Immunohistochemical and western blotting evaluation of the effect of the treatment on toll-like receptors on breast cancer cell lines with low (67nr) and high (4t1) metastatic potential

Aim: The aim of this study is to investigate the effects of Wortmannin and Thalidomide on Toll-Like Receptors (TLR) on breast cancer cell lines with low (67NR) and high (4T1) metastatic potential in vitro. Material and Method: Doubling time of breast cancer cell lines were calculated and by using MTT assay, IC50 values was determined as 2,5 μM for Wortmannin and 50 μM for Thalidomide. The cells were evaluated via TUNEL, indirect immunohistochemistry with anti-TLR2, anti-TLR4 anti-MyD88, anti-NFκB and anti- PI3K primary antibodies and Western Blotting methods, after the drug was administered on the 24th and 48th hours. Statistics were comparatively evaluated by using the One-way ANOVA test. Results: Calculating the % TUNEL index, the drug administered group had a statistically significant increase when compared to the control groups. The immunoreactivity intensities and H-Score values of anti-TLR2, anti-TLR4, anti- MyD88, anti-NFκB and anti-PI3K primary antibodies were calculated and it was observed that there was a statistically significant decrease in the drug administered groups when compared to the control groups. Using the Western Blotting method, the presence of TLR2, TLR4, NFκB and PI3K molecules was shown. Conclusion: It was first demonstrated by this study, that the effects on the 24th and 48th hour of the PI3K inhibitor Wortmannin and the angiogenesis inhibitor Thalidomide applications were effective on TLR signaling pathway and related molecules on 67NR and 4T1 breast cancer cell lines which have different metastatic potentials.