title: Valproic Acid Increases CD133 Positive Cells that Show Low Sensitivity to Cytostatics in Neuroblastoma.
abstract: Valproic acid (VPA) is a well-known antiepileptic drug that exhibits antitumor activities through its action as a histone deacetylase inhibitor. CD133 is considered to be a cancer stem cell marker in several tumors including neuroblastoma. CD133 transcription is strictly regulated by epigenetic modifications. We evaluated the epigenetic effects of treatment with 1mM VPA and its influence on the expression of CD133 in four human neuroblastoma cell lines. Chemoresistance and cell cycle of CD133+ and CD133- populations were examined by flow cytometry. We performed bisulfite conversion followed by methylation-sensitive high resolution melting analysis to assess the methylation status of CD133 promoters P1 and P3. Our results revealed that VPA induced CD133 expression that was associated with increased acetylation of histones H3 and H4. On treatment with VPA and cytostatics, CD133+ cells were mainly detected in the S and G2/M phases of the cell cycle and they showed less activated caspase-3 compared to CD133- cells. UKF-NB-3 neuroblastoma cells which express CD133 displayed higher colony and neurosphere formation capacities when treated with VPA, unlike IMR-32 which lacks for CD133 protein. Induction of CD133 in UKF-NB-3 was associated with increased expression of phosphorylated Akt and pluripotency transcription factors Nanog, Oct-4 and Sox2. VPA did not induce CD133 expression in cell lines with methylated P1 and P3 promoters, where the CD133 protein was not detected. Applying the demethylating agent 5-aza-2'-deoxycytidine to the cell lines with methylated promoters resulted in CD133 re-expression that was associated with a drop in P1 and P3 methylation level. In conclusion, CD133 expression in neuroblastoma can be regulated by histone acetylation and/or methylation of its CpG promoters. VPA can induce CD133+ cells which display high proliferation potential and low sensitivity to cytostatics in neuroblastoma. These results give new insight into the possible limitations to use VPA in cancer therapy.
authors.affiliation: Department of Pediatric Hematology and Oncology, 2nd Medical Faculty, Charles University and University Hospital Motol, Prague, Czech Republic.
authors.forename: Mohamed Ashraf
authors.initials: MA
authors.lastname: Khalil
authors.affiliation: Department of Pediatric Hematology and Oncology, 2nd Medical Faculty, Charles University and University Hospital Motol, Prague, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Hraběta
authors.affiliation: Department of Biochemistry, Faculty of Science, Prague, Czech Republic.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Groh
authors.affiliation: Institute of Experimental Medicine, Academy of Sciences, Prague, Czech Republic.|Institute of Biology and Medical Genetics, 1st Medical Faculty, Charles University, Prague, Czech Republic.
authors.forename: Pavel
authors.initials: P
authors.lastname: Procházka
authors.affiliation: Department of Pediatric Hematology and Oncology, 2nd Medical Faculty, Charles University and University Hospital Motol, Prague, Czech Republic.
authors.forename: Helena
authors.initials: H
authors.lastname: Doktorová
authors.affiliation: Department of Pediatric Hematology and Oncology, 2nd Medical Faculty, Charles University and University Hospital Motol, Prague, Czech Republic.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Eckschlager
mesh_terms: D000071916:AC133 Antigen / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D014408:Biomarkers, Tumor / Q000378:metabolism; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D054697:Cytostatic Agents / Q000494:pharmacology*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D009447:Neuroblastoma / Q000188:drug therapy*; D014635:Valproic Acid / Q000494:pharmacology*
chemical_list: D000071916:AC133 Antigen; D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; D054697:Cytostatic Agents; D014635:Valproic Acid; D053148:Caspase 3