Low-dose Diosbulbin-B (DB) Regulates Tumor-Intrinsic PD-L1/NLRP3 Signaling Pathway to Increase Cisplatin-Sensitivity in Gastric Cancer (GC)

Mapping Intimacies ◽

10.21203/rs.3.rs-56315/v1 ◽

2020 ◽

Author(s):

Chunfeng Li ◽

Junqiang Qiu ◽

Yingwei Xue

Keyword(s):

Gastric Cancer ◽

Cell Death ◽

Cell Viability ◽

Low Dose ◽

Apoptotic Cell ◽

Cell Counting ◽

Blue Staining ◽

Spheroid Formation ◽

Cisplatin Sensitivity

Abstract Background: Generation of cisplatin (DDP)-resistance by gastric cancer (GC) cells seriously limits the therapeutic efficacy of this drug in clinic, and recent data suggested that Diosbulbin-B (DB), the main anti-tumor compound of Dioscorea bulbifera L, was effective to improve cisplatin-sensitivity in GC cells. However, the underlying mechanisms are still largely unknown.Methods: Genes expressions were determined by Real-Time qPCR and Western Blot. Cell viability was evaluated by cell counting kit-8 and trypan blue staining assay. Annexin V-FITC/PI double staining assay was used to examine cell apoptosis. The Spheroid formation assay was used to evaluated cell stemness. Immunohistochemistry (IHC) was employed to examine the expressions and localization of Ki67 protein in mice tumor tissues.Results: Here we found that low-dose DB (12.5 μM) downregulated PD-L1 to activate NLRP3-medicated pyroptosis, and inhibited cancer stem cells (CSCs) properties, to sensitize cisplatin-resistant GC (CR-GC) cells to cisplatin. Mechanistically, the CR-GC cells were obtained, and either low-dose DB or cisplatin alone had little effects on cell viability in CR-GC cells, while low-dose DB significantly induced apoptotic cell death in cisplatin treated CR-GC cells. In addition, low-dose DB triggered cell pyroptosis in CR-GC cells stimulated with cisplatin, which were abrogated by silencing NLRP3. Next, CSCs tended to be enriched in CR-GC cells, instead of their parental cisplatin-sensitive GC (CS-GC) cells, and low-dose DB (12.5 μM) inhibited spheroid formation and stemness biomarkers (CD44, CD133, SOX2, OCT4 and Nanog) expressions to eliminate CSCs in CR-GC cells, which were reversed by upregulating programmed death ligand-1 (PD-L1). Furthermore, we proved that PD-L1 negatively regulated NLRP3 in CR-GC cells, and low-dose DB (12.5 μM) activated NLRP3-mediated pyroptotic cell death in cisplatin treated CR-GC cells by downregulating PD-L1. Also, low-dose DB aggravated the inhibiting effects of cisplatin on tumorigenesis of CR-GC cells in vivo. Conclusions: Collectively, low-dose DB (12.5 μM) regulated intrinsic PD-L1/NLRP3 pathway to improve cisplatin-sensitivity in CR-GC cells, and this study provided alternative therapy treatments for GC.

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Low-dose Diosbulbin-B (DB) activates tumor-intrinsic PD-L1/NLRP3 signaling pathway mediated pyroptotic cell death to increase cisplatin-sensitivity in gastric cancer (GC)

Cell & Bioscience ◽

10.1186/s13578-021-00548-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Chunfeng Li ◽

Junqiang Qiu ◽

Yingwei Xue

Keyword(s):

