Effects of bombesin on human small cell lung cancer cells: evidence for a subset of bombesin non-responsive cell lines

The effects of bombesin on three human small cell lung carcinoma cell (SCLC) lines (NCI-H69, NCI-H128, and NCI-H345) have been examined and compared to the effects of the peptide on the mouse fibroblast cell line Swiss 3T3, and the rat pituitary tumor cell line GH3W5. While all three SCLC lines expressed messenger RNA encoding pro-gastrin releasing peptide (GRP), only the NCI-H345 cells expressed detectable membrane receptors for GRP and responded to nanomolar concentrations of bombesin as shown by 125I-GRP binding, total inositol phosphate accumulation, and increased clonal growth in soft agarose. These data show that some SCLC lines are insensitive to bombesin and do not express detectable membrane receptors for GRP.     

Extracellular matrix proteins protect small cell lung cancer cells against apoptosis: a mechanism for small cell lung cancer growth and drug resistance in vivo.

Resistance to chemotherapy is a principal problem in the treatment of small cell lung cancer (SCLC). We show here that SCLC is surrounded by an extensive stroma of extracellular matrix (ECM) at both primary and metastatic sites. Adhesion of SCLC cells to ECM enhances tumorigenicity and confers resistance to chemotherapeutic agents as a result of beta1 integrin-stimulated tyrosine kinase activation suppressing chemotherapy-induced apoptosis. SCLC may create a specialized microenvironment, and the survival of cells bound to ECM could explain the partial responses and local recurrence of SCLC often seen clinically after chemotherapy. Strategies based on blocking beta1 integrin-mediated survival signals may represent a new therapeutic approach to improve the response to chemotherapy in SCLC.     

CCK antagonists interact with CCK-B receptors on human small cell lung cancer cells

The ability of cholecystokinin (CCK) receptor antagonists to interact with CCK receptors in small cell lung cancer (SCLC) cells was investigated. L-365,260, CCK-8, L-364,718, CBZ-CCK(27-32)-NH2 and proglumide analogue 10 inhibited specific 125I-CCK-8 binding to SCLC cells with IC50 values of 0.2, 2, 500, 100,000 and 500,000 nM, respectively. Gastrin-I and CCK-8 elevated the cytosolic Ca2+ when SCLC cells were loaded with Fura 2-AM. L-365,260 inhibited the cytosolic Ca2+ increase caused by 10 nM CCK-8 in a dose-dependent manner. The effects of 10 nM L-365,260 were reversed by high concentrations of CCK-8. These data indicate that L-365,260 functions as a reversible CCK-8 antagonist using SCLC cells.     

A new small cell lung cancer (SCLC)-specific marker discovered through antigenic subtraction of neuroblastoma cells

Small cell lung cancer (SCLC) is an aggressive form of lung cancer associated with cigarette smoking and presently accounts for approximately 20% of all lung cancer cases. SCLC cells derive from a neuroendocrine origin and therefore their antigenic profile coincides, to a great extent, with that of neuroendocrine cells. Multiple attempts to generate SCLC-specific MoAbs during the past decade have failed because all SCLC-specific MoAbs isolated also react against neuroendocrine tissues or normal immune cells. Cross-reactivity with normal antigens raises safety concerns due to the inevitable toxicity of such interactions and the dreaded effects. The concept of DIAAD trade mark ( Differential Immunization for Antigen and Antibody Discovery) provides for an immune response that can be effectively focused on cancer antigens. The object is to overcome obstacles resulting from an antigenic hierarchical pattern biased towards a response to dominant antigens in order to induce a robust immune response to cancer antigens. Cancer antigens are weak or nonimmunogenic molecules. Due to the fact that the immune system responds more strongly to immunodominant antigens than to weak immunogenic antigens, cancer cell proliferation is unencumbered. DIAAD employs protocols of induction of tolerance and immunity, conducted in sequential order to "biologically subtract" the immune response of dominant antigens expressed by normal cells. This biological subtraction is achieved in a laboratory animal by first eliminating the immune response to the normal cells or closely related cancer cells, followed by immunization of the same laboratory animal with diseased cells. This procedure directs the immune response exclusively towards antigens expressed by the diseased and not the normal cells. Our objective was to use DIAAD to generate monoclonal antibodies specific to SCLC antigens that are not shared by neuroendocrine cells by contrasting a pool of human SCLC cell lines with a pool of human neuroendocrine cancer cell lines. Four monoclonal antibodies reacted strongly and exclusively with SCLC cells and identified a membrane molecule comprising a single chain glycoprotein. Two of four antibodies were selected for a detailed analysis that revealed a narrow tissue specificity of antigen expressed by colon, lung, and pancreatic cancers (less than 20% staining was found on breast, ovarian and prostate cancer). These antibodies did not bind to various other cancers such as kidney, carcinoid, lymphoma, sarcoma, adrenal, liver, melanoma, seminoma, leiomyoma, basal cell cancer, or undifferentiated cancer. The epitope recognized by the selected MoAbs was destroyed with the removal of carbohydrates from SCLC cells. This result does not exclude the possibility of protein-carbohydrate cooperation in epitope recognition. However, it strongly suggests the pivotal role of carbohydrates in antibody binding to this molecule. Upon binding to the extracellular molecule on SCLC cells, the antibodies were shown to internalize. A low or insignificant level of internalization was recorded following incubation of the antibodies with neuroendocrine-derived tumors. The capacity of these antibodies to internalize upon binding the extracellular receptors renders them potential candidates for prodrug or immunotoxin-targeted therapeutics. In a qualitative experiment involving immunoaffinity purification, the SCLC antigen was shown to be differentially detected in sera of SCLC patients. Plans are being generated to explore the possible utility of this novel SCLC-specific antigen recognized by the above MoAbs as a new biomarker for early diagnosis of the disease, as well as for therapeutic intervention for SCLC.     

