Nur77, an orphan nuclear receptor, is involved in the development and progression of multiple tumors. In our previous study, we have shown that the protein level of Nur77 is elevated in colon tumors compared to adjacent normal tissues, highlighting its potential as a promising target for colorectal cancer therapy. Significantly, we have identified BI1071 as a Nur77-targeting compound that induces apoptosis in colorectal cancer cells. Based on the scaffold of BI1071, by substituting the indole group of BI1071 with a pyrrolyl group on one side, we rationally designed and synthesized a series of novel BI1071 analogues named SIM-C-PhCF3+Cl- targeting Nur77, and the structure-activity relationship of these BI1071 derivatives was summarized. From this series of compounds, A6 exhibited the strongest binding affinity to Nur77 (Kd = 0.40 ± 0.05 μM) and the most potent anti-proliferative activity against HCT116 and MC38 colorectal tumor cell lines, with IC50 values of 0.53 ± 0.06 μM and 0.16 ± 0.007 μM, respectively. Interestingly, unlike BI1071, which triggers Nur77-dependent apoptosis, compound A6 suppressed colon cancer cell proliferation predominantly by inducing Nur77-dependent mitotic arrest. Collectively, our findings provide a foundation for further investigation and development of Nur77-targeting antimitotic molecules toward colorectal cancer therapy.

The orphan nuclear receptor Nur77 is a multifunctional regulator involved in diverse cellular processes, including proliferation, survival, and apoptosis, through both transcription-dependent and -independent mechanisms. This regulatory complexity underscores the need for mechanistic studies in defined biological contexts. Here, we uncover a previously unrecognized non-genomic function of Nur77 at the centrosome that promotes mitotic progression in cancer cells. We show that Nur77 is phosphorylated at threonine 143 by cyclin-dependent kinase 1 (Cdk1), leading to its accumulation at the centrosome, where it binds the scaffold protein Cep192. This interaction is critical for maintaining centrosome integrity in tumor cells and facilitating the recruitment of Polo-like kinase 1 (PLK1), a key driver of centrosome maturation. Notably, Cdk1-mediated phosphorylation of Nur77 is aberrantly elevated in tumors, contributing to malignant proliferation through its mitotic role. Depletion of Nur77 or treatment with NMA39, a novel small-molecule Nur77 modulator that disrupts the Nur77-Cep192 interaction, results in mitotic arrest and cell death in tumor cells. These findings reveal a tumor-selective mitotic function of Nur77 and establish a mechanistic rationale for targeting phospho-Nur77 signaling as a cancer vulnerability.

Objective:Arsenic trioxide (ATO) is a clinically effective anticancer agent used in the treatment of leukemia. However, it exerts adverse effects on non-tumor cells, including bone marrow mesenchymal stem cells (BMSCs). This study aims to investigate the protective role and molecular mechanism of traditional Chinese medicine Astragalus polysaccharides (APS) in mitigating ATO-induced apoptosis in BMSCs. Methods:BMSCs exposed to ATO (0.5 μmol/L) were treated with APS (20, 40, 100, and 200 μg/mL). Cell viability, proliferation, and migration were assessed by using MTT, EdU staining, Transwell, and scratch wound healing assays. Apoptosis was evaluated via TUNEL assay, Hoechst 33258 staining, and flow cytometry. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential were measured by using DCFH-DA and JC-1 staining. Apoptotic protein expression was analyzed by Western blotting. Results:ATO exposure significantly inhibited the proliferation and migration of BMSCs and induced apoptosis, while APS markedly attenuated the apoptosis of BMSCs induced by ATO, and significantly improved cell proliferation and migration (P < 0.01). Mechanistically, APS effectively reduced ATO-induced ROS (P < 0.01), while the protein expression of Bcl-2-associated X protein (Bax) and cleaved Caspase-3 was significantly decreased (P < 0.05), and the protein expression of Bcl-2 was significantly increased (P < 0.01). In addition, APS markedly decreased the protein expression of c-Jun N-terminal kinase (Jnk) and p38 in ATO-activated BMSCs (P < 0.05), and significantly decreased the protein expression of p16 and p53 (P < 0.01), and increased the protein expression of phosphorylated protein kinase B (p-Akt) and phosphorylated extracellular signal-regulated kinase (p-Erk) (P < 0.01, 0.05). Conclusion:Our study reveals that APS exert significant protective effects against ATO-induced apoptosis in BMSCs. The mechanisms involve suppressing ROS generation, maintaining mitochondrial membrane stability, enhancing cell viability, migration, and proliferation, as well as inhibiting Jnk and p38 mitogen-activated protein kinase (p38 MAPK) signaling pathways. The findings highlight potential molecular targets and novel strategies for the clinical prevention and treatment of ATO-related toxicity .

