Selective ablation of Notch3 in HCC enhances doxorubicin's death promoting effect by a p53 dependent mechanism

Background/Aims:The functional roles of endogenous Notch3 and Notch1 for protecting human hepatocellular carcinoma (HCC) lines against doxorubicin-induced death have been investigated. We previously reported aberrant Notch3 and Notch4 up-regulation in HCC and we have extended these observations to include Notch1. Methods: Notch1 expression was assessed by immunohistochemistry and immunoblotting. Notch3 and Notch1 expression were ablated in multiple HCC lines by stable retroviral transduction of short hairpin RNAs (shRNAs). Effects on doxorubicin sensitivity were evaluated with respect to cell growth, expression of specific cell cycle effectors and multiple apoptotic parameters. Results: Notch3 depletion increased p53 expression, doxorubicin uptake, DNA damage, the apoptosis inducing effects of doxorubicin and also impeded the cell cycle progression of HCC cells. Ablating p53 expression in Notch3 knockdown (KD) cells largely abolished their enhanced doxorubicin sensitivity; and Notch3 KD in p53(-/-) Hep3B cells failed to influence their response to doxorubicin. Although up-regulated in most HCC, Notch1 (unlike Notch3) did not contribute to the doxorubicin resistance of HCC lines. Conclusions: Our in vitro results represent the first evidence that Notch3 silencing in combination with chemotherapeutics could conceivably provide a novel strategy for HCC treatment that deserves further exploration. (c) 2009 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.