The Mechanism Of Apoptosis Regulation Of Protein-tyrosine Kinase Etk In Small Cell Lung Cancer

BackgroundAt present,lung cancer is one of the most harmful malignant tumors to human health.The latest survey data of National Association on lung cancer showes that the incidence of lung cancer in China had been ranked first in all types of cancer.A quarter of all lung cancer patients are suffering from small cell lung cancer.Although small cell lung cancer patients are sensitive to the current clinical chemotherapy drugs （such as etoposide,cyclophosphamide,placlitaxel）,the effect of chemotherapy often is not satisfactory because of a vulnerable resistance.The recurrence rate of small cell lung cancer is high and clinical prognosis is poor.For the general 2-year survival rate is less than 5%and 90%of the patients in the more than five years after diagnosis in death,that the resistance to chemotherapy has become one of the issues in Small cell lung cancer treatment urgently is needed to be resolved in recent years.Study found that cancer cells escaping chemotherapy-induced apoptosis resistance to chemotherapy is a decisive factor.Therefore,the mechanism research of apoptosis in cancer cells escaping set out to become a key chemotherapy resistance.Whether the cells are apoptosis depends on the balance of Promoting cell apoptosis and inhibiting of apoptosis gene.When one or a variety of down-suppressor gene express over or promoting genes are inhibited,the cells would not be apoptosis.In many of apoptosis-related genes we have found some relate with resistance to cancer chemotherapy,such as the gene Bcl-2,P53.Bcl-2 gene is a kind ofproto-oncogenes,which can inhibt cell apoptosis.Gene transfection experiments have confirmed that bcl-2 protein can prevent a variety of factors-mediated apoptosis.The main mechanism of bcl-2 inhibition apoptosis may be as follows:â‘ as antioxidants,Bcl-2 regulates cell redox state and blocks effect on cell components of the damageâ‘¡Bcl-2 affects cell transmembrane and changes calcium distribution,which can act endogenously calcium of the endonuclease and glutamyltransferaseâ‘¢Bcl-2 suppress mitochondrial cytochrome C from therelease into the cytoplasm,mitochondrial pathway which cause DNA fragmentation in apoptosisâ‘£Bcl-2 protects RNase from damage and inhibit NDA fracture. However,Bcl-2 is not working alone,which could heterologous dimer formation with bax,the ratio of the two decisions is the key to apoptosis.Research also found that mutant p53 can inhibit Bcl-2 transcription initiation and canreduce the post-transcriptional mRNA stability,thereby Bcl-2 expression negative regulator.Bcl and Survivin structural are similarity with the promoter,there may be a common mechanism of transcriptional activation,which play synergistic anti-apoptotic effect. Another study showed that Bcl-2 could promote angiogenesis factor VEGF,bFGF, thus increase the viability of tumor cells.P53 gene coding P53 protein is well-known tumor suppressor gene.P53 protein consises of 393 amino acids,with specific activation of the transcription.When the DNA damage,the P53 protein in cells increases.It induced CKI-P21WAF1/CIP1 transcription,resulting in cells in the G1 phase to prevent DNA synthesis,meanwhile GADD45 gene actived by the CKI-P21WAF1/CIP1 transcription,repaire the damage of DNA.If G1 stagnation can not be achieved,the P53 gene induces cells apoptosis to prevent damage DNA passing to the offspring of the cell.The P53 gene mutation in 50 percent of the tumors.In the process of tumor,P53 is usually inactivated that the reason may be P53 gene deletion,mutation,MDM-2 High expression of inhibiting P53,certain tumor virus-encoded protein integration of P53 or P53 was prevented from entering the role of the nucleus.Btk kinase family is a large class of non-receptor tyrosine kinase which are close to T and B lymphocyte proliferation and differentiation.The epithelial and endothelial tyrosine kinase（Etk）,also known as Bmx（bone marrow X kinase）,is a cytoplasmic tyrosine kinase,which was first identified in human bone marrow cells.It is located in chromosomal band Xp22.2 between the DXS197 and DXS207 loci.Bmx cDNA consists of a long open reading frame of 675 amino acids containing a structure resembling the Tec family kinase pleckstrin homology domain（PH）,an SH3（Src homology 3） domain,an SH2（Src homology 2） domain,and a catalytic kinase domain.Etk/Bmxis the only Tec family kinasemember expressed in epithelial cells.It is commonly expressed in a variety of tissues and cell types,including salivary,lung, prostate,pancreas,intestine,colon and