The Suppressed Effect On The Tumor Cell Lines Of The Combination Of SPB And Gefitinib: In Vitro & In Vivo.

Tumor is a kind of disease that multiple factors,multiple steps and mulitiple genes changed,each medicine can play a part in only one or several target, if we can combined multiple inhibitors of tumor will get better effect, drug combination will become a tendency that tumor curement object.The ASCO meeting holded in US draw a new viewpoint that target treatment,drug combination and multiple target medicine combination et al.Such method will bring better effect and profit.The mechanism promote the tumor growth and carcinogenesis will uncertain to date, the gene phenotype modify and signal transduction have been study hot about the mechanisms of tumor growth and carcinogenesis.The gene phenotype modify can control the activation and inactivation of function gene so that activate selectively the specific hereditary information .Many studies have certified that tumor cells have the abnormality of gene phenotype states, the modify of acetylation and deacetylation is most major way that modify gene phenotype.Such sytle paritipate in the development and occurrence of many tumors. The mechanism that HDACIs cure tumor is a Hotpoint that interven the gene phenotype modify, summarize previous studies, we sum up several mechanism of HDACIs 1: induce the differentiation of tumor; 2: chang the cycle or tumor through impacting the P21WAF1,P53,Rb,myc et al that related to the cell cycle;3:induce the apoptosis of tumor;4:up-regulate the MHCⅠ,Ⅱ,CD40 molecules to regulate the immune function;5:suppress the vascularization of tumor ;6:revers the inverted phenotype;7:activat the silent gene;8:recover the sensitivity of drug-resistant tumor cells to drugs.The moleculer mechanism about the carcinogenesis ,the abnormality of signal transduction mediated by epidermal growth factor receptor is related to the development and occurrence of tumor.Many evidences have unclose that the overexpression of EGFR in many tumor coming from epithelium tissue,the activation of EGFR can activate the Ras/Raf/MAPK,PI-3K/Akt et al signal transduction pathway,in the end,such signal pathway influence the gene expression to induce the proliferation and differentiation biological effect.Gefitinib is a kind of micromolecule compound that selectively suppress the EGFR kinase,it can bind competivly the EGFR kinase to suppress the activation of EGFR kinase,it can block the signal transduction mediated by EGFR to suppress the proliferation and metastasis and induce the apoptosis of tumor,Gefitinib can specificly suppress the HER1 when low concentration ,it can suppress other PTK activation when high concentration ,so it has repressed activity to a series of tumor lines. Frist,we screen the optimal concentration of SPB and Gefitinib in vitro,1.0mM and higher concentration SPB have obviously suppression effect to three kinds of hametology lines (U937,HL-60,K562),such result consists with the effect on the tumor origined from epithelium tissue treated by SPB, compared with tumor lines origined from epithelium tissue ,the effect of hametology cell lines treated by SPB is more sensitive.The survival rate and the cell counts in U937,HL-60,K562 cell lines treated by Gefitinib only over 30g/L concentration show significant difference compared with control group,but the survival rate is over 75%,the cells number loss isn't obvious when the concentration is increased to 50g/L,the effect isn't consistant with the result in the cells origined from epithelium tissue treated by Gefitinib. The repressed effect is obviously when the concentration of Gefitinib was increased to 40g/L in the cell lines origined from epithelium tissue.We get the optimal drug concentration which suppression rate is obvious but survival rate is over 75%,as well as the conclusion that SPB is sensitive to hametology cells,but the Gefitinib is sensitive to cells line origined from epithelium tissue,we even found the synergistic inhibition between Gefitinib and SPB when A549 and SMMC-7721 treated.Interestingly,the Gefitinib is the inhibitor of EGFR,however,the EGFR is specific expressed in the cell origined from epithelium tissue,but it can suppress the proliferation of U937,HL-60,K562 cells though not so strong.Such result consist with conclusion that high concentration Gefitinib can suppress the other PTK activity in published literature ,morever other unusual mechanisms,It discover the that the gefitinib suppress the cell proliferation through not only RGFR pathway ,it can explain why low concentration Gefitinib cannot suppress the proliferation