Identification of HO-1/CO-induced tumor protective genes in HCC

Background and Aims: Induction or over-expression of the heme degrading enzyme heme oxygenase 1 (HO-1) has been shown to interfere with acute and chronic hepatitis in mice. On the other hand, HO-1 has been found to be over-expressed in tumors, including hepatocellular carcinoma (HCC). Here the anti-apoptotic HO-1 product CO protects tumor cells from apoptosis. Our aim was to identify mediators of CO-induced protection as specific targets for tumor therapy.

Methods: Freshly isolated primary mouse hepatocytes (PH) were incubated in the presence of the CO donor methylene chloride (MC) for 2 to 6 hours. Apoptosis was induced by actinomycin D (ActD)/TNF incubation. Cytotoxicity was measured by LDH release assay. Gene expression following CO-incubation was measured by Affymetrix arrays and verified by real time RT-PCR. Expression profiles were established for 52 HCC derived from Mdr2ko mice as well as from mouse and human hepatoma cell lines.

Results: The HO-1 product CO protected PH from induced apoptosis in a time- and dose-dependent manner. Affymetrix arrays identified 519 HO-1/CO regulated genes. Among those, several potentially cytoprotective factors were found induced. Bioinformatic analysis indicated the involvement of NFkB and AP1 transcription factor activation in HO-1/CO-mediated protection. Expression profiles of tumors and hepatoma cell lines revealed different compositions, even between tumors derived from the same individual. On the other hand, expression of some genes, including Myc, A20 and osteopontin, was found to be deregulated more frequently.

Conclusions: HCC seem to be very heterogenous in their gene expression profiles, while several genes are deregulated more frequently than others. Knockdown experiments will now determine the contribution of the identified genes or combinations of theses genes to tumor cell protection, e.g. against chemotherapy.