VCP inhibition induces rapid cell death through ER stress driven actin cytoskeletal collapse

VCP inhibition induces rapid cell death through ER stress driven actin cytoskeletal collapse | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article VCP inhibition induces rapid cell death through ER stress driven actin cytoskeletal collapse yao yuan, Jiamin Yao, Shaojuan Song, Liu Liu, Kaichao Wang, Lideng Cao, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9008638/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The endoplasmic reticulum (ER) maintains cellular proteostasis through the unfolded protein response (UPR), whereas excessive or unresolved stress can rapidly compromise cell viability. Beyond the well characterized transcriptional programs, how ER stress acutely remodels cellular architecture to induce cell death remains poorly understood. Here, we report the rational design of a nucleoside-based small molecule inhibitor of the AAA-ATPase VCP (NY-1), which forms a stable host-guest complex with β-cyclodextrin. NY-1 induces robust ER stress and a strikingly rapid form of cell death characterized by actin cytoskeletal collapse and synchronized membrane blebbing. We identify a non-transcriptional signaling pathway in which ER stress activated IRE1α remodels the 14-3-3 interactome, releasing and activating the phosphatase SSH1, thereby promoting cofilin dephosphorylation and F-actin disassembly. Chemical chaperone treatment restores cytoskeletal integrity and cell viability, demonstrating a direct causal link between ER proteostasis disruption and actin network collapse. These findings reveal an acute ER stress signaling axis controlling actin dynamics and establish NY-1 as a chemical probe for dissecting rapid cell death programs. Biological sciences/Cell biology/Cell adhesion Biological sciences/Cancer/Tumour-suppressor proteins Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The endoplasmic reticulum (ER) is a central hub for cellular proteostasis, coordinating protein folding, maturation, and trafficking [ 1 ] . Perturbations that overwhelm ER folding capacity activate the unfolded protein response (UPR), an adaptive signaling network that restores homeostasis by attenuating protein translation, inducing molecular chaperones, and enhancing degradation of misfolded proteins [ 2 ] . When ER stress is severe or unresolved, the UPR transitions from adaption to cell death, classically through PERK-CHOP mediated transcriptional programs or IRE1α dependent activation of pro-apoptotic signaling cascades such as JNK [ 3 – 5 ] . These canonical transcriptional and apoptotic outputs of the UPR have been extensively investigated. However, accumulating evidence indicates that ER stress is not confined to transcriptional and translational reprogramming, but can also elicit rapid and profound changes in cell morphology on a much shorter timescale. Acute perturbation of ER homeostasis by chemical stressors has been reported to induce cell rounding, surface blebbing, and actin cytoskeletal disassembly within minutes to hours, frequently preceding caspase activation or overt plasma membrane rupture [ 6 – 8 ] . These phenomena suggest that the ER communicates with the cytoskeleton not only through long term transcriptional remodeling but also through direct signaling events that can rapidly reshape cell architecture. Beyond its structural role, the actin cytoskeleton functions as a dynamic sensor and integrator of intracellular stress signals, and perturbation of actin dynamics is known to exert a decisive influence on cell fate decisions [ 9 , 10 ] . Despite these insights, whether and how ER proteostasis failure is directly coupled to actin cytoskeletal remodeling remains poorly defined at the molecular level. Valosin-containing protein (VCP/p97), an essential AAA-ATPase that drives ER-associated degradation (ERAD), plays a critical role in maintaining ER proteostasis by extracting misfolded proteins from the ER membrane [ 11 ] . Pharmacological inhibition of VCP rapidly leads to accumulation of unfolded proteins and acute ER stress, making VCP a powerful experimental entry point to interrogate early signaling events downstream of proteostasis collapse [ 12 , 13 ] . Using a newly synthesized small-molecule VCP inhibitor, we unexpectedly observed a rapid disassembly of filamentous actin accompanied by synchronized membrane blebbing and a rapid, non-apoptotic, necrosis-like form of cell death. These findings prompted us to hypothesize that ER stress can directly engage a cytoskeletal effector pathway that determines rapid cell demise. To enhance the bioavailability and cellular delivery of this VCP inhibitor, we further exploited host–guest supramolecular chemistry. Adamantane is a well-established guest for cyclodextrins, and β-cyclodextrin (β-CD) offers favorable aqueous solubility and stimulus-responsive drug release properties [ 14 – 16 ] . By self-assembly of an adamantane-containing VCP inhibitor (NY-1) with β-CD, we constructed a supramolecular nanoparticle (NY-1@β-CD) that markedly enhances cellular uptake and amplifies acute ER stress signaling. Using this system, we demonstrate that ER stress activated IRE1α remodels the 14-3-3 interactome, competitively releasing the actin phosphatase SSH1 from inhibitory sequestration. Activated SSH1 dephosphorylates cofilin, driving rapid F-actin disassembly and catastrophic cytoskeletal collapse. Chemical chaperone treatment restores actin integrity and cell survival, establishing a direct causal link between ER proteostasis failure and cytoskeletal failure. Moreover, NY-1@β-CD exhibits potent antitumor efficacy in vivo, outperforming the clinically used chemotherapeutic 5-fluorouracil. Together, our findings define an IRE1α 14-3-3/SSH1 signaling axis as a previously unrecognized structural effector arm of the ER stress response and reveal how organelle level proteostasis disruption can be translated into rapid mechanical failure of the cell. Materials and Methods Chemicals and reagents Chemicals used were 5-Fluorouracil (5-FU, HY-90006), Narciclasine (HY-16563), BAPTA-AM (HY-100545), VCP activator 1 (VCP-A, HY-157508), Z-VAD-FMK (Z-VAD, HY-16658B), Necrostatin-1 (Nec-1, HY-15760), Chloroquine (CQ, HY-17589A), MCC950 (HY-12815), Necrosulfonamide (NSA, HY-100573), EGTA (HY-D0861), EGTA-AM (HY-D0973), Tauroursodeoxycholate (TUDC, HY-19696), Ryanodine (HY-103306), and Anisomycin (HY-18982) from MedChemExpress (USA). Cell culture The cells Cal-27, A431, UM1, HN12, HN30, and HSC-3 were routinely cultured in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (Sigma, USA) at 37 ℃ in 5% CO 2 incubator. The cells H103 and H314 were cultured in DMEM/F12 (Gibco, USA) supplemented with 10% fetal bovine serum (Sigma, USA) and 0.5 µg/mL hydrocortisone (HY-N0583R, MedChemExpress, USA) at 37 ℃ in 5% CO 2 incubator. Synthesis and characterization of compounds All chemicals and solvents were commercially available laboratory grade and were used without further purification. Thin-layer chromatography (TLC) was performed on TLC aluminum sheet covered with silica gel 60 F254 (0.2 mm, Merck, Germany). Flash column chromatography (FC) was carried out with silica gel 60 (Haiyang chemical company, P. R. China) at 0.4 bar. NMR spectra were recorded on an AV II (Bruker, Germany) spectrometer at 600 MHz for 1H and 150 MHz for 13C. The J values are given in Hz, δ values in ppm are relative to Me4Si as internal standard. For NMR spectra measured in DMSO-d6, the chemical shift of the solvent peak was set to 2.50 ppm for 1H NMR and 39.50 ppm for 13C NMR. 1H-13C correlated (HMBC, HSQC), 1H-1H correlated (COSY, NOESY) NMR spectra were used for the assignment of the 1H/13C signals and identify the structural assignment of isomers. High resolution mass spectra were recorded on a LTQ mass spectrometer (Thermo, USA). Construction of NY-1@β-CD Prepare 50mM of NY-1 with DMSO and 4mg/ml of β-CD (TCI C0777) solution with double distilled water. Slowly add NY-1 to the stirred β-CD solution until the final concentration is 1mM. Stir at room temperature for 1 hour and then dialysis overnight to remove unreacted molecules. The control group was treated with a mixed solution of DMSO/β-CD of the same concentration. FITC-labeled β-CD and co-localization of lysosomal FITC reacted with β-CD in sodium bicarbonate solution and was used for the preparation of NY-1@β-CD nanoparticles after dialysis with ultrafiltration tubes. After treating the cells with fluorescently labeled nanoparticles, lysosomes and nuclei were fluorescently labeled using lysosome tracker Red (Beyotime, C1046) and Hoechst (Beyotime, C1011), and real-time imaging was performed by confocal microscopy. CCK8 assay Cells were seeded in 96-well plates (5 × 10 3 cells per well) and cultured for 24 h. Then cells were treated with varying concentrations (0, 1.25, 2.5, 5, 10 µM or 0, 5, 10, 20, 30, 40, 50, 60, 70, 80 µM) of NY-1、NY-2、NY-3 or 5-FU for 48 h; NY-1(10 µM), Narciclasine (2.5, 5, 10 nM), BAPTA-AM (0.5, 1, 2 µM), VCP-A (5, 10, 20 µM), Z-VAD-FMK (10 µM), Necrostatin-1 (Nec-1, 10 µM), Chloroquine (CQ, 10 µM), MCC950 (10 µM), Necrosulfonamide (NSA, 2.5 µM), and EGTA (500 µM) for 24 h; Add 10 µL CCK8 solution (Biosharp, China) to each well. And after incubating at 37 ℃ for 1 h, the absorbance at a wavelength of 450 nm was measured in triplicate or sextuplicate with multiple biological replicates with Varioskan Flash (Thermo Scientific, USA). Colony-formation assay Cells were seeded in 6-well plates (700 cells per well) and cultured for 24 h. Then cells were treated with NY-1 (2.5, 5 µM) or 5-FU (2.5, 5 µM) for 2 weeks. Change the medium every 3 days. Colonies were fixed with 10% formalin and stained with 1% crystal violet in 10% ethanol. Images were documented and colonies were counted using ImageJ software. Time lapse microscopy observation Cells were seeded in 6-well plates (2 × 105 cells per well). Then cells were treated with NY-1(10 µM) for 41 h. During this process, cells in the same area are photographed every 2–3 minutes to observe the continuous changes of the cells with a time lapse microscopy (Sartorius Incucyte, Germany). Scanning electron microscope Cells were seeded in 24-well plates (2 × 10 4 cells per well) with slides and cultured for 24 h. Then cells were treated with NY-1(10 µM) for 24–48 h. Next, all samples were cleaned with PBS twice and treated with 2.5% Glutaraldehyde EM Grade (Lilai, China) at 4°C overnight. A graded series of alcohol (30%, 50%, 75%, 85%, 95%, and 100%, respectively) was applied for sample dehydration for 15 min each. The dehydrated samples were naturally dried. Cell observations were under a scanning electron microscope (JEOL Ltd., Japan) with exposure parameters of 20 kV. Transmission electron microscope Cells were seeded in 60 mm diameter dish and cultured for 24 h to reach 50%–60% confluence. Then cells were treated with NY-1(10 µM) for 6 h and 24 h. Then cells were collected in a centrifuge tube, the supernatant was discarded, 0.5% glutaraldehyde fixing solution was slowly added along the tube wall with a pipette, and the cells were suspended, left for 5 min at 4℃. Then the cell suspension was transferred to a 1.5 mL apical bottom EP tube. Centrifuge at high speed (12000 rpm, 10 min), gently discard the supernatant, and slowly add 3% glutaraldehyde fixing solution along the tube wall with a straw. The samples were sent to Chengdu Lilai Biotechnology Co., Ltd. (China) to prepare and place on the copper mesh. The image of the copper mesh was collected by a transmission electron microscope (JEM-1400FLASH, JEOL). Staining of cell membrane with red fluorescent dye DiI Cells were seeded in 24-well plates (2 × 10 4 cells per well) and cultured for 24 h. Then cells were treated with NY-1(10 µM) for 24 h. Then, 10 µM DiI (Beyotime C1036, China) was added into the culture medium for 30 min incubation. The cells were observed and photographed under a fluorescence microscope (Thermo, Invitrogen EVOS M5000). F-actin detection assay Cells were seeded in 24-well plates (2 × 10 4 cells per well) with slides and cultured for 24 h. Then cells were treated with NY-1(10 µM) for 0–24 h; NY-1(10 µM) and Narciclasine (5 nM) or BAPTA-AM (1 µM) or VCP-A (10 µM) for 24 h. Cells were fixed with ice-cold methanol and wash with 0.1% Triton X-100 PBS for three times. After 1 h incubation with Actin-Tracker Green-488 (Byotime, China), 5 min incubation with DAPI (Servicebio, China) and three washes with 0.1% Triton X-100 PBS, samples were naturally dried, sealed and imaged with full tissue biopsy scanner (Olympus VS200, Germany). Average fluorescence intensity of F-actin analysis was performed using FlowJo 10.8.1. Immunofluorescence (IF) Cells were seeded in 24-well plates (2 × 10 4 cells per well) with slides and cultured for 24 h. Then cells were treated with NY-1(10 µM) for 24 h. Cells were fixed with ice-cold methanol and blocked with 5% BSA (Biofroxx GmbH, Germany) for 1 h and 0.5% Triton X-100 PBS for 5 min. After overnight incubation with primary antibodies at 4°C and three washes with PBS, samples were incubated with secondary antibodies for 1 h and DAPI for 5 min at room temperature. After three washes with PBS, samples were naturally dried, sealed and imaged with full tissue biopsy scanner (Olympus VS200, Germany). The primary antibodies used were YAP1 antibody (#14074, 1:200) from Cell Signaling Technology (USA); α-tubulin antibody (11224-1-AP, 1:200) from Proteintech (China). The secondary antibody was Donkey anti-Rabbit IgG Alexa Fluor 555 (Invitrogen, A-31572, 1:400). Average fluorescence intensity analysis was performed using FlowJo 10.8.1. Immunoblotting Cells were seeded in 6-well plates (2 × 10 5 cells per well) and cultured for 24 h. Then cells were treated with NY-1(10 µM) for 0–24 h; NY-1(10 µM), Narciclasine (2.5, 5 nM), BAPTA-AM (5 µM), VCP-A (10 µM) for 24 h; siVCP for 48 h. The cell lysates were obtained through RIPA buffer (50 mM Tris base, 1.0 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 1% cocktail) and quantified by Pierce™ BCA (bicinchoninic acid) protein assay kit (Thermo Scientific, USA) as previously described. The samples were processed to 12.5% or 7.5% sodium dodecyl sulfate polyacrylamide hydrogel electrophoresis (SDS-PAGE) (Yamei, China) at 80 V for 30 min and 120 V for 1 h and then transferred onto 0.22 µm polyvinylidene difluoride membrane (PVDF, MA, USA), using a wet transfer method at 300 mA for 75 or 90 min. The membranes were blocked with 5% skim milk in TBST for 1 h at room temperature and then incubated with primary antibodies overnight at 4 ℃. After washing with TBST for 15 min, membranes were incubated with goat anti-mouse/rabbit secondary antibody for 1 h at room temperature. Finally, the results were showed by BM Chemiluminescence Western Blotting kit (Roche, Switzerland). The primary antibodies used were FAK antibody (#3285, 1:1000), phospho-FAK antibody (#8556, 1:1000), YAP1 antibody (#14074, 1:1000), phospho-YAP1 antibody (#13008, 1:1000), Pan-TEAD antibody (#13295, 1:1000), CYR61 antibody (#39382, 1:1000), VCP antibody (#2648, 1:1000), cofilin antibody (#5175, 1:1000), phospho-cofilin antibody (#3313, 1:1000), IRE1α antibody (#3294, 1:1000), SSH1 antibody (13578, 1:1000) and XBP-1s antibody (40435, 1:1000) from Cell Signaling Technology (USA); phospho-IRE1α antibody (#ab124945, 1:1000) firm Abcam;14-3-3 antibody (#14503-1-AP, 1:1000), GAPDH antibody (60004-1-Ig, 1:10000) from Proteintech (China); The Goat anti-mouse/rabbit secondary antibody (ZB-2305 and ZB-2301, 1:1000) was purchased from ZSGB-BIO (China). RNA-seq The RNA-seq and analysis of Cal-27 and A431 cells treated with 10 µM NY-1 for 24 h was conducted. Total RNA was extracted using the Trizol™ reagent (Thermo Scientific, USA) according to the manufacturer’s protocol. The transcriptome sequencing and analysis were conducted by Berry Genomics (Beijing, China). GO enrichment analysis was carried out with topGO (version 3.8) software. KEGG enrichment analysis was carried out with KOBAS (version 3.0) software. siRNA transfection All siRNAs were synthesized by Shanghai GenePharma Co. Ltd. siRNAs were transfected using Lipofectamine RNAiMAX (Invitrogen, USA) according to the manufacturer’s instructions for 48 h. CCK8 assay and F-actin detection assay were executed as described in this article. The siRNAs were as follows: siRPS13: sense (5’ GCUCCUGGCAAGGGUAUUUTT3’), antisense (5’AAAUACCCUUGCCAGGAGCTT3’). siPRDX1: sense (5’ CAUCAAGCCUGAUGUCCAATT3’), antisense (5’UUGGACAUCAGGCUUGAUGTT 3’). siPRDX6: sense (5’ GCUCUGUGGUGCACACUGGGTT3’), antisense (5’ CCCAGUGUGCACCACAGAGCTT3’). siEEF2: sense (5’UCGAUCAUGAUAUUGCCCATT 3’), antisense (5’ UGGGCAAUAUCAUGAUCGATT3’). siVCP: sense (5’CCAACAGACCCAACAGCAUTT 3’), antisense (5’AUGCUGUUGGGUCUGUUGGTT3’). siRAB14: sense (5’ GCACCGUACAACUACUCUUTT3’), antisense (5’ AAGAGUAGUUGUACGGUGCTT3’). siENO1: sense (5’ CCAUGCCAGGGAGAUCUUUTT3’), antisense (5’ AAAGAUCUCCCUGGCAUGGTT3’). siIREα: sense (5’GCGAGAAGCAGCAGACUUUTT3’) antisense (5’AAAGUCUGCUGCUUCUCGCTT3’) siSSH1: sense (5’ GCAGGAAGGAUGCACAUAUT3’) antisense (5’ AUAUGUGCAUCCUUCCUGCTT 3’) Negative control: sense (5’UUCUCCGAACGUGUCACGUTT 3’), antisense (5’ACGUGACACGUUCGGAGAATT 3’). Drug target proteomic quantitative and data-independent acquisition (DIA) mass spectrometry analysis Cal-27 cells were seeded in 10 cm diameter dish and cultured for 24 h to reach 80%–95% confluence. After PBS washing for 3 times, the petri dish carrying cells was transported to Applied Protein Technology for sample processing and data analysis. Protein extraction and peptide enzymolysis: Cells were homogenized with precooled 1×PBS for protein extraction and Pierce™ BCA (bicinchoninic acid) protein assay kit (Thermo Scientific, USA) for protein quantification. The protein was treated with 10 µM NY-1 for 30 min to obtain the sample to be processed. The treated samples were pretreated with protease K (PK enzyme), and 15 µg of protein was taken for SDS-PAGE detection. After PK treatment, the samples were added with denaturant (UA/ DOC) and DTT to reach the final concentration of 20 mM, reacted at 30° for 2 h, and cooled to room temperature. Add appropriate amount of IAA until the final concentration is 25 mM, shake at 600 rpm for 1 min, avoid light at room temperature for 30 min, add appropriate amount of NH 4 HCO 3 buffer (50 mM) to dilute the UA/DOC concentration to less than 1.5 M. Then, 2 µg Trypsin was added into the sample at 37℃ for 16 h. Desalted and lyophilized, redissolved with 0.1% FA. The peptide concentration was determined by OD 280 . 