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Carrimycin is a macrolide antibiotic widely used in the pharmaceutical and clinical fields， with its primary composition consisting of three distinct derivatives of spiramycin. For decades， macrolides like carrimycin have been valued for their efficacy against Gram-positive bacteria and certain atypical pathogens. However， as research has progressed， recent studies have revealed that carrimycin， along with various other structurally modified spiramycin derivatives， exhibits significant antitumor activity in multiple experimental models. This finding suggests that the spiramycin molecular scaffold possesses intrinsic potential beyond its conventional antibacterial role. By strategically modifying the substituents attached to this core structure， it may be feasible to develop novel compounds with enhanced and more selective antitumor properties. Despite this promising outlook， research into the specific targets and comprehensive mechanisms underlying the antitumor effects of spiramycin derivatives remains notably insufficient. A major gap exists in the precise identification of their molecular targets within cancer cells and the detailed signaling pathways they modulate. This lack of mechanistic understanding poses a substantial barrier to the rational design， optimization， and clinical translation of spiramycin-based antitumor therapeutics. To systematically address this knowledge gap and elucidate the antitumor mechanism of this compound class， the present study adopted an activity-based protein profiling （ABPP） strategy. ABPP is a chemoproteomic approach that enables the direct identification of functionally active proteins that interact with small-molecule probes in a native biological context. As a first step， we designed and synthesized a novel， high-activity spiramycin derivative termed n-hexanoyl spiramycin （h-SPM）. Building upon the structure of h-SPM， we subsequently engineered and synthesized a structurally analogous activity-based probe. This probe was specifically functionalized with chemical handles （such as an alkyne group） compatible with ABPP methodologies， allowing for downstream bioorthogonal conjugation and enrichment steps. The experimental workflow began by incubating this active probe with live cancer cells. During this co-incubation period， the probe engaged with and covalently bound to its potential protein targets within the complex cellular environment. Following the interaction， cells were lysed， and the probe-labeled proteins were efficiently isolated and purified using affinity-based enrichment techniques-specifically via copper-catalyzed azide-alkyne cycloaddition （click chemistry） to immobilize them onto a solid support. The enriched protein pool was then subjected to in-depth analysis using liquid chromatography-mass spectrometry （LC-MS）. This analytical phase yielded detailed， proteome-wide information on the identities of proteins that interact with the h-SPM-based probe. To extract biological insights from the list of identified proteins， we performed comprehensive bioinformatic analysis using Gene Ontology （GO） enrichment. This systematic classification provided crucial information regarding the biological processes， molecular functions， and cellular components associated with the captured proteins. The functional annotations derived from GO analysis allowed us to evaluate and prioritize several promising candidate target proteins for further experimental validation. Through this integrated proteomic and bioinformatic approach， we successfully identified multiple potential cellular targets of h-SPM. Notable among these were amyloid precursor protein （APP） and low-density lipoprotein receptor （LDLR）， both of which are implicated in diverse cellular processes such as cell adhesion， signal transduction， and metabolic regulation. To confirm the biological relevance of these interactions， we employed Western Blotting experiments. These studies verified that the expression or post-translational modification states of APP and LDLR were altered in response to h-SPM treatment， thereby confirming their status as responsive molecular targets. After identifying and preliminarily validating APP as a key interactor， we proceeded to investigate its functional role in the drug's mechanism of action. Using short hairpin RNA （shRNA）-mediated protein knockdown， we generated cancer cell lines with significantly reduced APP expression. Comparative analysis of drug sensitivity between these knockdown cells and their wild-type counterparts revealed a marked attenuation of h-SPM's antitumor effects in the absence of APP. Complementary to this， cell staining assays， including immunofluorescence， were conducted to visualize morphological changes， alterations in cell viability， and the subcellular localization of relevant biomarkers following h-SPM treatment. Collectively， these functional experiments provided compelling evidence that APP plays a critical and indispensable role in mediating the antitumor activity of h-SPM. Our findings thus outline a preliminary model of the drug's mechanism， likely involving cellular pathways regulated