Hepatic stellate cells (HSCs) are the primary fibrogenic cells in the liver, and their activation plays a crucial role in the development and progression of hepatic fibrosis. Here, we report that retinoid X receptor-alpha (RXRα), a unique member of the nuclear receptor superfamily, is a key modulator of HSC activation and liver fibrosis. RXRα exerts its effects by modulating calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ)-mediated activation of AMP-activated protein kinase-alpha (AMPKα). In addition, we demonstrate that K-80003, which binds RXRα by a unique mechanism, effectively suppresses HSC activation, proliferation, and migration, thereby inhibiting liver fibrosis in the CCl4 and amylin liver NASH (AMLN) diet animal models. The effect is mediated by AMPKα activation, promoting mitophagy in HSCs. Mechanistically, K-80003 activates AMPKα by inducing RXRα to form condensates with CaMKKβ and AMPKα via a two-phase process. The formation of RXRα condensates is driven by its N-terminal intrinsic disorder region and requires phosphorylation by CaMKKβ. Our results reveal a crucial role of RXRα in liver fibrosis regulation through modulating mitochondrial activities in HSCs. Furthermore, they suggest that K-80003 and related RXRα modulators hold promise as therapeutic agents for fibrosis-related diseases.

Background/Objectives: Vasa and PL10 belong to the DEAD-box protein family, which plays crucial roles in various cellular functions, such as DNA replication, DNA repair, and RNA processing. Additionally, DEAD-box family genes have also been identified as being related to gonadal development in many species. However, the function of vasa and PL10 in abalone is poorly understood on a molecular level. Methods: In the present study, we individually isolated and characterized the vasa and PL10 orthologs in Haliotis discus hannai (Hdh-vasa and Hdh-PL10). We also characterized the mRNA distributions of vasa and PL10 in various tissues from adult organisms and different embryonic developmental stages using real-time PCR (RT-qPCR) techniques. Furthermore, spatial and temporal expression of Hdh-vasa and Hdh-PL10 throughout embryonic and larval development was examined by whole-mount in situ hybridization (WMISH). Results: The two predicted amino acid sequences contained all of the conserved motifs characterized by the DEAD-box family. Homology and phylogenetic analyses indicate that they belong to the vasa and PL10 subfamilies. We found that vasa and PL10 mRNA were not solely restricted to gonads but were widely expressed in various tissues. WMISH showed that Hdh-vasa and Hdh-PL10 largely overlapped, with both being maternally expressed and specifically localized to the micromere lineage cells during early cleavage stages. By the gastrulation stage, Hdh-vasa were expressed strongly in two bilaterally symmetrical paraxial clusters, but Hdh-PL10 was dispersed in entire endodermal region. Our results suggest that Hdh-vasa-expressing cells are located as a subpopulation of undifferentiated multipotent cells that express Hdh-PL10. As such, we infer that primordial germ cells are specified from these vasa-expressing cells at some point during development, and inductive signals (epigenesis) play an important role in specifying primordial germ cells (PGCs) in H. discus hannai. Conclusions: This study provides valuable insights into the molecular characteristics and expression patterns of Hdh-vasa and Hdh-PL10, contributing to a better understanding of their roles in germ cell specification and early embryonic development in H. discus hannai.

Acute promyelocytic leukemia (APL), a distinctive subtype of acute myeloid leukemia (AML), is characterized by the t(15;17) translocation forming the PML-RARα fusion protein. Recent studies have revealed a crucial role of retinoid X receptor α (RXRα) in PML-RARα’s tumorigenesis. This necessitates the development of dual RARα and RXRα targeting compounds for treating APL. Here, we developed a pair of brominated retinoid isomers, 5a and 5b, exhibiting RARα agonistic selectivity among the RAR subtypes and RXRα partial agonistic activities. In the treatment of APL cells, low doses (RARα activation range) of 5a and 5b degrade PML-RARα and strongly induce differentiation, while higher doses (RXRα activation range) induce G2/M arrest and apoptosis in both all-trans retinoic acid (ATRA)-sensitive and resistant cells. We replaced the bromine in 5a with chlorine or iodine to obtain compounds 7 or 8a. Interestingly, the chlorinated compound 7 tends to activate RXRα and induce G2/M arrest and apoptosis, while the iodinated compound 8a tends to activate RARα and induce differentiation. Together, our work underscores several advantages and characteristics of halogens in the rational design of RARα and RXRα ligands, offering three promising drug candidates for treating both ATRA-sensitive and resistant APL.