Gastric Cancer ◽

Cell Death ◽

Molecular Mechanisms ◽

Low Dose ◽

Apoptotic Cell ◽

Cell Counting ◽

Blue Staining ◽

Spheroid Formation ◽

Cisplatin Sensitivity

Abstract Background Emerging evidences suggests that Diosbulbin-B (DB) is effective to improve cisplatin (DDP)-sensitivity in gastric cancer (GC), but its molecular mechanisms were not fully delineated, and this study managed to investigate this issue. Methods Genes expressions were determined by Real-Time qPCR and Western Blot at transcriptional and translational levels. Cell proliferation and viability were evaluated by cell counting kit-8 (CCK-8) and trypan blue staining assay. Annexin V-FITC/PI double staining assay was used to examine cell apoptosis. The Spheroid formation assay was used to evaluated cell stemness. The xenograft tumor-bearing mice models were established, and the tumors were monitored and the immunohistochemistry (IHC) was employed to examine the expressions and localization of Ki67 protein in mice tumor tissues. Results Low-dose DB (12.5 μM) downregulated PD-L1 to activate NLRP3-mediated pyroptosis, and inhibited cancer stem cells (CSCs) properties, to sensitize cisplatin-resistant GC (CR-GC) cells to cisplatin. Mechanistically, the CR-GC cells were obtained, and either low-dose DB or cisplatin alone had little effects on cell viability in CR-GC cells, while low-dose DB significantly induced apoptotic cell death in cisplatin treated CR-GC cells. In addition, low-dose DB triggered cell pyroptosis in CR-GC cells co-treated with cisplatin, which were abrogated by silencing NLRP3. Next, CSCs tended to be enriched in CR-GC cells, instead of their parental cisplatin-sensitive GC (CS-GC) cells, and low-dose DB inhibited spheroid formation and stemness biomarkers (SOX2, OCT4 and Nanog) expressions to eliminate CSCs in CR-GC cells, which were reversed by upregulating programmed death ligand-1 (PD-L1). Furthermore, we proved that PD-L1 negatively regulated NLRP3 in CR-GC cells, and low-dose DB activated NLRP3-mediated pyroptotic cell death in cisplatin treated CR-GC cells by downregulating PD-L1. Also, low-dose DB aggravated the inhibiting effects of cisplatin on tumorigenesis of CR-GC cells in vivo. Conclusions Collectively, low-dose DB regulated intrinsic PD-L1/NLRP3 pathway to improve cisplatin-sensitivity in CR-GC cells, and this study provided alternative therapy treatments for GC.

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Overexpressed LncRNA ADAMTS9-AS2 inhibited gastric cancer (GC) development and sensitized chemoresistant GC cells to cisplatin by regulating miR-223-3p/NLRP3 axis

10.21203/rs.2.22861/v1 ◽

2020 ◽

Author(s):

Niansheng Ren ◽

Tao Jiang ◽

Chengbo Wang ◽

Shilin Xie ◽

Yanwei Xing ◽

...

Keyword(s):

Gastric Cancer ◽

Cell Death ◽

Cell Viability ◽

Pearson Correlation ◽

Luciferase Reporter ◽

Blue Staining ◽

Luciferase Reporter Gene ◽

Kaplan Meier ◽

Cisplatin Sensitivity ◽

Cancer Tissues

Abstract Background The role of LncRNA ADAMTS9-AS2 in the regulation of pathogenesis and chemoresistance of gastric cancer (GC) is largely unkonwn, and this study aimed to solve this problem. Methods Real-Time qPCR was used to examine LncRNA ADAMTS9-AS2 and miR-223-3p expressions, and pearson correlation analysis was conducted to analyse their correlations. Kaplan-Meier survival analysis was performed to evaluate prognosis of GC patients. Dual-luciferase reporter gene system and pull-down assay were employed to validate the target sites of LncRNA ADAMTS9-AS2, miR-223-3p and NLRP3 mRNA. Cell viability was evaluated by CCK-8 assay, trypan blue staining and colony formation assay. Cell apoptosis ratio was detected by FCM and TUNEL assay. Transwell assay for cell migration and Western Blot for protein expressions. ELISA was used to measure cytokines secretion. FISH was conducted to examine LncRNA ADAMTS9-AS2 expressions and localization in mice cancer tissues. Results LncRNA ADAMTS9-AS2 was low-expressed in GC tissues and cells compared to their normal counterparts. Besides, LncRNA ADAMTS9-AS2 inhibited miR-223-3p expressions in GC cells by acting as competing endogenous RNA, and the levels of LncRNA ADAMTS9-AS2 and miR-223-3p showed negative correlations in GC tissues. Of note, overexpression of LncRNA ADAMTS9-AS2 inhibited GC cell viability and motility by sponging miR-223-3p. In addition, the levels of LncRNA ADAMTS9-AS2 were lower, and miR-223-3p was higher in cisplatin-resistant GC (CR-GC) cells than their parental cisplatin-sensitive GC (CS-GC) cells. LncRNA ADAMTS9-AS2 overexpression enhanced the cytotoxic effects of cisplatin on CR-GC cells, which were reversed by overexpressing miR-223-3p. Furthermore, LncRNA ADAMTS9-AS2 increased NLRP3 expressions by targeting miR-223-3p, and upregulation of LncRNA ADAMTS9-AS2 triggered pyroptotic cell death in cisplatin treated CR-GC cells by activating NLRP3 inflammasome through downregulating miR-223-3p. Finally, the promoting effects of LncRNA ADAMTS9-AS2 overexpression on CR-GC cell death were abrogated by pyroptosis inhibitor NSA.Conclusions LncRNA ADAMTS9-AS2 acted as a tumor suppressor and enhanced cisplatin sensitivity in GC cells by regulating miR-223-3p/NLRP3 axis mediated pyroptosis.

