title: Natural resistance to ascorbic acid induced oxidative stress is mainly mediated by catalase activity in human cancer cells and catalase-silencing sensitizes to oxidative stress.
abstract: BACKGROUND
  Ascorbic acid demonstrates a cytotoxic effect by generating hydrogen peroxide, a reactive oxygen species (ROS) involved in oxidative cell stress. A panel of eleven human cancer cell lines, glioblastoma and carcinoma, were exposed to serial dilutions of ascorbic acid (5-100 mmol/L). The purpose of this study was to analyse the impact of catalase, an important hydrogen peroxide-detoxifying enzyme, on the resistance of cancer cells to ascorbic acid mediated oxidative stress.
  
  
  METHODS
  Effective concentration (EC(50)) values, which indicate the concentration of ascorbic acid that reduced the number of viable cells by 50%, were detected with the crystal violet assay. The level of intracellular catalase protein and enzyme activity was determined. Expression of catalase was silenced by catalase-specific short hairpin RNA (sh-RNA) in BT-20 breast carcinoma cells. Oxidative cell stress induced apoptosis was measured by a caspase luminescent assay.
  
  
  RESULTS
  The tested human cancer cell lines demonstrated obvious differences in their resistance to ascorbic acid mediated oxidative cell stress. Forty-five percent of the cell lines had an EC(50) > 20 mmol/L and fifty-five percent had an EC(50) < 20 mmol/L. With an EC(50) of 2.6-5.5 mmol/L, glioblastoma cells were the most susceptible cancer cell lines analysed in this study. A correlation between catalase activity and the susceptibility to ascorbic acid was observed. To study the possible protective role of catalase on the resistance of cancer cells to oxidative cell stress, the expression of catalase in the breast carcinoma cell line BT-20, which cells were highly resistant to the exposure to ascorbic acid (EC(50): 94,9 mmol/L), was silenced with specific sh-RNA. The effect was that catalase-silenced BT-20 cells (BT-20 KD-CAT) became more susceptible to high concentrations of ascorbic acid (50 and 100 mmol/L).
  
  
  CONCLUSIONS
  Fifty-five percent of the human cancer cell lines tested were unable to protect themselves against oxidative stress mediated by ascorbic acid induced hydrogen peroxide production. The antioxidative enzyme catalase is important to protect cancer cells against cytotoxic hydrogen peroxide. Silenced catalase expression increased the susceptibility of the formerly resistant cancer cell line BT-20 to oxidative stress.
authors.affiliation: Experimental Surgery, Department of Surgery, University of Würzburg Hospital, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Klingelhoeffer
authors.forename: Ulrike
authors.initials: U
authors.lastname: Kämmerer
authors.forename: Monika
authors.initials: M
authors.lastname: Koospal
authors.forename: Bettina
authors.initials: B
authors.lastname: Mühling
authors.forename: Manuela
authors.initials: M
authors.lastname: Schneider
authors.forename: Michaela
authors.initials: M
authors.lastname: Kapp
authors.forename: Alexander
authors.initials: A
authors.lastname: Kübler
authors.forename: Christoph-Thomas
authors.initials: CT
authors.lastname: Germer
authors.forename: Christoph
authors.initials: C
authors.lastname: Otto
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology / Q000627:therapeutic use; D000975:Antioxidants / Q000378:metabolism* / Q000494:pharmacology / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D001205:Ascorbic Acid / Q000494:pharmacology / Q000627:therapeutic use*; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D002277:Carcinoma / Q000188:drug therapy / Q000378:metabolism; D002374:Catalase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D020868:Gene Silencing; D005909:Glioblastoma / Q000188:drug therapy / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology / Q000627:therapeutic use*; D018384:Oxidative Stress / Q000187:drug effects*; D034741:RNA, Small Interfering / Q000378:metabolism; D014815:Vitamins / Q000494:pharmacology / Q000627:therapeutic use
chemical_list: D000970:Antineoplastic Agents; D000975:Antioxidants; D016877:Oxidants; D034741:RNA, Small Interfering; D014815:Vitamins; D006861:Hydrogen Peroxide; D002374:Catalase; D001205:Ascorbic Acid