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

doi:10.21203/rs.3.rs-1850889/v1

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

https://doi.org/10.21203/rs.3.rs-1850889/v1

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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 Ca²⁺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 Ca²⁺, 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

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% Er³⁺/ 20 mol% Yb³⁺ codoped NaYF₄ 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.

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% CO₂.

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 (2x10⁵ 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 Ca²⁺ 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.

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 Ca²⁺ 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.¹⁷Western 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.¹⁹Exposure 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.¹²Exposure 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).

UCNPs are currently being developed as fluorescent probes for biomedical applications. Evidence suggests 200µg/ml UCNPs have notable fluorescence imaging capabilities and biocompatibility, and NaYF₄:Yb³⁺/Er³⁺ (Tm³⁺) is the most efficient upconversion nanomaterial.²⁰The present study showed that 400µg/ml UCNPswere 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.²²Exposure 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 9.²⁶Caspase 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.¹⁴These data imply thatUCNPs 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.

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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No competing interests reported.

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

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Abstract

Figures

Introduction

Materials And Methods

Cell culture

MTT assay

Flow cytometry

Western blot analysis

Statistical analysis

Results

Discussion

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References

Additional Declarations

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