Cisplatin(CDDP) Induces Expression Of Lung Resistance Protein In Human Lung Cancer Cells By C-jun N-terminal Kinase Pathway

Background: Cisplatin(CDDP) is the first-line chemotherapy agent for lungcancer. Lots of patients are initially sensitive to it, but always relapse withmultidrug resistance. One of mechanism of cisplatin-resistance isintracellular drug accumulation reduction.Lung resistance protein(LRP) is akind of major vault protein, which was found in nuclear membranes andcytoplasmic vesicles. It is involved in drug transport in nuclear membranesand cytoplasmic vesicles. Preventing drug from entering nuclear andeliminating drug from cytoplasm by exocytosis by LRP could lead tointracellular drug accumulation reduction. Studies showed LRP was highlyexpressed in lung cancer tissue and was related to cell type、smoke andchemotherapeutics resistance. MAPK(Mitogen activated-protein kinase) isan important intracellular signal transduction, in charge of delivering signalfrom extracellular to intracellular, and regulating multiple physiologicalprocess,including metabolism、cell viability、cell division、apoptosis.JNK(c-jun N-terminal Kinase) is a main member of MAPK family. Studies showed JNK signal pathway involved in apoptosis, which played animportant role in apoptosis of chemotherapeutics. Based on above studies,we know inhibition of JNK activation could enhance apoptosis and LRP isclosely related to chemotherapeutics resistance, so we assume that theactivation of JNK signal pathway is relate to high expression of LRP. Andthere is no correlative news.Objective: To investigate the relationship between JNK pathway andLRP in mRNA and protein levels, and the effect of inactivation of JNK oncell inhibition and apoptosis, and to talk about the possible mechanism inA549and H446lung cancer cells.Methods: Firstly, subjects were lung adenocarcinoma cells A549andsmall cell lung cell H446.Secondly, cells were treated with CDDP in various concentrations for72h and expression of LRP mRNA was analyzed by Reverse transcriptasePCR; LRP、 JNK1/2、 P-JNK1/2was analyzed by Western Blot; cellinhibition was detected by CCK-8.Thirdly, cells were pretreated with SP600125(2、4μg/ml)for1h, thenCDDP(16μg/ml) was added into culture for72h.Cell Counting Kit-8wasused to investigate cell inhibition; Flow cytometry was used to detect theapoptosis rate; Expression of LRP mRNA was analyzed by Reversetranscriptase PCR; LRP、JNK1/2、P-JNK1/2was identified by Western Blot;Caspase-3Colorimetric Assay Kit was used to measure Caspase-3activity; Colony forming assay was used to detected to colony forming.Finally, statistical analysis.Results: Firstly, CDDP could upregulate the expression of LRP mRNAand LRP in dose-dependent fashion in A549and H446cells. The highestexpression was in8μg/ml and higher expression was in16μg/ml. Therewere significant difference between8、16μg/ml groups to controlgroup(p<0.05).Secondly, CDDP could phosphorylate JNK1/2inconcentration-dependent way. The highest expression was in16μg/ml inA549cells.Thirdly, JNK specific inhibitor SP600125could inhibit thephosphorylation of JNK1/2in2and4μg/ml for1h.Fourthly, pretreatment with SP600125could downregulate theexpression of LRP mRNA and LRP. There were significant differencebetween pretreatment with SP600125groups to CDDP group (p<0.05).Fifthly, pretreatment with SP600125in4μg/ml could enhance cellinhibition in A549cells, IC50was6.54μg/ml. IC50was9.41μg/ml in CDDPgroup.Sixthly, Cell apoptosis rate were8.37±2.92、13.10±0.86、15.18±0.90、30.01±6.36in control group、CDDP group、SP600125(2μg/ml)+CDDPgroup、SP600125(4μg/ml)+CDDP group. There were significant differencebetween SP600125+CDDP groups to CDDP group(p<0.05). Seventhly, pretreatment with SP600125in2and4μg/ml could rise theactivation of Caspase-3. There were significant difference betweenSP600125+CDDP groups to CDDP group.Eighthly, Colony forming rate were1、72%、51.2%、19.2%in controlgroup、 CDDP group、 SP600125(2μg/ml)+CDDP group、SP600125(4μg/ml)+CDDP group.Conclusion: Firstly, CDDP induced the expression of LRP and p-JNK1/2in dose-dependent manner in RNA and protein levels. Secondly, SP600125could inhibit the phosphorylation of JNK in dose-dependent fashion.Thirdly, Pretreatment with SP600125enhanced the sensitivity of CDDP onA549cells and promoted the apoptosis rate. At the same time, expression ofLRP mRNA and LRP were lower than CDDP alone. Fourthly, Inhibition ofJNK signal pathway promote apoptosis maybe by downregulating LRPexpression and enhancing the activity of Caspase-3.