or influenced by APP. In summary， this study achieves two significant objectives. First， it establishes and validates a robust， generalizable target-screening platform based on ABPP， specifically tailored for the investigation of spiramycin-class compounds. Second， and more importantly， it delivers novel biological insights by pinpointing specific protein targets such as APP. The identification of these targets provides invaluable mechanistic clues and a solid theoretical foundation for the future development of this family of compounds. This work effectively advances spiramycin derivatives from compounds with observed phenotypic activity toward agents with an emerging mechanistic understanding， thereby paving the way for more targeted drug design and informed combination therapy strategies in oncology. Future studies will focus on delineating the detailed downstream consequences of APP engagement by h-SPM and exploring the therapeutic potential of other identified targets in preclinical models. 可利霉素是一种在医药领域广泛使用的大环内酯类抗生素，主要成分为3种不同的螺旋霉素衍生物。然而随着相关研究的深入，近期发现可利霉素以及多种不同结构的螺旋霉素衍生物都具有一定的抗肿瘤活性，说明利用螺旋霉素作为母核，进行不同的取代基修饰，可能会得到更好的抗肿瘤活性药物。虽然螺旋霉素有着成为抗肿瘤药物的潜力，但是关于其作用靶点及完整机制的研究依然欠缺。为了揭示其抗肿瘤作用机制，本研究采用了基于活性的蛋白质组分析（ABPP）策略，设计并合成了一种高活性螺旋霉素衍生物正己酰螺旋霉素（h-SPM），进而依据其结构合成了一种结构相似且带有适用于ABPP实验官能团的活性分子探针。利用该探针与细胞蛋白质共孵育，使两者结合，从而捕获h-SPM潜在的药物靶点，随后对探针结合的蛋白质进行分离纯化与质谱分析，获得详细的靶点蛋白质信息。进一步通过基因本体（GO）分析挖掘这些蛋白质的性质和功能，得到明确的功能信息，并据此筛选出若干潜在靶点蛋白质。通过上述方法，从蛋白质谱的结果中鉴定出包括淀粉样前体蛋白（APP）、低密度脂蛋白受体（LDLR）在内的多个h-SPM潜在作用靶点，并采用免疫印迹（Western Blotting）方法确定这些蛋白质对h-SPM产生了响应。随后，通过短发夹RNA（shRNA）介导的蛋白质敲低及细胞染色等实验，证明蛋白质APP在该药物发挥作用的过程中扮演了关键角色，初步揭示了该类药物的抗肿瘤作用机制。本研究不仅建立了适用于螺旋霉素类化合物的靶点筛选方法，也为该类药物的后续开发提供了关键靶点线索与理论依据。
In the mammalian cerebellum, three types of astroglial cells-Bergmann glial cells (BGs), inner granule cell layer (IGL) astrocytes, and white matter (WM) astrocytes-arise in postnatal timing from two types of progenitors: Bergmann glia-like progenitors (BGLPs) and astrocyte-like progenitors (AsLPs). In contrast to AsLPs, which are commonly observed in other brain regions, BGLPs have not been well studied. Here, we investigate differentiation abilities, gene expression profiles and differentiation control mechanisms of BGLPs at postnatal stages. BGLPs and AsLPs decrease in number as development progresses from postnatal day 0 (P0), and are almost absent by P10. By utilizing an electroporation-based method to BGLPs, we found that P6 BGLPs differentiate into BGs and IGL astrocytes, but not into WM astrocytes, consistent with a previous report. However, P0 BGLPs were observed to differentiate into not only BGs and IGL astrocytes, but also WM astrocytes and a small number of molecular layer inhibitory neurons. By conducting spatial transcriptomic analysis with over 5000 probes (Xenium), we successfully identified distinct clusters corresponding to BGLPs at P0 and P6, respectively, and genes preferentially expressed in P0 and P6 BGLPs. In addition, upstream regulatory analysis using Enrichr identified Foxm1 and Nfia as candidate regulators that affect stage-specific properties of BGLPs. in vivo knockdown and overexpression experiments further demonstrated that precise regulation of Foxm1 and Nfia expression is important for proper progeny production from postnatal BGLPs. This study gives insights into understanding molecular nature and differentiation ability control of BGLPs during postnatal cerebellar development.
Casein kinase 2 (CK2) is a constitutively active serine/threonine kinase that orchestrates multiple oncogenic signaling networks, including PI3K/Akt, NF-κB, JAK/STAT3, Wnt/β-catenin, and DNA damage response pathways. Its broad involvement in cell survival, proliferation, and therapy resistance has positioned CK2 as a compelling target in oncology, particularly in breast cancer where CK2α and CK2β are frequently overexpressed and correlate with poor prognosis. Over the past decades, a wide range of CK2 inhibitors has been developed ranging from classical ATP-competitive scaffolds (TBB, DMAT, CX-4945) to highly selective second-generation chemical probes (SGC-CK2-1, AB668) and substrate-targeting peptides (CIGB-300). While two candidates, CX-4945 and CIGB-300, have advanced to clinical evaluation, major challenges persist, including limited selectivity, suboptimal pharmacokinetics, and reduced in vivo potency. Notably, kinome-wide profiling has revealed that several traditional inhibitors, including CX-4945, display substantial off-target activity, complicating the attribution of cellular phenotypes directly to CK2 inhibition. Emerging strategies including allosteric, αD-pocket-directed, bivalent, and substrate-targeting approaches have substantially improved selectivity and enabled more precise interrogation of CK2 biology. Preclinical evidence highlights strong antitumor effects of CK2 blockade in hormone-refractory and triple-negative breast cancer (TNBC), with additional potential to overcome endocrine and chemoresistance. This review integrates recent advances in CK2 biology, summarizes the evolution of CK2 inhibitor classes, and outlines the opportunities and remaining barriers for translating CK2 inhibition into effective cancer therapeutics.
Head and neck squamous cell carcinoma (HNSCC) remains a prevalent malignancy worldwide, posing significant health threats due to its high recurrence and metastatic potential. HPV-positive and negative HNSCC subtypes exhibit distinct prognostic profiles and their underlying pathogenic mechanisms remain poorly characterized. Four HNSCC cell lines were selected: two HPV-positive (UM-SCC-47 and UPCI-SCC-090) and two HPV-negative (FaDu and UM-SCC-4). Basal miR-106a expression was measured in HPV-positive and -negative cells, followed by RT-qPCR validation of miR-106a, HPV-E7, RUNX3 overexpression and knockdown efficiency. Functional assays included CCK-8 for proliferation, wound healing for migration, Transwell for invasion, and flow cytometry for apoptosis. RT-qPCR quantified HPV-E7, miR-106a, RUNX3, and TGF-β1 mRNA levels; RUNX3 and TGF-β1 protein expression was assessed via Western blot. Dual-luciferase reporter assays confirmed the direct targeting of miR-106a to RUNX3. Finally, xenograft nude mouse models assessed miR-106a's effects on tumor growth and downstream molecular regulation in HPV-positive and -negative HNSCC. Comparative analysis revealed that miR-106a was significantly upregulated in HPV-positive HNSCC cells compared to their HPV-negative counterparts. Functional assays demonstrated that miR-106a overexpression enhanced HNSCC cell proliferation, migration, and invasion while suppressing apoptosis, whereas ectopic expression of RUNX3 exerted opposing effects on these oncogenic phenotypes. Mechanistically, miR-106a overexpression transcriptionally downregulated RUNX3 and concurrently elevated TGF-β1 expression, while RUNX3 overexpression inversely suppressed TGF-β1 levels. Dual-luciferase reporter assays confirmed a direct binding interaction between miR-106a and the 3'UTR of RUNX3. Rescue experiments further established that HPV E7-driven oncogenic effects-enhanced proliferation, migration, invasion, and apoptosis suppression-were abrogated by miR-106a inhibition, concomitant with restored expression of RUNX3 and attenuated TGF-β1 signaling. In vivo studies corroborated these findings, showing that miR-106a overexpression accelerated tumor growth in xenograft models, accompanied by progressive RUNX3 downregulation and TGF-β1 upregulation, consistent with its in vitro regulatory axis. Our findings suggest that the E7/miR-106a/RUNX3/TGF-β1 axis modulates proliferation, migration, invasion, and apoptosis in HPV-positive versus negative HNSCC, implicating its pathogenic role in tumor progression.
Nitrogen (N) doping and amino functionalization markedly enhance the electron-donating capacity of graphene quantum dots (GQDs), thereby improving charge-transfer efficiency in amino-N-GQDs and yielding substantially superior photophysical performance compared with amino-free N-GQDs and N-free amino-GQDs. Further optimization was achieved through conjugation of amino-N-GQDs with sulfur- and nitrogen-rich polymers, polystyrene sulfonate and polyethylenimine, resulting in amino-N-GQD-polymer nanohybrids with significantly improved optical behavior. These hybrid nanostructures exhibited high quantum yields, excellent photostability, negligible reactive oxygen species generation, and strong two-photon luminescence, positioning them as promising contrast agents for nonlinear bioimaging. To enable molecular specificity, antibody functionalization was incorporated. When conjugated with anti-lipopolysaccharide or anti-TasA antibodies, the nanohybrids selectively targeted Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis), generating bright fluorescence, strong signal intensity, and high signal-to-noise ratios under two-photon excitation. Using a custom-built Ti:sapphire laser system operating at 970 nm (near-infrared-II region), imaging depths of up to 270 μm were achieved with ultralow excitation energies, 42.96 nJ pixel-1 for E. coli and 35.14 nJ pixel-1 for B. subtilis, acquired over 100 scans (total exposure = 0.666 s). The nanohybrids produced two-photon luminescence using only 1/49 and 1/36 of the energy required for cellular autofluorescence, corresponding to ∼2401- and ∼1296-fold signal enhancements, respectively. This remarkable efficiency supports deep, noninvasive imaging and underscores the potential of amino-N-GQD-polymer nanohybrids as versatile near-infrared-I/II-responsive probes for next-generation biomedical imaging applications.
Antibody-drug conjugates (ADCs) targeting Nectin4 have shown substantial clinical efficacy in the treatment of progressive or metastatic urothelial carcinoma (mUC). Accumulating evidence indicates that therapeutic responsiveness to ADCs is closely associated with the level of Nectin4 expression, which can be noninvasively evaluated using positron emission tomography (PET). However, our previously developed molecular probe, DN68, has displayed predominant renal excretion, a pharmacokinetic feature that may compromise its ability to reliably visualize primary mUC lesions. In the present study, we sought to develop novel molecular probes with reduced renal retention to improve PET imaging of mUC. To enhance hepatic metabolic processing, a lipid-soluble linker was introduced into the molecular probe precursor NOTA-DN96, thereby modulating its lipid-water partition coefficient and altering its in vivo clearance profile. The targeting characteristics of the novel precursor, radiolabeled with either [68Ga]Ga or [18F]AlF, were systematically assessed through in vitro cellular uptake assays and in vivo imaging studies using Nectin4-positive (Nectin4+) tumor-bearing models. Based on overall imaging performance, the optimal probe was subsequently selected for further validation in mUC models. Radiolabeling of [68Ga]Ga-NOTA-DN96 and [18F]AlF-NOTA-DN96 achieved efficiencies exceeding 95%, with radiochemical purity consistently greater than 99%. Cellular uptake assays demonstrated markedly higher accumulation of both tracers in MC38-Nectin4 cells (Nectin4+) compared with MC38 cells (Nectin4-negative, Nectin4-). In agreement with these results, biodistribution studies and PET imaging revealed pronounced and specific tracer accumulation in MC38-Nectin4 tumor models, whereas uptake in MC38 tumors remained substantially lower. The tumor-to-muscle (T/M) ratio increased progressively over time and reached its maximum at 120 min post-injection (p.i.). Moreover, tumor uptake of [68Ga]Ga-NOTA-DN96 was consistently higher than that of [18F]AlF-NOTA-DN96 across all tumor models examined. Notably, both tracers exhibited increased hepatic uptake accompanied by reduced renal accumulation, indicating a shift toward enhanced hepatobiliary clearance. Collectively, [68Ga]Ga-/[18F]AlF-NOTA-DN96 demonstrated excellent stability, high target specificity, and partial hepatobiliary elimination, highlighting their potential as promising PET probes for imaging Nectin4-expressing tumors. Their distinct pharmacokinetic characteristics also provided valuable insights for future probe design and optimization. These findings, therefore, established a foundation for the development of Nectin4-targeted PET imaging strategies in precision oncology.
