Expression of inducible nitric oxide synthase in bone metastases.

AIMS: The signalling molecule nitric oxide (NO), produced predominantly in cancer by the enzyme inducible NO synthase (iNOS), has been implicated in the pathophysiology of many human tumours. The increased NO concentrations found in many human cancers may facilitate both angiogenesis and tumour dissemination. NO also plays a concentration-dependent role in bone re-modelling by acting on osteoclasts. Although iNOS has been studied extensively in most primary tumours, there are no reports that have investigated its expression in metastatic bone disease. METHODS: An immunohistochemical study was performed using a monoclonal antibody to iNOS in 27 cases of breast, renal and lung bone metastases, biopsied at the time of treatment for pathological fracture. RESULTS: iNOS expression was found in 14 cases and was predominantly localised to tumour cells in the metastatic deposits. A significant difference was found between iNOS expression in metastases and adjacent bone (p<0.001), where immunostaining was rarely seen and confined to immune cells. No microscopic differences in bone architecture could be seen between iNOS positive and negative metastases, and no correlations were found between iNOS expression and clinico-pathological variables. CONCLUSIONS: iNOS expression is not a pathognomonic finding in bone metastasis. Its role in the behaviour of bone metastases requires further investigation.     

Immunohistochemical localization of inducible nitric oxide synthase and 3-nitrotyrosine in rat liver tumors induced by N-nitrosodiethylamine.

Human liver cancers have been associated mainly with chronic inflammations such as viral hepatitis B or C. This suggests that prolonged cell damage by chronic inflammation is critical in cancer development. Overproduction of nitric oxide (NO.) and its derivative (NOx, peroxynitrite) has been implicated as a cause of tissue damage by inflammation, thus contributing to tumor promotion. We have demonstrated the expression of the inducible isoform of nitric oxide synthase (iNOS) and 3-nitrotyrosine, a marker of peroxynitrite formation, by immunohistochemistry in preneoplastic and neoplastic rat liver tissues induced by continuous infusion of N-nitrosodiethylamine with mini-pumps. The preneoplastic lesions were characterized by proliferation of phenotypically altered hepatic foci (PAHF), dysplastic hepatocytes and oval cells. Histologically, the tumors were hepatocellular carcinomas (HCCs) of trabecular, (pseudo)glandular and solid types with or without cholangiocellular involvement. iNOS was located mainly in oval cells, capillary endothelial and muscular cells, epithelia of cholangiomas and glandular HCCs. 3-Nitrotyrosine was observed in the cytoplasms of PAHF and dysplastic hepatocytes in preneoplasias and in the cytoplasms of some living or apoptotic HCC cells, connective tissues, proteinaceous fluids, sinusoidal endothelia of tumorous hepatocytes and cholangiomas in tumors. From these observations, we suggest that: (i) chronic tissue damage by chemical carcinogens may act to induce iNOS and peroxynitrite formation; (ii) oval cells play a key role in development and/or growth of tumor tissues by producing NO. via iNOS, which may also cause tissue damage by peroxynitrite; (iii) iNOS can be considered as a phenotypic marker in cells of oval cell lineage and neovascularized capillaries in tumor tissues.     

Expression of cyclooxygenase-2 and inducible nitric oxide synthase in human ovarian tumors and tumor-associated macrophages