2 µg peptide was extracted, the appropriate amount of iRT standard peptide was added, and DIA mass spectrometry was performed. DIA mass spectrometry analysis: DIA analysis was chromatographically separated using the Vanquish Neo system (Thermo Scientific, USA) with nanoliter flow rate. Samples separated by nanoscale high performance liquid chromatography were analyzed by DIA mass spectrometry using Astral High resolution Mass Spectrometer (Thermo Scientific, USA). Detection mode: positive ion and parent ion scan range is 380–980 m/z, primary mass spectrometry resolution is 240,000 at 200 m/z, Normalized AGC Target is 500%, Maximum IT is 5 ms. MS2 adopts DIA data acquisition mode, 299 scanning Windows are set, Isolation Window is 2 m/z, HCD Collision Energy is 25 ev, Normalized AGC Target is 500%, Maximum IT value is 3 ms. Data analysis: DIA data is processed using Spectronaut software (Spectronaut™ 18.3.230830.50606) and the database is the same as that used for the data construction. The software parameters are set as follows: retention time prediction type is set to dynamic iRT, interference on MS2 level correction is set to enabled, cross run normalization is set to enabled, and all results must pass the Q Value cutoff parameter set to 0.01 (equivalent to FDR < 1%). The target protein set was analyzed through bioinformatic analysis (protein domain analysis, enrichment analysis, and Venn diagram analysis). DIA quantitative proteomic analysis Cal-27 cells were seeded in 10 cm diameter dish and cultured for 24 h to reach 50%–60% confluence. Then cells were treated with NY-1(10 µM) for 24 h. After PBS washing for 3 times, the cells were scraped and transferred to 1.5 mL EP tube. The tubes were transported to Applied Protein Technology for sample processing and data analysis. Protein extraction and peptide enzymolysis: Appropriate amount of SDT (4% SDS, 100 mM Tris-HCl, pH7.6) was added to each sample to extract protein, and the protein was quantified by BCA method. Take 15 µg protein from each sample and add an appropriate amount of 5X loading buffer, and bathe in boiling water for 5 min. SDS-PAGE electrophoresis (4%-20% prefabricated gradient adhesive, constant pressure 180 V, 45 min) and Coomassie bright blue R-250 staining were performed. Appropriate amount of protein was taken from all samples and mixed into Pool samples to be used as QC samples. All samples, including mixed Pool samples, were digested by trypsin using Filter aided proteome preparation (FASP). The peptide of the enzymolysis sample was dialyzed using C18 Cartridge, lyophilized and redissolved in 40 µL 0.1% formic acid solution. The peptide concentration of the sample was determined with OD 280 . An appropriate amount of iRT standard peptide was added to the enzymolysis peptide of each sample, and DIA mass spectrometry was performed by Astral High resolution Mass Spectrometer (Thermo Scientific, USA). DIA mass spectrometry analysis: same as Drug target proteomic quantitative and data-independent acquisition (DIA) mass spectrometry analysis. Data analysis: DIA data was processed by DIA-NN software. The software parameters were set as follows: the enzyme was trypsin, the max, miss, and cleavage site was 1, the fixed modification was Carbamidomethyl (C), and the dynamic modification was set to Oxidation(M) and Acetyl (Protein N-term). The protein identified by the database search must pass the set filtering parameter FDR < 1%. The protein set was analyzed through bioinformatic analysis (protein domain analysis, enrichment analysis, and KEGG analysis). Animals 4-week-old female BALB/c athymic nude mice were purchased from GemPharmatech LLC (China). All experiments were conducted in accordance with the guidelines outlined in the “Principles of Laboratory Animal Care” (NIH) and were approved by Medical Ethics Committee of West China Stomatology Hospital, Sichuan University. The animals had free access to sterilized water and food in a temperature-controlled room (22 ± 1 ℃) with a 12 h light/dark cycle in an SPF environment. They were fed adaptively for one week in this circumstance before the experiments. Cell line-derived xenograft (CDX) tumor assay The 4-week-old female BALB/c athymic nude mice were injected with 100 µL Cal-27 or A431 cell suspension (2×10 6 cells/mL) into the right flank. The weight of the mice and the size of the xenograft tumor were measured every other day. The calculation formula for the volume of the transplanted tumor was TV = π/6×length×(width) 2 . When the size of the xenograft tumor was close to 100 mm 3 , the mice were randomly grouped. Mice were intraperitoneally administered 100 µL 5-FU (15 mg/kg) or 100 µL NY-1 (5, 15 mg/kg) with equal volume of corn oil as the control once every 4 days. At the end of the experiment, all mice were sacrificed by cervical dislocation. The tumor tissues and organs were extracted from mice for histological analysis at the endpoint of the therapeutic study. Patient-derived xenograft (PDX) tumor assay The HNSCC PDX model was established by directly implanting fragments from patient’s HNSCC tissue into 4-week-old female BALB/c athymic nude mice. Briefly, surgically resected human HNSCC tissues were obtained and sliced into small fragments (~ 2 mm in size) to implant subcutaneously into the right flanks of the mice as the first generation of tumor (F1). After successfully establishing xenografts, they are extracted from donor mice and reimplanted into other recipient mice for further in vivo passaging. Following that, each generation was marked as second (F2), third (F3), and n generation (Fn). When the tumor volume reached approximately ~ 100 mm 3 , we started therapy same as CDX with the F3 generation of the PDX model. At the end of the experiment, all mice were sacrificed by cervical dislocation. The tumor tissues and organs were extracted from mice for histological analysis at the endpoint of the therapeutic study. Hematoxylin and eosin (H&E) staining The collected fresh tumor tissues, main organs (heart, liver, spleen, lung and kidney) and skin were fixed in 4% (W/V) paraformaldehyde solution for 12 h. After dehydration and paraffin embedding, the tissues were cut into 4 mm thick slices with a microtome (Leica, Germany) and placed on a glass slide. Then, the tissues were dewaxed by xylene for 20 min, dehydrated by gradient alcohol (100%~5 min, 100%~5 min, 95%~5 min, 80%~2 min, 70%~2 min), dyed with hematoxylin for 30 s–1 min, and washed with running water for 15 min. After that, they were dyed with eosin for 30 s–1 min, dehydrated with gradient alcohol (80%~2 min, 95%~2 min, 100%~5 min, 100%~5 min), and transparent with xylene for 20 min. After sealing with neutral resin, the tissues were observed with a full tissue biopsy scanner (Olympus VS200, Germany). Immunohistochemical (IHC) analysis After fixed, dehydrated, embedded, and sectioned, the tissues were deparaffinized with xylene for 20 min and dehydrated with gradient alcohol (100%~5 min, 100%~5 min, 95%~5 min, 80%~2 min, 70%~2 min). Then, the tissues were subjected to antigen retrieval, hydrogen peroxide and serum blocking for 30 min, and incubated with the primary antibody overnight at 4 ℃, washed with PBS, then incubated with the secondary antibody for 1 h at room temperature. After washing with PBS, DAB chromogenic solution (ZSGB-BIO, China) was used for tissue color development. Next, stain the tissues with hematoxylin for 30 s–1 min and rinse with running water for 15 min. After dehydration with gradient alcohol (80%~2 min, 95%~2 min, 100%~5 min, 100%~5 min), transparent with xylene for 20 min, and sealing with neutral resin, the tissues were observed with a full tissue biopsy scanner (Olympus VS200, Germany). The arithmetical mean proportion in the 6–8 regions of Ki67/CYR61-positive cells were counted by ImageJ software. The primary antibodies used were CYR61 antibody (#39382, 1:200) from Cell Signaling Technology (USA) and Ki67 antibody (ab15580, 1:100) from Abcam (UK). Goat anti-rabbit secondary antibody (ZB-2305 and PV-9001, 100 µL) was purchased from ZSGB-BIO (China). Statistical analysis Experiments were performed in triplicates, or otherwise as indicated. The data were shown as mean value ± SD (standard deviation) or mean value ± SEM (standard error of the mean). Statistical differences among different groups were assessed by one/two way ANOVA and Mann-Whitney test using the GraphPad Prism 9.5 software, which were considered statistically significant when p 0.05 was represented as “ns” meaning no significance. Results 1. Construction of NY-1@β-CD supramolecular nanoparticles induced rapid tumor cell death To synthesis the new molecular with satisfied anti-cancer efficiency, we conducted the process commenced with the preparation of compound 3 (Fig. 1 A, see the Supporting Information for the synthetic procedure). Considering that nucleobases have multiple hydrogen bond donors and acceptors, incorporating them into ligand molecules can potentially serve as binding sites, resulting in better affinity with proteins. Among them, uracil as a target for nucleophilic and electrophilic reagents shows interesting reactivity and its derivatives exhibit extremely diverse physiological activities. Therefore, 5-Fluorouracil (5-FU), which is widely used in oncology as an important anticancer agent, was reasonably selected the donor molecule to undergo Michael addition with compound 3 . The yield of final products including N1, N3 -di-adduct (NY-1), N1 -mono-adduct (NY-2) and N3 -mono-adduct (NY-3) (Fig. 1 A). All synthesized compounds were characterized by 1 H and 13 C NMR spectra, as well as high resolution mass spectra. 1 H- 13 C correlated (HMBC, HSQC), 1 H- 1 H correlated (COSY, NOESY) NMR spectra were used for the assignment of the 1 H and 13 C signals, identify the structural assignment of isomers. The 2D NMR data gave compelling evidence that compound NY-2 is the N1 -mono-adduct and compound NY-2 is N3 -mono-adduct. For details, see the Supporting Information for the synthetic procedure. For spectra, see the Supporting Information Fig. S1 A-Fig. S1 R . We firstly tested how three compounds (NY-1, NY-2, and NY-3) (Fig. 1 B) with an increased dose gradient inhibit the viability of eight squamous cell carcinoma (SCC) cell lines (cutaneous squamous cell carcinoma (cSCC) cell line A431, head and neck squamous cell carcinoma (HNSCC) cell lines Cal-27, UM1, HN12, HN30, HSC-3, H103, and H314). After treatment for 48 h, the cell viability was detected by CCK8 assay. As the dose increased, NY-1 demonstrated much higher inhibition rate of SCC cell viability compared to NY-2 and NY-3. More importantly, the NY-1 showed better anti-cancer effects compared with the donor molecules 5-FU (Fig. 1 C). The clone formation experiments further proved the significant anti-tumor efficacy of NY-1 ( Fig. 1 D and 1 E ) . To achieve rapid uptake of NY-1 by tumor cells and enhance the anti-tumor effect, we chose pH-response molecular β-cyclodextrin (β-CD) ( Fig. 1 F ) . β-CD and NY-1 were dissolved in the aqueous phase and the organic phase respectively, relying on host-guest recognition they can self-assemble after rapid mixing. The NY-1@β-CD nanoparticle exhibited uniformly dispersed spherical structure with a hydrated particle size of 170.08nm and PDI of 0.109, indicating the good dispersibility and stability of NY-1@β-CD(Fig. 1 G and 1 H). Energy dispersive spectroscopy (EDS) results confirmed the self-assemble of two components ( Fig. S1 S) . The zeta potential is -18mV, further indicating the satisfying stability (Fig. 1 I). After reacting with β-CD, the multiple NMR H 1 of NY-1 were significantly shifted, further indicating the effectively encapsulated of β-CD through host-guest recognition (Fig. 1 J ) . To ensure that NY-1@β-CD can be rapidly taken by tumor cells we first conducted cell TEM imaging after 3h treatment. We clearly observed that a monolayer membrane like endosome containing a large number of nanoparticles within the tumor cells (Fig. 1 K ) . To further illustrate the escape of NY-1@β-CD from endosomes and the release of NY-1, we labeled β-CD with FITC and conducted lysosomal fluorescence co-localization detection. The NY-1@β-CD showed significant co-localization with lysosome tracker at 1 hours, which was consistent with the TEM results. After 3 hours, their fluorescence signals separated, indicating successful escape from the endosome (Fig. 1 L ) . The cell live and dead staining and CCK-8 results further demonstrated the rapid cell death by NY-1@β-CD (Fig. 1 M and Fig. S1 T) . As expected, the NY-1@β-CD demonstrated better anti-tumor effect than NY-1 (Fig. 1 N ) . To identify the type of rapid cell death induced by NY-1@β-CD, multiple cell death inhibitors including apoptosis inhibitor Z-VAD-FMK (Z-VAD), necroptosis inhibitors Necrostatin-1 (Nec-1) and Necrosulfonamide (NSA), autophagy inhibitor Chloroquine (CQ), Ferroptosis inhibitor Ferrostatin-1 (Fer-1), and pyroptosis inhibitor MCC950 were employed. The decrease in cell viability induced by NY-1@β-CD could be restored by 10–20% with Z-VAD-FMK in both Cal-27 ( p < 0.001) and A431 ( p < 0.01) cells, suggesting that NY-1 could induce apoptosis in some cells (Fig. 1 O and Fig. S1 U) . MCC950 only restored about 10% of cell vitality in A431 cells ( p < 0.05), while Nec-1, NSA, CQ, and Fer-1, had no recovery ability on NY-1 treated cells (Fig. 1 O and Fig. S1 V) . Given that only a minor fraction of NY-1@β-CD treated cells underwent apoptosis, and that inhibitors of multiple canonical cell death pathways provided only limited protection (Fig. S1 D), these data suggest that NY-1@β-CD induces a rapid cytotoxic response that is largely independent of classical programmed cell death pathways. We therefore sought to define the mode and molecular mechanism underlying this unusually rapid form of cell death triggered by the supramolecular nanoparticle NY-1@β-CD. 2. NY-1 induces blebbing and actin depolymerization associated cell death To explore how cell death occurs, NY-1@β-CD treated Cal-27 cells were observed with a time lapse microscope. The progression of NY-1@β-CD-induced cell death can be classified into four stages (Fig. 2 A). The first stage of cell morphology change was that cell attachment lost and cell contracted tending to be round after NY-1 treatment. The cell membrane showed a subsequent change to form multiple small membrane blebbing (white arrow, < 5 µm) in the second stage. As the cell shrunk further, large membrane blebbing whose volume can even reach or exceed the size of the cell itself formed (red arrow, 10–30 µm) during the third stage. Eventually, the blebbing disappeared and the cells died in the fourth stage. More importantly, the live cell imaging showed that the blebbing occurred at 8h for NY-1 treatment, while occurring at 2h for NY-1@β-CD ( Fig. S2 A ), further illustrating the positive regulatory effect of nanoparticles on the of NY-1 anti-cancer ability. Moreover, in order to further observe the morphological changes of the cells during cell death, cells treated with NY-1@β-CD were observed by scanning electron microscopy (SEM). The presence of cell attachment losing and cell shrinkage (blue arrow), small membrane blebbing (white arrow), large membrane blebbing (red arrow) and membrane pores (green arrow, < 8 µm) were observed (Fig. 2 B). Multiple pores of different diameters in the cell membrane mean that the integrity of the cell membrane was destroyed. These pores may be traces of cell membrane damage left after the formation of small and large membrane blebbing. The SEM results further confirmed the time lapse microscope results. Blebbing occurs after the attachment losing and cell contraction, suggesting that it may be due to the great pressure inside the cells caused by volume reduction, and the cell membrane