title: PKI-587 and sorafenib alone and in combination on inhibition of liver cancer stem cell proliferation.
abstract: BACKGROUND
  Deregulated Ras/Raf/mitogen-activated protein kinase and PI3 K/AKT/mTOR signaling pathways are significant in hepatocellular carcinoma proliferation (HCC). In this study we evaluated differences in the antiproliferative effect of dual PI3 K/Akt/mTOR and Ras/Raf/mitogen-activated protein kinase inhibition of non liver cancer stem cell lines (PLC and HuH7) and liver cancer stem cell (LCSC) lines (CD133, CD44, CD24, and aldehyde dehydrogenase 1-positive cells).
  
  
  METHODS
  Flow cytometry was performed on the resulting tumors to identify the LCSC markers CD133, CD44, CD24, and aldehyde dehydrogenase 1. Methylthiazol tetrazolium assay was used to assess cellular proliferation. Finally, a Western blot assay was used to evaluate for inhibition of specific enzymes in these two signaling pathways.
  
  
  RESULTS
  Using flow cytometry, we found that LCSC contain 64.4% CD133 + cells, 83.2% CD44 + cells, and 96.4% CD24 + cells. PKI-587 and sorafenib caused inhibiton of LCSC and HCC cell proliferation. PLC cells were more sensitive to PKI-587 than LCSC or Huh7 (P < 0.001). Interestingly, HuH7 cells were more sensitive to sorafenib than LCSC or PLC cells. Additionally, combination therapy with PKI-587 and sorafenib caused significantly more inhibition than monotherapy in HuH7, PLC, and LCSC. Using the methylthiazol tetrazolium assay, we found that the LCSC proliferation was inhibited with sorafenib monotherapy 39% at 5 μM (P < 0.001; n = 12) and 67% by PKI-587 at 0.1 μM (P = 0.002, n = 12) compared with control. The combination of PKI-587 and sorafenib, however, synergistically inhibited LCSC proliferation by 86% (P = 0.002; n = 12).
  
  
  CONCLUSIONS
  LCSC (CD133+, CD44+, CD24+) were able to develop very aggressive tumors with low cell concentrations at 4 to 6 wk. Cells CD133+, CD44+, CD24+, which demonstrated at least moderate resistance to therapy in vitro. The combination of PKI-587 and sorafenib was better than either drug alone at inhibiting of LCSC and on HCC cell proliferation.
authors.affiliation: Department of Surgery, University of Kentucky, College of Medicine, Lexington, Kentucky. Electronic address: rgeda2@uky.edu.
authors.forename: Roberto
authors.initials: R
authors.lastname: Gedaly
authors.forename: Roberto
authors.initials: R
authors.lastname: Galuppo
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Musgrave
authors.forename: Paul
authors.initials: P
authors.lastname: Angulo
authors.forename: Jonathan
authors.initials: J
authors.lastname: Hundley
authors.forename: Malay
authors.initials: M
authors.lastname: Shah
authors.forename: Michael F
authors.initials: MF
authors.lastname: Daily
authors.forename: Changguo
authors.initials: C
authors.lastname: Chen
authors.forename: Donald A
authors.initials: DA
authors.lastname: Cohen
authors.forename: Brett T
authors.initials: BT
authors.lastname: Spear
authors.forename: B Mark
authors.initials: BM
authors.lastname: Evers
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004359:Drug Therapy, Combination; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D020935:MAP Kinase Signaling System / Q000187:drug effects; D009025:Morpholines / Q000494:pharmacology*; D014411:Neoplastic Stem Cells / Q000166:cytology / Q000187:drug effects*; D009536:Niacinamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D010671:Phenylurea Compounds / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D000077157:Sorafenib; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D014227:Triazines / Q000494:pharmacology*; D048490:raf Kinases / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D009025:Morpholines; D009536:Niacinamide; D010671:Phenylurea Compounds; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D016283:Proto-Oncogene Proteins p21(ras); D000077157:Sorafenib; D058570:TOR Serine-Threonine Kinases; D014227:Triazines; D048490:raf Kinases; C549060:gedatolisib; C546842:MTOR protein, human; C494918:AKT1 protein, human
keywords: Hepatocellular carcinoma; Liver cancer stem cells; PKI-587; Sorafenib