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Bradykinin Antagonist Dimer, CU201, Inhibits the Growth of Human Lung Cancer Cell Lines by a ‘‘Biased Agonist’’ Mechanism

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Bradykinin Antagonist Dimer, CU201, Inhibits the Growth of Human Lung Cancer Cell Lines by a ‘‘Biased Agonist’’ Mechanism Bradykinin antagonist dimer, CU201, inhibits the growth of human lung cancer cell lines by a ‘‘biased agonist’’ mechanism Daniel Chan*†‡, Lajos Gera*§, John Stewart*§, Barbara Helfrich*†, Marileila Verella-Garcia*†, Gary Johnson*¶, Anna Baron*ʈ, Jie Yang**, Theodore Puck*†,††, and Paul Bunn, Jr.*† *Lung Cancer Program, University of Colorado Cancer Center, the Departments of †Medicine, §Biochemistry, ¶Pharmacology, and ʈPreventive Medicine and Biostatistics, University of Colorado Health Sciences Center, Denver, CO 80262; and **National Jewish Research Center, and ††Eleanor Roosevelt Institute, Denver, CO 80206 Contributed by Theodore Puck, February 8, 2002 ␣ All small cell (SCLCs) and many non-small cell lung cancers (NSCLCs) kinase C- , calmodulin, and cell proliferation. Activation of G␣12,13 have neuroendocrine features including production of neuropeptides led to activation of c-Jun kinase and apoptosis (4, 12). and cell surface receptors creating autocrine and paracrine growth Some substance P derivatives (SPDs) were shown to inhibit loops. Neuropeptides bind to a family of 7-transmembrane receptors growth of several SCLC cell lines in vitro and in vivo (11, 13, 15, 16). and activate heterotrimeric G proteins consisting of G␣q and G␣12,13. These SPDs initially were reported to be broad-spectrum antago- Substance P derivatives (SPDs) induced apoptosis and inhibited nists, because they blocked the calcium response induced by growth of lung cancer cells by discoordinately inhibiting G␣q and multiple peptides of unrelated structures (5, 11, 15, 16). Subsequent ␣ stimulating G 12,13. However, these SPDs had low potency and short studies showed that SPDs inhibited G␣q activation while simulta- half-lives. In this report we show that a bradykinin antagonist dimer, neously stimulating G␣12,13 signaling (11, 16, 17). These compounds CU201, inhibited the growth of SCLC and NSCLC cell lines with or were termed ‘‘biased agonists,’’ because they inhibited proliferative without multidrug-resistant proteins and was 10-fold more potent signals and stimulated apoptotic signals. However, SPDs required with a longer plasma half-life than SPDs. Bradykinin agonists in either high concentrations to inhibit SCLC growth and did not inhibit the monomeric or dimeric form and monomeric bradykinin antagonist growth of NSCLCs. One of these SPDs, termed SPD-G, inhibited have no effect on lung cancer cell growth. The dimeric linking moiety the growth of SCLC tumors in nude mice and has completed phase of the two molecules was created, requiring a sufficient number of I testing with acceptable toxicity (18, 19). However, low potency and carbon chains to provide critical spacing between the two antago- a short half-life hinder further clinical development. These limita- 2؉ nists. CU201 inhibited intracellular Ca release in response to bra- tions led to the search for alternative compounds. dykinin, indicating blockage of the G␣q signal, and stimulated c-Jun The bradykinin (BK) receptor is expressed in most of the kinases, indicating stimulation of the G␣12,13 pathway. CU201-induced human lung cancer cell lines, and BK is one of the most potent apoptosis was preceded by unique changes in apparent nuclear DNA neuropeptides that causes intracellular calcium flux in these binding and by c-Jun kinase and caspase-3 activation. At the concen- SCLC and NSCLC cell lines (3). However, it shows only mod- tration at which CU201 inhibited the growth of the cancer cells, it had erate mitogenic effects. We discovered that a dimeric BK no effect on the growth of normal lung cells in vitro. CU201 and antagonist (CP-127, bradycor) and related dimers produced similar compounds offer hope of becoming a new form of targeted modest growth inhibition of SCLC cell lines at high concentra- therapy for tumors with neuroendocrine properties. tions (8). Subsequently, we synthesized a series of novel, potent BK antagonist monomers and dimers and have now shown that ung cancer is the leading cause of cancer death in men and one of the BK antagonist dimers, CU201, acts as a biased agonist, Lwomen in the U.S. (1). Small cell lung cancer (SCLC) comprises inhibits the growth of both SCLC and NSCLC cell lines by a 20% of lung cancer cases (2). Clinically, SCLCs are characterized unique mechanism, and has stability in serum. CU201 and by a propensity for early metastases and initial high response rates related compounds may be useful in the treatment of lung to chemotherapy and radiotherapy (2). Biologically, SCLCs are cancers and other cancers with neuroendocrine features. characterized by expression of neuroendocrine features including the production of neuropeptides coupled with expression of neu- Methods and Materials ͞ ropeptide receptors, creating autocrine paracrine growth loops Cell Lines and Culture Conditions. The NSCLC line NCI-H157, the (3–5). Many non-SCLCs (NSCLCs) and prostate, breast, and mesothelioma line H290, and