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Nitric oxide synthase expression is downregulated in basal cell carcinoma of the head and neck
British Journal of Oral and Maxillofacial Surgery (2000) 38, 633–636 © 2000 The British Association of Oral and Maxillofacial Surgeons doi: 10.1054/bjom.2000.0538
BRITISH JOURNAL OF ORAL
& M A X I L L O FAC I A L S U R G E RY
Nitric oxide synthase expression is downregulated in basal cell carcinoma of the head and neck P. A. Brennan,* T. Umar,† J. Bowden,‡ A. Hobkirk,§ A. V. Spedding,¶ B. Conroy,** G. Zaki,†† D. W. Macpherson‡‡ *BUPA Research Fellow, Maxillofacial Unit; †Specialist Registrar in Histopathology; ‡Specialist Registrar in Oral and Maxillofacial Surgery; §Medical Student; ¶Consultant Histopathologist, Queen Alexandra Hospital, Portsmouth; **Consultant Histopathologist, St Richard’s Hospital, Chichester; ††Consultant Oral and Maxillofacial Surgeon, Queen Alexandra Hospital, Portsmouth; ‡‡Consultant Oral and Maxillofacial Surgeon, St Richard’s Hospital, Chichester, West Sussex, UK SUMMARY. The small molecule nitric oxide (NO) has many actions, most of which are poorly understood. Recently, NO and related compounds have been implicated in skin damage caused by ultraviolet light although their exact role is not clear. We undertook an immunohistochemical study to assess the expression of type II NO synthase (NOS2) and type III (NOS3) in basal cell carcinomas (BCCs) of the head and neck. In all 48 cases studied, NOS2 was found in the basal cell layer of the skin at the tumour margin but it was significantly reduced in the tumour epithelial cells (P0.001). NOS3 was localized to the endothelium of the blood vessels in both skin and tumour in all cases, and it was not seen in the tumour epithelial cells. The results suggest that expression of NOS is down-regulated in basal cell carcinomas. © 2000 The British Association of Oral and Maxillofacial Surgeons
INTRODUCTION
reports of the distribution of NOS in these tumours. There are at least three NOS isoenzymes, each being the product of a separate gene.7 NOS1, also called nNOS as it was first found in neural tissue, is not present in normal keratinocytes,8 but has been detected in pigmented skin lesions including melanoma.9 NOS2, also named iNOS (immunological) is produced in response to cytokine release. It is found in many cells including macrophages, natural killer cells, and keratinocytes.8 NOS3, originally detected in the endothelium of blood vessels, is also found in keratinocytes, fibroblasts, arrector pili muscle and sweat glands.8 As the activity of NOS1 and NOS3 is dependent on raised local calcium concentrations, probably beyond 500 nM (from a resting level of 70–100 nM), their production of NO is transient.10 In this respect, most studies of NO in cancer have been directed at NOS2, which produces NO for many hours or days, after induction. There are still conflicting views about the role of NO in human cancer, compounded by the fact that it has both tumourpromoting and cytotoxic activities. Much of this controversy stems from studies of murine tumours, in which the production of NO by both tumour cells and macrophages is much greater in human cancers. A growing body of evidence suggests that the concentrations of NO found in human cancers are at least one to two orders of magnitude lower than that required for cytotoxicity.11 The result is that NO probably contributes to the development of human cancer by facilitating angiogenesis and dissemination.12
The small molecule nitric oxide (NO) has many biological actions. It is readily able to pass through cell membranes and influence enzymes and proteins in both the cytosol and the nucleus. These effects result from conformational changes in proteins (in a similar fashion to those seen when oxygen binds to haemoglobin) as well as direct chemical reactions with electron-accepting moieties such as thiols and transition metals. It may therefore act as a signalling molecule as well as having cytotoxic and possible genotoxic effects. The toxicity of NO is probably the result of the reaction of NO with the superoxide free radical (O2), to produce the powerful oxidant, peroxynitrite (ONOO9).1 What makes peroxynitrite particularly toxic is its remarkable stability as an anion at alkaline pH. This contrasts with NO itself, which has a half-life in biological systems that may be as short as 0.1 seconds.2 NO and related compounds have a complex role in homeostasis of the skin and in the