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.     

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.     

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.     

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.     

Bombesin-like peptides elevate cytosolic calcium in small cell lung cancer cells

The ability of bombesin-like peptides to elevate intracellular Ca2+ levels in small cell lung cancer cells was investigated using the fluorescent Ca2+ indicator Fura 2. Nanomolar concentrations of bombesin elevated cytosolic Ca2+ levels in the absence or presence of extracellular Ca2+. Potent bombesin receptor agonists, such as gastrin releasing peptide (GRP) or (GRP)14-27 elevated cytosolic Ca2+ levels whereas inactive compounds such as (D-Trp8)bombesin or (GRP)1-16 did not. Furthermore, the bombesin receptor antagonist (D-Arg1, D-Pro2, D-Trp7,9, Leu11) substance P (30 microM) had no effect on the Ca2+ levels by itself but antagonized the increase in Ca2+ caused by 10 nM or 100 nM bombesin. These data suggest that bombesin receptors may regulate the release of Ca2+ from intracellular organelles in small cell lung cancer cells.     

SR48692 is a neurotensin receptor antagonist which inhibits the growth of small cell lung cancer cells

Neurotensin (NT) is an autocrine growth factor for some small cell lung cancer (SCLC) cells. In this communication, the effects of a non-peptide NT receptor antagonist, SR48692, were investigated using SCLC cells. (3)H-SR48692 bound with high affinity (IC(50) = 20 nM) to NCI-H209 cells. Also, NT and SR48692 inhibited specific (125)I-NT binding with high affinity (IC(50) values of 2 and 200 nM). In contrast, the NT(2) receptor agonist, levocabastine, had little effect on specific (125)I-NT binding, second messenger production and proliferation using NCI-H209 cells. SR48692 (5 microM) antagonized the ability of NT (10 nM) to cause elevated cytosolic Ca2+ in Fura-2 AM loaded NCI-H209 cells. SR48692 antagonized the ability of NT to cause elevation of c-fos mRNA in these cells. Using a MTT proliferation assay, SR48692 inhibited NCI-H209 and H345 proliferation in a concentration-dependent manner. Using a clonogenic assay, 1 microM SR48692, reduced NCI-H209 colony number. Also, SR48692 (0.4 mg/kg per day) inhibited NCI-H209 xenograft proliferation in nude mice. These results suggest that SR48692 is a NT(1) receptor antagonist which inhibits SCLC growth.     

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.     

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.     

Chemotherapy of small cell lung cancer

The author summarizes the most recent information on small cell lung cancer. Reviews the epidemiology, prognostic markers and stages of small cell lung cancer Details the more frequently used combined therapeutic modalities, the criteria of the optimal therapeutic approaches and the achieved remission rates. Based on the most recent data, summarizes the new chemotherapeutic agents and their most effective combinations, the second line treatment and immunotherapy. Finally tries to answer the most frequently asked questions.     

Regulation of cholecystokinin secretion from a rat medullary thyroid carcinoma cell line: role of calcium, cyclic nucleotides, glucocorticoids, neurotensin, and calcitonin gene-related peptide.

CCK-secreting WE rat medullary thyroid carcinoma cell line resembles other calcitonin-producing (C-cell) lines in that calcium, cAMP, or agents which raise cAMP, dexamethasone, and beta-adrenergic agents all stimulate peptide secretion. Unlike other C-cell lines, the WE cells respond similarly to IBMX (3-isobutyl-1-methyl-xanthine, a phosphodiesterase inhibitor) in the presence and absence of forskolin, implying that these cells secrete substances that raise cAMP levels, whose effect is accentuated by IBMX. Both CGRP and neurotensin, peptides that may be secreted by these cells, caused a small, but significant, increase in CCK secretion. It is possible that these or other secreted substances that activate adenylate cyclase are responsible for the cell's high rate of CCK secretion. Their high rate of CCK synthesis and their regulated secretion suggest that these cells will be a good model for studies of CCK expression, biosynthesis, and processing.     

SCLC-J1, a novel small cell lung cancer cell line

Small cell lung cancer (SCLC) is a type of high-grade neuroendocrine carcinoma. It initially responds to chemotherapy but rapidly becomes chemoresistant and it is highly proliferative. The prognosis in SCLC is poor. We have established a novel SCLC cell line, SCLC-J1, from a malignant pleural effusion in a patient with advanced SCLC. SCLC-J1 cells express ganglioside GD2, CD276, and Delta-like protein 3. RB1 is lost.These features of the new SCLC cell line may be useful in understanding the cellular and molecular biology of SCLC and in designing better treatment.     

