title: An investigation of the molecular basis for the synergistic interaction of tirapazamine and cisplatin.
abstract: OBJECTIVE
  To determine if the observed tirapazamine (TPZ)-cisplatin synergistic cell kill was mediated at the cellular level by impairment of upregulation of key proteins involved in repair of DNA interstrand crosslinks. Cisplatin sensitivity has been shown to be dependent on the expression of the two DNA repair proteins ERCC1 and XPA. ERCC1 expression has also been shown to be upregulated by cisplatin exposure. Therefore, these studies were undertaken to determine if hypoxia-activated TPZ pretreatment inhibited the cells' normal protective response to cisplatin via inhibiting the upregulation of ERCC1 and/or XPA expression.
  
  
  METHODS
  Two different cell lines, one cisplatin sensitive and one cisplatin resistant, were treated with TPZ, cisplatin, both drugs together (which results in additive cytotoxicity), or TPZ followed by cisplatin (which results in synergistic cytotoxicity). All cells were exposed to 1 h of hypoxia to bioactivate the TPZ. Expression of ERCC1 and XPA were evaluated at the mRNA and protein level at 24 or 48 h after drug exposure.
  
  
  RESULTS
  In the cisplatin-sensitive non-small-cell lung cancer cell line, ERCC1 expression at the mRNA or protein level was not significantly altered in any of the treatment groups. In the cisplatin-resistant ovarian cancer cell line, ERCC1 expression was upregulated by TPZ, but not by cisplatin alone. The change in protein expression was less pronounced than the change in mRNA level. XPA expression was not significantly changed from baseline in either cell line at the mRNA or protein level.
  
  
  CONCLUSIONS
  In contrast to reports in the literature, this study did not demonstrate cisplatin inducing its own repair by upregulating the DNA crosslink repair proteins ERCC1 or XPA. Therefore, the TPZ-cisplatin synergism cannot be mediated through hypoxia-activated TPZ inhibiting this cellular protective response. TPZ alone, however, was able to alter repair protein expression, which may play a role in mediating its cytotoxicity.
authors.affiliation: Division of Radiation Biology, Department of Radiation Oncology, Stanford University School of Medicine, Stanford CA, 94305 USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Goldberg
authors.forename: J
authors.initials: J
authors.lastname: Evans
authors.forename: G
authors.initials: G
authors.lastname: Birrell
authors.forename: J M
authors.initials: JM
authors.lastname: Brown
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D015687:Cell Hypoxia / Q000502:physiology; D002945:Cisplatin / Q000494:pharmacology*; D004260:DNA Repair / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000187:drug effects* / Q000378:metabolism; D004357:Drug Synergism; D004720:Endonucleases; D006801:Humans; D011506:Proteins / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D000077704:Tirapazamine; D014227:Triazines / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects; D015854:Up-Regulation / Q000187:drug effects; D051760:Xeroderma Pigmentosum Group A Protein
chemical_list: D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D011506:Proteins; D012333:RNA, Messenger; D011838:Radiation-Sensitizing Agents; D014227:Triazines; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D000077704:Tirapazamine; C071447:ERCC1 protein, human; D004720:Endonucleases; D002945:Cisplatin