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Circ-RNF111 aggravates the malignancy of gastric cancer through miR-876-3p-dependent regulation of KLF12

World Journal of Surgical Oncology ◽

10.1186/s12957-021-02373-5 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Guoxian Wu ◽

Aimin Zhang ◽

Yinglin Yang ◽

Dongping Wu

Keyword(s):

Gastric Cancer ◽

Cell Cycle ◽

Cell Viability ◽

Colony Formation ◽

Xenograft Model ◽

Cell Counting ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Aberrant Expression

Abstract Background The aberrant expression of circular RNAs (circRNAs) plays vital roles in the advancement of human cancers, including gastric cancer (GC). In this study, the functions of circRNA ring finger protein 111 (circ-RNF111) in GC were investigated. Methods Quantitative real-time polymerase chain reaction (qRT-PCR) assay was performed for the levels of circ-RNF111, microRNA-876-3p (miR-876-3p) and krueppel-like factor 12 (KLF12) mRNA. RNase R assay was conducted for the feature of circ-RNF111. Cell Counting Kit-8 (CCK-8) assay, colony formation assay, wound-healing assay, and transwell assay were applied for cell viability, colony formation, migration, and invasion, respectively. Flow cytometry analysis was used to analyze cell apoptosis and cell cycle process. The glycolysis level was examined using specific commercial kits. Western blot assay was carried out to measure the protein levels of hexokinase 2 (HK-2) and KLF12. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were employed to verify the combination between miR-876-3p and circ-RNF111 or KLF12. Murine xenograft model was constructed for the role of circ-RNF111 in vivo. Immunohistochemistry (IHC) was used for KLF12 level. Results Circ-RNF111 was higher expressed in GC tissues and cells than normal tissues and cells. Silencing of circ-RNF111 restrained cell viability, colony formation, migration, invasion, cell cycle process and glycolysis and induced apoptosis in GC cells in vitro. Circ-RNF111 positively regulated KLF12 expression via absorbing miR-876-3p. MiR-876-3p downregulation reversed the impacts of circ-RNF111 silencing on GC cell malignant phenotypes. MiR-876-3p overexpression repressed GC cell growth, metastasis and glycolysis, inhibited apoptosis and arrested cell cycle, while KLF12 elevation weakened the effects. Besides, circ-RNF111 knockdown inhibited tumor growth in vivo. Conclusion Circ-RNF111 knockdown relieved the development of GC by regulating miR-876-3p/KLF12 axis.

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Knockdown of PGM1 enhances anticancer effects of orlistat in gastric cancer under glucose deprivation

Cancer Cell International ◽

10.1186/s12935-021-02193-3 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Bo Cao ◽

Huan Deng ◽

Hao Cui ◽

Ruiyang Zhao ◽

Hanghang Li ◽

...

Keyword(s):

Gastric Cancer ◽

Lipid Metabolism ◽

Cell Viability ◽

Glucose Deprivation ◽

Metabolic Reprogramming ◽

Cell Counting ◽

Tumor Node Metastasis Stage

Abstract Background Phosphoglucomutase 1 (PGM1) acts as an important regulator in glucose metabolism. However, the role of PGM1 in gastric cancer (GC) remains unclear. This study aims to investigate the role of PGM1 and develop novel regimens based on metabolic reprogramming in GC. Methods Correlation and enrichment analyses of PGM1 were conducted based on The Cancer Genome Atlas database. Data derived from the Kaplan–Meier Plotter database were analyzed to evaluate correlations between PGM1 expression and survival time of GC patients. Cell counting kit-8, 5-Ethynyl-2-deoxyuridine, flow cytometry assays, generation of subcutaneous tumor and lung metastasis mouse models were used to determine growth and metastasis in vitro and in vivo. Cell glycolysis was detected by a battery of glycolytic indicators, including lactate, pyruvic acid, ATP production and glucose uptake. Fatty Acid Synthase (FASN) activity and expression levels of lipid enzymes were determined to reflect on lipid metabolism. Results Correlation and enrichment analyses suggested that PGM1 was closely associated with cell viability, proliferation and metabolism. PGM1 was overexpressed in GC tissues and cell lines. High PGM1 expression served as an indicator of shorter survival for specific subpopulation of GC patients. It was also correlated with pathological tumor stage and pathological tumor node metastasis stage of GC. Under the glucose deprivation condition, knockdown of PGM1 significantly suppressed cell viability, proliferation and glycolysis, whereas lipid metabolism was enhanced. Orlistat, as a drug that was designed to inhibit FASN activity, effectively induced apoptosis and suppressed lipid metabolism in GC. However, orlistat conversely increased glycolytic levels. Orlistat exhibited more significant inhibitive effects on GC progression after knockdown of PGM1 under glucose deprivation due to combination of glycolysis and lipid metabolism both in vitro and in vivo. Conclusions Downregulation of PGM1 expression under glucose deprivation enhanced anti-cancer effects of orlistat. This combination application may serve as a novel strategy for GC treatment.

