The Association Of Cigarette Smoking With PDCD4 In Hepatocellular Carcinoma

Background and Objective:Hepatocellular carcinoma(HCC) is one of the ten most popular carcinomas in the world,and it had been authenticated by epidemiology materials that many factors work together to result in the happen of liver cancer.In our country,it has something to do with the infection of HBV and HCV,the taking in of AFB₁,the pollution of drinking water and so on,and this often happens in the same family.It seems that the environment factors more important than heredity factors.In Japan,both of drinking and smoking are the risk factors,and they can work together. In the cases of Italian,drinking takes 45%,HCV takes 30%,HBV takes 22%,there are almost six point two hundred thousand new cases every year,in which 42.5% happens in China,and this number is rising now.And during the progression of these patients,oxidative stress usually exists through the whole progression.Programmed Cell Death Factor 4(PDCD4),is one of the genes which has been found to be up-regulated after the initiation of apoptosis and some recent studies suggest that PDCD4 acts as a tumor suppressor gene.PDCD4 is found to be highly expressed in JB6 P^-(transformation-resistant) but not P⁺(transformation-susceptible) cells and reduction of PDCD4 protein by antisense PDCD4 results in acquisition of a P⁺ phenotype.This has also been confirmed in vivo experiments.Although a number of tumor suppressors target transcription,Pdcd4 is the first suppressor found to target translation.Pdcd4 interacts with translation initiation factors eIF4A and eIF4G to inhibit translation in a mRNA-specific fashion and consequently to inhibit pro-oncogenic events such as activation of activator protein-1(AP-1)-dependent transcription,anchorage-independent growth,and invasion.Inactivation of tumor suppressors contributes to oncogenesis.Although most tumor suppressors,including p53,are mutationally inactivated,others such as Pdcd4 is not.Pdcd4 expression is down-regulated with progression in a number of human cancer sites such as lung and colon.Some study reports that Akt and p70^S6K are activated by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate(TPA) and serum deprivation,so to result in the phosphorylation and degradation of Pdcd4 protein.In addition to Akt,p70^S6K signaling,active mitogen-activated protein/extracellular signal-regulated kinase(ERK) kinase(MEK)-ERK pathway is essential for tumor promoter-induced down-regulation.Cigarette smoking is the risk factor for a number of carcinoma,and this point has become popular,however,the mechanism is still keep unclear,particularly in terms of the contribution of water-soluble compounds in CS.Several studies have demonstrated that aqueous extracts of CS saline induce a distinct pattern of(oxidative) stress related effects in exposed cells,such as the formation of DNA strand breaks and the expression of stress response genes.In addition to this,CS has been shown to exert inflammatory activities,as indicated by the release of the chemotactic cytokine interleukin(IL)-8 from bronchial epithelial cells in response to CS.To our acknowledge,the hazard of smoking now is focus on the induce of cancer and other diseases in the lung and cadiovascular system.Although there are mass screenings showing that cigarette smoking is a risk factor for liver cancer,but now there are few study about the mechanism of smoking on hepatocellular carcinoma.We want to find out through this study whether CS has something to do with the expression of PDCD4 in liver,and if so,what is the mechanism.Methods:1.Statistics analysis of the patients:To evaluate the relationship of CS with PDCD4 in hepatocellular carcinoma,we chose patients as follow:1) male;2) age＞30;3) HBsAg(+);4) without metastasis through iconography test;5) underwent liver surgery and be sure that there were no carcinoma left in the liver;6) a diagnosis of hepatocellular carcinoma through pathology test and there were no carcinoma left on the cutting edge.Try to decrease the disturbance of other factors by being strict with the qualification of patients.Collect the carcinoma tissues and the normal tissues around the carcinoma getting through surgery,check out the expression of Pdcd4 in the carcinoma tissues and in the normal tissues around the carcinoma using western blotting.The amount of smoke exposure was calculated in pack-years:the product of the number of years an individual had smoked and the average number of cigarettes smoked per day,converted into a standard pack of 20 cigarettes.At the same time,we considered many other factors such as the size and number of carcinoma,whether they invasive the portal vein and hepatic veins,whether they invasive bile ducts,the situation of the background of the liver and the tissue differentiation of the carcinoma. Samples were coded afcer data collection,after that,all statistical analyses were performed with SPSS11.5 software.Statistical comparisons of the data were performed by the Mann-Whitney U test or Fisher's exact test.A difference was considered significant when P had a value below 0.05.2.The normal