This research aims to elucidate the mechanistic role of artesunate (ART) in enhancing the sensitivity of renal cell carcinoma (RCC) to sunitinib. To establish sunitinib-resistant RCC cell lines (786-O R and Caki-1 R), cells were treated with different concentrations of sunitinib and ART. The viability of the cells was measured through the cell counting kit-8 (CCK-8) assay. Tripartite motif-containing 24 (TRIM24) and paired box 6 (PAX6) expression were suppressed with lentiviral vectors, quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot (WB) analysis. TRIM24-PAX6 interaction was examined through co-immunoprecipitation (Co-IP) and deubiquitination assays. Additional assays included colony formation, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, WB detection of phosphorylated histone H2AX (γ-H2AX) for DNA damage, epithelial-mesenchymal transition (EMT) marker analysis, sphere formation, and stemness marker assessments. In vivo drug resistance was tested using a mouse subcutaneous xenograft model. ART enhanced sunitinib sensitivity in resistant RCC cells, reducing colony formation, inducing apoptosis, elevating γ-H2AX, and upregulating TRIM24. ART enhances sunitinib sensitivity in RCC cells by upregulating TRIM24 expression, which facilitates the ubiquitination of PAX6. This process leads to the suppression of EMT and stem cell-like transitions in RCC cells.

Bisphenol A (BPA) is a widely used industrial chemical with potential endocrine-disrupting effects on metabolic processes. This study investigates the impact of BPA on hepatic function and transcriptional regulation in mouse livers and AML12 cells. Male mice were exposed to low (5 g/kg) and high (50 g/kg) doses of BPA for six weeks. Transcriptomic analysis was performed on liver tissues, and histological examinations were conducted. AML12 cells were treated with varying BPA concentrations, and PPARG transcriptional activity was assessed using a luciferase reporter assay. Additionally, molecular docking, molecular dynamics (MD) simulations, drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), MM-PBSA calculations, and multi-species protein structure comparative analysis were employed to analyse the interaction between BPA and PPARG. Transcriptomic analysis revealed a decrease in differentially expressed genes with higher BPA doses, with low-dose exposure significantly downregulating hepatic Cpt1a mRNA levels. Histological examination indicated lipid vacuole formation at high doses without collagen deposition. BPA consistently inhibited PPARG activity in both MCF7 cells and mouse livers. BPA exposure disrupts hepatic lipid metabolism and PPARG activity, highlighting its role as an endocrine disruptor. Further research is needed to elucidate the long-term effects of BPA on liver health.

Drought stress is a major constraint on seed germination and crop productivity, particularly for drought-sensitive crops like common buckwheat (Fagopyrum esculentum). Exogenous melatonin has emerged as a promising strategy to mitigate drought stress by enhancing plant physiological and biochemical responses. However, its specific roles in regulating antioxidant defenses, osmotic adjustment, and plant compounds biosynthesis during buckwheat seed germination under drought stress remain poorly understood.

NPS-2143, as a CaSR allosteric antagonist, performs an important role in diverse cancers. However, the function and mechanism of NPS-2143 in human retinoblastoma have not been reported. Cell viability was evaluated by using cell counting kit-8, flow cytometry was used for apoptosis detection and western blotting was carried out to detect the expression of target genes. Small interference RNA (siRNA) was used to reduce CaSR expression. Bortezomib was used to suppress the NF-κB pathway. NPS-2143 (16.5 µM) suppressed the proliferation of Y-79 cells, and increased the apoptosis of Y-79 cells. NPS-2143 treatment inhibited the protein patterns of p-P65 NF-κB and p-IκBα. Additionally, si-CaSR transfection obviously decreased the proliferation of Y-79 cells, and increased the apoptosis of Y-79 cells. Moreover, the protein patterns of p-P65 NF-κB and p-IκBα were obviously decreased after si-CaSR transfection. Bortezomib treatment increased the apoptosis of Y-79 cells, and CaSR overexpression suppressed the apoptosis of Y-79 cells. The whole data indicated that NPS-2143 inhibited the viability of Y-79 cells, and induced apoptosis by modulating the NF-κB signaling pathways. Therefore, NPS-2143 has potential anti-retinoblastoma effect.

