Thymosin Al Down-Regulates the Growth of Human Non-Small Cell Lung Cancer Cells in Vitro and in Vivo Terry W

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[CANCER RESEARCH 53, 5214-5218, November 1, 1993] Thymosin al Down-regulates the Growth of Human Non-Small Cell Lung Cancer Cells in Vitro and in Vivo Terry W. Moody,1 MÃrelaFagarasan, Farah Zia, Mirjana Cesnjaj, and Allan L. Goldstein

Department of Biochemistry and Molecular Biology, The George Washington University School of Medicine and Health Sciences, Washington, DC 20037 Â¡T.W. M., M. F., E Z., A. L. G.I, and Laboratory of Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, NIH, Bethesda, Maryland 20892 [M. C.I

ABSTRACT increased survival especially in patients with nonbulky tumors (15): THNal in combination with interferon after cyclophosphamide treat The elicci of thymosin al (THNal) and its NH2-terminal fragment (THN1-14) and COOH-terminal fragment (THIS15-2") on non-small cell ment increased survival of mice with Lewis lung carcinomas (16). lung cancer (NSCLC) growth was evaluated. Using an anti-THNal anti These data suggest that THNal inhibits lung cancer proliferation. Here the effects of THN-like peptides on NSCLC cells were investi body, receptors were identified on NSCLC cells that were pretreated with IO-6 M THNal. | 'll| \niihidoMic acid was readily taken up by NSCLC gated. cells and THNal significantly increased the rate of arachidonic acid re lease. THN1-15 slightly stimulated but THN1"" and THNÃŸ4did not alter MATERIALS AND METHODS arachidonic acid release troni NCT-H1299 cells. In clonogenic growth assays in vitro, THNal (10~* M) significantly decreased NSCLC colony Cell Culture. NSCLC cells were cultured in RPMI 1640 containing 10% number whereas THN'-'4, THN1"8, and THNÃŸ4were less potent. Using heat-inactivated fetal bovine serum. When a monolayer formed, the adherent growth assays in vivo, THNal (10 /ig s.c./day) but not THN1-14, THN15-28, cells were washed with PBS and treated with trypsin/EDTA. The cells were or IIIN/ÃŽ-Iinhibited significantly NSCLC xenograft formation in nude pelleted and resuspended in serum supplemented medium and incubated at 37Â°Cin 5% COz/95% air (17). Routinely the cells were passed 1/1 weekly and mice. These data suggest that biologically active THNal receptors are present on NSCLC cells and that native THNal inhibits the growth of experiments were conducted when the cells were in exponential growth phase. human NSCLC. The cells utilized included NCI-H157 (squamous cell carcinoma) and NCI- H1299 (large cell carcinoma), NC1-H727 (lung carcinoid), and NCI-H838 and NCI-HI264 (adenocarcinoma). INTRODUCTION Immunocytochemistry. Antiserum (PR-1) to synthetic THNal-carbo- THNal,2 a 28-amino acid peptide which functions as a biological diimide-hemocyanin conjugates were elicited in rabbits. The antibodies were immunoaffinity purified (NSP) and PR-1 or NSP were used at 1:1000 dilution. response modifier by virtue of its immunomodulatory effects on NSCLC cells (IX IO4) were plated in Nunc chambers coated with fibronectin. T-cells, was initially isolated from a crude bovine thymus extract, After 4 days the media were changed. After 16 h the cells were treated with THN fraction 5 (1). THNal is derived from a 113-amino acid pre THN-like peptides for 30 min. The cells were washed twice with PBS con cursor protein, prothymosin a; the NH2-terminal 28-amino acid se taining 1% BSA, fixed with PBS containg 2% glutaraldehyde, and rinsed with quence of prothymosin a is identical to that of THNal (2, 3). By PBS containing 5% BSA and 4% normal goat serum. Subsequently the cells radioimmunoassay THN-like peptides have been detected in numer were treated with NSP or PR-1 at 25Â°C.After 16 h, the cells were washed twice ous tissues including the rat thymus, pituitary, and brain (4). In addi in PBS containing 5% BSA. The cells were treated with goat anti-rabbit serum tion to THNal, THNÃŸ4,a 43-amino acid peptide with pleotropic (1:60 dilution) for 60 min followed by incubation with PBS containing 5% effects as a biological response modifier, was isolated from THN BSA for 10 min. The washing with PBS/BSA was repeated three times. Then fraction 5 (5). THNÃŸ4, which has little sequence