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KUS121 attenuates the progression of monosodium iodoacetate-induced osteoarthritis in rats

Scientific Reports ◽

10.1038/s41598-021-95173-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Sachiko Iwai ◽

Hanako O. Ikeda ◽

Hisashi Mera ◽

Kohei Nishitani ◽

Motoo Saito ◽

...

Keyword(s):

Cell Death ◽

Er Stress ◽

Apoptotic Cell ◽

Intraperitoneal Administration ◽

Atp Depletion ◽

Knee Joints ◽

Cellular Protection ◽

Monosodium Iodoacetate

AbstractCurrently there is no effective treatment available for osteoarthritis (OA). We have recently developed Kyoto University Substances (KUSs), ATPase inhibitors specific for valosin-containing protein (VCP), as a novel class of medicine for cellular protection. KUSs suppressed intracellular ATP depletion, endoplasmic reticulum (ER) stress, and cell death. In this study, we investigated the effects of KUS121 on chondrocyte cell death. In cultured chondrocytes differentiated from ATDC5 cells, KUS121 suppressed the decline in ATP levels and apoptotic cell death under stress conditions induced by TNFα. KUS121 ameliorated TNFα-induced reduction of gene expression in chondrocytes, such as Sox9 and Col2α. KUS121 also suppressed ER stress and cell death in chondrocytes under tunicamycin load. Furthermore, intraperitoneal administration of KUS121 in vivo suppressed chondrocyte loss and proteoglycan reduction in knee joints of a monosodium iodoacetate-induced OA rat model. Moreover, intra-articular administration of KUS121 more prominently reduced the apoptosis of the affected chondrocytes. These results demonstrate that KUS121 protects chondrocytes from stress-induced cell death in vitro and in vivo, and indicate that KUS121 is a promising novel therapeutic agent to prevent the progression of OA.

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Pyrrolizidine Alkaloids Induce Cell Death in Human HepaRG Cells in a Structure-Dependent Manner

International Journal of Molecular Sciences ◽

10.3390/ijms22010202 ◽

2020 ◽

Vol 22 (1) ◽

pp. 202

Author(s):

Josephin Glück ◽

Julia Waizenegger ◽

Albert Braeuning ◽

Stefanie Hessel-Pras

Keyword(s):

Cell Death ◽

Cell Viability ◽

Pyrrolizidine Alkaloids ◽

Defense Mechanism ◽

Hepatoma Cell ◽

Hepatoma Cell Line ◽

Dependent Manner ◽

Human Hepatoma Cell

Pyrrolizidine alkaloids (PAs) are a group of secondary metabolites produced in various plant species as a defense mechanism against herbivores. PAs consist of a necine base, which is esterified with one or two necine acids. Humans are exposed to PAs by consumption of contaminated food. PA intoxication in humans causes acute and chronic hepatotoxicity. It is considered that enzymatic PA toxification in hepatocytes is structure-dependent. In this study, we aimed to elucidate the induction of PA-induced cell death associated with apoptosis activation. Therefore, 22 structurally different PAs were analyzed concerning the disturbance of cell viability in the metabolically competent human hepatoma cell line HepaRG. The chosen PAs represent the main necine base structures and the different esterification types. Open-chained and cyclic heliotridine- and retronecine-type diesters induced strong cytotoxic effects, while treatment of HepaRG with monoesters did not affect cell viability. For more detailed investigation of apoptosis induction, comprising caspase activation and gene expression analysis, 14 PA representatives were selected. The proapoptotic effects were in line with the potency observed in cell viability studies. In vitro data point towards a strong structure–activity relationship whose effectiveness needs to be investigated in vivo and can then be the basis for a structure-associated risk assessment.

