TLR4 induces tumor growth and inhibits paclitaxel activity in MyD88-positive human ovarian carcinoma in vitro

Abstract

In ovarian cancer patients, chemotherapy resistance is the principal factor restricting long-term treatment. Paclitaxel (Pac) has been previously reported to be a ligand to Toll-like receptor 4 (TLR4). It was determined that TLR4 signaling is divided into the following two pathways: Myeloid differentiation factor 88 (MyD88)-dependent and MyD88-independent. The present study investigated the effect of TLR4 ligation by Pac in MyD88-positive (MyD88⁺) and MyD88-negative (MyD88^-) human ovarian cancer cell lines. An RNA interference expression vector was specifically constructed to target TLR4 mRNA, which was stably transfected into the human ovarian cancer cell lines (SKOV3, OVCAR3, A2780 and 3AO). Cytokines, including interleukin (IL)-6 and IL-8, were detected. Cell proliferation and apoptosis were assessed in the cells transfected with scramble control and TLR4 shRNA to explore the possible functions of TLR4 in ovarian cancer cell growth. It was found that lipopolysaccharide and Pac significantly increase the secretion of IL-6 and IL-8 in the SKOV3 cell line. Similarly, Pac resulted in a significant upregulation of IL-6 and IL-8 in OVCAR3 cells, but not in A2780 and 3AO cells. These results suggested that in MyD88⁺ ovarian cancer cell lines, TLR4 depletion shows increased sensitivity to Pac treatment in inhibiting cell proliferation compared with in cells without TLR4 knockdown. On the contrary, such changes were not found in MyD88^- cells (A2780 and 3AO). TLR4 negatively regulates Pac chemotherapy, particularly in terms of cell proliferation, and TLR4 may be a novel treatment target in Pac-resistant ovarian cancer.

Keywords: MyD88; TLR4; chemotherapy; ovarian cancer; paclitaxel.

Figure 1

Expression of TLR4 and MyD88 in EOC cells at the mRNA level. TLR4 was expressed in all EOC cell lines; however, positive expression of MyD88 was identified in SKOV3 and OVCAR3 cells, while negative MyD88 expression was observed in A2780 and 3AO cells. TLR4, Toll-like receptor 4; MyD88, myeloid differentiation factor 88; EOC, epithelial ovarian cancer.

Figure 2

Levels of cytokines in tumor cell SNs. SNs were collected from tumor cells cultured at a density of 5×10⁵ cells/well following 36 h of treatment and tested for levels of IL-6. Three independent experiments were performed. ^*P<0.05, vs. NT/SKOV3 and ^**P<0.05, vs. NT/OVCAR3. SNs, supernatants; IL, interleukin; LPS, lipopolysaccharide; Pac, paclitaxel. NT, cells not treated with LPS or Pac.

Figure 3

Levels of cytokines in tumor cell SNs. SNs were collected from tumor cells cultured at a density of 5×10⁵ cells/well following 36 h of treatment and tested for levels of IL-8. Three independent experiments were performed. ^*P<0.05, vs. NT/SKOV3 and ^**P<0.05, vs. NT/OVCAR3. SNs, supernatants; IL, interleukin; LPS, lipopolysaccharide; Pac, paclitaxel. NT, cells not treated with LPS or Pac.

Figure 4

Effect of TLR4 knockdown in EOC cells using RT-PCR and western blot analysis. (A) Evaluation of mRNA expression of TLR4 in SKOV3, OVCAR3, A2780 and 3AO cell lines. (B) Western blot analysis for TLR4 protein expression in the four cell lines. shTLR4 cells were not treated with TLR4 interference vector transfection. TLR4, Toll-like receptor 4; EOC, epithelial ovarian cancer; RT-PCR, reverse transcription-polymerase chain reaction; NT, cells not treated with lipopolysaccharide or paclitaxel.

Figure 5

Levels of cytokines in tumor cell SNs. SNs were collected from tumor cells cultured at a density of 5×10⁵ cells/well following 36 h of treatment and tested for levels of IL-6. SNs, supernatants; IL, interleukin; LPS, lipopolysaccharide; Pac, paclitaxel. NT, cells not treated with LPS or Pac.

Figure 6

Levels of cytokines in tumor cell SNs. SNs were collected from tumor cells cultured at a density of 5×10⁵ cells/well following 36 h of treatment and tested for levels of IL-8. SNs, supernatants; IL, interleukin; LPS, lipopolysaccharide; Pac, paclitaxel. NT, cells not treated with LPS or Pac.

Figure 7

Following TLR4 knockdown, the apoptotic response of Pac (2 μM) treatment in MyD88⁺ and MyD88⁻ EOC cell lines was investigated. EOC cell lines were treated with 2 μmol/l Pac for 24 h and the levels of caspase-3/7 were measured using the Caspase-Glo 3/7 assay. Data are presented as the mean ± SD from at least three independent experiments. A significant increase was identified in caspase-3/7 activity following Pac treatment in SKOV3/shTLR4 cells compared with SKOV3 and SKOV3/shControl cells, as was the case with the OVCAR3 cells. ^*P<0.001, vs. wt/Pac and shControl/Pac. TLR4, Toll-like receptor 4; MyD88, myeloid differentiation factor 88; EOC, epithelial ovarian cancer; Pac, paclitaxel; wt/Pac, SKOV3/OVCAR3/A2780/3AO parental cells treated with Pac; shControl/Pac, SKOV3/OVCAR3/A2780/3AO shControl cells treated with Pac; shTLR4/Pac, SKOV3/OVCAR3/A2780/3AO shTLR4 cells treated with Pac.

Figure 8

Effect of TLR4 knockdown on EOC cell growth using MTT assay. SKOV3/shTLR4 cells were inhibited by ~60% in the presence of Pac at 2 μmol/l compared with parental SKOV3 (29%) and SKOV3/shControl (31%) cells. OVCAR3/shTLR4 cells showed a marked change compared with parental OVCAR3 and OVCAR3/shControl cells. However, no significant changes were identified among the parental, shControl and shTLR4 cells in the A2780 and 3AO cell lines. ^*P<0.001, vs. wt/Pac and shControl/Pac. TLR4, Toll-like receptor 4; EOC, epithelial ovarian cancer; Pac, paclitaxel; wt/Pac, SKOV3/OVCAR3/A2780/3AO parental cells treated with Pac; shControl/Pac, SKOV3/OVCAR3/A2780/3AO shControl cells treated with Pac; shTLR4/Pac, SKOV3/OVCAR3/A2780/3AO shTLR4 cells treated with Pac.