homology to the samples were treated with water for 3 min and this process was repeated 3 times. The samples were treated with Auroprobe LM and intense M (Amer- THNal, is an actin-sequestering peptide, stimulates terminal deoxy- sham) for light microscopic immunogold silver staining. The samples were nucleotidyltransferase, and stimulates luteinizing hormone-releasing then treated with water for 5 min twice and the cells were visualized for factor secretion from the rat hypothalamus resulting in increased immunostaining. luteinizing hormone secretion from the pituitary (5-7). Arachidonic Acid Release. NSCLC cells (5 x 10") were placed in 24-well THNal has been localized to the surface of mouse thymic epithelial plates coated with human fibronectin (20 fig) as described previously (11). cells using anti-THN antibodies (8). While the second messenger for After a monolayer of cells formed (5 days) [5,6,8,9,11,12,14,15-3H]arachi- THNal is not known, THN fraction 5 stimulated arachidonic acid donic acid (2.5 x IO5 cpm) was added. After 16 h the cells were washed twice in 1 ml of SIT medium (RPMI 1640 containing 3 X IO'8 MNa2SeO3, 5 /J.g/ml release from anterior pituitary cells (9). Also, THNal has sequence homology to VIP (10). THNal is reported to inhibit 125I-VIP binding insulin and 10 fig/ml transferrin) containing 0.2% fatty acid-free bovine serum albumin (S1T/BSA). New medium were added containing thymosin-like pep sites in rat blood mononuclear cells and liver plasma membranes with tides. After 40 min. 100 fil of media were removed from each well and placed low affinity (50% inhibitory concentration, 1 JAM).These studies sug in a scintillation vial, scintillation fluid added, and the samples were counted gest that THNal can interact with VIP receptors. Previously we found 125I-VIP binding sites on human lung cancer cells (11, 12). in a beta counter. Clonogenic Assay. NSCLC cell lines were harvested and tested in the THN fraction 5 prolongs survival of some lung cancer patients (13). agarose cloning system described previously (18). The base layer consisted of In postradiotherapy patients with NSCLC, synthetic THNal acceler 2 ml of 0.5% agarose in SIT medium containing 5% fetal bovine serum in ated reconstitution of thymic-dependent immunity (14). Also, THNal 6-well plates (Falcon, Oxnard, CA). The top layer consisted of 2 ml of SIT medium in 0.3% agarose, the THN-like peptides, and 1 X IO4 single viable cells/ml. For each cell line and peptide concentration, triplicate wells were Received 5/24/93; accepted 8/27/93. plated. After 2 weeks 1 ml of 0.1% p-iodonitrotetrazolium violet was added The costs of publication of this article were defrayed in part by the payment of page and after 16 h at 37Â°Cthe plates were screened for colony formation; the charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. number of colonies larger than 120 fÂ¿min diameter were counted. ' To whom requests for reprints should be addressed, at Biomarkers and Prevention Nude Mouse Assay. The ability of THNal to inhibit xenograft formation Res. Br.. NCI, 9610 Medical Center Dr., Bldg. C, Rm. 3(K). Rockville, MD 20850. in nude mice was investigated (19). Female athymic BALB/c nude mice, 4-5 2 The abbreviations used are: THNal, thymosin al; VIP, vasoactive intestinal peptide; weeks old, were housed in a pathogen-free temperature-controlled isolation PBS. phosphate-buffered saline; NSCLC, non-small cell lung cancer; BSA. bovine serum albumin; EOF, epidermal growth factor; cAMP. cyclic AMP; TGFa, transforming growth room and the diet consisted of autoclaved rodent chow and autoclaved water factor a. given ad libitum. NCI-H157 or H727 cells (1 X IO7) were injected into the 5214

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1993 American Association for Cancer Research. THYMOSIN ai DOWN-REGULATES GROWTH OF HUMAN NSCLC right flank of each mouse by s.c. injection. Palpable tumors were observed in <â€¢â€¢Â«â€¢ approximately 90% of the mice after 2 weeks. PBS (100 fil) or THN-like peptides (10 fig/day) were injected s.c. adjacent to the tumor. The tumor volume (height x width x depth) was determined weekly by calipers and recorded. When the tumor became necrotic, the growth studies were termi nated.