development of skin disorders.3 One intriguing possibility is that NO mediates the vasodilatation that accompanies human sunburn reactions.4 NO, peroxynitrite, and other nitrogen oxides are also released in human squamous cell carcinoma (SCC-13) cells irradiated with ultraviolet A.4 The release of NO is reduced by NG-mono-methyl-larginine (L-NMMA), an inhibitor of the enzyme NO synthase (NOS), which produces NO from l-arginine. NO is also to be protective against ultraviolet A-induced apoptosis, because it modulates the Bcl-2 family of proteins.5 Its exact role in the development of sun-induced skin tumours is therefore controversial. BCC is widely recognized as being caused by chronic exposure to sun,6 but to our knowledge, there are no
This study was funded by The British Association of Oral and Maxillofacial Surgeons and Leibinger, UK. 633
634
British Journal of Oral and Maxillofacial Surgery
As BCCs are usually slow-growing, and metastatic spread is exceptionally rare, we were interested to see whether these tumours expressed NOS2 and NOS3. We postulated that the expression of NOS2 in BCC would be lower than that found in more aggressive tumours such as squamous cell carcinoma (SCC). METHODS Forty-eight cases of BCCs completely excised from the head and neck (26 nodular and 22 morphoeic) were studied. Specimens were taken from patients aged between 58 and 90 years (mean average age, 72). There were 30 men and 18 women. Histological slides were prepared from previously formalin-fixed paraffin embedded specimens and initially stained with haematoxylin and eosin, and examined by a consultant histopathologist (AVS) both to confirm the diagnosis and to ensure that the invading margin of the tumour was present on the sections. Specimens not containing both tumour and adjacent unaffected skin were excluded. Multiple sections 4 m thick were obtained from each paraffin block and stained using monoclonal antibodies against both inducible NO synthase (NOS2) and the endothelial form (eNOS) (Transduction Laboratories, Lexington, KY, USA) as follows: Sections were placed on positively charged slides, heated for 40 minutes at 50°C, de-waxed in Solvo 800 for 10 minutes, re-hydrated in alcohol solutions, and placed in 5% hydrogen peroxide in absolute alcohol for 10 minutes to block endogenous peroxidase activity. The sections were allowed to drain for several minutes before being marked with a Pap Pen (Dako, Denmark). Rehydration was completed by placing them in absolute alcohol and finally in water. The slides were treated with a boiling solution of freshly prepared 0.05 M citrate buffer, pH 6.0 for 2 minutes in a pressure cooker. We have also tried to retrieve the antigen in a microwave but found the best results were obtained with the procedure described. The sections were treated with normal goat serum at a dilution of 1/10 with 1 drop/ml of avidin block for 20 minutes and incubated at room temperature for 1 hour with either NOS2 or NOS3 monoclonal antibodies (both at a dilution of 1/100) with 1 drop/ml of biotin block. They were rinsed in Tris-buffered saline (TBS) before being treated with biotinylated anti-mouse immunoglobulin (Dako) at a dilution of 1/200 for 30 minutes. After they had been rinsed again, the slides were treated with avidin–biotin–peroxidase complex (Dako) for a further 30 minutes before being washed again with TBS. Immunostaining was accomplished by developing them in diaminobenzidine (DAB) and the slides were then rinsed in distilled water and counter-stained with Mayer’s haematoxylin. Positive controls were slides of parotid tissue, the ducts of which stain intensely for NOS2.13 Negative controls were treated in the same manner but with the omission of the primary antibodies. Each case was scored by two independent observers (PB and TU) according to a scale that included intensity of staining (magnification 200) and the area of staining seen (magnification 40). Discrepancies between
