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Induction of apoptosis by a short-chain neuropeptide analog in small cell lung cancer
Peptides, Vol. 19, No. 9, pp. 1519 –1523, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/98 $19.00 1 .00
PII S0196-9781(98)00116-8
Induction of Apoptosis by a Short-Chain Neuropeptide Analog in Small Cell Lung Cancer RITA ROSATI,* MOHAMMED R. ADIL,* MUHAMMAD A. ALI,* JAMES ELIASON,* ANTAL OROSZ,† FERENC SEBESTYE´N† AND GREGORY P. KALEMKERIAN* 1 *Division of Hematology and Oncology, Wayne State University and the Barbara Ann Karmanos Cancer Institute, Detroit, MI 48201 †Tumor Biology Laboratory, Departments of Plant Physiology and Organic Chemistry, Eo¨tvo¨s Lora´nd University, Budapest, Hungary Received 20 May 1998; Accepted 2 July 1998 ROSATI, R., M. R. ADIL, M. A. ALI, J. ELIASON, A. OROSZ, F. SEBESTYE´N AND G. P. KALEMKERIAN. Induction of apoptosis by a short-chain neuropeptide analog in small cell lung cancer. PEPTIDES 19(9) 1519 –1523, 1998.—Small cell lung cancer (SCLC) cells express a variety of neuropeptides which act as autocrine growth factors. Although several neuropeptide analogs have been reported to antagonize SCLC proliferation, the development of these compounds has been limited by their low potency and the cytostatic nature of their effects. In the present study we evaluated the cytotoxic activity of four short-chain substance P analogs (NY3460, NY3238[-pHOPA], NY3238[Phe1], NY3238[Lys5]) against a panel of five SCLC cell lines. NY3460 was the most potent compound in all five SCLC cell lines (IC50 5 2.8 –3.7 mM) as assessed by a MTT growth inhibitory assay. NY3238[Phe1] was also relatively active in all cell lines (IC50 5 3.5–11.2 mM), while NY3238[Lys5] and NY3238[-pHOPA] were substantially less active. NY3460 was the only agent to induce an increase in the percentage of cells with subdiploid DNA content suggestive of apoptosis by flow cytometric DNA content analysis. The induction of apoptosis was confirmed by fluorescent microscopy in NCI-H69, NCI-H82, NCI-H446, and NCI-H510 cells after exposure to 5.0 mM NY3460 for 48 h. These findings suggest that NY3460 is a relatively potent cytotoxic inhibitor of SCLC growth, and that short-chain neuropeptide analogs deserve further evaluation as anti-SCLC agents. © 1998 Elsevier Science Inc. Small cell lung cancer
Substance P analogs
Cytotoxicity
LUNG cancer continues to be the leading cause of cancerrelated death in developed countries, with small cell lung cancer (SCLC) accounting for 20 –25% of all lung cancer deaths. SCLC is characterized by neuroendocrine differentiation, early metastatic potential and initial sensitivity to cytotoxic therapy. Unfortunately, the duration of response to treatment is usually short and most patients die with treatment-resistant disease (6). Recent advances in our understanding of the biology of SCLC have led to many novel therapeutic strategies aimed at improving the dismal prognosis of patients with this disease (5). SCLC cells secrete numerous hormones and neuropeptides which interact with specific receptors expressed on
Apoptosis
Experimental therapeutics
SCLC cell membranes and act as autocrine growth factors stimulating tumor cell proliferation (10,14). Based on these findings, a variety of neuropeptide antagonists have been developed in an attempt to inhibit the growth of SCLC cells. Substance P analogs have been found to inhibit both basal and neuropeptide-induced proliferation of SCLC cells (15), but only one of these analogs has been reported to induce apoptosis in selected SCLC cell lines (12,16). Numerous structural and chemical modifications, including shortening of the peptide chain, have been attempted in an effort to develop more potent and metabolically stable neuropeptide analogs (2,11,17). One short-chain substance P analog, Antagonist G, has undergone extensive preclinical evaluation
1 Requests for reprints should be addressed to Gregory P. Kalemkerian, MD, Harper Hospital, Hudson 515, 3990 John R., Detroit, MI 48201, USA Tel: (313) 745-2357; Fax: (313) 993-0559; E-mail: [email protected]