RESULTS ' Immunocytochemistry. Previously, we found that THNal antiserum recognized the COOH terminus of THNal whereas the NH2 terminus was essential for biological activity (4). Here NSCLC cells were fixed with glutaraldehyde and treated with a primary antibody (NSP or PR-1), followed by a secondary antibody (goat anti-rabbit serum) and silver enhancer. Using large cell carcinoma cells (NCI- H1299) optimal staining was observed using a 1:1000 dilution of NSP or PR-1. Cells did not stain in the absence of THNal antiserum (Fig. L4) or THNal treatment (Fig. IÃŸ).NCI-H1299 cells immunostained B if they were treated with l /ÃœMTHNal, followed by extensive wash ing, fixation, and treatment with primary and secondary antibody (Fig. 1C). These data suggest that THNal binds to NSCLC cell surface component. Similar data were obtained using adenocarcinoma (NCI- H838), squamous cell carcinoma (NCI-H157), and lung carcinoid cells (NCI-H727). Arachidonic Acid. Previously it was found that THN fraction 5 stimulated arachidonic acid release from pituitary cells (9). Here the effects of THN-like peptides on arachidonic acid release were inves tigated using NSCLC cells. THNal had little effect on arachidonic acid release at a 10~9 M concentration using NCI-HI299 cells but â€¢¿>â€¢* significantly stimulated arachidonic acid release at IO"6 Mconcentra tion (Fig. 2). THNal (10~5 M) increased the rate of arachidonic acid release approximately 3-fold and the half-maximal effective concen tration was 0.7 X 10~7 M. Table 1 shows that TON1'14 (IQ-6 M) slightly increased arachidonic acid release, respectively, whereas THN'5-2* and THNÃŸ4had no effect. Similar results were obtained using NCI-H727, 838, and 1264 cells. NSCLC Proliferation. The effect of THNal on NSCLC growth was investigated in vitro. Fig. 3 (top) shows that 150 NC1-H1299 colonies formed in the control culture. When 100 nMVIP was added the number of colonies formed increased significantly to 322 (data not shown). THNal inhibited NCI-HI299 colony formation in a dose- dependent manner with IO"7, IO"6, or W~5 but not IO"8 M THNal causing significant inhibition. Fig. 3 (middle) shows that numerous large viable colonies formed. In the presence of l JU.MTHNal [Fig. 3 (bottom)] the number of colonies was reduced as was the size. Similar data were obtained using NSCLC cell lines NCI-H157, H727, H838, and H1264. Table 2 shows that other THN-like peptides inhibited NCI-H838 Fig. l. Immunocytochemistry of NSCLC cell lines. NCI-H1299 cells were treated with colony formation. THN1"14 and THNI5~28 inhibited colony formation (B, C) or without (A) anti-THNal antisera after treatment with (C) or without (A, B) l ^LMTHNal. The cells were then silver stained as described in "Materials and Methods." by approximately 25% whereas THNÃŸ4was inactive. These data This experiment is representative of 2 others. suggest that NH2- and COOH-terminal fragments of THNal have some biological activity but neither fragment is as potent as is native THNal. in that if treatment was discontinued, the growth rate of the tumor Also, the effects of THNal on NSCLC growth was investigated in returned to normal values. vivo. Fig. 4 shows that tumors formed 1 week after s.c. injection of NCI-H157 cells into nude mice. Fig. 4 (top) shows that the NSCLC DISCUSSION xenografts grew exponentially and after 5 weeks the xenograft volume was 2306 mm1. When THN1-'4 or TON15-28 was administered s.c. Previously, THNal was postulated to prolong survival of NSCLC (10 ju,g/day) xenograft volume was reduced by approximately 20%. In patients via effects of the immune system (15). Here THNal inhibited contrast, THNal (10 fig) significantly reduced tumor volume by the growth of NSCLC cells in vitro and in vivo suggesting that THNa 1 approximately 75%. A dose-response curve for THNal was deter directly interacts with NSCLC cells. mined. Fig. 4 (bottom) shows that 10 or 1 /xg of THNal significantly (125I-Tyr")THNal binds with high affinity to THNal antiserum but decreased NCI-H727 tumor volume whereas 0.1 jug of THNal or 10 not NSCLC cells. Additional studies indicated that THNal and p.g of THNÃŸ4were ineffective. The effects of THNal were reversible THN1A~2I