observers were discussed with AVS to reach an agreed score. The intensity of staining was on the following scale: 0:no staining seen; 1; :mild staining; 2; : moderate staining and 3; :intense staining. The area of staining was measured as follows: 0:none; 1; : less than 25% of tissue stained; 2;: between 25 and 50% stained; 3; :between 50 and 75% stained and 4;:more than 75% stained. Therefore the minimum score when summed (area;intensity) was 0 and the maximum, 7. Tumours were considered ‘positive’ if more than 25% of their cells were stained. The results were analysed statistically using Quickstat Biomedical Software. The Smirnov two-sample test was used to compare the staining seen in the adjacent skin with that of the tumour. The level of significance for the tests was chosen to be P0.01. RESULTS NOS2 immunostaining was seen in the basal keratinocytes of the skin margins of the resected tumour in all 48 cases (Fig. 1A) and was given a mean average staining score of 4 (range 3–6). It was also found in a few cells, presumed to be macrophages, both in the underlying connective tissue and in the stroma of the tumour. NOS2 staining was found in nine nodular and seven morphoeic BCCs in fewer than 10% of tumour cells (and therefore was not considered ‘positive’). NOS2 staining was not seen in the tumour epithelial cells of the remaining 32 cases (Fig. 1B). No correlation was found between the 16 cases in which staining of the tumour cells was weak staining and clinicopathological variables. The difference in NOS2 staining between the skin at the resection margin and the tumour cells was significant (P0.001). NOS3 was found as expected in endothelium of blood vessels, basal keratinocytes, sweat glands, and arrector pili muscle in the skin margins. In the tumours, NOS3 was found in the endothelium blood vessels of all 48 cases (Fig. 2), but it was not expressed by the tumour cells in any specimen (P0.001) (Fig. 2). DISCUSSION This study shows that the distribution of both NOS2 and NOS3 is reduced in BCC when compared with skin, suggesting that NO production is also reduced in this tumour. This is in contrast to other more aggressive skin cancers including melanoma, in which greater expression of NOS2 has been found.14 Unfortunately, there are no reports of NOS expression in squamous cell carcinoma of the skin, with which we could compare our findings. We are therefore investigating the expression of NOS in squamous cell skin cancer and the results will be available shortly. The mechanism of this downregulation remains to be elucidated. The release of epidermal-derived growth factor (EDGF) by BCC cells is one possible explanation for our findings. EDGF promotes the growth of keratinocytes but inhibits the production of NO. As a reduction in NO allows proliferation of keratinocytes during
NOS2 expression is downregulated in BCC of the head and neck
635
Fig. 1 – A: Expression of NOS2 in the basal layers of the skin at the resection margin (original magnification 100). B: Taken from the same case as Fig. 1A, there is no staining of NOS2 by the tumour epithelial cells (original magnification 100).
Fig. 2 – Expression of NOS3 in endothelium of tumour blood vessels, but no expression in the tumour itself. (original magnification 100).
the process of wound healing,15 it could be postulated that the BCC cells proliferate as a result of reduced NO by this mechanism. Keratinocytes and skin fibroblasts also seem to be much more resistant to NO-mediated apoptosis and necrosis than cells in other sites.16 However, given the low or absent NOS2 expression in BCC cells, and the fact that the concentrations of NO produced in human cancers are probably insufficient to induce apoptosis in any event,12 this property is of academic interest only.
The low expression of NOS2 found in this study may also be one reason why BCCs rarely metastasize. While there are no data available at present on NOS expression in human SCCs, it is clear that NOS2 activity correlates with lymph node metastasis in oral SCCs17,18 and many other solid tumours. In addition to having a pivotal role in angiogenesis, NO increases vascular permeability and reduces leucocyte–endothelial (L/E) adhesiveness.19 Low L/E interaction in tumour vessels is considered to be one of the major limitations of host immune response against tumours. As both angiogenesis and L/E adhesiveness are thought to facilitate metastasis, the reduced NOS expression seen in our study may help to explain the low metastatic potential of BCC. Further research on the effects of increasing NO concentrations in BCC (for example by transfecting cells with a NOS2 vector) might lead to a better understanding of the pathology of both skin cancer and NO in this group of tumours.
Acknowledgements We thank the British Association of Oral and Maxillofacial Surgeons and Leibinger, UK for the funding of laboratory costs, Mrs Jayne Buckley for technical assistance and Mr Bernard Higgins, Medical Statistician, University of Portsmouth, for help with the statistical analysis.
