Signal Transduction Mechanism For AS₂O₃-Induced Non-small Cell Lung Cancer Cell Apoptosis

Lung cancer is a malignancy with a predominant morbidity and mortality.It gets to be a menace not only to the health but also to the existance of human being because of the fact that the elevation of the amplitude of its mortality being the first among all the tumors.The development of lung cancer is a complicated process,most likely it is the result of interaction between a group of environmental and hereditary factors.A vast amount of studies indicate that many gene.AS₂O₃ can inhibit the growth of various lines of human cancer cells,due at least in part to its specific effect on topoisomerase(topo)â…¡.A variety of differentiation-inducing agents,such as camptothecin,VP16,geranylacetone and all-trans-retinoic acid,were shown to induce apoptosis in tumor and normal cells.AS₂O₃ alone is only a modest inducer of differentiation of AML cells in primary culture.AS₂O₃ restored RA sensitivity to previously resistant NCI-H157 cells. Therefore,AS₂O₃ seems to act as a potent differentiation- and apoptosis-inducing agent in cancer cells.U937 cells.When U937 cells were treated with AS₂O₃ in the absence of serum,mitogen-activated protein(MAP) kinase activity was markedly increased after the start of treatment and elevated.Prior to the activation of MAP kinase,increased activities of Ras,Raf-1,and MAP kinase kinase were found,but these enzymes were transiently activated by the treatment with AS₂O₃.These results suggest that the signal was transmitted sequentially from Ras,Raf-1,and MAP kinase kinase to MAP kinase. That a pathway with the persistent activation of MAP kinase in U937 cells in response to AS203 is at least one of the signal transduction pathways involved in the induction of apoptosis.It has been reported that AS₂O₃ utilizes the extracellular signal-regulated kinase(ERK) cascade for signal transduction,leading not only to cell differentiation but also to apoptosis.It is thus postulated that the ERK cascade may control AS₂O₃ induced cell differentiation and apoptosis simultaneously.Caspases are cysteine proteases as the executioners of apoptosis.They activated, initiator Caspases activate downstream Caspases,resulting in an amplification of the Caspase cascade.Active Caspases cleave critical cellular protein substrates and systematically dismantle the cell.The availability and activation of Caspases are crucial steps in the commitment of a cell to die,are known to survive for the life of the organism,inhibition of apoptosis is a primary consideration in setting up cellular survival mechanisms.When in close proximity Caspases can autoactivate. Baculoviruses harbor yet another type of Caspase inhibitor,the inhibitor of apoptosis proteins(IAPs).These proteins are characterized by the presence of a homologous domain named the baculoviral IAP repeat(BIR) domain and IAPs.Survivin,a single BLR domain containing IAP,is upregulated in many common human tumors, implicating that the deregulation of IAPs contributes to human disease.Understanding of the mechanism by which IAPs inhibit apoptosis has come with the demonstration that XIAP can physically interact with and block Caspase-3,-7,and -9.Survivin,a novel inhibitor of apoptosis,expressing in a cell cycle-dependent manner,regulates the G(2)/M phase of the cell cycle by associating with mitotic spindle microtubules;it directly inhibits Caspase-3 and Caspase-7activity.During tumorigenesis,Survivin expression is inversely correlated with apoptosis and is positively correlated with proliferation and angiogenesis.Survivin expression up-regulation predicts short survival and poor prognosis in human cancers.Survivin targeting antisense nucleotide and Survivin mutants induce apoptosis,reduce tumor growth potential,and Sensitize tumor cells to chemotherapeutic drugs and X-irradiation in vitro and in vivo.These results suggest that Survivin may has the potential function as a new target for the diagnosis and treatment of cancer. Mitogen-activated protein kinases(MAPKs) participate in signaling cascades from transmission of extracellular signals into their intracellular targets,which regulate important biologic activities.The MAP kinase family members have been implicated in events necessary for proliferation,differentiation,apoptosis,and certain kinds of stress responses.Three major groups of Map kinases exist:the p38 Map kinase family,the