Single cell phospho-specific flow cytometry can detect dynamic changes of phospho-Stat1 level in lung cancer cells

Abstract Background Kejinyan decoction, as an experienced formula of Zhou Zhongying (the Master of Traditional Chinese Medicine) has been widely used in clinic for lung cancer treatment in China, while the anti-lung cancer mechanism of it is still remained to be elucidated. Herein, our basic study found that the survival of lung cancer xenograft mice was significantly prolonged after intragastrically administered high dose of Kejinyan decoction (3.8 g per kg BW) for 15 days. More importantly, we found that Kejinyan decoction inhibited the metastasis of lung cancer cells in vivo. Thus in this study, we aim to elucidate the anti-metastasis effects of Kejinyan decoction. Methods RNA-Seq was used to find out the gene regulation of Kejinyan decoction on the mice, flow cytometry assay was used to detect the immunocytes in the spleen, ELISA assay was used to detect the inflammatory factors in the serum and spleen, and immunofluorescence assay was used to detect the level of immune cells and the expression of glycol-metabolism related enzymes in situ. Also, we established a lung cancer orthotopic xenograft tumor model to assess the influence of Kejinyan decoction on the metastatic ability of lung cancer cells in vivo. Results GO analysis of gene sequencing of tumor tissue samples showed that Kejinyan decoction regulated immune response. Further flow cytometry analysis of splenic lymphocyte showed that Kejinyan decoction upregulated M1 macrophages and downregulated M2 macrophages, while the total level of macrophages changed little, which was verified by detection of CD68, F4/80, CD206, and CD86 in tumor tissue section. Moreover, detection of inflammatory cytokines showed that Kejinyan decoction downregulated TNF-α, IFN-γ, IL-6, as well as IL-4, IL-13 in tumor microenvironment. Further studies also showed that Kejinyan decoction had little effect on tumor hypoxia, but downregulated glycolysis in tumor tissues. More importantly, we found that Kejinyan decoction inhibited the metastasis of lung cancer cells in vivo. Conclusion Our findings conclude that Kejinyan decoction inhibited lung cancer cell metastasis through affecting macrophage polarization and energy reprogramming.

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Hematogenous dissemination of lung cancer cells during surgery: quantitative detection by flow cytometry and prognostic significance

Lung Cancer ◽

10.1016/s0169-5002(02)00102-2 ◽

2002 ◽

Vol 37 (3) ◽

pp. 293-301 ◽

Cited By ~ 28

Author(s):

Qianggang Dong ◽

Jinsu Huang ◽

Yunzhong Zhou ◽

Luping Li ◽

Guoliang Bao ◽

...

Keyword(s):

Lung Cancer ◽

Flow Cytometry ◽

Cancer Cells ◽

Prognostic Significance ◽

Lung Cancer Cells ◽

Quantitative Detection ◽

Hematogenous Dissemination

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Single-Cell Tracking of A549 Lung Cancer Cells Exposed to a Marine Toxin Reveals Correlations in Pedigree Tree Profiles

Frontiers in Oncology ◽

10.3389/fonc.2018.00260 ◽

2018 ◽

Vol 8 ◽

Cited By ~ 2

Author(s):

Mónica Suárez Korsnes ◽

Reinert Korsnes

Keyword(s):

Lung Cancer ◽

Single Cell ◽

Cancer Cells ◽

Cell Tracking ◽

Lung Cancer Cells ◽

Marine Toxin ◽

A549 Lung

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Application of TEM: Dynamic changes of AKAP95-Cx43 complex during G1 phase of lung cancer cells

10.21203/rs.3.rs-66232/v1 ◽

2020 ◽

Author(s):

Zifeng Deng ◽

Rong Liu ◽

Huiling Guo ◽

Luming Yao ◽

FengKai Ruan ◽

...

