FOXO4-DRI

OBJECTIVES: Endothelial cell dysfunction during aging is a key driver of vascular aging and related diseases; however, effective strategies to selectively eliminate senescent endothelial cells and restore vascular function remain lacking. FOXO4-DRI, a novel peptide-based intervention, specifically disrupts the interaction between FOXO4 and P53, thereby inducing apoptosis in senescent cells. This study innovatively focuses on the mechanism by which FOXO4-DRI induces apoptosis in senescent endothelial cells, demonstrating that it functions by activating the p53/BCL-2/Caspase-3 signaling pathway to promote selective apoptosis of these cells. FOXO4-DRI significantly improves vascular function and delays vascular aging. These findings not only enrich the molecular understanding of senescent cell clearance but also provide a novel strategy for precise targeting of endothelial cell senescence in therapeutic applications. MATERIALS AND METHODS: This study aims to analyze the vascular function and aging status of the aorta in naturally aged mice and progeroid model mice following FOXO4-DRI injection. Additionally, it investigates changes in endothelial cell function in senescent endothelial cells induced by oxygen-glucose deprivation (OGD), as well as the protein expression and interaction in the FOXO4-P53 signaling pathway. To assess the impact of FOXO4-DRI on endothelial cell senescence, the senescent endothelial cells were treated with FOXO4-DRI, followed by immunofluorescence and Western blotting experiments. RESULTS: Injection of FOXO4-DRI in both naturally aged and induced aging mice effectively suppressed aortic aging and improved aortic function. Additionally, we found that FOXO4-DRI alleviates endothelial cell senescence induced by OGD, thereby enhancing endothelial cell function. Through co-immunoprecipitation (CO-IP) experiments, we discovered that FOXO4-DRI prevents the binding of FOXO4 to P53, facilitating the phosphorylated P53 nuclear exclusion, which subsequently trigger BAX and cleaved caspase-3, leading to the apoptosis of senescent cells. Ultimately, this mechanism achieves the goal of inhibiting vascular aging. CONCLUSION: FOXO4-DRI promotes the nuclear export of phosphorylated P53 by inhibiting the binding of FOXO4 to P53 in endothelial cells, thereby facilitating the apoptosis of senescent endothelial cells and alleviating aging.

Male ageing is always accompanied by decreased fertility. The forkhead O (FOXO) transcription factor FOXO4 is reported to be highly expressed in senescent cells. Upon activation, it binds p53 in the nucleus, preventing senescent cell apoptosis and maintaining senescent cells in situ. Leydig cells play key roles in assisting spermatogenesis. Leydig cell senescence leads to deterioration of the microenvironment of the testes and impairs spermatogenesis. In this study, we observed that FOXO4-DRI, a specific FOXO4- p53 binding blocker, induced apoptosis in senescent Leydig cells, reduced the secretion of certain Senescence-Associated Secretory Phenotype and improved the proliferation of cocultured GC-1 SPG cells. In naturally aged mice, FOXO4-DRI-treated aged mice exhibited increased sperm quality and improved spermatogenesis.

Male late-onset hypogonadism is an age-related disease, the core mechanism of which is dysfunction of senescent Leydig cells. Recent studies have shown that elimination of senescent cells can restore proper homeostasis to aging tissue. In the present study, we found that the fork head box O (FOXO) transcription factor FOXO4 was specially expressed in human Leydig cells and that its translocation to the nucleus in the elderly was related to decreased testosterone synthesis. Using hydrogen peroxide-induced senescent TM3 Leydig cells as an in vitro model, we observed that FOXO4 maintains the viability of senescent Leydig cells and suppresses their apoptosis. By disrupting the FOXO4-p53 interaction, FOXO4-DRI, a specific FOXO4 blocker, selectively induced p53 nuclear exclusion and apoptosis in senescent Leydig cells. In naturally aged mice, FOXO4-DRI improved the testicular microenvironment and alleviated age-related testosterone secretion insufficiency. These findings reveal the therapeutic potential of FOXO4-DRI for the treatment of male late-onset hypogonadism.

