Development of High Affinity Camptothecin-Bombesin Conjugates That Have Targeted Cytotoxicity for Bombesin Receptor-containing Tumor Cells
2004; Elsevier BV; Volume: 279; Issue: 22 Linguagem: Inglês
10.1074/jbc.m401938200
ISSN1083-351X
AutoresTerry W. Moody, Samuel A. Mantey, Tapas K. Pradhan, Michael Schümann, Tomoo Nakagawa, Alfredo Martı́nez, Joseph A. Fuselier, David H. Coy, Robert T. Jensen,
Tópico(s)Pharmacological Receptor Mechanisms and Effects
ResumoMammalian bombesin (BN) receptors are among those most frequently overexpressed by a number of common tumors including prostate, breast, lung, and colon cancers. The aim of this study was to develop a camptothecin-bombesin (CPT-BN) conjugate that interacts with all classes of BN receptors and possibly functions as a prodrug via a labile linker with site-specific cytotoxicity for cancer cells bearing these receptors. CPT was coupled to analogs of [d-Tyr6,β-Ala11,Phe13,Nle14]BN-(6-14) (BA0) using carbamate linkers (L1 and L2) with built-in nucleophile-assisted releasing groups for intracellular cleavage of free cytotoxic agents. One conjugate, CPT-L2-BA3, bound to all three BN receptor classes with high affinity and functioned as a full agonist at each. 125ICPT-L2-BA3 was rapidly internalized by cells expressing each BN receptor class and, using fluorescent imaging, was found to co-localize with BN receptors initially and later to be internalized in cytoplasmic compartments. HPLC analysis of internalized ligand showed that 40% was intact, 25% was metabolized by releasing free CPT, and 35% was metabolized to other breakdown products. CPT-L2-BA3 inhibited the growth of NCI-H1299 non-small cell lung cancer cells in 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyl-2H-tetrazolium bromide (MTT) and clonal growth assays. CPT-L2-BA3 was cytotoxic in an MTT assay for cells transfected with each class of BN receptor; however, it had significantly less effect in cells lacking BN receptors. These results indicate that CTP-L2-BA3 is a potent agonist that is cytotoxic for cells overexpressing any of the three BN receptor classes and functions as a prodrug for receptor-mediated cytoxicity. It therefore should be a useful prototype to explore the effectiveness of tumor-specific cytotoxicity delivery using a receptor-mediated mechanism. Mammalian bombesin (BN) receptors are among those most frequently overexpressed by a number of common tumors including prostate, breast, lung, and colon cancers. The aim of this study was to develop a camptothecin-bombesin (CPT-BN) conjugate that interacts with all classes of BN receptors and possibly functions as a prodrug via a labile linker with site-specific cytotoxicity for cancer cells bearing these receptors. CPT was coupled to analogs of [d-Tyr6,β-Ala11,Phe13,Nle14]BN-(6-14) (BA0) using carbamate linkers (L1 and L2) with built-in nucleophile-assisted releasing groups for intracellular cleavage of free cytotoxic agents. One conjugate, CPT-L2-BA3, bound to all three BN receptor classes with high affinity and functioned as a full agonist at each. 125ICPT-L2-BA3 was rapidly internalized by cells expressing each BN receptor class and, using fluorescent imaging, was found to co-localize with BN receptors initially and later to be internalized in cytoplasmic compartments. HPLC analysis of internalized ligand showed that 40% was intact, 25% was metabolized by releasing free CPT, and 35% was metabolized to other breakdown products. CPT-L2-BA3 inhibited the growth of NCI-H1299 non-small cell lung cancer cells in 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyl-2H-tetrazolium bromide (MTT) and clonal growth assays. CPT-L2-BA3 was cytotoxic in an MTT assay for cells transfected with each class of BN receptor; however, it had significantly less effect