The pathological hallmarks of various neurodegenerative diseases including Parkinson’s disease and Alzheimer’s disease prominently feature the accumulation of misfolded proteins and neuroinflammation. Chaperone-mediated autophagy (CMA) has emerged as a distinct autophagic process that coordinates the lysosomal degradation of specific proteins bearing the pentapeptide motif Lys-Phe-Glu-Arg-Gln (KFERQ), a recognition target for the cytosolic chaperone HSC70. Beyond its role in protein quality control, recent research underscores the intimate interplay between CMA and immune regulation in neurodegeneration. In this review, we illuminate the molecular mechanisms and regulatory pathways governing CMA. We further discuss the potential roles of CMA in maintaining neuronal proteostasis and modulating neuroinflammation mediated by glial cells. Finally, we summarize the recent advancements in CMA modulators, emphasizing the significance of activating CMA for the therapeutic intervention in neurodegenerative diseases.

Supplementary Table S3. Z-10 and its derivatives.

Supplementary Methods. Description of additional methods and procedures used in the study.

PDF - 2226KB, Cell viability assays of A) breast cancer cell lines treated with increasing concentrations of the indicated IAP antagonists, B) cancer cells isolated from a patient mammary tumor fragment pretreated with vehicle or 5 μM of the indicated IAP antagonists for 4 h before treatment with TRAIL for a further 20 h, C) MDA-MB-231 cells pretreated with vehicle or 5 μM of the indicated IAP antagonists for 20 h before treatment with TRAIL for a further 20 h and D) MDA-MB-231 cells pretreated with vehicle, 100 μM 3-FC, 5 μM MLS-0390982 or both for 4 h before treatment with TRAIL for a further 20 h. E) Caspase-3/-7 activity assay with NB7 cells expressing empty vector (NB7+Empty Vector), caspase-8 or inactive caspase-8 (C360A) pretreated with 5 μM SBI-0636457 before treatment with 100 ng/mL TRAIL for 4 h. Activity is in relative units.

PDF - 53KB, Legend for Supplementary Figure 1.

Supplementary Table S2. Z compounds and their carboxyl derivatives.

The orphan nuclear receptor Nur77 is a critical regulator of the survival and death of tumor cells. The pro-death effect of Nur77 can be regulated by its interaction with Bcl-2, resulting in conversion of Bcl-2 from a survival to killer. As Bcl-2 is overexpressed in various cancers preventing them from apoptosis and promoting their resistance to chemotherapy, targeting the apoptotic pathway of Nur77/Bcl-2 may lead to new cancer therapeutics. Here, we report our identification of XS561 as a novel Nur77 ligand that induces apoptosis of tumor cells by activating the Nur77/Bcl-2 pathway. In vitro and animal studies revealed an apoptotic effect of XS561 in a range of tumor cell lines including MDA-MB-231 triple-negative breast cancer (TNBC) and MCF-7/LCC2 tamoxifen-resistant breast cancer (TAMR) in a Nur77-dependent manner. Mechanistic studies showed XS561 potently induced the translocation of Nur77 from the nucleus to mitochondria, resulting in mitochondria-related apoptosis. Interestingly, XS561-induced accumulation of Nur77 at mitochondria was associated with XS561 induction of Nur77 phase separation and the formation of Nur77/Bcl-2 condensates. Together, our studies identify XS561 as a new activator of the Nur77/Bcl-2 apoptotic pathway and reveal a role of phase separation in mediating the apoptotic effect of Nur77 at mitochondria.