is then squeezed and blebbed. To further clarify the origin of bubbles, the cell membrane was labeled with red dye DiI. We can obviously observe that blebbing membrane and cell membrane showed red fluorescence, indicating that they are with the same composition (Fig. 2 C, Video 1 ). Since some research conducted that the blebbing is associated with actin interference, we further conducted transmission electron microscopy (TEM) to clarify the microscopic changes of cells under the treatment of NY-1@β-CD. Just as we expected, significant cytoskeleton damage (marked by red dotted lines) was observed in the TEM analysis (Fig. 2 D ) . The proportion of cells exhibiting such damage in various regions, as observed through TEM, was calculated. Remarkably, cytoskeleton damage was identified in 51.39% of the cells ( p < 0.01). This finding suggests that cytoskeleton damage may play a crucial role in cell death. The primary components of the cytoskeleton include microfilaments and microtubules. Immunofluorescence assay was conducted to characterize and quantify microtubules (α-tubulin) in NY-1@β-CD treated Cal-27 cells, while no significant decrease of α-tubulin compared with control group ( Fig. S2 C ). In contrast, time lapse microscope exhibited the fracture of microfilaments (F-actin) after NY-1@β-CD treatment (Fig. 2 E). More importantly, the co-localization of the cell membrane with F-actin further indicated that blebbing is associated with actin interference (Fig. 2 F). Meanwhile, alterations F-actin in NY-1@β-CD treated cells were significant decreased just after 3h treatment ( Fig. 2 G and Fig. 2 H, Fig. S2 C and Fig. S2 D) . The same results also exhibited in WB results, as the downregulation of p-cofilin after NY-1@β-C treatment ( Fig. S2 E ). These findings suggest that NY-1 treatment may induce cytoskeleton damage, characterized by a depolymerization in F-actin filaments. The reduction of F-actin filaments caused by NY-1@β-CD treatment was significantly rescued by Narciclasine (Ncls) treatment, which induces F-actin polymerization through cofilin phosphorylation via Rho kinase signal pathway ( Fig. 2 I and Fig. 2 J, Fig. S2 F and Fig. S2 G) . More significantly, inhibition of cell viability induced by NY-1@β-CD treatment was significantly rescued by Ncls, indicating F-actin depolymerization is a key event contributing to cell death ( Fig. S2 H to 2J ). Above all, we demonstrated that NY-1@β-CD nanoparticles cause rapid cell death characterized by F-actin depolymerization by rapidly releasing NY-1 after 6h treatment. Thus, the direct target of NY-1 and the depolymerization mechanism of F-actin need further exploration. 3. NY-1 targets VCP to triggers F-actin depolymerization and cell death In order to clarify the direct target of NY-1, we firstly conducted drug target proteomic quantitative analysis and DIA mass spectrometry based on the limited trypsin digestion method were conducted on Cal-27 cells (Fig. 3 A). Proteins in the NY-1-treated groups (4918, 4913, and 4920) and control groups (4918, 4916, and 4909) corresponding to peptides in the NY-1-treated groups (73030, 73025, and 73033) and control groups (73032, 73020, and 73022) were identified. A p-value < 0.05 and a fold change (FC) 1.5 were established as the thresholds for significantly differential peptides. In comparison to the control group, a total of 7995 differentially expressed peptides were identified in Cal-27 cells treated with NY-1, comprising 2267 down-regulated peptides and 5728 up-regulated peptides. The underlying principle of the limited trypsin digestion method for target identification is predicated on the chemical's binding to target proteins, which subsequently obstructs the trypsin digestion sites on these proteins (Fig. 3 A ) . Consequently, this results in a simultaneous increase in the abundance of longer peptides (trypsin digestion) and a decrease in the abundance of the corresponding shorter peptides (semi-trypsin digestion) of the target proteins. Additionally, seven overlapping differentially expressed targets between trypsin and semi-trypsin digestion were identified using a threshold of p-value 1 (RPS13, ribosomal protein S13; PRDX1, peroxiredoxin-1; PRDX6, peroxiredoxin-6; EEF2, eukaryotic translation elongation factor 2; VCP, valosin-containing protein; RAB14, Ras related protein Rab-14; ENO1, enolase1) (Fig. 3 B). To identify the most promising target of NY-1, siRNAs targeting the overlapped seven genes- RPS13 , PRDX1 , PRDX6 , EEF2 , VCP , RAB14 , and ENO1 , were utilized to individually knock down the expression of seven proteins. Initially, alterations in cell death were examined following the knockdown of seven proteins. Most candidates resulted in a significant inhibition of cell viability in both cell lines, with VCP knockdown showing the greatest reduction in cell viability (Fig. 3 C). Moreover, we found a VCP activator, the NY-1-induced cell death was rescued by VCP-A in both Cal-27 and A431 cells (Fig. 3 D and Fig.S3A ). To further clarify that VCP is the direct target of NY-1, we further purified the VCP protein and conducted biolayer interferometry (BLI) experiments ( Fig.S3B ). The results showed that NY-1 exhibited excellent binding ability to the VCP protein, with a dissociation constant of 6.33*e −6 M (Fig.S3C) . Cellular thermal shift assay (CETSA) results further confirmed its target effect, as thermal stability of VCP protein enhanced after treatment with NY-1 ( Fig. 3 E and 3 F ) . These results strongly demonstrated that VCP is a direct target of NY-1 and is associated with the cytotoxic effects. After knocked down VCP, we observed the depolymerization of F-actin, which was similar to the NY-1@β-CD treatment, further illustrating the regulatory relationship between VCP and F-actin ( Fig. 3 G and Fig.S3C) . The data independent acquisition (DIA) quantitative proteomic analysis was conducted and monitored 2146 differentially expressed proteins were identified in Cal-27 cells treated with NY-1, comprising 811 down-regulated proteins and 1335 up-regulated proteins (Fig. 3 H). Protein domain analysis demonstrated that the differentially expressed proteins were primarily linked to cytoskeletal dynamics and cell adhesion which act in cell attachment (Fig. 3 I). Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the downregulated proteins revealed a significant enrichment in focal adhesion and cell adhesion signaling pathways which contribute to cell attachment (Fig. 3 J). Additionally, Gene Set Enrichment Analysis (GSEA) of the KEGG pathway enrichment at the RNA level between NY-1-treated Cal-27 and A431 cells identified significantly enriched pathways, with a notable downregulation observed in the F-actin FAK associated signaling (YAP1 signaling) ( Fig. S3D ). Knocking down VCP or directly targeting and inhibiting VCP with NY-1@β-CD can both achieve the inhibition of FAK, YAP and its downstream CYR61 in both Cal-27 and A431 cell lines ( Fig. 3 K-N ) . The nuclear locus of YAP was also significantly suppressed (Fig.S3E and Fig.S3F) . More importantly, the Ncls can also rescue the downregulation of pFAK, YAP, and CYR61, further demonstrated that NY-1@β-CD induces F-actin depolymerization related cell death by cell adhesion ( Fig. 3 O and Fig. 3 P ) . The above results confirmed that NY-1@β-CD can be rapidly taken up by cancer cells and release NY-1. NY-1 can target VCP directly to induce F-actin depolymerization and thus causes cells lose adhesion and death. The synergistic effect of nanoparticles and NY-1 can cause these events to occur rapidly within 6 hours to induce rapid cell death. Researches conducted that the activity of VCP protein is closely related to maintaining protein homeostasis, we further clarified the biological effects of VCP and its regulation on the depolymerization of F-actin. 4. NY-1 targets VCP and induces F-actin depolymerization through acute ER stress In order to clarify the changes of signal pathway caused by NY-1@β-CD, transcriptome sequencing and analysis (RNA-seq) was conducted on Cal-27 and A431 cells. A threshold of p-value 1 was employed to identify significantly differentially expressed genes (DEGs). Compared to the control group, 3099 and 3162 DEGs were identified in NY-1-treated Cal-27 and A431 cells, respectively (Fig. 4 A). Among these, 1838 genes were down-regulated and 1261 genes were up-regulated in Cal-27 cells, while 1523 genes were down-regulated and 1639 genes were up-regulated in A431 cells (Fig. 4 A). Subsequently, Venn diagrams were constructed to illustrate the overlap of up- and down-regulated DEGs between Cal-27 and A431 cells, using a threshold of q value 1. The analysis identified 894 co-up-regulated and 870 co-down-regulated genes common to both Cal-27 and A431 cells (Fig. 4 B). The substantial overlap of DEGs indicates a consistent pattern of RNA changes induced by NY-1 across different SCC cell lines. Gene Ontology (GO) enrichment analysis indicated that the up-regulated genes with overlapping expression were predominantly involved in the biological processes related to the protein folding and Endoplasmic Reticulum (ER) stress (Fig. 4 C). TEM detected that the significantly expanded of ER lumen after NY-1@β-CD treatment, further verifying the sequencing results (Fig. 4 D). To determine whether ER stress is causally linked to NY-1@β-CD induced cytotoxicity, cells were co-treated with the chemical chaperone tauroursodeoxycholic acid (TUDCA). ER stress alleviation by TUDCA significantly rescued cell viability and reduced LDH release, indicating that ER stress is a critical upstream event mediating NY-1@β-CD induced cell death ( Fig. 4 E and 4 F ) . Importantly, the extensive depolymerization of F-actin observed upon NY-1@β-CD treatment was effectively reversed by TUDCA, as demonstrated by both immunofluorescence staining and immunoblot analysis ( Fig. 4 G and 4 H ) . This ER stress–dependent regulation of actin integrity was further validated in fibroblasts, indicating that the phenomenon is not restricted to cancer cells (Fig. S4A) . Given the central role of actin dynamics in the observed phenotype, we next investigated regulators of actin filament turnover. Slingshot homolog 1 (SSH1), a phosphatase that activates cofilin by dephosphorylation and promotes F-actin disassembly, was markedly upregulated following NY-1@β-CD induced ER stress ( Fig. 4 I ) . Attenuation of ER stress by TUDCA suppressed SSH1 upregulation, suggesting that SSH1 activation is downstream of ER stress (Fig. S4B) . Functionally, knockdown of SSH1 significantly rescued F-actin depolymerization induced by NY-1@β-CD ( Fig. 4 J ) , establishing SSH1 as a necessary mediator of actin collapse. Among the three canonical UPR sensors, inhibition of IRE1α phosphorylation effectively suppressed SSH1 upregulation and restored F-actin organization ( Fig. 4 K and Fig. S4C) , implicating the IRE1α branch of the ER stress response in this process. Mechanistically, previous studies have shown that SSH1 activity is restrained through binding to the scaffolding protein 14-3-3, which maintains cofilin in its phosphorylated, inactive state. Co-immunoprecipitation assays revealed that NY-1@β-CD markedly disrupted the interaction between 14-3-3 and SSH1, whereas TUDCA treatment restored this association ( Fig. 4 L and 4 M ). These findings indicate that ER stress induced by NY-1 remodels the 14-3-3 interactome, thereby releasing SSH1 from inhibitory binding and enabling cofilin activation. Collectively, these data demonstrate that NY-1@β-CD induces acute ER stress through VCP inhibition, which activates the IRE1α pathway and reshapes 14-3-3 dependent protein interactions. This remodeling releases and activates SSH1, leading to cofilin-mediated F-actin depolymerization and rapid cytoskeletal collapse, ultimately driving cell death ( Fig. 4 N ) . 5. NY-1@β-CD induced rapid death exhibited promising in vivo anti-tumor activity Next, the significance of NY-1@β-CD for SCC treatment was explored in vivo . Since previous literature reports indicate that intraperitoneal administration of nanoparticles can achieve a greater tumor accumulation by avoiding the reticuloendothelial system cleaning compared with intravenous administration [ 17 ] , and in order to compare the therapeutic efficacy with the FDA approved drug 5-FU with intraperitoneal administration, we chose to inject the NY-1@β-CD intraperitoneally. We constructed cell line-derived xenograft (CDX) models by subcutaneously injecting Cal-27 (HNSCC cell line) and A431 (cSCC cell line) into BALB/c nude mice. Results showed that NY-1@β-CD (15 mg/kg/4 day i.p) significantly inhibited the progression in both xenografts in vivo (Cal-27, p < 0.01; A431, p < 0.0001), while no obvious tumor volume changes were observed in the 5-FU group at the same condition (Cal-27, ns; A431, ns) ( Fig. 5AB ). At the end of the experiment, tumors in the NY-1@β-CD group appeared visibly reduced ( Fig. S5AB ). To further evaluate the inhibition on CDX tumors, histological evaluation and Ki67 immunohistochemical (IHC) staining were performed (Fig. 5 C). Revealing significantly lower Ki67 positive rate in the NY-1@β-CD group compared to the control group (Cal-27, p < 0.0001; A431, p < 0.0001), not in the 5-FU group (Cal-27, ns; A431, ns) ( Fig. 5DE ). At the end of the experiment, no significant weight loss was observed ( Fig. S5CD ). Histological evaluation following NY-1@β-CD treatment showed minimal changes in the major organs (heart, kidney, liver, lung and spleen) ( Fig. S5EF ). These findings suggest that NY-1@β-CD demonstrates antitumor potential with minimal systemic toxicity in vivo in SCC CDX models. Meanwhile, we constructed HNSCC patient-derived xenograft (PDX) models which originated from different localization, PDX1 (derived from tongue abdominal and oral floor squamous cell carcinoma tissue) and PDX2 (derived from cheek squamous cell carcinoma tissue), respectively. The growth curve of the transplanted tumors showed that NY-1@β-CD treatment (15 mg/kg/4 day i.p) resulted in decreased tumor growth (PDX1, p < 0.01; PDX2, p < 0.001) ( Fig. 5FG ). At the end of the study, tumors in the NY-1@β-CD treatment group appeared visually smaller compared to those in the control group ( Fig. S5GH ). Ki67 immunohistochemical staining was performed to evaluate tumor proliferation ( Fig. 5 H ) , revealing a significantly lower Ki67-positive rate in the NY-1@β-CD treatment group (PDX1, p < 0.0001; PDX2, p < 0.0001) ( Fig. 5IJ ). Additionally, no changes in body weight were detected at the end of the experiment ( Fig. S5IJ ). Furthermore, no significant pathological alterations were observed in the organs (heart, kidney, liver, lung, and spleen) of NY-1@β-CD treated mice in both PDX models ( Fig. S5KL ). Overall, the results indicate that rapid and VCP inhibition targeted by NY-1@β-CD exhibits robust antitumor efficacy with minimal systemic toxicity in vivo within HNSCC PDX models. The regulatory effects of NY-1 treatment on the F-actin-FAK-YAP1 downstream pathway in vivo were further investigated. Immunohistochemical staining for CYR61 was conducted on tumor tissues obtained from CDX models (Fig. 5 K). The NY-1@β-CD treatment group exhibited significant inhibition of CYR61 expression in Cal-27 models ( p < 0.01) and A431 models ( p < 0.001) (Fig. 5 L). Additionally, CYR61 positive rates were assessed by immunohistochemical staining in PDX models (Fig. 5 M). Compared to the control group, the CYR61 positive rates were significantly reduced in both PDX1 ( p < 0.001) and PDX2 models ( p < 0.01) (Fig. 5 N). These findings suggest that the F-actin-FAK-YAP1 pathway is modulated following VCP inhibition and calcium dyshomeostasis in response to NY-1@β-CD treatment in vivo . Discussion ER stress regulation is an important mechanism in tumor treatment. The development of new drugs and new materials provides new strategies for regulating ER stress. In this study, we synthesized a potent anti-tumor small molecule NY-1, which is constructed with uracil and Ad through Michael addition reaction. Based on host-guest recognition, β-CD was introduced to construct PH-responsive supramolecular nanoparticles NY-1@β-CD. This supramolecular nanoparticle can be rapidly taken up by tumor cells and achieve lysosomal escape to release NY-1 within 3 hours, and rapidly induce tumor cells death which is characterized by F-actin depolymerization within 6 hours. Further mechanism studies revealed that NY-1 mainly can target VCP proteins and induce ER stress, the activation of IRE1α through ER stress can lead to the dissociation of SSH1 and 14-3-3. The dissociation of SSH1-14-3-3 complex further causing the cofilin mediated F-actin depolymerization. Further in vivo studies demonstrated that intraperitoneal administration of NY-1@β-CD showed significant efficacy in a variety of CDX and PDX models, and its effect was more significantly superior than the FDA-approved anti-tumor drug 5-FU. In conclusion, this supramolecular nanoparticles based on stress-effector targeted molecules provides a solution for future anti-tumor treatment. Declarations Acknowledgments The authors gratefully acknowledge support from the National Natural Science Foundation of China (NSFC) (Grant Nos. 82470984, 82271035, U25A6003, 82330029, 32200577), National Key Research and Development Program of China (Grant Nos. 2022YFC2402901), for financial support. All animal experiments are conducted in accordance with the guidelines outlined in the “Principles of Laboratory Animal Care” and were approved by Medical Ethics Committee of West China Stomatology Hospital, Sichuan University. Conflict of Interest Authors declare that they have no competing interests. 