| Background:The latest statistics data according to the global cancer morbidity shows that lung cancer is the most pathogenetic cancer all over the world nowadays.Among the developing countries,Chinese male are included to the most pathogenetic crowds, while the morbility of the female is middling all over the world,which is in the same manner with female in Australia and New Zealand.Recently,incidence of lung cancer in China is obviously increasing,incidence of small cell lung cancer(SCLC) accounts for 10%-25%of primary lung cancer,and the tumor differentiation grade is very low, even blood metastasis occurs in the earlier stage,and they end with a poor prognosis. Although sensitive to chemotherapy,SCLC can result in multidrug resistance(MDR) phenomenon,which lead to a failed chemotherapy course.MDR means that,once a tumor produces drug resistance to a chemotherapy drug,it will produce cross-resistance to other drugs with different structures and action mechanism,which is a special broad-spectrum drug resistance phenomenon.SCLC patients often produce MDR phenomenon easily,thus the chemotherapy effect is usually unideal, which leads to a high recurrence with SCLC and poor prognosis,and 2-year-survival rate is less than 5%,90%patients will die in 5 years after final diagnosis.Therefore, chemotherapy resistance has become one of the most emergency problems of SCLC clinical treatment.The studies showed that cell apoptosis induced by cancer cells escaping from hemotherapy drugs is the decisive factors to produce chemotherapy drug resistance, thus,to research on the mechanism of cancer cells escaping from apoptosis remains the key point of illuminating chemotherapy drug resistance.Whether cells will generate apoptosis is controlled by the balance between cancer cell apoptosis depressed genes and cancer cell apoptosis promoting genes,chemotherapy drugs could induce cancer cells apoptosis,but once one or some apoptosis depressed genes overexpress or apoptosis promoting genes is depressed,cells will not generate apoptosis,cancer cells escaping from chemotherapy drugs induction will produce drug resistance.Among the numerous apoptosis related genes,Bcl-2 family has a close relation with tumor chemotherapy drug resistance.Most members of Bcl-2 family are adjustor of cell chondriosome apoptosis,they can be divided into three groups:anti-apoptosis genes,including Bcl-2,Bcl-xL;apoptosis promoting genes, Bak,Bax;anti-apoptosi and apoptosis promoting two-ways regulation genes,Bid,Bik, Noxa and Bim.This study will concentrate on the anti-apoptosis genes Bcl-2, Bcl-xL and apoptosis depressed genes Bak and Bax.At present,Bcl-2 and Bcl-xL are the apoptosis depressed genes which may have a close relation with SCLC chemotherapy drug resistance.They can directly withstand outside apoptosis inducing irritation.Bax is a protein which can coprecipitate with Bcl-2,it is firstly discovered by Oltvai et al from human and mice B cells using immunoprecipitation et al methods in 1993,it has a feature to promote cell apoptosis,and remains a close relation with the generating and developing of tumor and MDR phenomenon.Bak(for bcl-2 homologus antagonist/ killer) is a new gene of Bcl-2 family cloned all alone by Chittenden,Farrow and Klefer in 1995,its expression can accelerate cell apoptosis.Though study on mechanism of Bcl-2 family apoptosis related genes act on cell apoptosis regulation has made a great progress,but the regulation pathway of upstream gene still remains unclear,and some study showed that PTK may play an important role in the regulation pathway of cell apoptosis.PTK includes two large categories,receptor species and non-receptor species. Btk family is a popular non-receptor PTK species which has a close relation with the generation and differentiation of T and B lymph cells recent years.Epithelial and endothelial tyrosine kinase(Etk),which is also called bone marrow X kinase(Bmx),is one of the important members of Btk family(Btk/AKT,ITK/EMT/TSK, tk/BMX,TEC).Etk is the only non-receptor specie PTK of Btk family which express in epithelial cells and vascular endothelial cells as well as in lymph and hematopoietic cells.Although Etk expresses low in epithelial cells,studies show that it plays an important role in the regulation of generation,differentiation,apoptosis of epithelial cells and genesis of tumor.Qiu confirmed that Etk take part in the differentiation of carcinoma of prostate cells which is mediated by IL-6.Guo et al found that Etk may join in the regulation of differentiation of nasal pharynx mucosa epithelium in some cases by immunohistochemistry technique.Our prophase study showed the expression positive rate of Etk in SCLC tissue was obviously higher than lung SqCa and adenocarcinoma of lung,and the expression of Etk has a close relation with apoptosis-related genes Bcl-2 and so on.To further approach the relation between Etk and SCLC cells apoptosis,we used ADM to treat SCLC NCI-H446 cells and Etk high expressing cell line NCI-H446WT,the result showed Etk resist to the cell apoptosis induced by ADM.Baced on the role Etk plays in the controlling of SCLC cell apoptosis,the experiment will further study on the expressing of Etk in SCLC cell and drug resistance cells,together with the relation between Etk and Bcl-2 family,to approach the role Etk plays in drug resistance generating