in cells lacking BN receptors. These results indicate that CTP-L2-BA3 is a potent agonist that is cytotoxic for cells overexpressing any of the three BN receptor classes and functions as a prodrug for receptor-mediated cytoxicity. It therefore should be a useful prototype to explore the effectiveness of tumor-specific cytotoxicity delivery using a receptor-mediated mechanism. In order to enhance tumor cytotoxicity and decrease side effects, there has been increased interest in the development of prodrugs that improve site-specific delivery of cytotoxic anti-cancer agents (1deGroot F.M. Damen E.W. Scheeren H.W. Curr. Med. Chem. 2001; 8: 1093-1122Crossref PubMed Scopus (116) Google Scholar, 2Dubowchik G.M. Walker M.A. Pharmacol. Ther. 1999; 83: 67-123Crossref PubMed Scopus (257) Google Scholar). Prodrug conjugates have been described utilizing antibodies directed against specific tumor-associated antigens, hydrophilic polymers, and peptide or steroid hormones that interact with receptors overexpressed or ectopically expressed on the tumor (1deGroot F.M. Damen E.W. Scheeren H.W. Curr. Med. Chem. 2001; 8: 1093-1122Crossref PubMed Scopus (116) Google Scholar, 2Dubowchik G.M. Walker M.A. Pharmacol. Ther. 1999; 83: 67-123Crossref PubMed Scopus (257) Google Scholar). The goal of the prodrug is to deliver the therapeutic agent to the target cell, at which time a tumor-specific process (enzyme activity, specific cellular degradation, etc.) will site-specifically release the active drug (1deGroot F.M. Damen E.W. Scheeren H.W. Curr. Med. Chem. 2001; 8: 1093-1122Crossref PubMed Scopus (116) Google Scholar). For peptide hormone receptor prodrugs, this requires coupling of the cytotoxic agent to a peptide hormone through a coupling mechanism that retains high affinity for the peptide hormone receptor and allows the cytotoxic drug to be released after receptor specific internalization and degradation (1deGroot F.M. Damen E.W. Scheeren H.W. Curr. Med. Chem. 2001; 8: 1093-1122Crossref PubMed Scopus (116) Google Scholar, 3Fuselier J.A. Sun L. Woltering S.N. Murphy W.A. Vasilevich N. Coy D.H. Bioorg. Med. Chem. Lett. 2003; 13: 799-803Crossref PubMed Scopus (29) Google Scholar, 4Langer M. Beck-Sickinger A.G. Curr. Med. Chem. Anti-Cancer Agents. 2001; 1: 71-93Crossref PubMed Scopus (109) Google Scholar). Peptide receptor ligands as vehicles to deliver cytotoxic agents are receiving considerable attention, because numerous studies using imaging methods or autoradiographic methods have shown many common malignant tumors either ectopically expressing large numbers of peptide receptors (4Langer M. Beck-Sickinger A.G. Curr. Med. Chem. Anti-Cancer Agents. 2001; 1: 71-93Crossref PubMed Scopus (109) Google Scholar, 5Reubi J.C. Wenger S. Schumuckli-Maurer J. Schaer J.C. Gugger M. Clin. Cancer Res. 2002; 8: 1139-1146PubMed Google Scholar, 6Kwekkeboom D. Krenning E.P. deJong M. J. Nucl. Med. 2000; 41: 1704-1713PubMed Google Scholar, 7Reubi J.C. Endocr. Rev. 2003; 24: 389-427Crossref PubMed Scopus (988) Google Scholar, 8Schally A.V. Nagy A. Life Sci. 2003; 72: 2305-2320Crossref PubMed Scopus (122) Google Scholar, 9Hoffman T.J. Quinn T.P. Volkert W.A. Nucl. Med. Biol. 2001; 28: 527-539Crossref PubMed Scopus (56) Google Scholar) or overexpressing them, thus allowing enhanced uptake of selective ligands for these receptors (6Kwekkeboom D. Krenning E.P. deJong M. J. Nucl. Med. 2000; 41: 1704-1713PubMed Google Scholar, 7Reubi J.C. Endocr. Rev. 2003; 24: 389-427Crossref PubMed Scopus (988) Google Scholar). The mammalian bombesin (BN) 1The abbreviations used are: BN, bombesin; CPT, camptothecin; GRP, gastrin-releasing peptide; BRS3, bombesin receptor subtype 3; hBRS3, human BRS3; GRPR, gastrin-releasing peptide receptor; hGRPR, human GRPR; NMBR, neuromedin B