The discovery of bifunctional degradation activating compounds (BiDACs) has led to the development of a new class of drugs that promote the clearance of their protein targets. BiDAC-induced ubiquitination is generally believed to direct cytosolic and nuclear proteins to proteolytic destruction by proteasomes. However, pathways that govern the degradation of other classes of BiDAC targets, such as integral membrane and intraorganellar proteins, have not been investigated in depth. In this study we use morphological profiling and CRISPR/Cas9 genetic screens to investigate the mechanisms by which BiDACs induce the degradation of plasma membrane receptor tyrosine kinases (RTKs) EGFR and Her2. We find that BiDAC-dependent ubiquitination triggers the trafficking of RTKs from the plasma membrane to lysosomes for degradation. Notably, functional proteasomes are required for endocytosis of RTKs upstream of the lysosome. Additionally, our screen uncovers a non-canonical function of the lysosome-associated arginine/lysine transporter PQLC2 in EGFR degradation. Our data show that BiDACs can target proteins to proteolytic machinery other than the proteasome and motivate further investigation of mechanisms that govern the degradation of diverse classes of BiDAC targets.

The development of acquired paclitaxel resistance poses a significant challenge in managing lung cancer clinically. Understanding the mechanism and developing effective strategies to counter paclitaxel resistance are highly desired. To explore the potential mechanisms of acquired paclitaxel resistance, we established a series of lung cancer cell lines exhibiting different levels of resistance to paclitaxel. Transcriptomic RNA-sequencing revealed a progressive decrease in alpha-2-macroglobulin (A2M) levels as paclitaxel resistance advanced in NCI-H446 cells. This was accompanied by the upregulation of known paclitaxel resistance inducers ABCB1, TMEM243, and ID1. A2M loss was further validated in paclitaxel-resistant A549 and HCC827 lung cancer cells. TCGA and CPTAC analyses demonstrated that A2M is downregulated in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), inversely correlating with tumor progression. Restoring A2M expression inhibited proliferation and invasion in paclitaxel-resistant lung cancer cells, suggesting its tumor-suppressing role in lung cancer. Notably, restoring A2M re-suppressed the expression of the paclitaxel resistance mediators (ABCB1, TMEM243 and ID1) in the drug-resistant cells, and re-sensitized them to paclitaxel. In summary, our data indicate that A2M is progressively lost during the development of paclitaxel resistance in lung cancer, and restoring A2M may help overcome this resistance. Thus, A2M deficiency may serve as both a predictor and a therapeutic target for paclitaxel resistance in lung cancer.

Cannabinoids have been used as anti-emetic agents in cancer. However, multiple studies suggest that cannabinoids present important anti-tumor actions as well. Estrogen receptor-positive (ER+) breast cancer is the most diagnosed breast cancer subtype, and despite the success of endocrine therapy, endocrine resistance development is a major challenge, demanding the discovery or implementation of alternative therapeutic approaches. In line with this, and following our previous work, the benefits of combining the aromatase inhibitors (AIs) used in the clinic, anastrozole (Ana), letrozole (Let), and exemestane (Exe), with cannabinol (CBN) were evaluated. Experiments were performed in MCF-7aro cells and spheroids to assess activity against specific molecular targets and underlying mechanisms of action. Among the three AIs studied, only the combination of CBN with Exe induced a significant beneficial impact on viability and growth of ER+ breast cancer cells and spheroids. Our results demonstrated that this combination was more effective than Exe in preventing the expression of aromatase and in modulating ERα and androgen receptor (AR) activity. In fact, the results revealed that CBN can prevent de novo synthesis of aromatase, surpass Exe's weak estrogen-like effect, and avoid the unfavorable overexpression of AR. By comparing these two therapeutic strategies, as well as the previously studied combination of Exe plus cannabidiol (CBD), differential transcriptome profiles were detected, which may help to better understand the mechanism of action of cannabinoids and disclose their full potential in breast cancer treatment. In conclusion, this study strengthens the hypothesis that cannabinoids are important anti-cancer agents with attractive co-adjuvant properties.

