Role Of MAPK Signal Pathways In Tobacco Smoke-induced Abnormal Pulmonary Neuroendocrine Differentiation And PEITC Intervention

Lung cancer is the most common cancer and the leading cause of cancer death worldwide.Studies have confirmed that smoking is the most important cause of lung cancer.It is estimated tobacco smoke(TS)accounts for about 90 % of men and 75%-80% of women with lung cancer.TS is causally associated with various types of cancers,especially lung cancer.Passive smoking has been identified as a human carcinogen.China is the largest producer and consumer of tobacco around the world,and smoking-caused lung cancer and other related diseases have become a major public health problem.There are four major histologic types of lung cancer,including squamous cell carcinoma,adenocarcinoma,small cell carcinoma,and large cell carcinoma.Several preneoplastic lesions of squamous cell carcinoma and adenocarcinoma have been identified.The precancerous lesions of small cell carcinoma and large cell neuroendocrine carcinoma remain unclear;the expression of its characteristic neuroendocrine markers has become key diagnostic indicators.MAPK signaling pathway play important role in biological process.In vitro and vivo studies have shown that TS exposure induces AP-1 and MAPK activation.To date,however,no studies have been done to investigate the effect of MAPK/AP-1 on TS-induced neuroendocrine changes of the lungs pulmonary epithelial cell.Phenethyl isothiocyanate(PEITC)is derived from cruciferous vegetables commonly consumed by man.Recently,several lines of evidence indicate that PEITC exhibits anti-inflammatory,anticarcinogenic,and antioxidant properties.PEITC exhibits chemopreventive action in humans against several types of cancer including lung,colon,bladder,and stomach shown through epidemiological studies.The present study aimed to investigate the effects of tobacco smoke on pulmonary neuroendocrine differentiation and the role of MAPK/AP-1 pathways in normal human bronchial epithelial(NHBE)cells and in the lungs of mice,by using in vitro and in vivo tobacco smoking models.Meanwhile,PEITC intervention of tobacco smoke-induced alterations in pulmonary neuroendocrine differentiation as well as MAPK/AP-1 pathways was examined.Findings from this research could provide new insights into the molecular mechanisms of tobacco smoke-associated pulmonary neuroendocrine tumors and its target prevention.Part I: Role of MAPK Pathways in Cigarette Smoke-induced NHBE Cells Abnormal Neuroendocrine Differentiation and PEITC InterventionObjective:By using in vitro cigarette smoking model,the present study aimed to investigate the effects of cigarette smoke on normal human bronchial epithelial(NHBE)cells neuroendocrine differentiation and alterations in MAPK/AP-1 pathways in NHBE cells.Meanwhile,PEITC intervention of cigarette smoke-induced alterations in NHBE cells neuroendocrine differentiation as well as MAPK/AP-1 pathways was examined.Methods:The effect of cigarette smoke on cell viability was examined by MTT assay.NHBE cells were treated with different concentrations of cigarette smoke extract alone or combine with inhibitors of MAPK for 7 days,the effects of tobacco smoke and inhibitors of MAPK on morphology of NHBE cells were examined.The m RNA expressions of neuroendocrine differentiation genes(Cg A,SYN,NSE,NCAM,CEA)and epithelial differentiation genes(E-cadherin?ZO-1?CK5?CK13)were determined by real-time PCR.The protein expressions of neuroendocrine differentiation and epithelial differentiation genes were measured by Western blot.Activation of MAPK(ERK1/2?JNK?p38)and AP-1(family members of Fos and Jun)pathways were analyzed by Western blot.The effect of PEITC alone or combine with cigarette smoke extract on NHBE cell viability was examined by MTT assay.The m RNA expressions of neuroendocrine differentiation genes and epithelial differentiation genes were determined by real-time PCR.The protein expressions of neuroendocrine differentiation and epithelial