the SCLC lines NCI-H345, -H69, gastrointestinal cancers also express peptide receptors and respond -H209, -H740, -H1048, -H510, and -H82 were provided by J. Minna to neuropeptide stimulation (6, 7). Autocrine growth stimulation by and A. Gazdar (University of Texas Southwestern Medical School, peptides led to the development of specific peptide inhibitors Dallas). The SCLC cell line SHP-77 was provided by A. Koros including monoclonal antibodies to growth factors or receptors and (University of Pittsburgh, Pittsburgh). The NSCLC line A549 and specific peptide antagonists (8–10). The low response rate to the normal lung fibroblast lines CCD-16LU and IMR90 were specific inhibitors was attributed to other peptides maintaining the obtained from the American Type Culture Collection. Cell lines autocrine loop when a single peptide was inhibited (8). were maintained in RPMI medium 1640 (GIBCO͞BRL Life Each peptide binds to cell surface receptors of appropriate Technologies, Grand Island, NY) supplemented with 10% FBS structure and activates a similar identical intracellular signaling pathway. This similarity created the possibility of developing com- pounds that would interfere with signals produced by multiple Abbreviations: SCLC, small cell lung cancer; NSCLC, non-SCLC; SPD, substance P derivative; BK, bradykinin; MTT, modified tetrazolium test; GRP, gastrin-releasing peptide; JNK, c-Jun peptides. In SCLC, each 7-transmembrane-spanning peptide re- N-terminal kinase. ceptor is coupled to both G␣q and G␣12,13 heterotrimeric G proteins ‡To whom reprint requests should be addressed at: University of Colorado Cancer Center, ␣ (8, 11–15). We and others showed that inhibition of the G q signal Box B171, 4200 E, 9th Avenue, Denver, CO 80262. E-mail: [email protected]. pathway (by transfection of GTPase-deficient G␣q genes or trans- ␤ The publication costs of this article were defrayed in part by page charge payment. This fection of catalytically inactive phospholipase C- genes) inhibited article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. inositol triphosphate release, calcium flux, activation of protein §1734 solely to indicate this fact. 4608–4613 ͉ PNAS ͉ April 2, 2002 ͉ vol. 99 ͉ no. 7 www.pnas.org͞cgi͞doi͞10.1073͞pnas.072077299 Downloaded by guest on September 26, 2021 Table 1. Effects of CU201, CU202, and SPD2 on cell cycle distribution and apoptosis in SCLC and NSCLC cell lines after 24-h exposure Cell line Agent %G1͞G0 %S %G2͞M %Apoptosis SHP-77 Control 43 36 21 0.0 CU201, 1.5 ␮M 47 40 13 53.0 CU202, 40 ␮M 51 30 19 0.2 SPD-D, 40 ␮M 50 37 13 26.0 A549 Control 52 35 13 0.0 CU201, 1.5 ␮M 47 39 14 0.14 CU202, 40 ␮M 52 34 14 0.04 SPD-D, 40 ␮M 55 37 8 2.8 Fig. 1. Structures of CU202, CU201, SPD2, SPD-D, CP126, and CP127. Hyp, trans-4-hydroxyproline; Igl, ␣-(2-indanyl) glycine; Oic, octahydroindole-2- were stained with saponin͞propidium iodide͞RNase solution carboxylic acid; SUIM, suberimidate linker; BMH, bismaleimidohexane; FDF, and analyzed by fluorescence-activated cell sorter using a 4-fluoro-D-phenylalanine; FDW, 5-fluoro-D-tryptophan. Coulter EPICS and MODFIT LT software (Verity Software House, Topsham, MA; ref. 25). (HyClone) or 10 nM hydrocortisone͞5.0 ␮g/ml insulin͞10 ␮g/ml Cytogenetic Analysis. To determine whether the two subpopula- transferrin͞10 nM 17-estradiol͞30 nM sodium selenite͞100 tions detected by DNA content analysis represented distinct units͞ml penicillin͞100 ␮g͞ml streptomycin (HITES medium, ref. aneuploid clones, karyotypic analyses using G-banding and 20). All cell lines were grown in 5% CO2 incubators with 100% humidity. spectral karyotyping techniques were performed according to techniques described previously (26). Synthesis of BK Antagonist Peptides. Peptide antagonists were Activation of Caspase-3. synthesized by the solid-phase method (21) using butyloxycarbonyl- After various treatments, cytosolic and amino acids and conventional side chain-blocking groups. Indanyl- mitochondrial fractions were isolated by differential centrifuga- tion. 35S-Labeled caspase-3, translated and purified as described glycine (D-␣-(2-indanyl) glycine, L-␣-(2-indanyl) glycine) was syn- thesized according to Gera and Stewart (22). Other unusual amino (27), was incubated with cytosolic and mitochondrial fractions acids, D-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid and octa- anddATPfor1hat37°C (27). At the end of the incubation period, the reaction was terminated, and the samples were hydroindole-2-carboxylic acid were purchased from Aldrich. Pep- ͞ tide amino acid analysis was characterized by thin layer chroma- submitted to 15% SDS PAGE. The gel was transferred to a tography and laser-desorption mass spectroscopy. nitrocellulose filter and exposed to Kodak X-Omat AR film for 16 h at room temperature. Synthesis of CU201 Dimers. Cross-linkers (dimethyl suberimi- date⅐2HCl, disuccinimidyl suberate, and bismaleimidohexane) of Detection of Apoptosis by Fluorescent Microscopy. After varying varying carbon-chain lengths were purchased from Pierce. Ho- treatments, cells were stained with propidium iodide and bis- modimers were cross-linked from monomers in solution as de- benzimide (Hoechst 33245) and examined for signs of apoptosis by scribed (23).
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