Excessive production of nitric oxide in rat solid tumor and its implication in rapid tumor growth.
BACKGROUND
Rapid tumor growth is caused by angiogenesis factors, growth factors, etc. We previously reported a possible connection between nitric oxide (NO) and enhanced vascular permeability in solid tumor. In the present experiment, the role of NO in solid tumor pathology was further investigated in animal tumor.


METHODS
To identify NO formed in solid tumor (AH136B) implanted in the feet of rats, electron paramagnetic resonance (EPR) spectroscopy was performed directly on the frozen tumor tissue at 110K by measuring endogenous nitrosyl iron-sulfur complexes, and by using exogenously added NO capturing agents, i.e., diethyldithiocarbamate (DETC)-Fe2+ and N-(dithiocarboxy) sarcosine (DTCS)-Fe2+ complexes. Induction of inducible isoform of nosymthase iNOS mRNA was examined with reverse transcriptase-polymerase chain reaction (RT-PCR) combined with Southern blot analysis. In addition, vascular permeability was assessed by measuring extravasation of 51Cr-labeled bovine serum albumin in solid tumor.


RESULTS
Strong EPR signals from NO adducts of DETC-Fe2+ and DTCS-Fe2+ as well as strong signals from NO-hemoglobin and dinitrosyl iron sulfur complex were generated by tumor. The signal height of NO-(DTCS)2-Fe2+ observed in AH136B solid tumor was increased as the tumor gained up to 1.75 g. Induction of iNOS mRNA expression was confirmed by the above methods. Enhanced vascular permeability was suppressed by NOS inhibitors N omega- monomethyl-L-arginine or S-methylisothiourea sulfate and augmented with administration of L-arginine.


CONCLUSIONS
Excessive NO production by iNOS in solid tumor was identified unequivocally by EPR spectroscopy. NO formed in solid tumor can be involved in enhanced vascular permeability and increased blood flow, and hence sustain tumor growth.
Doi
K
K
Department of Surgery II, Kumamoto University, School of Medicine, Japan.
Akaike
T
T
Horie
H
H
Noguchi
Y
Y
Fujii
S
S
Beppu
T
T
Ogawa
M
M
Maeda
H
H
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002199:Capillary Permeability; D002455:Cell Division / Q000502:physiology; D002860:Chromium Radioisotopes; D008969:Molecular Sequence Data; D009368:Neoplasm Transplantation; D009374:Neoplasms, Experimental / Q000378:metabolism* / Q000473:pathology*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012710:Serum Albumin, Bovine / Q000493:pharmacokinetics; D014158:Transcription, Genetic
D002860:Chromium Radioisotopes; D012333:RNA, Messenger; D012710:Serum Albumin, Bovine; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase