Water-soluble Yb 3+ , Er 3+ codoped NaYF 4 nanoparticles induced SGC-7901 cell death through mitochondrial dysfunction and ROS-mediated ER stress

Water-soluble Yb 3+ , Er 3+ codoped NaYF 4 nanoparticles induced SGC-7901 cell death through mitochondrial dysfunction and ROS-mediated ER stress | 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 Research Article Water-soluble Yb 3+ , Er 3+ codoped NaYF 4 nanoparticles induced SGC-7901 cell death through mitochondrial dysfunction and ROS-mediated ER stress Shaoqiang Sun, jingwei Mao, Chen Liu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1850889/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 Background： Nanoparticles are potential luminescent probes. The objective of this study was to examine the cytotoxicity and underlying mechanism of upconversion nanoparticles (UCNPs). Methods: The effects of 0-400μg/mL UCNPs on human gastric adenocarcinoma (SGC-7901) cells were investigated. Flow cytometry was used to evaluate reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), intracellular Ca 2+ levels, and apoptosis. Caspase-3 and 9 activities were measured using commercial kits. Cytochrome C (CytC) in the cytosol and B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), protein kinase B (Akt), phosphorylated-Akt (p-Akt), 78 kDa glucose-regulated protein (GRP78), 94 kDa glucose-regulated protein (GRP94), caspase-3, caspase- 9, calpain 1, and calpain 2 protein levels were detected using Western blotting. Results: Exposure to UCNPs inhibited the viability of SGC‑7901 cells vs. control (UNCP 0 µg/ml) in a concentration- and time-dependent manner. Exposure to UCNPs increased the proportion of SGC‑7901 cells in early apoptosis, and enhanced the Bax/Bcl-2 ratio, elevated ROS levels, decreased ΔΨm, increased intracellular Ca 2+ , induced apoptosis, increased CytC protein levels, decreased phosphorylated Akt protein levels, increased Caspase 3 and Caspade-9 activity and protein levels, and increased GRP-78, GRP-94, caplain 1 and caplain 2 protein levels in SGC‑7901 cells vs. control. Conclusions: UCNPs induced SGC-7901 cell death by promoting mitochondrial dysfunction and ROS-mediated ER stress, initiating the caspase 9/caspase 3 cascade. These findings provide valuable insights relevant to the development of effective anti-cancer therapies that target specific signaling pathways. water-soluble upconversion nanoparticles SGC-7901 cells reactive oxygen species mitochondrial dysfunction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The mortality rate associated with gastric cancer is the one of the highest among all gastrointestinal cancers. 1 Endoscopic techniques are key to the early diagnosis and treatment of gastric cancer; 2 however, the use of traditional endoscopic dye carries several disadvantages. 3 Biomedical nanotechnology is an alternative approach, and rare earth conversion nanomaterials are of particular interest. The near-infrared light excitation source has the advantages of large nanoparticle penetration depth while exerting minimal damage on biological tissue. 4 , 5 2 mol% Er 3+ / 20 mol% Yb 3+ codoped NaYF 4 upconverting nanoparticles (UCNPs) prepared with polyvinylpyrrolidone (PVP) as a surfactant and the facile solvothermal approach have excellent luminescent efficiency. 6 Nanoparticles have been tested for physical properties, in vitro imaging, and luminescent properties; however, the underlying molecular mechanisms of UCNPs in human gastric cell lines have yet to be investigated. Tumor cells have elevated reactive oxygen species (ROS) levels, redox imbalance, and increased oxidative stress. 7 In various tumor cells, elevated ROS levels mediate endoplasmic reticulum (ER) stress. 8 ER stress regulates several signaling cascades, including ER-associated protein degradation and glucose-regulated proteins (GRP). 9 In vitro, over-expression of antisense transcripts and ribozyme-based approaches demonstrated that 78kDa glucose-regulated protein (GRP78) and 94 kDa glucose-regulated protein (GRP94) may protect against cell death. 10 – 12 Similarly, curcumin may induce apoptosis in human lung carcinoma cells through GRP78 upregulation. 13 This study examined the cytotoxicity of UCNPs in gastric adenocarcinoma cells. Materials And Methods Cell culture The human gastric adenocarcinoma cell line SGC-7901 and the human gastric epithelial cell line GES-1 were obtained from the Key Laboratory of Blood Cancer Center, Ministry of Education, at Jilin University (Jilin, China). Cells were maintained in DMEM medium (Beijing Dingguo Changsheng Biotechnology, Co., Ltd., Beijing, China), supplemented with 10% FBS, penicillin (100 U/ml, Beyotime Institute of Biotechnology), and streptomycin (100 mg/ml, Beyotime Institute of Biotechnology) at 37˚C in 5% CO 2 . MTT assay An MTT assay (Beyotime Institute of Biotechnology) was used to assess GES-1 and SGC-7901 cell viability following exposure to UCNPs. 14 Cells were treated with 0, 50, 100, 150, 200 or 400 µg/ml UCNPs for 24, 48 or 72 h. Subsequently, MTT medium was removed, and150 µl DMSO (Beijing Dingguo Changsheng Biotechnology, Co., Ltd.) was added. Absorbance at 490 nm was recorded with a Synergy™ 4 Microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). Flow cytometry Flow cytometry with the Annexin V-FITC/propidium iodide (PI) double-labeling method was used to measure the apoptotic rate induced by UCNPs. SGC-7901 cells were treated with 0, 50, 100, 150, 200 or 400 µg/ml UCNPs for 24 h. Cells were incubated with Annexin V-FITC (5 µg/ml, 2019.05-2020.05) (Beyotime Institute of Biotechnology) at 5˚C for 15 min and incubated with PI (5 µg/ml, 2019.05-2020.05) at 5˚C for 5 min. Samples were analyzed on a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) with CELL-Quest software version 2.7 (Microsoft Corporation, Redmond, WA, USA). Measurement of ROS, mitochondrial membrane potential (ΔΨm), intracellular calcium, the triphosphate nick-end labeling (TUNEL) assay, and caspase activity SGC-7901 cells (2x10 5 cells/ml) were cultured in 6-well plates for 24 h and exposed to 0, 50, 100, 150, 200 or 400 µg/ml UCNPs for a further 24 h. ROS were studied by incubating cells with 10 µM dichloro-dihydro-fluorescein diacetate (DCFH-DA) (Beyotime Institute of Biotechnology) at 37˚C for 30 min. ΔΨm was measured by incubating cells with rhodamine 123 (Rh123; 10 µM) (Beyotime Institute of Biotechnology) at 37˚C for 30 min. Intracellular Ca 2+ was evaluated by incubating cells with Fluo-3/AM (8 µmol/l) at 37˚C for 45 min. Apoptosis was assessed using TUNEL cell apoptosis assay kits (Beyotime Institute of Biotechnology), according to the manufacturer’s instructions; fixed cells (4% paraformaldehyde) were incubated with TUNEL detection solution at 37˚C for 1h. Samples for the measurement of ROS, ΔΨm, intracellular calcium, and apoptosis were analyzed on a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) with CELL-Quest software version 2.7 (Microsoft Corporation, Redmond, WA, USA). Caspase-3 and 9 activities were measured using commercial kits (Beyotime Institute of Biotechnology), according to the manufacturers’ instructions. Cells were incubated with cell lysis buffer on ice for 2 h. Protein concentration was measured in supernatants. Cell lysates were incubated at room temperature for 1 h, and fluorescence intensity was determined by absorbance at 405 nm using a Synergy™ 4 Microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). Western blot analysis SGC-7901 cells exposed to 0, 50, 100, 150, 200 or 400 µg/ml UCNPs for 24 h were incubated with cell lysis buffer on ice for 2 h. Protein concentration was measured in supernatants. Samples were separated by 8% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Primary antibodies were CytC (cat no. 11940; 1:1,000), Bcl-2 (cat no. 3498; 1:500), Bax (cat no. 2774; 1:500), Akt (cat no. 5373; 1:500) and p-Akt (cat no. 5012; 1:1,000) (Cell Signaling Technology, Inc., Danvers, MA, USA). Secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-rabbit polyclonal secondary antibody (A0208, 1:10,000) and HRP-conjugated goat anti-mouse polyclonal secondary antibody (A0216, 1:10,000) (Beyotime Institute of Biotechnology). Detection was by enhanced chemiluminescence. Images were captured on Kodak radiographic film (Kodak, Rochester, NY, USA) in the dark. Gray-scale analysis of the western blot images was conducted with photoshop (Microsoft Corporation). Statistical analysis Statistical analysis was performed using SPSS v19.0 for Windows (IBM SPSS, Armonk, NY, USA). Data are expressed as mean ± standard deviation. Comparisons were performed with the Student's t-test. P < 0.05 was considered statistically significant. Results Exposure to 50-400 µg/ml UCNPs for 24 h, 48 h, or 72 h inhibited GES‑1 and SGC‑7901 cell viability vs. control (UNCP 0 µg/ml) in a concentration- and time-dependent manner. Exposure to 100-400 µg/ml UCNPs for 24 h or 48 h (P<0.05) or 50-400 µg/ml UCNPs for 72 h (P<0.01) significantly inhibited the viability of SGC‑7901 cells vs. control. Exposure to 400 µg/ml UCNPs for 24 h or 150-400 µg/ml UCNPs for 72 h significantly inhibited the viability of GES‑1 cells vs. control (P<0.05) ( Fig. 1 ). Flow cytometry revealed that exposure to 400 µg/ml UCNPs for 24 h induced SGC‑7901 cell death ( Fig. 2A and B ). Western blot analysis demonstrated that exposure to 50-400 µg/ml UCNPs for 24 h increased Bax and decreased Bcl-2 protein levels in SGC‑7901 cells vs. control, in a concentration-dependent manner. Exposure to 400 µg/ml UCNPs for 24 h significantly increased Bax protein levels (P<0.05) and exposure to 150-400 µg/ml UCNPs for 24 h (P<0.01) significantly decreased Bcl-2 protein levels in SGC‑7901 cells vs. control ( Fig. 2C ). Disruption of mitochondrial integrity is one indicator of early apoptosis. 15 Exposure to 50-400 µg/ml UCNPs for 24 h significantly decreased ΔΨm in SGC‑7901 cells vs. control in a concentration-dependent manner (P<0.05; Fig. 3A ). Exposure to 200 or 400 µg/ml UCNPs for 24 h significantly increased intracellular Ca 2+ in SGC‑7901 cells vs. control (P<0.05; Fig. 3B ). TUNEL results indicated that exposure to 200 or 400 µg/ml UCNPs for 24 h significantly induced apoptosis in SGC‑7901 cells compare to control (P<0.05; Fig. 3C ). Release of CytC usually accompanies a decrease inΔΨm. 16 Western blot analysis revealed that exposure to 400 µg/ml UCNPs for 24 h significantly increased CytC protein levels in SGC‑7901 cells vs. control (P<0.05; Fig. 3D ). The PI3K-AKT signaling pathway regulates tumor cell proliferation and resistance to chemotherapy. 17 Western blot analysis revealed that exposure to 400 µg/ml UCNPs for 24 h significantly decreased phosphorylated Akt protein levels in SGC‑7901 cells vs. control, which may have proapoptotic effects (P<0.05; Fig. 3E ). These results imply that UCNPs induced mitochondrial dysfunction and activated apoptosis-related signaling pathways in SGC-7901 cells. Caspase 3 is a crucial mediator of apoptosis, and caspase 9 may trigger the mitochondrial apoptotic pathway. 18 Exposure to 50-400 µg/ml UCNPs for 24 h significantly increased caspase 3 activity and protein levels in SGC‑7901 cells vs. control in a concentration-dependent manner (P<0.05), with the greatest increase seen in SGC‑7901 cells exposed to 400 µg/ml UCNPs (P<0.01). Exposure to 400 µg/ml UCNPs for 24 h significantly increased caspase 9 activity and protein levels in SGC‑7901 cells vs. control (P<0.05, Fig. 4 A -D ). These results imply that UCNPs regulated apoptosis-related protein expression and induced SGC-7901 cell death by triggering the mitochondrial pathway. ROS are essential for cell proliferation and cell death. 19 Exposure to 50-400 µg/ml UCNPs for 24 h significantly increased ROS levels in SGC‑7901 cells vs. control in a concentration-dependent manner (P<0.05), with the greatest increase seen in SGC‑7901 cells exposed to 400 µg/ml UCNPs (P<0.001; Fig. 5A ). Numerous studies have indicated that ROS induce cell death through activation of ER stress. 8,9 GRP mediates ER homeostasis. 12 Exposure to 400 µg/ml UCNPs increased GRP-78 and GRP-94 protein levels in SGC‑7901 cells vs. control in a time-dependent manner ( Fig. 5B ). Exposure to tunicamycin (TM), an inducer of ER stress, increased GRP-78 and GRP-94 protein levels in SGC‑7901 cells vs. control ( Fig. 5B ). Exposure to 400 µg/ml UCNPs increased caplain 1 and caplain 2 protein levels in SGC‑7901 cells vs. control in a time-dependent manner ( Fig. 5C ). These results imply that UCNPs induced apoptosis in SGC-7901 cells through mitochondrial dysfunction and ROS-mediated ER stress, subsequently promoting the caspase 9/caspase 3 cascade ( Fig. 5D ). Discussion UCNPs are currently being developed as fluorescent probes for biomedical applications. Evidence suggests 200µg/ml UCNPs have notable fluorescence imaging capabilities and biocompatibility, and NaYF 4 :Yb 3+ /Er 3+ (Tm 3+ ) is the most efficient upconversion nanomaterial. 20 The present study showed that 400µg/ml UCNPs were cytotoxic to SGC‑7901 cells and GEC-1 cells, and the apoptosis rate of SGC‑7901 cells was higher than GES‑1 cells, implying that SGC-7901 cells are more sensitive to UCNPs than GES-1 cells. Exposure to 400 µg/ml UCNPs for 24 hours increased the proportion of SGC‑7901 cells in early apoptosis. Apoptosis is a type of programmed cell death that can be initiated by the death receptor-mediated extrinsic pathway and/or the mitochondria-mediated intrinsic pathway. 21 ROS may trigger mitochondrial-mediated apoptosis. 22 Exposure to 50-400 µg/ml UCNPs for 24 h significantly increased ROS levels in SGC‑7901 cells vs. control in a concentration-dependent manner, with the greatest increase seen in SGC‑7901 cells exposed to 400 µg/ml UCNPs. Tumor cells have higher ROS levels and sensitivity compared to normal cells, which can result in tumor cell damage or death. 23 ROS levels peaked in SGC-7901 cells exposed to 400 µg/ml UCNPs, which corresponded with a decrease in ΔΨm, increase in intracellular Ca + , elevated cytoplasmic expression of CytC, and increased Bax and decreased Bcl-2 expression. These findings imply that SGC-7901 cell death occurred through the mitochondrial-mediated pathway. Dissipation of ΔΨm across the mitochondrial membrane is considered an early apoptosis signal that leads to irreversible damage. 24 . The BCL-2-protein family regulate CytC release. 25 CytC is a pro-apoptotic factor that binds with apoptotic protease activating factor-1 to activate pro-caspase 9. 26 Caspase 3 is involved in a cascade of reactions, which ultimately lead to apoptosis. The PI3K-AKT signaling pathway regulates tumor cell proliferation and resistance to chemotherapy. 27 UCNPs inhibit Akt phosphorylation at the Ser473 site. This mechanism promotes the mitochondrial apoptotic pathway and may underlie UCNP-induced SGC-7901 cell death. In the present study, exposure to 400 µg/ml UCNPs increased GRP-78, GRP-94, caplain 1 and caplain 2 protein levels in SGC‑7901 cells vs. control in a time-dependent manner, and exposure to TM, an inducer of ER stress, increased GRP-78 and GRP-94 protein levels in SGC‑7901 cells vs. control. Calpain 1 and 2 are two cysteine protease families that regulate pathological cell death. 14 These data imply that UCNPs induced SGC-7901 cell death through the activation of ER stress. In conclusion, UCNPs induced SGC-7901 cell death by promoting mitochondrial dysfunction and ROS-mediated ER stress, initiating the caspase 9/caspase 3 cascade. These findings provide valuable insights relevant to the development of effective anti-cancer therapies that target specific signaling pathways. Declarations Ethics approval and consent to participate The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Ethics Committee of Dalian medical university. Consent for publication Not applicable. Availability of data and materials ： The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding The department of Gastroenterology of the first hospital of Dalian medical University supported the study. Authors' contributions Author 1 (shaoqiang sun): Conceptualization, Methodology, Software, Investigation, Formal Analysis, Writing - Original Draft； Author 2(jingwei mao): Data Curation, Writing - Original Draft； Corresponding Author(chen liu): Conceptualization, Funding Acquisition, Resources, Supervision, Writing - Review & Editing. Acknowledgements Not applicable. 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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-1850889","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":128458092,"identity":"29574dd0-8b94-46bc-8b96-cef8b17da15b","order_by":0,"name":"Shaoqiang Sun","email":"","orcid":"","institution":"Dalian Med Univ","correspondingAuthor":false,"prefix":"","firstName":"Shaoqiang","middleName":"","lastName":"Sun","suffix":""},{"id":128458093,"identity":"75543289-b964-401c-ad3b-98cd0a77d964","order_by":1,"name":"jingwei Mao","email":"","orcid":"","institution":"Dalian Med Univ","correspondingAuthor":false,"prefix":"","firstName":"jingwei","middleName":"","lastName":"Mao","suffix":""},{"id":128458094,"identity":"99e78138-181c-4225-8540-0847130c10ca","order_by":2,"name":"Chen Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYBACA2YGBmYQg42ZsfHBBwMbO+K18LE3HzacUZCWTFgLA1SLHM+xNGmeD4cYGwhqYecx/FxQcYeBTSLH2NjG4AAzA/vhoxvwO4zHWHrGmWcgLYaPcwzu8DHwpKXdIKDFjJm37TDElhyDZ8wMEjxmRGj5B9ZiJm1hcJixgTgtDUAtIO8zEKeFrVia5xhQCyiQewzSktkI+cW+//DGzzw1hxnkm4FR+eOPjR0/++FjeLXAQH0DjMVGjPJRMApGwSgYBfgBACgrPqqnLZstAAAAAElFTkSuQmCC","orcid":"","institution":"Dalian Med Univ","correspondingAuthor":true,"prefix":"","firstName":"Chen","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2022-07-12 14:14:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1850889/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1850889/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":25325552,"identity":"15134144-b619-4351-a336-9dbfb4713a92","added_by":"auto","created_at":"2022-08-17 14:52:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":99434,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability in SGC-7901 and GES-1 cells treated with 0-400 µg/ml UCNPs for (A) 24 h, (B) 48 h, and (C) 72 h. \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01 and\u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001 vs. control (0 µg/ml UCNPs). 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UCNPs, upconversion nanoparticles; Bcl‑2, B‑cell lymphoma‑2; Bax, Bcl‑2 associated X protein.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-1850889/v1/59785c29bda548fe3b1998db.png"},{"id":25324506,"identity":"0323b573-8dd6-4688-9c32-851990743191","added_by":"auto","created_at":"2022-08-17 14:47:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":486668,"visible":true,"origin":"","legend":"\u003cp\u003eSGC-7901 cells treated with 0-400 µg/ml UCNPs for 24 h (A) ΔΨm ; (B) intracellular Ca\u003csup\u003e2+\u003c/sup\u003e; (C) TUNEL staining; (D) Western blot of CytC protein;\u0026nbsp;(E) Western blot of p-Akt and Akt proteins. \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01 and \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001 vs. control (0 µg/ml UCNPs). UCNPs, upconversion nanoparticles; ΔΨm, mitochondrial membrane potential; TUNEL, terminal deoxynucleotidyl transferase dUTP nick‑end labeling; CytC, cytochrome C; Akt, protein kinase B; p-Akt, phosphorylated-Akt.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-1850889/v1/0e5e2518d98999f22f0e3092.png"},{"id":25324504,"identity":"5995ae89-173b-4e1d-ade5-7bbc8ec59fdf","added_by":"auto","created_at":"2022-08-17 14:47:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":256392,"visible":true,"origin":"","legend":"\u003cp\u003eSGC-7901 cells treated with 0-400 µg/ml UCNPs for 24 h (A) Caspase 3 activity; (B) Caspase 9 activity; (C and D) Western blot of caspase 3 and caspase 9 proteins. \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05 and \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01 vs. control (0 µg/ml UCNPs). UCNPs, upconversion nanoparticles.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-1850889/v1/1956b2f7bd90d73e9a08b647.png"},{"id":25324502,"identity":"6c43927e-42af-4425-a6e4-0cfb2a2ee01d","added_by":"auto","created_at":"2022-08-17 14:47:59","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":434219,"visible":true,"origin":"","legend":"\u003cp\u003e(A) SGC-7901 cells treated with 0-400 µg/ml UCNPs for 24 h: ROS levels; (B and C) SGC-7901 cells treated with 400 µg/ml UCNPs for 6-24 h: Western blot of GRP‑78, GRP-94, calpain 1 and calpain 2 proteins. (D) Schematic of the underlying molecular mechanism of UCNPs-induced SGC-7901 apoptosis. \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01 and\u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001 vs. control (0 µg/ml UCNPs). UCNPs, upconversion nanoparticles; ROS, reactive oxygen species; ER, endoplasmic reticulum; GRP, glucose-regulated protein.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-1850889/v1/9d91d3a05b380ea704391fc0.jpeg"},{"id":32635116,"identity":"07360eb2-0e91-41c5-a9a2-0ebc6718ce49","added_by":"auto","created_at":"2023-02-08 07:59:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1758227,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1850889/v1/84371142-c873-48a4-9b67-0092d50baa4d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Water-soluble Yb 3+ , Er 3+ codoped NaYF 4 nanoparticles induced SGC-7901 cell death through mitochondrial dysfunction and ROS-mediated ER stress","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe mortality rate associated with gastric cancer is the one of the highest among all gastrointestinal cancers.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Endoscopic techniques are key to the early diagnosis and treatment of gastric cancer;\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e however, the use of traditional endoscopic dye carries several disadvantages.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Biomedical nanotechnology is an alternative approach, and rare earth conversion nanomaterials are of particular interest. The near-infrared light excitation source has the advantages of large nanoparticle penetration depth while exerting minimal damage on biological tissue.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e 2 mol% Er\u003csup\u003e3+\u003c/sup\u003e/ 20 mol% Yb\u003csup\u003e3+\u003c/sup\u003e codoped NaYF\u003csub\u003e4\u003c/sub\u003e upconverting nanoparticles (UCNPs) prepared with polyvinylpyrrolidone (PVP) as a surfactant and the facile solvothermal approach have excellent luminescent efficiency.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Nanoparticles have been tested for physical properties, in vitro imaging, and luminescent properties; however, the underlying molecular mechanisms of UCNPs in human gastric cell lines have yet to be investigated.\u003c/p\u003e \u003cp\u003eTumor cells have elevated reactive oxygen species (ROS) levels, redox imbalance, and increased oxidative stress.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e In various tumor cells, elevated ROS levels mediate endoplasmic reticulum (ER) stress.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e ER stress regulates several signaling cascades, including ER-associated protein degradation and glucose-regulated proteins (GRP).\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e In vitro, over-expression of antisense transcripts and ribozyme-based approaches demonstrated that 78kDa glucose-regulated protein (GRP78) and 94 kDa glucose-regulated protein (GRP94) may protect against cell death.\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Similarly, curcumin may induce apoptosis in human lung carcinoma cells through GRP78 upregulation.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e This study examined the cytotoxicity of UCNPs in gastric adenocarcinoma cells.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eThe human gastric adenocarcinoma cell line SGC-7901 and the human gastric epithelial cell line GES-1 were obtained from the Key Laboratory of Blood Cancer Center, Ministry of Education, at Jilin University (Jilin, China). Cells were maintained in DMEM medium (Beijing Dingguo Changsheng Biotechnology, Co., Ltd., Beijing, China), supplemented with 10% FBS, penicillin (100 U/ml, Beyotime Institute of Biotechnology), and streptomycin (100 mg/ml, Beyotime Institute of Biotechnology) at 37˚C in 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMTT assay\u003c/h2\u003e \u003cp\u003eAn MTT assay (Beyotime Institute of Biotechnology) was used to assess GES-1 and SGC-7901 cell viability following exposure to UCNPs.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Cells were treated with 0, 50, 100, 150, 200 or 400 \u0026micro;g/ml UCNPs for 24, 48 or 72 h. Subsequently, MTT medium was removed, and150 \u0026micro;l DMSO (Beijing Dingguo Changsheng Biotechnology, Co., Ltd.) was added. Absorbance at 490 nm was recorded with a Synergy\u0026trade; 4 Microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eFlow cytometry with the Annexin V-FITC/propidium iodide (PI) double-labeling method was used to measure the apoptotic rate induced by UCNPs. SGC-7901 cells were treated with 0, 50, 100, 150, 200 or 400 \u0026micro;g/ml UCNPs for 24 h. Cells were incubated with Annexin V-FITC (5 \u0026micro;g/ml, 2019.05-2020.05) (Beyotime Institute of Biotechnology) at 5˚C for 15 min and incubated with PI (5 \u0026micro;g/ml, 2019.05-2020.05) at 5˚C for 5 min. Samples were analyzed on a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) with CELL-Quest software version 2.7 (Microsoft Corporation, Redmond, WA, USA).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eMeasurement of ROS, mitochondrial membrane potential (ΔΨm), intracellular calcium, the triphosphate nick-end labeling (TUNEL) assay, and caspase activity\u003c/span\u003e \u003c/p\u003e \u003cp\u003eSGC-7901 cells (2x10\u003csup\u003e5\u003c/sup\u003e cells/ml) were cultured in 6-well plates for 24 h and exposed to 0, 50, 100, 150, 200 or 400 \u0026micro;g/ml UCNPs for a further 24 h. ROS were studied by incubating cells with 10 \u0026micro;M dichloro-dihydro-fluorescein diacetate (DCFH-DA) (Beyotime Institute of Biotechnology) at 37˚C for 30 min. ΔΨm was measured by incubating cells with rhodamine 123 (Rh123; 10 \u0026micro;M) (Beyotime Institute of Biotechnology) at 37˚C for 30 min. Intracellular Ca\u003csup\u003e2+\u003c/sup\u003e was evaluated by incubating cells with Fluo-3/AM (8 \u0026micro;mol/l) at 37˚C for 45 min. Apoptosis was assessed using TUNEL cell apoptosis assay kits (Beyotime Institute of Biotechnology), according to the manufacturer\u0026rsquo;s instructions; fixed cells (4% paraformaldehyde) were incubated with TUNEL detection solution at 37˚C for 1h. Samples for the measurement of ROS, ΔΨm, intracellular calcium, and apoptosis were analyzed on a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) with CELL-Quest software version 2.7 (Microsoft Corporation, Redmond, WA, USA). Caspase-3 and 9 activities were measured using commercial kits (Beyotime Institute of Biotechnology), according to the manufacturers\u0026rsquo; instructions. Cells were incubated with cell lysis buffer on ice for 2 h. Protein concentration was measured in supernatants. Cell lysates were incubated at room temperature for 1 h, and fluorescence intensity was determined by absorbance at 405 nm using a Synergy\u0026trade; 4 Microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eSGC-7901 cells exposed to 0, 50, 100, 150, 200 or 400 \u0026micro;g/ml UCNPs for 24 h were incubated with cell lysis buffer on ice for 2 h. Protein concentration was measured in supernatants. Samples were separated by 8% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Primary antibodies were CytC (cat no. 11940; 1:1,000), Bcl-2 (cat no. 3498; 1:500), Bax (cat no. 2774; 1:500), Akt (cat no. 5373; 1:500) and p-Akt (cat no. 5012; 1:1,000) (Cell Signaling Technology, Inc., Danvers, MA, USA). Secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-rabbit polyclonal secondary antibody (A0208, 1:10,000) and HRP-conjugated goat anti-mouse polyclonal secondary antibody (A0216, 1:10,000) (Beyotime Institute of Biotechnology). Detection was by enhanced chemiluminescence. Images were captured on Kodak radiographic film (Kodak, Rochester, NY, USA) in the dark. Gray-scale analysis of the western blot images was conducted with photoshop (Microsoft Corporation).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using SPSS v19.0 for Windows (IBM SPSS, Armonk, NY, USA). Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Comparisons were performed with the Student's t-test. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eExposure to 50-400\u0026nbsp;\u0026micro;g/ml\u0026nbsp;UCNPs\u0026nbsp;for 24 h, 48 h, or 72 h inhibited GES‑1 and SGC‑7901 cell viability vs. control (UNCP 0 \u0026micro;g/ml) in a concentration- and time-dependent manner.\u0026nbsp;Exposure to 100-400\u0026nbsp;\u0026micro;g/ml\u0026nbsp;UCNPs\u0026nbsp;for 24 h or 48 h (P\u0026lt;0.05) or\u0026nbsp;50-400\u0026nbsp;\u0026micro;g/ml\u0026nbsp;UCNPs\u0026nbsp;for 72 h (P\u0026lt;0.01) significantly inhibited the viability of SGC‑7901 cells vs. control.\u0026nbsp;Exposure to 400\u0026nbsp;\u0026micro;g/ml\u0026nbsp;UCNPs\u0026nbsp;for 24 h or\u0026nbsp;150-400\u0026nbsp;\u0026micro;g/ml\u0026nbsp;UCNPs\u0026nbsp;for 72 h significantly inhibited the viability of GES‑1 cells vs. control (P\u0026lt;0.05) (\u003cstrong\u003eFig. 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eFlow cytometry revealed that exposure to 400 \u0026micro;g/ml UCNPs\u0026nbsp;for 24 h\u0026nbsp;induced SGC‑7901 cell death (\u003cstrong\u003eFig. 2A\u003c/strong\u003e and \u003cstrong\u003eB\u003c/strong\u003e). Western blot analysis demonstrated that\u0026nbsp;exposure to 50-400\u0026nbsp;\u0026micro;g/ml\u0026nbsp;UCNPs\u0026nbsp;for 24 h increased Bax and decreased Bcl-2 protein levels in SGC‑7901 cells vs. control, in a concentration-dependent manner.\u0026nbsp;Exposure to 400\u0026nbsp;\u0026micro;g/ml\u0026nbsp;UCNPs\u0026nbsp;for 24 h significantly increased Bax protein levels (P\u0026lt;0.05)\u0026nbsp;and exposure to 150-400\u0026nbsp;\u0026micro;g/ml\u0026nbsp;UCNPs\u0026nbsp;for 24 h (P\u0026lt;0.01) significantly decreased Bcl-2 protein levels in SGC‑7901 cells vs. control ( \u003cstrong\u003eFig. 2C\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eDisruption of mitochondrial integrity is one indicator of early apoptosis.\u003csup\u003e15\u003c/sup\u003e Exposure to 50-400\u0026nbsp;\u0026micro;g/ml\u0026nbsp;UCNPs\u0026nbsp;for 24 h significantly decreased \u0026Delta;\u0026Psi;m in SGC‑7901 cells vs. control in a concentration-dependent manner (P\u0026lt;0.05;\u0026nbsp;\u003cstrong\u003eFig. 3A\u003c/strong\u003e).\u0026nbsp;Exposure to 200 or 400\u0026nbsp;\u0026micro;g/ml\u0026nbsp;UCNPs\u0026nbsp;for 24 h significantly increased intracellular\u0026nbsp;Ca\u003csup\u003e2+\u003c/sup\u003e in SGC‑7901 cells vs. control (P\u0026lt;0.05; \u003cstrong\u003eFig. 3B\u003c/strong\u003e).\u0026nbsp;TUNEL results indicated that\u0026nbsp;exposure to 200 or 400\u0026nbsp;\u0026micro;g/ml\u0026nbsp;UCNPs for 24 h significantly\u0026nbsp;induced apoptosis in SGC‑7901 cells compare to control (P\u0026lt;0.05; \u003cstrong\u003eFig. 3C\u003c/strong\u003e). Release of CytC usually accompanies a decrease in\u0026Delta;\u0026Psi;m.\u003csup\u003e16\u003c/sup\u003e Western blot analysis revealed that\u0026nbsp;exposure to 400\u0026nbsp;\u0026micro;g/ml\u0026nbsp;UCNPs\u0026nbsp;for 24 h significantly increased CytC protein levels in SGC‑7901 cells vs. control (P\u0026lt;0.05; \u003cstrong\u003eFig. 3D\u003c/strong\u003e). The PI3K-AKT signaling pathway regulates tumor cell proliferation and resistance to chemotherapy.\u003csup\u003e17\u0026nbsp;\u003c/sup\u003eWestern blot analysis revealed that\u0026nbsp;exposure to 400\u0026nbsp;\u0026micro;g/ml\u0026nbsp;UCNPs\u0026nbsp;for 24 h significantly decreased phosphorylated Akt protein levels in SGC‑7901 cells vs. control, which may have proapoptotic effects (P\u0026lt;0.05;\u003cstrong\u003e\u0026nbsp;Fig. 3E\u003c/strong\u003e). These results imply that UCNPs induced mitochondrial dysfunction and activated apoptosis-related signaling pathways in SGC-7901 cells.\u003c/p\u003e\n\u003cp\u003eCaspase 3 is a crucial mediator of apoptosis, and caspase 9 may trigger the mitochondrial apoptotic pathway.\u003csup\u003e18\u003c/sup\u003e Exposure to 50-400\u0026nbsp;\u0026micro;g/ml\u0026nbsp;UCNPs\u0026nbsp;for 24 h significantly increased caspase 3 activity and protein levels in SGC‑7901 cells vs. control in a concentration-dependent manner (P\u0026lt;0.05), with the greatest increase seen in SGC‑7901 cells exposed to 400 \u0026micro;g/ml\u0026nbsp;UCNPs (P\u0026lt;0.01).\u0026nbsp;Exposure to 400\u0026nbsp;\u0026micro;g/ml\u0026nbsp;UCNPs\u0026nbsp;for 24 h significantly increased caspase 9 activity and protein levels in SGC‑7901 cells vs. control (P\u0026lt;0.05, \u003cstrong\u003eFig. 4 A -D\u003c/strong\u003e). These results imply that UCNPs regulated apoptosis-related protein expression and induced SGC-7901 cell death by triggering the mitochondrial pathway.\u003c/p\u003e\n\u003cp\u003eROS are essential for cell proliferation and cell death.\u003csup\u003e19\u0026nbsp;\u003c/sup\u003eExposure to 50-400\u0026nbsp;\u0026micro;g/ml\u0026nbsp;UCNPs\u0026nbsp;for 24 h significantly increased ROS levels in SGC‑7901 cells vs. control in a concentration-dependent manner (P\u0026lt;0.05), with the greatest increase seen in SGC‑7901 cells exposed to 400 \u0026micro;g/ml\u0026nbsp;UCNPs\u0026nbsp;(P\u0026lt;0.001; \u003cstrong\u003eFig. 5A\u003c/strong\u003e). Numerous studies have indicated that ROS induce cell death through activation of ER stress.\u003csup\u003e8,9\u003c/sup\u003e GRP mediates ER homeostasis.\u003csup\u003e12\u0026nbsp;\u003c/sup\u003eExposure to 400\u0026nbsp;\u0026micro;g/ml\u0026nbsp;UCNPs increased\u0026nbsp;GRP-78 and GRP-94 protein levels in SGC‑7901 cells vs. control in a time-dependent manner (\u003cstrong\u003eFig. 5B\u003c/strong\u003e). Exposure to tunicamycin (TM), an inducer of ER stress, increased GRP-78 and GRP-94 protein levels in SGC‑7901 cells vs. control (\u003cstrong\u003eFig. 5B\u003c/strong\u003e). Exposure to 400\u0026nbsp;\u0026micro;g/ml\u0026nbsp;UCNPs increased\u0026nbsp;caplain 1 and caplain 2 protein levels in SGC‑7901 cells vs. control in a time-dependent manner (\u003cstrong\u003eFig. 5C\u003c/strong\u003e). These results imply that UCNPs induced apoptosis in\u0026nbsp;SGC-7901 cells through mitochondrial dysfunction and ROS-mediated ER stress, subsequently promoting the caspase 9/caspase 3 cascade (\u003cstrong\u003eFig. 5D\u003c/strong\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eUCNPs are currently being developed as fluorescent probes for biomedical applications. Evidence suggests 200\u0026micro;g/ml UCNPs have notable fluorescence imaging capabilities and biocompatibility, and NaYF\u003csub\u003e4\u003c/sub\u003e:Yb\u003csup\u003e3+\u003c/sup\u003e/Er\u003csup\u003e3+\u003c/sup\u003e (Tm\u003csup\u003e3+\u003c/sup\u003e) is the most efficient upconversion nanomaterial.\u003csup\u003e20 \u0026nbsp;\u003c/sup\u003eThe present study showed that 400\u0026micro;g/ml UCNPs\u003csup\u003e\u0026nbsp;\u003c/sup\u003ewere cytotoxic to SGC‑7901 cells and GEC-1 cells, and the apoptosis rate of SGC‑7901 cells was higher than GES‑1 cells, implying that SGC-7901 cells are more sensitive to UCNPs than GES-1 cells.\u003c/p\u003e\n\u003cp\u003eExposure to 400 \u0026micro;g/ml UCNPs for 24 hours increased the proportion of SGC‑7901 cells in early apoptosis. Apoptosis is a type of programmed cell death that can be initiated by the death receptor-mediated extrinsic pathway and/or the mitochondria-mediated intrinsic pathway.\u003csup\u003e21\u003c/sup\u003e ROS may trigger mitochondrial-mediated apoptosis.\u003csup\u003e22\u0026nbsp;\u003c/sup\u003eExposure to 50-400\u0026nbsp;\u0026micro;g/ml\u0026nbsp;UCNPs\u0026nbsp;for 24 h significantly increased ROS levels in SGC‑7901 cells vs. control in a concentration-dependent manner, with the greatest increase seen in SGC‑7901 cells exposed to 400 \u0026micro;g/ml\u0026nbsp;UCNPs. Tumor cells have higher ROS levels and sensitivity compared to normal cells, which can result in tumor cell damage or death. \u003csup\u003e23\u0026nbsp;\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eROS levels peaked in SGC-7901 cells exposed to 400 \u0026micro;g/ml UCNPs, which corresponded with a\u0026nbsp;decrease in \u0026Delta;\u0026Psi;m, increase in intracellular Ca\u003csup\u003e+\u003c/sup\u003e, elevated cytoplasmic expression of CytC, and increased Bax and decreased Bcl-2 expression. These findings imply that SGC-7901 cell death occurred through the mitochondrial-mediated pathway. Dissipation of \u0026Delta;\u0026Psi;m across the mitochondrial membrane is considered an early apoptosis signal that leads to irreversible damage. \u003csup\u003e24\u003c/sup\u003e. The BCL-2-protein family regulate CytC release.