METTL13, a dual-functional methyltransferase harboring protein N-terminal methyltransferase (NTMT) activity, has emerged as a pivotal regulator in carcinogenesis, tumor progression, and patient prognosis, thereby garnering substantial interest as a promising therapeutic target for cancers. Despite its clinical significance, no specific chemical probes or inhibitors have been reported to date, highlighting an urgent need for drug discovery efforts targeting this enzyme. Herein, we employed the bisubstrate inhibitor strategy to develop the chemical probe NT32 for METTL13, which showed selective against a panel of methyltransferases. Comprehensive investigations into the structural and functional impacts of NT32 on METTL13 were conducted via molecular docking, molecular dynamics (MD) simulations, and multi-dimensional analyses. Computational modeling revealed that NT32 could occupy both the SAM/SAH-binding pocket and the peptide substrate-binding site of METTL13 simultaneously, establishing a bivalent binding mode. Notably, MD simulations unveiled significant enhancements in both positive and negative residue correlations within the NT32-METTL13 complex compared to the wild-type enzyme. Specifically, interactions between NT32 and critical residues (Gly503, Asn614, Gly644, Glu645) strengthened the connectivity of Met692 with its surrounding residues. This structural reorganization was accompanied by augmented correlated motions between the Gly501-Val511 region (loop501-504-α2505-511) and the Leu613-Leu620 segment (β5613-615-loop616-618-α6619-620), culminating in a more compact and stable protein conformation. Furthermore, NT32 specifically targeted and stabilized METTL13 in cellular thermal shift assay. These findings not only provide mechanistic insights into NT32-METTL13 interactions but also pave the way for discovery of METTL13 probes or inhibitors.
To explore the molecular mechanism by which selenocystine (SeC) inhibits colon cancer cell growth in vitro. Colon cancer cells (RKO, HCT-116, and LoVo) were cultured and treated with 5, 10, or 20 μmol/L SeC for 24 h and 48 h. MTT assay was used to detect the cell viability, and wound healing assay was used to examine changes in cell migration. Flow cytometry with PI staining was used to analyze cell cycle arrest and apoptosis. Fluorescence probes were employed to monitor reactive oxygen species (ROS) generation, mitochondrial morphology and membrane potential, and the changes in ferroptosis were evaluated by detecting malondialdehyde (MDA), glutathione (GSH) and ferrous ion (Fe2+) levels; Western blotting was used to detect the changes in protein expressions. SeC at all the 3 doses significantly inhibited proliferation and migration of colon cancer cells, down-regulated the expression of cell cycle-related proteins CDK2 and CDK4 and activated the apoptotic proteins PARP and caspase-9. Western blotting showed that SeC decreased the expression of ferroptosis proteins FTH1 and xCT and increased the expression of DMT1. The levels of MDA and Fe2+ were increased and GSH level was decreased in SeC-treated cells. Fluorescence staining results showed that SeC treatment induced mitochondrial structure damages and promoted cellular ROS production. SeC treatment also increased phosphorylation of oxidative damage proteins and lowered the expression levels of NRF2 and HO-1 proteins. ROS scavenger significantly reversed the up-regulation of DMT1, PARP and p-H2A.X protein induced by SeC in colon cancer cells. SeC induces apoptosis and ferroptosis of colon cancer cells by promoting ROS generation and initiating oxidative damage, suggesting the potential of SeC as a potential chemotherapeutic agent for colon cancer. 目的: 评价硒代胱氨酸（SeC）体外抑制结肠癌细胞生长的分子机制。方法: 体外培养RKO、HCT-116、LoVo结肠癌细胞，SeC处理细胞24 h和48 h，分为正常对照组和SeC低、中、高剂量组（SeC为5、10、20 μmol/L）。MTT检测结肠癌细胞的活性。划痕实验检测结肠癌细胞迁移能力。流式细胞仪PI染色检测细胞周期阻滞和凋亡情况。荧光探针检测自由基产生、线粒体结构和膜电位变化。铁死亡标志物如丙二醛（MDA）、谷胱甘肽（GSH）、亚铁离子（Fe2+）分析铁死亡的水平，Western blotting检测蛋白表达情况。结果: SeC低、中、高剂量组均抑制结肠癌细胞增殖和迁移（P<0.05）;SeC不同剂量处理组中细胞周期相关蛋白CDK2、CDK4表达下调，并且凋亡蛋白PARP和Caspase9发生活化（P<0.05）;此外，Western blotting结果显示SeC诱导铁死亡蛋白FTH1，xCT表达下降和DMT1表达升高;SeC不同剂量处理组中MDA，Fe2+水平增加和GSH水平降低（P<0.05）。荧光结果显示线粒体结构损伤、活性氧（ROS）生成增多;Western blotting结果显示氧化损伤蛋白的磷酸化表达升高和NRF2/HO-1蛋白表达下调（P<0.05）;ROS清除剂显著逆转结肠癌细胞中SeC对DMT1、PARP、p-H2A.X蛋白的上调作用（P<0.05）。结论: SeC可通过诱导ROS生成启动氧化应激损伤诱导结肠癌细胞凋亡和铁死亡，进而抑制结肠癌细胞生长，是结肠癌潜在的化疗剂。.