differentiation genes were measured by Western blot.Activation of MAPK and AP-1pathways were analyzed by Western blot.Results:1.Cyotoxicity of tobacco smoke extract treatment in NHBE cellsNHBE cells were treated with different concentrations of cigarette smoke extract(CSE)for 7 days.The effect of tobacco smoke on cell viability was examined by MTT assay.The result showed that 0%-4% CSE did not have cell toxicities;however,CSE at 8% caused a significant decrease in cell viability in NHBE cells after 7 days exposure.Since 8% CSE was toxic to NHBE cells,0%-4% CSE were used for the following experiments.2.Effect of cigarette smoke extract on morphology of NHBE cellsNHBE cells were exposed to CSE(0%-4%)daily,the development of a neuronal-like phenotype characterized by the protrusion of cell filopodia was observed in a concentration dependent manner compared with control cells which showed cobble stone epithelial morphology after 7 days exposed.3.Cigarette smoke activated MAPK/AP-1 pathwayAfter NHBE cells were exposed to CSE(0%-4%)for 7 days,activation of MAPK and AP-1 pathways were analyzed by Western blot.We found that cigarette smoke significantly activate phosphorylated ERK1/2,JNK and p38,up-regulated the protein expression of Fos(c-Fos ? Fos B ? Fra-1 ? Fra-2)and c-Jun,and down-regulated the protein expression of Jun B and Jun D in NHBE cells.4.Cigarette smoke-induced NHBE cells abnormal neuroendocrine differentiationThe m RNA expressions of neuroendocrine differentiation genes and epithelial differentiation genes were determined by real-time PCR.The protein expressions of neuroendocrine differentiation and epithelial differentiation genes were measured by Western blot.The results showed that cigarette smoke dose-dependently induced up-regulation of m RNA and protein expression levels of neuroendocrine differentiation genes and down-regulation of m RNA and protein expression levels of epithelial differentiation genes.5.Effect of MAPK inhibitors on activation of MAPK/AP-1 pathwayWe pre-incubated NHBE cells with specific MAPK inhibitors before CSE treatment,and then the effects of MAPK inhibitors on activation of MAPK/AP-1 pathway were examined.ERK1/2(U0126,5?M)or p38(SB203580,10?M)suppressed cigarette smoke-triggered activation of MAPK/AP-1 pathways.SP600125(JNK inhibitor,10?M)was not able to significantly affect AP-1 activation.6.Role of MAPK signal pathways in cigarette smoke-induced abnormal neuroendocrine differentiation in NHBE cellsWe pre-incubated NHBE cells with specific MAPK inhibitors before CSE treatment for 7 days,and then the m RNA expressions of neuroendocrine differentiation genes and epithelial differentiation genes were determined by real-time PCR and the protein expressions of neuroendocrine differentiation and epithelial differentiation genes were measured by Western blot.The results showed that alterations in the expressions of these makers were blocked by inhibitor of ERK1/2(U0126)or p38(SB203580).SP600125(15?M)did not suppress the expressions of these makers.7.Effect of MAPK inhibitors on morphology of NHBE cellsWe pre-incubated NHBE cells with specific MAPK inhibitors before CSE treatment for 7 days.Pretreatment with the inhibitor of ERK1/2(U0126)or p38(SB203580)significantly attenuated CSE-induced phenotypical changes.However,pretreatment with SP600125(JNK inhibitor)was not able to significantly affect the morphological changes induced by CSE.8.Effect of PEITC on NHBE cells viabilityNHBE cells were exposed to CSE(0%-4%)alone or combination with PEITC for 7 days.MTT assay was performed to examine the effect of PEITC on cell viability.After 7 days exposure PEITC inhibited growth of NHBE cells in a concentration-dependent manner.2?M PEITC was used for following experiments.9.PEITC suppressed cigarette smoke triggered activation of MAPK pathwayNHBE cells were exposed to CSE alone or combination with PEITC for 7 days.We found that PEITC suppressed cigarette smoke-triggered activation of MAPK/AP-1 pathways.10.PEITC