substance-p--phe(5)-trp(7-9)-leu(11)- and Lung-Neoplasms

It is known that in human lung cancer samples, both small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) cells express the neurokinin-1 (NK-1) receptor; that after binding to the NK-1 receptor the peptide substance P (SP) elicits tumour cell proliferation and an antiapoptotic effect. By contrast, it has been demonstrated that non-peptide NK-1 receptor antagonists, after binding to the NK-1 receptor and hence by blocking the SP action in SCLC/NSCLC, exert an antiproliferative action (inhibit lung cancer cell proliferation and induce the death of tumour cells by apoptosis). It is also known that SP peptide NK-1 receptor antagonists also called SP analogue antagonists (broad-spectrum GPCR antagonists, broad-spectrum neuropeptide antagonists or synthetic analogues of SP), also exert antiproliferative actions against SCLC/NSCLC. However, the underlying mechanisms involved in this antiproliferative action remain unknown. By using competition assays with SP, here we demonstrate that the antiproliferative action exerted by the [D-Arg(1), D-Phe(5), D-Trp(7,9), Leu(11)] SP analogue on human H-69 SCLC and COR-L23 NSCLC cell lines, occurs at least through the NK-1 receptor.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Cell Line, Tumor; Cell Proliferation; Humans; Lung Neoplasms; Neurokinin-1 Receptor Antagonists; Receptors, Neurokinin-1; Substance P

[D-Arg1,D-Phe5,D-Trp7,9,Leu11]Substance P is a broad-spectrum neuropeptide growth factor antagonist that has exhibited in vitro activity against a range of human cancer cell lines. The fate of this compound in vivo following i.p. administration at 12 micrograms/g to nu/nu mice bearing the H69 small-cell lung cancer xenograft has been studied. Metabolism was confined to the C-terminus producing [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P acid and [D-Arg1,D-Phe5,D-Trp7,9]substance P(1-10). The peptide had a long half-life in plasma (45.9 min) and became widely distributed among the tissues studied with the highest accumulation observed in the liver (AUC 1102 micrograms/g x min) and the lowest in the brain (5 micrograms/g x min). Uptake into the tumor xenograft was poor (AUC 189 micrograms/g x min); however, uptake into the lungs was much greater (AUC 507 micrograms/g x min), offering encouragement that therapeutic concentrations may be targeted to primary lung tumors.

Topics: Animals; Antineoplastic Agents; Carcinoma, Small Cell; Female; Half-Life; Humans; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Transplantation; Substance P; Tissue Distribution; Transplantation, Heterologous; Tumor Cells, Cultured

Small cell lung cancer (SCLC) cell growth is sustained by multiple autocrine and paracrine growth loops involving neuropeptides. The bombesin family of peptides are autocrine growth factors in H345 SCLC cells and provide a paradigm for the study of growth factors and mitogenic signaling in SCLC cells. We show that bombesin (and other neuropeptides) stimulates protein tyrosine phosphorylation (particularly focal adhesion kinase) and protein tyrosine kinase (PTK) activity in intact SCLC cells. Furthermore, the broad spectrum neuropeptide receptor antagonist [D-Arg, D = Phe, D-Trp, Leu11]substance P inhibits all neuropeptide-mediated signals (including PTK activation), SCLC cell growth in vivo and in vitro, and also increases the natural rate of apoptosis seen in growing SCLC cell lines. Hence the effect of selective PTK inhibition on SCLC cell growth and apoptosis was examined. We show that selective inhibition of PTK activity, with genistein and (3,4,5-tri-hydroxyphenyl)-methylene(-propanedinitrile) tyrphostin-25 inhibits basal and neuropeptide-stimulated SCLC cell growth. Genistein and tyrphostin-25 also stimulate apoptosis in SCLC cells. Inhibition of proliferation in these cells is intimately linke to apoptosis, because these changes occurred without any effect on SCLC cell cycle kinetics, suggesting that apoptosis occurs independently of the cell cycle and that failure to progress through the cell cycle results in apoptosis. Because tyrphostin-25 fails to influence p53 or Bcl-2 expression in these cells, this mode of programmed cell death appears to be via a p53- and Bcl-2-independent mechanism. These results provide evidence that tyrosine phosphorylation is a mitogenic signal in SCLC cells and suggest that regulation of the level of protein tyrosine phosphorylation represents a critical determinant of whether SCLC cells survive and proliferate or die by apoptosis. Thus PTK inhibition may provide a novel therapeutic option in SCLC that has become resistant to conventional chemotherapeutic agents.