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and is currently in the early phases of clinical development (7). In the present study, the cytotoxic activity of four novel short-chain substance P analogs was evaluated in a heterogeneous panel of human SCLC cell lines. METHOD Cell Culture Five small cell carcinoma cell lines (NCI-H69, NCI-H82, NCI-H345, NCI-H446, NCI-H510) were obtained from American Type Culture Collection (Rockville, MD) and propagated in RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 5% fetal calf serum (FCS) (Sigma, St. Louis, MO), penicillin (100 U/ml), streptomycin (100 mg/ ml), and HITES [hydrocortisone (10 nM); insulin (15 mg/ ml); transferrin (100 mg/ml); 17-b-estradiol (10 nM); selenium (30 nM)] in a humidified 5% CO2 incubator at 37°C. All neuropeptide analogs were synthesized and supplied by two of us [A.O. and F.S.]. Vehicle-control cells for all experiments were exposed to 0.5% ethanol, which had no effect on basal SCLC cell proliferation. Growth Inhibition Assay Cells were seeded in 96-well plates with 200 ml/well and 5 wells per sample concentration at a density of 1– 4 3 104 cells/ml for 24 h prior to treatment with a range of concentrations of each neuropeptide analog up to 54 mM. Control cells were treated with 0.5% ethanol and blank wells were loaded with media only. After 5 days of neuropeptide exposure, 50 ml of 1.0 mg/ml MTT (Sigma Chemicals, St. Louis, MO) was added to each well and plates were incubated for 4 h at 37°C prior to centrifugation, removal of supernatant, and dissolution of formazan crystals in 200 ml of DMSO for 1 h at room temperature. Absorbance at 570 nm (A570) was determined for each well, and the survival fraction for each study condition was calculated from the ratio: (mean A570 treated cells–mean A570 blank wells)/ (mean A570 control cells–mean A570 blank wells). The IC50 was determined from dose-response data from at least three experiments with CalcuSyn software (Biosoft, Cambridge, UK). Flow Cytometry For DNA content analysis, 1 3 106 cells were exposed to either 0.5% ethanol or 5.0 mM of one of the neuropeptide analogs for 24 or 48 h prior to ethanol fixation and staining with 5.0 mg/ml propidium iodide (PI) 1 RNase A. All samples were analyzed using a Becton-Dickinson (San Jose, CA) FACScan flow cytometer. Relative DNA content (PI) was detected with bandpass filter 585/42 and the FACScan doublet discrimination circuit was employed to detect aggregates. Data were acquired in listmode, ungated to 105 events/sample, and analyzed using ModFit (Verity, Topsham, ME) and PC-LYSYS software. All experiments were repeated twice.
ROSATI ET AL. TABLE 1 AMINO ACID SEQUENCES OF SUBSTANCE P AND ANALOGS Name
Sequence
Substance P Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 Antagonist G Arg-DTrp-NMePhe*-DTrp-Leu-Met-NH2 † ‡ NY3460 DMePhe -DTrp-Phe-DTrp-Leu-c(CH2NH) Leu-NH2 NY3238 pHOPA§DTrp-Phe-DTrp-Leu-Leu-NH2 NY3238[2pHOPA]DTrp-Phe-DTrp-Leu-Leu-NH2 NY3238[Phe1] Phe-DTrp-Phe-DTrp-Leu-Leu-NH2 NY3238[Lys5] pHOPA§-DTrp-Phe-DTrp-Lys-Leu-NH2 Shaded areas 5 compounds used in present study. * NMe, Na-methyl-phenylalanine. † DMePhe, D-methyl-phenylalanine. ‡ c(CH2NH), pseudopeptide bond. § pHOPA, para-hydroxy-phenyl-acetic group.
Fluorescent Microscopy Cells (1–3 3 106) were exposed to either 0.5% ethanol or 5.0 mM of one of the neuropeptide analogs for up to 72 h prior to ethanol fixation and staining with 5.0 mg/ml PI 1 RNase A. Samples were analyzed on a Zeiss Laser Scanning Microscope LSM 310 with a 543 nm HENE laser. Experiments were repeated twice and representative data reported. RESULTS Growth Inhibition The amino acid sequences of substance P and the relevant neuropeptide analogs are shown in Table 1. The growth inhibitory activity of four of these analogs (NY3460, NY3238[-pHOPA], NY3238[Phe1], NY3238[Lys5]) was evaluated in a panel of five human SCLC cell lines (NCIH69, NCI-H82, NCI-H345, NCI-H446, NCI-H510). Unlike most prior studies, all experiments were performed in the presence of serum. Cell viability was determined by a standardized MTT assay after 5 days of exposure to each compound. NY3460 was the most potent inhibitor of growth in all five cell lines, with IC50 values of 2.8 –3.7 mM (Table 2). All five cell lines were also relatively sensitive to NY3238[Phe1] with IC50 values of 3.5–11.2 mM. The growth inhibition induced by both NY3460 and NY3238[Phe1] was clearly dose-dependent. In contrast, only two cell lines, NCI-H69 and NCI-H446, exhibited IC50 values below 20 mM after treatment with NY3238[Lys5], while all five SCLC cell lines were relatively resistant to NY3238[-pHOPA] (Table 2). Cell Cycle Analysis Flow cytometric DNA content analysis was performed in NCI-H82 and NCI-H69 SCLC cells after exposure to 5.0 mM of each compound for 48 h. Exposure to NY3460 resulted in a modest accumulation of cells in G1/G0 and a commensurate decrease in the proliferative cell fraction (S
SHORT-CHAIN NP ANALOGS IN SCLC
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GROWTH INHIBITORY ACTIVITY OF NEUROPEPTIDE ANALOGS AGAINST SCLC CELL LINES Neuropeptide iC50 (mM)* Cell line
NY3460
NY3238[2pHOPA]
NY3238[Phe1]
NY3238[Lys5]
NCI-H69 NCI-H82 NCI-H345 NCI-H446 NCI-H510
2.8 6 0.2 3.0 6 0.1 3.1 6 0.2 3.1 6 0.4 3.7 6 0.3
.50 23.4 6 0.6 .50 30.6 6 7.6 .50
10.8 6 1.2 4.9 6 0.8 6.7 6 1.2 3.5 6 0.9 11.2 6 2.9
14.7 6 3.1 27.5 6 0.7 41.4 6 0.2 10.5 6 0.7 40.8 6 2.7
* Values represent mean of at least two experimetns 6 SD.
1 G2/M) in both NCI-H82 (25%) and NCI-H69 (19%) cells. In addition, a substantial increase in the percentage of cells with hypodiploid DNA content, a finding that is suggestive of apoptosis, was noted in both NCI-H82 (4.3-fold) and NCI-H69 (3.1-fold) cells (Fig. 1). No substantial changes were noted in the cell cycle phase distribution or hypodiploid fraction after exposure to NY3238[-pHOPA], NY3238[Phe1], or NY3238[Lys5] for up to 48 h (Fig. 1).
Induction of Apoptosis To confirm the induction of apoptosis by NY3460, both treated and untreated SCLC cells were stained with propidium iodide and evaluated by fluorescent microscopy. All five SCLC cell lines were exposed to 5.0 mM each neuropeptide analog for 24 to 72 h prior to morphologic evaluation. After 48 h of exposure to NY3460, characteristic
FIG. 1. DNA content analysis. Single-parameter histograms of linear PI fluorescence indicating relative DNA content. Percentages indicate cells with reduced (less than diploid) DNA content, consistent with apoptosis, as determined by integration of the area under the curve within the marker limits. Upper left, control NCI-H82 SCLC cells; upper center, NCI-H82 cells treated with 5.0 mM of NY3460 for 48 h; upper right, NCI-H82 cells treated with 5.0 mM NY3238[Phe1] for 48 h; lower left, control NCI-H69 SCLC cells; lower center, NCI-H69 cells treated with 5.0 mM NY3460 for 48 h; lower right, NCI-H69 cells treated with 5.0 mM NY3238[Phe1] for 48 h.
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FIG. 2. Fluorescent micrographs of SCLC cells stained with propidium iodide after exposure to 0.5% ethanol (left column) or 5.0 mM NY3460 (right column) for 48 h. Note chromatin condensation and nuclear fragmentation consistent with apoptosis in all three cell lines following exposure to NY3460 (magnification 6003).
apoptotic features, including chromatin condensation and nuclear fragmentation, were clearly evident in the majority of NCI-H69, NCI-H82, and NCI-H446 cells (Fig. 2). NCIH510 cells exhibited a modest degree of apoptosis, while NCI-345 cells did not show any morphologic changes after up to 72 h of exposure to 5.0 mM NY3460 (data not shown). No morphologic evidence of apoptosis was seen in any of the SCLC cell lines after exposure to NY3238[-pHOPA], NY3238[Phe1], or NY3238[Lys5] (data not shown). These data suggest that NY3460, but not the other neuropeptide analogs studied, induced growth inhibition via apoptotic cell death in most SCLC cells. DISCUSSION Dramatic advances have been made over the past 20 years in our understanding of the cellular and molecular biology of SCLC. The identification of autocrine pathways that regulate the proliferation of SCLC cells is one of the most therapeutically promising of these advances. Thus far, neuropeptides reported to act as autocrine growth factors in SCLC include gastrin-releasing peptide, cholecystokinin, vasopressin, and bradykinin (14). These compounds stimulate calcium mobilization, tyrosine kinase activity and cellular proliferation at nanomolar concentrations in a variety of SCLC cell lines, including NCI-H69, NCI-H345, and
ROSATI ET AL.
NCI-H510 (15). Numerous studies have demonstrated that broad-spectrum neuropeptide analogs can antagonize neuropeptide-induced signal transduction pathway activation and inhibit both in vitro and in vivo growth of SCLC cells (1,2,13,16). Although substance P analogs can clearly inhibit SCLC growth in a cytostatic manner, the induction of apoptosis by these compounds has been reported in only two studies and only after exposure to relatively high concentrations (12,16). In the present study, the activity of several short-chain substance P analogs was evaluated in of a panel of human SCLC cell lines representing the genetic, phenotypic and biochemical variations seen in human SCLC tumors (3). NY3460 was the most potent inhibitor of growth in all cell lines tested, with IC50 values of 2.8 –3.7 mM and morphologic evidence of apoptosis in four of five cell lines. It has previously been reported that NY3460 inhibited the growth of NCI-H69 cells both in vitro (IC50 5 3.0 mM) and in an in vivo xenograft model (11). The activity of the other three analogs used in the present study has not been previously reported. In previous studies, apoptosis after exposure to the long-chain substance P analog, [DArg1, DPhe5, DTrp7,9, Leu11] substance P, was observed only after exposure to high concentrations of 25–50 mM in serum-free conditions (12,16). Antagonist G, a short-chain substance P analog currently in early clinical trials (Table 1), has been shown to inhibit proliferation of NCI-H69 and NCI-H345 cells in a cytostatic manner with an IC50 of 10 –20 mM (17), and to inhibit the growth of NCI-H69 xenografts in nude mice (7). In the present study, the induction of apoptosis by NY3460 at substantially lower concentrations and in the presence of serum suggests greater potential clinical relevance. Interestingly, the activity of the short-chain neuropeptide analogs does not appear to correlate with the degree of neuroendocrine differentiation