PDF file - 44K, S1. The predicted truncated merlin protein was not detected in Ben-Men-1 cells. S2. IC50 determination of AR-42 in normal meningeal cells. S3. AR-42 treatment increased protein acetylation and decreased p-AKT. S4. Normal meningeal cells proliferate with a doubling time of about three days. S5. Ben-Men-1-LucB and parental Ben-Men-1 cells exhibit similar sensitivities to AR-42. S6. Intracranial NF2-deficient KT21-MG1 xenograft tumors demonstrated features of malignant meningiomas. S7. Ben-Men-1-LucB xenografts strongly expressed vimentin, amesenchymal marker for meningiomas. S8. AR-42 treatment caused tumor regression, whereas AR-12 treatment slowed tumor growth over time. S9. MRI detected a small tumor in a Ben-Men-1-LucB tumorbearing mouse treated with AR-42 for three months. S10. MRI did not detect the residual tumor in an AR-42-treated mouse after removal from AR-42 diet for six months.

In this study, the optimal dietary protein levels for adult hybrid abalone (H. discus hannai & FEMALE; x H. fulgens & MALE;) were evaluated at two representative temperatures, and the interactive influences of dietary protein and water temperature on abalone meat quality were examined. Six isoenergetic and isolipidic diets containing graded levels of protein (15%, 20%, 25%, 30%, 35%, and 40%) were fed to the hybrid abalone for 90 days. Based on the specific growth rate, the optimum dietary protein levels for adult hybrid abalone using a quadratic curve model were estimated to be 28.07% and 26.47% of the diet at 23 degrees C (optimum temperature) and 27 degrees C (high temperature), respectively. The amount of crude protein in the abalone body increased linearly with the dietary protein levels and water temperature. Antioxidation enzyme activities of abalone muscle, including GPx and GST were significantly affected by the interaction of dietary protein levels and water temperature. As for texture analysis, the abalone kept at 23 degrees C exhibited higher meat hardness than those fed the same diets at 27 degrees C. The n-3 PUFAs (particularly DHA and EPA) in abalone meat were significantly influenced by dietary protein levels, and the highest content was observed in the 30% dietary protein group. These levels were not affected by water temperature. In addition, higher dietary protein levels and water temperatures increased the total contents of free amino acids and flavor amino acids (Asp + Glu + Gly + Ala + Arg + Tau). The cooking loss of abalone meat was reduced with increased protein levels and was significantly lower in the 23 degrees C group, along with a higher meat postmortem pH value. The collagen fiber ratio and collagen content of abalone meat were higher in the 23 degrees C group. In summary, for multiple diet feeding strategies in practical production of abalone aquaculture, the balance between the seasonally specific feeds cost benefits and the abalone meat quality should be carefully evaluated.

Transcriptome sequencing is an effective tool to reveal the essential genes and pathways underlying countless biotic and abiotic stress adaptation mechanisms. Although severely challenged by diverse environmental conditions, the Pacific abalone Haliotis discus hannai remains a high-value aquaculture mollusk and a Chinese predominantly cultured abalone species. Salinity is one of such environmental factors whose fluctuation could significantly affect the abalone’s cellular and molecular immune responses and result in high mortality and reduced growth rate during prolonged exposure. Meanwhile, hybrids have shown superiority in tolerating diverse environmental stresses over their purebred counterparts and have gained admiration in the Chinese abalone aquaculture industry. The objective of this study was to investigate the molecular and cellular mechanisms of low salinity adaptation in abalone. Therefore, this study used transcriptome analysis of the gill tissues and flow cytometric analysis of hemolymph of H. discus hannai (DD) and interspecific hybrid H. discus hannai ♀ x H. fulgens ♂ (DF) during low salinity exposure. Also, the survival and growth rate of the species under various salinities were assessed. The transcriptome data revealed that the differentially expressed genes (DEGs) were significantly enriched on the fluid shear stress and atherosclerosis (FSS) pathway. Meanwhile, the expression profiles of some essential genes involved in this pathway suggest that abalone significantly up-regulated calmodulin-4 (CaM-4) and heat-shock protein90 (HSP90), and significantly down-regulated tumor necrosis factor (TNF), bone morphogenetic protein-4 (BMP-4), and nuclear factor kappa B (NF-kB). Also, the hybrid DF showed significantly higher and sustained expression of CaM and HSP90, significantly higher phagocytosis, significantly lower hemocyte mortality, and significantly higher survival at low salinity, suggesting a more active molecular and hemocyte-mediated immune response and a more efficient capacity to tolerate low salinity than DD. Our study argues that the abalone CaM gene might be necessary to maintain ion equilibrium while HSP90 can offset the adverse changes caused by low salinity, thereby preventing damage to gill epithelial cells (ECs). The data reveal a potential molecular mechanism by which abalone responds to low salinity and confirms that hybridization could be a method for breeding more stress-resilient aquatic species.