cv i o

o o o

-8 -7 -6 -5 (Peptide), Log M

I -8 -7 -6 -5 (THN alpha 1) , Log M Fig. 2. Effect of THNa-1 on arachidonic acid release. NCI-H1299 cells were loaded with pHJarachidonic acid and the amount of radiolaheled arachidonic acid released was determined with increasing concentrations of THNal {â€¢).This experiment is represen tative of 2 others. Bars, SD.

Table 1 Effect of THN-like peptides on arachidonic acid release

Agent added 3H]Arachidonic acid released (cpm) None 688 Â± 90" THNal, 10-6M 1734 Â± 72fc THN'-'4. 1(T6 M 855 Â± 61 THNi5-2Â« 10-6M 602 Â± 51 THN/34, IO"6 M 608 Â±168 " Mean Â±SE of 4 determinations using NCI-HI299 cells.

bP< 0.01.

.v. Table 2 Effect of THN-like peptides on NSCLC growth in vitro

addedNoneTHNAgent no.54 Â±4Â° ^MjTHN'-'4(115-2Â« (1 44 Â±4 /IM) 40Â±3fc THN04 (1 (Â¿M) 52 Â±5 17Â±4C THNal (1 (Â¿M)Colony Fig. 3. NSCLC clonal growth assay. (A) The effect of THNal on NC1-H1299 colony " The mean Â±SEof NCI-H838 colonies (n = 3) was determined. formation was determined in the absence (â€¢)or presence (O) of increasing concentrations kP< 0.05. of THNal. The mean value Â±SE (bars) of 3 determinations is indicated: P < 0.05. *. CP < 0.01. Large viable NCI-H838 colonies formed (B) whereas the colony size and number was reduced in the presence of 1 ^tMTHNal (C).

Therefore a unique immunocytochemical approach was used to dem VIP (20); the VIP and EOF receptor proteins are composed of 459- onstrate interaction of THNal with NSCLC cells. Using this assay, and 1186-amino acid residues, respectively (21, 22). THNal was bound to NSCLC cells and fixed with glutaraldehyde (the The ability of THNal to alter second messenger production was NH2 terminus of THNal may bind to a receptor protein whereas the determined. THNal (10 fj..w)did not alter intracellular cAMP, whereas COOH terminus was in solution and consequently interacted with 10 nMVIP strongly increased cAMP levels (20). THNal did not alter THNal antibodies). Then THNal antiserum was added, followed by tyrosine phosphorylation whereas EOF (100 ng/ml) activated tyrosine goat anti-rabbit sera, and the bound antibodies were visualized by kinase activity causing phosphorylation of the A/r 170,000 EOF re silver staining. These data suggest that THNal may interact with a ceptor (23). Also, THNal had no effect on phosphatidylinositol turn NSCLC cell surface component, possibly a THNal receptor. Also, over or cytosolic calcium but did significantly stimulate arachidonic THNal can bind to the VIP receptor inasmuch as THNal inhibits acid release. The effect was dose dependent and THNal significantly 125I-VIP binding to NSCLC cells with low affinity (50% inhibitory stimulated [3H]arachidonic acid release. THN1"14 slightly stimulated concentration, 10 /iM3). Previously we found that almost all NSCLC arachidonic acid release whereas THNI5~2K and THNÃŸ4did not alter cell lines examined have high affinity binding sites for EOF (19) and arachidonic acid release. These data suggest that THNal may stimu late phospholipase A2 but not adenylate cyclase activity or tyrosine 1T. Moody, unpublished observations. kinase activity. Previously, it was found that NSCLC cells have cy- 5216

and was less effective if injected i.p. or i.v. It is possible that THNal is degraded by serum proteases and we are currently investigating the PBS half-life of THN-like peptides in the blood. Recently, we found that

