Darbepoetin inhibits proliferation of Huh-7 and Hep3B cells via up-regulation of the tumor-suppressor gene p53

In 2002 Aranesp (Darbepoetin) was licensed for patients with solid tumors suffering from chemotherapy-dependent anemia. As a better blood supply may favor tumor growth and spreading, we wanted to investigate whether there are direct effects of Darbepoetin on liver tumor cell lines or primary human hepatocytes. Human hepatoma cell-lines used were HepG2, Hep3B, SkHep1, Huh-7, AKN1, HCC-T and HCC-M. Primary human hepatocytes were isolated by collagenase perfusion according to the ethical guidelines of the MRI. Cells were stimulated with different concentrations of Darbepoetin (0, 0.15, 0.31, 0.63, 1.26, 2.5, 5, 10, 20 and 40 ng/ml). After 24 hours we measured viability by conversion of Alamar Blue substrate. HepG2, SkHep1, AKN1, HCC-T and HCC-M showed no significant reduction in viability. Surprisingly, Huh-7 and Hep3B cells showed less Alamar Blue conversion, dose-dependently from 5 ng/ml Darbepoetin on. However, no cellular damage could be detected at those concentrations as measured by LDH-release into the culture supernatant. FACS analysis and DNA laddering excluded apoptosis or necrosis as the cause for the reduced Alamar Blue conversion observed in Darbepoetin-treated Huh-7 and Hep3B cells. Interestingly, Western blot analysis revealed that Darbepoetin treatment increased p53 expression in both cell lines, causing growth arrest as observed early in senescence. Our data show that Darbepoetin causes reduced growth in the hepatic cell lines Huh-7 and Hep3B by up-regulation of the tumor-suppressor gene p53. If we manage to further analyze the underlying mechanisms Darbepoetin might be not only useful for patients with anemia but also be applied locally to suppress growth of certain tumors.

Hep3B - Huh-7 - darbepoetin - p53