301 RAGE IS REQUIRED FOR OVAL CELL ACTIVATION AND INFLAMMATION-ASSOCIATED MOUSE LIVER CARCINOGENESIS

Abstract

in this study we tested the hypothesis that due to its growth promoting function, SIRT1 expression may be a positive factor in the growth of HCC and therefore inhibiting its activity may be a means to limit the growth of HCC. Methods: HCC cell lines (Hep3B, HepG2, HuH7, HLE, HLF, HepKK1, skHep1) were screened for mRNA expression of SIRT1–7. To inhibit SIRT1, cells were either transduced with lentiviruses expressing shRNA sequences targeting SIRT1 or treated with small molecule inhibitors, sirtinol or cambinol. Genetic deletion was confirmed by RT-PCR and Western-blot. Effect of SIRT1 inhibition was monitored by changes in morphology, proliferation and cell cycle in vitro. Half a million wild type and SIRT1 knock-down HCC cells were transplanted under the liver capsule of Rag2gc−/− immunodeficient mice and tumor growth was monitored by bioluminescence. Results: SIRT1 mRNA and protein was significantly higher expressed in HCC cell lines compared to normal liver cells, whereas other family members, SIRT2–7, were expressed at equal or lower levels. Inhibition of SIRT1 in HepG2 and Hep3B cells either by shRNA or with sirtinol or cambinol altered cell morphology, impaired proliferation and in HepG2 resulted in a decrease of dedifferentiation markers AFP and glypican. SIRT1 inhibition in Hep3B cells resulted a senescent morphology and expression of SA b-gal. Intrahepatic tumors developed in 84% of mice injected with control cells, while only 33% of the animals developed tumors with SIRT1 knock-down cells. In addition, cambinol reduced xenograft growth in mice compared to vehicle treated controls. Conclusion: Targeted deletion of SIRT1 expression impairs the growth of HCC cells in vivo and vitro. Activation of SIRT1 may have a beneficial role in non-malignant liver tissue, however in malignant HCCs, inhibiting SIRT1 activity may be a novel therapeutic option.