636
British Journal of Oral and Maxillofacial Surgery
References 1. Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and the ugly. Am J Physiol 1996; 271: 1424–1437. 2. Gaston B, Drazen JM, Loscalzo J, Stamler JS. The biology of nitrogen oxides in the airways. Am J Resp Crit Care Med 1994; 149: 538–551. 3. Bruch-Gerharz D, Ruzicka T, Kolb-Bachofen V. Nitric oxide and its implications in skin homeostasis and disease – a review. Arch Dermatol Res. 1998; 290: 643–651. 4. Villiotou V, Deliconstantinos G. Nitric oxide, peroxynitrite and nitroso-compounds formation by ultraviolet A (UVA) irradiated human squamous cell carcinoma: potential role of nitric oxide in cancer prognosis. Anticancer Res 1995; 15: 931–934. 5. Suschek CV, Bruch-Gerharz D, Berendgi D et al. Nitric oxide fully protects against UVA-induced apoptosis in tight correlation with Bcl-2 up-regulation. J Biol Chem 1999; 274: 6130–6137. 6. Grossman D, Leffell DJ. The molecular basis of non melanoma skin cancer: new understanding. Arch Dermatol 1997; 133: 1263–1270. 7. Foerstermann U, Kleinert H. Nitric oxide synthase: expression and expressional control of the three isoforms. Naunyn-Schmeidebergs Arch Pharmacol 1995; 352: 351–364. 8. Shimizu Y, Sakai M, Umemura Y, Ueda H. Immunohistochemical localisation of nitric oxide synthase in normal human skin: expression of endothelial-type and inducible-type nitric oxide synthase in keratinocytes. J Dermatol 1997; 24: 80–87. 9. Ahmad B, Van Den Oord JJ. Expression of the neuronal isoform of nitric oxide synthase (nNOS) and its inhibitor, protein inhibitor of nNOS, in pigment cell lesions of the skin. Br J Dermatol 1999; 141: 12–19. 10. Schmidt HHHW, Pollock JS, Nakane M, Förstermann U, Murad F. Ca2;/calmodulin-regulated nitric oxide synthases. Cell Calcium 1992; 13: 427–434. 11. Albina JE, Reichner JS. Role of nitric oxide in mediation of macrophage cytotoxicity and apoptosis. Cancer Metastasis Rev 1998; 17: 39–53. 12. Thomsen LL, Miles DW. Role of nitric oxide in tumour progression: lessons from human tumours. Cancer Metastasis Rev 1998; 17: 107–118. 13. Brennan PA, Umar T, Buckley J, Zaki GA, Spedding AV, Langdon JD. Nitric oxide synthase expression in pleomorphic adenoma of the parotid. Br J Oral Maxillofac Surg 2000; 38: 338–342. 14. Tschugguel W, Pustelnik T, Lass H et al. Inducible nitric oxide synthase (iNOS) expression may predict distant metastasis in human melanoma. Br J Cancer 1999; 79: 1609–1612. 15. Heck DE, Laskin DI, Gardner CR, Laskin JD. Epidermal growth factor suppresses nitric oxide and hydrogen peroxide mediation by
16.
17. 18.
19.
keratinocytes: potential role for nitric oxide in the regulation of wound healing. J Biol Chem 1992; 267: 21 277–21 280. Krischel V, Bruch-Gerharz D, Suschek C, Kroncke KD, Ruzicka T, Kolb-Bachofen V. Biphasic effect of exogenous nitric oxide on proliferation and differentiation in skin derived keratinocytes but not fibroblasts. J Invest Dermatol 1998; 111: 286–291. Gallo O, Masini E, Morbidelli L et al. Role of nitric oxide in angiogenesis and tumour progression in head and neck cancer. J Natl Cancer Inst 1998; 90: 587–596. Brennan PA, Palacios-Callender M, Zaki GA, Spedding AV, Langdon JD. Type II nitric oxide synthase expression correlates with lymph node status in oral squamous cell carcinoma. Journal of Oral Pathology and Medicine (in press). Fukumura D, Yuan F, Endo M, Jain RK. Role of nitric oxide in tumour microcirculation: blood flow, vascular permeability and leukocyte-endothelial interactions. Am J Pathol 1997; 150: 713–725.
