Elisas

To evaluate in vitro secretion of IL-6 and IL-8 by EOC cell lines, 5 × 10⁵ cells were seeded into individual wells of a six-well plate. After 24 h of incubation, culture supernatants were collected and 2 ml of the culture medium with 10% FBS and various concentrations of DHMEQ (0, 5, and 10 μg ml⁻¹) were added to each well. After 4 h of incubation, culture supernatants were aspirated and 2 ml of culture medium with 10% FBS and the same DHMEQ concentration was added to each well. After a further 4 h of incubation, cells and supernatants were collected and tested for the presence of human IL-6 and IL-8 by ELISAs (BD Biosciences Pharmingen), and nuclear extracts were prepared. Reverse transcription–polymerase chain reaction was performed using TaqMan probes (Applied Biosystems) for IL-6, IL-8, and GAPDH. A relative quantitative method was applied for the target mRNA that was normalised to the level of control GAPDH mRNA.

All tissue samples of EOC were fixed in neutral-buffered formalin and embedded in paraffin. Plasmas were obtained from 37 EOC patients and 19 age-matched female healthy donors. The bloods taken from EOC 37 patients on the day of surgery were immediately centrifuged and plasmas were stored at −80 °C. The plasma of the healthy donors was similarly collected at the outpatient setting in the morning. Plasma human IL-6 and IL-8 was measured by ELISAs (BD Biosciences Pharmingen). Data were collected from clinical and pathological records with informed consent from patients following approval by the Institutional Review Board of Keio University School of Medicine (No. 20130118).