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Supramolecular Host-Guest Assemblies for Tunable and Modular Lysosome-Targeting Protein Degradation. ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 2025,64(33): Davis M E and M E Brewster. Cyclodextrin-based pharmaceutics: past, present and future. Nat Rev Drug Discov, 2004,3(12): 1023-35. Colby A H, J Kirsch, A N Patwa, et al. Radiolabeled Biodistribution of Expansile Nanoparticles: Intraperitoneal Administration Results in Tumor Specific Accumulation. ACS NANO, 2023,17(3): 2212-2221. Additional Declarations There is no duality of interest Supplementary Files songetalsupplementary.docx supplementary information Fulllengthwesternblots.pdf Full length western blot Video1.avi The video of the origin of bubbles Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9008638","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":600099151,"identity":"acf1958f-0ab1-40a0-8549-e02b9583fbe5","order_by":0,"name":"yao yuan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYBCDBDb2xsYHH0jTwnO42XAGSVoYJNLbpDmIUWpwI/nZY54amzw+yYcN0gwMdnK6DQS1pJkb8xxLK2aTTmwwLmBINjY7QFBLgpk0D9vhxDagluQZDAcStxHWkv5NmucfUIvkwYbDPMRpyTGT5m0DapFgbGwmSovkmTdlknP70hLbeBKbGWcYEOEXvuPp2yTefLNJnN9+/PmPDxV2cgS1KAAVMPEg3ElAOQjINzAwMP4gQuEoGAWjYBSMYAAAUspEqjlfByEAAAAASUVORK5CYII=","orcid":"","institution":"State Key Laboratory of Oral Diseases","correspondingAuthor":true,"prefix":"","firstName":"yao","middleName":"","lastName":"yuan","suffix":""},{"id":600099152,"identity":"42b8856e-3cb5-4c67-a066-51d90d63c9b9","order_by":1,"name":"Jiamin Yao","email":"","orcid":"","institution":"Sichuan university","correspondingAuthor":false,"prefix":"","firstName":"Jiamin","middleName":"","lastName":"Yao","suffix":""},{"id":600099153,"identity":"4b8c955f-bbf6-45b8-8eec-1b2263dad6ec","order_by":2,"name":"Shaojuan Song","email":"","orcid":"","institution":"Sichuan university","correspondingAuthor":false,"prefix":"","firstName":"Shaojuan","middleName":"","lastName":"Song","suffix":""},{"id":600099154,"identity":"3214985c-1b2b-4423-9eec-a061399b6218","order_by":3,"name":"Liu Liu","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Liu","middleName":"","lastName":"Liu","suffix":""},{"id":600099155,"identity":"a68f3be2-5dc7-4bd2-afe6-7f7226550bbb","order_by":4,"name":"Kaichao Wang","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Kaichao","middleName":"","lastName":"Wang","suffix":""},{"id":600099156,"identity":"bb88beb7-99c6-408f-8635-b1023fb32e1c","order_by":5,"name":"Lideng Cao","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Lideng","middleName":"","lastName":"Cao","suffix":""},{"id":600099157,"identity":"afad4957-2069-4082-b632-ecceffefc283","order_by":6,"name":"Qianming Chen","email":"","orcid":"https://orcid.org/0000-0002-5371-4432","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Qianming","middleName":"","lastName":"Chen","suffix":""},{"id":600099158,"identity":"04749059-2a49-47a2-8db1-7abfa4d76f18","order_by":7,"name":"Chongkui Sun","email":"","orcid":"","institution":"National Institutes of Health","correspondingAuthor":false,"prefix":"","firstName":"Chongkui","middleName":"","lastName":"Sun","suffix":""},{"id":600099159,"identity":"12b16e92-e8ea-418e-aa23-618b1fc5f7d0","order_by":8,"name":"Jiang Liu","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Jiang","middleName":"","lastName":"Liu","suffix":""},{"id":600099160,"identity":"419e2af5-56ad-46ad-bb68-5634d9a241df","order_by":9,"name":"Hang Zhao","email":"","orcid":"https://orcid.org/0000-0003-1268-0616","institution":"West China Hospital of Stomatology, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Hang","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2026-03-02 09:56:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9008638/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9008638/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104483932,"identity":"7287c3c8-a347-4819-b401-19336d565b4f","added_by":"auto","created_at":"2026-03-12 09:59:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5437629,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Synthesis and structures of \u003cstrong\u003eNY-1\u003c/strong\u003e (\u003cem\u003eN1,N3\u003c/em\u003e-di-adduct), \u003cstrong\u003eNY-2 \u003c/strong\u003e(\u003cem\u003eN1\u003c/em\u003e-mono-adduct), and \u003cstrong\u003eNY-3\u003c/strong\u003e (\u003cem\u003eN3\u003c/em\u003e-mono-adduct). Reagents and conditions: i) Et\u003csub\u003e3\u003c/sub\u003eN, dry THF, r.t., 1.5 h; ii) 5-FU, DBU, BHT, DMF, Ar protection, refluxed at 100 ℃, 24 h.\u003cstrong\u003e (B) \u003c/strong\u003eCCK8 assays showing the effect of 0-10 µM NY-1, NY-2, and NY-3 on cell viability of SCC cells. Data presented as mean ± SD, n = 3 biologically independent samples. \u003cstrong\u003e(C)\u003c/strong\u003e CCK8 assays showing the effect of 0-80 µM NY-1 and 5-FU on cell viability of SCC cells. Data presented as mean ± SD, n = 3 biologically independent samples. \u003cstrong\u003e(D) \u003c/strong\u003eRepresentative pictures of colony-formation assay showing the effect of NY-1 and 5-FU on cell proliferation of SCC cells \u003cem\u003ein vitro\u003c/em\u003e. \u003cstrong\u003e(E) \u003c/strong\u003eCloning efficiency of colony-formation assay showing the effect of NY-1 and 5-FU on cell proliferation of SCC cells \u003cem\u003ein vitro\u003c/em\u003e. Data presented as mean, n = 3 biologically independent samples. \u003cstrong\u003e(F)\u003c/strong\u003e Schematic diagram of NY-1 and β-CD host-guest identification.\u003cstrong\u003e (G) \u003c/strong\u003eTEM image of NY-1@β-CD.\u003cstrong\u003e (H)\u003c/strong\u003e DLS of NY-1@β-CD.\u003cstrong\u003e (I) \u003c/strong\u003eZeta potential of NY-1@β-CD. \u003cstrong\u003e(J)\u003c/strong\u003e The NMR spectra of NY-1 and NY-1@β-CD.\u003cstrong\u003e (K) \u003c/strong\u003eTEM images of cells taking up NY-1@β-CD being phagocytosed by lysosomes. \u003cstrong\u003e(L) \u003c/strong\u003eFluorescence co-localization map of NY-1@β-CD and lysosome.\u003cstrong\u003e (M) \u003c/strong\u003eCalcein-AM staining characterizes the number of living cells after NY-1 and NY-1@β-CD treatment. Data presented as mean ± SEM; n = 6. \u003cstrong\u003e\u0026nbsp;(N)\u003c/strong\u003e Comparison of the cell viability NY-1 effect of NY-1 and NY-1@β-CD for Cal-27 under the same concentration. \u003cstrong\u003e(O) \u003c/strong\u003eCCK8 assays showing the effect of NY-1@β-CD(10 µM), Z-VAD-FMK (10 µM), Nec-1 (10 µM), CQ (10 µM), Fer-1 (5 µM), MCC950 (10 µM), and NSA (2.5 µM) on cell viability of Cal-27.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9008638/v1/7f217321c32c0678d0c9f90e.png"},{"id":104483933,"identity":"23756ec2-ec4f-4260-96fb-cd5da31008b9","added_by":"auto","created_at":"2026-03-12 09:59:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1686250,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Images of living cells cell morphological changes of the four stages cell death with 10 µM NY-1@β-CD treatment. \u003cstrong\u003e(B)\u003c/strong\u003eScanning electron microscopy (SEM) showing cell morphological changes of the four stages cell death with 10 µM NY-1@β-CD treatment. Blue arrow:cell contraction, white arrow: small membrane blebbing, red arrow: large membrane blebbing, green arrow: membrane pore formation. \u003cstrong\u003e(C)\u003c/strong\u003e Staining of cell membrane showing the origination of cell blebbing, white arrow: small membrane blebbing.\u003cstrong\u003e (D)\u003c/strong\u003eTransmission electron microscope graph of Cal-27 cells treated with NY-1@β-CD (10 µM). Red dotted line: damaged cytoskeleton. Cytoskeleton damage rate was shown. Data presented as mean ± SEM; n = 6 different areas; significance was determined by Mann-Whitney test. ns, no significance. \u003cstrong\u003e(E) \u003c/strong\u003eImmunofluorescence showing the cleavage of F-actin. \u003cstrong\u003e(F) \u003c/strong\u003eCo-localization of the cell membrane (red) and F-actin (green).\u003cstrong\u003e (G and H) \u003c/strong\u003eImmunofluorescence showing effect of NY-1@β-CD (10 µM) on F-actin at different time. Mean fluorescence intensity per Cal-27 cell were shown. Data presented as mean ± SD; n = 8 different areas; significance was determined by Mann-Whitney test. ns, no significance.\u003cstrong\u003e (I and J) \u003c/strong\u003eImmunofluorescence showing effect of NY-1@β-CD (10 µM) and Narciclasine (5 nM) on F-actin at 24 h in Cal-27. Mean fluorescence intensity per Cal-27 cell were shown. Data presented as mean ± SD; n = 8 different areas; significance was determined by Mann-Whitney test. ns, no significance.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9008638/v1/001232a2192d4b868a26ce17.png"},{"id":104781327,"identity":"dd1ad2ad-fdea-4e02-b686-0b7a4ec0b12e","added_by":"auto","created_at":"2026-03-17 07:55:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4803895,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eFlow diagram of drug target proteomic quantitative and data-independent acquisition (DIA) mass spectrometry analysis. \u003cstrong\u003e(B) \u003c/strong\u003eVenn diagram showing overlap differentially expressed targets between semi-trypsin targets and trypsin targets after NY-1 treatment. \u003cstrong\u003e(C)\u003c/strong\u003e CCK8 assays showing the effect of siRPS13, siPRDX1, siPRDX6, siEEF2, siVCP, siRAB14, and siENO1 on cell viability of Cal-27 and A431 cells at 48 h. Data presented as mean ± SEM, n = 3 biologically independent samples, significance was determined by one-way ANOVA. ns, no significance. \u003cstrong\u003e(D)\u003c/strong\u003e CCK8 assays showing the effect of VCP activator for NY-1@β-CD inhibition effect.\u003cstrong\u003e (E) \u003c/strong\u003eThe CETSA of VCP in Cal-27 Cell lysis buffer.\u003cstrong\u003e (F)\u003c/strong\u003e The CETSA of VCP in Cal-27 cell supernatant. \u003cstrong\u003e(G) \u003c/strong\u003eF-actin detection assay showing effect of siVCP on F-actin at 48 h in Cal-27 cells. Data presented as mean ± SEM; n = 8 different areas; significance was determined by one-way ANOVA. ns, no significance. \u003cstrong\u003e(H) \u003c/strong\u003eVolcanic plot showing up/down-regulated differentially expressed proteins between 10 µM NY-1@β-CD treatment and control in Cal-27 cells. \u003cstrong\u003e(I) \u003c/strong\u003eDomain enrichment analysis showing differentially expressed proteins between 10 µM NY-1@β-CDtreatment and control in Cal-27 cells. \u003cstrong\u003e(J) \u003c/strong\u003eKEGG analysis showing the involving pathways of downregulated proteins between 10 µM NY-1@β-CD treatment and control in Cal-27 cells. \u003cstrong\u003e(K and L)\u003c/strong\u003e Immunoblotting showing effect of siVCP on VCP, FAK, p-FAK, YAP1, and p-YAP1 protein level in Cal-27 and A431 cells at 48 h. \u003cstrong\u003e(M and N)\u003c/strong\u003eImmunoblotting showing effect of NY-1@β-CD (10 µM) on FAK, p-FAK, YAP1, and CYR61 protein level in Cal-27 and A431 cells at 12, 15, 18, 21, 24 h. \u003cstrong\u003e(O and P)\u003c/strong\u003e Immunoblotting showing effect of NY-1@β-CD (10 µM) and Narciclasine (2.5 and 5 nM) on FAK, p-FAK, YAP1, and CYR61 protein level in Cal-27 and A431 cells.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9008638/v1/ba2d49764fbd566b6e5ab3b8.png"},{"id":104483935,"identity":"de068b0d-3a53-45f2-8230-b45c7b6a4938","added_by":"auto","created_at":"2026-03-12 09:59:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7319747,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eVolcanic plot showing up/down-regulated differentially expressed genes between 10 µM NY-1@β-CD treatment and control in Cal-27 and A431 cells. \u003cstrong\u003e(B)\u003c/strong\u003e Venn diagram showing overlap up/down-regulated differentially expressed genes between Cal-27 and A431 cells treated with NY-1(10 µM) for 24 h. \u003cstrong\u003e(C)\u003c/strong\u003e GO analysis showing the biological processes of overlap up/down-regulated differentially expressed genes between Cal-27 and A431 cells treated with NY-1(10 µM) for 24 h. \u003cstrong\u003e(D) \u003c/strong\u003eTransmission electron microscope graph of Cal-27 cells treated with NY-1(10 µM) for 6 h. Black arrow: normal endoplasmic reticulum. Blue arrow: expanded endoplasmic reticulum. ER width was shown. Data presented as mean ± SEM; n = 17 different areas; significance was determined by Mann-Whitney test. ns, no significance. \u003cstrong\u003e(E) \u003c/strong\u003eThe CCK8 assay of TUDCA reversed the cytotoxicity of NY-1@β-CD.\u003cstrong\u003e (F)\u003c/strong\u003e Cellular LDH assay showed TUDCA reversing NY-1@β-CD cytotoxicity. \u003cstrong\u003e(G) \u003c/strong\u003eF-actin detection assay showing effect of TUDCA on F-actin in Cal-27 cells. Data presented as mean ± SD; n = 4 different areas; significance was determined by one-way ANOVA. ns, no significance. \u003cstrong\u003e(H)\u003c/strong\u003e The activation of ER stress-related proteins and the reversal of this process by TUDCA for NY-1@β-CD. \u003cstrong\u003e(I) \u003c/strong\u003eThe upregulation of SSH-1 with the activation of ER stress related proteins for NY-1@β-CD. \u003cstrong\u003e(J)\u003c/strong\u003e F-actin detection assay showing effect of siSSH1 on F-actin in Cal-27 cells. Data presented as mean ± SD; n = 4 different areas; significance was determined by one-way ANOVA. ns, no significance. \u003cstrong\u003e(K) \u003c/strong\u003eWb results of the reversal of SSH1 activation by IRE1α phosphorylation inhibitors KIRA6.\u003cstrong\u003e (L) \u003c/strong\u003eThe IP results regarding the inhibition of the binding of 14-3-3 and SSH1 by NY-1@β-CD. \u003cstrong\u003e(M) \u003c/strong\u003eThe IP results regarding the reversed effect of inhibition the binding of 14-3-3 and SSH1 by NY-1@β-CD. \u003cstrong\u003e(N)\u003c/strong\u003e Schematic diagram of NY-1@β-CD release NY-1 and induce the depolymerization of F-actin.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9008638/v1/7dc96ff7d963fe40ec93befd.png"},{"id":104483938,"identity":"5a68cf62-727c-4fb1-9089-042b00b59f69","added_by":"auto","created_at":"2026-03-12 09:59:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8754640,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic diagrams of the construction and treatment of CDX and PDX models.\u003cstrong\u003e (B,C,G,H) \u003c/strong\u003eTumor growth curves of Cal-27 CDX (\u003cstrong\u003eB\u003c/strong\u003e), A431 CDX (\u003cstrong\u003eC\u003c/strong\u003e), PDX1 (\u003cstrong\u003eG\u003c/strong\u003e), and PDX2 (\u003cstrong\u003eH\u003c/strong\u003e) receiving 15 mg/kg NY-1@β-CD or 15 mg/kg 5-FU treatment by intraperitoneal injection. Data presented as mean ± SEM; n = 4 or 5 tumors per group; significance was determined by two-way ANOVA followed by Tukey’s multiple comparisons test. ns, no significance. \u003cstrong\u003e(D-F,I-K)\u003c/strong\u003eH\u0026amp;E staining and IHC staining (Ki67) of Cal-27 CDX (\u003cstrong\u003eD\u003c/strong\u003e), A431 CDX (\u003cstrong\u003eD\u003c/strong\u003e), PDX1 (\u003cstrong\u003eI\u003c/strong\u003e), and PDX2 (\u003cstrong\u003eI\u003c/strong\u003e) tumor tissues collected at the end of experiment with indicated treatment. Ki67 positive rates of Cal-27 CDX (\u003cstrong\u003eE\u003c/strong\u003e), A431 CDX \u003cstrong\u003e(F\u003c/strong\u003e), PDX1 (\u003cstrong\u003eJ\u003c/strong\u003e), and PDX2 (\u003cstrong\u003eK\u003c/strong\u003e) were shown. Data presented as mean ± SD; n = 8 different areas; significance was determined by one-way ANOVA. ns, no significance. \u003cstrong\u003e(L-O)\u003c/strong\u003e IHC staining (CYR61) of Cal-27 CDX (\u003cstrong\u003eL\u003c/strong\u003e), A431 CDX (\u003cstrong\u003eL\u003c/strong\u003e), PDX1 (\u003cstrong\u003eN\u003c/strong\u003e), and PDX2 (\u003cstrong\u003eN\u003c/strong\u003e) tumor tissues collected at the end of experiment with 15 mg/kg NY-1@β-CD or 15 mg/kg 5-FU treatment by intraperitoneal injection. CYR61 positive rates of Cal-27 CDX (\u003cstrong\u003eM\u003c/strong\u003e), A431 CDX (\u003cstrong\u003eM\u003c/strong\u003e), PDX1 (\u003cstrong\u003eO\u003c/strong\u003e), and PDX2 (\u003cstrong\u003eO\u003c/strong\u003e) were shown. Data presented as mean ± SD; n = 8 different areas; significance was determined by Mann-Whitney test. ns, no significance.