The Authors Peter A. Brennan FDSRCS, FRCS, FRCSI BUPA Research Fellow Maxillofacial Unit Tijjani Umar MB BS, FMCPath, MSc Specialist Registrar in Histopathology John Bowden FDSRCS, BM, MSc Specialist Registrar in Oral and Maxillofacial Surgery Andrew Hobkirk FDSRCS Medical Student Anne V. Spedding MRCPath Consultant Histopathologist Graeme Zaki FDSRCS, FRCS Consultant Oral and Maxillofacial Surgeon Queen Alexandra Hospital Portsmouth, UK Brian Conroy FRCPath Consultant Histopathologist D. W. Macpherson FDSRCS, FRCS Consultant Oral and Maxillofacial Surgeon St Richard’s Hospital Chichester West Sussex, UK Correspondence and requests for offprints to: Mr P. A. Brennan FDSRCS, FRCS, FRCSI, 11 Oxlease Close, Romsey, Hants SO51 7HA, UK. Tel: (work) ;44 (0)2393 286736 (direct); Fax: (work) ;44(0)2393 286089 Paper received 14 January 2000 Accepted 15 August 2000
BRITISH JOURNAL OF ORAL
& M A X I L L O FAC I A L S U R G E RY
Nitric oxide synthase expression is downregulated in basal cell carcinoma of the head and neck P. A. Brennan,* T. Umar,† J. Bowden,‡ A. Hobkirk,§ A. V. Spedding,¶ B. Conroy,** G. Zaki,†† D. W. Macpherson‡‡ *BUPA Research Fellow, Maxillofacial Unit; †Specialist Registrar in Histopathology; ‡Specialist Registrar in Oral and Maxillofacial Surgery; §Medical Student; ¶Consultant Histopathologist, Queen Alexandra Hospital, Portsmouth; **Consultant Histopathologist, St Richard’s Hospital, Chichester; ††Consultant Oral and Maxillofacial Surgeon, Queen Alexandra Hospital, Portsmouth; ‡‡Consultant Oral and Maxillofacial Surgeon, St Richard’s Hospital, Chichester, West Sussex, UK SUMMARY. The small molecule nitric oxide (NO) has many actions, most of which are poorly understood. Recently, NO and related compounds have been implicated in skin damage caused by ultraviolet light although their exact role is not clear. We undertook an immunohistochemical study to assess the expression of type II NO synthase (NOS2) and type III (NOS3) in basal cell carcinomas (BCCs) of the head and neck. In all 48 cases studied, NOS2 was found in the basal cell layer of the skin at the tumour margin but it was significantly reduced in the tumour epithelial cells (P0.001). NOS3 was localized to the endothelium of the blood vessels in both skin and tumour in all cases, and it was not seen in the tumour epithelial cells. The results suggest that expression of NOS is down-regulated in basal cell carcinomas. © 2000 The British Association of Oral and Maxillofacial Surgeons
INTRODUCTION
reports of the distribution of NOS in these tumours. There are at least three NOS isoenzymes, each being the product of a separate gene.7 NOS1, also called nNOS as it was first found in neural tissue, is not present in normal keratinocytes,8 but has been detected in pigmented skin lesions including melanoma.9 NOS2, also named iNOS (immunological) is produced in response to cytokine release. It is found in many cells including macrophages, natural killer cells, and keratinocytes.8 NOS3, originally detected in the endothelium of blood vessels, is also found in keratinocytes, fibroblasts, arrector pili muscle and sweat glands.8 As the activity of NOS1 and NOS3 is dependent on raised local calcium concentrations, probably beyond 500 nM (from a resting level of 70–100 nM), their production of NO is transient.10 In this respect, most studies of NO in cancer have been directed at NOS2, which produces NO for many hours or days, after induction. There are still conflicting views about the role of NO in human cancer, compounded by the fact that it has both tumourpromoting and cytotoxic activities. Much of this controversy stems from studies of murine tumours, in which the production of NO by both tumour cells and macrophages is much greater in human cancers. A growing body of evidence suggests that the concentrations of NO found in human cancers are at least one to two orders of magnitude lower than that required for cytotoxicity.11 The result is that NO probably contributes to the development of human cancer by facilitating angiogenesis and dissemination.12
