Inhibition Of Proliferation Of Human Non-Small Cell Lung Cancer A549 Cells By Physalin B And Its Mechanism Of Action

Objectives:Physalin B(PB),a major active steroidal component of P.alkekengi var.franchetii,exhibits a broad-spectrum of biological activities,such as anti-inflammatory,immunomodulatory,and antitumor effects.However,its effect on human NSCLC growth is still not well understood.Our previous studies have shown that the lung is the main target organ of PB.In order to further study the effects of physalin B on the proliferation inhibition,cycle regulation,apoptosis induction of human non-small cell lung cancer cell line(A549)and its relevant mechanisms,by which to provide a new method for the future drug development.Methods:(1)The effect of physalin B on A549 cells proliferation was detected by real-time cell analysis and statistics analysis,respectively;(2)The effect of physalin B on cell cycle and apoptosis rate was analyzed by flow cytometry;(3)Western blot and real-time PCR analysis were used to detected the protein and mRNA expression of p53,p21,CDC2 and cyclin B1 affected by physalin B,which revealed the mechanism of its influence on cell cycle;(4)Immunofluorescence assays were performed to determine the change of mitochondria morphology influenced by physalin B;(5)The energy metabolism of cells was measured by XF96 cell energy metabolism real-time analysis;(6)The effect of physalin B on mitochondrial membrane potential and reactive oxygen species was analyzed by flow cytometry;(7)Western blot was used to detect the expression of apoptosis-related proteins affected by physalin B.Results:(1)Physalin B inhibited the proliferation of these cells in a dose-and time-dependent manner,and compared with cis-dichlorodiamineplatinum(DDP,a positive control),physalin B could exert its effect more rapidly and was more effective at lower concentrations.(2)Physalin B inhibited A549 cell proliferation by arresting the cell cycle at the G2/M phase and subsequently inducing apoptosis with a p53-independent way.(3)Physalin B can down-regulate the expression of the respiratory chain complex subunit,and can cause mitochondrial respiratory function defects in A549 cells.(4)Physalin B induced cellular ROS production and reduced MMP in A549 cells.(5)Physalin B up-regulated the expression of cyt c and induced the activation of caspase-9,and-3 and the cleavage of poly(ADP-ribose)polymerase,indicating the involvement of a caspase signaling cascade;(6)The caspase inbibitor Z-VAD-fink significantly suppressed physalin B-indeced apoptosis.Conclusion:In this study,it was proved that physalin B induced an antiproliferative effect on human lung cancer A549 cells through mitochondrial disruption and down-regulation of CDC2/cyclin B1,leading to cell cyle arrest at the G2/M phase,loss of MMP,and subsequently apoptotic cell death.