- Email: [email protected]
Glutathione S-transferase π expression in matched human normal and malignant oral mucosa
Pergamon
,
Ora, Chcoi
PII:
Glutathione
817.00+0.00
SO964-1955(96)00060-7
S-Transferase n Expression Normal and Malignant Oral
in Matched Mucosa
Human
G. Sarkar, N. Nath, N.K. Shukla and R. Ralhan Department
of Biochemistry,
Institute
Rotary Nagar,
Cancer Hospital, All India New Delhi 110029, India
Institute
of Medical
Sciences,
Ansari
Over-expression of glutathione S-transferase x (GST-x) class isozyme is often associated with malignant transformation and/or drug resistance. To determine whether GST-K is implicated in betel and tobacco related oral carcinogenesis and/or drug resistance, its expression was studied in oral untreated primary squamous cell carcinomas (SCCs), recurrent tumours and matched normal oral tissues by immunohistochemical and immunoblotting techniques. The GST-x expression in primary tumours predominantly varied from mild to moderate levels and no significant difference in its expression was observed in the matched normal tissue surrounding these lesions. Mild to moderate levels of GST-n: expression in the oral mucosa of consumers of betel and tobacco observed in the matched normal tissues may support the physiological role of this detoxification enzyme in the metabolism of xenobiotics and elimination of toxic constituents of tobacco. The hallmark of the study is the significant increase in GST-x expression in recurrent oral SCCs, compared to the matched normal tissues, as well as primary oral tumours, suggesting its potential role as an indicator of prolonged exposure to carcinogens, or prognosis of the disease. :c 1997 Elsevier Science Ltd Key words: glutathione Oral Oncology,
S-transferase A, oral tissues, tobacco, human, squamous cell carcinoma
Vol. 33, No. 2, pp. 74-81,
1997
tive to the normal surrounding tissue, as well as in the serum of patients with cancer [ 14-241. The carcinogen-induced alterations in rat preneoplastic liver nodules are also associated with the development of broad spectrum resistance to hepatotoxins [25]. Cell lines selected for resistance to different antineoplastic agents have also been shown to exhibit an increase in GST-n activity, suggesting that changes in the expression of this enzyme in human tumours may be a marker of drug resistance [2629]. Gene transfer experiments have also shown that GST isozymes play a role in conferring resistance to drugs such as melphalan, cisplatin and doxorubicin [30-321. Inhibition of GST-n activity has been suggested as an effective approach to circumvent drug resistance. Studies on GST-rr expression in normal human tissues have shown that this isozyme is predominantly expressed in normal epithelial cells of the urinary, digestive and respiratory tracts, suggesting a possible role in detoxification and elimination of toxic substances [33]. However, there is a paucity of studies on the expression and role of GST-x in human normal and malignant oral mucosa. Oral squamous cell carcinoma (SCC) is the most common malignant neoplasm in South East Asia [34, 351. It is a disease of acquired occupational, nutritional and environmental insults. Chewing of betel quid containing lime, areca-
INTRODUCTION
The glutathione S-transferases (GSTs, EC2.5.1.18) constitute a multigene superfamily of cytosolic enzymes that catalyse the nucleophilic addition of glutathione to electrophilic centres of a wide variety of compounds, thereby playing an important role in protecting cells from xenobiotics [l-3]. These enzymes are involved in detoxification of cancer chemotherapeutic agents such as cisplatin, alkylating agents and doxorubicin [4-71. Enhanced formation of drug-GSH conjugates by the GSTs may reduce the sensitivity of neoplastic cells to the action of these drugs, thereby providing a mechanism for the emergence of drug-resistant tumours. On the basis of their structural, physicochemical, enzymatic and immunological properties, the GSTs are divided into four classes: CI, p. n and 8 [3, 81. The TI isozyme of GST has been associated with malignant transformation in rat [9-l l] and human tumours [12]. Studies in the Solt-Farber model of chemical carcinogenesis have shown that GST-P, a member of the K class is a useful marker for detection of preneoplastic and neoplastic cells [ 131. Over-expression of GST-rr has been observed in a wide variety of human tumours relaCorrespondence to R. R&an. Received 2i June 1996; provisionally accepted revised manuscript received 22 Aug. 1996.
24
July
1996;
74
Glutathione
S-Transferase
nut and tobacco, along with smoking, has been causally associated with the high incidence of oral cancer in India [36]. Remarkably, the location of tumours within the oral cavity of habitual tobacco-folded betel quid chewers correlates with the site where the quid is kept for prolonged time periods. Chronic exposure of the oral cavity of habitual tobacco quid chewers to a variety of carcinogens present in the quid may lead to overexpression of GST-rt. To understand the role of GST-rt as a marker of neoplastic transformation, progression, or drug resistance in oral malignancies, we initiated this study on the evaluation of the GST-x gene status and its expression in oral normal mucosa, primary, as well as recurrent SCCs, and matched normal mucosa. These results were correlated with clinicopathological parameters of the patients.
MATERIALS Tissue
AND
METHODS
specimens
Surgical specimens from 21 primary squamous cell carcinomas of the oral cavity, 22 recurrent oral SCCs, and their matched normal oral tissues were obtained from the Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, India. The oral cancer patients were divided into two groups: (i) primary oral tumours and, (ii) recurrent oral squamous cell carcinoma. Subjects enrolled in the cancer clinic at the onset of the primary tumours were included in group (i). These patients did not receive any treatment at the time of inclusion in the study. In group (ii), patients who enrolled with the recurrent stage of oral cancer were included. Biopsy specimens of normal oral tissues were obtained from cancer patients from normal tissue surrounding the tumour. A piece of the biopsy specimen was snap frozen and stored at -80°C for immunohistochemical analysis, while another was put in formalin for histopathological examination. The chnicopathological features of the patient namely, age, sex, tobacco- and betel-chewing history were recorded in each case. Histopathological studies included the histological grading of the lymphoventricular response, to establish histological differentiation of the tumours. Among the 21 primary oral tumour specimens examined, 16 were well differentiated SCCs, three moderately differentiated cases, one poorly differentiated carcinoma and one belonged to a group other than squamous cell carcinomas. In the recurrent oral SCC group, 15 were well differentiated, two moderately differentiated, three poorly differentiated and two belonged to a group of nonSCCs. Site distribution of oral cancer was also investigated. The buccal mucosa was found to be the commonest site (43.18%), followed by the lower alveolus (34.09%), the tongue (11.36%) and the soft palate (11.36%). Reagents
and DNA
probe
Sheep polyclonal GST-rr: antibody (The Binding Site, Birmingham, U.K.) is raised against human placental GST. Rabbit anti-goat IgG-HRP conjugate was kindly provided by Dr J. K. Batra, National Institute of Immunology, New Delhi, India. The cDNA for GST-rr: (cDNA-GST-x) cloned in pGEM4 (3.6 Kb plasmid) was obtained as a gift from Dr Jeffrey A. Moscow, National Institute of Health, Bethesda, Maryland, U.S.A. The cDNA-GST-rt is a 725 bp fragment,
75
R in Oral Cancer
subcloned at the EcoRI site in the cloning at its multiple cloning site (MCS).
