Hepatocellular carcinoma cell-specific peptide ligand for targeted drug delivery

Hepatocellular carcinoma is the fourth leading cause of cancer death worldwide. Novel treatment strategies derived from increased knowledge of molecular oncology are constantly being developed to cure this disease. Here, we used phage display to identify a novel peptide (SP94), which binds specifically to hepatocellular carcinoma cells. In vitro, the phage clone PC94 was shown to bind to hepatocellular carcinoma cell lines by ELISA and flow cytometry analysis. In vivo, PC94 homed specifically to tumor tissues but not to normal visceral organs in severe combined immunodeficient mice bearing human hepatocellular carcinoma xenografts. This homing ability could be competitively inhibited by synthetic peptide, SP94. Immunohistochemical staining confirmed that PC94 localized to tumor tissues and that it could not be detected in SP94-competed tumor tissues. In addition, PC94 recognized the tumor tissue but not nontumor tissue in surgical specimens from hepatocellular carcinoma patients, with a positive rate of 61.3% (19 of 31). With the conjugation of SP94 and liposomal doxorubicin, the targeted drug delivery system enhanced the therapeutic efficacy against hepatocellular carcinoma xenografts through enhanced tumor apoptosis and decreased tumor angiogenesis. Our results indicate that SP94 has the potential to improve the systemic treatment of patients with advanced hepatocellular carcinoma.     

新型多肽聚酰胺-胺型靶向药物载体的载药性能及细胞吸收和毒性

背景:前期研究通过噬菌体展示体内筛选方法获得了一条NCI-H460非小细胞肺癌特异结合的多肽(Lung cancer targetingpe ptide,LCTP),将该多肽与修饰的聚酰胺-胺型(Polyamidoamine,PAMAM)树枝状高分子材料连接制备了纳米靶向药物载体PAMAM-Ac-FITC-LCTP,该载体在体内外对非小细胞肺癌NCI-H460具有很好的靶向性。目的:在前期研究基础上,进一步研究PAMAM-Ac-FITC-LCTP靶向载体对阿霉素的包埋、释放及其细胞吸收和毒性性能。方法:以筛选到的多肽LCTP为靶向剂,构建了PAMAM-Ac-FITC-LCTP靶向载体。采用物理包埋法将PAMAM-Ac-FITC-LCTP与阿霉素连接,通过体外透析实验观察载体对药物的缓释功能,共聚焦显微镜观察细胞对药物的吸收。以游离阿霉素作为对照,MTT法观察载体载药后对NCI-H460细胞的作用。结果与结论:PAMAM-Ac-FITC-LCTP对阿霉素的最大包埋率为7.46%。载体对药物具有明显的缓释作用,离子浓度、pH和温度对药物的释放具有影响,说明PAMAM-Ac-FITC-LCTP主要是通过静电相互作用与阿霉素结合。PAMAM-Ac-FITC-LCTP/阿霉素短时间内较单独药物更高效进入NCI-H460细胞,而复合物24h的细胞毒性与阿霉素对细胞的毒性基本一致。以上结果说明PAMAM-Ac-FITC-LCTP可能是一个肿瘤治疗和诊断中很有用的药物靶向传输载体。     

Chloride channel-mediated brain glioma targeting of chlorotoxin-modified doxorubicine-loaded liposomes

The chlorotoxin (ClTx), a scorpion-derived peptide, binding to gliomas with high specificity, was firstly applied to establish the ClTx-modified doxorubicin (DOX)-loaded liposome delivery system for targeting the brain glioma and improving the anticancer efficacy. In vitro physicochemical characterization of the novel liposome system presented satisfactory size of 100 nm with uniform distribution, high encapsulation efficiency and adequate loading capacity of both fluorescent probe and anticancer drug. It was demonstrated quantitatively by the spectrophotofluorometry and flow cytometry and qualitatively by the confocal microscopy that ClTx highly facilitated the uptake of liposomes by three glioma cell lines and one endothelial cell line. In vitro cytotoxicity studies proved that the presence of ClTx increased the cytotoxicity against glioma cells and endothelial cells with various levels for different cell lines. In BALB/c mice bearing U87 tumor xenografts, biodistribution of DiR-loaded liposomes by body imaging and anti-glioma pharmacodynamics of DOX-loaded liposomes were investigated. The ClTx-modified liposomes showed more accumulation in the subcutaneous and intracranial tumors, higher tumor growth inhibition and lower blood toxicity in the armpit tumor model. The in vitro and in vivo results exhibited good correlation of glioma targeting of the ClTx-modified liposomes. Significantly, with the ClTx as the targeting ligand, the liposomes might serve as an applicable delivery system for brain glioma therapy or imaging.     

