Nitrative and oxidative DNA damage in intrahepatic cholangiocarcinoma patients in relation to tumor invasion

Abstract

Aim: Nitrative and oxidative DNA damage such as 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation has been implicated in initiation and/or promotion of inflammation-mediated carcinogenesis. The aim of this study is to clarify whether these DNA lesions participate in the progression of intrahepatic cholangiocarcinoma.

Methods: We investigated the relation of the formation of 8-nitroguanine and 8-oxodG and the expression of hypoxia-inducible factor-1alpha (HIF-1alpha) with tumor invasion in 37 patients with intra-hepatic cholangiocarcinoma.

Results: Immunohistochemical analyses revealed that 8-nitroguanine and 8-oxodG formation occurred to a much greater extent in cancerous tissues than in non-cancerous tissues. HIF-1alpha could be detected in cancerous tissues in all patients, suggesting low oxygen tension in the tumors. HIF-1alpha expression was correlated with inducible nitric oxide synthase (iNOS) expression (r = 0.369 and P = 0.025) and 8-oxodG formation (r = 0.398 and P = 0.015). Double immunofluorescence study revealed that iNOS and HIF-1alpha co-localized in cancerous tissues. Notably, the formation of 8-oxodG was correlated significantly with lymphatic invasion (r = 0.386 and P = 0.018). Moreover, 8-nitroguanine and 8-oxodG in non-cancerous tissues were associated significantly with neural invasion (P = 0.042 and P = 0.026, respectively). These results suggest that reciprocal activation between HIF-1alpha and iNOS mediates persistent DNA damage, which induces tumor invasiveness via mutations, resulting in poor prognosis.

Conclusion: The formation of 8-nitroguanine and 8-oxodG plays an important role in multiple steps of genetic changes leading to tumor progression, including invasiveness.

Figure 1

Localization of 8-oxodG and 8-nitroguanine in cancerous and noncancerous liver tissues in an ICC patient with a well-differentiated adenocarcinoma. Formation of 8-oxodG and 8-nitroguanine was assessed by immunohistochemistry using an immunoperoxidase method. Paraffin sections (6 μm thickness) were incubated with a rabbit polyclonal anti-8-nitroguanine antibody and a mouse monoclonal anti-8-oxodG antibody. The original magnification is 200×.

Figure 2

Localization of iNOS and HIF-1α in cancerous liver tissues in an ICC patient with poorly-differentiated adenocarcinoma. The expression of iNOS and HIF-1α was assessed by using double immunofluorescence technique. Paraffin sections were treated with anti-iNOS and anti-HIF-1α antibodies. The original magnification is 400×.

Figure 3

Overall survival curve of ICC patients. Thin line, neural invasion evaluated as -; thick line, neural invasion evaluated as + (Kaplan-Meier method).