title: Thymoquinone enhances cisplatin-response through direct tumor effects in a syngeneic mouse model of ovarian cancer.
abstract: BACKGROUND
  Ovarian cancer is the most lethal gynecologic malignancy characterized by the frequent development of resistance to platinum chemotherapy. Finding new drug combinations to overcome platinum resistance is a key clinical challenge. Thymoquinone (TQ) is a component of black seed oil that exerts multiple anti-tumorigenic effects on cells, including inhibition of NF-κB and promotion of DNA damage. We aimed to determine whether TQ enhances cisplatin cytotoxicity in cultured ovarian cancer cells and in an established murine syngeneic model of ovarian cancer.
  
  
  METHODS
  Ovarian cancer cell viability in vitro was measured by sulforhodamine B (SRB) assays, and drug interactions tested for synergism by isobologram analysis. ID8-NGL mouse ovarian cancer cells stably expressing an NF-κB reporter transgene were injected intra-peritoneally into C57BL/6 mice. After 30 day TQ and/or cisplatin treatment, we measured the following indices: tumor burden (ascites volume, number of peritoneal implants and mesenteric tumor mass); NF-κB reporter activity (luciferase assay); protein expression of the double-strand DNA break marker, pH2AX(ser139), the proliferation markers, Ki67/mib-1 and PCNA, and the apoptosis markers, cleaved caspase-3, cleaved PARP and Bax; and mRNA expression of NF-κB targets, TNF-α and IL-1β. Two-tailed Mann-Whitney tests were used for measuring differences between groups in mouse experiments.
  
  
  RESULTS
  In SRB assays, TQ and cisplatin synergized in ID8-NGL cells. In mice, cisplatin significantly reduced cell proliferation and increased apoptosis in tumors, resulting in decreased overall tumor burden. Combining TQ with cisplatin further decreased these indices, indicating co-operative effects between the drugs. TQ treatment promoted cisplatin-induced pH2AX expression in cultured cells and in tumors. While NF-κB inhibition by TQ induced anti-tumor effects in vitro, we made the unexpected observation that TQ alone increased both tumor NF-κB activity and formation of ascites in vivo.
  
  
  CONCLUSIONS
  TQ enhanced cisplatin-mediated cytoxicity in ovarian cancer cells in vitro and in a mouse syngeneic model, effects associated with increased DNA damage. However, our results strongly caution that TQ treatment alone may have an overall deleterious effect in the immunocompetent host through stimulation of ascites. Since TQ is a potential candidate for future clinical trials in ovarian cancer patients, this finding has considerable potential relevance to the clinic.
authors.affiliation: Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Vanderbilt University School of Medicine, Nashville, TN, USA. andrew.j.wilson@vanderbilt.edu.|Department of Obstetrics and Gynecology, Vanderbilt University Medical Center, B1100 Medical Center North, Nashville, TN, 37232, USA. andrew.j.wilson@vanderbilt.edu.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Wilson
authors.affiliation: Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Vanderbilt University School of Medicine, Nashville, TN, USA.
authors.forename: Jeanette
authors.initials: J
authors.lastname: Saskowski
authors.affiliation: Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: Whitney
authors.initials: W
authors.lastname: Barham
authors.affiliation: Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN, USA.|Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: Fiona
authors.initials: F
authors.lastname: Yull
authors.affiliation: Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Vanderbilt University School of Medicine, Nashville, TN, USA.|Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: Dineo
authors.initials: D
authors.lastname: Khabele
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D016227:Benzoquinones / Q000008:administration & dosage*; D053148:Caspase 3 / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000008:administration & dosage*; D004195:Disease Models, Animal; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D051379:Mice; D016328:NF-kappa B / Q000235:genetics; D010051:Ovarian Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
chemical_list: D016227:Benzoquinones; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D053148:Caspase 3; C003466:thymoquinone; D002945:Cisplatin