Screening For Novel Specific Small Molecule Peptides For Lung Cancer Cell And Its Application

BackgroundLung cancer is the leading malignant tumor with 26.9%increasing incidence per year in our country especially in city.China will be the biggest country with lung cancer patients in the world in 2025.Lung cancer is associated with high mortality and morbidity because most cases present as middle-advanced stage with partly metastasis after diagnosis.The long-term outlook for patients with advanced lung cancer is poor with median survival and is typically less than one year.Only about 20%of non-small lung cancer（NSCLC） patients response to anticancer drugs and have to give up chemotherapy with severe side-effect such as hematopoiesis inhibition.Moreover,there has been no effective way for screening or detection of early lung cancer up to now,in contrast to what have been established for prostate cancers and colon cancer.Obviously,new strategies are needed for early detection and treatment for lung cancer.Monoclone antibodies specific for antigen or receptor on cell surface generated by hybridoma technique,have been widely applied in the clinical diagnosis of breast cancer,colon cancer and prostate cancer.Moreover,molecular-targeting therapy has become a reality in treatment of lung cancer,breast cancer and lymphoma.For example,inhibitors of epidermal growth factor receptor,gefitinib and erlotinib,have recently been used to treat NSCLC refractory to traditional chemotherapy.Herceptin and Rituxan have been used for breast cancer and B cell leukemia.However,clinical application of monoclone antibodies in cancer treatment still remains several practical problems which affect the clinic therapeutic efficacy as follow:（1） Monoclone antibody may not permeate the target tumor mass effectively because of large molecule protein;（2） The non-specific aggregation of antibody in reticuloendothelial system may lead to a reduction of its anticancer potency;（3） Antimouse antibody or the chimera of antibody have potential immunogenicity.Recent studies have showed that small molecule peptide can retrieve a few shortage of antibody.Small molecule peptide has more advantage in the following aspects:（1） Effective to penetrate the tumor mass with less small molecule than a antibody,such as ten peptide have a molecular weight of approximately 1000 Da;（2） Easier and more constant to conjugated with toxin and radioactive agent than a antibody;（3） More stable and effective to protect amino acide from proteolysis by protein enzyme than a antibody; （4） Less to be phagocytosed by reticuloendothelial system;（5） Less likely to provoke an immunoresponse than proteins;（6） Much less cost in synthesis than antibody.We believe that small molecule peptide has more promising approach in cancer diagnosis and treatment.Small molecule peptide has basically been used to apply in cancer diagnosis and treatment in three aspects:（1） Small molecule peptide can capture metastatic cancer cell from circulation;（2） Small molecule peptide labelled with magnetic atom or fluorescein or radiation isotope will be molecule probes,for example,small molecule peptide RGD labelled with isotope can remarkably promote the sensitivity of MRI;（3） Small molecule peptide labelled with radiation isotope will be targeting radiative therapeutic agent for cance patients,for example,peptide containing RGD sequence labelled with ¹¹¹In or ^99mTc can dramatically inhibit tumor growth.Now there are two general screening methods for small molecule peptides:one is "one-bead one-peptide" combinatorial chemistry peptide library,another is phage-displaying peptide library."one-bead one-peptide" combinatorial chemistry peptide library was firstly introduced by Pro.Lam in 1991.The library is synthesized using the natural and unnatural amino acide on resin beads at random.According to the experiments,libraries with one peptide on one bead can be synthesized with 4 or 6 or 8 peptides length and loop or liner conformation.The efficiency of screening is very high because millions of peptides can be screened at one time.Phage-displaying peptide library is one of the main methods for peptides screening at present as well. However,"one-bead one-peptide" combinatorial chemistry peptide library can be added into unnatural amino acide randomly during the synthesis process and therefore peptides are generally resistant to proteolysis and more stable than the latter.So "one-bead one-peptide" combinatorial library is regard as more ideal method for small molecule peptide screening.Many scholars have successfully found some small molecule peptides bind specifically to cancer cells using "one-bead one-peptide" combinatorial libraries. Pennington et al（1996） identified two peptides,RU-1（LNIVSVNGRHX） and RX-1 （DNRIRLQAKXX） by scanning for prostate cancer cell among 1.5 million peptide beads.DeRoock et al（2001） found two peptides with six amino acid,RZ-3 （kmviywkag） and HYD-1（kikmviswkg）.These two peptides support tumor cell adhesion and can inhibit cancer cell adhesion to extracellular matrix protein.Mikawa et al（2004） identified a small molecular peptide NleDI/V/NIe can specifically adhere to bronchioloalveolar carcinoma cells.Aina et al（2005） reported a novel small molecule peptide cDGXGXXc by screening a cyclic random 8-mer library can specifically adhere to ovarian cancer cells. In this study,we intended to identify small molecule peptides specific for NSCLC A549 and SCLC DMS53 cells by screening for "one-bead one-peptide" combinatorial chemistry library.And applications of the small molecule peptide is also investigated in promoting cytopathological positive diagnosis rate of pleural fluid with lung cancer by synthesis the peptide on beads and is evaluated to be as specific molecular marker of NSCLC cells.The full text consists of the three following sections:Section one:A novel specific small molecule peptide for non-small cell lung cancer cell A549Aim:To screen small molecule peptide specific binding to non-small cell lung cancer cell（A549） using the "one-bead one-peptide" combinatorial library. Materials and Methods:A "one-bead one-peptide" combinatorial library with six amino acids was used to screen for specific binding peptides to non-small cell lung cancer cell（A549）.Beads with cell adhesion and growth（positive beads） were isolated,stripped,and microsequenced.The novel consensus peptides will be re-synthesized and further tested as follow:（1） Cell-type specificity was analysized by three independent experiments（A） A panel of cell lines including non-small-cell lung cancer A549,Calu-1 and other cancer cells were used to study their specificity to novel peptides;（B） Free peptides were used to block the binding between cancer cells and peptide beads;（C） Adhesion to a slide coated with free peptides was tested by twelve different cancer cells including A549 and Calu-1.（2） The strategies of site-directed deletion and alanine scanning were employed to determine the structure either length or specific amino acid for cell adhesion.（3） Expression profling of integrins for cell adhesion was investigated using restriction analysis of gene expression（RAGE）,flow cytometry and immunofluorescence staining technique. Antibody blocking assay was then used to confirm the binding site between A549 cells and small molecule peptides.Immunofluorescence technique was also used to observe the relationship between integrin a3 and focal adhesion kinase（FAK） and paxillin and vinculin which are all important cell adhesion associated signal transduction molecules.Results:（1） Twenty-nine positive beads binding to A549 cell were totally obtained after primary screening.Consensus peptide sequence of-NGXG-was identified by amino acid sequencing in ten beads.（2） Peptide cNGQGEQc was re-synthesized on beads and further studied for its cell specificity.Peptide cNGQGEQc was showed to be specific for cell attachment to non-small cell lung cancer cells including A549, Calu-1 and H178,but not to other cancer cell lines.（3） Both motif of-NGXG-and the length of peptide are very important for A549 adhesion.（4） Integrin a3 was presented on non-small cell lung cancer cells A549 and Calu-1 but not on small lung cancer cell by RAGE,flow cytometry and immunofluorescence staining technique. （5） In an blocking assay with anti-integrin antibodies（α1-6,v/β1-5）,cell adhesion of A549 to peptide beads was obviously inhibited by integfinα3 combining with anyβsubunits.（6） There is intensive coexpression relationship between integrinα3 and FAK and paxillin and vinculin during the binding process between small molecule peptide and A549.Conclusion:The results suggested that（1） small molecule peptide cNGQGEQc can bind specifically to non-small cell lung cancer cell A549 via integrinα3 on cell surface;（2） Integrinα3 represents a potential molecular marker and target for molecular therapy in non-small cell lung cancer.The novel small molecule peptide obtained in this study will be used as an ideal carder for molecular diagnosis and targeting therapy of lung cancer. Section two:A novel specific small molecule peptide for small cell lung cancer cell DMS-53Aim:To screen small molecule peptide specific binding to small cell lung cancer cell （DMS53） using the "one-bead one-peptide" combinatorial chemistry technology. Materials and Methods:A "one-bead one-peptide" combinatorial library with six amino acids was used to screen for small molecule peptide specific binding to small cell lung cancer cell（DMS53）.Beads with cell adhesion and growth（positive beads） were isolated,stripped,and microsequenced.The novel consensus peptides will be re-synthesized and further tested as follow:（1） Cell-type specificity was analysized by three independent experiments（A） A panel of cell lines including small cell lung cancer cell DMS53 and other cancer cells were used to study their specificity to novel peptides;（B） Free peptides were used to block the binding between cancer cells and peptide beads;（C） Adhesion to a slide coated with free peptides was tested by different cancer cells including small cell lung cancer cell（DMS53）.（2） The strategies of site-directed deletion and alanine scanning were employed to determine the structure either length or specific amino acid for cell adhesion.（3） Expression profling of integrins for cell adhesion was investigated using restriction analysis of gene expression（RAGE）,flow cytometry and immunofluorescence staining technique.Antibody blocking assay was then used to confirm the binding site between DMS53 cells and small molecule peptide.Results:（1） Thirty two positive beads binding to DMS53 were totally obtained after primary screening.Consensus peptide sequences of cXNGRXXc and cNGRXXXc were identified by amino acid sequencing in ten beads.Three representative peptides were re-synthesized on beads.Secondary screening showed cell adhesion percentage of cFNGRQQc was higher than the other two peptides.（2） cFNGRQQc was further studied for cell specificity,alanine scanning and site-directed deletion.The results showed that cFNGRQQc is specific for promoting cell adhesion to DMS53 but not other human cancer cell lines.（3） Both motif of-NGR-and the length of peptide are important for DMS53 attachment.（4） Integrinα5 andβ1 were presented on DMS53 by RAGE,flow cytometry and immunofluorescence staining technique.（5） In an antibody blocking assay,cell adhesion of DMS53 to peptide beads was not inhibited by antibodies including integrin,E-cadherin,NCAM and ICAM.Conclusion:The results suggested that（1） small molecule peptide cFNGRQQc can bind specifically to small cell lung cancer cell DMS53,but the binding site between the peptide and DMS53 is still unknown;（2） The binding site between the peptide cFNGRQQc and DMS53 is different from other peptide via integrin and may be not associated with adhesion molecules E-cadherin,NCAM and ICAM.The binding site on DMS53 surface for cFNGRQQc peptide need to be proven in the future.Section three:Application of specific small molecule peptide in lung cancerAim:To investigate the clinic significance of the specific small molecule peptide obtained in provious study for lung cancer in promoting cytopathological positive diagnosis rate of pleural fluid with lung cancer by synthesis the peptide on beads and is evaluated to be as specific molecular marker of NSCLC cells.Materials and Methods.（1） Small molecule peptide cNGQGEQc synthesized on resin beads was tested the the specificity in capturing A549 cells mixed with normal human blood.Small molecule peptide beads were then co-cultured with pleural fluid obtained from sixty-four cases including lung cancer,mesothelioma,gastric carcinoma,colon cancer,breast cancer,pneumonia and tuberculosis of pleura and so on.Capturing of cancer cells on the beads was accomplished using method as described above.Cells captured on beads were subsequently stripped off the beads and cell smears were prepared on slides for microscopic examination.（2） The small molecule peptide labelled with biotin or FITC was used to stain A549 cell as well as other cancer cells.The specificity of small molecule peptide binding to lung cancer cells was also tested in lung cancer tissues.Results:（1） Only one positive case was found.by routine cytology method,while five positive cases（10.81%） were present by routine cytologic method combining with capturing cancer cell with peptide beads.（2） Non-small lung cancer cells A549 and Calu-1 but not DMS53,were specifically recognised by peptide "cNGQGEQc".All the eighteen samples of adenocarcinoma and most of squemous cancinoma（7/9） of lung were immunostained with peptide but not in small cell lung cancer.Conclusion:（1） Small molecule peptide bead technology combining with routin cytology method can be used for isolating and enriching the scanty number of cancer cells in pleural effusion.（2） Small molecule peptide "cNGQGEQc" can specifically label the non-samll lung cancer and will be the novel helpful biological marker for NSCLC cells.