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Mito-Tempo suppresses autophagic flux via the PI3K/Akt/mTOR signaling pathway in neuroblastoma SH-SY5Y cells.

ABSTRACT: The generation of excessive mitochondrial reactive oxygen species (mtROS) is associated with glutamate-stimulated neurotoxicity and pathogenesis of Alzheimer's disease (AD). Impaired mitochondrial function is accompanied with oxidative stress that is a significant contributor to initiate autophagy, but the underlying mechanisms are not fully understood. The present study aimed to investigate the neuroprotective effects of Mito-Tempo on glutamate-induced neuroblastoma SH-SY5Y cell toxicity. SH-SY5Y cells were treated with 100 μM glutamate in the presence or absence of 50 and 100 μM Mito-Tempo for 24 h. Changes in cell viability were measured by MTT assay. Cytotoxicity and intracellular ROS accumulation were also evaluated using lactate dehydrogenase (LDH) activity assay and 2,7-dichlorofluorescein diacetate (DCFDA) Reactive Oxygen Species Assay kit, respectively. Mitochondrial membrane potential was analyzed by tetraethylbenzimidazoly-lcarbocyanine iodide (JC-1) staining. Expression of PI3K/AKT/mTOR pathway and autophagy markers, including LC3 (LC3-I/-II) and p62 (SQSTM1) were performed using Western blot analysis. Our results demonstrated that glutamate-exposed cells significantly increased cellular oxidative stress by enhancing ROS production. Glutamate treatment also increased LDH release follows the loss of mitochondrial membrane potential, caused cell viability loss. Treatment with Mito-Tempo not only attenuated the generation of ROS and improved mitochondrial membrane potential but also reduced the neurotoxicity of glutamate in a concentration-dependent manner, which leads to increased cell viability and decreased LDH release. Mito-Tempo has a greater protective effect by enhancing superoxide dismutase (SOD) activity and PI3K/AKT/mTOR phosphorylation. Moreover, Mito-Tempo treatment altered the autophagy process resulting in the decline in the ratio of the autophagy markers LC3-I/-II and p62 (SQSTM1). We propose that Mito-Tempo can improve neuronal properties against glutamate cytotoxicity through its direct free radical scavenging activity and inhibit excessive autophagy signaling pathway, therefore, allow for further studies to investigate the therapeutic potentials of Mito-Tempo in animal disease models and human.

SUBMITTER: Mukem S

PROVIDER: S-EPMC8239474 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Breaking resistance to chemotherapy is a major goal of combination therapy in many tumors, including advanced neuroblastoma. We recently demonstrated that increased activity of the PI3K/Akt network is associated with poor prognosis, thus providing an ideal target for chemosensitization. Here we show that targeted therapy using the PI3K/mTOR inhibitor NVP-BEZ235 significantly enhances doxorubicin-induced apoptosis in neuroblastoma cells. Importantly, this increase in apoptosis was dependent on scheduling: while pretreatment with the inhibitor reduced doxorubicin-induced apoptosis, the sensitizing effect in co-treatment could further be increased by delayed addition of the inhibitor post chemotherapy. Desensitization for doxorubicin-induced apoptosis seemed to be mediated by a combination of cell cycle-arrest and autophagy induction, whereas sensitization was found to occur at the level of mitochondria within one hour of NVP-BEZ235 posttreatment, leading to a rapid loss of mitochondrial membrane potential with subsequent cytochrome c release and caspase-3 activation. Within the relevant time span we observed marked alterations in a ?30 kDa protein associated with mitochondrial proteins and identified it as VDAC1/Porin protein, an integral part of the mitochondrial permeability transition pore complex. VDAC1 is negatively regulated by the PI3K/Akt pathway via GSK3? and inhibition of GSK3?, which is activated when Akt is blocked, ablated the sensitizing effect of NVP-BEZ235 posttreatment. Our findings show that cancer cells can be sensitized for chemotherapy induced cell death - at least in part - by NVP-BEZ235-mediated modulation of VDAC1. More generally, we show data that suggest that sequential dosing, in particular when multiple inhibitors of a single pathway are used in the optimal sequence, has important implications for the general design of combination therapies involving molecular targeted approaches towards the PI3K/Akt/mTOR signaling network.

Project description:BackgroundOsteosarcoma is a malignant tumor of bone and soft tissue in adolescents. Due to its tumor biological behavior pattern, osteosarcoma usually generates poor prognosis. Autophagy is an important self-defense mechanism in osteosarcoma.MethodsCell viability in IC50 testing and reverse assays was examined by the MTT assay. Cell apoptosis conditions were examined by flow cytometry, Hoechst 33,342 staining and apoptosis-related protein immunoblotting. Autophagy conditions were tested by autophagy-related protein immunoblotting, transmission electron microscopic observation and dual fluorescence autophagy flux detection. The possible targets of aloin were screened out by network pharmacology and bioinformatic methods. Osteosarcoma xenografts in nude BALB/c mice were the model for in vivo research on tumor suppression, autophagy induction, pathway signaling and toxicity tests. In vivo bioluminescence imaging systems, immunohistochemical assays, and gross tumor volume comparisons were applied as the main research methods in vivo.ResultsAloin induced osteosarcoma apoptosis in a dose-dependent manner. Its possible effects on the PI3K/AKT pathway were screened out by network pharmacology methods. Aloin increased autophagic flux in osteosarcoma by downregulating the PI3K/AKT pathway. Aloin promoted autophagic flux in the osteosarcoma cell lines HOS and MG63 in a dose-dependent manner by promoting autophagosome formation. Chloroquine reversed the apoptosis-promoting and autophagy-enhancing effects of aloin. Autophagy induced by starvation and rapamycin significantly enhanced the autophagic flux and apoptosis induced by aloin, which verified the role of the PI3K/AKT axis in the pharmacological action of aloin. Therapeutic effects, autophagy enhancement and regulatory effects on the PI3K/AKT/mTOR pathway were demonstrated in a nude mouse xenogeneic osteosarcoma transplantation model.ConclusionsAloin inhibited the proliferation of osteosarcoma by inhibiting the PI3K/AKT/mTOR pathway, increasing autophagic flux and promoting the apoptosis of osteosarcoma cells.

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Mito-Tempo suppresses autophagic flux via the PI3K/Akt/mTOR signaling pathway in neuroblastoma SH-SY5Y cells.

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