Nitric oxide synthase II gene disruption: implications for tumor growth and vascular endothelial growth factor production

The expression of a primary initiator of tumor angiogenic responses, vascular endothelial growth factor (VEGF), may be induced by nitric oxide (NO) in carcinoma cells. However, the net impact of NO on carcinogenesis remains unclear, because manipulation of NO levels has been shown to either stimulate or inhibit tumor growth. We have investigated the relationship between inducible NO synthase (NOS II), VEGF expression, and growth of B16-F1 melanoma over 14 days in wild-type (NOS II+/+) mice and in those in which the gene for NOS II has been deleted (NOS II-/-). B16-F1 tumor growth was measured as wet weight of the excised tissue. Tumor NOS II and VEGF localization were evaluated by immunohistochemistry, and VEGF mRNA levels were measured by Northern blot analysis. In NOS II+/+ mice inoculated with B16-F1 melanoma cells, macroscopic tumors were always observed at 14 days; however, 22% of NOS II-/- mice had no detectable tumor mass. Immunoreactive NOS II was detected in tumor cells of tumors grown in NOS II+/+ but not in NOS II-/- mice. Although immunoreactive VEGF was detected in the granules of tumor-associated mast cells from both NOS II+/+ and NOS II-/- mice, VEGF mRNA expression in tumors from NOS II-/- was half that in NOS II+/+ mice. Neither NOS II inhibition, exogenous NO, nor peroxynitrite influenced DNA synthesis in culture B16-F1 melanoma cells. The NO donor did not alter either VEGF mRNA levels or degranulation in cultures of the mast cell line RBL-2H3, but peroxynitrite increased both VEGF mRNA expression and degranulation. We conclude that host expression of NOS II contributes to induction of NOS II in the tumor and to melanoma growth in vivo, possibly by regulating the amount and availability of VEGF.     

Intact nitric oxide synthase II gene is required for interferon-beta-mediated suppression of growth and metastasis of pancreatic adenocarcinoma

Previous studies have shown that enforced expression of IFN-beta suppressed tumor growth and metastasis. In this report, we determined whether the induction of nitric oxide synthase II (NOS II) gene is required for IFN-beta-mediated antitumor activity using syngeneic mice with intact (NOS II+/+) or genetically disrupted (NOS II-/-) NOS II gene. PANC02-H7 highly metastatic murine pancreatic adenocarcinoma cells were transfected with an IFN-beta expression vector or a control pcDNA3 vector. The parental PANC02-H7, control vector-transfected, and IFN-beta-transfected cells were orthotopically implanted into the pancreas of syngeneic NOS II+/+ and NOS II-/- C57BL/6J mice. In NOS II+/+ C57BL/ 6J, both parental and control vector-transfected cells grew progressively in pancreas and produced numerous liver metastases and a large amount of malignant ascites, whereas IFN-beta-secreting cells did not. In NOS II-/- C57BL/6J mice, however, IFN-beta-secreting cells grew much more aggressively. Higher NO induction was detected in NOS II+/+ mice that received injections with IFN-beta-secreting cells than with the control cells, but it was not detected in NOS II-/- mice. These data suggested that IFN-beta secreted from tumor cells stimulates NO production by host cells and suppresses tumor growth and metastasis.     

Direct demonstration of negative regulation of tumor growth and metastasis by host-inducible nitric oxide synthase

Inducible nitric oxide synthase (NOS) II expression can be induced in the tumor bed, predominantly in host cells that infiltrate and surround a tumor. However, the impact of this physiological NOS II expression in host cells on tumor growth and metastasis remains unclear because of a lack of appropriate experimental approaches. In the present study, three NOS II-null (NOS II(-/-)) tumor cell lines, KX-dw1, KX-dw4, and KX-dw7, were established and verified using Southern, Northern, and Western blot analysis, and nitric oxide production assays. Cells from these lines were then s.c. and i.v. injected into NOS II(+/+) and NOS II(-/-) C57BL/6 mice. NOS II protein expression and enzyme activity were clearly detected in the tumors that formed in NOS II(+/+) mice but not in those that formed in NOS II(-/-) mice. Consistent with the absence of NOS II expression in the tumor stroma, KX-dw1, KX-dw4, and KX-dw7 cells grew much faster and produced many more experimental lung metastases in NOS II(-/-) mice than in NOS II(+/+) mice. Therefore, physiological expression of NOS II in host cells directly inhibits tumor growth and metastasis.     