carcinoma,cells.Although Etk/Bmx is expressed at a low level in epithelial cells,many studies have demonstrated that it plays an important role in their proliferation,differentiation,apoptosis and tumorigenesis.Lee et al.showed that Etk/Bmx was required for neurotrophic growth factor-induced androgen-independent growth of prostate cancer cells.Qiu et al.found that Etk/Bmx was involved in IL-6-inducd neuroendocrine differentiation of prostate cancer cells.Etk/Bmx also protect prostate cancer cells from radiation- and EGF-induced apoptosis and controls the proliferation and anchorageindependent growth of mammary epithelial cancer cells.Guo et al proved that Etk may participated in the nasopharynx epithelial differentiation in some cases.Through a series of the pathology and molecular biology experiments,we study tyrosine kinase Etk expression in small cell lung cancer tissue and the relationship between Etk and Bcl-2,P53.We further explore the role of Etk in the process of apoptosis.Objective:The research was to find if Etk is involved in apoptosis induced by Doxorubicin in small lung cancer cells.We also tried to detecte whether Etk is relevant with the expression levels of the apoptosis-related proteins,for example Bcl-2,P53.Material and Method1.Immunohistochemistry was performed to detect the Etk in lung tissures and Bcl-2,P53 in 71 cases of small cell lung cancer tissures.2.The Etk protein positive rate in NCI-H446 cells was detected by flow cytometry.3.Plamid Etk-WT and plamid Etk were transfencted into NCI-H446 cells through Lipofectamine^TM2000 and then transfectd cells were selected in G418 selective medium and those NCI-H446 cells stably expressing Etk were obtained.Then,the efficiency of protein Etk expressing was detected by cytometry.4.By MTT-assay we detected the sensitivity of NCI-H446 cells to doxorubicin and found the IC50.5.We compaire the apoptosis rate of NCI-H446 and NCI-H446 WT two kinds of cells by MTT-assay when cells are cultured by medium including doxorubicin.6.The changes of Bcl-2 and P53 expression after culturing Cells In Including semi-lethally doxorubicin medium for 24 hours were detected in NCI-H446 cells and NCI-H446-WT cells by Western Blot.7.Adopting SPSS11.5 count software package to deal with the date:The relationship analsis of the postive rate of Etk and P53 protein,Etk and Bcl-2 protein in small cell lung cancer was executed inχ²-test,The relationship analsis of the postive rate of Etk different in kinds of lung tissures was executed too,and the level of significance was P≤0.05.Results1.Etk expression was present in 54.71%cases of cancer.The positively-stained cancers include 34.09%in squamous carcinoma（SCC）,44.64%in adenocarcinoma（ADC） and 74.64%in small cell lung cancer（SCLC）.While negative staining was showed in either bronchial epithelium or alveolar cells.Etk was more frequently in SCLC than in either SCC or ADC（P<0.05）,but there is no statistical difference of Etk expression in SCC compared to that in ADC.2.Of the whole 71 cases of small cell lung cancer tissue,74.64%（53/71） were Etk positive,while the positive rate of Bcl-2 was 54.93%（39/71）,and the positive rate of P53 was 60.56%（43/71）,The statistical analysis showed that the positive rate difference between Etk and Bcl-2,Etk and p53 both were significant.3.The Etk positive rate of NCI-H446 was 26.31%,The positive rate of NCI-H446（plamid were transfencted ） was 28.56%,The positive rate of NCI-H446 WT（Etk-WT plamid were transfencted ） was 76.87%.We confirmed the expression of Etk in NCI-H446 WT is stronger than that in NCI-H446 cells by Western Blot.4.By MTT-assay we detected the sensitivity of NCI-H446 cells to doxorubicin and found the IC50,IC50=5.613μg/ml.5.The apoptosis rate of"NCI-H446 WT" was lower than that of"NCI-H446".6.By Western Bloting we found when doxorubicin effected upon cellls,the Bcl-2 increased stronger in "NCI-H446 WT" than that that in "NCI-H446".while the P53 increased slightlier in "NCI-H446 WT" than that that in "NCI-H446". Conclusion1.The positive rate of Etk express in small cell lung cancer tissues is higher than that in ADC,SCC,and nomal lung tissures.Etk may be regarded as a novel molecular marker for SCLC and the target for gene therapy in the future.2.Etk,Bcl-2 and P53 express highly in small cell lung cancer tissues.There are correlation between Etk and Bcl-2,there are also correlation between Etk and P53.3.The Etk can make a resistance to apoptosis induced by doxorubicin insma in small cell lung cancer cells.4.The Etk may promote Bcl-2 expression or inhibite p53 activity to suppress apoptosis in small cell lung cancer cells.