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9008638/v1/483b01c948c4c41b6a8c24ca.png"},{"id":105728053,"identity":"73fea847-4100-464d-9336-d4ecba12f7b4","added_by":"auto","created_at":"2026-03-30 11:08:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":27119100,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9008638/v1/93f92096-01c2-43a2-83f8-7647626f82a1.pdf"},{"id":104483936,"identity":"78130437-5247-4a79-9eb3-08e48cb0befb","added_by":"auto","created_at":"2026-03-12 09:59:32","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4284689,"visible":true,"origin":"","legend":"supplementary information","description":"","filename":"songetalsupplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-9008638/v1/c89a7ebd4c9de84ce4015df6.docx"},{"id":104780760,"identity":"cf2fdebd-5212-4f2e-8d25-110355709084","added_by":"auto","created_at":"2026-03-17 07:53:50","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":937321,"visible":true,"origin":"","legend":"Full length western blot","description":"","filename":"Fulllengthwesternblots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9008638/v1/ec1d02d6d0bb8b937851ee8a.pdf"},{"id":104483937,"identity":"bc990664-7cba-4bd5-8ff7-9799a0224631","added_by":"auto","created_at":"2026-03-12 09:59:32","extension":"avi","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":12564992,"visible":true,"origin":"","legend":"The video of the origin of bubbles","description":"","filename":"Video1.avi","url":"https://assets-eu.researchsquare.com/files/rs-9008638/v1/3108615d6772ea3a33ed969e.avi"}],"financialInterests":"There is no duality of interest","formattedTitle":"VCP inhibition induces rapid cell death through ER stress driven actin cytoskeletal collapse","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe endoplasmic reticulum (ER) is a central hub for cellular proteostasis, coordinating protein folding, maturation, and trafficking \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Perturbations that overwhelm ER folding capacity activate the unfolded protein response (UPR), an adaptive signaling network that restores homeostasis by attenuating protein translation, inducing molecular chaperones, and enhancing degradation of misfolded proteins \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. When ER stress is severe or unresolved, the UPR transitions from adaption to cell death, classically through PERK-CHOP mediated transcriptional programs or IRE1α dependent activation of pro-apoptotic signaling cascades such as JNK \u003csup\u003e[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. These canonical transcriptional and apoptotic outputs of the UPR have been extensively investigated.\u003c/p\u003e \u003cp\u003eHowever, accumulating evidence indicates that ER stress is not confined to transcriptional and translational reprogramming, but can also elicit rapid and profound changes in cell morphology on a much shorter timescale. Acute perturbation of ER homeostasis by chemical stressors has been reported to induce cell rounding, surface blebbing, and actin cytoskeletal disassembly within minutes to hours, frequently preceding caspase activation or overt plasma membrane rupture \u003csup\u003e[\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. These phenomena suggest that the ER communicates with the cytoskeleton not only through long term transcriptional remodeling but also through direct signaling events that can rapidly reshape cell architecture. Beyond its structural role, the actin cytoskeleton functions as a dynamic sensor and integrator of intracellular stress signals, and perturbation of actin dynamics is known to exert a decisive influence on cell fate decisions \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Despite these insights, whether and how ER proteostasis failure is directly coupled to actin cytoskeletal remodeling remains poorly defined at the molecular level.\u003c/p\u003e \u003cp\u003eValosin-containing protein (VCP/p97), an essential AAA-ATPase that drives ER-associated degradation (ERAD), plays a critical role in maintaining ER proteostasis by extracting misfolded proteins from the ER membrane \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Pharmacological inhibition of VCP rapidly leads to accumulation of unfolded proteins and acute ER stress, making VCP a powerful experimental entry point to interrogate early signaling events downstream of proteostasis collapse \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Using a newly synthesized small-molecule VCP inhibitor, we unexpectedly observed a rapid disassembly of filamentous actin accompanied by synchronized membrane blebbing and a rapid, non-apoptotic, necrosis-like form of cell death. These findings prompted us to hypothesize that ER stress can directly engage a cytoskeletal effector pathway that determines rapid cell demise.\u003c/p\u003e \u003cp\u003eTo enhance the bioavailability and cellular delivery of this VCP inhibitor, we further exploited host\u0026ndash;guest supramolecular chemistry. Adamantane is a well-established guest for cyclodextrins, and β-cyclodextrin (β-CD) offers favorable aqueous solubility and stimulus-responsive drug release properties \u003csup\u003e[\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. By self-assembly of an adamantane-containing VCP inhibitor (NY-1) with β-CD, we constructed a supramolecular nanoparticle (NY-1@β-CD) that markedly enhances cellular uptake and amplifies acute ER stress signaling. Using this system, we demonstrate that ER stress activated IRE1α remodels the 14-3-3 interactome, competitively releasing the actin phosphatase SSH1 from inhibitory sequestration. Activated SSH1 dephosphorylates cofilin, driving rapid F-actin disassembly and catastrophic cytoskeletal collapse. Chemical chaperone treatment restores actin integrity and cell survival, establishing a direct causal link between ER proteostasis failure and cytoskeletal failure. Moreover, NY-1@β-CD exhibits potent antitumor efficacy in vivo, outperforming the clinically used chemotherapeutic 5-fluorouracil. Together, our findings define an IRE1α 14-3-3/SSH1 signaling axis as a previously unrecognized structural effector arm of the ER stress response and reveal how organelle level proteostasis disruption can be translated into rapid mechanical failure of the cell.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and reagents\u003c/h2\u003e \u003cp\u003eChemicals used were 5-Fluorouracil (5-FU, HY-90006), Narciclasine (HY-16563), BAPTA-AM (HY-100545), VCP activator 1 (VCP-A, HY-157508), Z-VAD-FMK (Z-VAD, HY-16658B), Necrostatin-1 (Nec-1, HY-15760), Chloroquine (CQ, HY-17589A), MCC950 (HY-12815), Necrosulfonamide (NSA, HY-100573), EGTA (HY-D0861), EGTA-AM (HY-D0973), Tauroursodeoxycholate (TUDC, HY-19696), Ryanodine (HY-103306), and Anisomycin (HY-18982) from MedChemExpress (USA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eThe cells Cal-27, A431, UM1, HN12, HN30, and HSC-3 were routinely cultured in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (Sigma, USA) at 37 ℃ in 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. The cells H103 and H314 were cultured in DMEM/F12 (Gibco, USA) supplemented with 10% fetal bovine serum (Sigma, USA) and 0.5 \u0026micro;g/mL hydrocortisone (HY-N0583R, MedChemExpress, USA) at 37 ℃ in 5% CO\u003csub\u003e2\u003c/sub\u003e incubator.\u003c/p\u003e\n\u003ch3\u003eSynthesis and characterization of compounds\u003c/h3\u003e\n\u003cp\u003eAll chemicals and solvents were commercially available laboratory grade and were used without further purification. Thin-layer chromatography (TLC) was performed on TLC aluminum sheet covered with silica gel 60 F254 (0.2 mm, Merck, Germany). Flash column chromatography (FC) was carried out with silica gel 60 (Haiyang chemical company, P. R. China) at 0.4 bar. NMR spectra were recorded on an AV II (Bruker, Germany) spectrometer at 600 MHz for 1H and 150 MHz for 13C. The J values are given in Hz, δ values in ppm are relative to Me4Si as internal standard. For NMR spectra measured in DMSO-d6, the chemical shift of the solvent peak was set to 2.50 ppm for 1H NMR and 39.50 ppm for 13C NMR. 1H-13C correlated (HMBC, HSQC), 1H-1H correlated (COSY, NOESY) NMR spectra were used for the assignment of the 1H/13C signals and identify the structural assignment of isomers. High resolution mass spectra were recorded on a LTQ mass spectrometer (Thermo, USA).\u003c/p\u003e\n\u003ch3\u003eConstruction of NY-1@β-CD\u003c/h3\u003e\n\u003cp\u003ePrepare 50mM of NY-1 with DMSO and 4mg/ml of β-CD (TCI C0777) solution with double distilled water. Slowly add NY-1 to the stirred β-CD solution until the final concentration is 1mM. Stir at room temperature for 1 hour and then dialysis overnight to remove unreacted molecules. The control group was treated with a mixed solution of DMSO/β-CD of the same concentration.\u003c/p\u003e\n\u003ch3\u003eFITC-labeled β-CD and co-localization of lysosomal\u003c/h3\u003e\n\u003cp\u003eFITC reacted with β-CD in sodium bicarbonate solution and was used for the preparation of NY-1@β-CD nanoparticles after dialysis with ultrafiltration tubes. After treating the cells with fluorescently labeled nanoparticles, lysosomes and nuclei were fluorescently labeled using lysosome tracker Red (Beyotime, C1046) and Hoechst (Beyotime, C1011), and real-time imaging was performed by confocal microscopy.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCCK8 assay\u003c/h2\u003e \u003cp\u003eCells were seeded in 96-well plates (5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells per well) and cultured for 24 h. Then cells were treated with varying concentrations (0, 1.25, 2.5, 5, 10 \u0026micro;M or 0, 5, 10, 20, 30, 40, 50, 60, 70, 80 \u0026micro;M) of NY-1、NY-2、NY-3 or 5-FU for 48 h; NY-1(10 \u0026micro;M), Narciclasine (2.5, 5, 10 nM), BAPTA-AM (0.5, 1, 2 \u0026micro;M), VCP-A (5, 10, 20 \u0026micro;M), Z-VAD-FMK (10 \u0026micro;M), Necrostatin-1 (Nec-1, 10 \u0026micro;M), Chloroquine (CQ, 10 \u0026micro;M), MCC950 (10 \u0026micro;M), Necrosulfonamide (NSA, 2.5 \u0026micro;M), and EGTA (500 \u0026micro;M) for 24 h; Add 10 \u0026micro;L CCK8 solution (Biosharp, China) to each well. And after incubating at 37 ℃ for 1 h, the absorbance at a wavelength of 450 nm was measured in triplicate or sextuplicate with multiple biological replicates with Varioskan Flash (Thermo Scientific, USA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eColony-formation assay\u003c/h3\u003e\n\u003cp\u003eCells were seeded in 6-well plates (700 cells per well) and cultured for 24 h. Then cells were treated with NY-1 (2.5, 5 \u0026micro;M) or 5-FU (2.5, 5 \u0026micro;M) for 2 weeks. Change the medium every 3 days. Colonies were fixed with 10% formalin and stained with 1% crystal violet in 10% ethanol. Images were documented and colonies were counted using ImageJ software.\u003c/p\u003e\n\u003ch3\u003eTime lapse microscopy observation\u003c/h3\u003e\n\u003cp\u003eCells were seeded in 6-well plates (2 \u0026times; 105 cells per well). Then cells were treated with NY-1(10 \u0026micro;M) for 41 h. During this process, cells in the same area are photographed every 2\u0026ndash;3 minutes to observe the continuous changes of the cells with a time lapse microscopy (Sartorius Incucyte, Germany).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eScanning electron microscope\u003c/h2\u003e \u003cp\u003eCells were seeded in 24-well plates (2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well) with slides and cultured for 24 h. Then cells were treated with NY-1(10 \u0026micro;M) for 24\u0026ndash;48 h. Next, all samples were cleaned with PBS twice and treated with 2.5% Glutaraldehyde EM Grade (Lilai, China) at 4\u0026deg;C overnight. A graded series of alcohol (30%, 50%, 75%, 85%, 95%, and 100%, respectively) was applied for sample dehydration for 15 min each. The dehydrated samples were naturally dried. Cell observations were under a scanning electron microscope (JEOL Ltd., Japan) with exposure parameters of 20 kV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTransmission electron microscope\u003c/h2\u003e \u003cp\u003eCells were seeded in 60 mm diameter dish and cultured for 24 h to reach 50%\u0026ndash;60% confluence. Then cells were treated with NY-1(10 \u0026micro;M) for 6 h and 24 h. Then cells were collected in a centrifuge tube, the supernatant was discarded, 0.5% glutaraldehyde fixing solution was slowly added along the tube wall with a pipette, and the cells were suspended, left for 5 min at 4℃. Then the cell suspension was transferred to a 1.5 mL apical bottom EP tube. Centrifuge at high speed (12000 rpm, 10 min), gently discard the supernatant, and slowly add 3% glutaraldehyde fixing solution along the tube wall with a straw. The samples were sent to Chengdu Lilai Biotechnology Co., Ltd. (China) to prepare and place on the copper mesh. The image of the copper mesh was collected by a transmission electron microscope (JEM-1400FLASH, JEOL).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStaining of cell membrane with red fluorescent dye DiI\u003c/h2\u003e \u003cp\u003eCells were seeded in 24-well plates (2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well) and cultured for 24 h. Then cells were treated with NY-1(10 \u0026micro;M) for 24 h. Then, 10 \u0026micro;M DiI (Beyotime C1036, China) was added into the culture medium for 30 min incubation. The cells were observed and photographed under a fluorescence microscope (Thermo, Invitrogen EVOS M5000).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eF-actin detection assay\u003c/h2\u003e \u003cp\u003eCells were seeded in 24-well plates (2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well) with slides and cultured for 24 h. Then cells were treated with NY-1(10 \u0026micro;M) for 0\u0026ndash;24 h; NY-1(10 \u0026micro;M) and Narciclasine (5 nM) or BAPTA-AM (1 \u0026micro;M) or VCP-A (10 \u0026micro;M) for 24 h. Cells were fixed with ice-cold methanol and wash with 0.1% Triton X-100 PBS for three times. After 1 h incubation with Actin-Tracker Green-488 (Byotime, China), 5 min incubation with DAPI (Servicebio, China) and three washes with 0.1% Triton X-100 PBS, samples were naturally dried, sealed and imaged with full tissue biopsy scanner (Olympus VS200, Germany). Average fluorescence intensity of F-actin analysis was performed using FlowJo 10.8.1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence (IF)\u003c/h2\u003e \u003cp\u003eCells were seeded in 24-well plates (2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well) with slides and cultured for 24 h. Then cells were treated with NY-1(10 \u0026micro;M) for 24 h. Cells were fixed with ice-cold methanol and blocked with 5% BSA (Biofroxx GmbH, Germany) for 1 h and 0.5% Triton X-100 PBS for 5 min. After overnight incubation with primary antibodies at 4\u0026deg;C and three washes with PBS, samples were incubated with secondary antibodies for 1 h and DAPI for 5 min at room temperature. After three washes with PBS, samples were naturally dried, sealed and imaged with full tissue biopsy scanner (Olympus VS200, Germany). The primary antibodies used were YAP1 antibody (#14074, 1:200) from Cell Signaling Technology (USA); α-tubulin antibody (11224-1-AP, 1:200) from Proteintech (China). The secondary antibody was Donkey anti-Rabbit IgG Alexa Fluor 555 (Invitrogen, A-31572, 1:400). Average fluorescence intensity analysis was performed using FlowJo 10.8.1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblotting\u003c/h2\u003e \u003cp\u003eCells were seeded in 6-well plates (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well) and cultured for 24 h. Then cells were treated with NY-1(10 \u0026micro;M) for 0\u0026ndash;24 h; NY-1(10 \u0026micro;M), Narciclasine (2.5, 5 nM), BAPTA-AM (5 \u0026micro;M), VCP-A (10 \u0026micro;M) for 24 h; siVCP for 48 h. The cell lysates were obtained through RIPA buffer (50 mM Tris base, 1.0 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 1% cocktail) and quantified by Pierce\u0026trade; BCA (bicinchoninic acid) protein assay kit (Thermo Scientific, USA) as previously described. The samples were processed to 12.5% or 7.5% sodium dodecyl sulfate polyacrylamide hydrogel electrophoresis (SDS-PAGE) (Yamei, China) at 80 V for 30 min and 120 V for 1 h and then transferred onto 0.22 \u0026micro;m polyvinylidene difluoride membrane (PVDF, MA, USA), using a wet transfer method at 300 mA for 75 or 90 min. The membranes were blocked with 5% skim milk in TBST for 1 h at room temperature and then incubated with primary antibodies overnight at 4 ℃. After washing with TBST for 15 min, membranes were incubated with goat anti-mouse/rabbit secondary antibody for 1 h at room temperature. Finally, the results were showed by BM Chemiluminescence Western Blotting kit (Roche, Switzerland). The primary antibodies used were FAK antibody (#3285, 1:1000), phospho-FAK antibody (#8556, 1:1000), YAP1 antibody (#14074, 1:1000), phospho-YAP1 antibody (#13008, 1:1000), Pan-TEAD antibody (#13295, 1:1000), CYR61 antibody (#39382, 1:1000), VCP antibody (#2648, 1:1000), cofilin antibody (#5175, 1:1000), phospho-cofilin antibody (#3313, 1:1000), IRE1α antibody (#3294, 1:1000), SSH1 antibody (13578, 1:1000) and XBP-1s antibody (40435, 1:1000) from Cell Signaling Technology (USA); phospho-IRE1α antibody (#ab124945, 1:1000) firm Abcam;14-3-3 antibody (#14503-1-AP, 1:1000), GAPDH antibody (60004-1-Ig, 1:10000) from Proteintech (China); The Goat anti-mouse/rabbit secondary antibody (ZB-2305 and ZB-2301, 1:1000) was purchased from ZSGB-BIO (China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq\u003c/h2\u003e \u003cp\u003eThe RNA-seq and analysis of Cal-27 and A431 cells treated with 10 \u0026micro;M NY-1 for 24 h was conducted. Total RNA was extracted using the Trizol\u0026trade; reagent (Thermo Scientific, USA) according to the manufacturer\u0026rsquo;s protocol. The transcriptome sequencing and analysis were conducted by Berry Genomics (Beijing, China). GO enrichment analysis was carried out with topGO (version 3.8) software. KEGG enrichment analysis was carried out with KOBAS (version 3.0) software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003esiRNA transfection\u003c/h2\u003e \u003cp\u003eAll siRNAs were synthesized by Shanghai GenePharma Co. Ltd. siRNAs were transfected using Lipofectamine RNAiMAX (Invitrogen, USA) according to the manufacturer\u0026rsquo;s instructions for 48 h. CCK8 assay and F-actin detection assay were executed as described in this article. The siRNAs were as follows:\u003c/p\u003e \u003cp\u003esiRPS13: sense (5\u0026rsquo; GCUCCUGGCAAGGGUAUUUTT3\u0026rsquo;),\u003c/p\u003e \u003cp\u003eantisense (5\u0026rsquo;AAAUACCCUUGCCAGGAGCTT3\u0026rsquo;).