This study investigates whether and to what extent cyclooxygenase type-2 (COX-2) and inducible nitric oxide-synthase (iNOS), both known to have an immunosuppressive effect, are expressed in human ovarian tumors. Because COX-2 and iNOS can be expressed by activated macrophages, the presence of tumor-associated macrophages and the expression of COX-2 and iNOS by these tumor-associated macrophages were determined. The results obtained may provide insight into the function of COX-2 and iNOS expression by tumors. The expression of COX-2 and iNOS in tumor cells and macrophages was assessed in 18 malignant, 15 borderline, and 14 benign human ovarian tumors by immunohistochemical staining of frozen tissue sections. The intra- and peritumoral macrophages were stained using an anti-CD68 monoclonal antibody. Most of the malignant tumors (15 of 18), 10 of 15 borderline, and 9 of 14 benign tumors showed COX-2 expression in the epithelial cells, a result which indicates that COX-2 expression is not exclusive to malignancy. In addition, COX-2 staining was more intense in the epithelial cells of benign and borderline tumors than in malignant tumors. Weak iNOS staining was observed in 5 of 18 malignant, 4 of 15 borderline, and 5 of 14 benign tumors. The number of tumor-associated macrophages varied widely between the different tumors. The highest number of tumor-associated macrophages (> or =20/0.125 mm(2)) was observed in malignant tumors, whereas low to moderate intra- and peritumoral macrophage infiltration (5-20/0.125 mm(2)) was observed in the borderline and benign tumors. COX-2-positive tumor-associated macrophages were found in 3 of 18 malignant tumors, 7 of 15 borderline tumors, and 1 of 14 benign tumors. The number of COX-2-positive tumor-associated macrophages ranged from 3 to 30% of the total macrophage population. Some malignant (4 of 18), borderline (5 of 15), and benign (2 of 14) tumors contained iNOS-positive macrophages. Notable was that COX-2- and iNOS-positive macrophages were predominantly located in the tumor stroma, the regions between tumor and stroma, and in the lumina of the tumor when located in the tumor tissue. These data indicate that not only malignant but also borderline and benign ovarian tumors can exhibit increased levels of COX-2 and iNOS expression. In addition, a small proportion of the tumor-associated macrophages found in malignant, borderline, and benign tumors seems to be in an activated state, judged by their iNOS and COX-2 expression. This subpopulation of tumor-associated macrophages was invariably located in the tumor stroma or in the lumina of the tumor, specifically suggesting that macrophages outside the tumor can be tumor cytotoxic.     

Expression of inducible nitric oxide synthase in healthy pleura and in malignant mesothelioma

In this study we investigated the immunohistochemical expression of inducible nitric oxide synthase (iNOS) in a set of normal pleural mesothelial tissues, malignant mesotheliomas, mesothelioma cell lines and metastatic pleural adenocarcinomas. Furthermore, the expression of mRNA was assessed in four malignant mesothelioma cell lines in culture. Apoptosis and vascular density in malignant mesotheliomas was assessed by the TUNEL method and by immunohistochemistry with an antibody against FVIII-related antigen. Immunohistochemically mesothelial cells in non-neoplastic healthy pleural tissues were mostly negative for iNOS. Positivity for iNOS was observed in 28/38 (74%) and 24/25 (96%) of malignant mesotheliomas and metastatic pleural adenocarcinomas, respectively. Epithelial and mixed mesotheliomas expressed more often strong iNOS immunoreactivity compared to the sarcomatoid subtype (P = 0.023). Moreover, metastatic adenocarcinomas expressed more often iNOS positivity than mesotheliomas (P = 0.021). Experiments with the cell lines confirmed that malignant mesothelioma cells are capable of synthesizing iNOS. No significant association was found between iNOS expression and apoptosis or vascular density in malignant mesotheliomas. The higher expression of iNOS in the epithelial subtype of mesothelioma and pleural metastatic adenocarcinoma might be due to an increased sensitivity of these cell types to cytokine-mediated iNOS upregulation. The strong expression of iNOS suggests a putative role for NO in the growth and progression of these tumours.     