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Mitomycin C Induces Apoptosis in Rheumatoid Arthritis Fibroblast-Like Synoviocytes via a Mitochondrial-Mediated Pathway

Cellular Physiology and Biochemistry ◽

10.1159/000373938 ◽

2015 ◽

Vol 35 (3) ◽

pp. 1125-1136 ◽

Cited By ~ 21

Author(s):

Chuqi Yan ◽

Dechao Kong ◽

Dong Ge ◽

Yanming Zhang ◽

Xishan Zhang ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Cell Viability ◽

Mitomycin C ◽

Apoptotic Cell ◽

Chronic Inflammatory Disease ◽

Cell Counting ◽

Terminal Deoxynucleotidyl Transferase ◽

Control Treatment ◽

Induced Apoptosis ◽

Fibroblast Like Synoviocytes

Background/Aims: Rheumatoid arthritis (RA) is a systemic chronic inflammatory disease characterised by prominent synoviocyte hyperplasia and a potential imbalance between the growth and death of fibroblast-like synoviocytes (FLS). Mitomycin C (MMC) has previously been demonstrated to inhibit fibroblast proliferation and to induce fibroblast apoptosis. However, the effects of MMC on the proliferation and apoptosis of human RA FLS and the potential mechanisms underlying its effects remain unknown. Methods: Cell viability was determined using the Cell Counting Kit-8 assay. Apoptotic cell death was analysed via Annexin V-FITC/PI double staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling. The production of intracellular reactive oxygen species (ROS) was assessed via flow cytometry, and the changes in mitochondrial membrane potential (ΔΨm) were visualized based on JC-1 staining via fluorescence microscopy. The expression of apoptosis-related proteins was determined via Western blot. Results: Treatment with MMC significantly reduced cell viability and induced apoptosis in RA FLS. Furthermore, MMC exposure was found to stimulate the production of ROS and to disrupt the ΔΨm compared to the control treatment. Moreover, MMC increased the release of mitochondrial cytochrome c, the ratio of Bax/Bcl-2, the activation of caspase-9 and caspase-3, and the subsequent cleavage of poly(ADP-ribose) polymerase. Conclusion: Our findings suggest that MMC inhibits cell proliferation and induces apoptosis in RA FLS, and the mechanism underlying this MMC-induced apoptosis may involve a mitochondrial signalling pathway.

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NF-κB and AP-1 Activation by Nitric Oxide Attenuated Apoptotic Cell Death in RAW 264.7 Macrophages

Molecular Biology of the Cell ◽

10.1091/mbc.10.2.361 ◽

1999 ◽

Vol 10 (2) ◽

pp. 361-372 ◽

Cited By ~ 112

Author(s):

Andreas von Knethen ◽

Dagmar Callsen ◽

Bernhard Brüne

Keyword(s):

Nitric Oxide ◽

Cell Death ◽

Low Dose ◽

Apoptotic Cell ◽

Apoptotic Cell Death ◽

Raw 264.7 ◽

Raw 264.7 Macrophages ◽

Low Level ◽

Ifn Γ ◽

Cox 2

A toxic dose of the nitric oxide (NO) donorS-nitrosoglutathione (GSNO; 1 mM) promoted apoptotic cell death of RAW 264.7 macrophages, which was attenuated by cellular preactivation with a nontoxic dose of GSNO (200 μM) or with lipopolysaccharide, interferon-γ, and NG-monomethyl-l-arginine (LPS/IFN-γ/NMMA) for 15 h. Protection from apoptosis was achieved by expression of cyclooxygenase-2 (Cox-2). Here we investigated the underlying mechanisms leading to Cox-2 expression. LPS/IFN-γ/NMMA prestimulation activated nuclear factor (NF)-κB and promoted Cox-2 expression. Cox-2 induction by low-dose GSNO demanded activation of both NF-κB and activator protein-1 (AP-1). NF-κB supershift analysis implied an active p50/p65 heterodimer, and a luciferase reporter construct, containing four copies of the NF-κB site derived from the murine Cox-2 promoter, confirmed NF-κB activation after NO addition. An NF-κB decoy approach abrogated not only Cox-2 expression after low-dose NO or after LPS/IFN-γ/NMMA but also inducible protection. The importance of AP-1 for Cox-2 expression and cell protection by low-level NO was substantiated by using the extracellular signal-regulated kinase inhibitor PD98059, blocking NO-elicited Cox-2 expression, but leaving the cytokine signal unaltered. Transient transfection of a dominant-negative c-Jun mutant further attenuated Cox-2 expression by low-level NO. Whereas cytokine-mediated Cox-2 induction relies on NF-κB activation, a low-level NO–elicited Cox-2 response required activation of both NF-κB and AP-1.

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