Cancer arises and is resistant to therapy via intricate molecular networks that are poorly characterised. While individually, Cullin-3 (CUL3) and circular RNAs (circRNAs) have been reported to modulate cancer, their synergistic effect in the modulation of tyrosine kinase inhibitor (TKI) resistance is yet to be studied. An emerging circRNA-CUL3-TKI regulatory framework is highlighted as a potential contributor to oncogenesis and drug sensitivity in this review. We discuss how circRNA-associated networks may influence CUL3-dependent pathways implicated in tumour resistance to therapy by modulating autophagy, ferroptosis, stress-responses, and redox signalling. Exosomal circRNAs and circRNAs of the CUL3 gene itself are highlighted as dynamic mediators of resistance as well as biomarkers. How they interact with Kelch-like ECH-associated protein 1- Nuclear factor erythroid 2-related factor 2 (KEAP1-NRF2) signalling reveals that they enhance tumour survival under therapy pressure. By highlighting key processes of carcinogenesis and resistance, the circRNA-CUL3-TKI axis represents a testable therapeutic framework. Modeling circRNA networks, predicting TKI response, finding biomarkers, and developing personalised treatment plans are all made possible by applications of artificial intelligence and machine learning (AI/ML), as explored in this review. Antisense oligonucleotides, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based molecules, neddylation inhibitors or PROteolysis TArgeting Chimera (PROTACs) are examples of potential interventions that, when combined with AI/ML techniques, improve therapeutic efficacy and may inform future desensitisation strategies. These collectively emphasize the emerging applications for AI/ML in understanding the circRNA-CUL3-TKI crosstalk and developing methods to resensitize cancers that are resistant to therapy.
Multiple myeloma (MM) remains largely incurable despite major therapeutic advances, underscoring the need to define novel pathogenic mechanisms and druggable targets. Epitranscriptomic dysregulation, encompassing reversible chemical modifications on RNA, has emerged as a post-transcriptional regulatory layer that may contribute to MM biology. This focused review discusses the emerging roles of major RNA modifications and their regulators in MM pathogenesis, bone disease, drug resistance, and immune escape. We summarize representative experimental and translational studies on RNA-modifying enzymes, non-coding RNAs, and the bone marrow microenvironment, with emphasis on mechanisms directly validated in MM. Evidence derived from AML, solid tumors, or pan-cancer analyses is discussed as hypothesis-generating and requiring MM-specific validation. We summarize MM-supported evidence that m6A demethylases such as FTO and ALKBH5, as well as writers such as METTL3 and NSUN2, may regulate the stability and translation of disease-relevant transcripts. We also discuss emerging cross-cancer data on the m7G writer METTL1 as a hypothesis-generating framework that requires MM-specific validation. We delineate how RNA modification-dependent non-coding RNA networks and extracellular vesicle cargo remodel osteoclast and osteoblast function, linking the epitranscriptome to osteolytic bone disease. We further describe RNA modification-driven drug resistance circuits and immune escape pathways involving FTO, METTL3, H19, MALAT1, YTHDF1, and m5C-defined molecular subtypes. Finally, we summarize current epitranscriptomic therapeutic strategies, including small molecule inhibitors of writers, erasers, and readers, RNA-based therapeutics targeting pathogenic non-coding RNAs, and RNA modification-derived prognostic signatures for risk stratification. Collectively, this review discusses RNA-modification machinery as a potentially actionable regulatory layer in MM and outlines key challenges for clinical translation.