新型多肽聚酰胺-胺型靶向药物载体的载药性能及细胞吸收和毒性

背景：前期研究通过噬菌体展示体内筛选方法获得了一条NCI-H460非小细胞肺癌特异结合的多肽（Lung cancer targetingpe ptide,LCTP）,将该多肽与修饰的聚酰胺-胺型（Polyamidoamine,PAMAM）树枝状高分子材料连接制备了纳米靶向药物载体PAMAM-Ac-FITC-LCTP,该载体在体内外对非小细胞肺癌NCI-H460具有很好的靶向性。目的：在前期研究基础上,进一步研究PAMAM-Ac-FITC-LCTP靶向载体对阿霉素的包埋、释放及其细胞吸收和毒性性能。方法：以筛选到的多肽LCTP为靶向剂,构建了PAMAM-Ac-FITC-LCTP靶向载体。采用物理包埋法将PAMAM-Ac-FITC-LCTP与阿霉素连接,通过体外透析实验观察载体对药物的缓释功能,共聚焦显微镜观察细胞对药物的吸收。以游离阿霉素作为对照,MTT法观察载体载药后对NCI-H460细胞的作用。结果与结论：PAMAM-Ac-FITC-LCTP对阿霉素的最大包埋率为7.46%。载体对药物具有明显的缓释作用,离子浓度、pH和温度对药物的释放具有影响,说明PAMAM-Ac-FITC-LCTP主要是通过静电相互作用与阿霉素结合。PAMAM-Ac-FITC-LCTP/阿霉素短时间内较单独药物更高效进入NCI-H460细胞,而复合物24h的细胞毒性与阿霉素对细胞的毒性基本一致。以上结果说明PAMAM-Ac-FITC-LCTP可能是一个肿瘤治疗和诊断中很有用的药物靶向传输载体。     

Novel SRESPHP peptide mediates specific binding to primary medullary thyroid carcinoma after systemic injection

The efficient and specific introduction of genes into cancer cells in vivo remains a major challenge for current gene therapy modalities. Peptides possess appropriate properties to serve as tumor-targeting agents. Thus, finding new cancer-selective peptides directing gene transfer to neoplastic cells by reducing transduction of normal cells is a central goal for molecular targeting. We have previously reported identification of a peptide (HTFEPGV) that selectively binds to human medullary thyroid carcinoma (MTC)-derived TT cells in vitro and transplanted tumor xenografts in vivo, using phage display. In the present study, we have performed this approach in primary orthotopically growing murine MTCs of RET-C634R transgenic mice as a clinically relevant model for thyroid cancer by intravenous injection of a complex peptide library. Two rounds of screening on primary tumors yielded multiple copies of a phage that displays a cyclic 7-amino acid peptide, SRESPHP, with a 3000-fold increase in titer between rounds 1 and 2. The selected phage showed highly specific binding to the tumor after systemic administration, whereas binding to other organs such as lung, liver, kidney, and heart was reduced up to 90%. After tail vein injection, homing to the tumor was substantially reduced in the presence of synthetic SRESPHP peptide, indicating that tumor phage interaction strictly depends on the displayed peptide. Immunohistochemical analysis of paraffin sections from mouse tissues revealed direct binding of the SRESPHP peptide to MTC tissue. Moreover, this peptide also mediates binding to human MTC cells in vitro and in vivo, suggesting abundant expression of its cognate receptor in murine and human medullary thyroid carcinoma. Because the SRESPHP peptide is also efficiently internalized into MTC cells, it likely provides the basis for a new selective therapy of medullary thyroid carcinoma.     

Epithelial-neutrophil activating peptide (ENA-78) is an important angiogenic factor in non-small cell lung cancer.