舌鳞癌组织iNOSmRNA表达及其意义

背景及目的：近年的研究发现一氧化氮在肿瘤的发生、发展中起着重要的作用,在头颈肿瘤中检测出高水平的诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）表达,并与肿瘤的进展正相关,未见有关口腔癌中iNOS mRNA与其生物学行为相关的报道。本文拟研究舌鳞癌组织中iNOS mRNA表达与其侵袭性生长、颈淋巴结转移、预后的相关性。方法：原位杂交检测常规石蜡包埋的68例舌鳞癌活检组织中iNOS mRNA的表达;对所研究的舌鳞癌病例进行随访,获取有关预后资料;用原位杂交技术来分析iNOS mRNA与舌鳞癌侵袭性生长、颈淋巴结转移、预后的相关性。结果：①舌鳞癌浸润肌层组与无肌层浸润组iNOS mRNA阳性率分别为86．1％、48．0％,两组间差异有显著性（P＜0．05）,相关系数为0．3。②iNOS mRNA在舌鳞癌有颈淋巴结转移组阳性率为85．7％,无颈淋巴结转移组阳性率为62．5％,两组间差异有显著性（P＜0．05）,相关系数为0．3。③舌鳞部iNOS mRNA表达阳性组3年生存率显著低于iNOS mRNA表达阴性组（P＜0．05）。结论：①iNOS mRNA阳性表达与舌鳞癌的侵袭性生长、颈淋巴结转移正相关。②舌鳞癌组织中iNOS mRNA表达阳性的病例较iONS mRNA表达阴性的病例预后差。     

Expression of cyclooxygenase-2 and inducible nitric oxide synthase correlates with tumor angiogenesis in endometrial carcinoma

The present study evaluated the significance of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in 30 patients with endometrial carcinoma and the relationship of those molecular markers to tumor characteristics and microvessel density (MVD). Immunohistochemical expression of COX-2, iNOS, and CD34 was analyzed on paraffin-embedded tissue sections. The COX-2 and iNOS positive rates were 66.7% and 73.3%, respectively. The level of COX-2 expression was higher in grade II tumors than in grade III tumors (p < 0.05). The percentage of iNOS positivity was higher in patients with deep myometrial invasion than in patients without or less than 50% myometrial invasion (p < 0.05). There was significant correlation between positive COX-2 and positive iNOS expression (r = 0.601, p < 0.001). Both COX-2 and iNOS were significantly correlated with MVD (r = 0.02 p < 0.05; r = 0.599 p < 0.0001, respectively). The present findings suggest that combined expression of COX-2 and iNOS may play an important role in development and invasion of endometrial cancer and that this could be partially attributable to modulation of angiogenesis by COX-2 and iNOS.     

Expression of inducible nitric oxide synthase in human breast cancer depends on tumor grade

Expression of inducible nitric oxide synthase (iNOS) by tumor cells has been suggested to abrogate metastasis in several tumor models, whereas constitutive NOS expression correlated positively with tumor grade in human breast carcinoma. Whether or not expression of one of the various NOS isoforms could predict the prognosis of breast cancer, however, has not been established. In the present report we investigated the cellular distribution of NOS isoforms in a series of benign and malignant breast tumors and in normal breast tissue. Immunohistochemistry revealed that in samples of benign disease the number of iNOS+epithelial cells or total epithelial cells was 69±16% (n=50). In samples of grade II invasive ductal breast carcinomas the number of iNOS+ tumor cells or total tumor cells was 62±20 (n=40), compared to 12±9 (n=40) in samples of grade III carcinomas (P<0.0001). iNOS protein was also identifiable in most of the epithelial cells of normal breast tissue (n=4). In contrast, eNOS protein was restricted to vascular endothelial cells in all of the specimens studied. Since the presence of tumor cell iNOS protein is inversely related to the tumors metastatic potential, we conclude that endogenous tumor cell mediated iNOS expression might have an inhibitory effect on the metastatic process in breast cancer.     

Expression of inducible nitric oxide synthase (NOS) in bone marrow cells of myelodysplastic syndromes.

