Cyclooxygenase-2 Induction Plays An Anti-apoptotic Role In Human Bronchial Epithelial Cells After Vanadium Pentoxide Exposure

Lung cancer is the leading cause of death among various cancers for both men and women in the world.Incidence rates and mortality rates of lung cancer keep increasing year by year.Although the diagnostic techniques and surgical therapy for lung cancer has been notably improved,patients still suffer a lot from missing the early diagnosis and therapy.With the existing therapeutic efforts,patients with lung cancer have a 5-year survival rate of less than 15%and this statistic has changed minimally in the last 25 years. To address this problem we need to take more effective measures to prevent the disease and trying to find new therapeutic strategies.It is reported that 80～90%cancers are caused by the exposure of environmental carcinogen,especially the chemical carcinogen.Vanadium,as an environmental and occupational pollutant,is a fairly common element in the earth crust.It is widely used in industry and is released to the environment through the combustion of both fossil fuels and oil.Increased concentrations are found in soil and plants near industrial sites.Most vanadium production is consumed by steel industry.Vanadium is also used as a dye in pottery and ceramics and as a catalyst in,e.g., the production of sulphuric acid.Boilermakers are exposed to metal fume that contain vanadium too.Workers occupationally exposed to vanadium are at risk as respirable particulates may penetrate deep into the tracheobronchial tree.Vanadium is a potent toxic agent and carcinogen.The mechanisms involved in its toxicity and carcinogenesis are still unclear.Improper cell growth is believed to be involved in cancer development.Some studies have revealed genotoxic effects on human cells caused by vanadium and close relationship between vanadium and cancer.Among them,there were several studies showing that exposure to vanadium was associated with an increased risk of lung cancer. Nonetheless,the mechanisms by which vanadium causes human lung cancers remain to be intensively elucidated.Cyclooxygenase(COX)-2 converts arachidonic acid to PGH2(prostaglandin H2), which is acted on by various prostaglandin synthases to give rise to the individual bioactive PG species.Normally,COX-2 is not constitutively expressed in most cells and tissues, while it is inducible and expressed in response to cytokines,growth factors and other stimuli.Aberrant or excessive expression of COX-2 has been implicated in the pathogenesis of many disease processes,including carcinogenesis.It is overexpressed in a variety of different tumors,especially in lung cancer.Further investigation showed that COX-2 played a very important role in the mechanisms of lung cancer and the expression of COX-2 was associated with the prognosis and the responses to chemotherapy and radiotherapy in patients with lung cancer.COX-2 is known to have a significant role in apoptosis and cell survival.Overexpression of COX-2 has been linked to down regulation of apoptosis and thus to facilitation of malignant transformation in many cell types.There had been many studies which investigated the mechanisms of anti-apoptotic role for COX-2.One landmark study showed that forced expression of COX-2 was found to increase Bcl-2 expression and resistance to apoptosis.Subsequent studies in a variety of tumors suggested that both Bcl-2-dependent and independent pathways might be operative in COX-2-mediated resistance to apoptosis.A recent study identified survivin,an inhibitor of apoptosis protein,as a candidate protein that might be responsible for elevated apoptosis resistance in COX-2-overexpressing cells.Whether vanadium is able to induce COX-2 expression in human bronchial epithelial cells and,if it is,which signaling pathway mediates its induction,as well as what the role of COX-2 is in cell responses to exposure of vanadium have not yet been investigated.The present study documents that vanadium pentoxide can markedly induce COX-2 expression in human bronchial epithelial cells(Beas-2B) through NFAT-dependent pathway,and elevated COX-2 protein expression mediates the protection of Beas-2B cells from apoptosis caused by vanadium pentoxide.PartⅠ.Vanadium pentoxide can markedly induce COX-2 expression in human bronchial epithelial cells(Beas-2B) through NFAT-dependent pathwayIn this part,we tried to find out whether the transcription and expression of COX-2 were changed in human bronchial epithelial cells after exposed to vanadium pentoxide and