receptor; hNMBR, human NMBR; IP, inositol phosphate; NMB, neuromedin B; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; NSCLC, non-small cell lung cancer; BINAR, built-in nucleophile-assisted releasing; HA, hemagglutinin; TRITC, tetramethylrhodamine isothiocyanate; HPLC, high pressure liquid chromatography.1The abbreviations used are: BN, bombesin; CPT, camptothecin; GRP, gastrin-releasing peptide; BRS3, bombesin receptor subtype 3; hBRS3, human BRS3; GRPR, gastrin-releasing peptide receptor; hGRPR, human GRPR; NMBR, neuromedin B receptor; hNMBR, human NMBR; IP, inositol phosphate; NMB, neuromedin B; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; NSCLC, non-small cell lung cancer; BINAR, built-in nucleophile-assisted releasing; HA, hemagglutinin; TRITC, tetramethylrhodamine isothiocyanate; HPLC, high pressure liquid chromatography. family of receptors (gastrin-releasing peptide receptor (GRPR), neuromedin B receptor (NMBR), and the orphan receptor, bombesin receptor subtype 3 (BRS3)) are excellent candidates for possibly targeting cytotoxic agents to malignant neoplasms. Not only do many common tumors frequently possess and overexpress these receptors, but the BN family of receptors is one of the receptor families most frequently expressed by tumors, and the naturally occurring agonists for these receptors function as autocrine growth factors (7Reubi J.C. Endocr. Rev. 2003; 24: 389-427Crossref PubMed Scopus (988) Google Scholar, 8Schally A.V. Nagy A. Life Sci. 2003; 72: 2305-2320Crossref PubMed Scopus (122) Google Scholar, 10Moody T.W. Chan D. Fahrenkrug J. Jensen R.T. Curr. Pharm. Des. 2003; 9: 495-509Crossref PubMed Scopus (114) Google Scholar). BN receptors have been detected on 40-100% of prostate cancer, breast cancer, lung cancer, gastric cancer, malignant gliomas, colon cancer, and ovarian cancer (5Reubi J.C. Wenger S. Schumuckli-Maurer J. Schaer J.C. Gugger M. Clin. Cancer Res. 2002; 8: 1139-1146PubMed Google Scholar). Recently, a synthetic analog of BN has been described, [d-Tyr6,β-Ala11,Phe13,Nle14]BN-(6-14) (BA0), which functions as a universal ligand for all three mammalian BN receptors (11Mantey S.A. Weber H.C. Sainz E. Akeson M. Ryan R.R. Pradhan T.K. Searles R.P. Spindel E.R. Battey J.F. Coy D.H. Jensen R.T. J. Biol. Chem. 1997; 272: 26062-26071Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 12Pradhan T.K. Katsuno T. Taylor J.E. Kim S.H. Ryan R.R. Mantey S.A. Donohue P.J. Weber H.C. Sainz E. Battey J.F. Coy D.H. Jensen R.T. Eur. J. Pharmacol. 1998; 343: 275-287Crossref PubMed Scopus (89) Google Scholar). This analog binds with high affinity to each of the three BN receptor classes and is rapidly internalized by each class of BN receptors (11Mantey S.A. Weber H.C. Sainz E. Akeson M. Ryan R.R. Pradhan T.K. Searles R.P. Spindel E.R. Battey J.F. Coy D.H. Jensen R.T. J. Biol. Chem. 1997; 272: 26062-26071Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 12Pradhan T.K. Katsuno T. Taylor J.E. Kim S.H. Ryan R.R. Mantey S.A. Donohue P.J. Weber H.C. Sainz E. Battey J.F. Coy D.H. Jensen R.T. Eur. J. Pharmacol. 1998; 343: 275-287Crossref PubMed Scopus (89) Google Scholar). This is an important property because different tumors may possess different BN receptor classes (5Reubi J.C. Wenger S. Schumuckli-Maurer J. Schaer J.C. Gugger M. Clin. Cancer Res. 2002; 8: 1139-1146PubMed Google Scholar), and this ligand would still interact with high affinity with each. Furthermore, recently, a new novel carbamate linker system (3Fuselier J.A. Sun L. Woltering S.N. Murphy W.A. Vasilevich N. Coy D.H. Bioorg. Med. Chem. Lett. 2003; 13: 799-803Crossref PubMed Scopus (29) Google Scholar) has been described that allows the conjugation of a peptide to either the topoisomerase I inhibitor, camptothecin (CPT), or the tubulin-binding