\u003c/p\u003e \u003cp\u003esiPRDX1: sense (5\u0026rsquo; CAUCAAGCCUGAUGUCCAATT3\u0026rsquo;),\u003c/p\u003e \u003cp\u003eantisense (5\u0026rsquo;UUGGACAUCAGGCUUGAUGTT 3\u0026rsquo;).\u003c/p\u003e \u003cp\u003esiPRDX6: sense (5\u0026rsquo; GCUCUGUGGUGCACACUGGGTT3\u0026rsquo;),\u003c/p\u003e \u003cp\u003eantisense (5\u0026rsquo; CCCAGUGUGCACCACAGAGCTT3\u0026rsquo;).\u003c/p\u003e \u003cp\u003esiEEF2: sense (5\u0026rsquo;UCGAUCAUGAUAUUGCCCATT 3\u0026rsquo;),\u003c/p\u003e \u003cp\u003eantisense (5\u0026rsquo; UGGGCAAUAUCAUGAUCGATT3\u0026rsquo;).\u003c/p\u003e \u003cp\u003esiVCP: sense (5\u0026rsquo;CCAACAGACCCAACAGCAUTT 3\u0026rsquo;),\u003c/p\u003e \u003cp\u003eantisense (5\u0026rsquo;AUGCUGUUGGGUCUGUUGGTT3\u0026rsquo;).\u003c/p\u003e \u003cp\u003esiRAB14: sense (5\u0026rsquo; GCACCGUACAACUACUCUUTT3\u0026rsquo;),\u003c/p\u003e \u003cp\u003eantisense (5\u0026rsquo; AAGAGUAGUUGUACGGUGCTT3\u0026rsquo;).\u003c/p\u003e \u003cp\u003esiENO1: sense (5\u0026rsquo; CCAUGCCAGGGAGAUCUUUTT3\u0026rsquo;),\u003c/p\u003e \u003cp\u003eantisense (5\u0026rsquo; AAAGAUCUCCCUGGCAUGGTT3\u0026rsquo;).\u003c/p\u003e \u003cp\u003esiIREα: sense (5\u0026rsquo;GCGAGAAGCAGCAGACUUUTT3\u0026rsquo;)\u003c/p\u003e \u003cp\u003eantisense (5\u0026rsquo;AAAGUCUGCUGCUUCUCGCTT3\u0026rsquo;)\u003c/p\u003e \u003cp\u003esiSSH1: sense (5\u0026rsquo; GCAGGAAGGAUGCACAUAUT3\u0026rsquo;)\u003c/p\u003e \u003cp\u003eantisense (5\u0026rsquo; AUAUGUGCAUCCUUCCUGCTT 3\u0026rsquo;)\u003c/p\u003e \u003cp\u003eNegative control: sense (5\u0026rsquo;UUCUCCGAACGUGUCACGUTT 3\u0026rsquo;),\u003c/p\u003e \u003cp\u003eantisense (5\u0026rsquo;ACGUGACACGUUCGGAGAATT 3\u0026rsquo;).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eDrug target proteomic quantitative and data-independent acquisition (DIA) mass spectrometry analysis\u003c/h2\u003e \u003cp\u003eCal-27 cells were seeded in 10 cm diameter dish and cultured for 24 h to reach 80%\u0026ndash;95% confluence. After PBS washing for 3 times, the petri dish carrying cells was transported to Applied Protein Technology for sample processing and data analysis.\u003c/p\u003e \u003cp\u003eProtein extraction and peptide enzymolysis: Cells were homogenized with precooled 1\u0026times;PBS for protein extraction and Pierce\u0026trade; BCA (bicinchoninic acid) protein assay kit (Thermo Scientific, USA) for protein quantification. The protein was treated with 10 \u0026micro;M NY-1 for 30 min to obtain the sample to be processed. The treated samples were pretreated with protease K (PK enzyme), and 15 \u0026micro;g of protein was taken for SDS-PAGE detection. After PK treatment, the samples were added with denaturant (UA/ DOC) and DTT to reach the final concentration of 20 mM, reacted at 30\u0026deg; for 2 h, and cooled to room temperature. Add appropriate amount of IAA until the final concentration is 25 mM, shake at 600 rpm for 1 min, avoid light at room temperature for 30 min, add appropriate amount of NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e buffer (50 mM) to dilute the UA/DOC concentration to less than 1.5 M. Then, 2 \u0026micro;g Trypsin was added into the sample at 37℃ for 16 h. Desalted and lyophilized, redissolved with 0.1% FA. The peptide concentration was determined by OD\u003csub\u003e280\u003c/sub\u003e. 2 \u0026micro;g peptide was extracted, the appropriate amount of iRT standard peptide was added, and DIA mass spectrometry was performed.\u003c/p\u003e \u003cp\u003eDIA mass spectrometry analysis: DIA analysis was chromatographically separated using the Vanquish Neo system (Thermo Scientific, USA) with nanoliter flow rate. Samples separated by nanoscale high performance liquid chromatography were analyzed by DIA mass spectrometry using Astral High resolution Mass Spectrometer (Thermo Scientific, USA). Detection mode: positive ion and parent ion scan range is 380\u0026ndash;980 m/z, primary mass spectrometry resolution is 240,000 at 200 m/z, Normalized AGC Target is 500%, Maximum IT is 5 ms. MS2 adopts DIA data acquisition mode, 299 scanning Windows are set, Isolation Window is 2 m/z, HCD Collision Energy is 25 ev, Normalized AGC Target is 500%, Maximum IT value is 3 ms.\u003c/p\u003e \u003cp\u003eData analysis: DIA data is processed using Spectronaut software (Spectronaut\u0026trade; 18.3.230830.50606) and the database is the same as that used for the data construction. The software parameters are set as follows: retention time prediction type is set to dynamic iRT, interference on MS2 level correction is set to enabled, cross run normalization is set to enabled, and all results must pass the Q Value cutoff parameter set to 0.01 (equivalent to FDR\u0026thinsp;\u0026lt;\u0026thinsp;1%). The target protein set was analyzed through bioinformatic analysis (protein domain analysis, enrichment analysis, and Venn diagram analysis).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eDIA quantitative proteomic analysis\u003c/h2\u003e \u003cp\u003eCal-27 cells were seeded in 10 cm diameter dish and cultured for 24 h to reach 50%\u0026ndash;60% confluence. Then cells were treated with NY-1(10 \u0026micro;M) for 24 h. After PBS washing for 3 times, the cells were scraped and transferred to 1.5 mL EP tube. The tubes were transported to Applied Protein Technology for sample processing and data analysis.\u003c/p\u003e \u003cp\u003eProtein extraction and peptide enzymolysis: Appropriate amount of SDT (4% SDS, 100 mM Tris-HCl, pH7.6) was added to each sample to extract protein, and the protein was quantified by BCA method. Take 15 \u0026micro;g protein from each sample and add an appropriate amount of 5X loading buffer, and bathe in boiling water for 5 min. SDS-PAGE electrophoresis (4%-20% prefabricated gradient adhesive, constant pressure 180 V, 45 min) and Coomassie bright blue R-250 staining were performed. Appropriate amount of protein was taken from all samples and mixed into Pool samples to be used as QC samples. All samples, including mixed Pool samples, were digested by trypsin using Filter aided proteome preparation (FASP). The peptide of the enzymolysis sample was dialyzed using C18 Cartridge, lyophilized and redissolved in 40 \u0026micro;L 0.1% formic acid solution. The peptide concentration of the sample was determined with OD\u003csub\u003e280\u003c/sub\u003e. An appropriate amount of iRT standard peptide was added to the enzymolysis peptide of each sample, and DIA mass spectrometry was performed by Astral High resolution Mass Spectrometer (Thermo Scientific, USA).\u003c/p\u003e \u003cp\u003eDIA mass spectrometry analysis: same as Drug target proteomic quantitative and data-independent acquisition (DIA) mass spectrometry analysis.\u003c/p\u003e \u003cp\u003eData analysis: DIA data was processed by DIA-NN software. The software parameters were set as follows: the enzyme was trypsin, the max, miss, and cleavage site was 1, the fixed modification was Carbamidomethyl (C), and the dynamic modification was set to Oxidation(M) and Acetyl (Protein N-term). The protein identified by the database search must pass the set filtering parameter FDR\u0026thinsp;\u0026lt;\u0026thinsp;1%. The protein set was analyzed through bioinformatic analysis (protein domain analysis, enrichment analysis, and KEGG analysis).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e4-week-old female BALB/c athymic nude mice were purchased from GemPharmatech LLC (China). All experiments were conducted in accordance with the guidelines outlined in the \u0026ldquo;Principles of Laboratory Animal Care\u0026rdquo; (NIH) and were approved by Medical Ethics Committee of West China Stomatology Hospital, Sichuan University. The animals had free access to sterilized water and food in a temperature-controlled room (22\u0026thinsp;\u0026plusmn;\u0026thinsp;1 ℃) with a 12 h light/dark cycle in an SPF environment. They were fed adaptively for one week in this circumstance before the experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eCell line-derived xenograft (CDX) tumor assay\u003c/h2\u003e \u003cp\u003eThe 4-week-old female BALB/c athymic nude mice were injected with 100 \u0026micro;L Cal-27 or A431 cell suspension (2\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mL) into the right flank. The weight of the mice and the size of the xenograft tumor were measured every other day. The calculation formula for the volume of the transplanted tumor was TV\u0026thinsp;=\u0026thinsp;π/6\u0026times;length\u0026times;(width)\u003csup\u003e2\u003c/sup\u003e. When the size of the xenograft tumor was close to 100 mm\u003csup\u003e3\u003c/sup\u003e, the mice were randomly grouped. Mice were intraperitoneally administered 100 \u0026micro;L 5-FU (15 mg/kg) or 100 \u0026micro;L NY-1 (5, 15 mg/kg) with equal volume of corn oil as the control once every 4 days. At the end of the experiment, all mice were sacrificed by cervical dislocation. The tumor tissues and organs were extracted from mice for histological analysis at the endpoint of the therapeutic study.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003ePatient-derived xenograft (PDX) tumor assay\u003c/h2\u003e \u003cp\u003eThe HNSCC PDX model was established by directly implanting fragments from patient\u0026rsquo;s HNSCC tissue into 4-week-old female BALB/c athymic nude mice. Briefly, surgically resected human HNSCC tissues were obtained and sliced into small fragments (~\u0026thinsp;2 mm in size) to implant subcutaneously into the right flanks of the mice as the first generation of tumor (F1). After successfully establishing xenografts, they are extracted from donor mice and reimplanted into other recipient mice for further \u003cem\u003ein vivo\u003c/em\u003e passaging. Following that, each generation was marked as second (F2), third (F3), and n generation (Fn). When the tumor volume reached approximately\u0026thinsp;~\u0026thinsp;100 mm\u003csup\u003e3\u003c/sup\u003e, we started therapy same as CDX with the F3 generation of the PDX model. At the end of the experiment, all mice were sacrificed by cervical dislocation. The tumor tissues and organs were extracted from mice for histological analysis at the endpoint of the therapeutic study.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eHematoxylin and eosin (H\u0026amp;E) staining\u003c/h2\u003e \u003cp\u003eThe collected fresh tumor tissues, main organs (heart, liver, spleen, lung and kidney) and skin were fixed in 4% (W/V) paraformaldehyde solution for 12 h. After dehydration and paraffin embedding, the tissues were cut into 4 mm thick slices with a microtome (Leica, Germany) and placed on a glass slide. Then, the tissues were dewaxed by xylene for 20 min, dehydrated by gradient alcohol (100%~5 min, 100%~5 min, 95%~5 min, 80%~2 min, 70%~2 min), dyed with hematoxylin for 30 s\u0026ndash;1 min, and washed with running water for 15 min. After that, they were dyed with eosin for 30 s\u0026ndash;1 min, dehydrated with gradient alcohol (80%~2 min, 95%~2 min, 100%~5 min, 100%~5 min), and transparent with xylene for 20 min. After sealing with neutral resin, the tissues were observed with a full tissue biopsy scanner (Olympus VS200, Germany).\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eImmunohistochemical (IHC) analysis\u003c/h2\u003e \u003cp\u003eAfter fixed, dehydrated, embedded, and sectioned, the tissues were deparaffinized with xylene for 20 min and dehydrated with gradient alcohol (100%~5 min, 100%~5 min, 95%~5 min, 80%~2 min, 70%~2 min). Then, the tissues were subjected to antigen retrieval, hydrogen peroxide and serum blocking for 30 min, and incubated with the primary antibody overnight at 4 ℃, washed with PBS, then incubated with the secondary antibody for 1 h at room temperature. After washing with PBS, DAB chromogenic solution (ZSGB-BIO, China) was used for tissue color development. Next, stain the tissues with hematoxylin for 30 s\u0026ndash;1 min and rinse with running water for 15 min. After dehydration with gradient alcohol (80%~2 min, 95%~2 min, 100%~5 min, 100%~5 min), transparent with xylene for 20 min, and sealing with neutral resin, the tissues were observed with a full tissue biopsy scanner (Olympus VS200, Germany). The arithmetical mean proportion in the 6\u0026ndash;8 regions of Ki67/CYR61-positive cells were counted by ImageJ software. The primary antibodies used were CYR61 antibody (#39382, 1:200) from Cell Signaling Technology (USA) and Ki67 antibody (ab15580, 1:100) from Abcam (UK). Goat anti-rabbit secondary antibody (ZB-2305 and PV-9001, 100 \u0026micro;L) was purchased from ZSGB-BIO (China).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eExperiments were performed in triplicates, or otherwise as indicated. The data were shown as mean value\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (standard deviation) or mean value\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (standard error of the mean). Statistical differences among different groups were assessed by one/two way ANOVA and Mann-Whitney test using the GraphPad Prism 9.5 software, which were considered statistically significant when \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, while \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05 was represented as \u0026ldquo;ns\u0026rdquo; meaning no significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003e1. Construction of NY-1@β-CD supramolecular nanoparticles induced rapid tumor cell death\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo synthesis the new molecular with satisfied anti-cancer efficiency, we conducted the process commenced with the preparation of compound \u003cb\u003e3\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, see the Supporting Information for the synthetic procedure). Considering that nucleobases have multiple hydrogen bond donors and acceptors, incorporating them into ligand molecules can potentially serve as binding sites, resulting in better affinity with proteins. Among them, uracil as a target for nucleophilic and electrophilic reagents shows interesting reactivity and its derivatives exhibit extremely diverse physiological activities. Therefore, 5-Fluorouracil (5-FU), which is widely used in oncology as an important anticancer agent, was reasonably selected the donor molecule to undergo Michael addition with compound \u003cb\u003e3\u003c/b\u003e. The yield of final products including \u003cem\u003eN1, N3\u003c/em\u003e-di-adduct (NY-1), \u003cem\u003eN1\u003c/em\u003e-mono-adduct (NY-2) and \u003cem\u003eN3\u003c/em\u003e-mono-adduct (NY-3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). All synthesized compounds were characterized by \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR spectra, as well as high resolution mass spectra. \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC correlated (HMBC, HSQC), \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH correlated (COSY, NOESY) NMR spectra were used for the assignment of the \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC signals, identify the structural assignment of isomers. The 2D NMR data gave compelling evidence that compound \u003cb\u003eNY-2\u003c/b\u003e is the \u003cem\u003eN1\u003c/em\u003e-mono-adduct and compound \u003cb\u003eNY-2\u003c/b\u003e is \u003cem\u003eN3\u003c/em\u003e-mono-adduct. For details, see the Supporting Information for the synthetic procedure. For spectra, see the Supporting Information \u003cb\u003eFig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eR\u003c/b\u003e. We firstly tested how three compounds (NY-1, NY-2, and NY-3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) with an increased dose gradient inhibit the viability of eight squamous cell carcinoma (SCC) cell lines (cutaneous squamous cell carcinoma (cSCC) cell line A431, head and neck squamous cell carcinoma (HNSCC) cell lines Cal-27, UM1, HN12, HN30, HSC-3, H103, and H314). After treatment for 48 h, the cell viability was detected by CCK8 assay. As the dose increased, NY-1 demonstrated much higher inhibition rate of SCC cell viability compared to NY-2 and NY-3. More importantly, the NY-1 showed better anti-cancer effects compared with