The small molecule nitric oxide (NO) has many biological actions. It is readily able to pass through cell membranes and influence enzymes and proteins in both the cytosol and the nucleus. These effects result from conformational changes in proteins (in a similar fashion to those seen when oxygen binds to haemoglobin) as well as direct chemical reactions with electron-accepting moieties such as thiols and transition metals. It may therefore act as a signalling molecule as well as having cytotoxic and possible genotoxic effects. The toxicity of NO is probably the result of the reaction of NO with the superoxide free radical (O2), to produce the powerful oxidant, peroxynitrite (ONOO9).1 What makes peroxynitrite particularly toxic is its remarkable stability as an anion at alkaline pH. This contrasts with NO itself, which has a half-life in biological systems that may be as short as 0.1 seconds.2 NO and related compounds have a complex role in homeostasis of the skin and in the development of skin disorders.3 One intriguing possibility is that NO mediates the vasodilatation that accompanies human sunburn reactions.4 NO, peroxynitrite, and other nitrogen oxides are also released in human squamous cell carcinoma (SCC-13) cells irradiated with ultraviolet A.4 The release of NO is reduced by NG-mono-methyl-larginine (L-NMMA), an inhibitor of the enzyme NO synthase (NOS), which produces NO from l-arginine. NO is also to be protective against ultraviolet A-induced apoptosis, because it modulates the Bcl-2 family of proteins.5 Its exact role in the development of sun-induced skin tumours is therefore controversial. BCC is widely recognized as being caused by chronic exposure to sun,6 but to our knowledge, there are no
This study was funded by The British Association of Oral and Maxillofacial Surgeons and Leibinger, UK. 633
634
British Journal of Oral and Maxillofacial Surgery
As BCCs are usually slow-growing, and metastatic spread is exceptionally rare, we were interested to see whether these tumours expressed NOS2 and NOS3. We postulated that the expression of NOS2 in BCC would be lower than that found in more aggressive tumours such as squamous cell carcinoma (SCC). METHODS Forty-eight cases of BCCs completely excised from the head and neck (26 nodular and 22 morphoeic) were studied. Specimens were taken from patients aged between 58 and 90 years (mean average age, 72). There were 30 men and 18 women. Histological slides were prepared from previously formalin-fixed paraffin embedded specimens and initially stained with haematoxylin and eosin, and examined by a consultant histopathologist (AVS) both to confirm the diagnosis and to ensure that the invading margin of the tumour was present on the sections. Specimens not containing both tumour and adjacent unaffected skin were excluded. Multiple sections 4 m thick were obtained from each paraffin block and stained using monoclonal antibodies against both inducible NO synthase (NOS2) and the endothelial form (eNOS) (Transduction Laboratories, Lexington, KY, USA) as follows: Sections were placed on positively charged slides, heated for 40 minutes at 50°C, de-waxed in Solvo 800 for 10 minutes, re-hydrated in alcohol solutions, and placed in 5% hydrogen peroxide in absolute alcohol for 10 minutes to block endogenous peroxidase activity. The sections were allowed to drain for several minutes before being marked with a Pap Pen (Dako, Denmark). Rehydration was completed by placing them in absolute alcohol and finally in water. The slides were treated with a boiling solution of freshly prepared 0.05 M citrate buffer, pH 6.0 for 2 minutes in a pressure cooker. We have also tried to retrieve the antigen in a microwave but found the best results were obtained with the procedure described. The sections were treated with normal goat serum at a dilution of 1/10 with 1 drop/ml of avidin block for 20 minutes and incubated at room temperature for 1 hour with either NOS2 or NOS3 monoclonal antibodies (both at a dilution of 1/100) with 1 drop/ml of biotin block. They were rinsed in Tris-buffered saline (TBS) before being treated with biotinylated anti-mouse immunoglobulin (Dako) at a dilution of 1/200 for 30 minutes. After they had been rinsed again, the slides were treated with avidin–biotin–peroxidase complex (Dako) for a further 30 minutes before being washed again with TBS. Immunostaining was accomplished by developing them in diaminobenzidine (DAB) and the slides were then rinsed in distilled water and counter-stained with Mayer’s haematoxylin. Positive controls were slides of parotid tissue, the ducts of which stain intensely for NOS2.13 Negative controls were treated in the same manner but with the omission of the primary antibodies. Each case was scored by two independent observers (PB and TU) according to a scale that included intensity of staining (magnification 200) and the area of staining seen (magnification 40). Discrepancies between