Topics: Apoptosis; Bombesin; Carcinoma, Small Cell; Cell Adhesion Molecules; Cell Cycle; Cell Division; Enzyme Activation; Enzyme Inhibitors; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Genistein; Humans; Isoflavones; Kinetics; Lung Neoplasms; Necrosis; Neuropeptides; Nitriles; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Receptors, Neuropeptide; Substance P; Time Factors; Tumor Cells, Cultured; Tyrphostins

The broad spectrum antagonist [D-Arg1, D-Phe5, D-Trp7,9, Leu11] substance P has been shown previously to inhibit the growth of small cell lung cancer cells both in vitro and in vivo. To elucidate further the pathways involved in the growth inhibitory actions of [D-Arg1, D-Phe5, D-Trp7,9, Leu11] substance P we have examined the effect of this agent on cell viability and the induction of apoptosis in small cell and non-small cell lung cancer cells. Treatment of lung tumor cells with [D-Arg1, D-Phe5, D-Trp7,9, Leu11] substance P caused a concentration-dependent loss of cell viability which was accompanied by the onset of apoptosis, as defined by cytological criteria and DNA fragmentation. This effect occurred in both small cell and non-small cell lung cancer cells and was not dependent on de novo protein synthesis. Such findings indicate that the antiproliferative action of [D-Arg1, D-Phe5, D-Trp7,9, Leu11] substance P involves a signal transduction pathway for apoptosis.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Cell Line; Cell Survival; DNA Damage; DNA, Neoplasm; Flow Cytometry; Humans; Kinetics; Lung Neoplasms; Substance P; Time Factors; Tumor Cells, Cultured

Analogues of the neurotransmitter substance P (SP) can interact with neuropeptide receptors, and are reported to inhibit growth of small cell lung cancer cell lines (SCLC CLs). We found [D-Arg1, D-Phe5, D-Trp7,9, Leu11] substance P (D-Phe5SP) significantly inhibited DNA synthesis by 10/10 human tumour CLs; six SCLC, one N-SCLC (squamous), two ovarian and one squamous cervical carcinoma, with inhibition to 50% control levels (IC50) of 20-50 microM. There was dose dependent inhibition of colony forming efficiency (CFE) in 3/3 SCLC and 1/1 N-SCLC CL, IC50s of 0.5-6.5 microM in 5% serum. Exposure of SCLC CL HC12 to 100 microM D-Phe5SP for 1-4 h caused a progressive fall in viable cell number; surviving cells, grown in the absence of peptide, showed a decreased growth rate. During 1 week's exposure of two SCLC CLs to 20 microM D-Ph5SP, growth was slower than control cultures, while 50-100 microM completely inhibited growth. These inhibitory effects were partially reversed by increasing serum concentration from 5 to 20%, but not by SP, vasopressin, bombesin or insulin-like growth factor 1. There was some inhibition of CFE by 3/3 normal human bone marrows, IC50s of 30-80 microM, compared with 8 microM for HC12 in 20% FCS. Therefore D-Phe5SP appears to have more potent antiproliferative effects in tumour cells than normal cells, suggesting a role for this analogue in tumour treatment.