the donor molecules 5-FU (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The clone formation experiments further proved the significant anti-tumor efficacy of NY-1 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. To achieve rapid uptake of NY-1 by tumor cells and enhance the anti-tumor effect, we chose pH-response molecular β-cyclodextrin (β-CD) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e. β-CD and NY-1 were dissolved in the aqueous phase and the organic phase respectively, relying on host-guest recognition they can self-assemble after rapid mixing. The NY-1@β-CD nanoparticle exhibited uniformly dispersed spherical structure with a hydrated particle size of 170.08nm and PDI of 0.109, indicating the good dispersibility and stability of NY-1@β-CD(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Energy dispersive spectroscopy (EDS) results confirmed the self-assemble of two components (\u003cb\u003eFig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eS)\u003c/b\u003e. The zeta potential is -18mV, further indicating the satisfying stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). After reacting with β-CD, the multiple NMR H\u003csup\u003e1\u003c/sup\u003e of NY-1 were significantly shifted, further indicating the effectively encapsulated of β-CD through host-guest recognition (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ\u003cb\u003e)\u003c/b\u003e. To ensure that NY-1@β-CD can be rapidly taken by tumor cells we first conducted cell TEM imaging after 3h treatment. We clearly observed that a monolayer membrane like endosome containing a large number of nanoparticles within the tumor cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK\u003cb\u003e)\u003c/b\u003e. To further illustrate the escape of NY-1@β-CD from endosomes and the release of NY-1, we labeled β-CD with FITC and conducted lysosomal fluorescence co-localization detection. The NY-1@β-CD showed significant co-localization with lysosome tracker at 1 hours, which was consistent with the TEM results. After 3 hours, their fluorescence signals separated, indicating successful escape from the endosome (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL\u003cb\u003e)\u003c/b\u003e. The cell live and dead staining and CCK-8 results further demonstrated the rapid cell death by NY-1@β-CD (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM \u003cb\u003eand Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eT)\u003c/b\u003e. As expected, the NY-1@β-CD demonstrated better anti-tumor effect than NY-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eN\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo identify the type of rapid cell death induced by NY-1@β-CD, multiple cell death inhibitors including apoptosis inhibitor Z-VAD-FMK (Z-VAD), necroptosis inhibitors Necrostatin-1 (Nec-1) and Necrosulfonamide (NSA), autophagy inhibitor Chloroquine (CQ), Ferroptosis inhibitor Ferrostatin-1 (Fer-1), and pyroptosis inhibitor MCC950 were employed. The decrease in cell viability induced by NY-1@β-CD could be restored by 10\u0026ndash;20% with Z-VAD-FMK in both Cal-27 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and A431 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) cells, suggesting that NY-1 could induce apoptosis in some cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eO \u003cb\u003eand Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eU)\u003c/b\u003e. MCC950 only restored about 10% of cell vitality in A431 cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while Nec-1, NSA, CQ, and Fer-1, had no recovery ability on NY-1 treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eO \u003cb\u003eand Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eV)\u003c/b\u003e. Given that only a minor fraction of NY-1@β-CD treated cells underwent apoptosis, and that inhibitors of multiple canonical cell death pathways provided only limited protection (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD), these data suggest that NY-1@β-CD induces a rapid cytotoxic response that is largely independent of classical programmed cell death pathways.\u003c/p\u003e \u003cp\u003eWe therefore sought to define the mode and molecular mechanism underlying this unusually rapid form of cell death triggered by the supramolecular nanoparticle NY-1@β-CD.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2. NY-1 induces blebbing and actin depolymerization associated cell death\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo explore how cell death occurs, NY-1@β-CD treated Cal-27 cells were observed with a time lapse microscope. The progression of NY-1@β-CD-induced cell death can be classified into four stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The first stage of cell morphology change was that cell attachment lost and cell contracted tending to be round after NY-1 treatment. The cell membrane showed a subsequent change to form multiple small membrane blebbing (white arrow, \u0026lt;\u0026thinsp;5 \u0026micro;m) in the second stage. As the cell shrunk further, large membrane blebbing whose volume can even reach or exceed the size of the cell itself formed (red arrow, 10\u0026ndash;30 \u0026micro;m) during the third stage. Eventually, the blebbing disappeared and the cells died in the fourth stage. More importantly, the live cell imaging showed that the blebbing occurred at 8h for NY-1 treatment, while occurring at 2h for NY-1@β-CD (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA\u003c/b\u003e), further illustrating the positive regulatory effect of nanoparticles on the of NY-1 anti-cancer ability. Moreover, in order to further observe the morphological changes of the cells during cell death, cells treated with NY-1@β-CD were observed by scanning electron microscopy (SEM). The presence of cell attachment losing and cell shrinkage (blue arrow), small membrane blebbing (white arrow), large membrane blebbing (red arrow) and membrane pores (green arrow, \u0026lt; 8 \u0026micro;m) were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Multiple pores of different diameters in the cell membrane mean that the integrity of the cell membrane was destroyed. These pores may be traces of cell membrane damage left after the formation of small and large membrane blebbing. The SEM results further confirmed the time lapse microscope results. Blebbing occurs after the attachment losing and cell contraction, suggesting that it may be due to the great pressure inside the cells caused by volume reduction, and the cell membrane is then squeezed and blebbed. To further clarify the origin of bubbles, the cell membrane was labeled with red dye DiI. We can obviously observe that blebbing membrane and cell membrane showed red fluorescence, indicating that they are with the same composition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cb\u003eVideo 1\u003c/b\u003e). Since some research conducted that the blebbing is associated with actin interference, we further conducted transmission electron microscopy (TEM) to clarify the microscopic changes of cells under the treatment of NY-1@β-CD. Just as we expected, significant cytoskeleton damage (marked by red dotted lines) was observed in the TEM analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. The proportion of cells exhibiting such damage in various regions, as observed through TEM, was calculated. Remarkably, cytoskeleton damage was identified in 51.39% of the cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). This finding suggests that cytoskeleton damage may play a crucial role in cell death. The primary components of the cytoskeleton include microfilaments and microtubules. Immunofluorescence assay was conducted to characterize and quantify microtubules (α-tubulin) in NY-1@β-CD treated Cal-27 cells, while no significant decrease of α-tubulin compared with control group (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC\u003c/b\u003e). In contrast, time lapse microscope exhibited the fracture of microfilaments (F-actin) after NY-1@β-CD treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). More importantly, the co-localization of the cell membrane with F-actin further indicated that blebbing is associated with actin interference (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Meanwhile, alterations F-actin in NY-1@β-CD treated cells were significant decreased just after 3h treatment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, \u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC and Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eD)\u003c/b\u003e. The same results also exhibited in WB results, as the downregulation of p-cofilin after NY-1@β-C treatment (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eE\u003c/b\u003e). These findings suggest that NY-1 treatment may induce cytoskeleton damage, characterized by a depolymerization in F-actin filaments. The reduction of F-actin filaments caused by NY-1@β-CD treatment was significantly rescued by Narciclasine (Ncls) treatment, which induces F-actin polymerization through cofilin phosphorylation via Rho kinase signal pathway \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ, \u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eF and Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eG)\u003c/b\u003e. More significantly, inhibition of cell viability induced by NY-1@β-CD treatment was significantly rescued by Ncls, indicating F-actin depolymerization is a key event contributing to cell death (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eH to 2J\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAbove all, we demonstrated that NY-1@β-CD nanoparticles cause rapid cell death characterized by F-actin depolymerization by rapidly releasing NY-1 after 6h treatment. Thus, the direct target of NY-1 and the depolymerization mechanism of F-actin need further exploration.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3. NY-1 targets VCP to triggers F-actin depolymerization and cell death\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn order to clarify the direct target of NY-1, we firstly conducted drug target proteomic quantitative analysis and DIA mass spectrometry based on the limited trypsin digestion method were conducted on Cal-27 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Proteins in the NY-1-treated groups (4918, 4913, and 4920) and control groups (4918, 4916, and 4909) corresponding to peptides in the NY-1-treated groups (73030, 73025, and 73033) and control groups (73032, 73020, and 73022) were identified. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and a fold change (FC)\u0026thinsp;\u0026lt;\u0026thinsp;0.67 or FC\u0026thinsp;\u0026gt;\u0026thinsp;1.5 were established as the thresholds for significantly differential peptides. In comparison to the control group, a total of 7995 differentially expressed peptides were identified in Cal-27 cells treated with NY-1, comprising 2267 down-regulated peptides and 5728 up-regulated peptides. The underlying principle of the limited trypsin digestion method for target identification is predicated on the chemical's binding to target proteins, which subsequently obstructs the trypsin digestion sites on these proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Consequently, this results in a simultaneous increase in the abundance of longer peptides (trypsin digestion) and a decrease in the abundance of the corresponding shorter peptides (semi-trypsin digestion) of the target proteins. Additionally, seven overlapping differentially expressed targets between trypsin and semi-trypsin digestion were identified using a threshold of p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and |log2FC| \u0026gt; 1 (RPS13, ribosomal protein S13; PRDX1, peroxiredoxin-1; PRDX6, peroxiredoxin-6; EEF2, eukaryotic translation elongation factor 2; VCP, valosin-containing protein; RAB14, Ras related protein Rab-14; ENO1, enolase1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). To identify the most promising target of NY-1, siRNAs targeting the overlapped seven genes- \u003cem\u003eRPS13\u003c/em\u003e, \u003cem\u003ePRDX1\u003c/em\u003e, \u003cem\u003ePRDX6\u003c/em\u003e, \u003cem\u003eEEF2\u003c/em\u003e, \u003cem\u003eVCP\u003c/em\u003e, \u003cem\u003eRAB14\u003c/em\u003e, and \u003cem\u003eENO1\u003c/em\u003e, were utilized to individually knock down the expression of seven proteins. Initially, alterations in cell death were examined following the knockdown of seven proteins. Most candidates resulted in a significant inhibition of cell viability in both cell lines, with VCP knockdown showing the greatest reduction in cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Moreover, we found a VCP activator, the NY-1-induced cell death was rescued by VCP-A in both Cal-27 and A431 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD \u003cb\u003eand Fig.S3A\u003c/b\u003e). To further clarify that VCP is the direct target of NY-1, we further purified the VCP protein and conducted biolayer interferometry (BLI) experiments (\u003cb\u003eFig.S3B\u003c/b\u003e). The results showed that NY-1 exhibited excellent binding ability to the VCP protein, with a dissociation constant of 6.33*e\u003csup\u003e\u0026minus;6\u003c/sup\u003eM \u003cb\u003e(Fig.S3C)\u003c/b\u003e. Cellular thermal shift assay (CETSA) results further confirmed its target effect, as thermal stability of VCP protein enhanced after treatment with NY-1 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e. These results strongly demonstrated that VCP is a direct target of NY-1 and is associated with the cytotoxic effects. After knocked down VCP, we observed the depolymerization of F-actin, which was similar to the NY-1@β-CD treatment, further illustrating the regulatory relationship between VCP and F-actin \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG \u003cb\u003eand Fig.S3C)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe data independent acquisition (DIA) quantitative proteomic analysis was conducted and monitored 2146 differentially expressed proteins were identified in Cal-27 cells treated with NY-1, comprising 811 down-regulated proteins and 1335 up-regulated proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Protein domain analysis demonstrated that the differentially expressed proteins were primarily linked to cytoskeletal dynamics and cell adhesion which act in cell attachment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the downregulated proteins revealed a significant enrichment in focal adhesion and cell adhesion signaling pathways which contribute to cell attachment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). Additionally, Gene Set Enrichment Analysis (GSEA) of the KEGG pathway enrichment at the RNA level between NY-1-treated Cal-27 and A431 cells identified significantly enriched pathways, with a notable downregulation observed in the F-actin FAK associated signaling (YAP1 signaling) (\u003cb\u003eFig. S3D\u003c/b\u003e). Knocking down VCP or directly targeting and inhibiting VCP with NY-1@β-CD can both achieve the inhibition of FAK, YAP and its downstream CYR61 in both Cal-27 and A431 cell lines \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK-N\u003cb\u003e)\u003c/b\u003e. The nuclear locus of YAP was also significantly suppressed \u003cb\u003e(Fig.S3E and Fig.S3F)\u003c/b\u003e. More importantly, the Ncls can also rescue the downregulation of pFAK, YAP, and CYR61, further demonstrated that NY-1@β-CD induces F-actin depolymerization related cell death by cell adhesion \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eO \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eP\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eThe above results confirmed that NY-1@β-CD can be rapidly taken up by cancer cells and release NY-1. NY-1 can target VCP directly to induce F-actin depolymerization and thus causes cells lose adhesion and death. The synergistic effect of nanoparticles and NY-1 can cause these events to occur rapidly within 6 hours to induce rapid cell death. Researches conducted that the activity of VCP protein is closely related to maintaining protein homeostasis, we further clarified the biological effects of VCP and its regulation on the depolymerization of F-actin.