| BackgroundAccording to the latest global cancer statistics, the incidence of lung cancer ranks first in all malignant. So does the situation in China. More than one million people die from lung cancer. It is a serous threat to human health. More worrisome is clear upward trend in the incidence of lung cancer. This is related to the aging of population, the city’s industrialization, urbanization in rural areas, environmental pollution and unhealthy habits such as smoking. According to the morphology under microscope, lung cancer can be divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The latter include adenocarcinoma, squamous cell carcinoma and large cell lung cancer. SCLC accouts for about15%of primary lung cancers. It is poorly differentiated and easy to hematogenous metastasis at early stage, which leads to poor prognosis. Although sensitive to chemotherapy initially, SCLC develop multi-drug resistance (MDR) soon, which resulted in failure of chemotherapy and rapid progression. Recurrence and metastasis will be found in patients with early stage. Eventually died of multiple organ failure.5-year survival rate of SCLC is less than5%. Therefore, MDR is one of the problems to be conquered in the basic research and clinical treatment of SCLC.Multi-drug resistance is the main reason for the failure of chemotherapy treatment of malignant tumors. The tumors have acquired resistance to contact anticancer drugs regardless of function or structure not related to a variety of drugs. Multi-drug resistance mechanisms include the following factors. The first factor is pharmacological resistance. The resistance caused by the body itself and the influence of drugs. Such as enhanced metabolic activity of the drug into the body or drug activation barrier, insufficient blood supply of the tumor tissue, poor affinity and penetration between the drug and the organization. The second factor is microenvironment resistance. The survival and growth of tumor cells depends on the organ microenvironment, Organ microenvironment can influence gene expression by different drug sensitivity of tumor cells to chemotherapeutic agents resulting in. The third is apoptosis resistance which involved in the control of apoptosis genes was inhibited, such as P53, BCL-2, C-myc. In addition, biochemical resistance factor is also important.Changes in the genetic and biochemical characteristics of the tumor cells allows the cells to the emergence of drug resistance through different channels. ABCC membrane transporter protein super-family members, that is drug export pump, get the most extensively studied. The family include P-glycoprotein (P-gp), Multidrug resistance-associated protein (MRP), Lung resistance-related protein (LRP), and Glutathione S-transferase. These proteins are involved in multidrug resistance of tumor cells, but how to find and identify these proteins is important content. Subsequent study of its mechanism of action, changing its function by molecular biology techniques proteomics research is also important.Abnormal apoptosis:Apoptosis escape is the common characteristics of the tumor cells, that also play important role in SCLC chemotherapy resistant. In cell adhesion mediated drug, extracellular matrix proteins against cytotoxic drug-induced apoptosis signal. The extracellular matrix (ECM) mediated by β1integrin and activate the PI3K signaling pathway, upregulateing the target PKB of GSK3β expression and blocking apoptosis. CD9is the protein of the four transmembrane family members. CD9expressed higher in metastatic SCLC tissues, and the SCLC cell lines with cisplatin-resistant or etoposide-resistant was higher expression. The SCLC cell with CD9-positive resistant mediated β1integrin (β1integrin), and fibronectin protein (fibronectin) connection closer, by activating PI3K/AKT/mTOR signaling pathway, caused to chemotherapy-induced withered death not sensitive. The specificity using targeting CD9the monoclonal antibody ALB6small interference RNA (siRNA) can successfully trigger apoptosis in the resistant cells. The tumor suppressor gene p53protein response to DNA damage signal activation of growth arrest pathway and apoptosis pathway. Deletion or mutation of the p53gene in lung cancer patients with poor prognosis, often accompanied by radiotherapy and chemotherapy resistance. More than80%of the SCLC patients absented of wild-type p53protein, activity through the inhibition of cyclin-dependent kinase inhibitor p21wafl expression caused abnormal cell cycle checkpoint, eventually leading to uncontrolled cell proliferation, apoptosis blocked