| Background and ObjectiveLung cancer is is one of the highest incidence of malignancy in the world.Chemotherapy is one of the primary means for the treatment of lung cancer, and made someprogress. But in the past25years the5-year survival rate of lung cancer remains in the15%.The multi-drug resistance (MDR) is the main reasons for the failure of chemotherapy.Cisplatin (CDDP) is effective and widely used for lung cancer treatment, but its efficacy isunsatisfactory due to resistance.Cisplatin is a cycle-non-specific cytotoxic drugs. Various factors may contribute tocisplatin resistance, such as apoptosis of anti-cancer cells, increase in fingernail clippings inDNA damage repair process, ecrease in cellular uptake of drugs. However, the resistancemechanisms of CDDP to lung cancer have not yet been fully elucidated. Therefore, it isimportant to further study cisplatin resistance mechanisms and find new targets drug forimproving lung cancer chemotherapy efficacy and increasing5-year survival.Anti-apoptosis is one of the main mechanisms of cisplatin resistance. PI3K/AKTpathway is the most important anti-apoptotic pathways. We have found that theover-expression of AKT1protein and gene were closely related to cisplatin resistance inlung cancer cells. Cisplatin resistance can be reversed in A549/CDDP cell lines byinhibition of AKT1, and increase by activation of AKT1. LY294002, a PI3K inhibitor,significantly inhibited the expression of AKT1and enhanced the sensitivity to cisplatin inA549/CDDP cell lines.Ras gene is located upstream of the PI3K/AKT pathway. Previous studies have shownthat Ras-related upstream regulatory, such as EGFR, HER2, Grb2and guanylate exchangefactor (GEF, such as SOS1), are a substrate for SHP2. SHP2combined with these substrateto activate Ras,and combined with RasGAP to prolong the half-life of the RAS-GTP, so that the downstream signaling pathways, including PI3K/AKT pathway, keeped in sustainedactivation.Besides,we also found that inhibiting SHP2can also inhibit the expression ofAKT in breast cancer.It has been shown that cisplatin can be selectively activated bySHP2.Combined these, we speculate that SHP2may be a new cisplatin resistance-associated protein in lung cancer, and the mechanism may be that Ras regulate PI3K/AKTsignaling pathway.SHP2is a protein tyrosine phosphatase (PTP) containing two Src homology domain(Src homology-2, SH2). In the PTP superfamily, SHP2is the protein product of thePTPN11which is the first proto-oncogene. Tyrosine phosphorylation plays an importantrole in cellular signal transduction process, and controlled by the protein tyrosine kinase(PTK) and PTP.The imbalance between PTK and PTP will result in abnormal tyrosinephosphorylation which is closely related to many human diseases.It has been reported thatjuvenile myelomonocytic leukemia, myeloid leukemia and chronic myeloid-monocyticleukemia were resulted from SHP2activating mutations or sustained activation. It is alsoinvolved in H. pylori associated gastric cancer.There is high expression of SHP2in gastriccancer and breast cancer. Currently, the study of SHP2in cancer research mainly focusedon cancer occurs, cancer cell growth, invasion, migration and transformation, but that inlung cancer cisplatin resistance has not been reported.In this study, SHP2was detected in human lung cancer by tissue microarray andimmunohistochemical or immunocytochemical techniques. SHP2expression was increasedin H446cell line and decreased in H446/CDDP cell line by gene cloning and RNAinterference technology, and then cells were compared in cell biology and resistance tocisplatin before or after cisplatin, in order to explore the relationship between SHP2andresistance to cisplatin. We further analyzed the changes in the expression of SHP2、Ras、AKT1、pAKT1and survivin by Ras interference to elucidate the molecular mechanism.Methods1. SHP2expression was analyzed in lung cancer tissue， SCLC cell H446,adenocarcinoma cell SPC-A-1and cisplatin-induced MDR cells H446/CDDP, SPC-A-1/CDDP by tissue microarray and immunohistochemical or immunocytochemicaltechniques.2. The lentiviral vector containing SHP2or SHP2shRNA was constructed by genecloning