\u003c/p\u003e \u003cp\u003e \u003cb\u003e4. NY-1 targets VCP and induces F-actin depolymerization through acute ER stress\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn order to clarify the changes of signal pathway caused by NY-1@β-CD, transcriptome sequencing and analysis (RNA-seq) was conducted on Cal-27 and A431 cells. A threshold of p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and |log2FC| \u0026gt; 1 was employed to identify significantly differentially expressed genes (DEGs). Compared to the control group, 3099 and 3162 DEGs were identified in NY-1-treated Cal-27 and A431 cells, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Among these, 1838 genes were down-regulated and 1261 genes were up-regulated in Cal-27 cells, while 1523 genes were down-regulated and 1639 genes were up-regulated in A431 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Subsequently, Venn diagrams were constructed to illustrate the overlap of up- and down-regulated DEGs between Cal-27 and A431 cells, using a threshold of q value\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and |log2FC| \u0026gt; 1. The analysis identified 894 co-up-regulated and 870 co-down-regulated genes common to both Cal-27 and A431 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The substantial overlap of DEGs indicates a consistent pattern of RNA changes induced by NY-1 across different SCC cell lines. Gene Ontology (GO) enrichment analysis indicated that the up-regulated genes with overlapping expression were predominantly involved in the biological processes related to the protein folding and Endoplasmic Reticulum (ER) stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). TEM detected that the significantly expanded of ER lumen after NY-1@β-CD treatment, further verifying the sequencing results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). To determine whether ER stress is causally linked to NY-1@β-CD induced cytotoxicity, cells were co-treated with the chemical chaperone tauroursodeoxycholic acid (TUDCA). ER stress alleviation by TUDCA significantly rescued cell viability and reduced LDH release, indicating that ER stress is a critical upstream event mediating NY-1@β-CD induced cell death \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e. Importantly, the extensive depolymerization of F-actin observed upon NY-1@β-CD treatment was effectively reversed by TUDCA, as demonstrated by both immunofluorescence staining and immunoblot analysis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e. This ER stress\u0026ndash;dependent regulation of actin integrity was further validated in fibroblasts, indicating that the phenomenon is not restricted to cancer cells \u003cb\u003e(Fig. S4A)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven the central role of actin dynamics in the observed phenotype, we next investigated regulators of actin filament turnover. Slingshot homolog 1 (SSH1), a phosphatase that activates cofilin by dephosphorylation and promotes F-actin disassembly, was markedly upregulated following NY-1@β-CD induced ER stress \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI\u003cb\u003e)\u003c/b\u003e. Attenuation of ER stress by TUDCA suppressed SSH1 upregulation, suggesting that SSH1 activation is downstream of ER stress \u003cb\u003e(Fig. S4B)\u003c/b\u003e. Functionally, knockdown of SSH1 significantly rescued F-actin depolymerization induced by NY-1@β-CD \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ\u003cb\u003e)\u003c/b\u003e, establishing SSH1 as a necessary mediator of actin collapse. Among the three canonical UPR sensors, inhibition of IRE1α phosphorylation effectively suppressed SSH1 upregulation and restored F-actin organization \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK \u003cb\u003eand Fig. S4C)\u003c/b\u003e, implicating the IRE1α branch of the ER stress response in this process. Mechanistically, previous studies have shown that SSH1 activity is restrained through binding to the scaffolding protein 14-3-3, which maintains cofilin in its phosphorylated, inactive state. Co-immunoprecipitation assays revealed that NY-1@β-CD markedly disrupted the interaction between 14-3-3 and SSH1, whereas TUDCA treatment restored this association \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM\u003cb\u003e).\u003c/b\u003e These findings indicate that ER stress induced by NY-1 remodels the 14-3-3 interactome, thereby releasing SSH1 from inhibitory binding and enabling cofilin activation.\u003c/p\u003e \u003cp\u003eCollectively, these data demonstrate that NY-1@β-CD induces acute ER stress through VCP inhibition, which activates the IRE1α pathway and reshapes 14-3-3 dependent protein interactions. This remodeling releases and activates SSH1, leading to cofilin-mediated F-actin depolymerization and rapid cytoskeletal collapse, ultimately driving cell death \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eN\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e5. NY-1@β-CD induced rapid death exhibited promising in vivo anti-tumor activity\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNext, the significance of NY-1@β-CD for SCC treatment was explored \u003cem\u003ein vivo\u003c/em\u003e. Since previous literature reports indicate that intraperitoneal administration of nanoparticles can achieve a greater tumor accumulation by avoiding the reticuloendothelial system cleaning compared with intravenous administration\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, and in order to compare the therapeutic efficacy with the FDA approved drug 5-FU with intraperitoneal administration, we chose to inject the NY-1@β-CD intraperitoneally. We constructed cell line-derived xenograft (CDX) models by subcutaneously injecting Cal-27 (HNSCC cell line) and A431 (cSCC cell line) into BALB/c nude mice. Results showed that NY-1@β-CD (15 mg/kg/4 day i.p) significantly inhibited the progression in both xenografts \u003cem\u003ein vivo\u003c/em\u003e (Cal-27, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; A431, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), while no obvious tumor volume changes were observed in the 5-FU group at the same condition (Cal-27, ns; A431, ns) (\u003cb\u003eFig.\u0026nbsp;5AB\u003c/b\u003e). At the end of the experiment, tumors in the NY-1@β-CD group appeared visibly reduced (\u003cb\u003eFig. S5AB\u003c/b\u003e). To further evaluate the inhibition on CDX tumors, histological evaluation and Ki67 immunohistochemical (IHC) staining were performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Revealing significantly lower Ki67 positive rate in the NY-1@β-CD group compared to the control group (Cal-27, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; A431, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), not in the 5-FU group (Cal-27, ns; A431, ns) (\u003cb\u003eFig.\u0026nbsp;5DE\u003c/b\u003e). At the end of the experiment, no significant weight loss was observed (\u003cb\u003eFig. S5CD\u003c/b\u003e). Histological evaluation following NY-1@β-CD treatment showed minimal changes in the major organs (heart, kidney, liver, lung and spleen) (\u003cb\u003eFig. S5EF\u003c/b\u003e). These findings suggest that NY-1@β-CD demonstrates antitumor potential with minimal systemic toxicity \u003cem\u003ein vivo\u003c/em\u003e in SCC CDX models.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMeanwhile, we constructed HNSCC patient-derived xenograft (PDX) models which originated from different localization, PDX1 (derived from tongue abdominal and oral floor squamous cell carcinoma tissue) and PDX2 (derived from cheek squamous cell carcinoma tissue), respectively. The growth curve of the transplanted tumors showed that NY-1@β-CD treatment (15 mg/kg/4 day i.p) resulted in decreased tumor growth (PDX1, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; PDX2, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (\u003cb\u003eFig.\u0026nbsp;5FG\u003c/b\u003e). At the end of the study, tumors in the NY-1@β-CD treatment group appeared visually smaller compared to those in the control group (\u003cb\u003eFig. S5GH\u003c/b\u003e). Ki67 immunohistochemical staining was performed to evaluate tumor proliferation \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e, revealing a significantly lower Ki67-positive rate in the NY-1@β-CD treatment group (PDX1, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; PDX2, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (\u003cb\u003eFig.\u0026nbsp;5IJ\u003c/b\u003e). Additionally, no changes in body weight were detected at the end of the experiment (\u003cb\u003eFig. S5IJ\u003c/b\u003e). Furthermore, no significant pathological alterations were observed in the organs (heart, kidney, liver, lung, and spleen) of NY-1@β-CD treated mice in both PDX models (\u003cb\u003eFig. S5KL\u003c/b\u003e). Overall, the results indicate that rapid and VCP inhibition targeted by NY-1@β-CD exhibits robust antitumor efficacy with minimal systemic toxicity \u003cem\u003ein vivo\u003c/em\u003e within HNSCC PDX models.\u003c/p\u003e \u003cp\u003eThe regulatory effects of NY-1 treatment on the F-actin-FAK-YAP1 downstream pathway \u003cem\u003ein vivo\u003c/em\u003e were further investigated. Immunohistochemical staining for CYR61 was conducted on tumor tissues obtained from CDX models (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK). The NY-1@β-CD treatment group exhibited significant inhibition of CYR61 expression in Cal-27 models (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and A431 models (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL). Additionally, CYR61 positive rates were assessed by immunohistochemical staining in PDX models (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM). Compared to the control group, the CYR61 positive rates were significantly reduced in both PDX1 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and PDX2 models (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eN). These findings suggest that the F-actin-FAK-YAP1 pathway is modulated following VCP inhibition and calcium dyshomeostasis in response to NY-1@β-CD treatment \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eER stress regulation is an important mechanism in tumor treatment. The development of new drugs and new materials provides new strategies for regulating ER stress. In this study, we synthesized a potent anti-tumor small molecule NY-1, which is constructed with uracil and Ad through Michael addition reaction. Based on host-guest recognition, β-CD was introduced to construct PH-responsive supramolecular nanoparticles NY-1@β-CD. This supramolecular nanoparticle can be rapidly taken up by tumor cells and achieve lysosomal escape to release NY-1 within 3 hours, and rapidly induce tumor cells death which is characterized by F-actin depolymerization within 6 hours. Further mechanism studies revealed that NY-1 mainly can target VCP proteins and induce ER stress, the activation of IRE1α through ER stress can lead to the dissociation of SSH1 and 14-3-3. The dissociation of SSH1-14-3-3 complex further causing the cofilin mediated F-actin depolymerization. Further in vivo studies demonstrated that intraperitoneal administration of NY-1@β-CD showed significant efficacy in a variety of CDX and PDX models, and its effect was more significantly superior than the FDA-approved anti-tumor drug 5-FU. In conclusion, this supramolecular nanoparticles based on stress-effector targeted molecules provides a solution for future anti-tumor treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge support from the National Natural Science Foundation of China (NSFC) (Grant Nos. 82470984, 82271035, U25A6003, 82330029, 32200577), National Key Research and Development Program of China (Grant Nos. 2022YFC2402901), for financial support. All animal experiments are conducted in accordance with the guidelines outlined in the “Principles of Laboratory Animal Care” and were approved by Medical Ethics Committee of West China Stomatology Hospital, Sichuan University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll datasets on which the conclusions of the paper rely are available to readers.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eWalter P and D Ron. The unfolded protein response: from stress pathway to homeostatic regulation. Science, 2011,334(6059): 1081-6.\u003c/li\u003e\n \u003cli\u003eGenuth N R and A Dillin. Translational regulation in stress biology. NATURE CELL BIOLOGY, 2025,27(10): 1609-1621.\u003c/li\u003e\n \u003cli\u003eZinszner H, M Kuroda, X Wang, et al. CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum. Genes Dev, 1998,12(7): 982-95.\u003c/li\u003e\n \u003cli\u003eUrano F, X Wang, A Bertolotti, et al. Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1. Science, 2000,287(5453): 664-6.\u003c/li\u003e\n \u003cli\u003eMandula J K, S Chang, E Mohamed, et al. Ablation of the endoplasmic reticulum stress kinase PERK induces paraptosis and type I interferon to promote anti-tumor T cell responses. CANCER CELL, 2022,40(10): 1145-+.\u003c/li\u003e\n \u003cli\u003eChen Z M, P L Chen, J Y Li, et al. External strain on the plasma membrane is relayed to the endoplasmic reticulum by membrane contact sites and alters cellular energetics. SCIENCE ADVANCES, 2025,11(26):\u003c/li\u003e\n \u003cli\u003eTurishcheva E P, G A Ashniev, M S Vildanova, et al. Endoplasmic Reticulum Stress Inducer Dithiothreitol Affects the Morphology and Motility of Cultured Human Dermal Fibroblasts and Fibrosarcoma HT1080 Cell Line. RUSSIAN JOURNAL OF DEVELOPMENTAL BIOLOGY, 2023,54(5): 309-323.\u003c/li\u003e\n \u003cli\u003evan Vliet A R, F Giordano, S Gerlo, et al. The ER Stress Sensor PERK Coordinates ER-Plasma Membrane Contact Site Formation through Interaction with Filamin-A and F-Actin Remodeling. Molecular Cell, 2017,65(5): 885-899.e6.\u003c/li\u003e\n \u003cli\u003eBalasubramaniam L, S Monfared, A Ardaseva, et al. Dynamic forces shape the survival fate of eliminated cells. NATURE PHYSICS, 2025,21(2):\u003c/li\u003e\n \u003cli\u003eDupr\u0026eacute; L, I Castanon,and K Boztug. Immune-related actinopathies at the cross-road of immunodeficiency, autoimmunity and autoinflammation. NATURE REVIEWS IMMUNOLOGY, 2025,\u003c/li\u003e\n \u003cli\u003eMeyer H, M Bug,and S Bremer. Emerging functions of the VCP/p97 AAA-ATPase in the ubiquitin system. Nature Cell Biology, 2012,14(2): 117-123.\u003c/li\u003e\n \u003cli\u003eBaba O, F Zou, T Horie, et al. A novel VCP modulator, KUS121, attenuates atherosclerosis progression by maintaining intracellular ATP and mitigating ER stress in endothelial cells. EUROPEAN HEART JOURNAL, 2024,45(\u003c/li\u003e\n \u003cli\u003eZou F Q, O Baba, T Horie, et al. KUS121, a novel VCP modulator, attenuates atherosclerosis development by reducing ER stress and inhibiting glycolysis through the maintenance of ATP levels in endothelial cells. ATHEROSCLEROSIS, 2025,405(\u003c/li\u003e\n \u003cli\u003eTang M, J T Qiu, Y F Lu, et al. Molecular Recognition Driven Organelle Cross-Linking Induces Endoplasmic Reticulum Stress and Mitochondrial Dysfunction to Potentiate Cancer Immunotherapy. ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 2025,64(47):\u003c/li\u003e\n \u003cli\u003eChen X T, T T Wu, Y L Chen, et al. Supramolecular Host-Guest Assemblies for Tunable and Modular Lysosome-Targeting Protein Degradation. ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 2025,64(33):\u003c/li\u003e\n \u003cli\u003eDavis M E and M E Brewster. Cyclodextrin-based pharmaceutics: past, present and future. Nat Rev Drug Discov, 2004,3(12): 1023-35.\u003c/li\u003e\n \u003cli\u003eColby A H, J Kirsch, A N Patwa, et al. Radiolabeled Biodistribution of Expansile Nanoparticles: Intraperitoneal Administration Results in Tumor Specific Accumulation. ACS NANO, 2023,17(3): 2212-2221.\u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9008638/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9008638/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe endoplasmic reticulum (ER) maintains cellular proteostasis through the unfolded protein response (UPR), whereas excessive or unresolved stress can rapidly compromise cell viability. Beyond the well characterized transcriptional programs, how ER stress acutely remodels cellular architecture to induce cell death remains poorly understood. Here, we report the rational design of a nucleoside-based small molecule inhibitor of the AAA-ATPase VCP (NY-1), which forms a stable host-guest complex with β-cyclodextrin. NY-1 induces robust ER stress and a strikingly rapid form of cell death characterized by actin cytoskeletal collapse and synchronized membrane blebbing. We identify a non-transcriptional signaling pathway in which ER stress activated IRE1α remodels the 14-3-3 interactome, releasing and activating the phosphatase SSH1, thereby promoting cofilin dephosphorylation and F-actin disassembly. Chemical chaperone treatment restores cytoskeletal integrity and cell viability, demonstrating a direct causal link between ER proteostasis disruption and actin network collapse. These findings reveal an acute ER stress signaling axis controlling actin dynamics and establish NY-1 as a chemical probe for dissecting rapid cell death programs.\u003c/p\u003e","manuscriptTitle":"VCP inhibition induces rapid cell death through ER stress driven actin cytoskeletal collapse","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-12 09:59:23","doi":"10.21203/rs.3.rs-9008638/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"11cba30f-6595-4537-9a24-de6075b6ad83","owner":[],"postedDate":"March 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":63858094,"name":"Biological sciences/Cell biology/Cell adhesion"},{"id":63858095,"name":"Biological sciences/Cancer/Tumour-suppressor proteins"}],"tags":[],"updatedAt":"2026-03-24T10:20:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-12 09:59:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9008638","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9008638","identity":"rs-9008638","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}