| Objective 1. To clarify the metabolic state of cancer stem cells in small cell lung cancer(SCLC). 2. To reveal the major energy-generating pathways of cancer stem cells in SCLC. 3. To explore the effect of perturbing the major energy-generating pathways on stem-like characteristics of cancer stem cells in SCLC.Methods 1. Previous work from our laboratory showed that the stem cell population may be enriched in cells expressing the u PAR cell surface marker. In this study, we enriched the u PAR+ cells as stem cells and u PAR- cells as non-stem cells by flow cytometry in SCLC cell line H446. 2. Compare the energy metabolism of u PAR+ cells and u PAR- cells:(1) We measured the ATP contents in the u PAR+ cells and u PAR- cells using the ATP-based Cell Titer-Glo Luminescent Cell Viability kit.(2) The relative parameters of oxidative phosphorylation in the u PAR+ cells and u PAR- cells were detected: a. We compared the oxygen consumption rate and mitochondrial reserve capacity of cells; b. The reactive oxygen levels of cells were measured using the reactive oxygen species kit; c. The mitochondrial structures of cells were observed by transmission electron microscopy(TEM).(3) The relative parameters of glycolysis in u PAR+ cells and u PAR- cells were detected: a. We compared extracellular acidification rate and reserved cellular glycolytic potential of cells the using the Seahorse Extracellular Flux Analyzer XF24; b.We analyzed the glucose uptake of cells using the fluorescent 2-deoxy-glucose(2-DG) analog 2-NBDG; c. Lactate production of cells was measured using the L-lactate assay kit. 3. Investigate the major energy-generating pathways of cancer stem cells in SCLC:(1) Treating u PAR+ cells and u PAR- cells with glycolytic inhibitor 2-DG alone, compare the change of ATP content in u PAR+ cells and u PAR- cells.(2) Treating u PAR+ cells and u PAR- cells with oxidative phosphorylation inhibitoroligomycin alone,compare the change of ATP content in u PAR+ cells and u PAR- cells.(3) Treating u PAR+ cells and u PAR- cells with combination of oligomycin and 2-DG, compare the change of ATP content in u PAR + cells and u PAR- cells. 4. To explore the effect of oligomycin on the proliferation and self-renewal capacity of u PAR+ cells:(1) We studied the effect of oligomycin on the proliferation of u PAR+ cells through MTT assays.(2) We explored the effect of oligomycin on sphere-forming capacity of u PAR+ cells by sphere-forming assays.(3) We compared the effect of oligomycin on tumor-initiating by nude mice xenograft assays.Results 1. The comparison of energy metabolic state in cancer stem cells and non-stem cancer cells in SCLC.(1) ATP assays indicated that u PAR+ cells had decreased ATP contents when compared to u PAR- cells(P<0.05).(2) The oxygen consumption rate and the mitochondrial reserve capacity of u PAR+ cells were lower than that of u PAR- cells(P<0.05); ROS levels in the two groups of cells showed reactive oxygen species level of u PAR+ cells was also lower than that of u PAR- cells; but TEM results showed that mitochondrial structure of u PAR + cells and u PAR- cells had no significant difference.(3) The extracellular acidification rate and glycolysis reserve potential of u PAR+ cells was lower than that of u PAR- cells(P<0.05); Glucose uptake assays showed that the glucose uptake ability of u PAR+ cells was lower than that of u PARcells(P<0.05); Moreover, lactate production of u PAR+ cells was significantly decreased than that of u PAR- cells(P<0.05). 2. The comparison of the energy-generating pathways of cancer stem cells and non-stem cancer cells in SCLC(1) The change of ATP content in u PAR+ cells was higher than that in u PAR- cells after the application of 2-DG alone(P<0.05).(2) ATP depletion assays showed that u PAR+ cells were more sensitive to oligomycin.(3) When treating u PAR+ cells and u PAR- cells with combination of oligomycin and 2-DG, ATP content of u PAR + cells was higher than u PAR- cells. Furthermore,. the u PAR+ cells treated by oligomycin and 2-DG produced more ATP under hypoxic conditions than under normoxic conditions(P<0.05). However, this phenomenon was not observed in u PAR- cells. 3. The effect of inhibition of the major energy-generating pathway on proliferative and self-renewal capacities of cancer stem cells.(1) 2-DG affected proliferation of only u PAR- cells as measured in MTT assays, oligomycin inhibited proliferation of both u PAR+ cells and u PAR- cells.(2) Tumor sphere-forming assays showed that oligomycin disrupted the sphere morphology and size of u PAR+ cells, but 2-DG did not.(3) Nude mice experiments showed that the tumor formation rate of u PAR+ cells treated by oligomycin was lower than that of control group, but also the tumor volume and weight were lower compared with the control group.Conclusion 1. The energy metabolic state of cancer stem cells is different from that of non-stem cancer cells in SCLC. They are metabolically dormant tumor populations. 2. The energy-generating pathways in cancer stem cells differ from that in non-stem cancer cells. The cancer stem cells privilege oxidative phosphorylation for their energy demands. In hypoxic conditions, the cancer stem cells can produce partial ATP through mitochondrial substrate-level phosphorylation. 3. Inhibition of oxidative phosphorylation could decrease the stemness of cancer stem cells in SCLC. In summary, cancer stem cells in small cell lung cancer are a subgroup cells of relatively inactive energy metabolic state, oxidative phosphorylation is the major energy-producing pathways. Therefore, the strategies targeting glycolysis in the clinical can only kill non-stem cells, but not stem cells. Under hypoxic conditions, cancer stem cells in small cell lung cance can produce energy through mitochondrial substrate-level phosphorylation. At the same time, the inhibition ofoxidative phosphorylation can reduce the stemness of cancer stem cells in small cell lung cancer. Therefore, the research will be important for the development of related therapy targeting cancer stem cells metabolism. |
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