of the SCLC cell lines or the expression of specific neuropeptide receptors, findings which have also been reported for the activity of long-chain substance P analogs (8,12,15) and an anti-growth factor monoclonal antibody (18). Neuroendocrine differentiation is relatively high in so-called “classic” SCLC cell lines, such as NCI-H345, NCI-H510, and NCI-H69, and low to absent in ‘variant’ lines, such as NCI-H82 and NCI-H446 (3). However, the growth inhibitory activity of NY3460 is similar in all lines, and the activity of the other analogs evaluated is actually greatest in the ‘variant’ NCI-H82 and NCI-H446 cell lines. It has previously been reported that, despite being a potent inhibitor of SCLC proliferation, NY3460 was a less efficient inhibitor of bombesin:bombesin receptor binding than other short-chain analogs with less potent growth inhibitory activity (11). Neuropeptide analogs have also been shown to inhibit calcium mobilization and protein tyrosine kinase activity induced by a wide variety mitogens acting through distinct receptors (13,15,16). The targeting of a shared signal transduction
SHORT-CHAIN NP ANALOGS IN SCLC
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pathway would increase the therapeutic utility of such agents by allowing activity against SCLC tumors with varying neuropeptide growth factor and receptor phenotypes. Correlations between the structure and activity of neuropeptide antagonists have been difficult to extract from the available literature. In the present study, the hexapeptide analogs (NY3460, NY3238[Phe1]) were substantially more potent inhibitors of SCLC growth than the pentapeptide analogs (NY3238[-pHOPA], NY3238[Lys5]). This finding is most striking for NY3238[Phe1] and NY3238[-pHOPA], which differ only by the addition of an N-terminal phenylalanine residue in NY3238[Phe1]. In addition, NY3460, which induces apoptosis, and NY3238[Phe1], which does not, differ in only two respects, the N-terminal substitution of D-methyl-phenylalanine for phenylalanine and the insertion of a pseudopeptide bond between the C-terminal leucine residues in NY3460. The insertion of a pseudopep-
tide bond has also been shown to increase the growth inhibitory activity of long-chain neuropeptide analogs, but has not previously been associated with apoptosis (9). The pharmacologic stimulation of apoptotic pathways represents a rational approach to the prevention and treatment of cancer (4). Thus, the induction of apoptosis by NY3460 at relatively low concentrations suggests that such short-chain neuropeptide analogs may have potential utility for the treatment of SCLC. ACKNOWLEDGEMENTS We are grateful to Dr. Mary L. Varterasian for critical review of the manuscript; Ms. Linda Mayernik for technical assistance and figure production; and the Charlotte A. Woody Cancer Research Fund and the Webber Medical Advancement Fund for continued support. We also acknowledge the Analytical Cytometry and Confocal Imaging Core Facilities (supported in part by center grants P30CA22453 from the National Cancer Institute and P30ES06639 from the National Institutes of Environmental Health Sciences).
REFERENCES 1. Bepler, G.; Zeymer, U.; Mahmoud, S.; Fiskum, G.; Palaszynski, E.; Rotsch, M.; Willey, J.; Koros, A.; Cuttitta, F.; Moody, T. W. Substance P analogs function as bombesin receptor antagonists and inhibit small cell lung cancer clonal growth. Peptides. 9:1367–1372; 1989. 2. Bunn, P. A.; Chan, D.; Stewart, J.; Gera, L.; Tolley, R.; Jewett, P.; Tagawa, M.; Alford, C.; Mochzuki, T.; Yanaihara, N. Effects of neuropeptide analogs on calcium flux and proliferation of lung cancer cell lines. Cancer Res. 54:3602–3610; 1994. 3. Carney, D. N.; Gazdar, A. F.; Bepler, G.; Guccion, J.; Marangos, P. J.; Moody, T. W.; Zweig, M. H.; Minna, J. D. Establishment and identification of small cell lung cancer cell lines having classic and variant features. Cancer Res. 45:2913– 2923; 1985. 4. Dixon, S. C.; Soriano, B. J.; Lush, R. M.; Borner, M. M.; Figg, W. D. Apoptosis: its role in the development of malignancies and its potential as a novel therapeutic target. Ann. Pharmacother. 31:76 – 82; 1997. 5. Gazdar, A. F.; Carbone, D. P. The biology and molecular genetics of lung cancer. Austin: R.G. Landes; 1994. 6. Ihde, D. C. Chemotherapy of lung cancer. N. Engl. J. Med. 327:1434 –1441; 1992. 7. Jones, D. A.; Cummings, J.; Langdon, S. P.; Smyth, J. F. Preclinical studies on the broad-spectrum neuropeptide growth factor Antagonist G. Gen Pharmacol. 28:183–189; 1997. 8. Layton, J. E.; Scanlon, D. B.; Soveny, C.; Morstyn, G. Effects of bombesin antagonists on the growth of small cell lung cancer cells in vitro. Cancer Res. 48:4783– 4789; 1988. 9. Mahmoud, S.; Staley, J.; Taylor, J.; Bogden, A.; Moreau, J. P.; Coy, D.; Avis, I.; Cuttitta, F.; Mulshine, J.; Moody, T. W. [Psi13,14] bombesin analogs inhibit growth of small cell lung cancer in vitro and in vivo. Cancer Res. 51:1798 –1802; 1991. 10. Moody, T. W.; Cuttitta, F. Growth factor and peptide receptors in small cell lung cancer. Life Sci. 52:1161–1173; 1993.
