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1.
Biochem Pharmacol ; 156: 479-490, 2018 10.
Article in English | MEDLINE | ID: mdl-30222967

ABSTRACT

The liver kinase B1 (LKB1) gene is a tumor suppressor associated with the hereditary Peutz-Jeghers syndrome and frequently mutated in non-small cell lung cancer and in cervical cancer. Previous studies showed that the LKB1/AMPK axis is involved in regulation of cell death and survival under metabolic stress. By using isogenic pairs of cancer cell lines, we report here that the genetic loss of LKB1 was associated with increased intracellular levels of total choline containing metabolites and, under oxidative stress, it impaired maintenance of glutathione (GSH) levels. This resulted in markedly increased intracellular reactive oxygen species (ROS) levels and sensitivity to ROS-induced cell death. These effects were rescued by re-expression of LKB1 or pre-treatment with the anti-oxidant and GSH replenisher N-acetyl cysteine. This role of LKB1 in response to ROS-inducing agents was largely AMPK-dependent. Finally, we observed that LKB1 defective cells are highly sensitive to cisplatin and γ-irradiation in vitro, suggesting that LKB1 mutated tumors could be targeted by oxidative stress-inducing therapies.


Subject(s)
Cisplatin/pharmacology , Gamma Rays , Glutathione/metabolism , Hydrogen Peroxide/pharmacology , Oxidative Stress/drug effects , Protein Serine-Threonine Kinases/metabolism , AMP-Activated Protein Kinase Kinases , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/radiation effects , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/radiation effects , Humans , Magnetic Resonance Spectroscopy , Protein Serine-Threonine Kinases/genetics
2.
Leukemia ; 27(5): 1019-27, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23228943

ABSTRACT

The serine/threonine kinase AMP-activated protein kinase (AMPK) and its downstream effectors, including endothelial nitric oxide synthase and BCL-2, are hyperactivated in B-cell precursor-acute lymphoblastic leukemia (BCP-ALL) cells with MLL gene rearrangements. We investigated the role of activated AMPK in supporting leukemic cell survival and evaluated AMPK as a potential drug target. Exposure of leukemic cells to the commercial AMPK inhibitor compound C resulted in massive apoptosis only in cells with MLL gene rearrangements. These results were confirmed by targeting AMPK with specific short hairpin RNAs. Compound C-induced apoptosis was associated with mitochondrial membrane depolarization, reactive oxygen species production, cytochrome c release and caspases cleavage, indicating intrinsic apoptosis pathway activation. Treatment with low concentrations of compound C resulted in a strong antileukemic activity, together with cytochrome c release and cleavage of caspases and poly(ADP-ribose) polymerase, also in MLL-rearranged primary BCP-ALL samples. Moreover, AMPK inhibition in MLL-rearranged cell lines synergistically enhanced the antiproliferative effects of vincristine, daunorubicin, cytarabine, dexamethasone and L-asparaginase in most of the evaluated conditions. Taken together, these results indicate that the activation of the AMPK pathway directly contributes to the survival of MLL-rearranged BCP-ALL cells and AMPK inhibitors could represent a new therapeutic strategy for this high-risk leukemia.


Subject(s)
AMP-Activated Protein Kinases/antagonists & inhibitors , Apoptosis/drug effects , Myeloid-Lymphoid Leukemia Protein/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Pyrazoles/pharmacology , Pyrimidines/pharmacology , AMP-Activated Protein Kinases/physiology , Cell Cycle/drug effects , Cell Line, Tumor , Gene Rearrangement , Histone-Lysine N-Methyltransferase , Humans , Mitochondria/drug effects , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology
3.
Br J Ophthalmol ; 93(2): 244-8, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19174401