which transcriptional factor mediated COX-2 induction.First,Beas-2B cells were exposed to different concentrations of vanadium pentoxide for different time.The total RNAs were extracted from these exposed cells and were introduced to reverse transcription-polymerase chain reaction in order to find out the alteration tendency of COX-2 mRNA,as compared with the control.Second,the COX-2 expressions of the Beas-2B cells,which were transfected with COX-2 luciferase reporter plasmid,were measured using luciferase assay and western blot method after the cells were exposed to vanadium pentoxide.In order to further investigate the transcriptional factor for COX-2 expression in vanadium-exposed Beas-2B cells,the NFAT luciferase reporter plasmids were also transfected into Beas-2B cells and the activation of NFAT in vanadium-exposed Beas-2B cells were also measured using luciferase assay method.Finally,to find out whether the activation of NFAT is required for the COX-2 expression in vanadium-exposed Beas-2B,DN-NFAT,one eukaryotic expression construct which contains a deletion mutant of NFAT and can inhibit the transcription activity of all NFAT isoforms,was transfected into Beas-2B.Then COX-2 expression of the transfect was also detected in this NFAT-inhibition situation.The results showed that treatment of Beas-2B cells by vanadium pentoxide led to obvious COX-2 transcription and expression through RT-PCR and COX-2 luciferase reporter assay.This induction was not only dose-dependent,but also time-dependent.The expression of COX-2 protein was also detected by Western blot and the results showed that exposure to vanadium pentoxide could result in an increase of COX-2 expression in Beas-2B cells.Treatment of Beas-2B-NFAT-luc by vanadium pentoxide led to a marked activation of NFAT through NFAT luciferase reporter assay and this activation was also time and dose-dependent.Overexpression of DN-NFAT led to a dramatic inhibition of COX-2 expression induced by vanadium pentoxide through the COX-2-1uciferase reporter assay,which indicated that the COX-2 expression was NFAT dependent.PartⅡ.The COX-2 induced by vanadium pentoxide in human bronchial epithelial cells plays an anti-apoptotic role In partⅠ,we proved that the COX-2 expression in Beas-2B cells could be induced by vanadium in a time and dose-dependent manner through NFAT activation pathway.The purpose of this part was to find out the potential contribution of elevated COX-2 and activation of NFAT to the biological effects on Beas-2B cells by vanadium pentoxide and what effect the elevated COX-2 imposed on the apoptosis of the cells.Small interference RNAs of COX-2 and NFAT3 were constructed and transfected in Beas-2B cells to make the expression of these two proteins silence.The interference effects were proved by western blot assay after Beas-2B cells were exposed to vanadium pentoxide.The stable transfectants of Beas-2B cells including Beas-2B/DN-NFAT,Beas-2B/siCOX-2, Beas-2B/siNFAT3,and Beas-2B/Vector Control were exposed to vanadium pentoxide for 24 hours and were observed under microscopy.Cell apoptosis was determined by flow cytometry after the cells were stained by propidium iodide(PI).The result showed that siCOX-2 was able to knock down COX-2 expression in Beas-2B cells.Knockdown of COX-2 expression by siCOX-2 resulted in significant increases in the sensitivity of Beas-2B cells to vanadium-triggered cell death.Also, siNFAT3 was able to knock down NFAT3 in Beas-2B cells.Introductions of siNFAT3 and DN-NFAT were both able to lead to significant increases in the sensitivity of Beas-2B cells to vanadium-triggered cell death.Analysis of apoptosis using propidium iodide staining demonstrated that transfection of siCOX-2,siNFAT3 and DN-NFAT showed much more sensitive to apoptotic induction by vanadium pentoxide.The outcomes suggested that NFAT3 was a major NFAT isoform responsible for COX-2 induction,and more importantly,induction of COX-2 expression and activation of NFAT provided an inhibitory effect on apoptosis of Beas-2B cells caused by vanadium pentoxide.In summary,in this current study,we demonstrated that vanadium pentoxide was able to induce COX-2 expression in a NFAT-dependent way in Beas-2B cells,and the cell apoptosis of Beas-2B after vanadium pentoxide exposure was much more obvious when we blocked COX-2 expression and NFAT activation.From these findings,we anticipate that the carcinogenesis of vanadium to human bronchial cells may result from the inhibitory effect on cell apoptosis mediated by the NFAT-dependent induction of COX-2.