11. Orosz, A.; Schrett, J.; Nagy, J.; Bartha, L.; Scho¨n, I.; Nye´ki, O. New short-chain analogs of a substance-P antagonist inhibit proliferation of human small-cell lung cancer cells in vitro and in vivo. Int. J. Cancer 60:82– 87; 1995. 12. Reeve, J. G.; Bleehen, N. M. [D-Arg1, D-Phe5, D-Trp7,9, Leu11] substance P induces apoptosis in lung cancer cell lines in vitro. Biochem. Biophys. Res. Commun. 199:1313–1319; 1994. 13. Seckl, M. J.; Higgins, T.; Widmer, F.; Rozengurt, E. [DArg1, 5,7,9 DTrp , Leu11] substance P—a novel potent inhibitor of signal transduction and growth in vitro and in vivo in small cell lung cancer cells. Cancer Res. 57:51–54; 1997. 14. Sethi, T.; Rozengurt, E. Multiple neuropeptides stimulate clonal growth of small cell lung cancer: effects of bradykinin, vasopressin, cholecystokinin, galanin and neurotensin. Cancer Res. 51:3621–3623; 1991. 15. Sethi, T.; Langdon, S.; Smyth, J.; Rozengurt, E. Growth of small cell lung cancer cells: stimulation of multiple neuropeptides and inhibition by broad spectrum antagonists in vitro and in vivo. Cancer Res. 52:2737s–2742s; 1992. 16. Tallett, A.; Chilvers, E. R.; Hannah, S.; Dransfield, I.; Lawson, M. F.; Haslett, C.; Sethi, T.; Inhibition of neuropeptide-stimulated tyrosine phosphorylation and tyrosine kinase activity stimulates apoptosis in small cell lung cancer cells. Cancer Res. 56:4255– 4263; 1996. 17. Woll, P. J.; Rozengurt, E. A neuropeptide antagonist that inhibits the growth of small cell lung cancer in vitro. Cancer Res. 50:3968 –3973; 1990. 18. Yang, H. K.; Kelley, M. J.; Battey, J. F.; Mulshine, J.; Cuttitta, F.; Linnoila, R. I.; Simmons, A.; Johnson, B. E. Correlation of gastrin releasing peptide, gastrin releasing peptide receptor and neuromedin B receptor mRNA expression in small cell lung cancer cell lines with in vitro response to anti-bombesin monoclonal antibody. Proc. Am. Assoc. Cancer Res. 34:343; 1993.
PII S0196-9781(98)00116-8
Induction of Apoptosis by a Short-Chain Neuropeptide Analog in Small Cell Lung Cancer RITA ROSATI,* MOHAMMED R. ADIL,* MUHAMMAD A. ALI,* JAMES ELIASON,* ANTAL OROSZ,† FERENC SEBESTYE´N† AND GREGORY P. KALEMKERIAN* 1 *Division of Hematology and Oncology, Wayne State University and the Barbara Ann Karmanos Cancer Institute, Detroit, MI 48201 †Tumor Biology Laboratory, Departments of Plant Physiology and Organic Chemistry, Eo¨tvo¨s Lora´nd University, Budapest, Hungary Received 20 May 1998; Accepted 2 July 1998 ROSATI, R., M. R. ADIL, M. A. ALI, J. ELIASON, A. OROSZ, F. SEBESTYE´N AND G. P. KALEMKERIAN. Induction of apoptosis by a short-chain neuropeptide analog in small cell lung cancer. PEPTIDES 19(9) 1519 –1523, 1998.—Small cell lung cancer (SCLC) cells express a variety of neuropeptides which act as autocrine growth factors. Although several neuropeptide analogs have been reported to antagonize SCLC proliferation, the development of these compounds has been limited by their low potency and the cytostatic nature of their effects. In the present study we evaluated the cytotoxic activity of four short-chain substance P analogs (NY3460, NY3238[-pHOPA], NY3238[Phe1], NY3238[Lys5]) against a panel of five SCLC cell lines. NY3460 was the most potent compound in all five SCLC cell lines (IC50 5 2.8 –3.7 mM) as assessed by a MTT growth inhibitory assay. NY3238[Phe1] was also relatively active in all cell lines (IC50 5 3.5–11.2 mM), while NY3238[Lys5] and NY3238[-pHOPA] were substantially less active. NY3460 was the only agent to induce an increase in the percentage of cells with subdiploid DNA content suggestive of apoptosis by flow cytometric DNA content analysis. The induction of apoptosis was confirmed by fluorescent microscopy in NCI-H69, NCI-H82, NCI-H446, and NCI-H510 cells after exposure to 5.0 mM NY3460 for 48 h. These findings suggest that NY3460 is a relatively potent cytotoxic inhibitor of SCLC growth, and that short-chain neuropeptide analogs deserve further evaluation as anti-SCLC agents. © 1998 Elsevier Science Inc. Small cell lung cancer
Substance P analogs
Cytotoxicity
LUNG cancer continues to be the leading cause of cancerrelated death in developed countries, with small cell lung cancer (SCLC) accounting for 20 –25% of all lung cancer deaths. SCLC is characterized by neuroendocrine differentiation, early metastatic potential and initial sensitivity to cytotoxic therapy. Unfortunately, the duration of response to treatment is usually short and most patients die with treatment-resistant disease (6). Recent advances in our understanding of the biology of SCLC have led to many novel therapeutic strategies aimed at improving the dismal prognosis of patients with this disease (5). SCLC cells secrete numerous hormones and neuropeptides which interact with specific receptors expressed on