Nitric oxide (NO) is a biological mediator which is synthesized from L-arginine by a family of nitric oxide synthases (NOS). We have studied the expression of the inducible NOS (iNOS) by bone marrow cells from the patients with myelodysplastic syndromes (MDS) at the mRNA level by RT-PCR assay and at the protein level by immunohistochemical staining using a specific anti-iNOS monoclonal antibody. The iNOS message was present in 92% of bone marrow tissues from MDS patients (11 out of 12) by an examination using RT-PCR. Basically, iNOS message was negative or very weak in control (1/9) and AML (0/7) cases. This was supported by immunohistochemical findings that the iNOS was positive in most of the bone marrow samples from MDS patients (9 out of 12), while bone marrow cells of control (O out of 12) and AML (O out of 5) cases were basically negative. Double immunostaining for CD68 antigen, which is a marker for macrophage lineage cells, and iNOS was performed on MDS bone marrow sections. iNOS was dominantly localized to bone marrow macrophages, although a part of myeloid cells were also positively stained with anti-iNOS antibody in a part of cases. These results indicated that there is some in vivo induction of iNOS expression for local NO production that might be involved in the dysregulation of hematopoiesis in bone marrow of MDS.     

Increased level of exhaled nitric oxide and up-regulation of inducible nitric oxide synthase in patients with primary lung cancer.

Monocyte-macrophage series have an important role in host surveillance against cancer. The cytotoxic/cytostatic activity of macrophages is, to a great extent, attributed to the up-regulation of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO). Here, in 28 patients with primary lung cancer and 20 control subjects, we measured the concentration of exhaled NO and nitrite in epithelial lining fluid (ELF) using a chemiluminescence NO analyser, and studied NOS expression in alveolar macrophages (AM) and lung tissues by flow cytometry; immunohistochemical analysis was also undertaken. The mean fluorescence intensity (FI) of iNOS expression in AM was significantly increased in patients with lung cancer (tumour side 263.5 +/- 15.2 FI, normal side 232.4 +/- 18.6 FI; n = 28) compared with that in control subjects (27.3 +/- 3.2 FI; n = 20, P< 0.001). The level of exhaled NO from cancer patients (16.9 +/- 0.9 p.p.b.; n = 28) was significantly higher than that in the control group (6.0 +/- 0.5 p.p.b.; n = 20, P < 0.001). The level of nitrite was also significantly higher in ELF from cancer patients (tumour side 271.1 +/- 28.9 nM and normal side 257.4 +/- 19.6 nM vs control subjects 32.9 +/- 4.1 nM; P< 0.001). The intensity of iNOS expression in AM was correlated with the level of exhaled NO (rs = 0.73, n = 76, P< 0.001) and the nitrite released in ELF (rs = 0.56, n = 76, P< 0.001). The nitrite generation of cultured AM from patients with lung cancer was significantly enhanced compared with that of control subjects after culture for 24 h (tumour side 5.75 +/- 0.69 and normal side 5.68 +/- 0.58 microM per 106 cells vs control group 38.3 +/- 3.6 nM per 106 cells; P< 0.001). The distribution of iNOS was identified in AM, tumour-associated macrophages, endothelium, chondrocytes, airway epithelium of both lungs and malignant cells (adenocarcinoma and alveolar cell carcinoma) of cancer patients. cNOS was labelled in alveolar macrophages, endothelial cells and nerve elements from lung tissue. Our results indicate that, in patients with primary lung cancer, the production of NO from alveolar macrophages was increased as a result of the up-regulation of iNOS activity. The increased NO production was not specific to the tumour side and might be attributed to the tumour-associated non-specific immunological and inflammatory processes of the host.     