Keyword(s):

Lung Cancer ◽

Cyclin D1 ◽

Cancer Cells ◽

Connexin 43 ◽

Laser Scanning Microscopy ◽

Lung Cancer Cells ◽

G1 Phase ◽

Nuclear Pore ◽

Dynamic Changes ◽

Cyclin E1

Abstract BackgroundAKAP95(A-kinase anchoring protein) and Cx43 (connexin 43) express abnormally in lung cancer cells. As potential tumor therapeutic targets, specific process of bindings and dynamic changes of Cx43-AKAP95 complex in lung cancer cells may guide further treatments and detections of lung cancer. However, the process remains unclear now. We are aiming at investigating the dynamic changes of expression, localization, and binding of AKAP95 and Cx43, as well as their interaction with cyclin D1 and cyclin E1 in lung cancer cells during G1 phase.MethodsA549 and Beas-2B cells were arrested at preliminary stage(P), middle stage(M) and restriction point(R) of G1 phase and Western blot(WB), Confocal laser scanning microscopy (CLSM) and Transmission electron microscope (TEM) were used in our study to detect proteins to provide further evidence of the correlation of these proteins.Results: 1) AKAP95 carries Cx43 into the nucleus through the nuclear pore by binding to it during G1 phase. Some AKAP95 and Cx43 can aggregate and form larger protein aggregates. The process happens mainly during R stage. 2) AKAP95 and Cx43 mainly bind to cyclin D1 during P and M stage while bind to cyclin E1 during R stage respectively. Complexes of AKAP95-cyclin D1 and Cx43-cyclin D1 cannot enter nucleus while AKAP95-cyclin E1 and Cx43-cyclin E1 can.Conclusions1) Binding process of AKAP95-Cx43 complexes/ aggregates can be summarize as ‘bind and aggregate -target and enter nucleus- keep binding/aggregating in nucleus’. 2) Cyclin E1 was involved in the binding/aggregation and nuclear entry of AKAP95 and Cx43 while cyclin D1 only binds to them respectively in the cytoplasm.

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Imperatorin Targets MCL-1 to Sensitize CD133+ Lung Cancer Cells to γδ-T Cell-Mediated Cytotoxicity

Cellular Physiology and Biochemistry ◽

10.1159/000492874 ◽

2018 ◽

Vol 49 (1) ◽

pp. 235-244 ◽

Cited By ~ 2

Author(s):

Changxuan You ◽

Yu Yang ◽

Beili Gao

Keyword(s):

Lung Cancer ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Cancer Cells ◽

Γδ T Cells ◽

Lung Cancer Cells ◽

Enzyme Linked Immunosorbent Assay ◽

Γδ T Cell ◽

Cytochrome C Release

Background/Aims: CD133+ cancer cells display low sensitivity to anti-cancer treatment; thus, combination treatment with adjuvant drugs is required to improve the efficiency of cancer therapy. The aim of this study was to explore the effect of imperatorin, a linear furanocoumarin compound, on γδ T cell-mediated cytotoxicity against CD133+ lung cancer cells. Methods: CD133+ and CD133- subgroups from A549 and PC9 lung cancer cells were sorted by using flow cytometry. The cytotoxicity of γδ T cells against cancer cells was evaluated by measuring lactate dehydrogenase release. The concentration of tumor necrosis factor-related apoptosis-inducing ligand in the co-culture system was determined by using an enzyme-linked immunosorbent assay. Mitochondrial membrane potential, expression of death receptor 4 (DR4) and DR5 on the cell surface, and rate of apoptosis were measured by flow cytometry. Cytochrome c release and cellular protein expression were detected by western blot analysis. Results: Compared with CD133- cells, CD133+ cells were resistant to γδ T cell-mediated cytotoxicity. However, imperatorin significantly increased the sensitivity of CD133+ lung cancer cells to γδ T cell treatment in vitro and in vivo. Mechanically, we found that myeloid cell leukemia 1 (MCL-1), an important anti-apoptotic protein belonging to the Bcl-2 family, was overexpressed in CD133+ A549 and PC9 cells compared to their corresponding CD133- cells. Co-treatment with imperatorin and γδ T cells suppressed the expression of MCL-1, and thus promoted the mitochondrial apoptosis mediated by γδ T cells in CD133+ A549 and PC9 lung cancer cells. Conclusion: Up-regulated MCL-1 in CD133+ lung cancer cells is responsible for their resistance to γδ T cells. Furthermore, the combination of γδ T cells with imperatorin sensitized CD133+ lung cancer cells to γδ T cell-mediated cytotoxicity by targeting MCL-1.