vector pGEM4
Immunohistochemistry
Cryosections (5-8 pm thickness) of human oral tissue specimens were collected on slides and fixed in acetone for 10 min. Representative sections were stained with haematoxylin and eosin for histopathological analysis, while immunostaining was carried out on serial sections. For immunohistochemical analysis, the sections were incubated in methanol containing hydrogen peroxide (0.3% v/v) for 30 min to quench endogenous peroxidase activity. Subsequently, sections were incubated with 1% BSA for 20 min to block the non-specific binding, followed by incubation with anti-GST-n: (sheep polyclonal, 10 ng protein) for 16 h at 4°C. The secondary antibody, rabbit anti-goat IgG-HRP conjugate (10 ng) was added and sections were incubated for 1 h. After washing with PBS three times, the immunostaining was performed using diaminobenzidine as the substrate (50 mg dissolved in 98 ml PBS and 10 ~1 H,O,) for 7 min. All incubations were performed at room temperature in a moist chamber. After each step, the slides were washed twice with PBS. In negative controls, the primary antibody was replaced by PBS or non-immune mouse serum. Human placental tissue sections were used as the positive control. The level of GST-K expression in normal and cancerous oral tissues was assessed compared to the expression of GST-?r in human placenta (positive control), which was arbitrarily taken as moderate expression. The results were evaluated semiquantitatively and graded on an arbitrary 4point scale: + poor, equivocal or very weak staining; + + mild staining; + + + moderate staining; + + + + intense staining. The GST-r-t expression in human placental tissue sections was taken as moderate (+ + +).
Immunoblotting
Total cellular protein extracts (100 pg protein/lane) prepared from oral normal tissues, primary and recurrent SCCs were resolved on 10% polyacrylamide gels and proteins were transferred to nitrocellulose membranes. The membranes were treated with a blocking solution (5% nonfat milk in Tris-buffered saline, TBS containing O.l%, Tween 20), overnight at 4°C. Blots were probed for 2 h at 37’C with anti-GST-x antibody, membranes were washed three times with TBSiTween. Thereafter, membranes were incubated with rabbit anti-goat IgG-HRP conjugate for 1 h at 37X, washed, and the protein detected using diaminobenzidine as the chromogen, as described above.
Analysis
of GST-rr
gene status
by Southern
blotting
High molecular weight genomic DNA was prepared from human oral normal mucosa, primary as well as recurrent SCCs and placenta (positive control), by digestion with proteinase K and phenol/chloroform extraction [37]. The genomic DNA was digested with Hind111 and BamHI, subjected to electrophoresis using 0.8% agarose gels, transferred to nylon membrane, probed with a biotinylated cDNA GST-x probe and detected by a chemiluminescent method using lumigen-PPD reagent and autoradiography.
76
G. Sarkar et al.
Statistical analysis Comparison of GST-rt expression in primary and recurrent oral squamous cell carcinoma was performed using the chi-square test. McNemar’s chi-square test analysis was performed to compare the GST-rr: expression between the primary/recurrent cases and the matched normal tissues.
RESULTS The status of GST-rt gene and protein was evaluated in primary untreated oral SCCs (21 cases), recurrent tumours (22 cases) and matched normal oral tissues (31 cases) by southern hybridisation, immunoblotting and immunohistochemical analysis.
A significant with immunohistochemical observations. increase in GST-n: expression was seen in recurrent tumours (lanes F, G and H) and a marked increase was observed in primary tumours (lanes C, D and E). The GST-n expression in normal oral tissue ranged from mild to moderate levels (lanes A and B). Evaluation of GST-x gene status by southern blotting To determine if the increased levels of GST-rt are due to alterations at the genetic level, GST-n gene status was assessed in these tissue specimens. A representa-
Immunohistochemical analysis of GST-J-C Immunoreactivity of frozen oral tissue sections using antiGST-rt polyclonal antibody showed differential expression of the protein in normal tissues, primary and recurrent oral turnouts. The representative immunostained oral tissue sections are shown in Fig. 1. The normal tissue specimens were obtained from the patients with primary or recurrent oral SCCs from a site surrounding the lesions. Histopathological examination of haematoxylin and eosin stained sections showed features characteristic of normal oral tissue. However, mild to moderate expression of GST-rr was observed in these normal tissues (Fig. 1.a). The untreated primary oral SCCs showed moderate expression of GST-r-t (Fig. 1.b). The immunoreactivity in the epithelial cells was predominantly cytoplasmic. The hallmark of the study was the marked increase in GST-rt expression observed in recurrent oral tumours (Fig. 1.~). No staining was observed in control tissue sections of oral SCC treated with PBS or non-immune mouse serum in place of the primary antibody. Tissue sections of normal human placenta, used as positive control, showed moderate level of GST-x (data not shown). The results of immunohistochemical analysis of GST-rr expression in primary as well as recurrent SCCs and matched normal oral tissues are shown in Tables 1 and 2, respectively. In primary oral tumours, the enzyme expression varied from mild to moderate levels. In 11 out of 21 patients examined, there was poor to mild GST-lr expression, while 5 cases showed moderate expression (Table 1). The matched normal tissues did not show any significant difference in the expression of this protein. Out of 22 recurrent cases, GST-lr expression was intense in 7 (32.0%), moderate in 10 (45%), mild in 3 (14%) and poor in 2 (9%) cases (Table 2). However, in most recurrent cases GST-rc levels were substantially elevated above the levels in normal oral tissue from the same patient. Recurrent oral SCCs showed significant increase in GST-x expression as compared to the surrounding normal tissue (PC 0.05), as well as primary oral tumours (PC 0.001). Figure 2 gives a comparison of the level of GST-n expression in primary and recurrent oral SCCs. A positive association of GST-x expression in cancerous tissues was observed with beta1 quid and tobacco addiction. Detection of GST-rr by immunoblotting A representative immunoblot of oral normal tissue, primary as well as recurrent tumours, and a positive control (human placenta) is shown in Fig. 3. The data corroborate
Fig. 1. Immunohistochemical detection of GST-x sections using sheep polyclonal antibody. (a) Mild taining observed in normal oral tissue; (b) primary showing moderate expression of GST-x; (c) marked in GST-n expression in recurrent oral SCC (original cation a, b and c x400).
in frozen immunooral SCC increase magnifi-
Glutathione Table
1. Analysis
of GST-n
S-Transferase
expression
in primary
x in Oral Cancer oral
tumours
77 normal tissues
and matched
GST-x S. No.
