Research Of Mechanisms That CA916798Gene Participates In Cisplatin Resistance Through PI3K/AKT/mTOR Pathway

BackgroundLung cancer is one of the most common malignancies，its morbidity and mortality incitizens in our country are higher than other malignancies,and its morbidity is increasingyear by year【1】. Chemotherapy is the main treatment method to lung cancer. Multidrugresistance(MDR) Can lead to low sensitivity of lung cancer cells to chemotherapy drugs,reduced effect of chemotherapy treatment,and tumor recurrence,and it`s one of the mainreasons that cause high mortality of lung cancer.So the MDR problem is Urgently to besolved in clinic therapy.In chemotherapy drugs of lung ancer,Cis-dichlorodiamine platinum(CDDP)is one ofthe most important drugs,is part of first-line drugs of lung cancer. At present period,Inhibiting DNA synthesis【2】and Induced apoptosis【3】are taken for its anti-tumormechanisms.Although it is widely used in clinic theatment,its Curative effect is barelysatisfactory due to the Cisplatin resistance of lug cancer,and it`s also the major reason thatcauses the failure of Chemotherapy to lung cancer【4】.It`s generally considered that MDR iscaused by transformations of multiple structures,gene express and Signal path of tumorcells after they contact with chemotherapy drugs,and these transformations lead to changesof proliferation, metabolism, apoptosis and invasiveness,result in good growth of tomorcells in durgs.The mechanisms involve many mechanisms,such as classic mechanisms ofdrug-resistance mediated by P-170glycoprotein (P-gp), Lung resistance related protein（LRP）, multidrug resistance-associated protein（MRP） and nonclassic ones mediated byapoptosis，enzyme activity and the variation of pH in cells【5-10】. Domestic and foreignresearch show that the happening of MDR is a complicated process involved manygenes,and the mechanisms of MDR including Cisplatin–resistance are not fully illustrated so far.So searching for new drug-resistance genes, analyzing MDR mechanisms,andreversal of MDR of lung cancer chemotherapy are primary problems in front of anti-tumormedical workers.CA916798gene was discovered,reported and confirmed by our research group withprophase project funding by compare lung adenocarcinoma cells (SPC-A-1)andcorresponding Cisplatin-resistance cells(SPC-A-1/CDDP) with suppression subtractivehybridization technology(SSH).We found that its expression in SPC-A-1/CDDP is higherthan expression in SPC-A-1,and the CA916798gene is registered in GenBank EST【11】.Further research shows that:CA916798gene expressions in MDR lung cancer cells arehigher than those in parental cells,the effect of CA916798gene is similar to the effect ofclassic anti-apoptosis pathway(PI3K/AKT/mTOR pathway): inhibition of CA916798geneexpression or inhibition of PI3K/AKT/mTOR pathway can both reverse the drug-resistanceof MDR lung cancer cell A549/CDDP; up-regulation of CA916798gene expression orinhibition of PI3K/AKT/mTOR pathway can both engender drug-resistance of the H446cells,a kind of lung small cell carcinoma.These evidence preliminary prove CA916798geneis Cisplatin-resistance-related gene【12-15】,and it is maybe the substrate or target of thePI3K/AKT pathway.Objective:To research the relationship between PI3K/AKT/mTOR pathway and the CA916798gene,discuss the mechanisms of Cisplatin-resistance of CA916798gene so as to providetheoretical basis for reversal of drug-resistance.Method:1. Construct A549and A549/CDDP cells that sustained actively express AKT1orsustained deactively express AKT1by gene transfection technique.2. Observe the difference between growth curve and drug susceptibility in these cellstreat under Cisplatin, detect the expression of CA916798gene.3. Treat A549,A549/CDDP cells with LY294002or rapamycin,detect the expression ofCA916798,AKT1,mTOR and the drug susceptibility.Result:1. A549/CDDP is proved to be a MDR cell compaired with A549cell by checking itsmulti-drug susceptibility. 2. Transfect and construct these cell models successfully:A549with empty vector,A549-Myr-AKT with sustained high expression of AKT1, A549-AKT-K179M withsustained low expression of AKT1, A549/CDDP with empty vector, A549/CDDP-Myr-AKTwith sustained high expression of AKT1, A549/CDDP-AKT-K179M with sustained lowexpression of AKT1.3. Confirmed by Western Blot①Expression of total AKT1increase inA549-Myr-AKT cell、 A549/CDDP-Myr-AKT cell and A549-AKT-K179M cell、A549/CDDP-AKT-K179M cell；②Expression of pAKT1in A549-Myr-AKT is higher thanA549,the A549cell with empty vector is same，pAKT1expression in A549-AKT-K179MpAKT1is lower than A549③A549/CDDP-Myr-AKT cell、A549/CDDP with empty vector、A549/CDDP-AKT-K179M cell pAKT1expression are respectively higher, approximate andlower than A549/CDDP cell。4. Growth curve of these cells are close without Cisplatin.under treatment of Cisplatin,①Multiplication rate of A549-AKT-K179Mis lower than A549with empty vector，A549with empty vector is lower than A549-Myr-AKT；②Multiplication rate ofA549/CDDP-AKT-K179M is lower than A549/CDDP with empty vector， A549/CDDPwith empty vector is lower than A549/CDDP-Myr-AKT.5. It`s discovered that the mRNA and protein expression of CA916798remain highestlevel in A549-Myr-AKT cell,middle level in A549-empty vector cell,lowest level inA549-AKT-K179M cell by means of RT-PCT and WesternBlot.The same situation happensin each group of A549/CDDP cells.6. Examination about drug susceptibility of each group cells shows that the order ofthe IC50from high to low is: A549/CDDP-Myr-AKT cell、A549/CDDP empty vrctor cell、A549/CDDP-Myr-AKT cell、 A549-Myr-AKT cell、 A549empty vector cell，A549-AKT-K179M cell。7. The expression of AKT,pAKT,mTOR and CA916798in all cells and IC50aredecreased under the circumstance of LY294002;the expression of CA916798and IC50aredecreased as well in all cells when they treated by Rapamycin,but the expression ofAKT,pAKT,mTOR are unchanged. Conclusion:1.A549and A549/CDDP cells that sustained actively express AKT1or sustaineddeactively express AKT1were constructed successfully.2.Up-regulation or down-regulation of the expression of AKT1can cause thecorresponding change of the expression of CA916798gene.3.The mRNA and protein expression of CA916798gene are decreased under thecircumstance of LY294002or Rapamycin.4.Preliminary result shows CA916798gene lies downsteam of PI3K/AKT1/mTORpathway,and maybe is the substrate or target of PI3K/AKT/mTOR pathway.