and RNA interference technology.293FT cells used as the producer cell line weretransfected with vector which was transferred into H446or H446/CDDP cells.H446-SHP2-WT and H446/CDDP-SHP2-shRNA were selected with puromycin.SHP2mRNA and protein were determined by RT-PCR and Western blotting.3. The sensitivity of the cells to cisplatin was determined by CCK-8assay and shownin cell growth curve.4. Cell cycle and apoptosis influenced by cisplatin were analyzed by flow cytometry.5. The changes in the expression of SHP2、Ras、AKT1、pAKT1and survivin by RasRNA interference were analyzed by Western blotting.Results1. The total positive rates of SHP2was70.00%(56/80)in NSCLC. SHP2expressionwas positive in H446, SPC-A-1and cisplatin-induced MDR cells H446/CDDP,SPC-A-1/CDDP.2. Lentiviral expression vector and interference vector for SHP2were constructedsuccessfully. H446-SHP2-WT stable expression of SHP2and H446/CDDP-SHP2-shRNAsustained inhibition of SHP2were screened.3. IC50values of cisplatin were1.01μg/ml in no-load group and1.218μg/ml inH446-SHP2-WT. Resistance index was1.206. Resistance to cisplatin in H446-SHP2-WTwas increased by20.6%. IC50values of cisplatin were4.382μg/ml inH446/CDDP-SHP2-shRNA and11.92μg/ml in no-load group. Resistance index was2.72.Resistance to cisplatin in H446/CDDP-SHP2-shRNA was reversed by63.2%.The resultsindicated that the increased expression of SHP2reduce the sensitivity of H446cells tocisplatin, and the decreased expression of SHP2reversed resistance to cisplatin inH446/CDDP, which preliminarily confirmed that SHP2is a new cisplatinresistance-associated protein in lung cancer. 4. The degree of inhibition by cisplatin was lower in H446-SHP2-WT than in the H446and H446-SHP2-WT-no-load group, which was significantly (p <0.05) in the second dayand the third day. The degree of inhibition by cisplatin was higher inH446/CDDP-SHP2-shRNA than in the H446/CDDP and H446/CDDP-SHP2-shRNA-no-load group, which was significantly (p <0.05) after three days.We found that more cellsremained in the S phase under cisplatin. Apoptosis rate in H446-SHP2-WT was decreasedfrom49.43%to34.56%, while apoptosis rate in H446/CDDP-SHP2-shRNA was increasedfrom6.60%to16.75%by cisplatin.The results further confirmed that SHP2is a newcisplatin resistance-associated protein in lung cancer and might exert its influence throughregulation of apoptosis.5. The expression of SHP2was found no significant difference between H446andH446/CDDP. The activity of SHP2was significantly higher in H446/CDDP than H446(ODvalues were0.488±0.015and0.249±0.011, p <0.05).The results showed that theresistance of H446/CDDP to cisplatin was related to the activation of the SHP2.6.All the expression of Ras,AKT1,pAKT1and survivin was higher in H446/CDDP andH446/CDDP-SHP2-shRNA-no-load group than in H446, which was the same as theexpression of SHP2. All the expression could be decreased by inhibiting SHP2.The resultsindicated that SHP2might locate upstream of Ras、AKT1、survivin and had a positiveregulatory role in the PI3K/AKT1pathway. The expression of pAKT1and survivin wasincreased by cisplatin in H446、 H446/CDDP and H446/CDDP-SHP2-shRNA-no-loadgroup,while the expression of pAKT1and survivin was not influenced by cisplatin inH446/CDDP-SHP2-shRNA. The results indicated that cisplatin might activate SHP2andinhibit apoptosis through SHP2/Ras/PI3K/AKT1/survivin in drug resistance.7. The expression of Ras was decreased by cisplatin in Ras siRNA group, while nosignificant changes in expression of SHP2in H446、H446-SHP2-WT and H446/CDDP. Theresults indicated that SHP2might locate upstream of Ras. The expression of AKT1andpAKT1was higher in H446-SHP2-WT and H446/CDDP interferenced with Ras RNA,which indicated that there might be other pathway increasing the expression of AKT1andpAKT1. Conculsion:1. There is high expression rate of SHP2in human lung cancer.2. SHP2is a new cisplatin resistance-associated protein in lung cancer.Its influence isrelated to the expression levels and activity.3. SHP2inhibits apoptosis through SHP2/Ras/PI3K/AKT1/survivin pathway. SHP2has a positive regulatory role in the pathway. |
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