reversed cigarette smoke induced NHBE cells abnormal neuroendocrine differentiationNHBE cells were exposed to CSE alone or combine with PEITC for 7 days and the effects of PEITC on expressions of neuroendocrine differentiation genes and epithelial differentiation genes were observed.PEITC reversed cigarette smoke induced up-regulation of m RNA and protein expression levels of neuroendocrine differentiation genes and down-regulation of m RNA and protein expression levels of epithelial differentiation genes.11.Effect of PEITC on morphology of NHBE cellsCigarette smoke treatment of NHBE cells for 7 days induced cellular morphology changes,and PEITC restored cell morphology features.Conclusions: The present study has revealed for the first time that cigarette smoke induces neuroendocrine differentiation in NHBE cells.MAPK/AP-1 pathways play an important role in the cigarette smoke-induced neuroendocrine differentiation in NHBE cells.PEITC effectively attenuated cigarette smoke-induced neuroendocrine differentiation in NHBE cells through modulation of MAPK/AP-1 pathways.Our data shed new light on the molecular mechanism of tobacco smoke-induced pulmonary neuroendocrine tumors and targeted intervention.Part?: Role of MAPK Signal Pathways in Tobacco Smoke-induced Abnormal Pulmonary Neuroendocrine Differentiation in BALB/c mice and PEITC InterventionObjective:By using in vivo tobacco smoking(TS)model,the present study aimed to investigate the effects of TS on neuroendocrine differentiation and the role os MAPK/AP-1 pathways in BALB/c mice lung.Meanwhile,PEITC intervention of tobacco smoke-induced alterations in pulmonary neuroendocrine differentiation as well as MAPK/AP-1 pathways was examined.Methods: Eight-week-old male BALB/c mice weighing 18-22 g were purchased from the Animal Research Center of Nanjing Medical University.Mice were exposed to TS in a smoking apparatus.TS was generated by a smoke machine that smoked the cigarettes and pumped the mainstream cigarette smoke from burning cigarettes at a constant rate(each cigarette took 5 min to burn out).The smoke was delivered to whole-body exposure chambers with target concentration of total particulate matter(TPM)of 85 mg/m3.Animals were exposed for 6 hours daily for 12 consecutive weeks.Male BALB/c mice were treated with TS alone or in combination with MAPK inhibitors.The effects of TS and MAPK inhibitors on body weight of mice were examined.The m RNA expressions of neuroendocrine differentiation genes(Cg A,SYN,NSE,NCAM,CEA)and epithelial differentiation genes(E-cadherin?ZO-1?CK5?CK13)were determined by real-time PCR.The protein expressions of neuroendocrine differentiation and epithelial differentiation genes were measured by Western blot.Activation of MAPK(ERK1/2?JNK?p38)and AP-1(Fos and Jun family members)pathways were analyzed by Western blot.The effects of PEITC alone or combination with tobacco smoke on body weight of mice were examined.The m RNA expressions of neuroendocrine differentiation genes and epithelial differentiation genes were determined by real-time PCR.The protein expressions of neuroendocrine differentiation and epithelial differentiation genes were measured by Western blot.Activation of MAPK and AP-1 pathways were analyzed by Western blot.Results:1.TS reduced body weight of miceAnimals were exposed for 6 hours daily for 12 consecutive weeks.Mice were weighed weekly.Compared to filtered air controls;body weight of mice exposed to cigarette smoke was markedly reduced after 12 weeks TS exposure.2.TS significantly activated MAPK and AP-1 pathways in the lungs of miceAnimals were exposed for 6 hours daily for 12 consecutive weeks.Activation of MAPK and AP-1 pathways were analyzed by Western blot.TS significantly activated phosphorylated ERK1/2,JNK,p38,up-regulated the protein expression of Fos(c-Fos?Fos B?Fra-1)and c-Jun?Jun B,and down-regulated the protein expression of Jun D in the lungs of mice.3.TS induced abnormal pulmonary neuroendocrine differentiation in BALB/c miceAnimals