agent, combretastatin. This novel linker system (3Fuselier J.A. Sun L. Woltering S.N. Murphy W.A. Vasilevich N. Coy D.H. Bioorg. Med. Chem. Lett. 2003; 13: 799-803Crossref PubMed Scopus (29) Google Scholar) contains a built-in nucleophile-assisted releasing (BINAR) group that enables fine timing of intracellular cleavage rates of free cytotoxic agents containing reactive hydroxyl groups such as CPT or combretastatin (3Fuselier J.A. Sun L. Woltering S.N. Murphy W.A. Vasilevich N. Coy D.H. Bioorg. Med. Chem. Lett. 2003; 13: 799-803Crossref PubMed Scopus (29) Google Scholar). In the present study, we have synthesized a number of CPT analogs coupled to universal bombesin agonists through different carbamate linkers (L1 and L2) to identify a potential general BN receptor cytotoxic prodrug. We have identified one analog, CPT-L2-BA3, which shows high affinity for all three mammalian BN receptors, is fully biologically active at each receptor, and is rapidly internalized by each receptor subtype. Furthermore, CPT-L2-BA3 is cytotoxic for NCI-H1299 non-small cell lung cancer (NSCLC) cells, which possess native GRP receptors (13Moody T.W. Zia F. Venugopal R. Fagarasan M. Oie H. Hu V. J. Cell. Biochem. Suppl. 1996; 24: 247-256Crossref PubMed Scopus (34) Google Scholar) and demonstrate greater cytoxicity for Balb/c 3T3 cells containing GRP receptors than those lacking these receptors, suggesting site-selective cytotoxicity. Materials—The following cells and materials were obtained from the sources indicated: Balb/c 3T3 (mouse fibroblast), NCI-H345 (human small cell lung cancer), NCI-H1299 (human NSCLC), and HEK 293 (human embryonic kidney) cells from ATCC (Manassas, VA); Dulbecco's minimum essential medium, phosphate-buffered saline, RPMI 1640, trypsin-EDTA, and fetal bovine serum from Biofluids (Rockville, MD); G418 sulfate from Invitrogen; Na125I (2200 Ci/mmol) and myo-[2-3H]inositol (20 Ci/mmol) from Amersham Biosciences; camptothecin, formic acid, ammonium formate, disodium tetraborate, 4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4-H-pyran, 2-dimethylaminoisopropylchloride hydrochloride, 1-methyl-2-pyrrolidinone, dimethylaminopyridine, soybean trypsin inhibitor, bacitracin, leupeptin, 4-(2-aminoethyl)-benzenesulfonyl fluoride, poly-l-lysine, protease inhibitor mixture, and Triton X-100 from Sigma; 1,2,4,6-tetrachloro-3α,6α-diphenylglycouril (IODO-GEN) from Pierce; Fura-2/AM from Calbiochem; AG 1-X8 resin from Bio-Rad; BN, GRP, NMB, and [Tyr4]BN from Bachem (Torrence, CA); paraformaldehyde (16% stock solution) from Electron Microscopy Sciences (Ft. Washington, PA); bovine serum albumin from ICN Pharmaceutical Inc. (Aurora, OH); HA antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); secondary antibodies and donkey serum from Jackson ImmunoResearch Laboratories, West Grove, PA; and Vectorshield from Vector (Burlingame, CA). The mammalian expression vector, pcDNA3, custom primers, and restriction endonucleases (BamHI, HindIII, XbaI, and EcoRI) were from Invitrogen. The ExSite PCR-based site-directed mutagenesis kit was from Stratagene (La Jolla, CA). Cell Culture—NCI-H345 and NCI-H1299 cells were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (Invitrogen). NCI-H1299 cells were split weekly 1:20 with trypsin-EDTA. NCI-H345 cells were diluted 1:1 into new media. Balb/c 3T3 cells stably expressing human BRS3 receptors (hBRS3), human NMB receptors (hNMBR), or human GRP receptors (hGRPR) made as described previously (11Mantey S.A. Weber H.C. Sainz E. Akeson M. Ryan R.R. Pradhan T.K. Searles R.P. Spindel E.R. Battey J.F. Coy D.H. Jensen R.T. J. Biol. Chem. 1997; 272: 26062-26071Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 14Benya R.V. Kusui T. Pradhan T.K. Battey J.F. Jensen R.T. Mol. Pharmacol. 