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Effect of Proteasome Inhibitor Bortezomib on MDR1 Gene Expression in K562 Cell

Author: ZhaoJia
Tutor: ZuoJinSong
School: Dalian Medical University
Course: Internal Medicine
Keywords: Bortezomib K562 cells mdr1 Reversal of drug resistance Promoter
CLC: R733.7
Type: Master's thesis
Year: 2011
Downloads: 1
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Abstract


Objective: Overexpression of the human multidrug resistance gene 1 (MDR1) plays a key role in multidrug resistance (MDR) in hematological malignancies. This study was designed to investigate inhibitory effects of proteasome inhibitor bortezomib on MDR1 expression in K562 cells,and the luciferase reporter gene plasmids were constructed to elucidate the mechanisms which bortezomib reverses the drug resistance .Methods: 1. Selection of K562 cells resistant to adriamycin (K562/ADM). 2. Detection of growth inhibition and drug resistance capacity with MTT assay. 3. Growth inhibition of K562/S, K562/ADM was analyzed by measuring drug cytotoxicity at a series of concentrations to find the appropriate concentrations for treating cells. 4. The expression of mdr1 in K562/S and K562/ADM cells was detected by reverse transcription PCR (RT-PCR) with and without bortezomib at a concentration of 5nmol/L and 10nmol/L, respectively. 5. The expression of mdr1 in K562/ADM cells with and without 10nmol/L bortezomib for 24 hours was detected by flow cytometry. 6. A series of PGL4.15 luciferase reporter gene plasmids were constructed containing 5’-truncated mdr1 promoter (including -1499bp~ +187bp, -505bp ~+187 bp, -227 bp ~+187 bp,- 121bp ~+187 bp,-38 bp ~+187 bp and -10 bp ~+187 bp). The constructs were testified by double enzyme digestion and DNA sequencing.Results:1. K562/ADM cells were established and the fold of multiple drug resistance is 20.4.2. Cytotoxicity of bortezomib on K562/S, K562/ADM cells were confirmed, moreover, it was positively correlated with drug concentrations (P<0.05). According to the linear regression equation, the IC5 value of K562/S and K562/ADM were 3.99nm/L and 7.16nm/L, respectively.3. Reverse transcription PCR (RT-PCR) found that mdr1 expression was down-regulated by bortezomib. FCM assay confirmed that bortezomib could decrease mdr1 expression in K562/ADM.4. Restriction endonuclease digestion and DNA sequencing confirmed plasmids pGL4 containing mdr1 promoter correct, according to GenBank.Conclusion: K562 cells resistant to adriamycin (K562/ADM) were selected successfully. This study verified that the bortezomib can down-regulate mdr1 expression at transcription level. Luciferase reporter gene plasmids containing mdr1 promoter was successfully constructed for further elucidating molecular-mechanism mdr1 by bortezomib.

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CLC: > Medicine, health > Oncology > Hematopoietic and lymphoid neoplasms > Leukemia
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