extracellular signal-regulated kinase(ERk) family,and the c-Jun NH2-terminal kinase (JNK) kinase.Map kinase signaling cascades are activated by a variety of different cellular stimuli(stress,cytokine) and mediate diverse responses,The ERK pathway is activated in response to several cytokines and growth factors,and primarily mediates mitogenic and antiapoptotic signals.It has been suggested that the excessive activation of the ERK-kinase cascade,which is generally known to play a role in cell survival,is an event necessary for AS₂O₃-mediated apoptosis.Such a hyperexcitation of the ERK-kinase cascade is due to the activation of an upper signal component Ras and the concomitant downregulation of the protein kinase A activity that may negatively regulate the Raf-1 function.However,signal transduction leading to AS₂O₃ mediated cell differentiation has not been explored.In order to characterize the mechanisms of apoptosis-inducing effect of AS₂O₃ on NCI-H157 cells,we examined the expression of Survivin and role of ERK pathway in AS₂O₃-induced apoptosis.Methods1,Assays for the proliferationThe NCI-H157 cell proliferation was determined by MTT.2,Assay for apoptosisThe apoptosis of NCI-H157 cell was assessed by morphological analysis.Cell Cycle Analysis Using a Becton Dickinson FACScan flow cytometer(Cambridge,MA) using a commercially available software program.3,Western Blot analysis Survivin,Caspase-3 and p-ERK were analyzed by Western blot 4,RT-PCRSurvivin mRNA mRNA expression was detected by semi-quantitative RT-PCR.5,Statistical Analysis.The significance of differences between experimental conditions was determined using the two-tailed Student t testResults1.AS₂O₃ induced apoptosis in a dose and time- dependent fashion in NCI-H157 cells.2.Proliferation of NCI-H157 cells was inhibited by AS₂O₃ and the IC₅0 at 24h, 48h and 72h were 11.6μmol/L,8.1μmol/L and 5.8μmol/L,respectively.Apoptosis of NCI-H157 cells were induced when the cells were treated with AS₂O₃ at concentrations of 5μmol/L and higher.cell hypodiploid percentage at 24h,48h and 72h was 13.7 %,20.5%及37.3%by flow cytometry analyses.respectively Compared with control,treatment with AS₂O₃ at 5μmol/L for 12 h,24 h and 48h resulted in decrease of Survivin protein expression.The cleaved active subunits of Caspase-3 were observed following AS₂O₃ treatment from 24h to 72h.3.We found that in NCI-H157 acute promyelocytic cell line treatment with the MEK inhibitors PD98059 greatly enhances apoptotic cell death induced by AS₂O₃ alone.Combined treatment results in downregulation of the Survivin in NCI-H157 cell line.The levels of p-ERK were determined by Western Blot analysis at 5 minute after treatment with AS₂O₃ alone.DiscussionsSomeone reports that AS₂O₃ induced apoptosis of HL60,ML1 and U937 cell in high concentration,and induced differentiation toward monocyte/macrophage-like cells in low concentration.Our results showed that AS₂O₃ alone induced apoptosis of NCI-H157 cells and NCI-H157 in the concentration of more than 5μmol/L,but there is no apoptosis in less than 5μmol/L.Some observations were reported that MEK1/2 inhibitors potentiate the antitumor activity of various cytotoxic agents,including ara-C, cisplatin,and paclitaxel,suggests a possible role for MEK1/2 inhibitors in the treatment of human malignancies.At present,mechanism of AS₂O₃ is still unknown.It possibly actived APK,inhibit Topoâ…¡activity,and up-regulated Tiaml or down-regulated the express of c-myc and bcl-2 mRNA to induce apoptosis.To explore details involved in the process,apoptosis-related protein Survivin was determined by Western Blot and RT-PCR,Caspase-3 also was tested by Western Blot. We found that exposure of NCI-H157 cells to AS₂O₃ resulted in down-regulation of Survivin and Caspase-3 protein,down-regulation of Survivin mRNA was also detected.Furthermore,Survivin downregulation and activation of Caspase-3 became more marked in coadministration of AS₂O₃ with PD98059,the results indicates MEK/ERK signaling pathway mediates progess of AS₂O₃-induced apoptosis involved in Survivin downregulation in the post-transcription level.Conclusions1.AS₂O₃ inhibited NCI-H157 cell proliferation in a time- and dose-dependent manner and induced NCI-H157 cell apoptosis2.AS₂O₃ induced NCI-H157 cell apoptosis accompany with Survivin downregulation and activation of Caspase-3.3.MEK/ERK signaling pathway is negative regulation in AS₂O₃- induced apoptosis in NCI-H157 cell.