ÃŒTHNJ-1Â« THNamide, which has an amidated COOH terminus and is resistant to THN15-28 degradation by carboxypeptidases, strongly inhibits NSCLC growth. VIP, which has an amidated COOH terminus (28), is also resistant to degradation by carboxypeptidases. It remains to be determined if THN-like peptides function as auto crine growth factors in NSCLC. By radioimmunoassay, THN-like peptides are present in NSCLC extracts and conditioned medium.3 Previously, TGFa mRNA was found in NSCLC cells and TGFa THNalpha 1 immunoreactivity was found in conditioned medium (29). TGFa binds with high affinity to EOF receptors, causes tyrosine kinase activity, and stimulates growth (23). Because EOF receptor mono clonal antibodies inhibit the growth of NSCLC, TGFa may be a 3 4 postitive autocrine growth factor (19). Currently, we are investigating Time, Weeks if THNal is a negative autocrine growth factor for NSCLC. THNal may also be effective at slowing the growth of other cancers. In small cell lung cancer, VIP elevates cAMP levels, stimu lates early oncogene expression of c-fos, and increases the secretion B rate of bombesin-like peptides resulting in increased small cell lung cancer growth (30). THNal (10 JAM)inhibited the clonal growth of n i PBS several small cell lung cancer cell lines (20). o 10 ug THN beta4 In summary, THNal stimulates arachidonic acid release and inhib 0.1ug THN alphal its NSCLC growth in vitro and in vivo. Therefore THNal may func tion as a regulatory peptide in NSCLC.