Neurotensin elevates cytosolic calcium in small cell lung cancer cells

The ability of neurotensin (NT) to elevate cytosolic Ca²⁺ in small cell lung cancer (SCLC) cells was investigated using the fluorescent Ca²⁺ indicator Fura 2-AM. Using SCLC cell line NCI-H345, NT elevated cytosolic Ca²⁺ levels in a concentration-dependent manner. Using a 10 nM dose, NT and C-terminal fragments such as NT(8–13) but not N-terminal fragments such as NT(1–8) elevated the cytosolic Ca²⁺ levels. Because EGTA (5 mM) did not affect the NT response, NT may cause release of Ca²⁺ from intracellular stores. These data indicate that SCLC NT receptors may use Ca²⁺ as a second messenger.     

Cholecystokinin elevates cytosolic calcium in small cell lung cancer cells

The ability of cholecystokinin (CCK) to elevate intracellular Ca2+ levels in small cell lung cancer cells was investigated using the fluorescent Ca2+ indicator Fura 2. CCK-8 elevated the cytosolic Ca2+ levels in cell line NCI-H345 in a dose dependent manner. Nanomolar concentration of CCK-8 elevated cytosolic Ca2+ levels in the absence or presence of extracellular Ca2+. Potent CCK agonists such as gastrin-1 and nonsulfated CCK-8 but not inactive compounds such as CCK-27-32-NH2 elevated cytosolic Ca2+ levels. These data suggest that CCK receptors may regulate the release of Ca2+ from intracellular organelles in small cell lung cancer cells.     

Vasopressin elevates cytosolic calcium in small cell lung cancer cells

The ability of vasopressin to elevate cytosolic Ca²⁺ in small cell lung cancer (SCLC) cells was investigated. Ten nanomolar vasopressin elevated the cytosolic Ca²⁺ in 6 of 8 SCLC cell lines that were loaded with Fura-2 AM. Using SCLC cell line NCI-H345, the effect of vasopressin was dose dependent, being maximal at 100 nM, where the cytosolic Ca²⁺ was elevated from 150 to 210 nM. Because addition of 1 mM EGTA had no effect on the vasopressin response, vasopressin released Ca²⁺ from intracellular pools. Also, oxytocin weakly elevated the cytosolic Ca²⁺. The response to vasopressin was strongly blocked by [(β-mercapto-β,β-cyclopentamethylene propionic acid)¹,O-MeTyr²,Arg⁸]vasopressin and weakly blocked by [(β-mercapto-β,β-cyclopentamethylene propionic acid)¹,O-MeTyr²,Orn⁸]vasotocin. These data suggest that V₁ vasopressin receptors are present on SCLC cells.     

Characterization of glycoprotein ligands for P-selectin on a human small cell lung cancer cell line NCI-H345.

