Reversal of multidrug resistance in MCF-7 breast adenocarcinoma cell line by silencing interleukin 6 with RNA interference

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Tezin Türü: Yüksek Lisans

Tezin Yürütüldüğü Kurum: Orta Doğu Teknik Üniversitesi, Fen Bilimleri Enstitüsü, Fen Bilimleri Enstitüsü, Türkiye

Tezin Onay Tarihi: 2013

Öğrenci: NEŞE ÇAKMAK

Danışman: UFUK GÜNDÜZ

Özet:

Multidrug resistance (MDR) in cancer is characterized by development of resistance to several unrelated drugs upon long time administration of a certain type of chemotherapeutic agent. In doxorubicin resistant MCF-7 cell line, resistance is developed mainly by upregulation of MDR1 gene which encodes an ABC transporter protein known as P-glycoprotein. Interleukin 6 (IL-6) is a cytokine which acts as a growth factor for certain cell types including some cancer cells. IL-6 is found at high levels in cancer patients having poor prognosis and resistance to certain chemotherapeutic agent. It is known that in MCF7 cell line, IL-6 induces overexpression of MDR1 gene by activating its transcription factor, C/EBPβ and confers resistance against Doxorubicin. In this study, the aim is silencing IL6 and to see the effects on MDR. Effects of IL-6 silencing on the levels of IL6, MDR1 and MRP1 genes are determined by qRT-PCR. Further confirmation of IL6 silencing is done by checking secreted IL-6 amount in cell culture media. Drug accumulation assay is done for evaluation of IL6 silencing on P-gp function. Moreover, to show that silencing of this particular gene sensitizes cells to chemotherapeutic drug Doxorubicin, XTT cytotoxicity assay is done. Results indicate that IL6 is successfully silenced by applying RNA interference which is observed by 36% decrease in IL-6 level to cell culture media. In correlation, IL6 mRNA level decreased 0.5 fold 24 hours after treatment. In addition, MDR1gene is downregulated by 45% as a consequence of interruption of IL-6 signaling in cell. MRP1, another ABC transporter coding gene, which is not connected to IL-6 signaling pathway, is not affected by silencing showing that reversal of multidrug resistance is only dependent on decreased MDR1 expression. Cytotoxicity assays show that cells are sensitized to Doxorubicin in an extent of 65% It is demonstrated that silencing IL6 gene makes Doxorubicin resistant MCF-7 cells significantly more susceptible to drug.