*1 Ug THN alpha 1 REFERENCES

1. Goldstein, A. L., Low, T. L. K., McAdoo, M, McClure, J., Thurman, G. B., Rossio, J., Lai, C. Y, Chang, D., Wang, S. S., Harvey, C., Ramer, A. H. and Meienhofcr. J. Thymosin al: isolation and sequence analysis of an immunologically active thymic *lOug THN alpha 1 polypeptide. Proc. Nati. Acad. Sci. USA, 74: 725-729. 1977. 2. Grangou-Laxaridis. M., Clinton. M.. Goodall, G. J., and Horecker, B. L. Prolhymosin a and parathymosin: amino acid sequences deduced from the cloned rat spleen cDNAs. Arch. Biochem. Biophys.. 26.?: 305-310, 1988. 2345 3. Eschenfeldt, W. H., and Berger. S. L. The human prothymosin a gene is polymorphic Time, weeks and induced upon growth stimulation: evidence using a cloned cDNA. Proc. Nati. Acad. Sci. USA, 83: 9403-9407. 1986. Fig. 4. NSCLC xenograft formation. (Top) NCI-H157 tumors formed after 2 weeks and 4. Palaszynski, E. W., Moody, T. W., O'Donohue, T. L., and Goldstein, A. L. Thymosin the mice were given daily injections of PBS (O), or 10 ug (s.c.) THN'-'4 (A), THN15-28 al-like peptides: localization and biochemical characterization in the rat brain and (A), and THNal (â€¢).(Bottom) Nude mice bearing NCI-H727 tumors were given daily pituitary gland. Peptides, 4: 463-469, 1983. s.c. injections of PBS (â€¢),10 ngTHNÃŸ4 (O), 0.1 /Â¿gTHNal (A), l /ig THNal (A), and 5. Low. T. L. K., and Goldstein, A. L. Chemical characterization of thymosin (34. J. Biol. 10 (ig THNal (â€¢).The mean tumor volume Â±SD of 3 determinations is indicated; P < Chem., 257: 1000-1008, 1982. 0.05, â€¢¿. 6. Rebar, R. W.. Miyake, A., Low, T. K. L.. and Goldstein, A. L. Thymosin stimulates secretion of luteinizing hormone releasing factor. Science (Washington DC), 214: 669-671, 1981. 7. Sanders, M. S.. Goldstein. A. L., and Wang, Y-L. Thymosin ÃŸ4(Fxpeptide) is a potent clooxygenase activity resulting in prostaglandin E2 production (24). It regulator of actin polymerization in living cells. Proc. Nati. Acad. Sci. USA, 89: remains to be determined if THNal alters prostaglandin levels in 4678-4682, 1992. 8. Rabien. N., Auger. C. and Monier. J. C. Immunolocalization of thymosin ori, thy- NSCLC cells. mopoietin and thymulin in mouse thymic epithelial cells at different stages of culture: THN-like peptides inhibited the growth of NSCLC cells in vitro. A light and electron microscopic study. Immunology. 63: 721-727, 1988. The effect was dose dependent and THNal (l JU.M)significantly 9. Spangelo, B. L.. Judd. A. M., Ross, P. C.. Login, I. S., Jarvis, D., Badamchian, M., inhibited colony formation. THN1"14 (1 pt,M)significantly inhibited Goldstein, A. L., and MacLeod, R. M. Thymosin fraction 5 stimulates prolactin and NSCLC growth but was less potent than was THNal. THN15~28 growth hormone release from anterior pituitary cells in vitro. Endocrinology. 727: 2035-2043, 1987. caused a slight inhibition of growth whereas THNÃŸ4was inactive. 10. Calvo, J. R., Guerrero, J. M.. and Goberna. R. Interactions of thymic peptide thymosin Previously it was found that THN1^14 was more potent at stimulating al with VIP receptors in rat intestinal epithelial cells: Comparison with PHI and T-cell function in aging mice than was THN15-28 (25). VIP (100 nM) secretin. Gen. Pharmacol.. 20: 503-505. 1989. 11. Lee, M., Jensen, R. T.. Huang. S. C.. Bepler, G., Korman, L., and Moody. T. W. significantly increased NSCLC colony formation whereas if VIP plus Vasoactive intestinal polypeptide binds with high affinity to non-small cell lung THNal was added there were few colonies.1 These data suggest that cancer cells and elevates cyclic AMP levels. Peptides, 77: 1205-1209, 1990. 12. Shaffer. M. M., Carney. D. N., Korman, L. Y, Lebovic. G. S., and Moody, T. W. the stimulatory effects of VIP on colony formation are overwhelmed High affinity binding of VIP to human lung cancer cell lines. Peptides, fi: by THNal. It remains to be determined if THNal alters NSCLC 1101-1106, 1987. 13. Cohen. M. H., Chretien, P. B., Inde, D. C., Fossieck, B. E., Makuch. R., Bunn, P. A., oncogene expression. NSCLC cells have amplification and increased Johnston. A. V, Shackney, S. E., Matthews, M. J., Lipson, S. D., Kenady. D. E., and expression of the c-myc and k-ras oncogenes (26, 27). Minna. J. D. Thymosin fraction 5 and intensive combination chemotherapy prolong THN-like peptides inhibited the growth of NSCLC in vivo. THNal ing the survival of patients with small-cell lung cancer. JAMA. 2-*7: 1813-1821, 1979. inhibited NSCLC growth in a dose-dependent manner and the half- 14. Schulof, R. S.. Lloyd, J. J., Cox, J., Palaszynski, S. R., McClure, J. E., Incefy, G., and maximal concentration was 1 fig (0.04 mg/kg). In contrast, THN1"14, Goldstein. A. L. An evaluation of two different schedules of synthtic thymosin al THN15-28, and THNÃŸ4had little effect. Preliminary data3 indicate administration in patients with lung cancerâ€”preliminary results. In: M. Chirogos and A. L. Goldstein (eds.), Thymic Hormones and Lymphokines, pp. 601-613. New York: that THNal was most effective if injected s.c. adjacent to the tumor Raven Press, 1984. 5217

