Figure 2

Immunohistochemical localization and Western blot analysis of TLR2, TLR4 and MyD88 in serous papillary ovarian carcinoma. The immunoreactivity for TLR2 was moderate in the surface epithelium of OC + Mel (B) compared to OC animals (A) (arrows). Weak immunoreactivity for TLR2 was found in OC + EtOH animals (C) but was more pronounced in OC + EtOH + Mel animals (D). TLR4 immunoreactivity was intense in the surface epithelium, but not in the stroma, of the OC (E) and OC + EtOH groups (G) (arrows). Weak immunoreactivity for TLR4 was observed in the OC + Mel group (F) (arrow), and moderate TLR4 reaction was detected in the epithelium of the OC + EtOH + Mel group (H) (arrow). Moderate MyD88 reaction was observed in the epithelium of the OC group (I) (arrow), in contrast to very weak immunoreactivity for MyD88 in the OC + Mel group (J) (arrow). The OC + EtOH (K) and OC + EtOH + Mel groups (L) displayed moderate and weak signals for MyD88, respectively (arrows). Bar = 20 μm. Negative controls were used. (M) Representative TLR2, TLR4, and MyD88 expression profiles in extracts (70 μg protein) pooled from 10 samples/group (upper panel). (N–P) Extracts obtained from individual rats were used for densitometric analysis of the TLR2, TLR4, and MyD88 levels following normalization to the β-actin. All results are expressed as the means ± SD (n = 10 animals/group). ^aP < 0.05 vs. OC; ^bP < 0.05 vs. OC + Mel and ^cP < 0.05 vs. OC + EtOH. (Q) Schematic representation of the TLR2 and TLR4 signaling pathways via MyD88 and IRAK intracellular cascade, leading to NFkB activation, in a typical ovarian carcinoma cell. TLR2 is upregulated by mel and downregulated by EtOH + Mel, whereas TLR4 is downregulated by mel. Mel therapy also negatively regulates MyD88 expression. mel: melatonin; EtOH: ethanol; IRAK: IL-1 receptor-associated kinase; TRAF6: TNF receptor-associated factor 6; TAK1: TGF-B-activated kinase 1; NFkB: nuclear factor kappa B.