Age/sex (years)
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
50 44 40 45 55 40 52 50 57 50 60 50 60 16 50 70 56 56 60 44 85
F F F M M M F M M M M M M M M M M M M F M
Histopathological grading
Addiction
cc. t.c. t.s. t.s. t.s. t.c.
t.c. t.s.
well
t.c. cc.
tive Southern blot showing GST-x gene status in norma1 and malignant oral tissues examined is shown in Fig. 4. No apparent change (amplification or deletion) was visible in any of the samples, suggesting that the overexpression of the protein is not due to gene amplification. of GST-n
2. Analysis
expression
+++ ++ ii ++ -I-++ ++ f +-I-+ + + ++ ++ + +++ ++ + ++ f-k ++ ++
staining; + + + + , intensive staining.
DISCUSSION Oral cancer is a major cause of morbidity and mortality in India. Over 80% of malignant lesions of the mouth and oropharynx are squamous cell carcinomas of the oral mucosa. Currently, the primary treatment for oral cancer is surgery to remove the cancer and any involved cervical lymph
in recurrent
oral
tumours
and
normal tissues
matched
GST-x S. No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Age/Sex (years) 65 42 52 60 52 55 67 70 64 60 50 60 75 53 37 63 46 60 50 50 38 47
F F M M M M F M M F F F M M M M M M M M F M
Histopathological Grading
Addiction
t.c. + t.s. t.c. t.c. t.s. t.s. t.c. t.c. + t.s. t.s. t.c. t.c. t.c. t.c. + t.s. t.c. t.c. t.s. t.c. t.c. + t.s.
+, poor, equvocal or very weak staining; + + , mild staining; t.c., tobacco chewing; f.s., tobacco smoking.
oral
+++
ND ND + ++ ++ + +++ ++ ND ND ND ND ND
others well well well well mod. well well
+, poor, equivocal or very weak staining; + + , mild staining; + + + , moderate t.c., tobacco chewing; t.s., tobacco smoking.
Table
+++ ++ ++ ++ ++ +++ ++ ii+
WC41
t.c. t.c.
Primary see
Normal
mod. well poor well well well well well mod well well
t.c. f.S. t.c.
expression
poor well others well others well mod. poor well well well well poor well well well well well mod. well well well
+ + + , moderate
expression
Normal
+++ +++ ++ ++ +++ ++ +++ + ii ND ND ND ++ ++ + + ++ ii +++ ND ND ++
statning; + + + + , intensive staining.
Primary see ++-I-+ -I-+++ +++ -I-++ +-I-+ ++++ ii+ ++-Ii-k++ 4-f ii+ +++ ++-I-+ +++ +++-I + ++ +++ +++ + ++ -I-+++
oral
G. Sarkar et al.
78
60
m
Primary
Poor
Mild
Moderate
-I
Intensive
Fig. 2. Comparison of GST-x expression in primary and recurrent oral SCC. The results have been evaluated semiquantitatively and graded on an arbitrary 4-point scale; + poor, equivocal or very weah staining; ++ mild staining; + + + moderate staining and + + + + intense staining.
nodes, followed by radiotherapy. Normally, the patients reporting to the clinics are in the advanced stages and palliative chemotherapy is the choice of treatment. However, the rate of success of these chemotherapeutic treatments is not predictable by the markers that are currently available. Given the established role of GST-a isoenzyme in drug detoxification in vitro and in cell culture, it is important to
kDa -200
-43
-29
-18’4
-14.3 Fig. 3. Immunoblot analysis of GST-x in oral tissues. Total celhdar protein extracts (100 pg protein/lane) prepared fi-om normal oral tissue, primary and recurrent oral SCC were resolved on a 10% polyacrylamide gel, transferred to nitrocehdose membrane, probed with GST-r polyclonal antibody and detected using diaminobenzidine as chromogen. Lanes A and B normal oral tissue; lanes C, D and E primary oral cancer; lanes F, G and H recurrent oral cancer.
Fig. 4. Southern blot analysis of the GST-z gene expression in oral tissues. Genomic DNA was extracted ikom normal tissue, primary and recurrent oral SCC and placenta (positive control) digested with Hi&III and BamHl, subjected to electrophoresis, transferred to nylon membrane, probed with biotinylated cDNA GST-s probe and detected by chemildnescent method. Lane A, normal oral tissue; lane B, primary oral cancer, lane C, recurrent oral cancer; lane D, placenta.
consider it as a possible prognostic marker. GSTs have also been used as markers for malignant and premalignant changes in a number of tissues [38]. Glutathione and GSTs have been implicated as important factors in tobacco related oral tumorigenesis as well as resistance to a number of anticancer agents. To determine whether GST-rt is implicated in betel and tobacco related oral carcinogenesis and/or drug resistance, the GST-rt protein expression was studied in oral untreated primary SCCs, recurrent tumours and matched normal oral tissue from the same patient. The GST-x expression in primary tumours predominantly varied from mild to moderate and no significant difference in its expression was observed in the matched normal tissue surrounding the lesions. A low level of GST-n. expression was observed in the oral mucosa of patients who did not consume betel and tobacco, although the number of such cases in this study was small. However, a low incidence of oral cancer among non-consumers of betel and tobacco has been reported by several other workers [39, 401. The marked expression of GST-rt by the nonpathological tissues of patients having primary tumours suggests that this isoenzyme may be a marker of increased carcinogen exposure but not
Glutathione
S-Transferase
necessarily a marker of oral cancer. Sasano et al. [41] could not observe any significant correlation between the amount or pattern of GST-rt expression in oesophageal carcinoma and the clinicopathological findings in these cases, while the non-pathological tissues in their series also showed marked increase in GST-rc levels, suggesting the potential of GST-rr as a plausible marker of increased carcinogen exposure. Alternatively, the mild to moderate levels of GST-x expression, observed in the matched normal tissues in our studies, may also support the proposed physiological role of this detoxification enzyme in the metabolism of xenobiotics and elimination of toxic substances [33]. The association between betel quid chewing and oral cancer has generated ample interest among researchers. In India, there is an unequivocal relationship between chewing of tobacco and the incidence of oral cancer [42, 431. Exposure of mammalian oral mucosal surface repeatedly to benzo(x)pyrenes and nitrosamines present in tobacco [44], and nitrosamines derived from areca nut [45], predisposes the whole field of tissue to the development of multiple primary malignant foci [46]. Carcinogenicity of betel quid has been shown to be considerably augmented by the presence of nut alkaloids (areca catechu), resulting in the stimulation of collagen synthesis by fibroblasts in vitro [47], and also by the use of lime or betel which, perhaps, releases the DNAdamaging reactive oxygen species and elicits reparative hyperplasia of the mucosa in response to cytotoxicity [48, 491. Thus, overexpression of GST-x by habitual tobacco chewers might be a marker of increased carcinogen exposure. A striking feature of this study is the significant increase in expression of GST-rt in recurrent oral tumours, as compared to primary oral turnours. Of the 22 patients with recurrent oral tumours, 5 patients who were non tobacco consumers at the time of development of recurrent tumours showed GST-rt levels comparable to those who were tobacco consumers. These patients had been habitual consumers of tobacco (heavy smokers/tobacco chewers), but had abstained from this habit at the time of development of primary tumours. The high level of GST-x expression in these patients may thus be due to the earlier