Increased inducible nitric oxide synthase in lung carcinoma of smokers

BACKGROUND: Cigarette smoking is well known to play an important role in the development of lung cancer. Inducible nitric oxide synthase (iNOS) can either promote or inhibit cell proliferation and growth, which makes its role in the development of malignant tumors controversial. The relation between cigarette smoking and iNOS in human lung cancer is unknown. METHODS: The study examined the levels of iNOS/NO in nonsmall-cell lung cancer (NSCLC) tissues of smokers and nonsmokers and in NSCLC cells (NCI-H23) treated by 4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent tobacco-specific carcinogen. RESULTS: The level of iNOS/NO was significantly higher in lung cancer tissues of smokers than that of nonsmokers. Unlike iNOS/NO, the activity of caspase-3 was reduced in the former compared with the latter. The expression of the cleaved caspase-3 was deceased in NCI-H23 cells treated with S-Nitroso-N-acetylpenicillamine (SNAP), an NO donor, whereas treatment with NG-methyl-L-arginine (NMA), an NO inhibitor, caused an increase in cleaved caspase-3. Consistent with the change in caspase-3, SNAP treatment inhibited cell death induced by UCN01, a potent cell death-inducer. NMA treatment greatly enhanced the sensitivity of the cells to UCN01. Further, the cells treated by NNK showed an increase in iNOS protein, accompanied by an elevation of cell proliferation. CONCLUSIONS: The study demonstrates that cigarette smoking promotes the level of iNOS/NO but suppresses the activity of caspase-3, which may lead to the proliferation and growth of lung cancer cells.     

诱导型一氧化氮合酶与Bcl-2在鼻咽癌中的表达及意义

目的 探讨鼻咽癌组织中诱导型一氧化氮合酶（iNOS）与滤泡型B细胞淋巴瘤／白血病-2（Bcl-2）的表达情况及其意义。方法 采用免疫组织化学法检测50例鼻咽癌石蜡标本和15例慢性炎症的鼻咽黏膜标本中iNOS和Bcl-2蛋白表达情况。结果 慢性炎症的鼻咽黏膜中iNOS无表达,Bcl-2有表达,但局限于上皮的基底细胞层。在鼻咽癌组织中,iNOS阳性表达者细胞浆和胞核内出现棕黄色细颗粒,且胞核染色比胞浆更强;Bcl-2阳性表达者细胞浆或核膜上出现棕黄色细颗粒。鼻咽癌组织iNOS的阳性表达率为74.0％（37／50）,Bcl-2为82.0％（41／50）,与慢性炎症的鼻咽黏膜比较,差异均有统计学意义（P＜0.05）。在鼻咽癌组织中,iNOS与Bcl-2蛋白表达强度呈正相关（r=0.945,P＜0.05）;iNOS的表达与T分期及有无淋巴结转移有关,与年龄和性别无关;Bcl 2的表达与临床病理特征无关。结论 iNOS与Bcl 2的表达与鼻咽癌的发生、发展相关,进一步研究两者的关系对探索鼻咽癌新的治疗手段有重要意义。     

诱导型一氧化氮合酶在鼻咽癌中的表达及意义

背景与目的：诱导型一氧化氮合酶（iNOS）在许多肿瘤中呈高表达，被认为与肿瘤的发生、发展、浸润及转移有关，但iNOS在鼻咽癌（NPC）肿瘤组织中的表达及其作用，国内尚未见报道，国外（PUBMED）也仅可查到两条与iNOS在NPC中表达有关的信息，然而这两项研究并未对iNOS表达与NPC的生物学行为进行探讨。本研究应用免疫组化法检测iNOS在鼻咽正常粘膜和NPC中的表达情况，探讨iNOS与NPC发生发展浸润和转移的关系。方法：选取鼻咽活检石蜡标本50例，其中低分化鳞癌40例，正常鼻咽粘膜标本10例作对照。石蜡切片免疫组织化学染色，光镜下观察iNOS的蛋白表达情况。结果：正常鼻咽组织中，iNOS蛋白无表达；在NPC组织中，iNOS免疫组化阳性者细胞胞质和胞核内出现棕黄色细颗粒，且胞核染色比胞质更强。在NPC组织中iNOS蛋白表达明显上调，其阳性表达率分别为67．5％（27／40），与正常鼻咽组织比较（0／10），差异具有显著性（P〈0．05）；有淋巴结转者iNOS的表达高于无淋巴结转移者（72．41％vs54．55％），T3/4期NPC的iNOS表达高于T1/2期NPC（73．33％vs50．00％），差异均具有显著性（P〈0．05）。结论：①在NPC组织中，iNOS蛋白表达定位于细胞胞质和胞核，且胞核表达比胞质更强。②在NPC组织中，iNOS蛋白表达明显上调。iNOS的过度表达可能与NPC的发生、发展、侵袭和转移有关，选择性iNOS抑制剂可能有助于NPC的治疗。     

