2­D08 mediates notable anticancer effects through multiple cellular pathways in uterine leiomyosarcoma cells.

2',3',4'­trihydroxyflavone (2­D08), a SUMO E2 inhibitor, has several biological functions, including anticancer activity, but its effects on uterine leiomyosarcoma (Ut­LMS) are unknown. The anticancer activity of 2­D08 was explored in an in vitro model using SK­LMS­1 and SK­UT­1B cells (human Ut­LMS cells). Treatment with 2­D08 inhibited cell viability in a dose­ and time­dependent manner and significantly inhibited the colony­forming ability of Ut­LMS cells. In SK­UT­1B cells treated with 2­D08, flow cytometric analysis revealed a slight increase in apoptotic rates, while cell cycle progression remained unaffected. Western blotting revealed elevated levels of RIP1, indicating induction of necrosis, but LC3B levels remained unchanged, suggesting no effect on autophagy. A lactate dehydrogenase (LDH) assay confirmed increased LDH release, further supporting the induction of apoptosis and necrosis by 2­D08 in SK­UT­1B cells. 2­D08­induced production of reactive oxygen species and apoptosis progression were observed in SK­LMS­1 cells. Using Ki67 staining and bromodeoxyuridine assays, it was found that 2­D08 suppressed proliferation in SK­LMS­1 cells, while treatment for 48 h led to cell­cycle arrest. 2­D08 upregulated p21 protein expression in SK­LMS­1 cells and promoted apoptosis through caspase­3. Evaluation of α­SM­actin, calponin 1 and TAGLN expression indicated that 2­D08 did not directly initiate smooth muscle phenotypic switching in SK­LMS­1 cells. Transcriptome analysis on 2­D08­treated SK­LMS­1 cells identified significant differences in gene expression and suggested that 2­D08 modulates cell­cycle­ and apoptosis­related pathways. The analysis identified several differentially expressed genes and significant enrichment for biological processes related to DNA replication and molecular functions associated with the apoptotic process. It was concluded that 2­D08 exerts antitumor effects in Ut­LMS cells by modulating multiple signaling pathways and that 2­D08 may be a promising candidate for the treatment of human Ut­LMS. The present study expanded and developed knowledge regarding Ut­LMS management and indicated that 2­D08 represents a notable finding in the exploration of fresh treatment options for such cancerous tumors.

2-D08 treatment regulates C2C12 myoblast proliferation and differentiation via the Erk1/2 and proteasome signaling pathways.

SUMOylation is one of the post-translational modifications that involves the covalent attachment of the small ubiquitin-like modifier (SUMO) to the substrate. SUMOylation regulates multiple biological processes, including myoblast proliferation, differentiation, and apoptosis. 2-D08 is a synthetically available flavone, which acts as a potent cell-permeable SUMOylation inhibitor. Its mechanism of action involves preventing the transfer of SUMO from the E2 thioester to the substrate without influencing SUMO-activating enzyme E1 (SAE-1/2) or E2 Ubc9-SUMO thioester formation. However, both the effects and mechanisms of 2-D08 on C2C12 myoblast cells remain unclear. In the present study, we found that treatment with 2-D08 inhibits C2C12 cell proliferation and differentiation. We confirmed that 2-D08 significantly hampers the viability of C2C12 cells. Additionally, it inhibited myogenic differentiation, decreasing myosin heavy chain (MHC), MyoD, and myogenin expression. Furthermore, we confirmed that 2-D08-mediated anti-myogenic effects impair myoblast differentiation and myotube formation, reducing the number of MHC-positive C2C12 cells. In addition, we found that 2-D08 induces the activation of ErK1/2 and the degradation of MyoD and myogenin in C2C12 cells. Taken together, these results indicated that 2-D08 treatment results in the deregulated proliferation and differentiation of myoblasts. However, further research is needed to investigate the long-term effects of 2-D08 on skeletal muscles.

2-D08 as a SUMOylation inhibitor induced ROS accumulation mediates apoptosis of acute myeloid leukemia cells possibly through the deSUMOylation of NOX2.

Acute myeloid leukemia (AML) is a heterogeneous clonal hematopoietic malignancy with poor survival and frequent relapse. Recently, a posttranslational modification of proteins with small ubiquitin-like modifiers (SUMO) has been notably implicated in a wide spectrum of diseases, especially cancers. Ubc9, as the sole E2-conjugating enzyme in SUMOylation cascade, particularly has been associated with adverse clinical outcomes. 2-D08, a small molecular agent, functions by blocking the transfer of SUMO from the Ubc9 thioester to SUMO substrates without any effects on other individual steps in this process. However, both the effects and mechanisms of 2-D08 on AML cells are still unknown. In this study, we found that 2-D08 significantly suppressed cell viability and colony formation ability. Additionally, it induced mitochondrial-mediated apoptosis with dramatic accumulation of the reactive oxygen species (ROS), which could be almost completely rescued by the ROS scavenger N-acetylcysteine (NAC). Furthermore, we confirmed that the fatal accumulation of ROS was due to its aberrant generation instead of defective scavenging. In summary, our results suggest that 2-D08, as a specific SUMOylation inhibitor, induces ROS accumulation-mediated intrinsic apoptosis of AML cells possibly through deSUMOylation of NOX2. Therefore, 2-D08 might be a promising therapeutic agent for the treatment of AML in the future.

