\nfrom persistent endometritis (31). In neoplastic cells however ,\ninflammatory background resulting in ROS overproduction, lead to\ngenomic instability and enhance mutation formation. As neoplasms\nare fast growing, cellular loss due to enhanced DNAmutations is\nnot an issue. This genetic instability also increases the occurrence\nof ‘favourable’ mutations that may result in tumour resistance to\nchemotherapy or radiotherapy. 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J Physiol Pharmacol 2015; 66:\n449-462.\nReceived: October 21, 2015\nAccepted: May 25, 2016\nAuthor ’s address: Assoc. Prof. Kazimierz Pitynski,\nDepartment of Gynaecology and Oncology , Jagiellonian\nUniversity Medical College, 23 Kopernika S treet, 31-501\nCracow, Poland.\nE-mail: pitynski@wp.pl\n402","source_license":"CC0","license_restricted":false}