RESUMO
Mechanisms underlying the depletion of NAD+ and accumulation of reactive oxygen species (ROS) in aging and age-related disorders remain poorly defined. We show that reverse electron transfer (RET) at mitochondrial complex I, which causes increased ROS production and NAD+ to NADH conversion and thus lowered NAD+ /NADH ratio, is active during aging. Genetic or pharmacological inhibition of RET decreases ROS production and increases NAD+ /NADH ratio, extending the lifespan of normal flies. The lifespan-extending effect of RET inhibition is dependent on NAD+ -dependent Sirtuin, highlighting the importance of NAD+ /NADH rebalance, and on longevity-associated Foxo and autophagy pathways. RET and RET-induced ROS and NAD+ /NADH ratio changes are prominent in human induced pluripotent stem cell (iPSC) model and fly models of Alzheimer's disease (AD). Genetic or pharmacological inhibition of RET prevents the accumulation of faulty translation products resulting from inadequate ribosome-mediated quality control, rescues relevant disease phenotypes, and extends the lifespan of Drosophila and mouse AD models. Deregulated RET is therefore a conserved feature of aging, and inhibition of RET may open new therapeutic opportunities in the context of aging and age-related diseases including AD.
Assuntos
Doença de Alzheimer , Células-Tronco Pluripotentes Induzidas , Camundongos , Animais , Humanos , NAD , Espécies Reativas de Oxigênio/metabolismo , Elétrons , Células-Tronco Pluripotentes Induzidas/metabolismo , Envelhecimento/genética , Envelhecimento/metabolismo , Doença de Alzheimer/genética , Drosophila/genética , Drosophila/metabolismoRESUMO
The cross-talk between apoptosis and autophagy influences anticancer drug sensitivity and cellular death in various cancer cell lines. However, the fundamental mechanisms behind this phenomenon are still unidentified. We demonstrated anti-cancerous role of cisplatin (CP) and morin hydrate (Mh) as an individual and/or in combination (CP-Mh) in hepatoma cells and tumor model. Exposure of CP resulted in the production of intracellular reactive oxygen species (ROS)-mediated cellular vacuolization, expansion of mitochondria membrane and activation of endoplasmic reticulum (ER)-stress. Consequently, Cyt c translocation led to the increase of Bax/Bcl-2 ratio, which simultaneously triggered caspase-mediated cellular apoptosis. In addition, CP-induced PARP-1 activation led to ADP-ribosylation of HMGB1, which consequently developed autophagy as evident by the LC3I/II ratio. Chemically-induced inhibition of autophagy marked by increased cell death signified a protective role of autophagy against CP treatment. CP-Mh abrogates the PARP-1 expression and significantly reduced HMGB1-cytoplasmic translocation with subsequent inhibition of the HMGB1-Beclin1 complex formation. In the absence of PARP-1, a reduced HMGB1 mediated autophagy was observed followed by induced caspase-dependent apoptosis. To confirm the role of PARP-1-HMGB1 signaling in autophagy, we used the PARP-1 inhibitor, 4-amino-1,8-naphthalimide (ANI), HMGB1 inhibitor, ethyl pyruvate (EP), autophagy inhibitors, 3-methyl adenine (3-MA) and bafilomycin (baf) and small interfering RNAs (siRNA) to target Atg5 in combination of CP and Mh. Exposure to these inhibitors enhanced the sensitivity of HepG2 cells to CP. Collectively, our findings indicate that CP-Mh in combination served as a prominent regulator of autophagy and significant inducer of apoptosis that maintains a homeostatic balance towards HepG2 cells and the subcutaneous tumor model.
Assuntos
Autofagia/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Cisplatino/farmacologia , Flavonoides/farmacologia , Neoplasias Hepáticas/patologia , Animais , Autofagia/genética , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Cisplatino/administração & dosagem , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Sinergismo Farmacológico , Quimioterapia Combinada , Flavonoides/administração & dosagem , Proteína HMGB1/fisiologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Poli(ADP-Ribose) Polimerase-1/fisiologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Carvacrol is a monoterpenoid flavonoid found abundantly in thyme plants. Its physiochemical instability and partial solubility in water is the principal limitation for its industrial use. Hence, we made a carvacrol nanoemulsion (CANE) using ultrasonication method and characterized it by dynamic light scattering (DLS) technique which revealed a negative surface charge (-29.89 mV) with 99.1 nm average droplet size. CANE effectively induced apoptosis in doxorubicin-resistant A549 lung carcinoma cells (A549DR) evident by the elevated expression of apoptotic proteins such as Bax, Cytochrome C, and Cleaved caspase 3 and 9. Also, CANE displayed cell senescence leading to cell cycle arrest by reducing CDK2, CDK4, CDK6, Cyclin E, Cyclin D1 and enhancing p21 protein expression. In addition, a potential role of CANE in the inhibition of autophagy was noted by evaluating the reduced conversion of LC-3 I to II. Beside this, a down-regulation of important autophagy markers ATG5 and ATG7 and upregulation of p62 were detected in response to CANE. We conclude that the synthesized CANE has potential to cause cell senescence, cell cycle arrest, autophagy inhibition and apoptosis in A549DR cells and could be used as a potential candidate for lung cancer therapy.