Genetic disruption of host nitric oxide synthase II gene impairs melanoma-induced angiogenesis and suppresses pleural effusion

Our previous study showed that genetic disruption of nitric oxide (NO) synthase II (NOS II) expression inhibits the metastatic ability of non-immunogenic B16 melanoma cells in syngeneic mice. In the present study, the mechanisms for this metastasis suppression were determined. B16-BL6 and B16-F10 murine melanoma cells were injected i.v. into syngeneic wild-type (NOS II(+/+)) and NOS II-null (NOS II(-/-)) C57BL/6 mice. Both melanoma cells produced less and smaller experimental pulmonary metastases in NOS II(-/-) mice than in NOS II(+/+) mice. Moreover, less metastatic pleural effusion was observed in NOS II(-/-) mice than in NOS II(+/+) mice. Immunohistochemical analyses indicated that absence of NOS II expression was correlated with decreased vascular endothelial growth factor expression and tumor-associated vascular formation. After activation with lipopolysaccharide and IFN-gamma, neither melanoma cell line produced detectable levels of NO. Our data demonstrate that tumor-induced expression of host NOS II enhances melanoma metastasis and pleural effusion, at least in part, through regulation of vascular formation and vascular permeability.     

内皮型一氧化氮合酶来源的NO调节肿瘤血管的生成

背景与目的:内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)来源的一氧化氮(nitric oxicle,NO)广泛表达于肿瘤组织,调节着肿瘤血管的生长,但研究结果并不一致。本研究中探讨NO对肿瘤血管形成的作用及其机制。方法:将C57BL/6小鼠随机分为3组:NO组小鼠右胁下接种eNOS基因转染的Lewis肺癌细胞;eNOS拮抗组小鼠接种Lewis肺癌细胞后腹腔注射eNOS拮抗剂L-NAME;对照组接种Lewis肺癌细胞后注射同等体积的生理盐水。处理3周后,测定血浆内NO含量,计数外周血中内皮祖细胞(EPC)数;取肿瘤组织测定每高倍视野下(HPF)血管密度、EPC细胞数量和血管内皮细胞生长因子及其受体复合物(VEGF-VEGFR)的表达。结果:接种Lewis细胞4周后,对照组肿瘤体积为(3022±401)mm3,而L-NAME组和eNOS组分别为(1204±97)mm3和(1824±239)mm3,三组比较有显著性差异(P<0.01)。eNOS基因转染组肿瘤组织内eNOS蛋白表达和NO的生成显著高于对照组,但肿瘤组织中EPC数量[(48±19)/HPF]、血管密...     

卵巢肿瘤一氧化氮合酶的活性研究

目的　探讨一氧化氮合酶 (nitricoxidesynthase ,NOS)与卵巢肿瘤间的关系 ,并观察其在卵巢肿瘤组织中的分布。方法　NOS的比活性用分光光度法 ,NOS在组织中的分布用NADPH 黄递酶组化染色法。结果　 (1)卵巢恶性肿瘤组织中NOS活性较正常卵巢组织和卵巢良性肿瘤组织显著增高 (P <0 0 1)。 (2 )卵巢良性肿瘤和正常卵巢组织中NOS含量差异无显著性 (P >0 0 5 )。 (3)NOS活性随卵巢恶性肿瘤组织分化程度降低而增高 (P <0 0 1)。 (4)恶性肿瘤患者血清中NOS活性高于正常对照。 (5 )在卵巢恶性肿瘤组织中NADPH 黄递酶染色呈阳性 ,而间质呈阴性 ;癌组织中染色深度高于正常卵巢及卵巢良性肿瘤组织 (P <0 0 1)。 (6 )NOS主要分布于癌细胞的胞膜和胞浆 ,胞核基本不着色。结论　一氧化氮 (nitricoxide,NO)合成的增多 ,可能与卵巢恶性肿瘤生长及恶性行为有关 ,卵巢恶性肿瘤组织中NOS含量增高是NO合成增多的重要原因之一。     

Genetic disruption of host interferon-gamma drastically enhances the metastasis of pancreatic adenocarcinoma through impaired expression of inducible nitric oxide synthase.