Topics: Analysis of Variance; Antineoplastic Combined Chemotherapy Protocols; Bombesin; Bone Marrow; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Cell Division; Cell Line; Colony-Forming Units Assay; DNA; Dose-Response Relationship, Drug; Female; Fibroblasts; Humans; In Vitro Techniques; Insulin-Like Growth Factor I; Lung Neoplasms; Ovarian Neoplasms; Substance P; Time Factors; Vasopressins

The proliferation of small cell lung cancer (SCLC) cells appears sustained by multiple autocrine and paracrine circuits involving Ca2+ mobilizing neuropeptides. Consequently, broad spectrum neuropeptide antagonists which inhibit SCLC growth in vitro have been suggested as potential anticancer agents. Here we evaluated this hypothesis using xenografts of WX322 cells, a SCLC cell line that responds to multiple Ca2+ mobilizing neuropeptides. The broad spectrum neuropeptide antagonists [Arg6,D-Trp7,9,MePhe8]substance P(6-11) and [D-Arg1,D-Phe5,Trp7,9Leu11[substance P were shown to inhibit the growth of WX322 xenografts in nude mice. Similar results were obtained with xenografts of the SCLC cell line H69. The results indicate that broad spectrum neuropeptide antagonists can inhibit the growth of SCLC in vivo and suggest that these antagonists could be useful in the treatment of SCLC.

Topics: Animals; Calcium; Carcinoma, Small Cell; Drug Screening Assays, Antitumor; Female; Humans; Lung Neoplasms; Mice; Mice, Nude; Peptide Fragments; Signal Transduction; Substance P; Tumor Cells, Cultured

In the search for novel antiproliferative agents for small cell lung cancer (SCLC), we found the neuropeptide antagonist [Arg6, D-Trp7,9,MePhe8]substance P(6-11) to be effective in vitro. In murine Swiss 3T3 cells [Arg6,D-Trp7,9,MePhe8]substance P(6-11) was identified as a potent inhibitor of vasopressin-stimulated DNA synthesis which also blocks [3H]vasopressin binding to specific cell-surface receptors. It was a less potent antagonist of gastrin-releasing peptide and bradykinin in these cells but did not block the effects of other mitogens. In SCLC cell lines, [Arg6,D-Trp7,9,MePhe8]substance P(6-11) inhibited colony-formation in soft agarose and growth in liquid culture in a dose-dependent manner. It also blocked receptor-mediated Ca2+ mobilization induced by vasopressin, bradykinin, cholecystokinin, galanin, gastrin-releasing peptide, and neurotensin. We suggest that broad-spectrum neuropeptide antagonists can block multiple autocrine and paracrine growth loops in SCLC and could be useful therapeutic agents.

Topics: Animals; Bradykinin; Calcium; Carcinoma, Small Cell; Cell Division; Cells, Cultured; DNA, Neoplasm; Drug Screening Assays, Antitumor; Gastrin-Releasing Peptide; Humans; Lung Neoplasms; Mice; Peptide Fragments; Peptides; Recombinant Proteins; Substance P; Tumor Cells, Cultured; Vasopressins

In the search for a more potent bombesin antagonist, we found [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P to be effective in mouse fibroblasts and to inhibit the growth of small cell lung cancer, a tumor that secretes bombesin-like peptides that may act as autocrine growth factors. In murine Swiss 3T3 cells, [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P proved to be a bombesin antagonist as judged by the following criteria: (i) inhibition of DNA synthesis induced by gastrin-releasing peptide and other bombesin-like peptides; (ii) inhibition of 125I-labeled gastrin-releasing peptide binding to the bombesin/gastrin-releasing peptide receptor; (iii) reduction in cross-linking of the Mr 75,000-85,000 protein putatively a component of the bombesin/gastrin-releasing peptide receptor; (iv) blocking of early cellular events that precede mitogenesis--calcium mobilization and inhibition of epidermal growth factor binding. [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P was 5-fold more potent than the antagonist [D-Arg1,D-Pro2,D-Trp7,9,Leu11]substance P. [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P also inhibits mitogenesis induced by vasopressin but not that induced by a variety of other mitogens. Both antagonists reversibly inhibited the growth of small cell lung cancer in vitro in a concentration-dependent manner. Peptide antagonists could, therefore, have far-reaching therapeutic implications.

Topics: Amino Acid Sequence; Animals; Binding, Competitive; Bombesin; Carcinoma, Small Cell; DNA Replication; Epidermal Growth Factor; Gastrin-Releasing Peptide; Lung Neoplasms; Mice; Mitosis; Molecular Weight; Peptides; Substance P; Tumor Cells, Cultured