Apoptosis
Experimental therapeutics
SCLC cell membranes and act as autocrine growth factors stimulating tumor cell proliferation (10,14). Based on these findings, a variety of neuropeptide antagonists have been developed in an attempt to inhibit the growth of SCLC cells. Substance P analogs have been found to inhibit both basal and neuropeptide-induced proliferation of SCLC cells (15), but only one of these analogs has been reported to induce apoptosis in selected SCLC cell lines (12,16). Numerous structural and chemical modifications, including shortening of the peptide chain, have been attempted in an effort to develop more potent and metabolically stable neuropeptide analogs (2,11,17). One short-chain substance P analog, Antagonist G, has undergone extensive preclinical evaluation
1 Requests for reprints should be addressed to Gregory P. Kalemkerian, MD, Harper Hospital, Hudson 515, 3990 John R., Detroit, MI 48201, USA Tel: (313) 745-2357; Fax: (313) 993-0559; E-mail: [email protected]
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and is currently in the early phases of clinical development (7). In the present study, the cytotoxic activity of four novel short-chain substance P analogs was evaluated in a heterogeneous panel of human SCLC cell lines. METHOD Cell Culture Five small cell carcinoma cell lines (NCI-H69, NCI-H82, NCI-H345, NCI-H446, NCI-H510) were obtained from American Type Culture Collection (Rockville, MD) and propagated in RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 5% fetal calf serum (FCS) (Sigma, St. Louis, MO), penicillin (100 U/ml), streptomycin (100 mg/ ml), and HITES [hydrocortisone (10 nM); insulin (15 mg/ ml); transferrin (100 mg/ml); 17-b-estradiol (10 nM); selenium (30 nM)] in a humidified 5% CO2 incubator at 37°C. All neuropeptide analogs were synthesized and supplied by two of us [A.O. and F.S.]. Vehicle-control cells for all experiments were exposed to 0.5% ethanol, which had no effect on basal SCLC cell proliferation. Growth Inhibition Assay Cells were seeded in 96-well plates with 200 ml/well and 5 wells per sample concentration at a density of 1– 4 3 104 cells/ml for 24 h prior to treatment with a range of concentrations of each neuropeptide analog up to 54 mM. Control cells were treated with 0.5% ethanol and blank wells were loaded with media only. After 5 days of neuropeptide exposure, 50 ml of 1.0 mg/ml MTT (Sigma Chemicals, St. Louis, MO) was added to each well and plates were incubated for 4 h at 37°C prior to centrifugation, removal of supernatant, and dissolution of formazan crystals in 200 ml of DMSO for 1 h at room temperature. Absorbance at 570 nm (A570) was determined for each well, and the survival fraction for each study condition was calculated from the ratio: (mean A570 treated cells–mean A570 blank wells)/ (mean A570 control cells–mean A570 blank wells). The IC50 was determined from dose-response data from at least three experiments with CalcuSyn software (Biosoft, Cambridge, UK). Flow Cytometry For DNA content analysis, 1 3 106 cells were exposed to either 0.5% ethanol or 5.0 mM of one of the neuropeptide analogs for 24 or 48 h prior to ethanol fixation and staining with 5.0 mg/ml propidium iodide (PI) 1 RNase A. All samples were analyzed using a Becton-Dickinson (San Jose, CA) FACScan flow cytometer. Relative DNA content (PI) was detected with bandpass filter 585/42 and the FACScan doublet discrimination circuit was employed to detect aggregates. Data were acquired in listmode, ungated to 105 events/sample, and analyzed using ModFit (Verity, Topsham, ME) and PC-LYSYS software. All experiments were repeated twice.
ROSATI ET AL. TABLE 1 AMINO ACID SEQUENCES OF SUBSTANCE P AND ANALOGS Name
Sequence
Substance P Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 Antagonist G Arg-DTrp-NMePhe*-DTrp-Leu-Met-NH2 † ‡ NY3460 DMePhe -DTrp-Phe-DTrp-Leu-c(CH2NH) Leu-NH2 NY3238 pHOPA§DTrp-Phe-DTrp-Leu-Leu-NH2 NY3238[2pHOPA]DTrp-Phe-DTrp-Leu-Leu-NH2 NY3238[Phe1] Phe-DTrp-Phe-DTrp-Leu-Leu-NH2 NY3238[Lys5] pHOPA§-DTrp-Phe-DTrp-Lys-Leu-NH2 Shaded areas 5 compounds used in present study. * NMe, Na-methyl-phenylalanine. † DMePhe, D-methyl-phenylalanine. ‡ c(CH2NH), pseudopeptide bond. § pHOPA, para-hydroxy-phenyl-acetic group.
