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: 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 01/2014; 2014:260210. · 2.71 Impact Factor
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ABSTRACT: Background Aberrant AKT activation contributes to gastric cancer cell survival and chemotherapy resistance, however its regulation is poorly understood. microRNAs have been established to be important regulators in gastric carcinogenesis. Here, we showed the functional role and putative target of let-7b and let-7 g (let-7b/g) in gastric carcinogenesis.Methods The expression of let-7b/g in gastric cancer cell lines and primary tumors were evaluated by miRNA qRT-PCR. The putative target gene of let-7b/g was explored by TargetScan followed by further validation. Functional analyses including MTT proliferation, monolayer colony formation, cell invasion assays and in vivo study were performed in both ectopic expression and knockdown approaches.Resultslet-7b/g was found down-regulated in gastric cancer and its downregulation was associated with poor survival and correlated with lymph node metastasis. let-7b/g inhibited AKT2 expression by directly binding to its 3¿UTR, reduced p-AKT (S473) activation and suppressed expression of the downstream effector pS6. AKT2 mRNA expression showed negative correlation with the expression of let-7b/g in primary tumors. Short interfering RNA (siRNA) mediated knockdown of AKT2 phenocopied the tumor-suppressive effects of let-7b/g. Moreover, AKT2 re-expression partly abrogated the growth-inhibitory effect of let-7b/g.Conclusion In conclusion, our findings reveal decreased let-7b/g contributes to aberrant AKT activation in gastric tumorigenesis and provide a potential therapeutic strategy for gastric cancer.Journal of translational medicine. 10/2014; 12(1):281.
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ABSTRACT: Akt2 overexpression correlates with chemoresistance of colorectal cancer (CRC). However, the cellular functions and precise signals elicited by Akt2 in LSCC have not been elucidated. Here, we transfected a CRC cell line HCT116 with Akt-2 targeted shRNA in order to establish a cell line with Akt2 knockdown. In vitro experiments showed that knockdown Akt2 in HCT116 cells was associated with decrease in cell proliferation as well as enhanced cell apoptosis. Furthermore, our results demonstrated that Akt2 knockdown correlated with elevated chemosensitivity of HCT116 cells to paclitaxel. Importantly, we found that knockdown of AKt2 resulted in downregulation of MDR-1 and MRP-1. Our findings may lead to a better understanding of the biological effect of Akt2 and may provide mechanistic insights for developing potential therapeutic strategies targeting AKt2.Cell biochemistry and biophysics. 08/2014;