Korean J Obstet Gynecol KJOG Korean Journal of Obstetrics and Gynecology 1738-5628 Korean Society of Obstetrics and Gynecology 10.5468/kjog.2010.53.4.330 Original Article MyD88 expression and anti-apoptotic signals of paclitaxel in epithelial ovarian cancer cells Suh Dong Soo M.D. Jo Moo Sung M.D. Yu Shin Ae M.D. Kim Ki Hyung M.D. Yoon Man Soo M.D. Department of Obstetrics and Gynecology, Pusan National University School of Medicine, Busan, Korea. Corresponding author (ghkim@pusan.ac.kr) 04 2010 30 04 2010 53 4 330 338 11 12 2009 25 03 2010 Copyright © 2010 Korean Society of Obstetrics and Gynecology 2010 Objective

The objectives of this study was to evaluate the correlation between myeloid differentiation protein 88 (MyD88) expression and paclitaxel effects on epithelial ovarian cancer cells and to evaluate whether paclitaxel had anti-apoptotic signals.

 Methods

Epithelial ovarian cancer cells isolated from ascites and established cell lines were treated with increasing concentrations of paclitaxel (0.2 to 20 µM) for 24 and 48 hours and cell viability was determined using the CellTiter 96 AQueous One Solution Cell Proliferation Assay. Cytokine profiling was performed from culture supernatants using the Luminex 200 system. Nuclear factor-kappaB (NF-κB) activity was determined using a Luciferase reporter system. Levels of phospho-extracellular signal-regulated kinase (p-ERK) were measured by Western blot analysis.

 Results

A strong signal for MyD88 expression was observed in R182, 01-19b and SKOV3 cells (MyD88-positive). A2780, R454 and 01-28 cells showed low levels of MyD88 (MyD88-negative). Paclitaxel effectively decreased cell viability in MyD88-negative A2780, R454, 01-28 cells after 24 and 48 hours (57%, 49%, 42% and 35%, 28%, 29%, respectively). MyD88-positive cells were resistant to paclitaxel. There was a significant increase in caspase-3/7 activity following paclitaxel treatment in MyD88-negative cells. No significant change in caspase-3/7 activity was detected in MyD88-positive cells. Paclitaxel induced NF-κB activation and enhanced the secretion of interleukin-6 (IL-6) and IL-8 in a dose dependent manner and induced ERK phosphorylation on MyD88-positive cells.

 Conclusion

Paclitaxel treatment for MyD88-positive ovarian cancer could have detrimental effects due to the paclitaxel-induced enhancement of NF-κB, ERK activities and pro-inflammatory cytokine production, which promote chemoresistance and tumor progression.

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Western blot analysis for MyD88 protein expression from epithelial ovarian cancer cells.

Paclitaxel significantly decreases the number of viable MyD88-negative ovarian cancer cells (*P=0.000; General linear model repeated measures followed by Turkey method). The viability (in percentage, normalized to untreated cells) of MyD88-positive (R182, 01-19b, SKOV3) and -negative (A2780, R454, 01-28) cell lines after treatment with increasing concentrations of paclitaxel (0.2, 2, 20 µM) for 24 (A) and 48 (B) hours (P=0.000; General linear model repeated measures followed by Turkey method).

Paclitaxel significantly induces apoptosis in MyD88-negative A2780 cells. Cells were treated with 2 µM paclitaxel for 24 hours. Activity of caspase-3/7 was measured using Caspase-Glo assay (*P=0.000; General linear model repeated measures followed by Turkey method).

Differential effects of paclitaxel on nuclear factor-kappaB (NF-κB) activation in MyD88-positive R182 cells and -negative A2780 cells. Cells were transfected with a luciferase reporter plasmid activated by NF-κB and treated with 2 µM paclitaxel. NF-κB activity was measured as luminescence. Arrow indicates paclitaxel treatment.

Cytokine profiles after paclitaxel treatment in MyD88-positive R182 cells. Cells were treated with paclitaxel (0.2, 2, 20 µM) for 48 hours and the levels of secreted cytokines were determined using Luminex 200 system (P=0.000; General linear model repeated measures followed by Turkey method).

(A) Effects of paclitaxel on extracellular signal-regulated kinase (ERK) activation in MyD88-positive and -negative cells. Cells were treated with paclitaxel (0.2, 2, 20 µM) for 24 hours and levels of phospho-ERK (p-ERK) and total ERK (t-ERK) were determined by Western blotting. (B) cell viability after paclitaxel at different times in MyD88-negative A2780 cells (*P=0.000; General linear model repeated measures followed by Turkey method). (C) In vitro growth pattern in cells with constitutive low ERK and high ERK.

Simplified model of MyD88-mediated toll like receptor (TLR) signaling in epithelial ovarian cancer cells.

NF-κB: nuclear factor-kappaB.