Exploring the Structural Diversity in Inhibitors of α-Synuclein Amyloidogenic Folding, Aggregation, and Neurotoxicity.

Aggregation of α-Synuclein (αS) protein to amyloid fibrils is a neuropathological hallmark of Parkinson's disease (PD). Growing evidence suggests that extracellular αS aggregation plays a pivotal role in neurodegeneration found in PD in addition to the intracellular αS aggregates in Lewy bodies (LB). Here, we identified and compared a diverse set of molecules capable of mitigating protein aggregation and exogenous toxicity of αSA53T, a more aggregation-prone αS mutant found in familial PD. For the first time, we investigated the αS anti-amyloid activity of semi-synthetic flavonoid 2', 3', 4' trihydroxyflavone or 2-D08, which was compared with natural flavones myricetin and transilitin, as well as such structurally diverse polyphenols as honokiol and punicalagin. Additionally, two novel synthetic compounds with a dibenzyl imidazolidine scaffold, Compound 1 and Compound 2, were also investigated as they exhibited favorable binding with αSA53T. All seven compounds inhibited αSA53T aggregation as demonstrated by Thioflavin T fluorescence assays, with modified fibril morphology observed by transmission electron microscopy. Ion mobility-mass spectrometry (IM-MS) was used to monitor the structural conversion of native αSA53T into amyloidogenic conformations and all seven compounds preserved the native unfolded conformations of αSA53T following 48 h incubation. The presence of each test compound in a 1:2 molar ratio was also shown to inhibit the neurotoxicity of preincubated αSA53T using phaeochromocytoma (PC12) cell viability assays. Among the seven tested compounds 2-D08, honokiol, and the synthetic Compound 2 demonstrated the highest inhibition of aggregation, coupled with neuroprotection from preincubated αSA53T in vitro. Molecular docking predicted that all compounds bound near the lysine-rich region of the N-terminus of αSA53T, where the flavonoids and honokiol predominantly interacted with Lys 23. Overall, these findings highlight that (i) restricted vicinal trihydroxylation in the flavone B-ring is more effective in stabilizing the native αS conformations, thus blocking amyloidogenic aggregation, than dihydroxylation aggregation in both A and B-ring, and (ii) honokiol, punicalagin, and the synthetic imidazolidine Compound 2 also inhibit αS amyloidogenic aggregation by stabilizing its native conformations. This diverse set of molecules acting on a singular pathological target with predicted binding to αSA53T in the folding-prone N-terminal region may contribute toward novel drug-design for PD.

Structure-activity relationships for flavone interactions with amyloid ß reveal a novel anti-aggregatory and neuroprotective effect of 2',3',4'-trihydroxyflavone (2-D08).

Naturally-occurring flavonoids have well documented anti-aggregatory and neuroprotective properties against the hallmark toxic protein in Alzheimer's disease, amyloid ß (Aß). However the extensive diversity of flavonoids has limited the insight into the precise structure-activity relationships that confer such bioactive properties against the Aß protein. In the present study we have characterised the Aß binding properties, anti-aggregatory and neuroprotective effects of a discreet set of flavones, including the recently described novel protein sumoylation inhibitor 2',3',4'-trihydroxyflavone (2-D08). Quercetin, transilitin, jaceosidin, nobiletin and 2-D08 were incubated with human Aß1-42 for 48h in vitro and effects on Aß fibrillisation kinetics and morphology measured using Thioflavin T (ThT) and electron microscopy respectively, in addition to effects on neuronal PC12 cell viability. Of the flavones studied, only quercetin, transilitin and 2-D08 significantly inhibited Aß1-42 aggregation and toxicity in PC12 cells. Of those, 2-D08 was the most effective inhibitor. The strong anti-amyloid activity of 2-D08 indicates that extensive hydroxylation in the B ring is the most important determinant of activity against ß amyloid within the flavone scaffold. The lack of efficacy of jaceosidin and nobiletin indicate that extension of B ring hydroxylation with methoxyl groups result in an incremental loss of anti-fibrillar and neuroprotective activity, highlighting the constraint to vicinal hydroxyl groups in the B ring for effective inhibition of aggregation. These findings reveal further structural insights into anti-amyloid bioactivity of flavonoids in addition to a novel and efficacious anti-aggregatory and neuroprotective effect of the semi-synthetic flavone and sumoylation inhibitor 2',3',4'-trihydroxyflavone (2-D08). Such modified flavones may facilitate drug development targeting multiple pathways in neurodegenerative disease.