1995; 47: 10-20PubMed Google Scholar, 15Benya R.V. Wada E. Battey J.F. Fathi Z. Wang L.H. Mantey S.A. Coy D.H. Jensen R.T. Mol. Pharmacol. 1992; 42: 1058-1068PubMed Google Scholar) were grown in Dulbecco's modified Eagle's cell medium supplemented with 300 mg/liter G418 sulfate. HEK 293 cells were cultured in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. The cells (Balb/c 3T3 (mouse fibroblast), NCI-H345 (human SCLC), NCI-H1299 (human NSCLC), and HEK 293 (human embryonic kidney) cells were from ATCC, were mycoplasma-free, and were used when they were in the exponential growth phase after incubation at 37 °C in 5% CO2, 95% air. Preparation of Peptides—The peptide portions of the peptide-cytotoxin conjugates were synthesized by solid phase methods using the standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) protection strategy (3Fuselier J.A. Sun L. Woltering S.N. Murphy W.A. Vasilevich N. Coy D.H. Bioorg. Med. Chem. Lett. 2003; 13: 799-803Crossref PubMed Scopus (29) Google Scholar). Sequences were assembled on Rink amide resin (Advanced ChemTech, Louisville, KY), and Trt group protection was employed for the imidazole NH of His, Boc for the indole NH of Trp, and tert-butyl group for the OH groups of Ser and Tyr. Bromoacetic acid was coupled to the N terminus of the protected peptide, which was then reacted with a 5 m excess of tertiary butyloxycarbonyl-1-N-methylamino,2-aminoethane (2 h). CPT (Roche Applied Science) was activated at its position 20 OH group by treating a suspension in anhydrous dichloromethane with dimethyaminopyridine (4 eq) and a 20% solution of phosgene in toluene (4 eq) (4 h) followed by removal of excess solvent and phosgene in vacuo. A 3 m excess of the resulting yellow oil was added to the above resin in anhydrous dichloromethane and allowed to react overnight. After washing and drying of the resin, the camptothecin-peptide conjugate was cleaved using the standard acid mixture, trifluoroacetic acid/H2O/ethanedithiol/triisopropylsilane, 95:2:2:1 (2 h), followed by evaporation of the trifluoroacetic acid and precipitation and washing of the peptide with ether. Conjugates were purified to >97% purity by preparative HPLC (C18 silica) and characterized by mass spectrometry and amino acid analysis. For the compounds shown in Fig. 1, BA0 had a calculated mass of 1133.3 and was found to have a mass of 1132.7; CPT-L1-BA1 had a calculated mass of 1661.8 and was found to have a mass of 1666.3; CPT-L2-BA1 had a calculated mass of 1675.5 and was found to have a mass of 1676.1; CPT-L1-BA2 (mixture of polymers) had an average mass of 3000; and CPT-L2-BA3 had a calculated mass of 1709.9 and was found to have a mass of 1709.1. Preparation of 125I-BA0, 125I-L2-BA3, 125I-CPT-L2-BA3, 125I-[d-Tyr0]NMB, and 125I-[Tyr4]BN—These radioligands, with specific activities of 2200 Ci/mmol, were prepared as previously described (11Mantey S.A. Weber H.C. Sainz E. Akeson M. Ryan R.R. Pradhan T.K. Searles R.P. Spindel E.R. Battey J.F. Coy D.H. Jensen R.T. J. Biol. Chem. 1997; 272: 26062-26071Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 12Pradhan T.K. Katsuno T. Taylor J.E. Kim S.H. Ryan R.R. Mantey S.A. Donohue P.J. Weber H.C. Sainz E. Battey J.F. Coy D.H. Jensen R.T. Eur. J. Pharmacol. 