Synergistic induction of the inducible nitric oxide synthase (NOS II) gene requires a combination of interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). In this study, we determined whether the induction of IFN-gamma was required for NOS II-mediated antitumor activity in vivo. Highly metastatic H7 murine pancreatic adenocarcinoma cells were implanted into the subcutis, footpad, and pancreas of syngeneic IFN-gamma(+/+) and IFN-gamma(-/-) mice. These cells grew and produced metastases and ascites in IFN-gamma(+/+) mice. In sharp contrast, the same tumor cells grew much more aggressively, metastasized more extensively, and produced a larger amount of malignant ascites in IFN-gamma(-/-) mice. Also, induction of IFN-gamma correlated with NOS II gene expression and NO production in IFN-gamma(+/+) injected with the tumor cells but not in IFN-gamma(-/-) mice or IFN-gamma(+/+) mice without tumor challenge. In vitro, only LPS plus IFN-gamma induced a high level of NO production and cytotoxicity against H7 cells. These data suggested that the tumor cells stimulated IFN-gamma secretion from host cells, which in turn stimulated NO production by host cells and suppressed tumor growth and metastasis.     

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.     

Effect of cyclooxygenase and nitric oxide synthase inhibitors on tumor growth in mouse tumor models with and without cancer cachexia related to prostanoids

The potential interaction between cyclooxygenase (Cox) and NO metabolic pathways in the control of local tumor growth was evaluated. Mice bearing either a sarcoma-derived tumor (C57B1; MCG 101) or a malignant melanoma (C3H/HeN; K1735-M2) were used. These models were principally different because they demonstrate, in tumor hosts, conditions with and without cancer cachexia, seemingly related to high and low production of prostanoids, respectively. Cox inhibitors (Cox-1 and Cox-2) decreased tumor growth by 35-40% in MCG 101-bearing mice but had no such effect on melanoma-bearing mice, despite the expression of the Cox-2 protein in melanoma cells. Indomethacin reduced prostanoid production in both tumor (MCG 101) and host tissues and reduced tumor cell proliferation, mainly in vivo. Nitric oxide synthase (NOS) inhibitors (N(omega)-nitro-L-arginine methyl ester and N(omega)-nitro-L-arginine) reduced tumor growth in vivo by approximately 50% in both tumor models. Tumor growth reduction, related to NOS inhibition, was unrelated to prostanoid production and was an in vivo phenomenon in both tumor models. Specific inhibitors of inducible NOS activity, unexpectedly, had no effect in any tumor model, although inducible NOS protein was present in tumor tissues in large amounts. A combination of Cox and NOS inhibitors had no additive effect on tumor growth (MCG 101). Cox inhibition increased tumor tissue (MCG 101) expression of cNOS mRNA but had no significant effect on tumor tissue expression of the transferrin receptor, vascular endothelial growth factor, or basic fibroblast growth factor. NOS inhibition increased tumor tissue content of cNOS mRNA but showed as well a trend to increase mRNA content of the transferrin receptor and vascular endothelial growth factor. Our results suggest that NOS inhibitors can decrease the local growth of tumors that are either responsive or unresponsive to Cox inhibition. This effect may reflect cross-talk between Cox and NOS pathways within or among tumor cells, or it may represent unrelated effects on tumor and host cells. Whether NO inhibition may be used therapeutically in clinical tumors that are unresponsive to eicosanoid intervention remains to be evaluated.     

Use of retroviral vectors encoding murine inducible nitric oxide synthase gene to suppress tumorigenicity and cancer metastasis of murine melanoma

The purpose of this study was to determine whether retrovirus-mediated transfer of murine macrophage inducible nitric oxide synthase (iNOS) can produce inhibition of tumorigenicity and metastasis. Retroviral vectors encoding macrophage iNOS constructed in pLXSN, a retroviral vector with the iNOS gene under the control of a long terminal repeat promoter, were stably transfected into PA317 cells. Medium harvested from confluent monolayers of the virus-producing cell lines was used for infection of the murine K-1735 melanoma cells. Expression of iNOS was confirmed by northern and Western blot analyses. Functional iNOS protein expression was confirmed by bioassay of nitrite accumulation in the culture supernatant. Cells infected by a control iNOS-negative retrovirus produced fast-growing subcutaneous tumors and many lung metastases in nude mice, whereas iNOS-transduced cells produced slow-growing tumors and few lung metastases, showing that the infection of murine tumor cells by retroviruses harboring the iNOS gene can suppress tumorigenicity and metastasis.     

