Identification of a novel FOXO3 agonist that protects against alcohol induced liver injury.

Alcohol-related liver disease (ALD) is a global healthcare concern which caused by excessive alcohol consumption with limited treatment options. The pathogenesis of ALD is complex and involves in hepatocyte damage, hepatic inflammation, increased gut permeability and microbiome dysbiosis. FOXO3 is a well-recognized transcription factor which associated with longevity via promoting antioxidant stress response, preventing senescence and cell death, and inhibiting inflammation. We and many others have reported that FOXO3-/- mice develop more severe liver injury in response to alcohol. In the present study, we aimed to develop compounds that activate FOXO3 and further investigate their effects in alcohol induced liver injury. Through virtual screening, we discovered series of small molecular compounds that showed high affinity to FOXO3. We confirmed effects of compounds on FOXO3 target gene expression, as well as antioxidant and anti-apoptotic effects in vitro. Subsequently we evaluated the protective efficacy of compounds in alcohol induced liver injury in vivo. As a result, the leading compound we identified, 214991, activated downstream target genes expression of FOXO3, inhibited intracellular ROS accumulation and cell apoptosis induced by H2O2 and sorafenib. By using Lieber-DeCarli alcohol feeding mouse model, 214991 showed protective effects against alcohol-induced liver inflammation, macrophage and neutrophil infiltration, and steatosis. These findings not only reinforce the potential of FOXO3 as a valuable target for therapeutic intervention of ALD, but also suggested that compound 214991 as a promising candidate for the development of innovative therapeutic strategies of ALD.

Cytosolic and mitochondrial ROS production resulted in apoptosis induction in breast cancer cells treated with Crocin: The role of FOXO3a, PTEN and AKT signaling.

Different groups have reported the Crocin anticancer activity. We previously showed Crocin-induced apoptosis in rat model of breast and gastric cancers, through the increased Bax/Bcl-2 ratio and caspases activity, as well as the cell cycle arrest in a p53-dependent manner. Since Crocin antioxidant activity has been shown under different conditions, it is interesting to elucidate its apoptotic mechanism. Here, we treated two breast cancer cell lines, MCF-7 and MDA-MB-231, with Crocin. MTT and ROS assays, cell cycle arrest, Bax/Bcl-2 ratio and caspase3 activity were determined. PARP cleavage and expression of some proteins were studied using Western blotting and immunofluorescence. The results indicated stepwise ROS generation in cytosol and mitochondria after Crocin treatment. Attenuating the early ROS level, using diphenyleneiodonium, diminished the sequent mitochondrial damage (decreasing Δψ) and downstream apoptotic signaling. Crocin induced ROS production, FOXO3a expression and nuclear translocation, and then, elevation of the expression of FOXO3a target genes (Bim and PTEN) and caspase-3 activation. Application of N-acetylcysteine blocked AKT/FOXO3a/Bim signaling. FOXO3a knockdown resulted in a decrease of Bim, PTEN and caspase 3, after Crocin treatment. PTEN knockdown caused a decrease in FOXO3a, Bim and caspase 3, in addition to an increase in p-AKT and p-FOXO3a, after Crocin treatment. In conclusion, Crocin induced apoptosis in MCF-7 and MDA-MB-231 human breast cancer cells. The ROS-activated FOXO3a cascade plays a central role in this process. FOXO3a-mediated upregulation of PTEN exerted a further inhibition of the AKT survival pathway. These data provide a new insight into applications of Crocin for cancer therapy.

Mst1 knockdown alleviates cardiac lipotoxicity and inhibits the development of diabetic cardiomyopathy in db/db mice.

Diabetic cardiomyopathy (DCM) accounts for increasing deaths of diabetic patients, and effective therapeutic targets are urgently needed. Myocardial lipotoxicity, which is caused by cardiac non-oxidative metabolic fatty acids and cardiotoxic fatty acid metabolites accumulation, has gained more attention to explain the increasing prevalence of DCM. However, whether mammalian Ste20-like kinase 1 (Mst1) plays a role in lipotoxicity in type 2 diabetes-induced cardiomyopathy has not yet been illustrated. Here, we found that Mst1 expression was elevated transcriptionally in the hearts of type 2 diabetes mellitus mice and palmitic acid-treated neonatal rat ventricular myocytes. Adeno-associated virus 9 (AAV9)-mediated Mst1 silencing in db/db mouse hearts significantly alleviated cardiac dysfunction and fibrosis. Notably, Mst1 knockdown in db/db mouse hearts decreased lipotoxic apoptosis and inflammatory response. Mst1 knockdown exerted protective effects through inactivation of MAPK/ERK kinase kinase 1 (MEKK1)/c-Jun N-terminal kinase (JNK) signaling pathway. Moreover, lipotoxicity induced Mst1 expression through promoting the binding of forkhead box O3 (FoxO3) and Mst1 promoter. Conclusively, we elucidated for the first time that Mst1 expression is regulated by FOXO3 under lipotoxicity stimulation and downregulation of Mst1 protects db/db mice from lipotoxic cardiac injury through MEKK1/JNK signaling inhibition, indicating that Mst1 abrogation may be a potential treatment strategy for DCM in type 2 diabetic patients.

FoxO3 reverses 5-fluorouracil resistance in human colorectal cancer cells by inhibiting the Nrf2/TR1 signaling pathway.