Bcl-2 protein is an important factor in the regulation of apoptosis. This study focused on the role of Bcl-2 protein in this process by evaluating the growth inhibition effect of EBE on glioma cells C6 and U251 and the possible mechanism of inducing apoptosis. The apoptosis rate was measured by flow cytometry and the growth inhibition effect of EBE was evaluated by cell proliferation assay. The gene expression changes of C6 and U251 cells treated with EBE were analyzed by qPCR, and the chemical components of EBE were identified by high performance liquid chromatography (HPLC). All experiments were statistically analyzed to ensure the reliability of the results. The results showed that EBE significantly inhibited the growth of C6 and U251 cells and effectively induced apoptosis. In C6 cells, after EBE treatment, the expression level of Bcl-2 gene was significantly decreased, while the expression of pro-apoptotic gene was significantly increased. Similar results were observed in U251 cells, further confirming the inhibitory effect of EBE on Bcl-2 protein. Chemical composition analysis confirmed the existence of multiple components in EBE that exert anticancer effects. The study showed that the extract of Erigeron breviscapus promoted the apoptosis of glioma cells by down-regulating the expression of Bcl-2 protein. This discovery provides a new idea for the treatment of glioma and lays a foundation for the development of Bcl-2 related targeted therapy strategies.

Amnesia is a common feature of neurodegenerative disorders. The seeds of Buchholzia coriacea have been traditionally employed to treat various diseases, including inflammatory conditions, pain, infections, and neurological disorders.

Ribosome binding protein 1 (RRBP1) was reported to play a regulatory role in certain cancers. It was related to poor prognosis and drug resistance in multiple myeloma (MM), but its mechanism has not been reported before. Hence, its effect and the specific mechanism on MM cells were explored.

Multiple sclerosis is a chronic inflammatory and neurodegenerative disorder of the central nervous system. Despite ongoing research, effective treatments remain limited, especially during progressive phase. Saponins extracted from the stem and leaf of Panax notoginseng (PNSL) demonstrate a superior anti-inflammatory effect by inhibiting NO production in LPS-induced BV2 cells. Ginsenoside Rb3, the primary active and most abundant component in PNSL, has been demonstrated to mitigate inflammation-induced damage. However, whether Rb3 mitigates demyelination by inhibiting neuroinflammation had not been previously reported. In this study, biochemical and histological assays revealed that ginsenoside Rb3 effectively mitigated Cuprizone-induced demyelination and attenuated aberrant microglial activation and reactive astrogliosis within the demyelinated areas. Mechanistic investigations demonstrated that Rb3 suppresses glial cell activation and consequently mitigates inflammatory responses by inhibiting the secretion of TNF-α, IL-6, and IL-1β. TNF receptor-associated factor 6 (TRAF6) is activated by K63-linked polyubiquitination, which leads to downstream activation of the inhibitor of nuclear factor-κB kinase (IKK) complex and mitogen-activated protein kinases (MAPKs). Furthermore, Rb3 was found to inhibit the activation of nuclear factor-κB (NF-κB) and MAPKs, as evidenced by the dephosphorylation of NF-κB p65 and the MAPKs p38 and JNK. Further investigation revealed that Rb3 binds to TRAF6 at residues 69 and 88, thereby inhibiting its K63-linked polyubiquitination. Conversely, the TRAF6 mutation at E69Q or R88N abolished the inhibition effects of Rb3 on K63-linked ubiquitination of TRAF6 and subsequent downstream signaling activation. Meta-analysis showed that Rb3 exerts its anti-inflammatory effects primarily by inhibiting the NF-κB pathway. Collectively, it is concluded that Rb3 alleviates demyelination and inhibits inflammation through bound to TRAF6 to prevent its K63-linked ubiquitination and subsequent activation of NF-κB. In this study, we have for the first time elucidated that dual mechanism by which Rb3 inhibits both NF-κB and MAPK pathways to exert its anti-inflammatory effects. This study demonstrates that Rb3 shows promising preclinical therapeutic potential. Additionally, TRAF6 represents a potential therapeutic target for MS treatment.

