IMMUNOCYTOCHEMICAL EVALUATION OF TOLL-LIKE RECEPTORS ON BREAST CANCER CELL LINES UNDER THE TREATMENT OF WORTMANNIN AND THALIDOMIDE IN VITRO
DOI:
https://doi.org/10.29121/granthaalayah.v10.i4.2022.4556Keywords:
Breast cancer, TLrs, PI3K, Wortmannin, Thalidomide, 67NR, 4T1, ImmunocytochemistryAbstract [English]
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.
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References
Agnese DM, Calvano JE, Hahm S, Coyle SM, Corbett SA, Calvano SE, Lowry SF, (2002). Human Toll-like receptor 4 mutations but not CD14 polypmorphism are associated with an increased risk of gramnegative infections. The Journal of Infectious Diseases, 186(10),1522-152. https://doi.org/10.1086/344893
Akter R, Hossain MZ, Kleve MG, Gealt MA. (2012). Wortmannin induces MCF-7 breast cancer cell death via the apoptotic pathway, involving chromatin condensation, generation of reactive oxygen species, and membrane blebbing. 13(4),103-13. https://doi.org/10.2147/BCTT.S31712
Arbibe L, Mira JP, Teusch N, Kline L, Guha M, Mackman N, et al. (2000). Toll-like receptor 2-mediated NF-kappa B activation requires a Rac1-dependent pathway. https://doi.org/10.1038/82797
Basith S, Manavalan B, Yoo TH, Kim SG, Choi S. (2012). Roles of toll-like receptors in cancer : a double-edged sword for defense and offense. Journal of Occupational Medicine and Toxicology. 35(8),1297-316. https://doi.org/10.1007/s12272-012-0802-7
Becker S. (2015). A historic and scientific review of breast cancer : The next global healthcare challenge. International Journal of Gynaecology and Obstetrics. https://doi.org/10.1016/j.ijgo.2015.03.015
Bhatelia K, Singh K, Singh R. (2014). TLRs : linking inflammation and breast cancer. Cellular Signalling. 26(11), 2350-7. https://doi.org/10.1016/j.cellsig.2014.07.035
Biswas SK, Lopez-Collazo E. (2009). Endotoxin tolerance : new mechanisms, molecules and clinical significance. Trends in Immunology. 30,475-87. https://doi.org/10.1016/j.it.2009.07.009
Carmeliet P. (2005). VEGF as à key mediator of angiogenesis in cancer. Oncology. 69(3),4-10. https://doi.org/10.1159/000088478
Cook KM, Figg WD. (2010). Angiogenesis inhibitors : current strategies and futurs prospects. A Caner Journal for Clinicians, 60(4),222-43. https://doi.org/10.3322/caac.20075
DeSantis CE, Bray F, Ferlay J, Lortet-Tieulent J, Anderson BO, Jemal A. Dexter DL, Kowalski HM, Blazar BA, Fligiel Z, Vogel R, (1978). Heterogeneity of tumor cells from a single mouse mammary tumor. National Libarry of Medicine, 38(10), 3174-3181. https://pubmed.ncbi.nlm.nih.gov/210930/
Friis S, Kesminiene A, Espina C, Auvinen A, Straif K, Schüz J. (2015). European Code against Cancer 4th Edition : Medical exposures, including hormone therapy, and cancer. Cancer Epidemiology, 107-19. https://doi.org/10.1016/j.canep.2015.08.003
Fukata M, Chen A, Vamadevan AS, Cohen J, Breglio K, Krishnareddy S et al. (2007). Toll-like receptor-4 promotes the development of colitis-associated colorectal tumors. Gastroenterology. 133(6),1869-81. https://doi.org/10.1053/j.gastro.2007.09.008
Hirsch FR, Varella-Garcia M, Bunn PA Jr, Di Maria MV, Veve R, Bremmes RM, et al., (2003). Epidermal growth factor receptor in non-small-cell lung carcinomas : Correlation between gene copy number and protein expression and impact on prognosis. Journal of Clinical Oncology, 21(20), 3798-807. https://doi.org/10.1200/JCO.2003.11.069
Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. (2011). Global cancer statistics. A Cancer Journal for Clinicians, 61(2),69-90. https://doi.org/10.3322/caac.20107