were exposed for 6 hours daily for 12 consecutive weeks.TS dose-dependently induced up-regulation of m RNA and protein expression levels of neuroendocrine differentiation genes and down-regulation of m RNA and protein expression levels of epithelial differentiation genes.4.Effect of MAPK inhibitors on body weight of miceMice were treated with TS alone or in combination with p38 inhibitor(SB203580,1mg/kg),ERK1/2 inhibitor(U0126,0.5mg/kg),JNK inhibitor(SP600125,1mg/kg).Animals were exposed for 6 hours daily for 12 consecutive weeks.SB203580 prevented TS-induced decrease of body weight.U0126 and SP600125(JNK inhibitor)were not able to significantly affect TS-induced decrease of body weight.5.Effect of MAPK inhibitors on activation of MAPK and AP-1 pathwaysBALB/c mice were treated with TS alone or in combination with p38 inhibitor(SB203580,1mg/kg),ERK1/2 inhibitor(U0126,0.5mg/kg),JNK inhibitor(SP600125,1mg/kg).Animals were exposed for 6 hours daily for 12 consecutive weeks.Activation of MAPK and AP-1 pathways were analyzed by Western blot.SB203580 suppressed TS-triggered activation of p38 MAPK and AP-1 pathways.U0126 and SP600125 suppressed TS-triggered activation of ERK1/2 and JNK MAPK pathways,respective;while U0126 and SP600125 were not able to significantly affect AP-1 activation.6.Role of MAPK pathways in TS-induced abnormal neuroendocrine differentiation in BALB/c miceBALB/c mice were treated with TS alone or in combination with p38 inhibitor(SB203580,1mg/kg),ERK1/2 inhibitor(U0126,0.5mg/kg),JNK inhibitor(SP600125,1mg/kg).Animals were exposed for 6 hours daily for 12 consecutive weeks.The m RNA expressions of neuroendocrine differentiation genes(Cg A?SYN?NSE)and epithelial differentiation genes(E-cadherin?ZO-1?CK5)were determined by real-time PCR and the protein expressions of neuroendocrine differentiation and epithelial differentiation genes were measured by Western blot.The results showed that the alterations in the expressions of these makers were prevented by p38 inhibitor(SB203580).ERK1/2(U0126)or SP600125(15?M)did not suppress the expressions of these makers.7.PEITC prevented TS-induced decrease of body weight of miceBALB/c mice were treated with TS alone or in combination with PEITC(500mg/kg BW and 1000 mg BW).Animals were exposed for 6 hours daily for 12 consecutive weeks.PEITC prevented tobacco smoke-induced decrease of body weight.8.PEITC suppressed TS-triggered MAPK and AP-1 pathways activationBALB/c mice were treated with TS alone or in combination with PEITC(500mg/kg BW and 1000 mg BW).Animals were exposed for 6 hours daily for 12 consecutive weeks.Activation of MAPK and AP-1pathways were analyzed by Western blot.PEITC(1000mg BW)suppressed tobacco smoke-triggered activation of MAPK and AP-1 pathways.9.PEITC reversed TS-induced BALB/c mice abnormal neuroendocrine differentiationBALB/c mice were treated with TS alone or in combination with PEITC(500mg/kg BW and 1000 mg BW).Animals were exposed for 6 hours daily for 12 consecutive weeks.The m RNA expressions of neuroendocrine differentiation genesand epithelial differentiation genes were determined by real-time PCR.The results showed that TS-induced alterations in the expressions of these makers were attenuated by PEITC.The protein expressions of neuroendocrine differentiation genes(Cg A?SYN?NSE?CEA)and epithelial differentiation genes(E-cadherin?ZO-1?CK5)were measured by Western blot.The results showed that the expressions of these makers were blocked by PEITC(1000mg BW).Conclusions: The present study has revealed for the first time that tobacco smoke induces pulmonary neuroendocrine differentiation in BALB/c mice.P38 MAPK pathway plays an important role in TS-induced neuroendocrine differentiation in BALB/c mice.PEITC effectively attenuated tobacco smoke-induced neuroendocrine differentiation in BALB/c mice lung through modulation of MAPK/AP-1 pathways.Our data shed new light on the molecular mechanism of TS-induced pulmonary neuroendocrine tumors and targeted intervention.