of SCLC cells,so it will afford new theory support for drug resistance problems which is most easily occurs in the treatment of SCLC clinically.Objects:1:This experiment choose H69 and H69AR as object to analyse the discrepancy in morphology,cell cycle and the sensitivity to chemotherapy drugs of the two cell lines.2:To study on the Etk expression discrepancy of H69 and H69AR at mRNA and protein levels,using RNAi technique to silence the Etk gene of H69AR cell line,then detecting the effect of silencing,and observe the change of drug resistance of H69AR cell line.3:To analyse the expression difference of Bcl-2 family proteins(Bcl-2,Bcl-xL,Bak and Bax) between H69 and H69AR,together with the expressing relation between Etk protein and Bcl-2 family proteins.Methods:1:To observe the discrepancy of morphology and cell cycle of H69AR and H69 cell line by light microscope.2:Using CCK8 to detect the discrepancy of drug resistance coefficient of H69 and H69AR cell lines,to authenticate the drug resistance of H69AR to ADM and cross-resistance to other chemotherapy drugs.3:Real time quantity PCR,flow cytometry and Western blot techniques were used to analyse the discrepancy of Etk level between the two cell lines.4:After real time quantitity PCR,flow cytometry and Western blot techniques,it was found that Etk level was higher in H69AR than in H69 cells,later by Lipofectamine2000 EtksiRNA was transfected into H69AR cells,then Etk silencing was carried out,and detect the effect of gene silencing using flow cytometry and Western blot techniques.5:CCK8 detects the change of drug resistance with H69AR cells after Etk silencing.6:Flow cytometry and Western blot detect the expression changes of protein Bcl-2,Bcl-xL,Bak and Bax in H69 and H69AR cell lines.7:IP technique detects the relation between protein Bcl-xL and Etk of Bcl-2 family.8:Statistics analysis using SPSS13.0.cell cycle distribution,protein content assay,gray scale value redup each for three times(n=3),data is shown with(?)±s,the detailed statistic method adopt with One-Way ANOVA,P＜0.05 stands for significant difference.Protein concentration standard curve and cell survival rate curve are got by Excel mapping.Results1:Compared with parental H69 cells,the growth rate of H69AR cells slower, cell cycle distribution show that G0/G1 increases from 51.07±1.69 to 68.24±1.74(%), G2/M decreases from 17.4±1.08 to 2.98±1.1,and shows significant difference (p＜0.05).2:CCK8 drug resistance spectrum analysis show that,the drug resistance multiple of H69AR cell line to ADM is 63.88,in addition,the cell line show a cross-resistance with ither chemotherapy drugs such as TAX,resistance coefficient is 5.626.3:Compared with parent H69 cells,after real time quantitation PCR,Etk and P-Etk levels in H69AR cells at mRNA level increased notably,from8.426×10-6 to 5.514×10-5.Etk level in H69AR cells at protein level increases notably too.Flow cytometry shows Etk increases from 46±4.4 to 92.83±2.8.Western blot grey scale value analysis shows an increase of Etk from 0.56±0.014 to 0.99±0.038,show a significant difference(p＜0.05).4:Etk content of H69AR cell line is successfully decreased by gene silencing technique,flow cytometry and Western blot assay show an obvious decrease of Etk in H69AR cells after Etk silencing,the data of flow cytometry show a decrease of Etk level from 77.7±2.9 to 6.9±1.8,Western blot gray scale value show a decrease of Etk level from 0.4457±0.005 to 0.2925±0.0038,show a significant difference(p＜0.05).5:The CCK8 data shows an obvious decrease of the drug resistance of Etk silencing H69AR cell to ADM,drug resistance index decreases from 63.88 to 7.26.6:At protein level,compared with H69 cells,flow cytometry show an significant addition of Bcl-2(from 86.6±0.83 to 93.3±1.93) and Bcl-xL(from 28.1±1.32 to 45.7±1.4) of H69AR cells,and a notable degrade of Bak(from 28. 6±0.96 to 9.7±0.42) and Bax(rom 19.8±0.78 to 9.7±0.42) level,and shows significant difference(p＜0.05).The gray scale value of western blot show a notable increase of Bcl-2(from 0.736±0.02 to 0.907) and Bcl-xL(from 0.571±0.007 to 0.714±0.009) in H69AR cells.,while the expressing level of Bcl-2(from 0.427±0.005 to 0.264±0.009) and Bcl-xL(from 0.306±0.009 to 0.214±0.007) indicate an obvious degrade,show a significant difference(p＜0.05).7:According to the IP data,Etk and Bcl-xL of Bcl-2 family are interactive proteins,and further confirmed that H69AR cell an obvious addition of Bcl-xL and Etk in H69AR cells compared with H69 cells.Conclusions:1:Human SCLC drug resistant cell line H69AR has MDR.2:Compared with SCLC cell line H69,Etk level of SCLC ADM resistant cell line H69AR increases obviously,but after degrading the Etk level of H69AR by gene silencing techniques,the sensitivity to ADM of H69AR increases greatly,to further confirm that Etk may stand up to chemotherapeutics induced cell apoptosis,and may play an important role in SCLC drug resistance generating course.3:The drug resistance of H69AR generates from increasing of Etk level, increasing expression of protein Bcl-2 and Bcl-xL of Bcl-2 family and decreasing expression of protein Bak and Bax,the data from co-immunoprecipitation shows that, Etk and Bcl-xLof Bcl-2 family are interactive proteins,they may play an important role in the generating of SCLC drug resistance.
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