Knockdown of AKT2 expression by RNA interference inhibits proliferation, enhances apoptosis, and increases chemosensitivity to the anticancer drug VM-26 in U87 glioma cells
Department of Neurosurgery, Changzheng Hospital, Second Military Medical University, No. 415 FengYang Road, Shanghai 200003, People's Republic of China. Brain research
(Impact Factor: 2.84).
07/2012; 1469:1-9. DOI: 10.1016/j.brainres.2012.06.043
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.
Available from: PubMed Central
- "The poor prognosis of those patients is owing to the intrinsic resistance of the GBM cells to apoptosis . Therefore, induction of apoptosis in glioblastoma cells has come to be appreciated as targets for the management of GBM . "
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ABSTRACT: Hitherto, limited clinical impact has been achieved in the treatment of glioblastoma (GBMs). Although phytochemicals found in medicinal herbs can provide mankind with new therapeutic remedies, single agent intervention has failed to bring the expected outcome in clinical trials. Therefore, combinations of several agents at once are gaining increasing attractiveness. In the present study, we investigated the effects of crude alkaloid (CAERS) and flavonoid (CFEZO) extracts prepared from medicinal herbs, Rhazya stricta and Zingiber officinale, respectively, on the growth of human GBM cell line, U251. R. stricta and Z. officinale are traditionally used in folkloric medicine and have antioxidant, anticarcinogenic, and free radical scavenging properties. Combination of CAERS and CFEZO treatments synergistically suppressed proliferation and colony formation and effectively induced morphological and biochemical features of apoptosis in U251 cells. Apoptosis induction was mediated by release of mitochondrial cytochrome c, increased Bax : Bcl-2 ratio, enhanced activities of caspase-3 and -9, and PARP-1 cleavage. CAERS and CFEZO treatments decreased expression levels of nuclear NF-κBp65, survivin, XIAP, and cyclin D1 and increased expression level of p53, p21, and Noxa. These results suggest that combination of CAERS and CFEZO provides a useful foundation for studying and developing novel chemotherapeutic agents for the treatment of GBM.
BioMed Research International 07/2014; 2014:260210. DOI:10.1155/2014/260210 · 1.58 Impact Factor
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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; 169(6). DOI:10.1007/s12010-013-0098-1 · 1.74 Impact Factor
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ABSTRACT: To characterize the phenotype of Akt2/low-density-lipoprotein receptor double knockout (Akt2/LDLr dKO) mice with respect to insulin resistance and features of atherosclerotic plaque progression.
Metabolic profile and atherosclerotic plaque progression were compared between LDLr KO mice and Akt2/LDLr dKO mice. Total cholesterol, glucose and insulin levels were significantly higher and oral glucose tolerance test was more impaired in Akt2/LDLr dKO mice than in LDLr KO mice. Although atherosclerotic plaques at both the carotid artery and the aortic root of Akt2/LDLr dKO mice were significantly smaller (p<0.05) compared with LDLr KO controls, plaque composition in these mice was more complex, showing 34-50% reduced collagen content (p<0.01), 1.4-fold larger necrotic cores (p<0.05) and 6-fold more TUNEL positive cells (p<0.01). In situ zymography revealed a more than two-fold higher gelatinolytic activity in Akt2/LDLr dKO mice (p<0.05). In vitro analyses showed that deletion of Akt2 caused decreased migration, proliferation and collagen content of vascular smooth muscle cells (VSMCs) and disturbed the balance of metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase (TIMP) mRNA expression in macrophages and VSMCs.
Akt2/LDLr dKO mice develop insulin resistance and complex atherosclerotic lesions. These phenotypic characteristics make Akt2/LDLr dKO mice an interesting mouse model to study the effects of insulin resistance on the development and progression of atherosclerosis.
Cardiovascular Research 11/2013; 101(2). DOI:10.1093/cvr/cvt252 · 5.94 Impact Factor
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