TRPV2 is the least studied member of the vanilloid TRP subfamily despite its emerging relevance in cancer metastasis, pain, and inflammation. Although several small-molecule TRPV2 modulators have been reported, including the natural products piperlongumine (PL) and cannabidiol, all lack selectivity, complicating the interpretation of phenotypic readouts and functional insights into the role of the channel in health and disease. Here, we report a series of PL-based derivatives rationally designed to maintain TRPV2 antagonism while eliminating covalent off-target activity associated with the electrophilic groups present in PL. Using electrophysiological and calcium fluorescence imaging assays in HEK293T cells and DRG nociceptors, we identified HKC54 as the most potent TRPV2 antagonist to date (IC50 = 0.4 μM), displaying ∼50-fold selectivity over TRPV1 and ∼70-fold selectivity over TRPA1. Cellular thermal shift assays demonstrated direct TRPV2 engagement, and molecular dynamics and docking studies suggest a near-identical binding mode of the derivatives to PL. To assess proteome-wide selectivity, we pursued an unbiased chemoproteomic strategy and developed photoaffinity probes derived from PL and noncovalent derivative HKC22. Whereas the PL-based probe labeled many established covalent and noncovalent PL targets (e.g., GSTP1, GSTO1, STAT3, and KEAP1), no off-targets were detected for HKC22, suggesting high selectivity for TRPV2. Finally, PL derivatives inhibited cancer cell migration in vitro and suppressed metastasis in vivo, underscoring the therapeutic potential of selective TRPV2 antagonists.
Tumor-immune interactions in the cancer microenvironment have a major influence on the development of colorectal cancer (CRC), as well as immune evasion by the CRC tumor. Chemokine CXCL9 (which signals through the CXCR3 receptor) has been implicated in the immune recruitment of cells and progression of tumors; however, the role of CXCL9 in CRC is poorly understood. To explore the mechanistic role of CXCL9 in CRC cell proliferation, migration, invasion, survival, and immune modulation, we utilized CRC cell lines HCT116 and HT-29. Knocking down CXCL9 expression using siRNA decreased CRC cell proliferation, EdU incorporation, clonogenic survival, and anchorage-independent growth. The restoration of CXCL9 expression partially reversed these observations. In migration and invasion assays, CXCL9 increased motility and epithelial-mesenchymal transition (EMT) of CRC cells via the regulation of E-cadherin, N-cadherin, and vimentin expression. Silencing CXCL9 expression (due to the CXCL9 siRNA) resulted in increased apoptosis and G0/G1 cell cycle arrest, whereas restoring CXCL9 expression allowed CRC cells to transition to S-phase. Mechanistically, CXCL9 maintained redox homeostasis by preventing accumulation of reactive oxygen species (ROS), and activating pro-survival signaling pathways (AKT, ERK1/2, and NF-κB). Importantly, CXCL9 positively regulated immune checkpoint molecules (PD-L1 and IDO1) at both mRNA and protein levels. In co-culture experiments, CXCL9 promoted CD8+ T-cell chemotaxis through CXCR3 and enhanced T-cell-mediated cytotoxicity, effector molecule expression, and pro-inflammatory cytokine secretion under the tested in vitro conditions. Together, these in vitro findings suggest that CXCL9 contributes to CRC cell proliferation, migration, invasion, redox regulation, immune checkpoint-associated signaling, and CD8+ T cell responses in simplified co-culture models. The novelty of this study lies in the combined evaluation of tumor-cell phenotypes, PD-L1/IDO1-associated signaling, and CD8+ T cell responses within the same CXCL9-modulated CRC cell-line system. Further validation using patient-derived samples, in vivo models, and clinically annotated cohorts is required before broader conclusions regarding the CRC tumor microenvironment or therapeutic targeting can be made.
Molecular glues, compounds that bind cooperatively at protein-protein interfaces (PPIs), are revolutionizing chemical biology and drug discovery, allowing the modulation of traditional "undruggable" targets. Here, we focus on a native regulatory PPI between the scaffolding protein 14-3-3 and C-RAF, a key component of the MAPK signaling pathway. Extensive drug discovery efforts have focused on the MAPK pathway due to its central role in oncology and developmental disorders (RASopathies). However, the modulation of its protein complexes is underexplored. C-RAF activity is regulated on multiple levels including dimerization, phosphorylation, and complex formation with 14-3-3, which prevents C-RAF activation by binding to a C-RAF sequence centered on phospho-serine 259. We used a fragment-merging approach to design molecular glues that bound to the composite surface of this 14-3-3/C-RAFpS259 complex. Molecular glues stabilized the inhibitory complex up to 300-fold; their glue-based mechanism of action was confirmed by crystallography and biophysical studies. Selectivity among the other RAF isoforms and other RAF phosphorylation sites was evaluated. The best compounds showed excellent selectivity among a broad panel of 80 14-3-3 clients. Cellular assays demonstrated on-target engagement, enhanced phosphorylation levels of C-RAFpS259, and reduced levels of RAF dimerization and ERK phosphorylation. Overall, this approach enabled chemical biology studies for a C-RAF site that was intrinsically disordered prior to 14-3-3 binding and had not been targeted previously. These molecular glues will be useful chemical probes and starting points for drug discovery efforts to modulate native PPI stabilization in the MAPK pathway with applications in oncology and RASopathies.