and caused disease progression. poor prognosis and resistance to chemotherapy related. Bcl-2gene is located on chromosome18q21, encoding the relative molecular mass of26,000protein, Bcl-2gene family members is a regulation of apoptosis. In SCLC often Bcl-2expression, which can also be found in the increased expression of Bcl-2in vitro induced resistant SCLC cell lines.Guo used microRNA microarray to analysis the differences microRNA in SCLC multidrug resistant cell line H69AR and sensitive cell lines H69. The results showed that61miRNAs are presented significanty including up-regulation of24miRNAs and down-regulation of37miRNA. Among these miRNAs,48of61differentially expressed miRNAs were firstly reported to be closely associated with drug resistance, including miR-134, miR-379and miR-495. Following transfection of H69AR cells with the mimics of miR-134, miR-379and miR-495, respectively, the sensitivity to Cisplatin, Etoposides and Doxorubicin was significantly increased. MRPA/ABCC1was the target of miR-134. After transfecting of H69AR cells with the mimics of miR-134, that the MRPA/ABCC1protein was considerably decreased, and the sensitivity to Cisplatin, Etoposides and Doxorubicin was also increased. Homeobox genes are critical genes that modulate biological processes, such as cell differentiation, proliferation and apoptosis. They involved in embryonic development and ontogeny. Studies have shown that homeobox genes can regulate apoptosis and multi-drug resistance of cancers. Xiao analyzed the differences of gene expression between H69 and H69AR by using cDNA microarray. The studies showed that HOXA1expression in SCLC cell lines H69was4.16-fold of that in multi-drug resistant cell line H69AR. The result was verified by using quantitive real-time PCR and Western Blot. Forced HOXA1expression increased chemo-sensitivity of H69AR cells, reduction of HOXA1expression in H69cell increased its chemo-resistance. HOXA1is the target genes of miR-100. After transfection of miR-100inhibitor into H69AR cell, miR-100expression reduced, but HOXA1protein levels increased. Up-regulation of miR-100in H69cell resulted in increase of resistance to ADM, DDP and VP-16. Epithelial and endothelial tyrosine kinase (Ekt) is one of the important members of the Btk family. Studies found that plays an important role in epithelial proliferation, differentiation, apoptosis and tumorigenesis. Zhow Confirmed that the Etk expression levels in H69AR cell was significantly higher than in H69cells by real-time PCR and western blotting. After the gene silencing to reduce Etk level in H69AR cells, the sensitivity to doxorubicin increased significantly. That further confirmed the Etk resistance to chemotherapeutic and apoptosis, and may play important role in the process of small cell lung cancer drug resistance.The relationship between the abnormal of membrane protein drug pump expression and multi-drug resistant. The multi-drug resistant formatiing are related membrane proteins, such as the over-expression of the ABCC family members of P-gp, MDR-related protein (MDR1, MRP1, MRP2). That the P-gp and MRP-1express higher in the resistant SCLC tissues and cultured SCLC cell lines in vitro. The formation of the MDR is related to LRP high expression. LRP can mediated DNA-targeting chemotherapy drugs cisplatin, carboplatin and other alkylating agents, such as block into the nucleus, and played the role of intermediate checkpoints. Import drugs into the nucleus, or re-transported out of the drug that had entered into the nuclear and the cytoplasm So that the drug was the atrioventricular distribution, and through exocytosis mechanism washout cells and reduce the concentration of the drug in the cell, ultimately resulting in the resistance. Scholars confirmed FZD1of GLI1and FRZB resistant SCLC cell lines and present patients of the drug phenomenon in significantly high expression from gene chip. FZD1is involved. In the curl protein gene encoding that is transmembrane Wnt receptor.10gene (FZD1-FZD10) are involved in the Wnt receptor curl Protein encoding. That plays a very important role in the development of malignant tumors occur. The gene chip confirmed that the FZD1expressied higher emerged both in vitro H69AR cells or in vivo. FZD1involving curl protein of Wnt receptor coding genes is a reversal of the SCLC multi-drug resistance ideal target. GLI is an important transcription factor of the Sonic hedgehog (Shh) signaling pathway. Shh signaling pathway is abnormally activated in pancreatic cancer, basal cell carcinoma, SCLC tumor. Shh signaling pathway mediated the target cells by ultimating Patched (PTC) on the cell membrane and two transcription originals and activate a transcription factor GLI. Chemotherapy drugs stimulation the upstream gene of the Shh signaling pathway, and triggering the biological function change in the downstream transcription factor. GLI1as the end of the Shh signaling pathway, illustrated fully that the GLI1not only involved in tumor development and involved in tumor cell resistance to chemotherapeutic drugs. FRZB is a negative regulator of the Wnt signaling pathway. The study found that FRZB was significantly higher expressed in resistant SCLC cell lines and present patients of the drug phenomenon. The resistant cell line was significantly higher expression than the parental cell line, indicating that the gene is involved in tumor resistance. These gene changes involved in the drug resistance of tumor cells as well as how to avoid these resistance genes play the role still requires a deep level of research.Relationship between the abnormal enzyme system with multi-drug resistant cells. Topoisomerase (TOP) is the main cell DNA replication and transcription ribozyme. Top inhibitor is one of the most commonly used chemotherapy drugs SCLC. Down-regulation expression levels of the Top as well as the change of expression is a the SCLC Top inhibitor resistant reason. A new study found that liposome transfect of synthetic siRNA can effectively inhibit human small cell lung cancer cell line H446Top Ⅰ expression and significantly improve the sensitivity of VP16. Top Ⅰ levels decreased after transfection of the cell line, the same time the expression of elevated levels of the Top Ⅱ. The experiments of the vivo and the in vitro have confirmed that Top I inhibitors and Top II inhibitor combined application has obvious synergies. Lawson et screening SCLC multi-drug resistance-related genes by cDNA microarray analysis, and changed candidate target gene expression levels, observe the SCLC drug sensitivity. The results showed high expression of DNA polymerase β and neuroendocrine transcription factor NKX may caused SCLC resistant to etoposide, also confirmed this conclusion in a the SCLC tissue microarray results.The relationship between cell repair system with multi-drug resistance. Recent studies show that the DNA mismatch repair gene (MMR) plays a very important role in SCLC acquired drug resistance. Scholars have found that the MMR genes MLH1and MSH2downregulation may occur with the SCLC and its MDR. The specific mechanism of MMR genes downregulated is not yet clear, May go with histone acetylation, phosphorylation or to start hypermethylation and that caused by MMR gene silencing.Proteomics is a hot spot in the clinical oncology and basic research. Have an important role in for understanding tumor mechanisms, diagnosis and treatment and other multifaceted. Proteomics is the subject that used large-scale application technology for protein separation and identification. It is also a high-throughput protein screening technology, including structural proteomics and functional proteomics, exploring the essence of life and its laws from the overall protein level. Proteomics technologies include protein separation technology, identification technology and bio-informatics technology. Available cells, tissues, blood, body fluids samples can be used for the proteomics research. In recent years, Laser capture micro-dissection technology applications, both to ensure the tumor tissue drawn from the availability of a sufficient amount of a single cell components, but also to maintain the cells in the original form. The key technology of protein separation is two-dimensional polyacry lamide gel electrophoresis (2D-PAGE). Its principle is based on isoelectric point and the different molecular weight, by way of electrophoresis to protein separation.The role of identification technology for protein is the Mass spectrometry. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALD-TOF-FMS) was most commonly used. The principle of MS analysis is converted the protein to ions using the ionization source, then separating protein molecule with proportion of M/Z in the electric field and the magnetic field by means of the mass spectrometer. The separated ions were collected through the ion detector, determine the M/Z value of ion, analysis and identify the unknown protein. The advantage of MS is high-resolution, high-sensitivity, high-throughput and direct detection of complex biological samples, etc. That has become the special technology of proteomics research.Bioinformatics is also an important part of the proteomics research. In the study of proteomics, bio-information technology plays an important role in constructing and analyzing of2-DE maps, database search and construction. Proteome database is the logo and the basis of the level of proteome research. Proteomics must be combined with bioinformatics to extract valuable information in the complexity of the data, provide effective guidelines for research.ObjectivesTo analysis the differences express protein in SCLC multi-drug resistant cell line H69AR and sensitive cell lines H69, and select the significantly different express protein DJ-1and confilin-1(CFL-1) for further study. Drug sensitivity of the expression levels of the two proteins through RNA interference technology down and observe resistant strains Doxorubicin (ADM), of Cisplatin (DDP), Etoposide (VP-16) changes. Observe the relationship between the express of DJ-1and CFL-1in SCLC tissues with the clinicopathological features. To explore the possibility of biomarkers as SCLC therapeutic targets and prognosis. Further enrich the SCLC multidrug resistance molecular mechanisms, and provide theoretical and experimental basis for clinical treatment.Materials and methods1. Analyzed the differences of protein expression between small cell lung cancer cell line H69and its multi-drug resistant cell line H69ARInvestigated the difference of protein expression between SCLC multi-drug resistant cell line H69AR and sensitive cell lines H69,2-DE and Matrix-assisted laser adsorption ionization time-of-flight mass spectrometry (MALD-TOF-FMS) were used in the study. And selected the significantly different express protein DJ-1and confilin-1(CFL-1) for further study. Using the Western Blot techniques to verify the differential expression of the two proteins in the two cell lines.2. Down regulation DJ-1or CFL-1expression in the cell lines of H69AR by siRNAWe synthesized of DJ-1-siRNA, CFL-1-siRNA and control siRNA, and transfected into H69AR cells by the liposomes2000. Total protein was extracted after24h of cell culture. Detecting of the differences of DJ-1or CFL-1protein expression in the group, that transfected DJ-1-siRNA, CFL-1-siRNA and control siRNA, using western blotting technology.3. Analysis of the effect of multidrug resistance in H69AR cell lines after down regulation the DJ-1or CFL-1expression.Chemo-sensitivity to different concentration of ADM, DDP and VP-16were determined by using CCK-8assay in H69AR-DJ1-siRNA394cells and H69AR-CFL1-siRNA523cells.4. The expression of DJ-1and CFL-1in SCLC tissues and clinical significance116cases of SCLC paraffin-embedded tissues were collected in our study. The average age of the patients was58.92±10.09, and followed up for0-128months. Male101cases, female15cases.58cases aged less then60years,58cases were60years or more. Clinical limited stage72cases, extensive stage46cases;95cases undergoing chemotherapy,21cases abcent chemotherapy. DJ-1and CFL-1expression were detected by immunohistochemistry stain in SCLC tissue, observed the relationship between the DJ-1or CFL-1expression levels and clinicopathological characteristics and prognosis.5. Statistical analysisData are represented as mean±s.e.m. All statistical analyses were carried out with SPSS13.0software. Factorial experiment ANOVA was used to analyze results of CCK-8experiment. The association between DJ-1and CFL-1expression and clinicopathological features were analyzed by Chi-square test. Survival curves were obtained by Kaplain-Meier method. Prognostic factors were examined by univariate and multivariate analyses (Cox proportional hazards model). P value <0.05was considered significant.Results1. Differentially expressed protein screening of SCLC in the multi-drug resistant cell line H69AR and sensitive cell line H69We adopted a2-DE analysis to quantitatively compare the protein profilings of SCLC mutidrug resistant cell ling H69AR and cell line H69. The differently expressed proteins were more than2fold changes between the two groups.25protein spots in H69AR were higher than H69, but10spots were less.25higher proteins spots were slected to perform protein identification by MALDI-TOF-MS. A total of16proteins were successfully identified including molecular chaperones (HSPB1), Glucose metabolism enzyme (IMPDH2, Alpha-enolase), Cytoskeletal proteins (Cytoskeletal9, Cytoskeletal1, Cofilin-1), Calcium-binding protein (Annexin-A2, Sorcin, S100A6), Cell cycle and apoptosis-related proteins (14-3-3epsilon, PCNA, Stathmin), others (Protein DJ-1, PPIA, GIPC1, vinculin).The different expression of DJ-1and CFL-1between