We report here the role of the CXC chemokine, epithelial neutrophil activating peptide (ENA-78), as an angiogenic factor in human non-small cell lung cancer (NSCLC). In freshly isolated human specimens of NSCLC, elevated levels of ENA-78 were found that strongly correlated with the vascularity of the tumors. In a SCID mouse model of human NSCLC tumorigenesis, expression of ENA-78 in developing tumors correlated with tumor growth in two different NSCLC cell lines. Furthermore, passive immunization of NSCLC tumor-bearing mice with neutralizing anti-ENA-78 antibodies reduced tumor growth, tumor vascularity, and spontaneous metastases, while having no effect on the proliferation of NSCLC cells either in vitro or in vivo. These findings suggest that ENA-78 is an important angiogenic factor in human NSCLC.     

用噬菌体展示技术筛选喉癌细胞靶向肽的研究

目的筛选人源喉癌Hep-2细胞株特异结合的短肽,作为喉癌靶向治疗的载体。方法体外培养Hep-2细胞株作为靶细胞,人正常喉黏膜上皮细胞为吸附细胞;用噬菌体展示十二肽库进行3轮差减筛选,随机挑取10个噬菌体克隆进行测序;采用酶联免疫吸附（enzyme linked immunosorbent assay,ELISA）法鉴定噬菌体与Hep-2细胞的结合活性;通过免疫荧光鉴定喉癌细胞特异性结合肽（F2）噬菌体阳性克隆与喉癌细胞结合的特异性。结果经过3轮筛选后,噬菌体在靶细胞Hep-2上出现明显富集;ELISA分析鉴定显示5个阳性克隆能与Hep-2细胞特异结合,其中F2噬菌体克隆对喉癌细胞的结合靶向性明显高于对照细胞（P〈0.05）;免疫荧光显色显示,F2能特异性地与喉癌细胞结合。结论利用噬菌体展示肽库技术,可以成功筛选到F2,其可能成为喉癌靶向治疗的载体。     

Antitumor activity of an epithelial cell adhesion molecule targeted nanovesicular drug delivery system

Site-specific delivery of anticancer agents to tumors represents a promising therapeutic strategy because it increases efficacy and reduces toxicity to normal tissues compared with untargeted drugs. Sterically stabilized immunoliposomes (SIL), guided by antibodies that specifically bind to well internalizing antigens on the tumor cell surface, are effective nanoscale delivery systems capable of accumulating large quantities of anticancer agents at the tumor site. The epithelial cell adhesion molecule (EpCAM) holds major promise as a target for antibody-based cancer therapy due to its abundant expression in many solid tumors and its limited distribution in normal tissues. We generated EpCAM-directed immunoliposomes by covalently coupling the humanized single-chain Fv antibody fragment 4D5MOCB to the surface of sterically stabilized liposomes loaded with the anticancer agent doxorubicin. In vitro, the doxorubicin-loaded immunoliposomes (SIL-Dox) showed efficient cell binding and internalization and were significantly more cytotoxic against EpCAM-positive tumor cells than nontargeted liposomes (SL-Dox). In athymic mice bearing established human tumor xenografts, pharmacokinetic and biodistribution analysis of SIL-Dox revealed long circulation times in the blood with a half-life of 11 h and effective time-dependent tumor localization, resulting in up to 15% injected dose per gram tissue. These favorable pharmacokinetic properties translated into potent antitumor activity, which resulted in significant growth inhibition (compared with control mice), and was more pronounced than that of doxorubicin alone and nontargeted SL-Dox at low, nontoxic doses. Our data show the promise of EpCAM-directed nanovesicular drug delivery for targeted therapy of solid tumors.     

人前列腺癌细胞特异性结合短肽的筛选

目的获得与人前列腺癌细胞系PC3M细胞特异结合的短肽，作为人前列腺癌靶向治疗的先导化合物。方法以PC3M细胞为靶细胞，正常人前列腺细为吸附细胞对噬菌体7肽库进行差减筛选，用细胞ELISA、免疫荧光细胞化学胞染色鉴定阳性噬菌体克隆并测序。结果经三轮筛选，从随机挑选的10个噬菌体克隆中得到8个能特异性与PC3M细胞结合，而不与正常人前列腺细胞结合的阳性克隆。其氨基酸序列有一定同源性。结论得到多个序列不同的特异性结合PC3M细胞的噬菌体克隆。本实验获得的短肽具有一定的肿瘤亲合力和特异性，为针对不同位点的靶向药物设计提供了实验依据。     

Identification and characterization of novel human glioma-specific peptides to potentiate tumor-specific gene delivery