habit of tobacco consumption or due to the treatment (chemotherapy/radiotherapy) received at the time of primary tumours. The recurrent oral tumours are often chemoresistant and show only partial response to combination chemotherapy (cisplatin 50 mg/m*, bleomycin 15 mgim2, methotrexate 40 mg/m’ iv.) for 4-6 weeks, which may partially suggest a role for this enzyme in multidrug resistance. Van der Zee et al., [50] in their study on ovarian cancers, found that the determination of P-gp, GST-x, c-erb B-2 and ~53 does not permit more accurate predictions of response to chemotherapy. Expression of GST-x in liver cirrhosis, colonic adenomatous polyps or dysplasia of the uterine cervix [40] indicate that it may be a useful marker, not only for some cancers, but also for high risk precancerous lesions [ 141. Studies on variability of GST-tt isoenzyme patterns in matched normal and cancerous human breast tissue have shown that, in most cases the average level of GST-x was substantially elevated in the cancer tissues above the levels in normal breast tissue from the same patient. Gilbert et al., [51] reported that a high GST-K level in the tumour was a
x in Oral Cancer
79
significant predictor of early recurrence and death in nodenegative breast cancer patients. Studies on colorectal tumours [52] indicate that GST subclass levels in colorectal adenocarcinomas are not related to clinicopathological parameters and that GST enzyme activity has a prognostic value for the overall survival of the patients. Yellin et aZ., [53] in their study on the relationship of glutathione and GST-x to cisplatin in human head and neck carcinoma cell lines, observed no relationship between GST-x gene amplification, mRNA expression and drug resistance, despite the capacity of cisplatin to induce GST-tr enzyme activity. The finding of increased a-class GST and mdr gene expression in ras-transfected rat liver epithelial cells [54], suggests that these genes may be regulated by cellular oncogenes and that GST-n: may be useful in screening for malignant transformation in tumours. Since drug resistance remains a major obstacle in the effective treatment of cancer, interest has also been focussed on the elucidation of a molecular mechanism of resistance. Although our knowledge as to which factors are responsible for a regulated coexpression of resistance mechanism is little, one possibility proposed is that the resistance factors belong to a set of genes, which is controlled by a general regulatory mechanism. The c-fos/c-jun protein complex which binds specifically to AP-1 affects the transcriptional expression of several cellular genes. Interestingly, P-glycoprotein and glutathioneS-transferase contain the AP-1 motif, thus these genes may also be regulated by the cellular oncogenes c-fos and c-jun [55, 561. In conclusion, our preliminary data on the expression of GST-rt in matched normal and cancerous oral tissues indicate that markedly increased levels of GST-x in primary oral tumours and significantly higher levels in recurrent oral SCCs may be an index for the prognosis of the disease. Changes in GST-rt levels may also account for a certain pattern of drug resistance and may be a potential route for the modulation of such resistance. However, the exact role of this GST isoenzyme in the resistance of a human neoplasm to a particular anticancer drug will be subject to more extensive clinical studies which relate changes in gene expression to clinical outcome.
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cells. Journul qj Biology und Chernlrtr~,, 1986, 261, 1554415549. Cowan, K. H., Batist, G., Tulpule, A., Sinha, B. K. and Myers, C. E., Similar biochemical changes associated with multidrug resistance in human breast cancer cells and carcinogens induced resistance to xenobiotics in rats. Proceedings of Ihe Nurionul Academy of Sciences, ll.S.A., 1986, 83, 93289332. Fairchild, C. R., Ivy, S. I’. and Kao-Shan, C. S., et al., Isolation of amplified and overexpressed DNA sequences from adriamycin resistant human breast cancer cells. Cancer Research, 1987, 47, 5141-5148. Nakagawa, K., Saijo, N. and Tsuchida, S., er al., Glutathione S-transferase x as a determinant of drug resistance in transfectant cell lines. Journul of Biology and Chemistry, 1990, 265, 4296-4302. Puchalski, R. B. and Fahl, W. E., Expression of recombinant glutathione. S-transferase pi, ya or ybi confers resistance to alkylating agents. Proceedings y/ the National Academy of Sciences, U.S.A., 1990, 87, 2443-2449. Moscow, J. A., Townsend, A. J. and Kowan, K. H., Elevation of the pi class of glutathione S-transferase activity in human breast cancer cells by transfection of the GST pi gene and its effect on sensitivity to toxins. Molecular Pharmucolog)~, 1989, 36, 22-28. Terrier, I’., Townsend, A. J., Coindrc, J. M., Triche, T. J. and Cowan, K. H., An immunohistochemical study of pi class glutathione S-transferase expression in normal human tissue. Americun Journal of Pathology, 1990, 137, 845-853. Pindborg, J. J., Epidemiology of oral cancer. In Oral Cancer and Precancer. John Wright, Bristol, 1980, l-l 1. Johnson, N. W., A global view of the epidemiology of oral cancer. In Risk Markers for Oral Disease, Oral Cancer, Detection of Patients and Lesions at Risk, Vol. 2, ed. N. Johnson. Cambridge University Press, Cambridge, 1991, pp. 3-26. Gupta, I’. C., Mehta, F. S. and Daftary, D. K., Incidence rates of oral cancer and natural history of oral precancerous lesions in a 10 year follow-up study of Indian villagers. Communit? Den&l Oral Epidemiology, 1980, 8, 283-333. Sambrook, J., Fritsch, F. F. and Maniatis, T. Molecular Cloning. A Labratoy Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1969. Hall, A., Foster, S., Proctor, S. J. and Cattan, A. R., Purification and characterization of a pi class glutathione Stransferase from human leukaemic cells. Brirish Journal qf Haematolog~, 1990, 76, 494-500. Rana Singh, A. W., Wamakulasuriya, K. A. A. S. and Johnson, N. W., Low prevalence of expression of ~53 oncoprotein in oral carcinomas from Sri Lanka associated with betel and tobacco chewing. Oral Oncology,, European Journal of‘ Cancer, 1993,29B, 147-150. Wamakulasuriya, K. A. A. S., Smoking and chewing habits in Sri Lanka: implications for oral cancer and precancer. In Control of Tobacco Related Cancers and Other Diseases, eds P. C. Hamner, I’. R. Murti, P. C. Gupta. Oxford University Press, Bombay, 1992, pp. 113-l 18. Sasano, H., Mirazaki, S., Shiga, K., Goukon, Y., Nishihara, T. and Nagura, H., GST in human oesophageal carcinoma. Anticanwr Resrarch, 1993, 13, 363-368. Daftary, D. K., The situation in high risk areas of the world. In Risk Markers for Oral Diseases, Vol. 2, ed. N. W. Johnson. Cambridge University Press, Cambridge. Gupta, P. C., Mehta, F. S., Pindborg, J. J., Aghi, M. B., Bhonsle, R. B. and Murti, P. R., An educational intervention study for tobacco chewing and smoking habits among Indian villagers. In Smoking and Health, eds A.M. Hisamichi and S. Tominaga. Excetpta Medica, Amsterdam, p. 623. Hecht, S. S. and Hoffmann, D., N-nitroso compounds and tobacco induced cancers in man. Inlernational Agency ,for Research in Cancer Scientific Publications, 199 1, 105, 54461. Prokopczyk, B., Rivenson, A., Bertinat, P., Brunneman, K. D. and Hoffmann, D., 3-(methylnitrosamino) proplonitrile: occurrence in saliva of betel quid chewers, carcinogenicity, and DNA methylation in F344 rats. Cancer Research, 1987, 47, 467-471.
