Establishment And Preliminary Evaluation Of A Tobacco-induced Cell Malignant Transformation Model For Cancer Prevention Drug Research

Background:Cancer is the result of gene and environment interactions,which seriously threatens human health and life.Chemoprevention is an effective anti-cancer strategy,aiming to reduce the risk of cancer,to delay the development of cancer and to prevent the recurrence of cancer by using drugs.However,there is no experimental platform for the study of cancer chemoprevention.Bhas 42 cell transformation assay is an effective method to analyse cancerogens with great advantages.Therefore,it has potential to be applied for the research of cancer chemoprevention drugs.The carcinogen was used to induce transformation of cells,and then the effect of drugs on the transformation process were observed.To develop the model as an experimental platform for the study of cancer chemprevention drugs.Bai He Gu Jin Fang is a traditional formula designed to treat lung and kidney yin deficiency.It has the effect of nourishing yin and clearing heat,cooling blood to stop bleeding,moistening the lung and relieving cough.The chemical composition of TCMs in the formula were reported had cancer chemoprophylaxis.Therefore,it has the potential value of studying the chemoprevention.Objective:To establish a cell model suitable for researching cancer chemprevention drugs,and preliminarily evaluate the model's stability and feasibility based on the cigarette smoke condensate(CSC)inducing Bhas 42 cell transformation assay.At the same time,to study the chemprevention of Bai He Gu Jin Fang based on the cell model.Methods:1.Preparation and Consistency evaluation of CSC:collecting 7 brands of blend tobacco,and the smoke particulates of cigarettes were collected on a Cambridge filter pad from cigarettes smoked.The particulates collected on Cambridge filter were extracted in organic solvent.The ingredients and similarity of extracts were analyzed by GC-MS and the fingerprints technique.2.Establishment of cell malignant transformation model induced by CSC and research on its transformation mechanism:the effects of different concentrations of CSC on cell proliferation were observed by cck-8.Bhas 42 cells were inoculated into 6 holes cell culture plate and induced by CSC for 72h.After three weeks,the number of transforming foci were calculated.The transformed cells were separated from the foci and named T-Bhas 42.ELISA and RT-PCR were performed to explore the changes of a-SMA protein,TGF-?1,Smad4 and ?-SMA gene expression in Bhas 42 cell and T-Bhas 42 cell.The cell-cycle distribution was analyzed by FCM.3.Feasibility study of using cell malignant transformation model to research cancer chemprevention drugs:The cells began to interven with four effective monomers including salicylic acid,curcumin,sodium selenite,and ferulic acid.After three weeks and the number of transforming foci were calculated similarly.To investigate the effect of four effective monomers on the formation of foci.4.Based on the cell model to investigate the role of Bai He Gu Jin Fang in cancer prevention:According to the pharmacopoeia prepareing extract of TCM with water extraction and alcohol precipitation method.The cells were intervened with single potion,after three weeks,we stained and calculated transforming foci.The effects of combined drugs on the formation of the foci were also compared,including Radix Rehmannia+ Angelica,Radix Rehmannia+ ophiopogon root,Angelica+ ophiopogon root,Radix Rehmannia+ Angelica+ white Peony root(cooling blood to stop bleeding),fritillaria +Liquorice +Platycodon(moistening the lung and relieving cough),Lily+dried Radix Rehmannia+ ophiopogon root+ scrophulariae(nourishing yin and clearing heat).To verification of results by vivo experiments,model of transplanted tumor on mice was used to study the anticancer effect of compound in vivo.After two weeks,the tumor tissue,spleen and thymus were stripped to calculate tumor inhibitory rate,spleen index and thymus index.Results:1.The preparation process of CSC was stable,and the yield of one cigarettes was 8.95�1.78 ?g.GC-MS results showed that the main compositions of CSC prepared is nicotine(64.69%)and Triacetate(22.81%),and the similarity value between different brands was not less than 0.98.The CSC is suitable for cell modeling for its good stability and consistency.2.Bhas 42 cells induced by CSC could form obvious ? type foci,significantly different from the control group.The results of ELISA showed that the expression of a-SMA decreased obviously in T-bhas 42 cells(P<0.05).The results of RT-PCR showed that the relative expression levels of TGF-?1,Smad 4and ?-SMA in the T-bhas 42 cells were all decreased,which were significantly different compared with Bhas 42 cells(P<0.05).The results of FCM showed that the S-phase cell fraction increased,and the G0/G1-phase cell fraction decreased after transformation(P<0.05).It proved that CSC may induce Bhas 42 cells to form T-bhas 42 cells with proliferation and growth advantage by regulating TGF-? pathway.3.Effective monomer intervention experiments showed that 5 ?M of salicylic acid,high doses of ferulic acid(20 ?M and 40 ?M),a certain concentration of curcumin and sodium selenite could inhibitthe formation of foci,and the foci were mainly ? type and ? type foci,which were significantly different compared with control group.It proved that Bhas 42 cell transformation model induced by CSC could be used in the investigation of cancer chemopreventive drugs.3.Bai He Gu Jin Fang intervention experiment shows that the combination of 1.2 mg/mL Radix Rehmannia and Angelica could obviously decrease the number of foci,which were significantly different compared with control group P<0.05).Experiment of transplanted tumor on mice showed that Radix Rehmannia and Angelica could inhibit the occurrence and development of tumor,while Bai He Gu Jin Fang had no effect on the process.The conclusion presents the significance of the cell model on screening cancer chemoprevention.Conclusion:Bhas 42 cell transformation model induced by CSC can be used in the study of cancer chemopreventive drugs,and this model is proved has certain stability and feasibility by using effective monomer to evaluate it.Based on the cell model,the cancer chemical prevention drugs were screened out from Bai He Gu Jin Fang,and the reliability of the model was preliminarily verified through animal experiments.