Glioblastomas account for approximately 20% of all primary brain tumors in adults. Glioblastoma multiforme (GBM) is a highly malignant tumor. In spite of advances in surgery, chemotherapy, and radiotherapy, the life expectancy of the patient with glioblastoma is approximately 11 months. To enhance glioma-specific gene delivery, we employed a 12-mer phage display peptide library to isolate phages that bind specifically to human glioma cell lines. Here, we report the isolation and functional characterization of novel glioma-specific peptides that target transgenes specifically to a wide array of human glioblastomas in vitro and in vivo. One of the isolated peptides, tentatively denoted as MG11, is demonstrated to be glioma specific and gives an in vitro-binding enrichment of more than 5-fold for glioma cells when compared with nonglioma cells. Intravenous injection of phages bearing the MG11 peptide-binding motif enables the phages to home specifically to glioma xenografts. Most significantly, when Lissamine rhodamine-labeled MG11 peptide is injected intratumorally, it targets specifically to glioma xenografts instead of non-glioma-derived xenografts. In summary, our results suggest that the MG11 peptide is able to target specifically to tumors of glial origin, which would allow the design of applications related to the diagnosis and treatment of human gliomas.     

Targeting ceramide synthase 6–dependent metastasis-prone phenotype in lung cancer cells

Sphingolipids make up a family of molecules associated with an array of biological functions, including cell death and migration. Sphingolipids are often altered in cancer, though how these alterations lead to tumor formation and progression is largely unknown. Here, we analyzed non–small-cell lung cancer (NSCLC) specimens and cell lines and determined that ceramide synthase 6 (CERS6) is markedly overexpressed compared with controls. Elevated CERS6 expression was due in part to reduction of microRNA-101 (miR-101) and was associated with increased invasion and poor prognosis. CERS6 knockdown in NSCLC cells altered the ceramide profile, resulting in decreased cell migration and invasion in vitro, and decreased the frequency of RAC1-positive lamellipodia formation while CERS6 overexpression promoted it. In murine models, CERS6 knockdown in transplanted NSCLC cells attenuated lung metastasis. Furthermore, combined treatment with l-α-dimyristoylphosphatidylcholine liposome and the glucosylceramide synthase inhibitor D-PDMP induced cell death in association with ceramide accumulation and promoted cancer cell apoptosis and tumor regression in murine models. Together, these results indicate that CERS6-dependent ceramide synthesis and maintenance of ceramide in the cellular membrane are essential for lamellipodia formation and metastasis. Moreover, these results suggest that targeting this homeostasis has potential as a therapeutic strategy for CERS6-overexpressing NSCLC.     

P-selectin mediates adhesion of platelets to neuroblastoma and small cell lung cancer.

Activated platelets and stimulated endothelial cells express P-selectin, an integral membrane protein receptor that binds monocytes and neutrophils. P-selectin mediates adhesion to glycoproteins with carbohydrate structures containing sialyl-Lewis X. Since many carcinoma cells also express these carbohydrate structures and are known to interact with platelets, we asked whether P-selectin may mediate this interaction. Both small cell lung cancer and neuroblastoma cell lines bound to activated platelets, and this interaction was blocked with inhibitory anti-P-selectin antibodies and by pretreatment of these cancer cells with neuraminidase or trypsin. Platelet binding to the small cell lung cancer cells was not inhibited with anti-GP IIb-IIIa antibody or Arg-Gly-Asp-Ser peptide. Pretreatment of the neuroblastoma cells with inhibitors of N-linked carbohydrate biosynthesis had little effect on binding to P-selectin, indicating that relevant carbohydrate ligand(s) may be O-linked. In addition, lipospheres containing P-selectin specifically bound to cryostat sections derived from a small cell lung tumor and two neuroblastoma tumors, but not to sections of normal lung. These observations demonstrate that P-selectin mediates binding of platelets to small cell lung cancer and to neuroblastoma and suggest a possible role for this lectin in metastasis.     