liver cells L02 and hepatoma carcinoma cells HepG2 were cultured, check out the expression of PDCD4 in this two kinds of cells through western blot by using special antibody for human PDCD4 protein.Cigarette smoke extract(CSE) was prepared by a modification of the method before.In brief,two cigarettes without filters were combusted with a modified syringe-driven apparatus.The smoke was bubbled through 50 ml of serum-free DMEM,and the resulting suspension was adjusted to pH 7.4 and then filtered through a 0.20μm pore filter to remove bacteria and large particles,using 5%CSE to stimulate L02 cells for different times.Using MTT to test the activity and proliferation of the cells.Tested the changes of the transcription of PDCD4 through RT-PCR.Using 1%,5%,10%CSE to treat human liver cells L02 for 4 hours and treat L02 cells with 5%CSE for different times(1h,4h,16h,24h) After that,we harvested the cells and collected the protein, checked out the changes of the expression of PDCD4 by using special antibody for human PDCD4 protein.Through using Cycloheximide(CHX) to inhibit the new protein synthesis,and connected with 5%CSE,to check out whether the function of CSE is working on the translation period or after that.Pretreating L02 cells usig special blocking agents for pathway:Ro31-8425 for pan-PKC,PD98059 for MEK,LY294002 for PI3K,SB203580 for p38 MAPK,SP600125 for JNK,and then stimulated L02 cells with 5%CSE for 4 hours,harvested the cells and collected the protein extract,checked out the changes of the expression of PDCD4 using special antibody for it to test the which pathway takes the main role on the function CSE had to PDCD4.Result:1.Through statistics analysis of the clinical patients we found that:in the 68,male, hepatocellular carcinoma patients without metastasis we chose,there are no significant difference between the carcinoma tissues from both smokers group and non-smokers group(P>0.05).When coming to the difference between the smokers group and nonsmokers group,we got the significant difference of the content of PDCD4 between the normal tissues from both groups(P< 0.05).In the smokers group, the content of PDCD4 in the cancer tissues was apparent few to that in the normal tissues,while in the nonsmokers group,we had the more significant results (P<0.01).When comparing the difference of other factors,we found the patient's age of the smokers group was lower than that of non-smokers group.The severity of the fibrosis in the background liver of the smokers group was less than those of the non-smokers group.There is no significant difference between the extent of the invasion situation of the portal vein,hepatic veins and bile ducts.2.By comparing the normal liver cells L02 with carcinoma cells HepG2,we found the expression of PDCD4 in HepG2 was less than that in L02 cells.This changes have significant difference(P<0.05).By using 5%CSE to treat human liver cells L02 for different times and tested through MTT,we found that 5%CSE didn't influence the activity of L02 apparently.RT-PCR showed that there was no apparent difference in the transcription of PDCD4 with the stimulation of CSE.Using different concentration of CSE or treat L02 cells for different times,testing through western blot,we found that CSE could down-regulate the expression of PDCD4,and this changes had concentration and time dependence.Through using Cycloheximide(CHX) to inhibit the new protein synthesis,and connected with 5%CSE,we found the protein level of PDCD4 decreased greatly when both of CHX and CSE work.This prompts that the influence CSE had to PDCD4 maybe posttranslation,perhaps through speeding up the degradation of PDCD4.By using special blocking agents for some pathway which might we guess might have some relationship with the degradation of PDCD4, Ro31-8425 for PKC,PD98059 for MEK,LY294002 for PI3K,SB203580 for p38 MAPK,SP600125 for JNK,to pretreat L02 cells,then used 5%CSE to treat the cells for 2 hours,after that,test the changes of the expression of PDCD4 through western blot.We found Ro31-8425,PD98059,LY294002 all can change the function that CSE had to the expression of PDCD4 apparently,while SB203580,SP600125 seems not.Conclusion:1.The expression of PDCD4 is decrease in hepatocellular carcinoma.There is not significant difference about the expression of PDCD4 in the carcinoma tissues between the smokers group and the non-smokers group,prompting that PDCD4,as a carcinoma suppressor,decreases to some extend and then the hepatocellular carcinoma will happen.When comparing the difference of PDCD4 in the normal tissues around the carcinoma,the smokers group had a more apparent decrease than non-smokers group,which might mean the worse prognosis.2.The expression of PDCD4 of carcinoma cells HepG2decreases greatly than that of the normal liver cells L02.3.CSE can decrease the expression of PDCD4 in L02 cells,but not influent the transcription,which prompts that the inhibition function CSE had on PDCD4 is posttranslation.4.CSE might active PI3K-Akt-p70^S6K pathway and PKC,which can phosphorylate PDCD4 and result in the degradation of PDCD4.MEK-ERK pathway might be essential in this process.