环氧合酶-2和诱导型一氧化氮合酶在宫颈癌组织中的表达及其意义

目的 :探讨环氧合酶 2 (COX 2 )与诱导型一氧化氮合酶 (iNOS)的表达与人宫颈癌发生发展的关系。方法 :应用免疫组织化学方法及逆转录聚合酶链反应 (RT PCR)技术检测 2 5例宫颈癌组织中COX 2和iNOS蛋白及mRNA的表达。结果 :宫颈癌组织中COX 2、iNOSmRNA的表达与正常组织比较均明显上调 ;其蛋白阳性表达率分别为 6 0 %和 80 % ;COX 2蛋白的阳性表达与组织学分级呈负相关 (r=- 0 4 2 0 ,P <0 0 5 )。淋巴结转移阳性组COX 2的表达高于阴性组 ,差异具有显著性 (P <0 0 5 )。iNOS蛋白的阳性表达与宫颈癌的临床分期及肿瘤分化程度有关 ,COX 2与iNOS蛋白阳性表达呈正相关 (r =0 4 5 6 ,P <0 0 1)。结论 :COX 2及iNOS的共同高表达可能参与宫颈癌的侵袭和转移 ,选择性COX 2抑制剂联合iNOS抑制剂的协同作用可能有助于晚期宫颈癌的治疗。     

Expression of endothelial nitric oxide synthase is induced by estrogen with glycogen synthase 3beta phosphorylation in MCF-7 cells

The endothelial nitric oxide synthase (eNOS) gene is induced by a variety of extracellular signals and NOS plays a key role in many physiological as well as pathological processes, including tumorgenesis. Some studies showed a positive correlation between the level of NOS protein and progression of malignancy in human breast cancer. In this study, we examined eNOS mRNA expression in human breast cancer cell lines. MCF-7 cells, which showed an estrogen receptor positive phenotype, were treated with estradiol or LiCl, a selective inhibitor of glycogen synthase kinase (GSK)-3beta. Both estradiol and LiCl enhanced the expression of eNOS mRNA with the phosphorylation of GSK-3beta, but not Akt. The induction was completely suppressed by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002, but not by PD98059, MEK-1 inhibitor nor rapamycin, p70S6 kinase inhibitor. We conclude that the estradiol-induced eNOS expression is modulated by PI3-kinase-dependent GSK-3beta pathway.     

诱导型一氧化氮合酶和碱性成纤维细胞生长因子在声门上喉鳞癌中的表达及意义

背景与目的：目前,有关诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS)和碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF)的表达与声门上型喉鳞癌颈淋巴结转移关系的研究很少。本文旨在探讨iNOS和bFGF在声门上喉鳞癌颈淋巴结转移等生物学行为中的作用。方法：应用iNOS和bFGF的多克隆抗体,以LSAB法对64例声门上喉鳞癌（实验组）和33例喉粘膜慢性炎症组织（对照组）进行对照研究,同时应用病理图像分析系统进行定量检测。结果：（1）iNOS和bFGF的阳性染色部位集中于细胞浆,bFGF也可表达于细胞核和细胞间质。（2）实验组iNOS和bFGF的表达明显高于对照组。（3）颈淋巴结阳性组的表达明显高于颈淋巴结阴性组,但初治阴性随诊阳性组与初诊阳性组的差异无统计学意义。结论：iNOS和bFGF的表达与声门上喉鳞癌关系密切,测定iNOS和bFGF的表达有助于预测声门上喉鳞癌淋巴结转移情况。     

Therapy of cancer metastasis by activation of the inducible nitric oxide synthase