Interleukin-13 receptor-targeted nanovesicles are a potential therapy for glioblastoma multiforme

The difficulties associated with treatment of malignant brain tumors are well documented. For example, local infiltration of high-grade astrocytomas prevents the complete resection of all malignant cells. It is, therefore, critical to develop delivery systems for chemotherapeutic agents that ablate individual cancer cells without causing diffuse damage to surrounding brain tissue. Here, we describe sterically stable human interleukin-13 (IL-13)-conjugated liposomes, which efficiently bind to the brain cancer cells that overexpress the IL-13 receptor alpha2 protein. The conjugated liposomes bind to glioblastoma multiforme tissue specimens but not to normal cortex. Conjugating the liposomes with human IL-13 allows for specific binding to glioma cells and uptake of the liposomes via endocytosis. Delivering doxorubicin to glioma cells by IL-13-conjugated liposomes results in enhanced cytotoxicity and increased accumulation and retention of drug in the glioma cells compared with delivery of free drug. The therapeutic potential and targeting efficacy of the IL-13-conjugated liposomes carrying doxorubicin was tested in vivo using a s.c. glioma tumor mouse model. Animals receiving i.p. injections of IL-13-conjugated liposomes carrying doxorubicin for 7 weeks had a mean tumor volume of 37 mm3 compared with a mean volume of 192 mm3 in animals injected with nontargeted liposomes. These results strongly suggest that IL-13-conjugated liposomes carrying cytotoxic agents are a feasible approach for creating a nanovesicle drug delivery system for brain tumor therapy.     

Interferon-gamma-inducible protein 10 (IP-10) is an angiostatic factor that inhibits human non-small cell lung cancer (NSCLC) tumorigenesis and spontaneous metastases

The success of solid tumor growth and metastasis is dependent upon angiogenesis. Neovascularization within the tumor is regulated, in part, by a dual and opposing system of angiogenic and angiostatic factors. We now report that IP-10, a recently described angiostatic factor, as a potent angiostatic factor that regulates non-small cell lung cancer (NSCLC)-derived angiogenesis, tumor growth, and spontaneous metastasis. We initially found significantly elevated levels of IP-10 in freshly isolated human NSCLC samples of squamous cell carcinoma (SCCA). In contrast, levels of IP-10 were equivalent in either normal lung tissue or adenocarcinoma specimens. The neoplastic cells in specimens of SCCA were the predominant cells that appeared to express IP-10 by immunolocalization. Neutralization of IP-10 in SCCA tumor specimens resulted in enhanced tumor-derived angiogenic activity. Using a model of human NSCLC tumorigenesis in SCID mice, we found that NSCLC tumor growth was inversely correlated with levels of plasma or tumor- associated IP-10. IP-10 in vitro functioned as neither an autocrine growth factor nor as an inhibitor of proliferation of the NSCLC cell lines. Reconstitution of intratumor IP-10 for a period of 8 wk resulted in a significant inhibition of tumor growth, tumor-associated angiogenic activity and neovascularization, and spontaneous lung metastases, whereas, neutralization of IP-10 for 10 wk augmented tumor growth. These findings support the notion that tumor-derived IP-10 is an important endogenous angiostatic factor in NSCLC.     

Inhibition of fibroblast to myofibroblast transition by halofuginone contributes to the chemotherapy-mediated antitumoral effect

Stromal myofibroblasts play an important role in tumor progression. The transition of fibroblasts to myofibroblasts is characterized by expression of smooth muscle genes and profuse synthesis of extracellular matrix proteins. We evaluated the efficacy of targeting fibroblast-to-myofibroblast transition with halofuginone on tumor progression in prostate cancer and Wilms' tumor xenografts. In both xenografts, low doses of halofuginone treatment, independent of the route of administration, resulted in a trend toward inhibition in tumor development. Moreover, halofuginone synergizes with low dose of docetaxel in prostate cancer and vincristine and dactinomycin in Wilms' tumor xenografts, resulting in significant reduction in tumor volume and weight comparable to the effect observed by high doses of the respective chemotherapies. In prostate cancer and Wilms' tumor xenografts, halofuginone, but not the respective chemotherapies, inhibited the synthesis of collagen type I, alpha-smooth muscle actin, transgelin, and cytoglobin, all of which are characteristics of activated myofibroblasts. Halofuginone, as the respective chemotherapies, increased the synthesis of Wilms' tumor suppressor gene product (WT-1) and prostate apoptosis response gene-4 (Par-4), resulting in apoptosis/necrosis. These results suggest that targeting the fibroblast-to-myofibroblast transition with halofuginone may synergize with low doses of chemotherapy in achieving a significant antitumoral effect, avoiding the need of high-dose chemotherapy and its toxicity without impairing treatment efficacy.     