John T, Liu G, Tsao MS. (2009). Overview of molecular testing in non-small-cell lung cancer : Mutational analysis, gene copy number, protein expression and other biomarkers of EGFR for the prediction of response to tyrosine kinase inhibitors. Oncogene, 14-23. https://doi.org/10.1038/onc.2009.197
Kim S, and Karin M. (2011). Role of TLR2-dependent inflammation in metastatic progression. Ann. The New York Academy of Science, 191-206. https://doi.org/10.1111/j.1749-6632.2010.05882.x
Laird MH, Rhee SH, Perkins DJ, Medvedev AE, Piao W, Fenton MJ, et al. (2009). TLR4/MyD88/PI3K interactions regulate TLR4 signaling. Journal of Leukocyte Blology, 85,966-77. https://doi.org/10.1189/jlb.1208763
Mehmeti M, Allaoui R, Bergenfelz C, Saal LH, Ethier SP, Johansson ME, Jirström K, Leandersson K. (2015). Expression of functional toll like receptor 4 in estrogen receptor/progesterone receptor-negative breast cancer. Breast Cancer Research, 17(1),130. https://doi.org/10.1186/s13058-015-0640-x
Nakanishi C, Toi M. (2005). Nuclear factor-κB inhibitors as sensitizers to anticancer drugs. Nature Reviews Cancer. 5(4), 297-309. https://doi.org/10.1038/nrc1588
Ozgul M, Turkoz Uluer E, Tanriover G, Inan S. (2016). Evaluation of the Apoptotic Effects of Wortmannin and Thalidomide on Breast Cancer Cell Lines. Turkish Journal of Biochemistry.
Rakoff-Nahoum S, Medzhitov R. (2007). Regulation of spontaneous intestinal tumorigenesis through the adaptor protein MyD88. Science, 124-7. https://doi.org/10.1126/science.1140488
Rakoff-Nahoum S, Medzhitov R. (2009). Toll-like receptors and cancer. Nature Reviews Cancer, 9,57-63. https://doi.org/10.1038/nrc2541
Robert J. (2015). Textbook of Cell Signaling in Cancer. Springer. https://doi.org/10.1007/978-3-319-14340-8
Ruse M, Knaus UG. (2006). New players in TLR-mediated innate immunity : PI3K and small Rho GTPases. 34,33-48. https://doi.org/10.1385/IR:34:1:33
Thike AA, Chng MJ, Fook-Chong S, Tan PH. (2001). Immunohistochemical expression of hormone receptors in invasive breast carcinoma : correlation of results of H-score with pathological parameters. Pathology, 33(1), 21-5. https://doi.org/10.1080/00313020120034858
Troutman TD, Bazan JF, Pasare C. (2012). Toll-like receptors, signaling adapters and regulation of the pro-inflammatory response by PI3K. 11(19),3559-67. https://doi.org/10.4161/cc.21572
Underhill D M, Ozinsky A, (2002). Toll-like receptors : key mediators of microbe detection. Current Opinion in Immunology, 14 (1), 103- 110. https://doi.org/10.1016/S0952-7915(01)00304-1
Xie W, Wang Y, Huang Y, Yang H, Wang J, Hu Z. (2009). Toll-like receptor 2 mediates invasion via activating NF-kappaB in MDA-MB-231 breast cancer cells. Biochemical and Biophysical Research Communications, 1027-32. https://doi.org/10.1016/j.bbrc.2009.01.009
Yang H, Zhou H, Feng P, Zhou X, Wen H, Xie X, et al. (2010). Reduced expression of Toll-like receptor 4 inhibits human breast cancer cells proliferation and inflammatory cytokines secretion. Journal of Experimental and Clinical Cancer research. https://doi.org/10.1186/1756-9966-29-92
Yuan TL, Cantley LC. (2008). PI3K pathway alterations in cancer : variations on a theme. Oncogene. 5497-5510. https://doi.org/10.1038/onc.2008.245
Yun J, Lv YG, Yao Q, Wang L, Li YP, Yi J. (2012). Wortmannin inhibits proliferation and induces apoptosis of MCF-7 breast cancer cells. 33(4),367-9. https://article.imrpress.com/journal/EJGO/33/4/pii/1631087281572-1634603826/367-369.pdf
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