5-fluorouracil (5-FU) is widely used in chemotherapy for colorectal cancer (CRC), but a high rate of chemoresistance reduces its effectiveness in clinical treatment. We found remarkably decreased expression of forkhead box 3 (FoxO3) protein, a tumor inhibitor, in 5-FU-resistant SW620 and HCT-8 (SW620/5-FU and HCT-8/5-FU) cells. Moreover, FoxO3 overexpression sensitized SW620/5-FU and HCT-8/5-FU cells to 5-FU. Mechanistically, FoxO3 inhibited the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway by directly binding to Keap1 promoter. Thioredoxin reductase 1 (TR1), a pivotal target gene of Nrf2, was observed to promote 5-FU resistance by reducing intracellular ROS levels. Clinical data also revealed that significant upregulation of TR1 was associated with poor outcome in CRC patients. Auranofin (AUR), a FoxO3 agonist and TR1 inhibitor, enhanced the sensitivity of HCT-8/5-FU and SW620/5-FU cells to 5-FU in vitro and in vivo. Taken together, our results suggest that FoxO3 could reverse 5-FU resistance in CRC via inhibiting the Nrf2/TR1 signaling pathway, and increasing the level of intracellular reactive oxygen species. Chemotherapeutic agents targeting FoxO3 and/or TR1, including AUR, might be promising adjuvant sensitizers to reverse chemoresistance in 5-FU-resistant CRC.

Depigmenting Effect of Resveratrol Is Dependent on FOXO3a Activation without SIRT1 Activation.

Resveratrol exhibits not only anti-melanogenic property by inhibiting microphthalmia-associated transcription factor (MITF), but also anti-aging property by activating sirtuin-1 (SIRT1). In this study, the relationship between depigmenting effect of resveratrol and SIRT1/forkhead box O (FOXO) 3a activation and was investigated. Resveratrol suppressed melanogenesis by the downregulation of MITF and tyrosinase via ERK pathway. Results showed that the expression of both SIRT1 and FOXO3a were increased. It is reported that SIRT1 is critical regulator of FOXO-mediated transcription in response to oxidative stress. However in our study, FOXO3a activation appeared earlier than that of SIRT1. Furthermore, the effect of resveratrol on the levels of MITF and tyrosinase was suppressed when melanocytes were pre-treated with SP600125 (JNK inhibitor). However, pre-treatment with SIRT1 inhibitor (EX527, or sirtinol) did not affect the levels of MITF and tyrosinase. Therefore, resveratrol inhibits melanogenesis through the activation of FOXO3a but not by the activation of SIRT1. Although SIRT1 activation by resveratrol is a well-known mechanism of resveratrol-induced antiaging effects, our study showed that not SIRT1 but FOXO3a activation is involved in depigmenting effects of resveratrol.

The tubulin inhibitor MG-2477 induces autophagy-regulated cell death, ROS accumulation and activation of FOXO3 in neuroblastoma.

Neuroblastoma is the most frequent extra-cranial solid tumor in children with still high mortality in stage M. Here we studied the tubulin-inhibitor MG-2477 as a possible therapeutic agent for neuroblastoma therapy and uncovered that MG-2477 induces death in neuroblastoma cells independent of PKB-activation status and stage. MG-2477 triggers within 30 minutes extensive autophagosome-formation that finally leads to cell death associated with mitotic catastrophe. Autophagy is critical for MG-2477-induced death and is regulated by the BH3-only protein PMAIP1/NOXA which sequesters the anti-apoptotic BCL2-protein BCLXL and thereby displaces and activates the autophagy-regulator BECN1/beclin1. Knockdown of NOXA or overexpression of its pro-survival binding partners MCL1 and BCLXL counteracts MG-2477-induced cell death. MG-2477 also rapidly induces the repression of the anti-apoptotic protein Survivin, which promotes autophagy and cell death. We further observed the accumulation of reactive oxygen species (ROS) that triggers autophagy induction suggesting a change of the PI3 kinase-III/BECN1 complex and activates the transcription factor FOXO3, which contributes to final cell death induction. The combined data suggest that MG-2477 induces a sequential process of ROS-accumulation, autophagy and FOXO3-activation that leads to cell death in neuroblastoma cells.

Forkhead box O3 plays a role in skeletal muscle atrophy through expression of E3 ubiquitin ligases MuRF-1 and atrogin-1 in Cushing's syndrome.

Cushing's syndrome is caused by overproduction of the adrenocorticotropic hormone (ACTH), which stimulates the adrenal grand to make cortisol. Skeletal muscle wasting occurs in pathophysiological response to Cushing's syndrome. The forkhead box (FOX) protein family has been implicated as a key regulator of muscle loss under conditions such as diabetes and sepsis. However, the mechanistic role of the FOXO family in ACTH-induced muscle atrophy is not understood. We hypothesized that FOXO3a plays a role in muscle atrophy through expression of the E3 ubiquitin ligases, muscle RING finger protein-1 (MuRF-1), and atrogin-1 in Cushing's syndrome. For establishment of a Cushing's syndrome animal model, Sprague-Dawley rats were implanted with osmotic minipumps containing ACTH (40 ng·kg-1·day-1). ACTH infusion significantly reduced muscle weight. In ACTH-infused rats, MuRF-1, atrogin-1, and FOXO3a were upregulated and the FOXO3a promoter was targeted by the glucocorticoid receptor (GR). Transcriptional activity and expression of FOXO3a were significantly decreased by the GR antagonist RU486. Treatment with RU486 reduced MuRF-1 and atrogin-1 expression in accordance with reduced enrichment of FOXO3a and Pol II on the promoters. Knockdown of FOXO3a prevented dexamethasone-induced MuRF-1 and atrogin-1 expression. These results indicate that FOXO3a plays a role in muscle atrophy through expression of MuRF-1 and atrogin-1 in Cushing's syndrome.