Currently, limited information is available on the molecular basis of the biosynthesis of essential oil in the Monarda citriodora plant. Given the pivotal role of the MEP pathway in the biosynthesis of monoterpenes, in the present study, DXS genes have been functionally characterized from M. citriodora, for the first time. The CDS corresponding to four McDXS genes (1-4) were cloned, and their deduced proteins displayed distinct phylogenetic positioning. Using a bacterial complementation test, we demonstrated that all four McDXS genes encode functional DXS proteins. Based on the results obtained from phylogenetic analysis, tissue-specific expression analysis, and accumulation of monoterpenes, McDXS2 was identified as the candidate gene involved in the biosynthesis of monoterpenes of essential oil in M. citriodora. Transient overexpression and silencing of McDXS2 significantly modified the content of volatile monoterpenes in M. citriodora. Constitutive expression of McDXS2 in Nicotiana tabacum resulted in increased biosynthesis of specialized diterpenoids. Further, the exogenous treatment of MeJA, ABA, and GA3 modulated the expression of McDXS2, and the content of the components of essential oil in M. citriodora. McDXS2 promoter activity was primarily restricted to the glandular trichomes of M. citriodora. The present work demonstrates that McDXS2 is primarily involved in the specialized terpenoid biosynthesis in M. citriodora.

Storing 'Rocha' pear treated with 1-methylcyclopropene (1-MCP) in controlled atmosphere is a common commercial strategy to extend pear storage time and prevent postharvest disorders. However, this strategy represents a challenge to the fruit industry because 1-MCP treatment obstructs the normal fruit ripening, potentially affecting the quality to consumers. To explore possible mechanisms to reactivate ripening, 'Rocha' pears treated with 1-MCP were exposed to 2 and 4 mM 1-naphthaleneacetic acid (1-NAA) and stored at 20 ± 2 °C for 15 days. Typical ripening indicators, such as firmness, skin color, ethylene and aroma volatiles production, sugar content, and the genetic expression of ethylene-related enzymes (ACS and ACO) and receptors (PcETR1, PcETR2, and PcETR5) were determined over the 15 days of storage. A PCA analysis incorporating both physiological and biochemical data showed that 1-NAA promoted the recovery of ripening capacity in 1-MCP treated pears. Treating pears with 1-NAA led to increased activity of genes like PcACS1, PcACS4, and PcETR2, which are involved in ethylene signalling and production. This resulted in higher levels of ethylene and compounds associated with ripening, as well as softer texture, more yellow color, and higher sucrose content. The boost in ethylene-related gene activity likely heightened ethylene sensitivity and production in the treated pears. Consequently, these fruits showed accelerated softening, color change, and aroma development. This suggests that 1-NAA treatment can reverse the ripening inhibition caused by 1-MCP, possibly by enhancing ethylene sensitivity and production. This mechanism could enable consistent ripening of 'Rocha' pears after they are taken out of cold storage, and it may have similar effects on other fruits.

Toxic heavy metals seriously affect plant growth and human health. Among the heavy metals, cadmium (Cd) and lead (Pb) are serious pollutants. Dianthus spiculifolius has strong tolerance to, and an ability to accumulate, heavy metals. Therefore, it has potential applications as a heavy metal hyperaccumulator. Gamma glutamylcyclotransferase (GGCT) is a key enzyme in maintaining glutathione homeostasis, and it plays a role in plant growth and development and in responses to various stresses. Previously, DsGGCT2-1 was identified as a gene showing significantly increased transcript levels in response to Cd and Pb by transcriptome analysis. In this study, DsGGCT2-1 was confirmed to increase the Cd and Pb tolerance of transgenic yeast, Arabidopsis, and Dianthus, decrease the their accumulation in Dianthus. Overexpression of DsGGCT2-1 in D. spiculifolius plants resulted in increased GGCT activity, higher glutamate (Glu), and glutathione (GSH) content. The results suggest that more Glu is synthesized to maintain GSH homeostasis through the activity of GGCT2-1 in the glutamyl cycle, and the generated GSH is used to chelate with toxic heavy metals, and reduce the toxicity of heavy metals in the cytoplasm. These findings will be useful for devising strategies to remediate heavy metal-polluted soils, and for breeding plants that tolerate heavy metals.

