Abstract:

Objective To discuss the effect of hypoxia inducible factor-1α （HIF-1α）/ reactive oxygen species （ROS） on the apoptosis and invasion of the non-small cell lung cancer A549 cells，and to clarify the related mechanism. Methods The A549 cells were cultured under hypoxia condition for 12， 24， 48 and 72 h to construct the hypoxia cell model. The expression levels of HIF-1α mRNA in the cells at different treatment time were detected by real-time fluorescence quantitative PCR（RT-qPCR） method， and the proliferation abilities of the cells were detected by CCK-8 assay；the hypoxia cell model was verified， and the optimal induction time was determined.The A549 cells were divided into control group（21%O₂）， model group（hypoxia induction）， NAC group（20 mmol·L^－1 NAC）， NAC+YC-1 group（20 mmol·L^－1 NAC+100 μmol·L^－1 YC-1） and YC-1 （100 μmol·L^－1 YC-1）group. The expression levels of HIF-1α and FPR1 mRNA and proteins were detected by RT-qPCR and Western blotting methods； the ROS levels were detected by flow cytometry； the autophagosome structure in the cells was observed by transmission electron microscope； the expressions of microtubule-associated protein light china 3 Ⅱ（LC3-Ⅱ） in the cells in various groups were detected by immunofluorescence； the apoptotic rates of the cells in various groups were detected by flow cytometry； and the number of invasion cells was detected by Transwell chamber assay. Results With the prolongation of hypoxia induction time， the HIF-1α mRNA expression levels in the A549 cells and cell proliferation abilities were increased（P<0.05 or P<0.01）， and the optimal hypoxia induction time was 24 h. The results of RT-qPCR and Western blotting methods showed that compared with control group，the expression levels of HIF-1α and FPR1 mRNA and proteins in the cells in model group were significantly increased（P<0.01）； compared with model group，the expression levels of HIF-1α and FPR1 mRNA and proteins in the cells in NAC group， NAC+YC-1 group and YC-1 group were significantly decreased（P<0.01）.Compared with control group，the level of ROS in the cells in model group was significantly increased （P<0.01）； compared with model group，the ROS levels in the cells in NAC group， NAC+YC-1 group， and YC-1 group were significantly decreased（P<0.01）；compared with NAC group，the ROS level in the cells in NAC+YC-1 group was significantly decreased（P<0.01）.The transmission electron microscope results showed that the organelle structure of the cells in control group was intact and no autophagosome structure was seen； a large number of autophagosomes were seen in the cells in model group， obvious vacuoles were visible， and intracellular organelles were degraded； compared with model group， the number of autophagosomes in the cells in NAC group， NAC+YC-1 group and YC-1 group were decreased，among them the autophagosomes in the cells in NAC+YC-1 group were the least. The immunofluorescence assay results showed that the expression intensity of LC3-Ⅱ in the cells in model group was significantly increased compared with control group， and the expression intensities of LC3-Ⅱ in the cells in NAC group， NAC+YC-1 and YC-1 group were signficantly decreased compared with model group.Compared with control group， the apoptotic rate of the cells in model group was significantly decreased（P<0.01）； compared with model group， the apoptotic rates of the cells in NAC group， NAC+YC-1 group and YC-1 group were significantly increased（P<0.01）； compared with NAC group，the apoptotic rate of the cells in NAC+YC-1 group was increased（P<0.01）.The results of Transwell chamber assay showed that compared with control group the number of the invasion cells in model group was significantly increased（P<0.01）； compared with model group， the numbers of invasion cells in NAC group， NAC+YC-1 group and YC-1 group were significantly decreased （P<0.01）；compared with NAC group，the number of the in vasion cells in NAC+YC-1 group was decreased（P<0.01）. Conclusion Inhibition of HIF-1α/ROS under hypoxia condition can promote the apoptosis and inhibit cell invasion of cancer cells，and its mechanism may be realted to inhibiting the autophagy of lung cancer cells.

Key words: Hypoxia, Hypoxia inducible factor-1α, Reactive oxygen species, Autophagy, Cancer，non-small cell lung