15. Schulof. R. S.. Lloyd. M. J., Cleary, P. A., Palaszynski. S. R.. Mai, D. A., Cox, !. W., peptide. Neuron. 8: 811-819, 1992. Alabaster. O.. and Goldstein. A. L. A randomized trial to evaluate the immunorestor- 23. Moody, T. W., Lee, M., Kris, R. M.. BelliÂ«.F.. Bepler, G., Oie, H., and Gazdar, A. F. alive properties of synthetic thymosin al in patients with lung cancer. J. Biol. Lung carcinoid cell lines have bombesin-like peptides and EOF receptors. J. Cell. Response Modif.. 4: 147-158. 1985. Biochem.. 43: 139-147. 1990. 16. Garaci. E.. Mastino, A.. Pica. F.. and Favilli. C. Combination treatment using thy 24. Hubbard, W. C., Alley, M. C., Gray, G. N., Green, K. C., McLemore, T. L., and Boyd, mosin a 1 and Interferon after cyclophosphamide is able to cure Lewis lung carcinoma M. R. Evidence for prostanoid biosynthesis as a biochemical feature of certain in mice. Cancer Immunol. Immunother.. 32: 154-161), 1990. subclasses of non-small cell carcinomas of the lung as determined in established eell 17. Carney, D. N., Gazdar, A. F., Bepler, G., Guccion. J. G., Marangos, P. J., Moody. T. lines derived from human lung tumors. Cancer Res.. 49: 826-832, 1989. W., Zweig. M. H., and Minna, J. D. Establishment and identification of small cell lung 25. Frasca. D., Adorini, L., Mancini, C., and Doria. G. Reconstitution of T cell function cancer cell lines having classic and variant features. Cancer Res.. 45: 2913â€”2923. in aging mice by thymosin al. Immunopharmacology. //: 155-163, 1986. 1985. 26. Little, C. D.. Nau. M. M.. Carney, D. N.. Gazdar, A. F.. and Minna. J. D. Amplification 18. Mahmoud. S.. Staley. J., Taylor, J.. Bogden, A.. Moreau, J. P., Coy, D., Avis, I., and expression of the c-myc oncogene in human lung cancer cell lines. Nature Cuttilta, F.. Mulshine. J. L., and Moody, T. W. (Psi-I3,14)bombesin analogues inhibit (Lond.), 306: 194-196, 1983. growth of small cell lung cancer in vitro and in uYÂ«.Cancer Res., 5/: 1798-1802. 1991. 27. Rodenhuis, S., Van de Wetering, M. L.. Mooi, W. J., Evers, S. G., Van Zandwijk, N., and Bos, J. L. Mutational activation of the K-ras oncogene: a possible pathogenetic 19. Lee. M., Draoui, M., Zia, F., Gazdar, A. F., Oie. H.. Tarr. C.. Bello!, F., Kris, R.. and factor in adenocarcinoma of the lung. N. Engl. J. Med., 317: 929-935, 1987. Moody, T. W. Epidermal growth factor receptor monoclonal antibodies inhibit the growth of lung cancer cell lines. J. Nati. Cancer Inst. Monogr., 13: 117-123, 1992. 28. Said, S. I., and Mutt. V. Polypeptide with broad biological activity: isolation from the small intestine. Science (Washington DC), 6Â°:1217-1218. 1970. 20. Moody, T. W., Zia, F., Goldstein. A., Naylor, P., Sarin. E.. Brenneman, D., Koros, A. M. C., Reubi, J. C.. Fridkin. M.. and Gozes. I. VIP analogues inhibit small cell lung 29. Imanishi. K.. Yamaguchi. K.. Km.in.um. M.. Kyo. E.. Hozumi, T.. and Abe. K. cancer growth. Biomed. Res., 13: 131-136, 1993. Inhibition of growth of human lung adenocarcinoma cell lines by anti-transforming 21. Hunter, T., and Cooper. J. A. Protein tyrosine-kinascs. Annu. Rev. Biochem., 54: growth factor-a monoclonal antibody. J. Nail. Cancer Inst.. 81: 220-223, 1989. 897-930, 1985. 30. Korman, L. Y.. Carney. D. N., Citron. M. L.. and Moody, T. W. Secretin/VIP stimu 22. Ishihara, T., Shigemoto. R.. Mori. K.. Takahashi. K.. and Nagata, S. Functional lated secretion of bombesin-like peptides from human small cell lung cancer. Cancer expression and tissue distribution of a novel receptor for vasoactive intestinal poly- Res., 46: 1214-1218, 1986.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1993 American Association for Cancer Research. Thymosin α1 Down-regulates the Growth of Human Non-Small Cell Lung Cancer Cells in Vitro and in Vivo

Terry W. Moody, Mirela Fagarasan, Farah Zia, et al.

Cancer Res 1993;53:5214-5218.

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