Knockdown of AKT2 expression by RNA interference inhibits proliferation, enhances apoptosis, and increases chemosensitivity to the anticancer drug VM-26 in U87 glioma cells.
ABSTRACT The AKT2 kinase (protein kinas Bβ) is frequently overexpressed in malignant gliomas. In this study, the human glioblastoma cell line U87 was stably transfected with a lentivirus vector expressing a short hairpin RNA (shRNA) targeting AKT2. Knockdown of AKT2 by the shRNA inhibited U87 cell proliferation and increased the rate of apoptosis. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blot analysis revealed that cells stably underexpressing AKT2 showed lower expression of the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2) and enhanced expression of the apoptosis effector caspase-3 compared to U87 cells stably transfected with a control vector. Furthermore, expression levels of AKT2 were correlated with the IC50 of the antitumor drug VM-26 (teniposide); the VM-26 IC50 was reduced from 6.46±0.42μg/ml in control glioma cells to 1.15±0.22μg/ml in U87 cells underexpressing AKT2. Combined AKT2 knockdown and VM-26 treatment inhibited cell proliferation in vitro more effectively than either treatment alone. Knockdown of AKT2 expression was associated with decreased expression of the multidrug resistance-associated protein 1 (MRP1) without affecting MRP1 mRNA expression. However, the mRNA and protein levels of MDR1 (p-glycoprotein) were unaffected by AKT2 knockdown. These results indicate that inhibition of AKT2 expression may be an effective means for overcoming AKT2-associated chemoresistance in human malignant glioma cells and may represent a potential gene-targeting approach to treat glioma.
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ABSTRACT: RNA interference (RNAi) is an evolutionary conserved gene regulation pathway that has emerged as an important discovery in the field of molecular biology. One of the important advantages of RNAi in therapy is that it brings about efficient downregulation of gene expression by targeting complementary transcripts in comparison with other antisense-based techniques. RNAi can be can be achieved by introducing chemically synthesized small interfering RNAs (siRNAs) into a cell system. A more stable knockdown effect can be brought about by the use of plasmid or viral vectors encoding the siRNA. RNAi has been used in reverse genetics to understand the function of specific genes and also as a therapeutic tool in treating human diseases. This review provides a brief insight into the therapeutic applications of RNAi against debilitating diseases.Applied biochemistry and biotechnology 01/2013; · 1.94 Impact Factor