The Study On The Relationship Between The Expression Of TC-1Gene And The Anti-anoikis Ability Of The Tumor Cells From The Malignant Pleural Effusion Of Human Lung Adenocarcinoma

Anoikis，named by Frisch SM in1994, was a special apoptosis of theepithelial cells when they lost the contract with the extracellular matrix （ECM）[1]. As a self-protection mechanism of human body, anoikis could prevent theepithelial cells from ectopic growing in other tissue or distant organ aftertransporting through the piping system or cavities of human body whendetched from their ECM, which keeps the body in a steady state. However, apart of the carcinoma cells, originated from epithelial cell, could grow in theECM of other tissue or distant organ into a metastasis[2], even when the newECM was absolutely not suitable for the survival of the normal epithelial cellsfrom their primary place. This phenomenon indicated that the carcinoma cellsmight possess the ability of anoikis resistant. This “anti-apoptosis” is widelyconsidered to be one of the important mechanisms of the metastasis.Pleural metastasis of lung cancer would cause the accumulation ofpleural effusion in the pleural cavity, which was named malignant pleuraleffusion （MPE）. A large number of tumor cells could survive in the MPE underthe circumstance of suspension-style, and then implant and grow in the pleural.This phenomenon indicated that the tumor cells in MPE possessed the ability of anti-anoikis and was one of typical anti-anoikis cells in human body.The mechanism of “anoikis/anti-anoikis” is so complex to be elucidatedclearly till now. Lots of studies indicated that there were many factors respondedfor the mechanism of “anoikis/anti-anoikis” and involved many signaltransduction pathways. TC-1gene （C8orf4）, a newly found gene, is relevant toinflammation and tumor. It was first found for its abnormal high expressionlevel[3]in the carcinoma of thyroid and also in the suspension cultured humanlung adenocarcinoma cell line A549. However, the correlation between theexpression of TC-1gene and the development as well as the metastasis ofhuman lung cancer is still far from discovered at present, and it still need someway to explore the relationship between the expression level of TC-1gene andthe anti-anoikis ability of the human lung cancer cells.Objective: To set up an appropriate method for the isolation and purification ofthe tumor cell from MPE and establish the model of human anti-anoikis tumorcells from the MPE of human lung adenocarcinoma in vitro. In this modelmorphology and biological characteristics study would be performed in thetumor cells from the MPE of human lung adenocarcinoma and compared withthe tumor cells from the primary lung cancer site. The relationship between theexpression of TC-1gene and the anti-anoikis ability of the tumor cells from theMPE of human lung adenocarcinoma would be discussedMethods:1: MPE samples were obtained from the MPE in patients whosufferered the lung adenocarcinoma. Model of human anti-anoikis tumor cellsfrom the MPE of lung adenocarcinoma cell was established by usingpoly-hydroxyethyl methacrylate （Poly-HEMA）resin processed petri dishes,which prevented tumor cells from adherent, and then isolated and purified bydensity gradient centrifugation. 2: Compared with the cells from primary site, morphology and biologicalcharacteristics study were performed in the tumor cells from the MPE of humanlung adenocarcinoma. The morphology of the tumor cells of the two groups （theprimary site tumor cells; the MPE tumor cells） were observed with scan electronmicroscopy and optical microscope, the cell viability was evaluated with Methylthiazolyl tetrazolium （MTT） chromatometry assay and cell growth curve wasgenerated. The tumor cells doubling time were calculated according to the cellgrowth curve; and the flow cytometry （FCM） was used to detect apoptosispercentage and phase distribution of cell cycles.3: The expression of the TC-1in the tumor cells of the two groups wasdetected by immunohistochemistry staining and western blot analysis, and theinhibit rate （IR%） of PD176974, an inhibitor of the TC-1gene associatedpathway, was evaluated with MTT chromatometry assay and the cell growthcurve were generated to measure the effectiveness of PD176974on the twogroups cells.Results:1: Tumor cells could be effectively isolated and purified from the MPEby using of twice density gradient centrifugation. The model of humananti-anoikis tumor cells from the MPE of human lung adenocarcinoma in vitrowas established successfully, and the suspension culture time of the anoikisresistance cells was about4weeks.2: Biology and morphology studies of the resistance tumor cells showedthat：（1）. The primary site tumor cells growth curve falled into three phases,including latency phase, logarithmic phase and the platform phase, however, thegrowth curve of the tumor cells isolated from the MPE was a low andapproximate straight line. Cell doubling time was（115.74±15.38）h, indicatingthat the cells cell proliferation was low.（2）. Cell cycle distribution detection showed that cell cycle of the tumor cells from the MPE including:（58.95±4.13）%cells in G₀/G₁phase,（26.2±3.7）%in S phase and （14.85±1.3）%in G₂/M phase, which meaned that nearly60%of cells was in the quiescentstage.（3）. The number of viable cells in the primary site tumor group and MPEgroup were more than90%of the total cells and the apoptosis and necrosis ratewas less than10%. Suspended for48hours, the live rate of primary site tumorcell was only （38.9±6.9）%, and the apoptosis and necrosis cell was greaterthan60%.（4）. Compared with primary site tumor cells, cell membrane of tumorcells from the MPE increased under SEM; cells cilia reduced, and cells were in aspherical sample and plate shape; A small amount of tumor cells from the MPEshowed pre-apoptosis changes in apoptotic vesicle.3:（1）. The immunohistochemistry and western blot showed thatsignificantly higher TC-1gene expressed occurred in the tumor cells from theMPE than in the primary site.（2）. PD176074, a TC-1pathway inhibitor, couldpromote the apoptosis or necrosis in the tumor cells from the MPE,and theinhibition rate was54.43%. However, PD176074had no effect on adherentcultured primary site tumor cells.Conclusion: The model of human anti-anoikis tumor cells from the MPE ofhuman lung adenocarcinoma in vitro was established successfully. Primary sitetumor cells of lung adenocarcinoma and MPE metastatic tumor cells showeddifferent biological and morphological characteristics. The expression of TC-1gene correlatd with anti-anoikis ability of the tumor cells from the MPE.