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Archive > Volume 31 Issue 2 > 2024,31(2):146-153. DOI:10.3872/j.issn.1007-385X.2024.02.005 Prev Next
Basic Research
Atractylodin induces programmed necrosis of non-small cell lung cancer A549 cells and inhibits xenograft growth in nude mice by activating the RIPK1/RIPK3/MLKL signaling pathway
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Abstract:
To investigate the influence of atractylodin (ATR) on programmed death of non-small cell lung cancer (NSCLC) A549 cells and the growth of xenografts in nude mice by regulating receptor-interacting protein kinase (RIPK) 1/RIPK3/mixed lineage kinase domain like (MLKL) signaling pathway. Methods: A549 cells were treated with 0~160 μmol/L ATR, and the cell viability was detected by MTT method to determine the concentration of subsequent experiments. A549 cells were treated with ATR and/or RIPK1 inhibitor necrostatin-1 (Nec-1) and caspase inhibitor Z-VAD-FMK, to verify whether ATR induced programmed necrosis in A549 cells. A549 cells were divided into the control group, the ATR-L, ATR-M and ATR-H group (treated with 0, 10, 20 and 40 μmol/L ATR, respectively) and the ATR+Nec-1 group (treated with 40 μmol/L atractylodin and 50 μmol/L Nec-1). After 24 h of treatment, PI single staining and Hoechst33342/PI double staining were used to detect cell death; transmission electron microscopy (TEM) was used to observe the morphology of cell death; DCFH-DA fluorescent probe was used to detect intracellular ROS level; JC-1 staining was used to detect mitochondrial membrane potential, and WB method was used to detect the expression level of RIPK1/RIPK3/MLKL signaling pathway-related proteins in cells. A xenograft model of A549 cells was constructed in nude mice, and 10 mg/kg ATR (dissolved in corn oil) was administered to nude mice by gavage for 5 weeks to observe the effect of atractylodin on xenograft growth. The expression level of RIPK1/RIPK3/MLKL signaling pathway-related proteins in xenograft tissues was detected by WB method. Results: 10-160 μmol/L ATR could significantly inhibit the proliferation of A549 cells, and the concentrations of 10, 20 and 40 μmol/L were selected for follow-up experiments. The survival rate of A549 cells in the ATR group was significantly lower than that in the control group (P<0.01) and ATR+Nec-1 group (P<0.01), while the cell survival rate in the ATR+z-VAD group was significantly lower than that in the z-VAD group (P<0.01), indicating that ATR could induce programmed necrosis of A549 cells instead of apoptosis. Compared with the control group, A549 cells in the ATR-treated groups were swollen; the mitochondria were vacuolated; the inner ridge disappeared, the cell contents leaked outward, and the nuclei were aggregated, showing necrotic characteristics. The mortality rate, ROS level, expression levels of p-RIPK1, p-RIPK3 and p-MLKL in the ATR-L group, ATR-M group and ATR-H group A549 cells increased significantly, while the mitochondrial membrane potential decreased significantly (all P<0.01), all of which were concentration-dependent. Compared with the ATR-H group, the mortality rate, ROS level, and expression levels of p-RIPK1, p-RIPK3 and p-MLKL in the ATR+Nec-1 group decreased, while the mitochondrial membrane potential increased significantly (all P<0.01). The results of nude mouse xenograft experiment showed that compared with the control group, the volume and mass of xenografts were decreased (P<0.05 or P<0.01), and the protein expression levels of p-RIPK1, p-RIPK3 and p-MLKL in the tumor tissues in the ATR group increased significantly (all P<0.01). Conclusion: ATR may induce programmed necrosis of A549 cells by activating the RIPK1/RIPK3/MLKL signaling pathway, and inhibit the growth of A549 cells and their nude mouse xenografts.
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