Studies On The Mechanisms Of Senescence And Apoptosis Induced By Pseudolaric Acid B In Humannon-small Cell Lung Cancer Cells

Pseudolaric acid B(PAB),a diterpene acid isolated from the root bark of Pseudolarix kaempferi Gondon,has shown to exert anti-tumor effects via inducing eell cycle arrest and apoptosis in several cancer eell lines.Here we rqported that PAB induced a mitotic catastrophe in human non-small cell lung cancer(NSCLS)A549 cells,which resulted in senescence without apoptosis or necrosis.Besides,PAB-induced senescent cells exhibited higher glucose utiliation.In this study,we investigated the mechalism between glucose metabolism and tumor suppression in vitro.Three human NSCLS cell lines(A549,H460 and H1299 cells)were examined.Cell growth inhibition was assessed with MTT assay.Cell eycle distribution and glucose uptake were determined using a flow cytometer with PI and 2-NBDG staining,respectively.Cell nuclear morphology after DAPI staining was observed under a fluorescence microscope.Senescent cells were detected using SA-?-Gal staining.Apoptotic,senescent and glycolytic protein expressions were examined using Western blot analysis.ATP and lactate levels were measured by commercial assay kits.The expression of p53 or p21 in the cells was downregulated by siRNAs using siRNA-Mate.The utilization of glucose was blocked by 2-Deoxy-D-glucose(2-DG)treatment.Treatment with PAB(5-80?M)inhibited the growth of A549 cells in dose-and time-dependent manners.Prolonged treatment with PAB(20 ?M)caused G2/M arrest at day 1 followed by mitotic catastrophe from day 2,which eventually resulted in cell senescence between days 3 and 4 without cell death(apoptosis or necrosis).Knockdown of p53 expression with siRNA significantly suppressed PAB-induced senescence in A549 cells(p53 wild).Furthermore,PAB-induced senescence was also observed in NSCLS H460 cells(p53 wild),but not in NSCLS H1299 cells(p53 null).In this study,senescent A549 cells increased glucose utilization with PAB treatment.Furthernore,p53 or p21 inhibition decreased the uptake and metabolism of glucose while elevated PAB-induced apoptosis.Accordingly,PAB-treated A549 cells were more sensitive to undergoing apoptosis when glucose utilization was disturbed.Previous results also displayed that PAB caused senescence in NSCLS H460 cells(p53 wild),but not in NSCLS H1299 cells(p53 null).This study suggested that the utilization of glucose was enhanced in H460 cells but decreased in H1299 cells.The anti-tumor action of PAB against NSCLS A549 cells in vitro involves the induction of senescence through activation of the p53 pathway.Senescent cells enhanced the utilization of glucose by PAB treatment.However,p53 inhibition induced apoptosis with lower glucose utilization.Collectively,these findings unveiled the mechanism that blocked glucose utilization contributed to the switch from senescence to apoptosis.