Increased Oxidative Phosphorylation Is Required for Stemness Maintenance in Liver Cancer Stem Cells from Hepatocellular Carcinoma Cell Line HCCLM3 Cells
<p>Enrichment and characterization of liver cancer stem cells (LCSCs) from HCCLM3 cells. (<b>A</b>) Morphological characteristics of HCCLM3 and sphere-forming cells (SFCs); process of sphere formation from a single P3 SFC (scale bar = 50 μm). (<b>B</b>) Comparison of the clonogenicity of HCCLM3 cells and SFCs in vitro. (<b>C</b>) Detection of the surface markers CD133 and CD44. (<b>D</b>) Detection of stemness genes. (<b>E</b>) Drug resistance analysis of SFCs and HCCLM3 cells. HCCLM3 cells and SFCs were treated with 5-FU and sorafenib for 12 h. Cell survival was determined by a CCK8 assay. Relative values are presented as the means ± standard deviation (SD) of three independent experiments. <span class="html-italic">n</span> = 3; * <span class="html-italic">p</span> < 0.05 and ** <span class="html-italic">p</span> < 0.01.</p> "> Figure 2
<p>Comparison of glycolysis between HCCLM3 and LCSCs. (<b>A</b>) Comparison of glycolytic genes. (<b>B</b>) Comparison of 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose (2-NBDG) uptake. (<b>C</b>) Comparison of the glycolytic protein hexokinase 2 (HK2). (<b>D</b>) Comparison of the glycolytic protein lactate dehydrogenase A (LDHA). The values are presented as the means ± SD of three independent experiments. <span class="html-italic">n</span> = 3; * <span class="html-italic">p</span> < 0.05; ** <span class="html-italic">p</span> < 0.01; and *** <span class="html-italic">p</span> < 0.001.</p> "> Figure 3
<p>Comparison of oxidative phosphorylation (OXPHOS) between HCCLM3 and LCSCs. (<b>A</b>) Comparison of pyruvate levels. (<b>B</b>) Comparison of the pyruvate dehydrogenase complex (PDHC) expression. (<b>C</b>) Measurement of the mtDNA copy number. (<b>D</b>) Measurement of the mitochondrial mass. (<b>E</b>) Measurement of the reactive oxygen species (ROS) level. The values are presented as the means ± SD of three independent experiments. <span class="html-italic">n</span> = 3; * <span class="html-italic">p</span> < 0.05; ** <span class="html-italic">p</span> < 0.01; and *** <span class="html-italic">p</span> < 0.001.</p> "> Figure 4
<p>Assessment of LCSC metabolism and stemness gene expression after 2-DG treatment. (<b>A</b>) Detection of stemness genes after 2-DG treatment. (<b>B</b>) Detection of the surface markers CD133 and CD44 after 2-DG treatment. (<b>C</b>) Comparison of PDHC expression after 2-DG treatment. (<b>D</b>) Comparison of pyruvate levels after 2-DG treatment. (<b>E</b>) Measurement of the mitochondrial mass after 2-DG treatment. (<b>F</b>) Measurement of the ROS level after 2-DG treatment. The values are presented as the means ± SD of three independent experiments. <span class="html-italic">n</span> = 3; * <span class="html-italic">p</span> < 0.05; ** <span class="html-italic">p</span> < 0.001.</p> "> Figure 5
<p>Assessment of mitochondrial biogenesis and stemness gene expression in LCSCs after Mdivi-1 treatment. (<b>A</b>) Measurement of the mtDNA copy number after Mdivi-1 treatment. (<b>B</b>) Measurement of the mitochondrial mass after Mdivi-1 treatment. (<b>C</b>) Measurement of the ROS level after Mdivi-1 treatment. (<b>D</b>) Detection of stemness genes after Mdivi-1 treatment. (<b>E</b>) Detection of the surface markers CD133 and CD44 after Mdivi-1 treatment. The values are presented as the means ± SD of three independent experiments. <span class="html-italic">n</span> = 4; * <span class="html-italic">p</span> < 0.05; and ** <span class="html-italic">p</span> < 0.01.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Enriched Sphere-Forming Cells Exhibit Stemness Properties
2.2. Liver Cancer Stem Cells Exhibit More Robust Glycolysis than Non-Stemness Cells
2.3. Liver Cancer Stem Cells Exhibit More Robust Oxidative Phosphorylation than Non-Stemness Cells
2.4. 2-DG Increases Liver Cancer Stem Cell Stemness by Upregulating Oxidative Phosphorylation
2.5. Mdivi-1 Reduces Liver Cancer Stem Cells Stemness by Inhibiting Oxidative Phosphorylation
3. Discussion
4. Materials and Methods
4.1. Cell Culture and Enrichment
4.2. Sphere Formation Assay
4.3. Colony Formation Assay
4.4. Flow Cytometry
4.5. Chemotherapeutic Sensitivity Assay
4.6. Quantitative Real-Time PCR
4.7. Western Blot Analysis
4.8. 2-DG Treatment
4.9. Mdivi-1 Treatment
4.10. Determination of Pyruvate Levels
4.11. Statistical Analysis
Author Contributions
Funding
Conflicts of Interest
References
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Liu, G.; Luo, Q.; Li, H.; Liu, Q.; Ju, Y.; Song, G. Increased Oxidative Phosphorylation Is Required for Stemness Maintenance in Liver Cancer Stem Cells from Hepatocellular Carcinoma Cell Line HCCLM3 Cells. Int. J. Mol. Sci. 2020, 21, 5276. https://doi.org/10.3390/ijms21155276
Liu G, Luo Q, Li H, Liu Q, Ju Y, Song G. Increased Oxidative Phosphorylation Is Required for Stemness Maintenance in Liver Cancer Stem Cells from Hepatocellular Carcinoma Cell Line HCCLM3 Cells. International Journal of Molecular Sciences. 2020; 21(15):5276. https://doi.org/10.3390/ijms21155276
Chicago/Turabian StyleLiu, Ge, Qing Luo, Hong Li, Qiuping Liu, Yang Ju, and Guanbin Song. 2020. "Increased Oxidative Phosphorylation Is Required for Stemness Maintenance in Liver Cancer Stem Cells from Hepatocellular Carcinoma Cell Line HCCLM3 Cells" International Journal of Molecular Sciences 21, no. 15: 5276. https://doi.org/10.3390/ijms21155276
APA StyleLiu, G., Luo, Q., Li, H., Liu, Q., Ju, Y., & Song, G. (2020). Increased Oxidative Phosphorylation Is Required for Stemness Maintenance in Liver Cancer Stem Cells from Hepatocellular Carcinoma Cell Line HCCLM3 Cells. International Journal of Molecular Sciences, 21(15), 5276. https://doi.org/10.3390/ijms21155276