\u003csup\u003e25\u003c/sup\u003e CytC is a pro-apoptotic factor that binds with apoptotic protease activating factor-1 to activate pro-caspase 9.\u003csup\u003e26\u0026nbsp;\u003c/sup\u003eCaspase 3 is involved in a cascade of reactions, which ultimately lead to apoptosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe PI3K-AKT signaling pathway regulates tumor cell proliferation and resistance to chemotherapy.\u003csup\u003e27\u003c/sup\u003e UCNPs inhibit Akt phosphorylation at the Ser473 site. This mechanism promotes the mitochondrial apoptotic pathway and may underlie UCNP-induced SGC-7901 cell death.\u0026nbsp;In the present study, exposure to 400\u0026nbsp;\u0026micro;g/ml\u0026nbsp;UCNPs increased\u0026nbsp;GRP-78, GRP-94, caplain 1 and caplain 2 protein levels in SGC‑7901 cells vs. control in a time-dependent manner, and exposure to TM, an inducer of ER stress, increased GRP-78 and GRP-94 protein levels in SGC‑7901 cells vs. control. Calpain 1 and 2 are two cysteine protease families that regulate pathological cell death.\u003csup\u003e14\u0026nbsp;\u003c/sup\u003eThese data imply that\u003csup\u003e\u0026nbsp;\u003c/sup\u003eUCNPs induced SGC-7901 cell death through the activation of ER stress.\u003c/p\u003e\n\u003cp\u003eIn conclusion, UCNPs induced SGC-7901 cell death by promoting mitochondrial dysfunction and ROS-mediated ER stress, initiating the caspase 9/caspase 3 cascade. These findings provide valuable insights relevant to the development of effective anti-cancer therapies that target specific signaling pathways.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Ethics Committee of\u0026nbsp;Dalian medical university.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe department of Gastroenterology of the first hospital of Dalian medical University supported the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthor 1 (shaoqiang sun): Conceptualization, Methodology, Software, Investigation, Formal Analysis, Writing - Original Draft；\u003c/p\u003e\n\u003cp\u003eAuthor 2(jingwei mao): Data Curation, Writing - Original Draft；\u003c/p\u003e\n\u003cp\u003eCorresponding Author(chen liu): Conceptualization, Funding Acquisition, Resources, Supervision, Writing - Review \u0026amp; Editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSUNG H, FERLAY J, SIEGEL R L, et al. 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J Cell Mol Med 9: 59-71, 2005.\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":"water-soluble upconversion nanoparticles, SGC-7901 cells, reactive oxygen species, mitochondrial dysfunction","lastPublishedDoi":"10.21203/rs.3.rs-1850889/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1850889/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground：\u003c/strong\u003eNanoparticles are potential luminescent probes. The objective of this study was to examine the cytotoxicity and underlying mechanism of upconversion nanoparticles (UCNPs).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e The effects of 0-400μg/mL UCNPs on human gastric adenocarcinoma (SGC-7901) cells were investigated. Flow cytometry was used to evaluate reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), intracellular Ca\u003csup\u003e2+ \u003c/sup\u003elevels, and apoptosis. Caspase-3 and 9 activities were measured using commercial kits. Cytochrome C (CytC) in the cytosol and B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), protein kinase B (Akt), phosphorylated-Akt (p-Akt), 78 kDa glucose-regulated protein (GRP78), 94 kDa glucose-regulated protein (GRP94), caspase-3, caspase- 9, calpain 1, and calpain 2 protein levels were detected using Western blotting. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eExposure to UCNPs inhibited the viability of SGC‑7901 cells vs. control (UNCP 0 µg/ml) in a concentration- and time-dependent manner. Exposure to UCNPs increased the proportion of SGC‑7901 cells in early apoptosis, and enhanced the Bax/Bcl-2 ratio, elevated ROS levels, decreased ΔΨm, increased intracellular Ca\u003csup\u003e2+\u003c/sup\u003e, induced apoptosis, increased CytC protein levels, decreased phosphorylated Akt protein levels, increased Caspase 3 and Caspade-9 activity and protein levels, and increased GRP-78, GRP-94, caplain 1 and caplain 2 protein levels in SGC‑7901 cells vs. control. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e UCNPs induced SGC-7901 cell death by promoting mitochondrial dysfunction and ROS-mediated ER stress, initiating the caspase 9/caspase 3 cascade. These findings provide valuable insights relevant to the development of effective anti-cancer therapies that target specific signaling pathways.\u003c/p\u003e","manuscriptTitle":"Water-soluble Yb 3+ , Er 3+ codoped NaYF 4 nanoparticles induced SGC-7901 cell death through mitochondrial dysfunction and ROS-mediated ER stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-08-17 14:47:57","doi":"10.21203/rs.3.rs-1850889/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":"8a81cacf-00d2-4df9-94d3-d9afbed03e0e","owner":[],"postedDate":"August 17th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-02-08T07:59:40+00:00","versionOfRecord":[],"versionCreatedAt":"2022-08-17 14:47:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1850889","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1850889","identity":"rs-1850889","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}