Research of toll like reseptors in resistance mechanism against bortezomibe in Multiple Myeloma

RESEARCH OF TOLL LIKE RECEPTORS IN RESISTANCE MECHANISM AGAINST BORTEZOMIBE IN MULTIPLE MYELOMA Multiple myeloma (MM) is a hematologic malignancy that is characterized as an abnormal proliferation and invasion of plasma cells into the bone marrow. The most effective treatment method currently available for MM therapy is chemotherapeutic regimens in which the proteasome inhibitor bortezomibin is involved. However, resistance mechanism against bortezomib causes failure of treatment. Despite the study of many mechanisms known to play a role in the resistance mechanism in the literature, the role of TLR in drug resistance has not yet been elucidated. Therefore, this thesis study aimed to investigate the role of TLRs in the resistance mechanism against bortezomib in MM cell lines. For this purpose, bortezomib-resistant (KMS-20) and sensitive (KMS-28BM) MM cell lines were used. Firstly, it was determined the dose and time dependent effects of bortezomib for two cell lines on cell viability by using MTT test. Secondly, cell lines were treated with different concentrations of bortezomib for 12, 24 and 48 hours, followed by total RNA isolation followed by cDNA synthesis. mRNA levels of TLR 2, 3, 4, 7, 9 and MyD88 genes were determined by real-time RT-PCR using primers specific for each gene and TaqMan probes. IC50 doses in the 12, 24 and 48 hours for KMS-20 cell line were, respectively; 31.62 nM; 15.85nM; 5.89 nM and IC50 doses in the 12, 24 and 48 hours for KMS-28BM cell line were, respectively; 11.84 nM; 5.30nM; 3.66 nM. The resistance index (RI) of KMS-20 cell line for 12, 24 and 48 hours were 2.67; 2.99 and 1.61 respectively. When gene expression levels were examined, TLR2 expression levels were found to be significantly decreased in resistant cells compared to sensitive cells, depending on dose and time. While TLR3 and TLR4 gene expression showed dose and time dependent decrease in sensitive cells, they were completely suppressed in resistant cells. While the TLR7 expression showed a dose-dependent and time-dependent decrease in the sensitive cell, it was shown a significantly increase in the resistant cell. TLR9 showed a significant decrease in dose and time in sensitive cells, while a significant decrease in only high concentrations in resistant cells. A significant increase in only 48 hours in the MyD88 resistant cell showed, while a significantly decrease showed at 24 and 48 hours in sensitive cells. Finally, when all gene expression levels in resistant and sensitive cells are compared, the decrease or complete suppression of TLR2, TLR3 and TLR4 mRNA levels, but the increase of TLR7 and MyD88 mRNA levels in resistant cells may play a role in the resistance mechanism against bortezomib. Keywords: Multiple myeloma, Drug Resistance, Toll Like Receptors, qRT-PCR, Bortezomib.