P-selectin (CD62P) is a cell adhesion molecule expressed on stimulated endothelial cells and on activated platelets. It interacts with PSGL-1 (P-selectin glycoprotein ligand-1; CD162) on leukocytes and mediates recruitment of leukocytes during inflammation. P-selectin also binds to several types of cancer cells in vitro and facilitates growth and metastasis of colon carcinoma in vivo. Here we show that P-selectin, but not E-selectin, binds to NCI-H345 cells, a cell line derived from a human small cell lung cancer. EDTA or P7 (a leukocyte adhesion blocking mAb to P-selectin), but not PL5 (a leukocyte adhesion blocking mAb to PSGL-1), can inhibit this binding. P-selectin affinity chromatography can precipitate a approximately 110-kDa major band and a approximately 220-kDa minor band from [3H]-glucosamine-labeled NCI-H345 cells. No expression of PSGL-1 protein and mRNA can be detected in NCI-H345 cells. Taken together, these results suggest that NCI-H345 cells express glycoprotein ligands for P-selectin that are distinct from leukocyte PSGL-1.     

Bombesin-related peptides induce calcium mobilization in a subset of human small cell lung cancer cell lines

To examine the biochemical basis for growth factor-induced responses in human lung cancer cells, we used the quin2 technique to study the effect of the amphibian peptide bombesin and its congeners including mammalian gastrin-releasing peptide (GRP) on the intracellular free calcium level [Ca2+]i in small cell lung cancer cell lines. In five of eleven cell lines tested, Tyr4-bombesin or GRP elicited a rapid and transient increase in [Ca2+]i. The response was seen with as little as 1 nM ligand, was not affected by membrane depolarization, and derived in part from internal calcium stores. Desensitization to a second addition of active bombesin congeners occurs subsequent to initial addition of Tyr4-bombesin. Structure-activity analysis showed the carboxyl-terminal octapeptide was the active portion of the peptide. Analogs in which the carboxyl terminus was oxidized or deamidated were inactive. Ranatensin, litorin, alytesin, and GRP, but not physalaemin, were as active as Tyr4-bombesin. A monoclonal antibody to the carboxyl terminus of bombesin selectively blocked the increased [Ca2+]i elicited by Tyr4-bombesin. These studies suggest that bombesin congeners can act on some small cell lung cancer cell lines by a pathway utilizing increased [Ca2+]i.     

Sigma ligands inhibit the growth of small cell lung cancer cells

The effects of sigma ligands on small cell lung cancer (SCLC) cells were investigated. 125I-N-(2-(piperidino)ethyl)-2-iodobenazmide (2-IBP) bound with high affinity to SCLC cell line NCI-H209 and NCI-N417. Specific 125I-2-IBP binding was inhibited with high affinity by ifendipine, haloperidol, (2-piperidinyl-aminoethyl)-4-iodobenzamide (IPAB) and 1,3-ditolylguanidine (DTG) with IC50 values of 3, 10, 15 and 90 nM respectively. In vitro, 10 microM 2-IBP, haloperidol or IPAB inhibited NCI-N417 proliferation using a MTT or clonogenic assay. In vivo, 4 mg/kg IPAB or 2-IBP inhibited NCI-N417 xenograft proliferation. 125I-2-IBP localized to the SCLC tumors after subcutaneous injection. These results suggest that sigma ligands may be utilized to localize and inhibit the proliferation of SCLC tumors.     

Markers of small cell lung cancer

Lung cancer is the number one cause of cancer death; however, no specific serum biomarker is available till date for detection of early lung cancer. Despite good initial response to chemotherapy, small-cell lung cancer (SCLC) has a poor prognosis. Therefore, it is important to identify molecular markers that might influence survival and may serve as potential therapeutic targets. The review aims to summarize the current knowledge of serum biomarkers in SCLC to improve diagnostic efficiency in the detection of tumor progression in lung cancer. The current knowledge on the known serum cytokines and tumor biomarkers of SCLC is emphasized. Recent findings in the search for novel diagnostic and therapeutic molecular markers using the emerging genomic technology for detecting lung cancer are also described. It is believed that implementing these new research techniques will facilitate and improve early detection, prognostication and better treatment of SCLC.     

