title: Selective ablation of Notch3 in HCC enhances doxorubicin's death promoting effect by a p53 dependent mechanism.
abstract: OBJECTIVE
  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.
authors.affiliation: Center for Applied Biomedical Research (CRBA), S.Orsola-Malpighi University Hospital, Bologna, Italy. catia.giovannini@aosp.bo.it
authors.forename: Catia
authors.initials: C
authors.lastname: Giovannini
authors.forename: Laura
authors.initials: L
authors.lastname: Gramantieri
authors.forename: Pasquale
authors.initials: P
authors.lastname: Chieco
authors.forename: Manuela
authors.initials: M
authors.lastname: Minguzzi
authors.forename: Federica
authors.initials: F
authors.lastname: Lago
authors.forename: Simona
authors.initials: S
authors.lastname: Pianetti
authors.forename: Eric
authors.initials: E
authors.lastname: Ramazzotti
authors.forename: Kenneth B
authors.initials: KB
authors.lastname: Marcu
authors.forename: Luigi
authors.initials: L
authors.lastname: Bolondi
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D004249:DNA Damage / Q000187:drug effects / Q000502:physiology; D004317:Doxorubicin / Q000494:pharmacology / Q000627:therapeutic use*; D019008:Drug Resistance, Neoplasm / Q000502:physiology; D005260:Female; D017353:Gene Deletion; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D008297:Male; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism; D000071656:Receptor, Notch3; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000502:physiology; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
chemical_list: D000903:Antibiotics, Antineoplastic; C497761:NOTCH1 protein, human; C499374:NOTCH3 protein, human; D051881:Receptor, Notch1; D000071656:Receptor, Notch3; D051880:Receptors, Notch; D016159:Tumor Suppressor Protein p53; D004317:Doxorubicin