1998; 343: 275-287Crossref PubMed Scopus (89) Google Scholar) or as described below. Briefly, 0.8 μg of IODO-GEN solution (0.01 μg/μl in chloroform) was added to a 5-ml plastic test tube, dried under nitrogen, and washed with 100 μl of 0.5 m potassium phosphate solution (pH 7.4 except for 125I-L2-BA3 and 125I-CPT-L2-BA3, which required pH 7.0). To this tube, 20 μl of potassium phosphate of the appropriate pH, 8 μg of peptide in 4 μl of water, and 2 mCi (20 μl) of Na125I were added and incubated for 6 min at room temperature (Na125I (2200 Ci/mmol) and myo-[2-3H]inositol (20 Ci/mmol) were from Amersham Biosciences). The incubation was stopped with 300 μl of water. For 125I-[d-Tyr0]NMB and 125I-[Tyr4]BN, which have COOH-terminal methionine groups, 300 μl of 1.5 m dithiothreitol was added and heated for 1 h at 80 °C. The radiolabeled peptides were separated using a C18 Sep-Pak (Waters Associates, Milford, MA) and further purified by reverse-phase high performance liquid chromatography on a C18 column. The fractions with the highest radioactivity and binding were neutralized with 0.2 m Tris buffer (pH 9.5) and stored with 0.5% bovine serum albumin (w/v) at -20 °C. Binding of 125I-Labeled BN-related Peptides to Various Cells—Binding was performed as described previously (11Mantey S.A. Weber H.C. Sainz E. Akeson M. Ryan R.R. Pradhan T.K. Searles R.P. Spindel E.R. Battey J.F. Coy D.H. Jensen R.T. J. Biol. Chem. 1997; 272: 26062-26071Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 12Pradhan T.K. Katsuno T. Taylor J.E. Kim S.H. Ryan R.R. Mantey S.A. Donohue P.J. Weber H.C. Sainz E. Battey J.F. Coy D.H. Jensen R.T. Eur. J. Pharmacol. 1998; 343: 275-287Crossref PubMed Scopus (89) Google Scholar, 14Benya R.V. Kusui T. Pradhan T.K. Battey J.F. Jensen R.T. Mol. Pharmacol. 1995; 47: 10-20PubMed Google Scholar). The standard binding buffer contained 24.5 mm HEPES (pH 7.4), 98 mm NaCl, 6 mm KCl, 5 mm MgCl2, 2.5 mm NaH2PO4, 5 mm sodium pyruvate, 5 mm sodium fumarate, 0.01% (w/v) soybean trypsin inhibitor, 1% amino acid mixture, 0.2% (w/v) bovine serum albumin, and 0.05% (w/v). Balb/c 3T3 cells stably expressing hGRPR (0.3 × 106), hNMBR (0.03 × 106), hBRS3 (0.3 × 106), or NCI-H1299 cells were incubated with 50 pm 125I-labeled ligand at 22 °C for 60 min. Aliquots (100 μl) were removed and centrifuged through 300 μl of incubation buffer in 400-μl Microfuge tubes at 10,000 × g for 1 min using a Beckman Microfuge B. The pellets were washed twice with buffer and counted for radioactivity in a γ counter. The nonsaturable binding was the amount of radioactivity associated with cells in incubations containing 50 pm radioligand (2200 Ci/mmol) and 1 μm unlabeled ligand. Nonsaturable binding was <10% of total binding in all the experiments. Receptor affinities (Ki) were determined using a least-square, curve-fitting program (KaleidaGraph) and the Cheng-Prusoff equation (16Cheng Y. Prusoff W.H. Biochem. Pharmacol. 1973; 22: 3099-3108Crossref PubMed Scopus (12259) Google Scholar). Internalization—Balb/c 3T3 cells stably transfected with hGRPR, hNMBR, or hBRS3 were incubated with radioligands as stated under "Experimental Procedures." Internalization experiments were performed as described previously (14Benya R.V. Kusui T. Pradhan T.K. Battey J.F. Jensen R.T. Mol. Pharmacol. 1995; 47: 10-20PubMed Google Scholar, 15Benya R.V. Wada E. Battey J.F. Fathi Z. Wang L.H. Mantey S.A. Coy D.H. Jensen R.T. Mol. Pharmacol. 1992; 42: 1058-1068PubMed Google Scholar). Briefly, after the indicated incubation times, 100-μl samples were added to a 1.5-ml Microfuge tube with 1 ml of 4 °C acid-stripping solution containing 0.2 m acetic acid (pH 2.5) and 0.5 m NaCl to remove the surface-bound radioligand. After 5 min, the cells were pelleted, the supernatant was removed, and the cells were washed twice with incubation buffer. In all cases, parallel incubations were conducted in the presence of 1 μm unlabeled ligand to determine changes in nonsaturable binding. Results are expressed as the percentage of saturable 125I-ligand added that is surface-bound (acid-stripped) or internalized (not acid-stripped). The internalization of 125I-CPT-BN conjugates was investigated in human NCI-H1299 NSCLC cells that naturally possess hGRPR (13Moody T.W. Zia F. Venugopal R. Fagarasan M. Oie H. Hu V. J. Cell. Biochem. Suppl. 