High expression of endothelial cell nitric oxide synthase in peritumoral microvessels predicts increased disease-free survival in colorectal cancer
Introduction
Nitric oxide (NO) is generated from l-arginine by three nitric oxide synthase (NOS) isoenzymes, neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial cell NOS (ecNOS) [1], [2]. NO is involved in neurotransmission, immune responses, blood pressure regulation and angiogenesis as well as regulation of apoptosis. Previous studies have revealed that different NOS isoenzymes are expressed by human cancers and by cancer cell lines [3], [4], [5], [6], [7], [8], [9], [10], [11]. Recent studies have, moreover, shown that the human breast cancer (MCF-7) cell line expresses ecNOS, but not other NOS isoforms, in a p53-regulated fashion and that NO affects apoptosis of these cells [8].
In a retrospective material of premenopausal women with breast cancer we have previously documented that the frequency of peritumoral microvessels expressing ecNOS (peritumoral ecNOS-expressing microvessel density: PEMVD) is a significant and independent prognostic factor for overall and disease-free survival [9]. This may suggest that NO is involved in protecting endothelial cells from invading tumor cells. Such a hypothesis concurs with observations showing that nitric oxide (NO) produced by endothelial cells can kill tumor cells [12] and reduce tumor cell adhesion to endothelial cells [13]. Moreover, in an animal model, inhibition of NO-generation in liver sinusoids has been found to result in an increased number of liver metastases [14].
In order to see if these findings were of more general significance, we have now completed a retrospective study encompassing 186 patients with colorectal cancers. All tumors included in the study represented Duke's stage B.
Section snippets
Materials and methods
Tumor material. The material comprised 186 colorectal cancer specimens (Duke's stage B) from Hvidovre Hospital. Specimens from the proximal and distal ends of resected tumors were routinely fixed in 10% buffered formalin and embedded in paraffin.
Immunocytochemistry. Five-micrometer deparaffinized sections were pretreated for 30 min with methanol containing 0.03% H2O2 to inhibit endogenous peroxidase activity and subsequently microwaved in 10 mM sodium citrate buffer, pH 6, as described [9].
Results
There were 108 male and 78 female patients, the median age was 71 years (range 35–88 years). Hundred and twenty-four cancers were localized to the colon while 62 cancers occurred in the rectum. The median observation time was 8.0 years (range 6.9–9.1 years). There were 89 deaths (64 deaths at 5 years with four deaths within 1 month), 21 distant metastases, 31 local recurrences and 43 patients with both types of recurrences.
Sections from the proximal and distal ends of each specimen were
Discussion
Our data show that a high density of ecNOS-positive peritumoral microvessels is associated with a significantly lowered chance for recurrences in colorectal cancer patients. Our data indicate that a high PEMVD had greatest impact on absence of distant metastases while the effect on local recurrences was less. We have considered death of all causes at 5 years as a secondary end-point. Cancer specific death is not available since most patients are no longer autopsied in Denmark. Thus, since most
Acknowledgements
Grant support was from the Danish Cancer Society. We thank B. Traasdahl for expert technical assistance.
References (16)
Mammalian nitric oxide synthases
Biochim. Biophys. Acta
(1999)- et al.
Expression of nitric oxide synthase in gastric cancer
Cancer Lett.
(1999) - et al.
Endogenous endothelial cell nitric oxide synthase modulates apoptosis in cultured breast cancer cells and is transcriptionally regulated by p53
J. Biol. Chem.
(1999) - et al.
Nitric oxide: physiology, pathophysiology, and pharmacology
Pharmacol. Rev.
(1991) - et al.
Relaxin activates the l-arginine-nitric oxide pathway in human breast cancer
Cancer Res.
(1995) - et al.
Cytoplasmic localization of endothelial constitutive nitric oxide synthase in endometrial carcinomas
Tumour Biol.
(1997) - et al.
Expression of two types of nitric oxide synthase mRNA in human neuroblastoma cell lines
J. Neurochem.
(1994) - et al.
Selective expression of inducible nitric oxide synthase in human prostate carcinoma
Cancer
(1998)