Fluorescent Microscopy Cells (1–3 3 106) were exposed to either 0.5% ethanol or 5.0 mM of one of the neuropeptide analogs for up to 72 h prior to ethanol fixation and staining with 5.0 mg/ml PI 1 RNase A. Samples were analyzed on a Zeiss Laser Scanning Microscope LSM 310 with a 543 nm HENE laser. Experiments were repeated twice and representative data reported. RESULTS Growth Inhibition The amino acid sequences of substance P and the relevant neuropeptide analogs are shown in Table 1. The growth inhibitory activity of four of these analogs (NY3460, NY3238[-pHOPA], NY3238[Phe1], NY3238[Lys5]) was evaluated in a panel of five human SCLC cell lines (NCIH69, NCI-H82, NCI-H345, NCI-H446, NCI-H510). Unlike most prior studies, all experiments were performed in the presence of serum. Cell viability was determined by a standardized MTT assay after 5 days of exposure to each compound. NY3460 was the most potent inhibitor of growth in all five cell lines, with IC50 values of 2.8 –3.7 mM (Table 2). All five cell lines were also relatively sensitive to NY3238[Phe1] with IC50 values of 3.5–11.2 mM. The growth inhibition induced by both NY3460 and NY3238[Phe1] was clearly dose-dependent. In contrast, only two cell lines, NCI-H69 and NCI-H446, exhibited IC50 values below 20 mM after treatment with NY3238[Lys5], while all five SCLC cell lines were relatively resistant to NY3238[-pHOPA] (Table 2). Cell Cycle Analysis Flow cytometric DNA content analysis was performed in NCI-H82 and NCI-H69 SCLC cells after exposure to 5.0 mM of each compound for 48 h. Exposure to NY3460 resulted in a modest accumulation of cells in G1/G0 and a commensurate decrease in the proliferative cell fraction (S
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1521 TABLE 2
GROWTH INHIBITORY ACTIVITY OF NEUROPEPTIDE ANALOGS AGAINST SCLC CELL LINES Neuropeptide iC50 (mM)* Cell line
NY3460
NY3238[2pHOPA]
NY3238[Phe1]
NY3238[Lys5]
NCI-H69 NCI-H82 NCI-H345 NCI-H446 NCI-H510
2.8 6 0.2 3.0 6 0.1 3.1 6 0.2 3.1 6 0.4 3.7 6 0.3
.50 23.4 6 0.6 .50 30.6 6 7.6 .50
10.8 6 1.2 4.9 6 0.8 6.7 6 1.2 3.5 6 0.9 11.2 6 2.9
14.7 6 3.1 27.5 6 0.7 41.4 6 0.2 10.5 6 0.7 40.8 6 2.7
* Values represent mean of at least two experimetns 6 SD.
1 G2/M) in both NCI-H82 (25%) and NCI-H69 (19%) cells. In addition, a substantial increase in the percentage of cells with hypodiploid DNA content, a finding that is suggestive of apoptosis, was noted in both NCI-H82 (4.3-fold) and NCI-H69 (3.1-fold) cells (Fig. 1). No substantial changes were noted in the cell cycle phase distribution or hypodiploid fraction after exposure to NY3238[-pHOPA], NY3238[Phe1], or NY3238[Lys5] for up to 48 h (Fig. 1).
Induction of Apoptosis To confirm the induction of apoptosis by NY3460, both treated and untreated SCLC cells were stained with propidium iodide and evaluated by fluorescent microscopy. All five SCLC cell lines were exposed to 5.0 mM each neuropeptide analog for 24 to 72 h prior to morphologic evaluation. After 48 h of exposure to NY3460, characteristic
FIG. 1. DNA content analysis. Single-parameter histograms of linear PI fluorescence indicating relative DNA content. Percentages indicate cells with reduced (less than diploid) DNA content, consistent with apoptosis, as determined by integration of the area under the curve within the marker limits. Upper left, control NCI-H82 SCLC cells; upper center, NCI-H82 cells treated with 5.0 mM of NY3460 for 48 h; upper right, NCI-H82 cells treated with 5.0 mM NY3238[Phe1] for 48 h; lower left, control NCI-H69 SCLC cells; lower center, NCI-H69 cells treated with 5.0 mM NY3460 for 48 h; lower right, NCI-H69 cells treated with 5.0 mM NY3238[Phe1] for 48 h.
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FIG. 2. Fluorescent micrographs of SCLC cells stained with propidium iodide after exposure to 0.5% ethanol (left column) or 5.0 mM NY3460 (right column) for 48 h. Note chromatin condensation and nuclear fragmentation consistent with apoptosis in all three cell lines following exposure to NY3460 (magnification 6003).
apoptotic features, including chromatin condensation and nuclear fragmentation, were clearly evident in the majority of NCI-H69, NCI-H82, and NCI-H446 cells (Fig. 2). NCIH510 cells exhibited a modest degree of apoptosis, while NCI-345 cells did not show any morphologic changes after up to 72 h of exposure to 5.0 mM NY3460 (data not shown). No morphologic evidence of apoptosis was seen in any of the SCLC cell lines after exposure to NY3238[-pHOPA], NY3238[Phe1], or NY3238[Lys5] (data not shown). These data suggest that NY3460, but not the other neuropeptide analogs studied, induced growth inhibition via apoptotic cell death in most SCLC cells. DISCUSSION Dramatic advances have been made over the past 20 years in our understanding of the cellular and molecular biology of SCLC. The identification of autocrine pathways that regulate the proliferation of SCLC cells is one of the most therapeutically promising of these advances. Thus far, neuropeptides reported to act as autocrine growth factors in SCLC include gastrin-releasing peptide, cholecystokinin, vasopressin, and bradykinin (14). These compounds stimulate calcium mobilization, tyrosine kinase activity and cellular proliferation at nanomolar concentrations in a variety of SCLC cell lines, including NCI-H69, NCI-H345, and
ROSATI ET AL.