1996; 24: 247-256Crossref PubMed Scopus (34) Google Scholar). 125I-CPT-L2-BA3 (50 pm) was incubated with NCI-H1299 NSCLC cells for 2 h at 4 °C. Two sets of cells were washed three times in receptor binding buffer to remove free peptide. One set was then treated with 0.5 m acetic acid and 0.15 m NaCl for 5 min at 4 °C to remove peptide bound to the cell surface. The supernatant was counted in a γ counter. A second set of cells was treated with 0.2 n NaOH to determine total binding at 4 °C. Also, two sets of cells were incubated at 37 °C for 5 min and washed three times in receptor binding buffer to remove free peptide. One set was then treated with 0.5 m acetic acid and 0.15 m NaCl for 5 min at 4 °C to remove peptide bound to the cell surface. The supernatant was counted in a γ counter. A second set of cells was treated with 0.2 n NaOH to determine total binding at 37 °C. Characterization of Internalized Radioactivity—Balb/c 3T3 cells with no transfected bombesin receptors and Balb/c 3T3 cells stably transfected with hGRPR (2 × 106/ml) were incubated at 37 °C for 15 min with 0.4 nm 125I-CPT-L2-BA3 in standard binding buffer containing 25 μg/ml leupeptin, 0.02% (w/v) bacitracin, 10 μg/ml antipain, 50 μg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride, and 2 μl/ml of protease inhibitor mixture (Sigma). Cells were then washed with standard binding buffer with protease inhibitors at 4 °C and underwent acid stripping to remove surface-bound ligand performed for 5 min at 4 °C with 0.2 m acetic acid (pH 2.5) and 0.5 m NaCl, as described under "Internalization." After centrifugation, cells were resuspended in 1 ml of standard binding buffer with protease inhibitors containing 1% Triton X-100 (pH 6), and the resuspension was sonicated to solubilize the radioactivity. Nine hundred μl of the sonicated mixture was injected on an HPLC (Waters model 510, Milford, MA) equipped with a C-18 column and eluted with a linear gradient increasing at 0.8% acetonitrile in trifluoroacetic acid (0.1%) per min starting at 12% acetonitrile and ending at 80%. One-ml fractions were collected, and radioactivity in each fraction was determined by counting in a Packard γ counter. Measurement of [3H]Inositol Phosphates ([3H]IP)—Changes in total [3H]IP were measured as described previously (14Benya R.V. Kusui T. Pradhan T.K. Battey J.F. Jensen R.T. Mol. Pharmacol. 1995; 47: 10-20PubMed Google Scholar, 15Benya R.V. Wada E. Battey J.F. Fathi Z. Wang L.H. Mantey S.A. Coy D.H. Jensen R.T. Mol. Pharmacol. 1992; 42: 1058-1068PubMed Google Scholar). Briefly, hBRS3-, hGRPR-, or hNMBR-transfected Balb/c 3T3 cells were subcultured into 24-well plates (5 × 104 cells/well) in regular propagation medium and then incubated for 24 h at 37 °C in a 5% CO2 atmosphere. The cells were then incubated with 3 μCi/ml myo-[2-3H]inositol in growth medium supplemented with 2% fetal bovine serum for an additional 24 h. Before assay, the 24-well plates were washed by incubating for 30 min at 37 °C with 1 ml/well of phosphate-buffered saline (pH 7.0) containing 20 mm lithium chloride. The wash buffer was aspirated and replaced with 500 μl of IP assay buffer containing 135 mm sodium chloride, 20 mm HEPES (pH 7.4), 2 mm calcium chloride, 1.2 mm magnesium sulfate, 1 mm EGTA, 20 mm lithium chloride, 11.1 mm glucose, 0.05% bovine serum albumin (w/v) and incubated with or without any of the peptides studied. After 60 min of incubation at 37 °C, the experiments were terminated by the addition of 1 ml of ice-cold 1% (v/v) hydrochloric acid in methanol. Total [3H]IP was isolated by anion exchange chromatography as described previously (14Benya R.V. Kusui T. Pradhan T.K. Battey J.F. Jensen R.T. Mol. Pharmacol. 1995; 47: 10-20PubMed Google Scholar, 15Benya R.V. Wada E. Battey J.F. Fathi Z. Wang L.H. Mantey S.A. Coy D.H. Jensen R.T. Mol. Pharmacol. 