In response to chilling, plants undergo a variety of metabolic changes, including structural modifications of sphingolipids, which have an important but poorly understood effect on cold tolerance. Here, we used biochemical, molecular, cell biological, and genetic approaches to investigate the mechanism of cold-induced sphingolipid regulation. Chilling stress affected sphingolipid metabolism in Arabidopsis thaliana, resulting in increased long-chain base (LCB) unsaturation by promoting the expression of the sphingoid LCB ∆8 desaturase gene SLD1. The sld1-1 mutant showed decreased Chl content, reduced plasma membrane fluidity, and growth inhibition under chilling stress. LCB ∆8 double-bond formation during cold stress was regulated by the jasmonate (JA) pathway, and the loss of the JA receptor CORONATINE INSENSITIVE1 (COI1) caused a decrease in LCB unsaturation. The JA pathway master transcription factor MYC2 directly targeted the SLD1 promoter to regulate its transcription. MYC2 also regulates the expression of C-REPEAT BINDING FACTORs (CBFs) and INDUCER OF CBF EXPRESSION1 (ICE1), which play pivotal roles in the cold stress response. Our results reveal the mechanism by which the JA pathway mediates sphingolipid metabolism during cold stress, providing insights into the underlying mechanism of lipid function in cold tolerance.

Pancreatic cancer is one of the deadliest malignant tumours worldwide. Despite the developments in the treatments of pancreatic cancer, survival rates remain at a low level, and the mechanisms underlying the aggressive course of the cancer are not fully understood. VISTA is an immune checkpoint and has recently become a significant target in cancer treatment; however, the roles of VISTA in the development of pancreatic cancer have largely remained unknown. Histone deacetylase inhibitors (HDACi) have been reported to reverse the epithelial-mesenchymal transition (EMT) and may enhance the efficacy of anti-PD-1 therapy. The PD-L1/PD-1 immune checkpoint targeted by this therapy shares structural similarity with VISTA. Moreover, combination therapy of vorinostat and anti-PD-1 has been shown to significantly reduce tumour growth by suppressing the transcription factor c-Myc. Therefore, in this study, we aim to investigate the genes that are associated with EMT and explore the potential mechanism involving Twist1, a proto-oncogene, and VISTA in pancreatic cancer. We also sought to determine the synergistic effects of an HDACi, vorinostat, in combination with Twist1-siRNA on VISTA expression in pancreatic cancer cells' viability and proliferation. Our results revealed that Twist1 blockade in combination with vorinostat in pancreatic cancer cells suppresses EMT-associated genes and the immune checkpoint VISTA compared to treatments administered alone. As a result, identifying the genes associated with EMT in pancreatic cancer and understanding the role of Twist1 in this process is a crucial step to contribute to the identification of new targets for pancreatic cancer treatment and the improvement of existing treatment strategies.

Seed germination is a critical developmental stage in the lifecycle of plants, and its regulation is essential for ensuring crop productivity, particularly under adverse environmental conditions. Here, we find that the Arabidopsis thaliana Pre-mRNA PROCESSING FACTOR 21 (PRP21) is crucial for regulating the abscisic acid (ABA) response and seed germination. Our RNA deep sequencing and poly(A) tag sequencing analyses reveal that PRP21 is involved in pre-mRNA splicing, genome-wide gene expression, and mRNA 3' end processing, highlighting its multifunctional role in gene regulation. Furthermore, PRP21 interacts with various splicing factors and small nuclear ribonucleoproteins, confirming its involvement in spliceosome assembly. Additionally, we demonstrate that PRP21 negatively regulates the expression of ABA-responsive genes, such as ABA INSENSITIVE 3 (ABI3), ABA INSENSITIVE 5 (ABI5), EARLY METHIONINE-LABELED 1 (EM1), and EM6, thereby modulating ABA response and seed germination. Our findings underscore the importance of PRP21 in coordinating transcriptional and post-transcriptional processes and provide insights into the molecular mechanisms underlying seed germination, potentially guiding crop improvement for stress tolerance.