H69AR and H69cells identified in proteomic study was further validated by Western blot analysis. H69AR cells had increased expressed level of DJ-1and CFL-1compared to H69cells.2. Selection of efficient siRNA for interference the DJ-1and CFL-1expressionWe designed and synthesized three pairs of siRNA oligonucleotides targeting DJ-1and CFL-1respectively, included DJ-1-homo-394, DJ-1-homo-483, DJ-1-homo-612, CFL-1-homo-326, CFL-1-homo-523, CFL-1-homo-579. Transfected them and control siRNA into H69AR cells by using lipofectamin2000. Study showed that sharply down express of DJ-1and CFL-1in cells that transfected DJ-1-homo-394-siRNA and CFL-1-homo-523-siRNA, and select DJ-1-homo-394-siRNA and CFL-1-homo-523-siRNA for fellow research.3. Down-regulation DJ-1and CFL-1expression and the effect of SCLC multi drug resistanceAfter interfered, DJ-1and CFL-1expression level decreased sharply in H69ARcells. CKK-8assay results showed that, after treatment of ADM, DDP, VP-16, the survival rate of H69AR-DJ-1-siRNA394decreased significantly compared with that mock control. Similarly, the survival rate of H69AR-CFL-1-siRNA394decreased significantly compared with that mock control.4. Relationship between DJ-1and CFL-1expression and clinical pathological features in SCLC tissues.Positive DJ-1expression in SCLC tissues was located in the cytoplasm. Its positive rate was51.7%(60/116). Positive rate of DJ-1expression in male patients was52.5%(53/101), while in female ones46.7%(7/15). There was no significant difference between them (χ2=0.176> P=0.674). Positive rate of DJ-1expression in patients younger than60-year-old was53.4%(31/58), while in over60-years old50.0%(29/58). There was no significant difference between them (x2=0.138, P=0.710). Positive rate of DJ-1expression in patients in limited stage was52.8%(38/72), while in extensive stage50.0%(22/44). There was significant difference between them (χ2=0.084, P=0.771). Positive rate of DJ-1expression in95patients undergone chemotherapy was50.5%(48/95), while in21patients with no chemotherapy51.7%(12/21). There was significant difference between them (χ2=0.302, P=0.583). Positive rate of DJ-1expression in survival patients was15.4%(2/13), while in dead57.1%(52/93). There was significant difference between them (χ2=7.946, P=0.005).Positive CFL-1expression in SCLC tissues was located in the cytoplasm nucleus. Its positive rate was51.7%(60/116) in all cases. Positive rate of DJ-1expression in male patients was52.5%(53/101), while in female ones46.7%(7/15). There was no significant difference between them (χ2=0.176, P=0.674). Positive rate of CFL-1expression in patients younger than60-year-old was53.4%(31/58), while in over56-years old50.0%(29/58). There was no significant difference between them (χ2=0.138, P=0.710). Positive rate of CFL-1expression in patients in limited stage was52.8%(38/72), while in extensive stage50.0%(22/44). There was significant difference between them (χ2=0.084, P=0.771). Positive rate of CFL-1expression in 95patients undergone chemotherapy was50.5%(48/95), while in21patients with no chemotherapy51.7%(12/21). There was significant difference between them (χ2=0.302, P=0.583). Positive rate of CFL-1expression in survival patients was15.4%(2/13), while in dead57.1%(52/93). There was significant difference between them (χ2=7.946, P=0.005).5. Survival analysis of SCLC patients(1)COX regression model By using stepwise COX regression model analysis, DJ-1over-express (HR=2.509, P<0.001,95%CI:1.622-3.880) and CLF-1over-express (HR=2.000, P=0.001,95%CI:1.304-3.068) were high death risk for SCLC patients. Chemotherapy is also a clearly factor for progression (HR=0.488, P=0.012,95%CI:0.278-0.856).(2) Estimation of survival timeWe used Kaplan-Meier method to estimate the survival time of patients. DJ-1expression was one independent poor prognosis factor in SCLC patients(HR=2.960, P=0.001,95%CI:1.519-5.767).Conclusions1. Two dimensional gel electrophoresis and mass spectrometry analysis of proteomic technology can be used for screening out SCLC multidrug resistance-associated protein.2. DJ-1and CFL-1expressed higher in SCLC multidrug resistant cell line H69AR and lower in the drug-sensitive cell line H69. Down regulated the expression of DJ-1and CFL-1can cause H69AR drug sensitivity change, DJ-1and CFL-1involved in drug resistance in SCLC.3. DJ-1and CFL-1expression in SCLC tissue were related to survival time of patients. DJ-1and CFL-1were expected to be prognostic indicators and targets of the drug treatment of patients with SCLC. |
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