1992; 42: 1058-1068PubMed Google Scholar). Briefly, samples were loaded onto Dowex AG1-X8 anion exchange resin columns, washed with 5 ml of distilled water to remove free [3H]inositol, and then washed with 2 ml of 5 mm disodium tetraborate, 60 mm sodium formate solution to remove [3H]glycerophosphorylinositol. Two ml of 1 mm ammonium formate, 100 mm formic acid solution were added to the columns to elute total [3H]IP. Each eluate was mixed with scintillation mixture and measured for radioactivity in a scintillation counter. Cytosolic Calcium [Ca2+]i Measurement—The ability of the CPT-BN conjugates to alter cytosolic [Ca2+]i was investigated as described previously (17Moody T.W. Venugopal R. Zia F. Patierno S. Leban J.J. McDermed J. Life Sci. 1995; 56: 521-529Crossref PubMed Scopus (26) Google Scholar). NCI-H1299 cells and NCI-H345 cells were harvested (2.5 × 106/ml) and incubated with 5 μm Fura-2/AM at 37 °C for 30 min. The cells, which contained loaded Fura-2, were centrifuged at 1500 × g for 10 min and resuspended at the same concentration in new SIT medium (RPMI 1640 containing 30 nm sodium selenite, 5 μg/ml bovine insulin, and 10 μg/ml transferrin). After two washes with the same medium, cells were placed in a Delta PTI Scan 1 spectrofluorometer (Photon Technology International, South Brunswick, NJ) equipped with a magnetic stirring mechanism and temperature (37 °C)-regulated cuvette holder. The fluorescence intensity was continuously monitored at dual excitation wavelengths of 340 and 380 nm, using an emission wavelength of 510 nm prior to and after the addition of BN-like peptides. Proliferation Assays—Growth studies were performed using the 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyl-2H-tetrazolium bromide (MTT) assay. Growth studies in vitro were conducted using MTT (Sigma) colorimetric assays. NCI-H1299 cells, hGRPR-transfected Balb/c 3T3 cells, or Balb/c 3T3 cells (104/well) were placed in SIT medium (100 μl), and various concentrations of CPT-L2-BA3 were added. After 4 days, 15 μl (1 mg/ml) of MTT was added, and after another 4 h, 150 μl of Me2SO was added. After 16 h, the optical density at 570 nm was determined using an ELISA reader. The proliferation rates were calculated from the OD readings with various concentrations of CPT-L2-BA3 using the untreated cells as 100%. Clonogenic Assays—The effects of CPT-BN-containing peptides on the growth of NCI-H1299 and NCI-H345 cells were investigated using a clonogenic assay (18Mahmoud S. Staley J. Taylor J. Bogden A. Moreau J.P. Coy D. Avis I. Cuttitta F. Mulshine J.L. Moody T.W. Cancer Res. 1991; 51: 1798-1802PubMed Google Scholar). The base layer consisted of 3 ml of 0.5% agarose in SIT medium containing 5% fetal bovine serum in 6-well plates. The top layer consisted of 3 ml of SIT medium in 0.3% agarose (FMC Corp., Rockford, ME), CPT-BN conjugates, and 5 × 104 NCI-H1299 cells. Triplicate wells were plated; after 2 weeks, 1 ml of 0.1% p-iodonitrotetrazolium violet was added; and after 16 h at 37 °C, the plates were screened for colony formation. The number of colonies larger than 50 μm in diameter were counted using an Omnicon image analysis system. Preparation of HA-GRPR—The cDNA of the mouse GRPR used was identical to that described previously (14Benya R.V. Kusui T. Pradhan T.K. Battey J.F. Jensen R.T. Mol. Pharmacol. 1995; 47: 10-20PubMed Google Scholar, 19Benya R.V. Fathi Z. Pradhan T. Battey J.F. Kusui T. Jensen R.T. Mol. Pharmacol. 1994; 46: 235-245PubMed Google Scholar). A sequence encoding the HA epitope tag (YPYDVPDYA) was inserted between the first (Met) and second (Ala) amino acid residue of the GRPR using the ExSite PCR-based site-directed
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