title: Liver cancer cells are sensitive to Lanatoside C induced cell death independent of their PTEN status.
abstract: BACKGROUND
  Hepatocellular carcinoma is the second deadliest cancer with limited treatment options. Loss of PTEN causes the P13K/Akt pathway to be hyperactive which contributes to cell survival and resistance to therapeutics in various cancers, including the liver cancer. Hence molecules targeting this pathway present good therapeutic strategies for liver cancer.
  
  
  OBJECTIVE
  It was previously reported that Cardiac glycosides possessed antitumor activity by inducing apoptosis of multiple cancer cells through oxidative stress. However, whether Cardiac glycoside Lanatoside C can induce oxidative stress in liver cancer cells and induce cell death both in vitro and in vivo remains unknown.
  
  
  METHODS
  Cell viability was measured by SRB assay. Cell death analysis was investigated by propidium iodide staining with flow cytometry and PARP cleavage. DCFH-DA staining and cytometry were used for intracellular ROS measurement. Protein levels were analyzed by western blot analysis. Antitumor activity was investigated on mice xenografts in vivo.
  
  
  RESULTS
  In this study, we found that Cardiac glycosides, particularly Lanatoside C from Digitalis ferruginea could significantly inhibit PTEN protein adequate Huh7 and PTEN deficient Mahlavu human liver cancer cell proliferation by the induction of apoptosis and G2/M arrest in the cells. Lanatoside C was further shown to induce oxidative stress and alter ERK and Akt pathways. Consequently, JNK1 activation resulted in extrinsic apoptotic pathway stimulation in both cells while JNK2 activation involved in the inhibition of cell survival only in PTEN deficient cells. Furthermore, nude mice xenografts followed by MRI showed that Lanatoside C caused a significant decrease in the tumor size. In this study apoptosis induction by Lanatoside C was characterized through ROS altered ERK and Akt pathways in both PTEN adequate epithelial and deficient mesenchymal liver cancer cells.
  
  
  CONCLUSIONS
  The results indicated that Lanatoside C could be contemplated in liver cancer therapeutics, particularly in PTEN deficient tumors. This is due to Lanatoside C's stress inducing action on ERK and Akt pathways through differential activation of JNK1 and JNK2 by GSK3β.
authors.affiliation: Department of Molecular Biology and Genetics, Faculty of Science, Bilkent University, Ankara, Turkey. Electronic address: durmaz@bilkent.edu.tr.
authors.forename: Irem
authors.initials: I
authors.lastname: Durmaz
authors.affiliation: Department of Molecular Biology and Genetics, Faculty of Science, Bilkent University, Ankara, Turkey. Electronic address: bilget@bilkent.edu.tr.
authors.forename: Ebru Bilget
authors.initials: EB
authors.lastname: Guven
authors.affiliation: Cancer Systems Biology Laboratory, Graduate School of Informatics, METU, 06800 Ankara, Turkey. Electronic address: ersahin@metu.edu.tr.
authors.forename: Tulin
authors.initials: T
authors.lastname: Ersahin
authors.affiliation: Izmir Biomedicine and Genome Center, Faculty of Medicine, Dokuz Eylul University, Izmir, Turkey. Electronic address: mehmet.ozturk@deu.edu.tr.
authors.forename: Mehmet
authors.initials: M
authors.lastname: Ozturk
authors.affiliation: Department of Pharmacognosy, Faculty of Pharmacy, Near East University, Lefkosa (Nicosia), N. Cyprus. Electronic address: icalis664@gmail.com.
authors.forename: Ihsan
authors.initials: I
authors.lastname: Calis
authors.affiliation: Department of Molecular Biology and Genetics, Faculty of Science, Bilkent University, Ankara, Turkey; Cancer Systems Biology Laboratory, Graduate School of Informatics, METU, 06800 Ankara, Turkey. Electronic address: rengul@metu.edu.tr.
authors.forename: Rengul
authors.initials: R
authors.lastname: Cetin-Atalay
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D006528:Carcinoma, Hepatocellular / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004070:Digitalis / Q000737:chemistry; D006801:Humans; D007799:Lanatosides / Q000494:pharmacology*; D008113:Liver Neoplasms / Q000473:pathology*; D051379:Mice; D008819:Mice, Nude; D018384:Oxidative Stress; D051059:PTEN Phosphohydrolase / Q000378:metabolism*; D015398:Signal Transduction; D023041:Xenograft Model Antitumor Assays
chemical_list: D007799:Lanatosides; C018548:lanatoside C; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human
keywords: Akt; ERK; Hepatocellular carcinoma; Lanatoside C; Oxidative stress; PTEN