Gene expression and cellular sources of inducible nitric oxide synthase during tumor promotion

The present studies examined the temporal sequence of induciblenitric oxide synthase (iNOS) gene expression and the cellularsources of iNOS protein and of 3-nitrotyrosine, as a markerof production of nitric oxide-derived reactive nitrogen intermediatesduring murine multi-stage carcino-genesis. Levels of iNOS mRNAin dorsal skin isolated from acetone-treated female Sencar micewere 2.5-fold higher than iNOS gene expression detected in cutaneoustissue isolated from Sencar mice at 1,3,6,10,16 and 22 weeksafter exposure to a single topical application of 25 nmol 7,12-dimethylbenz[     

Polymorphisms of the endothelial nitric oxide synthase gene in breast cancer

Summary Nitric oxide (NO) is known to be critically involved in breast carcinogenesis. Genetic polymorphisms of the gene encoding for endothelial nitric oxide synthase (Nos3), the enzyme catalyzing the production of the NO, are known to predispose to malignant disease. Whether these polymorphisms also influence breast cancer risk is unknown. In the present case–control study, we ascertained 2 polymorphisms of the Nos3 gene cluster (Nos3 exon 7 Glu298Asp and a 27-base pair repeat in intron 4 of Nos3) in 269 Caucasian patients with breast cancer and 244 healthy controls using pyrosequencing and PCR, respectively. Presence of the exon 7 Nos3 polymorphism predisposed women to breast cancer (p=0.03, Odds ratio [95% Confidence Intervals]=1.9 [1.1–3.6]), but was not associated with any clinico-pathological parameters. No significant associations were ascertained with respect to the intron 4 Nos3 polymorphism. In our series, presence of the mutant exon7 Nos3 polymorphism was associated with an increased risk for breast cancer in Caucasian women.     

一氧化氮合酶在结肠腺癌中的表达及其与血管形成的关系

目的：通过检测结肠腺癌组织中ｉＮＯＳ、ｅＮＯＳ和ＣＤ３４的表达,探讨ＮＯＳ与结肠腺癌血管形成的关系。方法：利用免疫组织化学ＳＰ法检测ｉＮＯＳ、ｅＮＯＳ和ＣＤ３４在结肠癌不同病理分期中的表达,并观察其与肿瘤微血管密度的关系。结果：ｉＮＯＳ阳性率５６．７％;ｅＮＯＳ阳性率５３．３％;在结肠癌不同病理分期中,两种ＮＯＳ表达具有差异性（Ｐ〈０．０５）,同时微血管密度亦有差异（Ｐ〈０．０５）,尤其是Ｄｕｋｅ     

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.     

Endothelial nitric oxide synthase gene polymorphisms and genetic susceptibility to prostate cancer.

The endothelial cell-specific form of nitric oxide synthases (ecNOS) is localized at 7q35-q36 and is involved in vascular development and tumour growth in human prostate cancer. We have conducted a case-control study to investigate the prevalence of two polymorphisms at intron 4 (ecNOS4a/b) and exon 7 (Glu-Asp298) of ecNOS gene in 125 prostate cancer (PCa) patients and in 153 controls. We observed that the a-allele (aa or ab genotypes from ecNOS4a/b) was over-presented in the group of PCa with Gleason histological grade >or=7 (P=0.041). With regard to the Glu-Asp298 polymorphism, patients with the T-allele were younger than patients with no T-allele (P=0.037), and a statistically significant difference was noted in the Glu-Asp298 genotype distribution between cases with advanced disease and cases with localized disease (P=0.0013). When comparing cases and controls with logistic regression analysis we observed that the presence of the a-allele is associated with prostate cancer risk (odds ratio (OR) 1.83; 95% confidence interval (CI) 1.06-3.17; P=0.029), to high histological grade (Gleason >or=7) of PCa (OR 2.18; 95% CI 0.95-4.98; P=0.062) and with the risk of progression of the cancer disease (OR 2.85; 95% CI 1.19-6.82; P=0.018). Furthermore, we found that carriers with the combination of the a-allele (aa and ab ecNOS4a/b genotypes) and T-allele (GT and TT from Glu-Asp298) have a threefold increase in prostate cancer risk (OR 3.13; 95% CI 1.41-6.91, P=0.004). In summary, we have identified an NO-related genetic risk factor for prostate cancer that may help in understanding the molecular mechanism involved in the individual susceptibility to prostate cancer.     