The process of cancer metastasis consists of multiple sequential and highly selective steps. The vast majority of tumor cells that enter the circulation die rapidly; only a few survive to produce metastases. This survival is not random. Metastases are clonal in origin and are produced by specialized subpopulations of cells that preexist in a heterogeneous primary tumor. Experimental studies concluded that metastatic cells survive in the circulation whereas nonmetastatic cells do not. In part, this difference is due to an inverse correlation between expression of endogenous inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO) and metastatic potential. Direct evidence for the role of iNOS in metastasis has been provided by our data on transfection of highly metastatic murine K-1735 clone 4 (C4.P) cells which express low levels of iNOS, with a functional iNOS (C4.L8), inactive mutated iNOS (C4.S2), or neomycin resistance (C4.Neo) genes in medium containing 3 mM of the specific iNOS inhibitor N^G-L-methyl arginine (NMA). C4.P, C4.Neo, and C4.S2 cells were highly metastatic, whereas C4.L8 cells were not. Moreover, C4.L8 cells produced slow-growing subcutaneous tumors in nude mice, whereas the other 3 cell lines produced fast-growing tumors. In vitro studies indicated that the expression of iNOS in C4.L8.5 cells was associated with either cytostasis or cytolysis via apoptosis, depending upon NO output. The tumor cells producing high levels of NO underwent autocytolysis and produced cytolysis of bystander cells under both in vitro and in vivo conditions. Multiple i.v. injections of liposomes containing a synthetic lipopeptide upregulated iNOS expression in murine M5076 reticulum sarcoma cells growing as hepatic metastases. The induction of iNOS was associated with the complete regression of the lesions. Transfection of interferon- suppressed tumor formation and eradicated metastases, which was apparently linked to iNOS expression and NO production in host cells such as macrophage. Besides mediating cell death, NO produced tumor suppression by regulating expression of genes related to metastasis, e.g., survival, invasion, and angiogenesis. Suppression of metastasis can be achieved through use of immunomodulators that induce iNOS expression in tumor lesions or by the direct delivery of the iNOS gene to tumor cells or host cells through liposome and/or viral vectors.     

Immunostimulatory effects of low-dose cyclophosphamide are controlled by inducible nitric oxide synthase

Cyclophosphamide is a widely used chemotherapeutic drug that was recently applied as either an antiangiogenic/antivasculogenic or an immunostimulatory agent in combination with cancer immunotherapies. It has been previously shown that cyclophosphamide augments the efficacy of antitumor immune responses by depleting CD4+ CD25+ T regulatory cells and increasing both T-lymphocyte proliferation and T memory cells. Furthermore, cyclophosphamide was shown to mediate killing of circulating endothelial progenitors. However, the molecular basis for these observations has not yet been elucidated. We show here that the cyclophosphamide-mediated inhibition of inducible nitric oxide synthase is directly linked to its immunostimulatory but not to its antivasculogenic effects. Moreover, combined application of cyclophosphamide with a novel, oral DNA vaccine targeting platelet-derived growth factor B (PDGF-B), overexpressed by proliferating endothelial cells in the tumor vasculature, not only completely inhibited the growth of different tumor types but also led to tumor rejections in mice. These findings provide a new rationale at the molecular level for the combination of chemotherapy and immunotherapy in cancer treatment.     

Frequent expression of inducible nitric oxide synthase in esophageal squamous cell carcinomas.

Nitric oxide (NO) plays important biological roles in cardiovascular, nervous and immune systems, and is synthesized by nitric oxide synthase (NOS). Intracellular NO is known to cause DNA damage as a mutagen. We examined the expression of cytokine-inducible NOS (iNOS) in human esophageal squamous cell carcinomas. Weak iNOS immunoreactivity was seen in the basal and parabasal layers of non-neoplastic esophageal stratified squamous epithelium. iNOS expression was detected in 50 (87.7%) of the 57 esophageal squamous cell carcinomas, regardless of the depth of tumor invasion, histological differentiation and lymph node status. Early-stage cancers, i.e. mucosal squamous cell carcinomas, also showed significant iNOS expression. We speculate that increased iNOS expression is associated with the carcinogenesis of human esophageal cancer.