Detection Of S100B In The Serum Of Patients With Brain Metastasis Of Lung Cancer And Involvement Of Its Specific Mechanism In Brain Metastasis

Background:Lung cancer is a malignant tumor of global morbidity and mortality in thehighest， the incidence rate showed an increasing trend， has become one of themajor diseases of the threat to human life and health. Approximately20%-50%of lung cancer can develop brain metastasis；Prolong survival in patients withimproved testing method to the diagnosis of brain metastasis increased graduallyas the comprehensive treatment of lung cancer progress. Adenocarcinoma of theincidence of brain metastasis highest，followed by small cell lung cancer. Brainmetastasis is an important cause of lung cancer deaths related to natural survivaltime of only1to2months. In the clinical treatment of brain metastasis fromlung cancer treatments such as surgery，radiotherapy，chemotherapy，etc. are incontinuous improvement， but the treatment effect is still unsatisfactory， themain reason is the mechanism of brain metastasis of lung cancer is still unclear.Therefore， further study and perfect the mechanism of lung cancer brainmetastasis development to find new therapeutic targets，it is expected to improvethe efficacy of treatment，to extend the survival of patients.Brain is a highly specialized environment with a unique cellularcomposition， surrounded by the blood-brain barrier and lacks lymphaticdrainage. According to the classical hypothesis of Paget’s，tumor cells like seedsand the metastasis target organ like soil，the smooth growth of the tumor cellsmetastasis depends on the seed and soil suitable for the conditions of seed development and growth. In other words，environmental factors in terms of thetumor cells is very important，so successful brain metastasis cannot be separatedfrom the brain microenvironment，involving a series of molecular biologymolecular interaction. However，the development of mechanisms of brainmetastasis and related molecules is still poorly understood. Our previous studyfound high S100B expression in the brain-specific metastatic NSCLC linePC14/B，suggested S100B is closely correlated with brain metastasis in NSCLC.The subject by the2009National Natural Science Foundation (project name:the role and mechanism of S100B in brain metastasis of lung cancer itemnumber:30973488).Prevoiusly，we have understands of S100B in the brain oflung cancer specific role in the transfer process occurred by transfering S100BRNAi to lung adenocarcinoma cell line PC14/B，and observing its biologicalfunction in brain metastasis of lung cancer.Objective:The objective of this study is to in-depth understands of S100B in the brainof lung cancer specific role in the transfer process occurred by transferingS100B to lung adenocarcinoma cell line PC14of S100B，and observing PC14growth and proliferation，and migration of the invasive ability of reveal S100Bbiological function，and provide new ideas for in-depth study of lung cancerwith brain metastasis.Methods:（1） Collection of clinical diagnosis of brain metastasis with and without brainmetastasis from lung adenocarcinoma patients with serum，to examinethe content of S100B in serum S100B’s role in the early diagnosis ofbrain metastasis of lung cancer；（2） transfection of S100B to PC14cells，construct stable cell lines G418selection and identify the transfection effect by RT-PCR and Western blot；（3） Determine the OD value of PC14/S100B and control group PC14cells bycell growth curve of MTT assay，to draw cell growth curves，in order todetect cell proliferation capacity by MTT assay；（4） To detect changes in the cell cycle and apoptosis by flow cytometry；（5） To detect cells transfected with changes in migration and invasion byTranswell invasion chamber；（6） By laser scanning confocal and Western blot method to investigate themechanism in PC14cell migration and invasion.Results:（1） Lung cancer patients with brain metastasis compared to the serum of lungcancer patients with brain metastasis，of S100B content was significantlyincreased（0.048μg/l vs0.015μg/l）（2） Successfully construct stable transfected cell lines PC14/S100B，and byRT-PCR and Western blot identity；（3）By MTT assay，PC14/S100B cells has a higher ability of cell proliferationthan the control group of PC14；（4） Migration and invasion were enhanced in Transwell results were83.4±5.6vs.53.2±3.9and53.4±4.7vs.31.2±3.0(P <0.01)（5） Flow cytometry was used to find the result that cell apoptosis ofPC14/S100B cells was inhibited much more than the control groupPC14cell，the results were3.40±0.44%and10.90±0.67%(P<0.05)，and the cell PC14/S100B cell cycle G1�'S phase transitionwas observed by PC14/S100B cells；（6） PC14/S100B cell apparent invasive pseudopodia，the cells change shapefrom round to spindle; Western blot results showed that the expression ofIQGAP1than the control group，PC14cells was significantly increased (IOD value117.64vs.20.569).Conclusion:In the present study，the clinical serum samples collected after the ELISAdetection of S100B content，the results show that newly diagnosed lung cancerpatients with brain metastasis than those without brain metastasis weresignificantly increased， the results suggest that S100B in the early diagnosis ofbrain metastasis of lung cancer patients with significance. Second，transfectionof S100B PC14cell proliferation capacity to improve，apoptosis reduced，indicating that the proliferation of S100B anti-apoptotic role， while theproportion of cells in G1phase decreased， increase in the proportion of Sphase， these results suggest， S100B protein can promote cellproliferation accelerated from G1to S phase transition； transwell experimentsconfirmed that S100B after transfection of PC14cell migration and invasionwere significantly enhanced， cell shape from round to spindle change，andinvasive pseudopodia，and further Western blot analysis showed an increase inthe expression level of the scaffold protein IQGAP1. The IQGAP1preciselyinduced the generation of invasive pseudopodia， and sports an important factorin the tumor cells， thereby indirectly mediated tumor cell motility，migrationand invasion. The enhancement of these capabilities，it is lung cancer with brainmetastasis occurred in the“necessary conditions”. Therefore，we speculate thatthe S100B protein to improve the capacity of the lung cancer cell proliferation，inhibit apoptosis， thereby promoting the rapid tumor cells to proliferateindefinitely S100B protein can indirectly activate the transcription andexpression of IQGAP1.As a cytoskeletal protein，IQGAP1not only to combinethe MAPK pathway of signaling molecules，directly or indirectly affect themitogen-activated protein kinase signal response of the waterfall cascading，and can affect cell adhesion，of IQGAP1also induce tumor cell invasiveness pseudo-enough generation and movement， thereby promoting tumor cellmotility，migration and invasion. Therefore，we believe that S100B promote themalignant behavior of lung cancer cells，and regulate the cytoskeleton throughIQGAP1and lung cancer cells enhanced migration， invasion，and thus play animportant role in the development of brain metastasis. S100B is expected tobecome the new marker of brain metastasis of lung cancer screening，a newtarget for treatment and the new prognosis factor.