ABSTRACT

BACKGROUND: Vascular endothelial growth factor (VEGF) has been shown to play a major role in the pathological neovascularisation that occurs in degenerative retinal diseases like age-related macular degeneration (AMD). Although several approaches to attenuate VEGF show significant promise, repeated treatments are required to achieve therapeutic benefits. As lentiviruses efficiently and stably infect resting cells, a human immunodeficiency virus type 1 (HIV-1)-based vector was used for the delivery and long-term endogenous expression of a short hairpin RNA (shRNA) specific for VEGF in postmitotic human retinal pigment epithelium (RPE) cells. METHODS: An HIV-1 vector expressing a shRNA targeting VEGF was developed and adopted to transduce RPE cell cultures, in both normoxic and hypoxic conditions in vitro. Intracellular VEGF expression was analysed by western blotting, and the release of VEGF in culture supernatants was determined by ELISA. RESULTS: At least 90% of RPE cells were successfully transduced by HIV-1 virions. Inhibition of VEGF expression and reduction by 95% of VEGF release in transduced cells were achieved. Moreover, shRNA-VEGF effectively and specifically prevented hypoxia-induced VEGF upregulation. CONCLUSION: HIV-1-mediated delivery of a shRNA-VEGF leading to gene expression knockdown could represent a novel therapeutic strategy against neovascularisation-related eye diseases.


Subject(s)
Gene Knockdown Techniques/methods , HIV-1/genetics , Retinal Pigment Epithelium/metabolism , Vascular Endothelial Growth Factor A/metabolism , Cells, Cultured , Epithelial Cells/cytology , Epithelial Cells/metabolism , Gene Silencing , Genetic Vectors , Humans , Inverted Repeat Sequences/genetics , RNA, Small Interfering/genetics , Retinal Pigment Epithelium/cytology , Vascular Endothelial Growth Factor A/genetics
4.
J Cell Physiol ; 207(3): 711-21, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16518856

ABSTRACT

The LGI1 gene has been implicated in the malignant progression of glioblastoma and it has also been genetically linked to a form of partial epilepsy (ADLTE). In this study, we investigated the relevance of LGI1 expression for neuroblastoma cells. The analysis of two cell lines (SH-SY5Y and SK-N-BE) revealed unpredictably low levels of LGI1 and stable cell transfection with LGI1 cDNA yielded moderate increases of LGI1 expression. Neuroblastoma cell clones exhibited impaired cell growth and survival ability in relation to LGI1 levels. The process of growth inhibition could be discerned under experimental conditions of low cell density, since conditions of elevated cell density, which enhance the requirement for survival stimuli, resulted in massive cellular death. At high cell density, spontaneous apoptosis of LGI1 cells was clearly shown by the release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria and by phosphatydil serine exposure and nuclear fragmentation. Activation of apoptotic effectors caspase-3/7 also occurred, however, the broad caspase inhibitor Z-VAD-FMK substantially failed to block cell death. Thus the possibility that LGI1-triggered apoptosis may involve initiator caspases linked to activation of death receptors, appears unlikely. The decreased ratio of Bcl-2 to Bax suggests that apoptosis is initiated by the intrinsic mitochondrial pathway through the release of caspase-dependent and -independent apoptogenic molecules. This study provides the first evidence that LGI1 controls neuronal cell survival, suggesting its role in the development of the nervous system in relation to the pathogenesis of neuroblastoma and ADLTE.


Subject(s)
Apoptosis , Gene Expression Regulation, Neoplastic , Neuroblastoma/metabolism , Neuroblastoma/pathology , Proteins/metabolism , Up-Regulation , Active Transport, Cell Nucleus , Apoptosis Inducing Factor/metabolism , Caspase 3 , Caspase 7 , Caspases/metabolism , Cell Division , Cell Line, Tumor , Cell Membrane/metabolism , Cell Nucleus/metabolism , Cytochromes/metabolism , Cytosol/metabolism , Enzyme Activation , Humans , Intracellular Signaling Peptides and Proteins , Neuroblastoma/genetics , Phosphoserine/metabolism , Proteins/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , RNA, Messenger/genetics , Receptors, Tumor Necrosis Factor/metabolism , bcl-2-Associated X Protein/genetics
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