Bacterial cell populations are phenotypically heterogenous, including, e.g., exponentially growing cells, besides nonreplicating persisters that tolerate antibiotics, or so-called "viable-but non-culturable" cells. The lack of biomarkers and the dynamic, reversible nature of these cellular growth phenotypes make it challenging to differentiate them and study their underpinning molecular characteristics. Here, we present step-by-step protocols for our recently developed technique termed "Cellular Phenotypic Profiling and backTracing" which uses index sorting to connect the experimentally determined growth fate of single cells with their phenotypic characterization using fluorescent probes. The method is illustrated using the opportunistic Gram-positive pathogen Staphylococcus aureus grown under dormancy-inducing low pH conditions and a live-dead staining combination. The method is easily adaptable to other species of interest and may accommodate a broad variety of fluorescent probes for cellular phenotypic profiling.
This investigation seeks to examine the relationship between exosomal Insulin-like Growth Factor Binding Protein Acid Labile Subunit (IGFALS) gene expression, immune infiltration, and clinical outcomes in individuals with hepatocellular carcinoma (HCC). Clinical data and IGFALS expression levels were obtained from the TCGA and GEO databases. Immunohistochemistry was performed to confirm IGFALS expression in both HCC and adjacent non-tumor tissues. To validate survival analyses, restricted cubic spline models were used to explore associations between overall survival (OS) and the expression of IGFALS in liver hepatocellular carcinoma (LIHC). Gene set enrichment analysis (GSEA) identified IGFALS-associated pathways, while Gene set enrichment analysis (ssGSEA) evaluated IGFALS-immune cell infiltration correlations. Functional characterization included proliferation, migration/invasion, molecular profiling, and apoptosis assays. Compared to normal tissues, IGFALS expression levels were notably decreased in tumor tissues. A notable link was detected between IGFALS expression and multiple clinical factors, including gender, weight, residual tumor, adjacent hepatic tissue inflammation, vascular invasion, AFP, BCLC, tumor size, multinodular, TACE, and satellite lesion in HCC. Reduced IGFALS expression in HCC was correlated with decreased OS. Moreover, the IGFALS level in malignant tumor cells post-immunotherapy was observed to be markedly higher than that in the pre-treatment phase. A strong association between IGFALS and immune infiltration levels was also established. At the same time, in vitro experiments also verified the function of the IGFALS gene. The exosomal IGFALS gene holds potential as a prospective indicator for assessing the outcome of individuals with HCC.
R-loops are three-stranded nucleic acid structures formed by a DNA-RNA hybrid and a displaced single-stranded DNA. They regulate transcription, replication, and DNA repair, but their dysregulation causes genomic instability and inflammation, contributing to brain diseases. The nervous system exhibits selective vulnerability to R-loop stress due to ultra-long gene transcription, post-mitotic longevity, and high metabolic demands. This review synthesizes current literature from PubMed, Scopus, Web of Science, and Embase (2010-2026) on R-loop biology, with a focus on brain-specific mechanisms, regulatory factors (SETX, ZPR1, METTL3, TDP-43/FUS), and disease models. In neurodegeneration, R-loop accumulation drives repeat expansion disorders (Fragile X, Huntington's disease) and loss-of-function SETX mutations (AOA2), whereas gain-of-function SETX (L389S) causes pathological R-loop depletion in ALS4, disrupting TGF-β signaling. TDP-43/FUS and SMN are integral to R-loop resolution, unifying ALS/FTD and SMA. In brain cancers, METTL3-mediated m6A modification of TERRA stabilizes telomeric R-loops in ALT-positive neuroblastoma, creating a therapeutic vulnerability to METTL3 inhibitors (STM2457, STC-15). Glioma stem cells rely on m6A-modified circPOLR2B to regulate R-loop formation and malignancy. Clinical-stage agents (EP102, TUG1ASO, ATX-559) and R-loop-derived prognostic signatures (RLPI) are emerging, but translation is hindered by a lack of non-invasive biomarkers and the dual physiological/pathological roles of R-loops. R-loops are central to brain disease pathogenesis, offering promising therapeutic targets. Future research should prioritize precision R-loop modulators, non-invasive biomarkers, and combinatorial strategies.
Nucleus pulposus (NP) cell apoptosis and extracellular matrix (ECM) degradation constitute the two major pathological hallmarks of intervertebral disc degeneration (IVDD). Inhibiting these deleterious processes represents an effective strategy for attenuating IVDD progression. Sirtuin 7 (SIRT7), a member of the sirtuin family, plays a critical role in modulating gene expression, mediating cellular stress adaptation, and facilitating DNA repair. While SIRT7 has demonstrated therapeutic potential across diverse pathological contexts, its specific contribution to IVDD pathogenesis remains elusive. This study aimed to delineate the functional contribution of SIRT7 to IVDD progression and unravel its molecular mechanisms. We quantified SIRT7 levels by immunohistochemistry (IHC) in degenerative human and rat NP tissues, and by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in tert-butyl hydroperoxide (TBHP)-treated NP cells. To evaluate the protective capacity of SIRT7 overexpression, we conducted multifaceted analyses encompassing oxidative stress markers, apoptotic indices, ECM turnover, and nuclear factor kappa B (NF-κB) cascade activity in TBHP-challenged NP cells, utilizing reactive oxygen species detection probes, mitochondrial membrane potential indicators, Hoechst 33342 nuclear staining, qRT-PCR, western blotting, and immunofluorescence techniques. Its therapeutic potential was subsequently validated through magnetic resonance imaging and comprehensive histopathological evaluation (hematoxylin and eosin, Safranin O/Fast Green, and Masson trichrome staining) combined with IHC in a rat IVDD puncture model. SIRT7 was consistently downregulated in degenerated human and rat NP tissues as well as TBHP-treated NP cells, concomitant with elevated NF-κB pathway activation. SIRT7 overexpression in TBHP-stimulated NP cells effectively attenuated oxidative stress, apoptosis, and ECM degradation. Mechanistically, SIRT7 overexpression may exert inhibitory effects on NF-κB signaling. Consistently, SIRT7 overexpression in the rat IVDD model decreased NF-κB activity, reduced NP cell apoptosis and ECM depletion, eventually ameliorating disc degeneration. Our findings demonstrate that SIRT7 expression declines progressively during IVDD development. SIRT7 overexpression protects against NP cell apoptosis and ECM degradation, and this protective effect correlates with inhibition of the NF-κB pathway. These findings suggest tha SIRT7 is a guardian of NP homeostasis and highlight its substantial promise as a molecular target for IVDD therapeutics.