Nanostructured lipid carriers as multifunctional nanomedicine platform for pulmonary co-delivery of anticancer drugs and siRNA

We developed, synthesized, and tested a multifunctional nanostructured lipid nanocarrier-based system (NLCS) for efficient delivery of an anticancer drug and siRNA directly into the lungs by inhalation. The system contains: (1) nanostructured lipid carriers (NLC); (2) anticancer drug (doxorubicin or paclitaxel); (3) siRNA targeted to MRP1 mRNA as a suppressor of pump drug resistance; (4) siRNA targeted to BCL2 mRNA as a suppressor of nonpump cellular resistance and (5) a modified synthetic analog of luteinizing hormone-releasing hormone (LHRH) as a targeting moiety specific to the receptors that are overexpressed in the plasma membrane of lung cancer cells. The NLCS was tested in vitro using human lung cancer cells and in vivo utilizing mouse orthotopic model of human lung cancer. After inhalation, the proposed NLCS effectively delivered its payload into lung cancer cells leaving healthy lung tissues intact and also significantly decreasing the exposure of healthy organs when compared with intravenous injection. The NLCS showed enhanced antitumor activity when compared with intravenous treatment. The data obtained demonstrated high efficiency of proposed NLCS for tumor-targeted local delivery by inhalation of anticancer drugs and mixture of siRNAs specifically to lung cancer cells and, as a result, efficient suppression of tumor growth and prevention of adverse side effects on healthy organs.     

LyP-1-conjugated PEGylated liposomes: A carrier system for targeted therapy of lymphatic metastatic tumor

The application of liposomes in targeted therapy of lymphatic metastatic tumors has been hampered by the low uptake rate of liposome by metastatic lymph nodes. In this report, LyP-1, a peptide that can specifically bind tumor cells, tumor lymphatics and tumor-associated macrophages, was conjugated to liposomes for targeting and treating lymphatic metastatic tumors. Firstly, LyP-1-conjugated PEGylated liposomes loaded with fluorescein or doxorubicin (DOX) were prepared and showed satisfactory vesicle size and size distribution. The in vitro cellular uptake and in vivo near-infrared fluorescence imaging results showed that LyP-1 modification increased liposome uptake by tumor cells and metastatic lymph nodes, but did not increase uptake by normal lymph nodes. The immunofluorescence analysis evidenced that LyP-1-conjugated liposomes were distributed adjacent to tumor lymphatics and tumor-associated macrophages in metastatic lymph nodes. The pharmacodynamic study suggested that compared with unmodified liposomes, LyP-1-conjugated DOX-loaded liposomes exhibited enhanced inhibition effect on tumor cells in vitro and lymphatic metastatic tumors in vivo. Pathological examination showed that liposomal DOX caused reduced tissue damage to injection site compared with DOX solution. In summary, LyP-1-conjugated PEGylated liposomes could be targeted to metastatic lymph nodes based on their specific binding to tumor cells, tumor lymphatics and tumor-associated macrophages. They are a safe and effective drug delivery system of antineoplastic agents for targeted therapy of lymphatic metastatic tumors.     

CD10/NEP in non-small cell lung carcinomas. Relationship to cellular proliferation.

The cell surface metalloproteinase CD10/neutral endopeptidase 24.11 (NEP) hydrolyzes a variety of peptide substrates and reduces cellular responses to specific peptide hormones. Because CD10/NEP modulates peptide-mediated proliferation of small cell carcinomas of the lung (SCLC) and normal fetal bronchial epithelium, we evaluated the enzyme's expression in non-small cell lung carcinomas (NSCLC). Bronchoalveolar and large cell carcinoma cell lines had low levels of CD10/NEP expression whereas squamous, adenosquamous, and adenocarcinoma cell lines had higher and more variable levels of the cell surface enzyme. Regional variations in CD10/NEP immunostaining in primary NSCLC specimens prompted us to correlate CD10/NEP expression with cell growth. In primary carcinomas of the lung, clonal NSCLC cell lines and SV40-transformed fetal airway epithelium, subsets of cells expressed primarily CD10/NEP or the proliferating cell nuclear antigen (PCNA). Cultured airway epithelial cells had the lowest levels of CD10/NEP expression when the highest percentage of cells were actively dividing; in addition, these cells grew more rapidly when cell surface CD10/NEP was inhibited. NSCLC cell lines had receptors for a variety of mitogenic peptides known to be CD10/NEP substrates, underscoring the functional significance of growth-related variability in CD10/NEP expression.