Dysregulation of deubiquitination is essential for cancer growth. However, the role of 26S proteasome non-ATPase regulatory subunit 14 (PSMD14) in the progression of triple-negative breast cancer (TNBC) remains to be determined. Gain- and loss-of-function experiments showed that silencing PSMD14 notably attenuated the growth, invasion, and metastasis of TNBC cells in vitro and in vivo. Overexpression of PSMD14 produced the opposite results. Mechanistically, PSMD14 decreased K63-linked ubiquitination on SF3B4 protein to de-ubiquitin and stabilize SF3B4 protein. Then, SF3B4/HNRNPC complex bound to FADS1 mRNA and promoted exon inclusion in the target mRNA through m6A site on FADS1 mRNA recognized by HNRNPC, thereby up-regulating the expression of FADS1 and activating Akt/mTOR signaling. Exogenous arachidonic acid supplementation combined with PSMD14 knockdown induced synthetic lethality, which was further confirmed in TNBC organoid (PDO) and TNBC patient-derived xenograft (PDX) mouse models. Overall, our findings reveal an oncogenic role of PSMD14 in TNBC progression, which indicates a potential biomarker and ferroptosis-mediated therapeutic strategy for TNBC.

RAS is the most frequently mutated oncogene in cancer. RAS proteins show high sequence similarities in their G-domains but are significantly different in their C-terminal hypervariable regions (HVR). These regions interact with the cell membrane via lipid anchors that result from posttranslational modifications (PTM) of cysteine residues. KRAS4b is unique as it has only one cysteine that undergoes PTM, C185. Small molecule covalent modification of C185 would block any form of prenylation and subsequently inhibit attachment of KRAS4b to the cell membrane, blocking its biological activity. We translated this concept to the discovery and development of disulfide tethering screen hits into irreversible covalent modifiers of C185. These compounds inhibited proliferation of KRAS4b-driven mouse embryonic fibroblasts, but not cells driven by N-myristoylated KRAS4b that harbor a C185S mutation and are not dependent on C185 prenylation. Top-down proteomics was used to confirm target engagement in cells. These compounds bind in a pocket formed when the HVR folds back between helix 3 and 4 in the G-domain (HVR-α3-α4). This interaction can happen in the absence of small molecules as predicted by molecular dynamics simulations and is stabilized in the presence of C185 binders as confirmed by small-angle X-ray scattering and solution NMR. NOESY-HSQC, an NMR approach that measures internuclear distances of 6 Å or less, and structure analysis identified the critical residues and interactions that define the HVR-α3-α4 pocket. Further development of compounds that bind to this pocket could be the basis of a new approach to targeting KRAS cancers.

PROteolysis Targeting Chimera (PROTAC) technology is an innovative and potent approach for achieving targeted protein degradation (TPD). Within bromodomain-containing proteins, various degraders targeting BET family-related targets, for example, BRD4, were developed in the last years. On the other hand, a limited number of PROTACs acting against non-BET proteins were reported so far. Among them, BRD9 was recently linked to oncogenic roles in the tumorigenesis processes, especially in sarcomas and leukemias. Herein, we describe the design and synthesis of a focused collection of new BRD9-targeting degraders based on the [1,2,4]triazolo[4,3-a]quinoxaline heterocyclic scaffold employing two distinct E3 ubiquitin ligase ligands. Through in silico evaluation, synthesis, binding affinity determination, and in vitro analysis, we identified two new VHL-based PROTACs (2 and 9), which showed remarkable degradation of the protein of interest and antiproliferative activity in acute myeloid leukemia (AML) cells. Notably, compound 9 exhibited selective degradation of BRD9 over BRD7. These results enlarge and differentiate the pool of heterobifunctional molecules able to degrade BRD9 through the proteasome machinery, providing a promising reference for the discovery of new tools to further explore both the involvement of this epigenetic regulatory factor in tumor processes and to evaluate novel strategies for AML treatment.