Glutathione
S-Transferase
46.
Slaughter, D. I’., Southwick, H. W. and Smejkal, W., “Field cancerization” in oral stratified squamous epithelium: Clinical implications of muticentric origin. Cancer, 1953, 6, 963-968. 47. Caniff, J. I’. and Harvey, W., The aetiology of oral sub mucous fibrosis. The stimulation of collagen synthesis by extracts of Arecanuts. hternutional Journal qf Oral Surger~~, 1981, 10, 163-167. 48. Nair, U. J., Friesen, M., Richard, I., MacLennan, R., Thomas, S. J. and Bartsch, H., Effect of lime composition on the formation of reactive, oxygen species from Arecanut extract in vitro. Carcinogenesis, 1990, 11, 2145-2148. 49. Nair, U. J., Obe, G., Friesen, M., Goldberg, M. T. and Bartsch, H., Role of lime in the generation of reactive oxygen species from betel quid ingredients. En~ironnwntul Health Perspwtiwr,
50.
51.
1992,
98,
203-205.
Van der Zee, A. G., Hollema, H. and Sourmeijer, A. J., et ai., GSTpi, c-erb-2 and ~53 as prognostic factors in ovarian carcinomas. Journal of Clinical Onrologv, 1995, 13, 70-78. Gilbert, L., Elwood, L. J. and Merino, M., er al., A pilot study of II class glutathione S-transferase expression in breast cancer: correlation with estrogen receptor expression and prognosis in node negative breast cancer. Journal of Chid Onc~olo~~~, 1993,11,45-58.
rr in Oral
Cancer
81
52. Mulder, T. P. J., Verspaget, H. W. and Cornelis, F. M., et al., Glutathione S-transferase II in colorectal tumors is predictive for overall survival. Cancer Reseurch, 1995, 85, 2696-2702. 53. Yellin, S. A., Davidson, B. J., Pinto, J. T., Sacks, I’. G., Qiao, C. and Schantz, S. I’., Relationship of glutathione and glutathione S-transferase to cisplatin sensitivity in human head and neck squamous carcinoma cell lines. Crrncer Letrers, 1994, 85, 345-35 1. 54. Burt, R. K., Garfield, S., Johnson, K. and Thorgeirsson, S. S., Transformation of rat liver epithelial cells with V-H-ras or v-raf causes expression of MDR-1, glutathione S-transferase P and increased resistance to cytotoxic chemicals. Cmcinogenesis, 1988, 9, 2329-2332. 55. Angel, I’. and Karin, M., The role of iun, Fos and the Ap-1 complex in cell proliferation and transformation. Biodwmistr~ mu/ Bioph.rlGcs
Ac /(I, 1991,
1072,
129-157.
56. Teeter, L. D., Eckersberg, T., Tsai, Y. and Kuo, M., Analysis of the Chinese hamster p-glycoprotein multidrug resistance gene pgp-1 reveals that the AR-1 site is essential for full promoter activity. Cell Grm~h Dij/erenriation, 1991, 2, 429-437. Acknowledgements-This
grant
from
the Department
study was supported by a research of Science and Technology, India.
,
Ora, Chcoi
PII:
Glutathione
817.00+0.00
SO964-1955(96)00060-7
S-Transferase n Expression Normal and Malignant Oral
in Matched Mucosa
Human
G. Sarkar, N. Nath, N.K. Shukla and R. Ralhan Department
of Biochemistry,
Institute
Rotary Nagar,
Cancer Hospital, All India New Delhi 110029, India
Institute
of Medical
Sciences,
Ansari
Over-expression of glutathione S-transferase x (GST-x) class isozyme is often associated with malignant transformation and/or drug resistance. To determine whether GST-K is implicated in betel and tobacco related oral carcinogenesis and/or drug resistance, its expression was studied in oral untreated primary squamous cell carcinomas (SCCs), recurrent tumours and matched normal oral tissues by immunohistochemical and immunoblotting techniques. The GST-x expression in primary tumours predominantly varied from mild to moderate levels and no significant difference in its expression was observed in the matched normal tissue surrounding these lesions. Mild to moderate levels of GST-n: expression in the oral mucosa of consumers of betel and tobacco observed in the matched normal tissues may support the physiological role of this detoxification enzyme in the metabolism of xenobiotics and elimination of toxic constituents of tobacco. The hallmark of the study is the significant increase in GST-x expression in recurrent oral SCCs, compared to the matched normal tissues, as well as primary oral tumours, suggesting its potential role as an indicator of prolonged exposure to carcinogens, or prognosis of the disease. :c 1997 Elsevier Science Ltd Key words: glutathione Oral Oncology,
S-transferase A, oral tissues, tobacco, human, squamous cell carcinoma
Vol. 33, No. 2, pp. 74-81,
1997
tive to the normal surrounding tissue, as well as in the serum of patients with cancer [ 14-241. The carcinogen-induced alterations in rat preneoplastic liver nodules are also associated with the development of broad spectrum resistance to hepatotoxins [25]. Cell lines selected for resistance to different antineoplastic agents have also been shown to exhibit an increase in GST-n activity, suggesting that changes in the expression of this enzyme in human tumours may be a marker of drug resistance [2629]. Gene transfer experiments have also shown that GST isozymes play a role in conferring resistance to drugs such as melphalan, cisplatin and doxorubicin [30-321. Inhibition of GST-n activity has been suggested as an effective approach to circumvent drug resistance. Studies on GST-rr expression in normal human tissues have shown that this isozyme is predominantly expressed in normal epithelial cells of the urinary, digestive and respiratory tracts, suggesting a possible role in detoxification and elimination of toxic substances [33]. However, there is a paucity of studies on the expression and role of GST-x in human normal and malignant oral mucosa. Oral squamous cell carcinoma (SCC) is the most common malignant neoplasm in South East Asia [34, 351. It is a disease of acquired occupational, nutritional and environmental insults. Chewing of betel quid containing lime, areca-
INTRODUCTION
The glutathione S-transferases (GSTs, EC2.5.1.18) constitute a multigene superfamily of cytosolic enzymes that catalyse the nucleophilic addition of glutathione to electrophilic centres of a wide variety of compounds, thereby playing an important role in protecting cells from xenobiotics [l-3]. These enzymes are involved in detoxification of cancer chemotherapeutic agents such as cisplatin, alkylating agents and doxorubicin [4-71. Enhanced formation of drug-GSH conjugates by the GSTs may reduce the sensitivity of neoplastic cells to the action of these drugs, thereby providing a mechanism for the emergence of drug-resistant tumours. On the basis of their structural, physicochemical, enzymatic and immunological properties, the GSTs are divided into four classes: CI, p. n and 8 [3, 81. The TI isozyme of GST has been associated with malignant transformation in rat [9-l l] and human tumours [12]. Studies in the Solt-Farber model of chemical carcinogenesis have shown that GST-P, a member of the K class is a useful marker for detection of preneoplastic and neoplastic cells [ 131. Over-expression of GST-rr has been observed in a wide variety of human tumours relaCorrespondence to R. R&an. Received 2i June 1996; provisionally accepted revised manuscript received 22 Aug. 1996.
