Nitric oxide in Liver Cancer

Abstract

Nitric oxide (NO) has been implicated in the pathogenesis of liver cancer or hepatocellular carcinoma (HCC). However, the exact role(s) of NO and the expression pattern of its enzymes, nitric oxide synthases (NOS), remain unclear. Thus, this study was designed to investigate the expression and regulation of NOS in both human and animal HCC samples. An animal model of HCC was established and this allowed the tracking of NOS expression during the progression of HCC. NOS selective inhibitors and NO donors were administered to determine the possible role(s) of NO in HCC in an animal model. An animal model for HCC was established with the administration of diethlynitrosamine (DEN) in drinking water. This treatment resulted in the development of cirrhosis in the liver prior to the development of visible foci, similar to the progress of HCC in humans. DEN was given to male Sprague-Dawley rats ad libitum for a period of 4, 6, 8, 10, 12, 14 and 16 weeks. Apart from visible foci formations, glutathione S-transferase-p (GST-p), a commonly used marker for neoplasms, staining was carried out to confirm the development of neoplastic lesions. With time, DEN-treated rats were found to have increased NO and NOS, Rac2 and caveolin-1 expression. However, eNOS and iNOS, the two isoforms of NOS studied, showed differential expression activities which were most distinct at week 10. To further investigate the role of NO in the progression of HCC, NOS selective inhibitors and NO donors were administrated to rats after 10 weeks of DEN treatment. Again the levels of NO and NOS expression and activities were studied. NOS selective inhibitors, N-nitro-L-arginine (L-NNA) and N-1-iminoethly-L-lysine (L-NIL), were selected for their high inhibitory effects against eNOS and iNOS respectively. NO donors, isosorbide dinitrate (ISDN) and molsidomine were selected as these are currently used in prescription drugs for humans. Interestingly, NOS inhibitors exacerbated HCC progression, which implies a protective role for eNOS. NO donor treatments on the other hand, showed decreased visible foci, cirrhotic lesions and GST-p positive staining. This implies NO plays a protective role in HCC progression in our animal model for HCC. In conclusion, we found that the different isoforms of NOS, iNOS and eNOS, are expressed differently in human liver diseases. During the progression to neoplasm as studied in our HCC model, iNOS and eNOS play different roles as suggested by the differential expression and activity patterns. Administration of NOS inhibitors and NO donors more clearly defined the role of NO in foci formation. NOS inhibitors did not alleviate HCC formation. In contrast, the systemic and liver specific NO donors, both showed dramatic improvement in reducing neoplasm formation. This could be used as a platform for examining the therapeutic potential of NO donors in the treatment of HCC.