NCI-H510 (15). Numerous studies have demonstrated that broad-spectrum neuropeptide analogs can antagonize neuropeptide-induced signal transduction pathway activation and inhibit both in vitro and in vivo growth of SCLC cells (1,2,13,16). Although substance P analogs can clearly inhibit SCLC growth in a cytostatic manner, the induction of apoptosis by these compounds has been reported in only two studies and only after exposure to relatively high concentrations (12,16). In the present study, the activity of several short-chain substance P analogs was evaluated in of a panel of human SCLC cell lines representing the genetic, phenotypic and biochemical variations seen in human SCLC tumors (3). NY3460 was the most potent inhibitor of growth in all cell lines tested, with IC50 values of 2.8 –3.7 mM and morphologic evidence of apoptosis in four of five cell lines. It has previously been reported that NY3460 inhibited the growth of NCI-H69 cells both in vitro (IC50 5 3.0 mM) and in an in vivo xenograft model (11). The activity of the other three analogs used in the present study has not been previously reported. In previous studies, apoptosis after exposure to the long-chain substance P analog, [DArg1, DPhe5, DTrp7,9, Leu11] substance P, was observed only after exposure to high concentrations of 25–50 mM in serum-free conditions (12,16). Antagonist G, a short-chain substance P analog currently in early clinical trials (Table 1), has been shown to inhibit proliferation of NCI-H69 and NCI-H345 cells in a cytostatic manner with an IC50 of 10 –20 mM (17), and to inhibit the growth of NCI-H69 xenografts in nude mice (7). In the present study, the induction of apoptosis by NY3460 at substantially lower concentrations and in the presence of serum suggests greater potential clinical relevance. Interestingly, the activity of the short-chain neuropeptide analogs does not appear to correlate with the degree of neuroendocrine differentiation of the SCLC cell lines or the expression of specific neuropeptide receptors, findings which have also been reported for the activity of long-chain substance P analogs (8,12,15) and an anti-growth factor monoclonal antibody (18). Neuroendocrine differentiation is relatively high in so-called “classic” SCLC cell lines, such as NCI-H345, NCI-H510, and NCI-H69, and low to absent in ‘variant’ lines, such as NCI-H82 and NCI-H446 (3). However, the growth inhibitory activity of NY3460 is similar in all lines, and the activity of the other analogs evaluated is actually greatest in the ‘variant’ NCI-H82 and NCI-H446 cell lines. It has previously been reported that, despite being a potent inhibitor of SCLC proliferation, NY3460 was a less efficient inhibitor of bombesin:bombesin receptor binding than other short-chain analogs with less potent growth inhibitory activity (11). Neuropeptide analogs have also been shown to inhibit calcium mobilization and protein tyrosine kinase activity induced by a wide variety mitogens acting through distinct receptors (13,15,16). The targeting of a shared signal transduction
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pathway would increase the therapeutic utility of such agents by allowing activity against SCLC tumors with varying neuropeptide growth factor and receptor phenotypes. Correlations between the structure and activity of neuropeptide antagonists have been difficult to extract from the available literature. In the present study, the hexapeptide analogs (NY3460, NY3238[Phe1]) were substantially more potent inhibitors of SCLC growth than the pentapeptide analogs (NY3238[-pHOPA], NY3238[Lys5]). This finding is most striking for NY3238[Phe1] and NY3238[-pHOPA], which differ only by the addition of an N-terminal phenylalanine residue in NY3238[Phe1]. In addition, NY3460, which induces apoptosis, and NY3238[Phe1], which does not, differ in only two respects, the N-terminal substitution of D-methyl-phenylalanine for phenylalanine and the insertion of a pseudopeptide bond between the C-terminal leucine residues in NY3460. The insertion of a pseudopep-
tide bond has also been shown to increase the growth inhibitory activity of long-chain neuropeptide analogs, but has not previously been associated with apoptosis (9). The pharmacologic stimulation of apoptotic pathways represents a rational approach to the prevention and treatment of cancer (4). Thus, the induction of apoptosis by NY3460 at relatively low concentrations suggests that such short-chain neuropeptide analogs may have potential utility for the treatment of SCLC. ACKNOWLEDGEMENTS We are grateful to Dr. Mary L. Varterasian for critical review of the manuscript; Ms. Linda Mayernik for technical assistance and figure production; and the Charlotte A. Woody Cancer Research Fund and the Webber Medical Advancement Fund for continued support. We also acknowledge the Analytical Cytometry and Confocal Imaging Core Facilities (supported in part by center grants P30CA22453 from the National Cancer Institute and P30ES06639 from the National Institutes of Environmental Health Sciences).
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