24
July
1996;
74
Glutathione
S-Transferase
nut and tobacco, along with smoking, has been causally associated with the high incidence of oral cancer in India [36]. Remarkably, the location of tumours within the oral cavity of habitual tobacco-folded betel quid chewers correlates with the site where the quid is kept for prolonged time periods. Chronic exposure of the oral cavity of habitual tobacco quid chewers to a variety of carcinogens present in the quid may lead to overexpression of GST-rt. To understand the role of GST-rt as a marker of neoplastic transformation, progression, or drug resistance in oral malignancies, we initiated this study on the evaluation of the GST-x gene status and its expression in oral normal mucosa, primary, as well as recurrent SCCs, and matched normal mucosa. These results were correlated with clinicopathological parameters of the patients.
MATERIALS Tissue
AND
METHODS
specimens
Surgical specimens from 21 primary squamous cell carcinomas of the oral cavity, 22 recurrent oral SCCs, and their matched normal oral tissues were obtained from the Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, India. The oral cancer patients were divided into two groups: (i) primary oral tumours and, (ii) recurrent oral squamous cell carcinoma. Subjects enrolled in the cancer clinic at the onset of the primary tumours were included in group (i). These patients did not receive any treatment at the time of inclusion in the study. In group (ii), patients who enrolled with the recurrent stage of oral cancer were included. Biopsy specimens of normal oral tissues were obtained from cancer patients from normal tissue surrounding the tumour. A piece of the biopsy specimen was snap frozen and stored at -80°C for immunohistochemical analysis, while another was put in formalin for histopathological examination. The chnicopathological features of the patient namely, age, sex, tobacco- and betel-chewing history were recorded in each case. Histopathological studies included the histological grading of the lymphoventricular response, to establish histological differentiation of the tumours. Among the 21 primary oral tumour specimens examined, 16 were well differentiated SCCs, three moderately differentiated cases, one poorly differentiated carcinoma and one belonged to a group other than squamous cell carcinomas. In the recurrent oral SCC group, 15 were well differentiated, two moderately differentiated, three poorly differentiated and two belonged to a group of nonSCCs. Site distribution of oral cancer was also investigated. The buccal mucosa was found to be the commonest site (43.18%), followed by the lower alveolus (34.09%), the tongue (11.36%) and the soft palate (11.36%). Reagents
and DNA
probe
Sheep polyclonal GST-rr: antibody (The Binding Site, Birmingham, U.K.) is raised against human placental GST. Rabbit anti-goat IgG-HRP conjugate was kindly provided by Dr J. K. Batra, National Institute of Immunology, New Delhi, India. The cDNA for GST-rr: (cDNA-GST-x) cloned in pGEM4 (3.6 Kb plasmid) was obtained as a gift from Dr Jeffrey A. Moscow, National Institute of Health, Bethesda, Maryland, U.S.A. The cDNA-GST-rt is a 725 bp fragment,
75
R in Oral Cancer
subcloned at the EcoRI site in the cloning at its multiple cloning site (MCS).
vector pGEM4
Immunohistochemistry
Cryosections (5-8 pm thickness) of human oral tissue specimens were collected on slides and fixed in acetone for 10 min. Representative sections were stained with haematoxylin and eosin for histopathological analysis, while immunostaining was carried out on serial sections. For immunohistochemical analysis, the sections were incubated in methanol containing hydrogen peroxide (0.3% v/v) for 30 min to quench endogenous peroxidase activity. Subsequently, sections were incubated with 1% BSA for 20 min to block the non-specific binding, followed by incubation with anti-GST-n: (sheep polyclonal, 10 ng protein) for 16 h at 4°C. The secondary antibody, rabbit anti-goat IgG-HRP conjugate (10 ng) was added and sections were incubated for 1 h. After washing with PBS three times, the immunostaining was performed using diaminobenzidine as the substrate (50 mg dissolved in 98 ml PBS and 10 ~1 H,O,) for 7 min. All incubations were performed at room temperature in a moist chamber. After each step, the slides were washed twice with PBS. In negative controls, the primary antibody was replaced by PBS or non-immune mouse serum. Human placental tissue sections were used as the positive control. The level of GST-K expression in normal and cancerous oral tissues was assessed compared to the expression of GST-?r in human placenta (positive control), which was arbitrarily taken as moderate expression. The results were evaluated semiquantitatively and graded on an arbitrary 4point scale: + poor, equivocal or very weak staining; + + mild staining; + + + moderate staining; + + + + intense staining. The GST-r-t expression in human placental tissue sections was taken as moderate (+ + +).
Immunoblotting
Total cellular protein extracts (100 pg protein/lane) prepared from oral normal tissues, primary and recurrent SCCs were resolved on 10% polyacrylamide gels and proteins were transferred to nitrocellulose membranes. The membranes were treated with a blocking solution (5% nonfat milk in Tris-buffered saline, TBS containing O.l%, Tween 20), overnight at 4°C. Blots were probed for 2 h at 37’C with anti-GST-x antibody, membranes were washed three times with TBSiTween. Thereafter, membranes were incubated with rabbit anti-goat IgG-HRP conjugate for 1 h at 37X, washed, and the protein detected using diaminobenzidine as the chromogen, as described above.
Analysis
of GST-rr
gene status
by Southern
blotting
High molecular weight genomic DNA was prepared from human oral normal mucosa, primary as well as recurrent SCCs and placenta (positive control), by digestion with proteinase K and phenol/chloroform extraction [37]. The genomic DNA was digested with Hind111 and BamHI, subjected to electrophoresis using 0.8% agarose gels, transferred to nylon membrane, probed with a biotinylated cDNA GST-x probe and detected by a chemiluminescent method using lumigen-PPD reagent and autoradiography.
76
G. Sarkar et al.
Statistical analysis Comparison of GST-rt expression in primary and recurrent oral squamous cell carcinoma was performed using the chi-square test. McNemar’s chi-square test analysis was performed to compare the GST-rr: expression between the primary/recurrent cases and the matched normal tissues.