丁酸钠对结肠癌 HT-29细胞的增殖抑制作用及 iNOS表达的影响

背景与目的：研究证明丁酸钠在体外对多种肿瘤细胞有抑制作用。本研究观察丁酸钠对结肠癌细胞株HT-29的生长抑制情况,以及对诱导型一氧化氮合成酶(iNOS)表达水平及其对一氧化氮(NO)分泌的影响。方法：采用不同浓度的丁酸钠对HT-29细胞进行干预．分别运用MTT法检测HT-29细胞株的增殖抑制情况,免疫细胞化学染色技术(SP法)对HT-29细胞胞浆内iNOS蛋白进行染色,图像分析系统检测胞浆内iNOS吸光度值(A值),Griess法检测HT-29细胞NO的分泌。结果：丁酸钠对HT-29细胞株增殖作用呈浓度、时间依赖性。在不同作用时间下,丁酸钠的IC50值不同(12h为15．4mmol／L．24h为5．7mmol／L,36h为2．5mmol／L,48h为0．9mmol／L)。丁酸钠也降低了HT-29细胞胞浆内iNOS的表达以及NO的分泌,亦呈浓度、时间依赖性。结论：丁酸钠能够抑制iNOS的表达水平,减少NO的分泌。这可能是它抑制结肠癌HT-29细胞增殖的作用机制之一。     

Mammary tumor latency is increased in mice lacking the inducible nitric oxide synthase

Nitric oxide (NO) and its metabolites are implicated in carcinogenesis and metastasis. Both stimulatory and inhibitory effects of NO have been reported in relation to breast cancer and its role in the development of malignancies and metastasis remains uncertain. We have used the polyomavirus middle T antigen (PyV-mT) targeted to the mouse mammary gland and bred into an inducible NO synthase (iNOS)-deficient C57Bl/6 strain to examine a role for nitric oxide in modulating tumors that develop in the complex environment of the whole animal. The development of hyperplasias was delayed to the extent that the earliest palpable tumors arose 2-4 weeks later in PyV-mT/iNOS(-/-) mice compared with PyV-mT/iNOS(+/+) mice, identifying a role for iNOS in early events in mammary tumor formation. Tumors that did develop in PyV-mT/iNOS(-/-) mice were characteristically well differentiated and had a cribriform pattern. Other tumors were myoepithelial adenocarcinomas with uniform nuclear size. In contrast, mice capable of iNOS activity typically developed solid nodular adenocarcinomas with a high mitotic index and pleomorphic nuclei. No significant effect of iNOS deficiency was found on vascular density in hyperplasias or tumors by examining CD31-positive vessels. The infiltration of lesions by macrophages, cells capable of significant NO production, remained unchanged in PyV-mT/iNOS(-/-) mice. Metastatic potential was retained by PyV-mT-transformed epithelium in the absence of iNOS, indicating that NO production by iNOS is not essential for this process. These results indicate a role for iNOS in tumorigenesis, particularly in the regulation of early events.     

Inducible nitric oxide synthase (iNOS) expression may predict distant metastasis in human melanoma

Expression of inducible nitric oxide synthase (iNOS) and its cellular localization was investigated in subcutaneous or lymph node metastases of human melanoma. Immunohistochemistry revealed that iNOS expression was limited to melanoma cells. In samples of patients without distant metastases, the number of iNOS+ tumour cells/total tumour cells was 55% +/- 17% (n = 12) compared with 9% +/- 8% when distant metastases of lung, liver or brain occurred within an observation period of 3 years (n = 10) (P < 0.001). Western blotting confirmed the expression of iNOS protein in select cases. Notably, iNOS is expressed in regional melanoma metastases and its expression is inversely related to the tumour's metastatic potential. Thus, iNOS expression may have predictive value for the development of distant metastases of human melanoma.