Camptothecin-somatostatin conjugates inhibit the growth of small cell lung cancer cells

The effects of camptothecin-somatostatin (CPT-SS) conjugates were investigated on small cell lung cancer (SCLC) cells. CPT was coupled to a SS agonist (SSA), c(Cys-Phe-DTrp-Lys-Thr-Cys)Thr-NH2 using the built in nucleophile assisted-releasing group (L1) N-methyl-aminoethyl-Gly-Dser-Nle-Dtyr-Dser or (L2) aminoethyl-Gly-Dser-Nle-Dtyr-Dser. The resulting CPT-L1-SSA and CPT-L2-SSA inhibited the specific binding of [125I-Tyr11]SS to NCI-H69 cell membranes with IC50 values of 0.2 and 2.1 nM, respectively. [125I]CPT-L1-SSA was internalized by SCLC cells at 37 degrees C but not at 4 degrees C. CPT-L1-SSA and CPT-L2-SSA inhibited in a dose-dependent manner the increase in adenylylcyclase activity caused by 25 microM forskolin. In vitro, 0.3 microM CPT-L1-SSA half-maximally inhibited the clonal growth of SCLC cells and 1 microM CPT-L1-SSA strongly inhibited 3H-thymidine incorporation into DNA and trypan-blue exclusion. These results suggest that CPT conjugated peptides such as CPT-L1-SSA may prove useful for exploring the efficacy of receptor-directed cytotoxicity to inhibit the proliferation of SCLC cells.     

High affinity binding of cholecystokinin to small cell lung cancer cells

The binding of 125I-Bolton Hunter-cholecystokinin octapeptide (125I-BH-CCK-8) to small cell lung cancer cell lines was investigated. 125I-BH-CCK-8 bound with high affinity (Kd = 2.4 nM) to an apparent single class of sites (1700/cell) using cell line NCI-H209. Binding was time dependent and the ratio of specific/nonspecific binding was 8/1. Pharmacology studies indicated that gastrin, caerulein, CCK-33 and nonsulfated CCK-8 were potent inhibitors of specific 125I-BH-CCK-8 binding whereas CCK-26-32-NH2 was not. Because CCK receptors are present on small cell lung cancer cells, CCK may function as a regulatory peptide in this disease.     

Targeting Stealth liposomes in a murine model of human small cell lung cancer.

Tumor accumulation and therapeutic activity of Stealth liposomes loaded with doxorubicin (DXR) were examined in Balb/c nude mice xenografts inoculated subcutaneously with the human small cell lung cancer (SCLC) cell line, H69. Mice were treated with non-targeted liposomes (SL) or liposomes targeted with antagonist G coupled to the liposome surface (SLG). SLG showed 30-44-fold higher binding to H69 cells harvested from H69 xenografts than SL. At 48 and 72 h post injection, tumor accumulation of [(125)I]tyraminylinulin-containing liposomes was shown to be dependent on liposome size but independent of the presence of the targeting ligand. Maximum tumor uptake of either SLG or SL ranged from 2 to 4% of injected dose/g of tissue. In therapeutic studies, mice received three weekly injections of 3 or 6 mg free DXR/kg or 3 or 10 mg liposomal DXR/kg at initial tumor volumes of either 7 or 33 mm(3). The therapeutic efficacy of DXR-containing SL or SLG was significantly improved over free DXR, but SLG did not improve anti-tumor efficacy relative to SL. Stealth liposomes containing DXR have potential as a therapy against human SCLC tumors.     

Serum markers in small cell lung cancer: Opportunities for improvement

Lung cancer is one of the leading causes of death from malignancy worldwide. In particular small cell lung cancers, which comprise about 15–20% of all lung cancers, are extremely aggressive and cure rates are extremely low. Therefore, new treatment modalities are needed and detection at an early stage of disease, as well as adequate monitoring of treatment response is essential in order to improve outcome. In this respect, the use of non-invasive tools for screening and monitoring has gained increasing interest and the clinical applicability of reliable, tumor-related substances that can be detected as tumor markers in easily accessible body fluids is subject of intense investigation. Some of these indicators, such as high LDH levels in serum as a reflection of the disease, have been in use for a long time as a general tumor marker. To allow for improved monitoring of the efficacy of new therapeutic modalities and for accurate subtyping, there is a strong need for specific and sensitive markers that are more directly related to the biology and behavior of small cell lung cancer. In this review the current status of these potential markers, like CEA, NSE, ProGRP, CK-BB, SCC, CgA, NCAM and several cytokeratins will be critically analyzed with respect to their performance in blood based assays. Based on known cleavage sites for cytoplasmic and extracellular proteases, a prediction of stable fragments can be obtained and used for optimal test design. Furthermore, insight into the synthesis of specific splice variants and neo-epitopes resulting from protein modification and cleavage, offers further opportunities for improvement of tumor assays.