The Anticancer Effects And Mechanisms Research Of Attar Extracted From Cyperus Rotundus On Inducing Apoptosis Of A549 Cells

Objective:This study aimed at the effects and mechanisms of the anticancer attar?ACA?extracted from cyperus rotundus by supercritical CO₂ fluid extraction on inducing apoptosis of lung cancer A549 cells in vitro.Methods:?1?ACA was extracted from cyperus rotundus by supercritical CO₂ fluid extraction.?2?Dose-effect and time-effect relationship of ACA for killing A549 cells were measured by MTT assay and lactate dehydrogenase?LDH?activity.?3?Toxicity of ACA was evaluated by normal lung tissue culture.?4?Hoechst 33342/PI and Annexin V-EGFP/PI double staining were applied for morphological analysis respectively.?5?Intracellular Ca²⁺concentration was detected by Fura-2 AM assay.?6?The levels of malondialdehyde?MAD?and superoxide dismutase?SOD?activity were tested to demonstrate that ACA lead to oxidative damage of A549 cells.?7?The level of ATP was detected to testify that ACA could inhibit energy metabolism of cells.?8?Mitochondrial membrane potential????was tested by Rhodamine 123?Rh 123?fluorescent staining.?9?Expression levels of apoptosis related protein Bcl-2 and Bax were detected by Western blot method.The activity of Caspase-9 was measured by microplate reader.Results:?1?ACA could extremely decrease viability of A549 cells in dose and time dependent manner.ACA?286?g/mL for 24 h or 200?g/mL for 48 h?can almost completely kill A549 cells,but positive control durg cisplatin?DDP?can not reach the maximal effect.The result showed that ACA has strong efficacy to induce apoptosis of A549 cell in a short time.?2?ACA increased extracellular LDH levels.?3?Toxic effect of ACA on normal lung tissue might be within the acceptable range.?4?Hoechst 33342/PI and Annexin V-EGFP/PI double staining showed that ACA induces apoptosis of A549 cells.?5?ACA resulted in intracellular calcium overload.?6?ACA facilitated oxidative damage by means of significantly increasing MDA levels and inhabiting SOD activity.?7?ACA inhibited energy metabolism of cells.?8?ACA damaged the construction and function of mitochondria and resulted in??dissipation.?9?ACA elevated the ratio of Bax/Bcl-2 and increased activity of caspase-9.Conclusion:ACA increases intracellular Ca²⁺concentration,facilitates oxidative damage,inhibits energy metabolism,damages construction and function of mitochondria,elevates the ratio of Bax/Bcl-2and activity of caspase-9.As a result,ACA induces apoptosis of A549cells by the mitochondrial apoptosis pathway.As an anti-lung cancer potential candidate,ACA presents a very good developmental prospect.