of hamtology cell lines.We conclude the possible mechanism that combining such two drugs through analyzing such two drugs mechanism of action:①enhance the arrest the cycle ,arrest the tumor cell in G1 stage;②suppress the angiogenesis around the tumor tissue and metastasis;③regulate the expression of P21WAF1,P53,CDK4 gene and protein that related to the apoptosis and cycle;④synergistic inhibit the signal transduction .We consider the essentiality and rationality to combining such two drugs according to the analysis and previous expriment experience. P38MAPK is a family member in the mitogen-activated protein kinase, MAPK cascade is a predominant signal transduction system in cells, it adjust the growth,differentiation,cleavage,death and function synchronization among cells process.We detected the expression of phospho-P38MAPK and gene,protein and apoptosis when suppress the P38MAPK activity with Westernblot assay to determine if the drug combination of SPB and Gefitinib can adjust the survival state through the P38MAPK signal transduction pathway.We found the drug combination of SPB and Gefitinib can get better effect in tumor cell origined from epithelium tissue than hematological cells,the effect of drug combination isn't so obvious in hematological cells compared with SPB or Gefitinib only(P>0.05).So we chose two kinds of cells including the A549 and SMMC-7721 cell lines to determine the action and mechanism of drug combination in vitro.We found the drug combination of SPB and Gefitinib can increase the apoptosis rate of SMMC-7721 and A549 cell, compared with SPB or Gefitinib only,the difference shows significant(P<0.05), but the extent of SPB and gefitinib under optimal concentration induced apoptosisisn't so obvious,drug combination increase only the apoptosis rate from 7-8% to 11-12% compared with SPB and Gefitinib only,it elucidate that the mechanims of the drug under optimal concentration is suppression of proliferation but not inducing apoptosis of tumor cells. SPB and Gefitinib can both arrest such two kinds of tumor cells in G1 stage to suppress the proliferation of cell.The drug combination of SPB and Gefitinib can arrest cycle in G1 stage obviously compared with SPB and Gefitinib only(P<0.05). SPB and Gefitinib only can also enhance the expression of P53 and P21 protein, suppress the expression of CDK4/CyclinD1. The expression tendency of these protein is more obvious in drug combination group compared with SPB and Gefitinib group(P<0.05),the expression of P53﹑P21 gene and protein decreased, CDK4/CyclinD1 increased when the P38MAPK kinase activated by P79350, such expriment delinate the drug combination of SPB and Gefitinib increase the expression of P53﹑P21,decrease the CDK4/CyclinD1 compound though P38MAPK signal transduction pathway.It certifies that P38MAPK signal transduction pathway affect te proliferation mediated by cell cycle,we can adjust the change of the cycle an apoptosis through changing the related gene and protein.Different literatures have different viewpoints about the influnce on the gene and protein by SPB and Gefitinib,some thought the SPB adjust cell cycle independ on P53,some thought Gefitinib can arrest G2/M stage.Different studies draw different conclusions because of different drug concentrations and cell lines.Such two drugs are both concentration and time dependent style according to previous literatures.Our results consist with above conclusions,some studies thought drug combination of HDACI and Gefitinib is sequence-dependent style,we don't testify such conclusion,we detected the suppressed effect that SPB 12h prior to Gefitinib, such partly consisted with published literatures.To authenticate the suppressed effect after metabolism and the influnce by immunization and endosecretory ,we chose the nake mices injected from right armpit as object in vivo.We chose concentration that screened in preliminary experiment,the SPB and Gefitinib only can suppress the preliferation of human A549 cell line in vivo,they can suppress tumor weight and volume in that nick mice that tumor tissue has formed, moreever lengthen the survival time and increase the survival rate .We detected the expression of P53 and CDK4 protein with immunohischemistry methods,drug combination can enhance the expression of such protein ,such consist with the results in vitro,we also detected the suppressed effect with the drug concentration according to related published literatures.We didn't screen the concentration grade in vivo and compare the effect under the same concentration between in vitro & vivo, we need to investigate the experiment further.