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.     

Multiple gastrin-releasing peptide gene-associated peptides are produced by a human small cell lung cancer line

Products of the gastrin-releasing peptide gene were isolated from culture medium supernatant of a small cell lung cancer line, NCI-H345, by several (high performance liquid chromatography) HPLC steps. The column eluates were monitored by immunoassay and absorbance profiles. Gastrin-releasing peptide was identified in HPLC eluates by a specific radioimmunoassay. Two carboxyl-terminal gastrin-releasing peptide gene-associated peptides were identified by a radioimmunoassay specific for their predicted carboxyl terminus. The amino termini of these two peptides were determined by microsequence analysis. The shorter peptide was revealed to be a fragment of the larger peptide. Expression of an alternate mRNA was shown by isolation and characterization of a novel tetradecapeptide. Amino acid analysis, microsequence analysis, and mass spectral analysis confirmed that the structure was Ser-Leu-Leu-Gln-Val-Leu-Asn-Val-Lys-Glu-Gly-Thr-Pro-Ser. This peptide represents the carboxyl terminus of a peptide resulting from alternate processing of gastrin releasing peptide mRNA. This mRNA contains a 19-base deletion, creating a frame shift. A radioiodinated synthetic analog of this peptide (Tyr-Leu-Val-Asp-Ser-Leu-Leu-Gln-Val-Leu-Asn-Val-Lys-Glu-Gly-Thr-Pro-Ser ) bound specifically to a small cell cancer line with high affinity, suggesting possible biological activity of the isolated peptide.     

Plasma galanin, vasopressin, and oxytocin in patients with Addison's disease.

Galanin is colocalized with adrenocorticotrophin (ACTH) in the human pituitary and with corticotrophin releasing hormone, arginine, vasopressin, and oxytocin in the hypothalamus. Galanin, vasopressin, and oxytocin influence the secretion of pituitary ACTH. The aim of this study was to investigate if the endogenous stimulation of ACTH release in Addison's disease was reflected in plasma galanin, vasopressin, and oxytocin. ACTH, galanin, vasopressin, and oxytocin were measured in plasma from 14 patients with Addison's disease, one patient with Nelson's syndrome, and 14 healthy controls. Eight patients had elevated plasma ACTH whereas six patients and all controls had ACTH levels within the reference-range. There was no difference in galanin or vasopressin between patients and controls or between samples with low or high ACTH concentrations. In contrast, oxytocin was higher in patients with elevated plasma ACTH compared to patients and controls with normal or low ACTH. No relation was found between galanin or oxytocin and age or sex. A tendency towards lower vasopressin with increasing age was found among the men (p=0.057). The highest ACTH and galanin levels were found in the patient with Nelson's syndrome. In conclusion, increased plasma ACTH was not reflected in elevated plasma galanin or vasopressin. In contrast, elevated ACTH levels were accompanied by higher oxytocin levels.     

Oncogene amplification and chromosomal abnormalities in small cell lung cancer

Twelve cell lines isolated from patients with small cell lung cancer have been studied for amplification of the three characterised members of the myc proto-oncogene family (c-myc, N-myc, and L-myc) and for abnormalities of chromosome 3. Ten of these lines were being studied for the first time. Ten of the 12 small cell lung cancer cell lines had amplification of one member of the myc proto-oncogene family. Amplification of c-myc was observed in only one small cell lung line--a "morphological variant". One "classic" small cell lung cancer line expressed c-myc but had no obvious amplification of the gene. N-myc and L-myc were more commonly amplified than c-myc. Chromosomal abnormalities (mainly deletions) in chromosome 3 were observed in all small cell lung carcinoma cell lines examined. When the small cell lung carcinoma lines were grouped according to "classic" or "variant" characteristics, it was found that the "classics" had deletions of the short arm of chromosome 3, whereas the "biochemical variants" had deletions of the long arm of chromosome 3. The extent of the deletions varied between cell lines. For the deletion in the short arm of chromosome 3 the minimum common region of overlap was assigned to bands 3p23-3p24.     

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.