observers were discussed with AVS to reach an agreed score. The intensity of staining was on the following scale: 0:no staining seen; 1; :mild staining; 2; : moderate staining and 3; :intense staining. The area of staining was measured as follows: 0:none; 1; : less than 25% of tissue stained; 2;: between 25 and 50% stained; 3; :between 50 and 75% stained and 4;:more than 75% stained. Therefore the minimum score when summed (area;intensity) was 0 and the maximum, 7. Tumours were considered ‘positive’ if more than 25% of their cells were stained. The results were analysed statistically using Quickstat Biomedical Software. The Smirnov two-sample test was used to compare the staining seen in the adjacent skin with that of the tumour. The level of significance for the tests was chosen to be P0.01. RESULTS NOS2 immunostaining was seen in the basal keratinocytes of the skin margins of the resected tumour in all 48 cases (Fig. 1A) and was given a mean average staining score of 4 (range 3–6). It was also found in a few cells, presumed to be macrophages, both in the underlying connective tissue and in the stroma of the tumour. NOS2 staining was found in nine nodular and seven morphoeic BCCs in fewer than 10% of tumour cells (and therefore was not considered ‘positive’). NOS2 staining was not seen in the tumour epithelial cells of the remaining 32 cases (Fig. 1B). No correlation was found between the 16 cases in which staining of the tumour cells was weak staining and clinicopathological variables. The difference in NOS2 staining between the skin at the resection margin and the tumour cells was significant (P0.001). NOS3 was found as expected in endothelium of blood vessels, basal keratinocytes, sweat glands, and arrector pili muscle in the skin margins. In the tumours, NOS3 was found in the endothelium blood vessels of all 48 cases (Fig. 2), but it was not expressed by the tumour cells in any specimen (P0.001) (Fig. 2). DISCUSSION This study shows that the distribution of both NOS2 and NOS3 is reduced in BCC when compared with skin, suggesting that NO production is also reduced in this tumour. This is in contrast to other more aggressive skin cancers including melanoma, in which greater expression of NOS2 has been found.14 Unfortunately, there are no reports of NOS expression in squamous cell carcinoma of the skin, with which we could compare our findings. We are therefore investigating the expression of NOS in squamous cell skin cancer and the results will be available shortly. The mechanism of this downregulation remains to be elucidated. The release of epidermal-derived growth factor (EDGF) by BCC cells is one possible explanation for our findings. EDGF promotes the growth of keratinocytes but inhibits the production of NO. As a reduction in NO allows proliferation of keratinocytes during
NOS2 expression is downregulated in BCC of the head and neck
635
Fig. 1 – A: Expression of NOS2 in the basal layers of the skin at the resection margin (original magnification 100). B: Taken from the same case as Fig. 1A, there is no staining of NOS2 by the tumour epithelial cells (original magnification 100).
Fig. 2 – Expression of NOS3 in endothelium of tumour blood vessels, but no expression in the tumour itself. (original magnification 100).
the process of wound healing,15 it could be postulated that the BCC cells proliferate as a result of reduced NO by this mechanism. Keratinocytes and skin fibroblasts also seem to be much more resistant to NO-mediated apoptosis and necrosis than cells in other sites.16 However, given the low or absent NOS2 expression in BCC cells, and the fact that the concentrations of NO produced in human cancers are probably insufficient to induce apoptosis in any event,12 this property is of academic interest only.
The low expression of NOS2 found in this study may also be one reason why BCCs rarely metastasize. While there are no data available at present on NOS expression in human SCCs, it is clear that NOS2 activity correlates with lymph node metastasis in oral SCCs17,18 and many other solid tumours. In addition to having a pivotal role in angiogenesis, NO increases vascular permeability and reduces leucocyte–endothelial (L/E) adhesiveness.19 Low L/E interaction in tumour vessels is considered to be one of the major limitations of host immune response against tumours. As both angiogenesis and L/E adhesiveness are thought to facilitate metastasis, the reduced NOS expression seen in our study may help to explain the low metastatic potential of BCC. Further research on the effects of increasing NO concentrations in BCC (for example by transfecting cells with a NOS2 vector) might lead to a better understanding of the pathology of both skin cancer and NO in this group of tumours.
Acknowledgements We thank the British Association of Oral and Maxillofacial Surgeons and Leibinger, UK for the funding of laboratory costs, Mrs Jayne Buckley for technical assistance and Mr Bernard Higgins, Medical Statistician, University of Portsmouth, for help with the statistical analysis.