RESULTS The status of GST-rt gene and protein was evaluated in primary untreated oral SCCs (21 cases), recurrent tumours (22 cases) and matched normal oral tissues (31 cases) by southern hybridisation, immunoblotting and immunohistochemical analysis.
A significant with immunohistochemical observations. increase in GST-n: expression was seen in recurrent tumours (lanes F, G and H) and a marked increase was observed in primary tumours (lanes C, D and E). The GST-n expression in normal oral tissue ranged from mild to moderate levels (lanes A and B). Evaluation of GST-x gene status by southern blotting To determine if the increased levels of GST-rt are due to alterations at the genetic level, GST-n gene status was assessed in these tissue specimens. A representa-
Immunohistochemical analysis of GST-J-C Immunoreactivity of frozen oral tissue sections using antiGST-rt polyclonal antibody showed differential expression of the protein in normal tissues, primary and recurrent oral turnouts. The representative immunostained oral tissue sections are shown in Fig. 1. The normal tissue specimens were obtained from the patients with primary or recurrent oral SCCs from a site surrounding the lesions. Histopathological examination of haematoxylin and eosin stained sections showed features characteristic of normal oral tissue. However, mild to moderate expression of GST-rr was observed in these normal tissues (Fig. 1.a). The untreated primary oral SCCs showed moderate expression of GST-r-t (Fig. 1.b). The immunoreactivity in the epithelial cells was predominantly cytoplasmic. The hallmark of the study was the marked increase in GST-rt expression observed in recurrent oral tumours (Fig. 1.~). No staining was observed in control tissue sections of oral SCC treated with PBS or non-immune mouse serum in place of the primary antibody. Tissue sections of normal human placenta, used as positive control, showed moderate level of GST-x (data not shown). The results of immunohistochemical analysis of GST-rr expression in primary as well as recurrent SCCs and matched normal oral tissues are shown in Tables 1 and 2, respectively. In primary oral tumours, the enzyme expression varied from mild to moderate levels. In 11 out of 21 patients examined, there was poor to mild GST-lr expression, while 5 cases showed moderate expression (Table 1). The matched normal tissues did not show any significant difference in the expression of this protein. Out of 22 recurrent cases, GST-lr expression was intense in 7 (32.0%), moderate in 10 (45%), mild in 3 (14%) and poor in 2 (9%) cases (Table 2). However, in most recurrent cases GST-rc levels were substantially elevated above the levels in normal oral tissue from the same patient. Recurrent oral SCCs showed significant increase in GST-x expression as compared to the surrounding normal tissue (PC 0.05), as well as primary oral tumours (PC 0.001). Figure 2 gives a comparison of the level of GST-n expression in primary and recurrent oral SCCs. A positive association of GST-x expression in cancerous tissues was observed with beta1 quid and tobacco addiction. Detection of GST-rr by immunoblotting A representative immunoblot of oral normal tissue, primary as well as recurrent tumours, and a positive control (human placenta) is shown in Fig. 3. The data corroborate
Fig. 1. Immunohistochemical detection of GST-x sections using sheep polyclonal antibody. (a) Mild taining observed in normal oral tissue; (b) primary showing moderate expression of GST-x; (c) marked in GST-n expression in recurrent oral SCC (original cation a, b and c x400).
in frozen immunooral SCC increase magnifi-
Glutathione Table
1. Analysis
of GST-n
S-Transferase
expression
in primary
x in Oral Cancer oral
tumours
77 normal tissues
and matched
GST-x S. No.
Age/sex (years)
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
50 44 40 45 55 40 52 50 57 50 60 50 60 16 50 70 56 56 60 44 85
F F F M M M F M M M M M M M M M M M M F M
Histopathological grading
Addiction
cc. t.c. t.s. t.s. t.s. t.c.
t.c. t.s.
well
t.c. cc.
tive Southern blot showing GST-x gene status in norma1 and malignant oral tissues examined is shown in Fig. 4. No apparent change (amplification or deletion) was visible in any of the samples, suggesting that the overexpression of the protein is not due to gene amplification. of GST-n
2. Analysis
expression
+++ ++ ii ++ -I-++ ++ f +-I-+ + + ++ ++ + +++ ++ + ++ f-k ++ ++
staining; + + + + , intensive staining.
DISCUSSION Oral cancer is a major cause of morbidity and mortality in India. Over 80% of malignant lesions of the mouth and oropharynx are squamous cell carcinomas of the oral mucosa. Currently, the primary treatment for oral cancer is surgery to remove the cancer and any involved cervical lymph
in recurrent
oral
tumours
and
normal tissues
matched
GST-x S. No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Age/Sex (years) 65 42 52 60 52 55 67 70 64 60 50 60 75 53 37 63 46 60 50 50 38 47
F F M M M M F M M F F F M M M M M M M M F M
Histopathological Grading
Addiction
t.c. + t.s. t.c. t.c. t.s. t.s. t.c. t.c. + t.s. t.s. t.c. t.c. t.c. t.c. + t.s. t.c. t.c. t.s. t.c. t.c. + t.s.
+, poor, equvocal or very weak staining; + + , mild staining; t.c., tobacco chewing; f.s., tobacco smoking.
oral
+++
ND ND + ++ ++ + +++ ++ ND ND ND ND ND
others well well well well mod. well well
+, poor, equivocal or very weak staining; + + , mild staining; + + + , moderate t.c., tobacco chewing; t.s., tobacco smoking.
Table
+++ ++ ++ ++ ++ +++ ++ ii+
WC41
t.c. t.c.
Primary see
Normal
mod. well poor well well well well well mod well well
t.c. f.S. t.c.
expression
poor well others well others well mod. poor well well well well poor well well well well well mod. well well well
+ + + , moderate
expression
Normal
+++ +++ ++ ++ +++ ++ +++ + ii ND ND ND ++ ++ + + ++ ii +++ ND ND ++
statning; + + + + , intensive staining.
Primary see ++-I-+ -I-+++ +++ -I-++ +-I-+ ++++ ii+ ++-Ii-k++ 4-f ii+ +++ ++-I-+ +++ +++-I + ++ +++ +++ + ++ -I-+++
oral
G. Sarkar et al.
78
60
m
Primary
Poor
Mild
Moderate
-I
Intensive
Fig. 2. Comparison of GST-x expression in primary and recurrent oral SCC. The results have been evaluated semiquantitatively and graded on an arbitrary 4-point scale; + poor, equivocal or very weah staining; ++ mild staining; + + + moderate staining and + + + + intense staining.
nodes, followed by radiotherapy. Normally, the patients reporting to the clinics are in the advanced stages and palliative chemotherapy is the choice of treatment. However, the rate of success of these chemotherapeutic treatments is not predictable by the markers that are currently available. Given the established role of GST-a isoenzyme in drug detoxification in vitro and in cell culture, it is important to
kDa -200
-43
-29
-18’4
-14.3 Fig. 3. Immunoblot analysis of GST-x in oral tissues. Total celhdar protein extracts (100 pg protein/lane) prepared fi-om normal oral tissue, primary and recurrent oral SCC were resolved on a 10% polyacrylamide gel, transferred to nitrocehdose membrane, probed with GST-r polyclonal antibody and detected using diaminobenzidine as chromogen. Lanes A and B normal oral tissue; lanes C, D and E primary oral cancer; lanes F, G and H recurrent oral cancer.