Temozolomide (TMZ) is still the first-line drug for glioblastoma (GBM) treatment though tumor cell resistance remains a major challenge. TMZ administration may induce cellular senescence (CSEN), which exerts a dual regulatory effect on tumor progression, and evidence has suggested that CSEN is closely associated with mitochondrial dysfunction. Acylglycerol kinase (AGK), a mitochondrial membrane kinase, has been reported to participate in regulating mitochondrial function and the production of reactive oxygen species (ROS). Here, we aimed to investigate the role of AGK in GBM progression and TMZ resistance, assessed whether CSEN mediates these effects, and investigated the therapeutic potential of senolytic agents. Firstly, the analysis of TCGA data revealed that the increased expression of AGK was correlated with a poor prognosis of GBM patients. Secondly, through cell experiments of AGK-knockdown and overexpression, we found that AGK suppression inhibited the proliferation of tumor cells while paradoxically promoting TMZ resistance. Mechanistically, AGK suppression amplified TMZ-induced CSEN through increasing mitochondrial ROS (mtROS) and decreasing membrane potential. Conversely, Mito-TEMPO, a mtROS scavenger, and FOXO4-DRI, a senolytic agent, both enhanced the therapeutic efficacy of TMZ by eliminating these senescent cells via apoptosis. Furthermore, the clinical analysis linked AGK levels, CSEN, and prognosis of GBM patients treated with TMZ. In conclusion, our results establish senescence induction as a novel mechanism for AGK-mediated TMZ sensitization, suggesting that co-targeting AGK and CSEN represents a promising strategy to enhance TMZ therapy.

Cancer cell radioresistance is the primary cause of the decreased curability of non-small cell lung cancer (NSCLC) observed in patients receiving definitive radiotherapy (RT). Following RT, a set of microenvironmental stress responses is triggered, including cell senescence. However, cell senescence is often ignored in designing effective strategies to resolve cancer cell radioresistance. Herein, we identify the senescence-like characteristics of cancer-associated fibroblasts (CAFs) after RT and clarify the formidable ability of senescence-like CAFs in promoting NSCLC cell proliferation and radioresistance through the JAK/STAT pathway. Specific induction of senescence-like CAF apoptosis using FOXO4-DRI, a FOXO4-p53-interfering peptide, resulted in remarkable effects on radiosensitizing NSCLC cells in vitro and in vivo. In addition, in this study, we also uncovered an obvious therapeutic effect of FOXO4-DRI on alleviating radiation-induced pulmonary fibrosis (RIPF) by targeting senescence-like fibroblasts in vivo. In conclusion, by targeting senescence, we offer a strategy that simultaneously decreases radioresistance of NSCLC and the incidence of RIPF.

Autologous chondrocyte implantation (ACI) is a procedure used to treat articular cartilage injuries and prevent the onset of post-traumatic osteoarthritis. In vitro expansion of chondrocytes, a necessary step in ACI, results in the generation of senescent cells that adversely affect the quality and quantity of newly formed cartilage. Recently, a senolytic peptide, fork head box O transcription factor 4-D-Retro-Inverso (FOXO4-DRI), was reported to selectively kill the senescent fibroblasts. In this study, we hypothesized that FOXO4-DRI treatment could remove the senescent cells in the expanded chondrocytes, thus enhancing their potential in generating high-quality cartilage. To simulate the in vitro expansion for ACI, chondrocytes isolated from healthy donors were expanded to population doubling level (PDL) 9, representing chondrocytes ready for implantation. Cells at PDL3 were also used to serve as the minimally expanded control. Results showed that the treatment of FOXO4-DRI removed more than half of the cells in PDL9 but did not significantly affect the cell number of PDL3 chondrocytes. Compared to the untreated control, the senescence level in FOXO4-DRI treated PDL9 chondrocytes was significantly reduced. Based on the result from standard pellet culture, FOXO4-DRI pre-treatment did not enhance the chondrogenic potential of PDL9 chondrocytes. However, the cartilage tissue generated from FOXO4-DRI pretreated PDL9 cells displayed lower expression of senescence-relevant secretory factors than that from the untreated control group. Taken together, FOXO4-DRI is able to remove the senescent cells in PDL9 chondrocytes, but its utility in promoting cartilage formation from the in vitro expanded chondrocytes needs further investigation.