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Effects of Dynamin-related Protein 1 Regulated Mitochondrial Dynamic Changes on Invasion and Metastasis of Lung Cancer Cells

Journal of Cancer ◽

10.7150/jca.29756 ◽

2019 ◽

Vol 10 (17) ◽

pp. 4045-4053 ◽

Cited By ~ 7

Author(s):

Jie-Tao Ma ◽

Xiang-Yan Zhang ◽

Rui Cao ◽

Li Sun ◽

Wei Jing ◽

...

Keyword(s):

Lung Cancer ◽

Cancer Cells ◽

Lung Cancer Cells ◽

Related Protein ◽

Invasion And Metastasis ◽

Dynamic Changes ◽

Mitochondrial Dynamic

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2-(6-Hydroxyhexylthio)-5,8-dimethoxy-1,4-naphthoquinone Induces Apoptosis through ROS-Mediated MAPK, STAT3, and NF-κB Signalling Pathways in Lung Cancer A549 Cells

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2020/7375862 ◽

2020 ◽

Vol 2020 ◽

pp. 1-13

Author(s):

Gui-Nan Shen ◽

Cheng Wang ◽

Ying-Hua Luo ◽

Jia-Ru Wang ◽

Rui Wang ◽

...

Keyword(s):

Lung Cancer ◽

Flow Cytometry ◽

Western Blot ◽

Cancer Cells ◽

A549 Cell ◽

A549 Cells ◽

Cyclin B1 ◽

Lung Cancer Cells ◽

Signalling Pathways ◽

Intrinsic Pathway

Two novel compounds, 2-(2-hydroxyethylthio)-5,8-dimethoxy-1,4-naphthoquinone (HEDMNQ) and 2-(6-hydroxyhexylthio)-5,8-dimethoxy-1,4-naphthoquinone (HHDMNQ), were synthesized to investigate the kill effects and mechanism of 1,4-naphthoquinone derivatives in lung cancer cells. The results of the CCK-8 assay showed that HEDMNQ and HHDMNQ had significant cytotoxic effects on A549, NCI-H23, and NCI-H460 NSCLC cells. Flow cytometry and western blot results indicated that HHDMNQ induced A549 cell cycle arrest at the G2/M phase by decreasing the expression levels of cyclin-dependent kinase 1/2 and cyclin B1. Fluorescence microscopy and flow cytometry results indicated that HHDMNQ could induce A549 cell apoptosis, and western blot analysis showed that HHDMNQ induced apoptosis through regulating the mitochondria pathway, as well as the MAPK, STAT3, and NF-κB signalling pathways. Flow cytometry results showed that intracellular reactive oxygen species (ROS) levels were increased after HHDMNQ treatment, and western blot showed that ROS could modulate the intrinsic pathway and MAPK, STAT3, and NF-κB signalling pathways. These effects were blocked by the ROS inhibitor N-acetyl-L-cysteine in A549 cells. Our findings suggest that compared with HEDMNQ, HHDMNQ had the stronger ability to inhibit the cell viability of lung cancer cells and induce apoptosis by regulating the ROS-mediated intrinsic pathway and MAPK/STAT3/NF-κB signalling pathways. Thus, HHDMNQ might be a potential antitumour compound for treating lung cancer.

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