Fig. 4. Southern blot analysis of the GST-z gene expression in oral tissues. Genomic DNA was extracted ikom normal tissue, primary and recurrent oral SCC and placenta (positive control) digested with Hi&III and BamHl, subjected to electrophoresis, transferred to nylon membrane, probed with biotinylated cDNA GST-s probe and detected by chemildnescent method. Lane A, normal oral tissue; lane B, primary oral cancer, lane C, recurrent oral cancer; lane D, placenta.
consider it as a possible prognostic marker. GSTs have also been used as markers for malignant and premalignant changes in a number of tissues [38]. Glutathione and GSTs have been implicated as important factors in tobacco related oral tumorigenesis as well as resistance to a number of anticancer agents. To determine whether GST-rt is implicated in betel and tobacco related oral carcinogenesis and/or drug resistance, the GST-rt protein expression was studied in oral untreated primary SCCs, recurrent tumours and matched normal oral tissue from the same patient. The GST-x expression in primary tumours predominantly varied from mild to moderate and no significant difference in its expression was observed in the matched normal tissue surrounding the lesions. A low level of GST-n. expression was observed in the oral mucosa of patients who did not consume betel and tobacco, although the number of such cases in this study was small. However, a low incidence of oral cancer among non-consumers of betel and tobacco has been reported by several other workers [39, 401. The marked expression of GST-rt by the nonpathological tissues of patients having primary tumours suggests that this isoenzyme may be a marker of increased carcinogen exposure but not
Glutathione
S-Transferase
necessarily a marker of oral cancer. Sasano et al. [41] could not observe any significant correlation between the amount or pattern of GST-rt expression in oesophageal carcinoma and the clinicopathological findings in these cases, while the non-pathological tissues in their series also showed marked increase in GST-rc levels, suggesting the potential of GST-rr as a plausible marker of increased carcinogen exposure. Alternatively, the mild to moderate levels of GST-x expression, observed in the matched normal tissues in our studies, may also support the proposed physiological role of this detoxification enzyme in the metabolism of xenobiotics and elimination of toxic substances [33]. The association between betel quid chewing and oral cancer has generated ample interest among researchers. In India, there is an unequivocal relationship between chewing of tobacco and the incidence of oral cancer [42, 431. Exposure of mammalian oral mucosal surface repeatedly to benzo(x)pyrenes and nitrosamines present in tobacco [44], and nitrosamines derived from areca nut [45], predisposes the whole field of tissue to the development of multiple primary malignant foci [46]. Carcinogenicity of betel quid has been shown to be considerably augmented by the presence of nut alkaloids (areca catechu), resulting in the stimulation of collagen synthesis by fibroblasts in vitro [47], and also by the use of lime or betel which, perhaps, releases the DNAdamaging reactive oxygen species and elicits reparative hyperplasia of the mucosa in response to cytotoxicity [48, 491. Thus, overexpression of GST-x by habitual tobacco chewers might be a marker of increased carcinogen exposure. A striking feature of this study is the significant increase in expression of GST-rt in recurrent oral tumours, as compared to primary oral turnours. Of the 22 patients with recurrent oral tumours, 5 patients who were non tobacco consumers at the time of development of recurrent tumours showed GST-rt levels comparable to those who were tobacco consumers. These patients had been habitual consumers of tobacco (heavy smokers/tobacco chewers), but had abstained from this habit at the time of development of primary tumours. The high level of GST-x expression in these patients may thus be due to the earlier habit of tobacco consumption or due to the treatment (chemotherapy/radiotherapy) received at the time of primary tumours. The recurrent oral tumours are often chemoresistant and show only partial response to combination chemotherapy (cisplatin 50 mg/m*, bleomycin 15 mgim2, methotrexate 40 mg/m’ iv.) for 4-6 weeks, which may partially suggest a role for this enzyme in multidrug resistance. Van der Zee et al., [50] in their study on ovarian cancers, found that the determination of P-gp, GST-x, c-erb B-2 and ~53 does not permit more accurate predictions of response to chemotherapy. Expression of GST-x in liver cirrhosis, colonic adenomatous polyps or dysplasia of the uterine cervix [40] indicate that it may be a useful marker, not only for some cancers, but also for high risk precancerous lesions [ 141. Studies on variability of GST-tt isoenzyme patterns in matched normal and cancerous human breast tissue have shown that, in most cases the average level of GST-x was substantially elevated in the cancer tissues above the levels in normal breast tissue from the same patient. Gilbert et al., [51] reported that a high GST-K level in the tumour was a
x in Oral Cancer
79
significant predictor of early recurrence and death in nodenegative breast cancer patients. Studies on colorectal tumours [52] indicate that GST subclass levels in colorectal adenocarcinomas are not related to clinicopathological parameters and that GST enzyme activity has a prognostic value for the overall survival of the patients. Yellin et aZ., [53] in their study on the relationship of glutathione and GST-x to cisplatin in human head and neck carcinoma cell lines, observed no relationship between GST-x gene amplification, mRNA expression and drug resistance, despite the capacity of cisplatin to induce GST-tr enzyme activity. The finding of increased a-class GST and mdr gene expression in ras-transfected rat liver epithelial cells [54], suggests that these genes may be regulated by cellular oncogenes and that GST-n: may be useful in screening for malignant transformation in tumours. Since drug resistance remains a major obstacle in the effective treatment of cancer, interest has also been focussed on the elucidation of a molecular mechanism of resistance. Although our knowledge as to which factors are responsible for a regulated coexpression of resistance mechanism is little, one possibility proposed is that the resistance factors belong to a set of genes, which is controlled by a general regulatory mechanism. The c-fos/c-jun protein complex which binds specifically to AP-1 affects the transcriptional expression of several cellular genes. Interestingly, P-glycoprotein and glutathioneS-transferase contain the AP-1 motif, thus these genes may also be regulated by the cellular oncogenes c-fos and c-jun [55, 561. In conclusion, our preliminary data on the expression of GST-rt in matched normal and cancerous oral tissues indicate that markedly increased levels of GST-x in primary oral tumours and significantly higher levels in recurrent oral SCCs may be an index for the prognosis of the disease. Changes in GST-rt levels may also account for a certain pattern of drug resistance and may be a potential route for the modulation of such resistance. However, the exact role of this GST isoenzyme in the resistance of a human neoplasm to a particular anticancer drug will be subject to more extensive clinical studies which relate changes in gene expression to clinical outcome.
1.
Habig, W. H., Pabst, M. J. and Jakoby, W. B., Glutathione Stransferases. Journal qf Biology and Chemistry, 1974, 249,
2.
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the Department
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