IMMUNOCYTOCHEMICAL EVALUATION OF TOLL-LIKE RECEPTORS ON BREAST CANCER CELL LINES UNDER THE TREATMENT OF WORTMANNIN AND THALIDOMIDE IN VITRO

Abstract

Aim: The aim of this study was to investigate the effects of the PI3K inhibitor Wortmannin and the angiogenesis inhibitor Thalidomide on Toll-Like Receptors (TLR) on breast cancer cell lines which have non (67NR) and high (4T1) metastatic potential using immunocytochemical technique.
 Material and Method: The cells were evaluated using avidin-biotin-peroxidase indirect immunocytochemistry method. Anti-TLR2, anti-TLR4, anti-MyD88, anti-PI3K and anti-NFκB primary antibodies were performed after 24h and 48h administrations of Wortmannin and Thalidomide. The distribution of immunocytochemical intensities of primary antibodies were graded semi-quantitatively as mild (1), moderate (2), strong (3) and very strong (4). The mean values of the staining intensities and the percentage of positively stained cells were calculated using the H-Score. Statistics were comparatively evaluated by using the One-way ANOVA test. Significance was defined as p<0.05.
 Results: On the control group of 67NR breast cancer cell line, immunoreactivity of TLR2 was seen as very strong. While immunoreactivity of MyD88 was seen as strong, immunoreactivities of TLR4, NFκB and PI3K were observed as moderate in this group. On the control group of 4T1 breast cancer cell line, immunoreactivities of MyD88 and NFκB were seen as strong, while immunoreactivities of TLR2, TLR4 and PI3K were observed as moderate/strong in this group.
 Conclusion: It was demonstrated by this study, that the effects on the 24th and 48th hour of the Wortmannin acts as an inhibitor of PI3K and Thalidomide acts as an inhibitor of NFκB were effective on TLR signaling pathway and related molecules on 67NR and 4T1 breast cancer cell lines which have different metastatic properties. It was concluded that these drugs have an important role as inhibitors in cancer signaling pathways included invasion and metastasis. As future expectation, they might be used therapeutically in addition to classical treatments on cancer treatment.