Stem cell-derived and plant-derived exosomes are emerging as promising therapeutic agents in cutaneous repair, regeneration, and rejuvenation. They facilitate wound healing and skin revitalization through multifaceted mechanisms, including immunomodulation, promotion of cellular differentiation, and stimulation of angiogenesis. Additionally, their ability to modulate collagen production and remodeling underscores their potential in addressing skin aging and improving cosmetic outcomes. Consequently, exosome-based therapies show promise for a range of conditions, from challenging wounds and skin aging to pigmentary disorders, hair loss, certain immune-mediated dermatoses. To ensure a comprehensive and unbiased synthesis of the current evidence, this systematic review was conducted following a structured methodology, encompassing a search across multiple major databases over a defined 20-year period. This review systematically outlines the roles and applications of commonly employed plant exosomes and stem cell exosomes in recent years' advancements in skin repair and cosmetic dermatology. By synthesizing the current understanding of their mechanisms and clinical potential, this review aims to highlight viable therapeutic strategies that bridge the gap between medical dermatology and aesthetic medicine.
Osteoarthritis (OA) is a degenerative joint disease characterized by progressive destruction of articular cartilage. Isoquercitrin (ISO), a natural flavonoid, has well-documented anti-inflammatory and antioxidant properties. This study investigates the chondroprotective effect of ISO in OA, with a particular emphasis on its regulation of P53-mediated ferroptosis. In tert‑butyl hydroperoxide (TBHP)-induced chondrocytes, ISO treatment effectively ameliorated extracellular matrix (ECM) metabolic imbalance by upregulating the expression of COL2A1 and ACAN, while downregulating MMP13 and ADAMTS5. Concurrently, ISO reduced intracellular levels of reactive oxygen species (ROS), lipid peroxidation, and iron accumulation, as measured by fluorescent probes DCFH-DA, BODIPY 581/591 C11, and FerroOrange, respectively. Furthermore, ISO significantly suppressed P53 protein expression while enhancing the levels of SLC7A11 and GPX4, which were key regulators of ferroptosis resistance. Network pharmacology analysis, molecular docking, and cellular thermal shift assay (CETSA) collectively identified P53 as a direct molecular target of ISO. In vivo, daily administration of ISO markedly attenuated cartilage degeneration and ferroptotic damage in a rat model of OA induced by anterior cruciate ligament transection (ACLT), as confirmed by histopathological evaluation. Notably, these protective effects were partially reversed upon co-treatment with nutlin-3, a pharmacological activator of P53. Collectively, these findings demonstrate that ISO alleviates OA pathology by inhibiting P53-dependent ferroptosis, highlighting its potential as a dietary supplement for the prevention and management of OA.
Primary samples, including freshly isolated cells, tissues, and clinical specimens, preserve native physiological states and microenvironments, making them especially valuable for understanding biological systems in health and disease. Proximity labeling (PL) has emerged as a powerful strategy for interrogating molecular interaction networks in situ, but conventional enzyme-based approaches are often difficult to apply to primary samples due to their reliance on genetic manipulation and sustained exogenous expression. Photocatalytic proximity labeling (PPL) provides a non-genetic alternative in which small-molecule photocatalysts activate labeling probes under light irradiation, enabling temporally gated and spatially localized covalent tagging of proximal biomolecules in a wide range of contexts. This review summarizes recent developments in the application of PPL to primary samples, spanning organelle-resolved proteomics, cell surface protein interaction profiling, cell-cell interaction analysis in tissues and in vivo, and emerging immune-engineering applications. While a growing diversity of photocatalytic systems and probe chemistries has been reported, their evaluation and deployment in primary cells and tissues remain at an early stage, reflecting the heightened demands for efficiency, specificity, and robustness in native biological contexts. We discuss how advances in reaction chemistry, catalyst targeting, and long-wavelength activation are beginning to address these challenges, and we outline key opportunities and limitations for extending PPL toward broader use in primary samples. Continued development of primary-oriented photocatalytic toolkits is expected to facilitate more direct interrogation of native biological systems, providing valuable insights into cellular organization, tissue-level communication, and disease-associated molecular remodeling.