Nur77, an orphan nuclear receptor, is implicated in regulating diverse cellular biological processes including apoptosis and inflammation. We previously identified BI1071 (DIM-C-pPhCF3+MeSO3-), an oxidized methanesulfonate salt of (4-CF3-Ph-C-DIM), was a direct ligand of Nur77, which could activate the Nur77-Bcl-2 apoptotic pathway. To obtain more effective compounds targeting the Nur77-mediated apoptotic pathway, we designed and synthesized a series of BI1071 analogs by introducing various substituent groups in the indolyl-rings of BI1071. Structure-activity relationship study identified A11, B5 and B15 as improved analogs with stronger binding affinity to Nur77 and enhanced apoptotic activity compared to BI1071. Nur77-binding studies demonstrated that A11, B5 and B15 bind to Nur77 with a Kd of 34 nM, 19 nM and 16 nM, respectively. Furthermore, mechanism studies showed that A11, B5 and B15 induced apoptosis through utilizing the Nur77-Bcl-2 pathway.

The hybrid abalone Haliotis discus hannai female x H. fulgens male (DF) shows obvious growth advantages compared with one of its parents, H. discus hannai (DD); however, the reasons for these advantages are not fully understood. Using a video surveillance system, the current study showed that the feeding behaviors of DD and DF exhibited an obvious circadian rhythm, with significantly more feeding at night by DD and DF than during the day. In addition, the amount of time feeding by DF during the day and at night was significantly higher compared with that of DD, especially at ZT8 (20:00), ZT12 (24:00), ZT16 (04:00), and ZT20 (08:00), with the proportion of time feeding by DF increasing 2.22-, 1.60-, 2.71-, and 2.40-fold compared with those of DD, respectively. The maximum values of pepsase activity in DD and DF, of alpha-amylase activity in DD, and of alginate lyase activity in DF coincided with the peak feeding period. Relative to the changes in expression levels of neuropeptide Y receptor (NPYR) in the intestine, diurnal changes in NPYR expression levels in cerebral ganglia were consistent with the changes in feeding behavior, being highest at 24:00 h in both DD and DF and then declining. There was no significant difference in the circadian expression levels of cholecystokinin receptor (CCKR) in the cerebral ganglia, whereas, in the intestinal tract, peak CCKR expression levels coincided with its anorexigenic effect; the expression levels of CCKR in the intestinal tract exhibited a significant circadian rhythm in DD. These results explain the growth advantage of DF over DD in terms of feeding behavior and digestive physiology, and support the hypothesis that diurnal changes in feeding-related genes in the nervous and digestive systems are correlated with the initiation of abalone feeding behavior. Our findings also provide guidance for developing a suitable feeding strategy for, and improving the efficiency of, the production of abalone.

RXRα, a unique and important nuclear receptor, plays a vital role in various biological and pathological pathways, including growth, differentiation, and apoptosis. We recently reported a transcription-independent function of RXRα in cancer cells in which RXRα is phosphorylated by Cdk1 at the onset of mitosis, resulting in its translocation to the centrosome, where the phosphorylated RXRα (p-RXRα) interacts with polo-like kinase 1 (PLK1) to promote centrosome maturation and mitotic progression. Significantly, we also identified that a small molecule XS-060 binds to RXRα and selectively inhibits the p-RXRα/PLK1 interaction to induce mitotic arrest and catastrophe in cancer cells. Here, we report our design, synthesis, and biological evaluation of a series of XS-060 analogs as RXRα-targeted anti-mitotic agents. Our results identified B10 as an improved anti-mitotic agent. B10 bound to RXRα (Kd = 3.04 ± 0.58 μM) and inhibited the growth of cervical cancer cells (HeLa, IC50 = 1.46 ± 0.10 μM) and hepatoma cells (HepG2, IC50 = 3.89 ± 0.45 μM and SK-hep-1, IC50 = 5.74 ± 0.50 μM) with low cytotoxicity to nonmalignant cells(LO2, IC50 > 50 μM). Furthermore, our mechanistic studies confirmed that B10 acted as an anticancer agent by inhibiting the p-RXRα/PLK1 pathway. These results provide a basis for further investigation and optimization of RXRα-targeted anti-mitotic molecules for cancer therapy.