636
British Journal of Oral and Maxillofacial Surgery
References 1. Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and the ugly. Am J Physiol 1996; 271: 1424–1437. 2. Gaston B, Drazen JM, Loscalzo J, Stamler JS. The biology of nitrogen oxides in the airways. Am J Resp Crit Care Med 1994; 149: 538–551. 3. Bruch-Gerharz D, Ruzicka T, Kolb-Bachofen V. Nitric oxide and its implications in skin homeostasis and disease – a review. Arch Dermatol Res. 1998; 290: 643–651. 4. Villiotou V, Deliconstantinos G. Nitric oxide, peroxynitrite and nitroso-compounds formation by ultraviolet A (UVA) irradiated human squamous cell carcinoma: potential role of nitric oxide in cancer prognosis. Anticancer Res 1995; 15: 931–934. 5. Suschek CV, Bruch-Gerharz D, Berendgi D et al. Nitric oxide fully protects against UVA-induced apoptosis in tight correlation with Bcl-2 up-regulation. J Biol Chem 1999; 274: 6130–6137. 6. Grossman D, Leffell DJ. The molecular basis of non melanoma skin cancer: new understanding. Arch Dermatol 1997; 133: 1263–1270. 7. Foerstermann U, Kleinert H. Nitric oxide synthase: expression and expressional control of the three isoforms. Naunyn-Schmeidebergs Arch Pharmacol 1995; 352: 351–364. 8. Shimizu Y, Sakai M, Umemura Y, Ueda H. Immunohistochemical localisation of nitric oxide synthase in normal human skin: expression of endothelial-type and inducible-type nitric oxide synthase in keratinocytes. J Dermatol 1997; 24: 80–87. 9. Ahmad B, Van Den Oord JJ. Expression of the neuronal isoform of nitric oxide synthase (nNOS) and its inhibitor, protein inhibitor of nNOS, in pigment cell lesions of the skin. Br J Dermatol 1999; 141: 12–19. 10. Schmidt HHHW, Pollock JS, Nakane M, Förstermann U, Murad F. Ca2;/calmodulin-regulated nitric oxide synthases. Cell Calcium 1992; 13: 427–434. 11. Albina JE, Reichner JS. Role of nitric oxide in mediation of macrophage cytotoxicity and apoptosis. Cancer Metastasis Rev 1998; 17: 39–53. 12. Thomsen LL, Miles DW. Role of nitric oxide in tumour progression: lessons from human tumours. Cancer Metastasis Rev 1998; 17: 107–118. 13. Brennan PA, Umar T, Buckley J, Zaki GA, Spedding AV, Langdon JD. Nitric oxide synthase expression in pleomorphic adenoma of the parotid. Br J Oral Maxillofac Surg 2000; 38: 338–342. 14. Tschugguel W, Pustelnik T, Lass H et al. Inducible nitric oxide synthase (iNOS) expression may predict distant metastasis in human melanoma. Br J Cancer 1999; 79: 1609–1612. 15. Heck DE, Laskin DI, Gardner CR, Laskin JD. Epidermal growth factor suppresses nitric oxide and hydrogen peroxide mediation by
16.
17. 18.
19.
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The Authors Peter A. Brennan FDSRCS, FRCS, FRCSI BUPA Research Fellow Maxillofacial Unit Tijjani Umar MB BS, FMCPath, MSc Specialist Registrar in Histopathology John Bowden FDSRCS, BM, MSc Specialist Registrar in Oral and Maxillofacial Surgery Andrew Hobkirk FDSRCS Medical Student Anne V. Spedding MRCPath Consultant Histopathologist Graeme Zaki FDSRCS, FRCS Consultant Oral and Maxillofacial Surgeon Queen Alexandra Hospital Portsmouth, UK Brian Conroy FRCPath Consultant Histopathologist D. W. Macpherson FDSRCS, FRCS Consultant Oral and Maxillofacial Surgeon St Richard’s Hospital Chichester West Sussex, UK Correspondence and requests for offprints to: Mr P. A. Brennan FDSRCS, FRCS, FRCSI, 11 Oxlease Close, Romsey, Hants SO51 7HA, UK. Tel: (work) ;44 (0)2393 286736 (direct); Fax: (work) ;44(0)2393 286089 Paper received 14 January 2000 Accepted 15 August 2000