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VIP-ellipticine derivatives inhibit the growth of breast cancer cells
Life Sciences 71 (2002) 1005 – 1014 www.elsevier.com/locate/lifescie
VIP-ellipticine derivatives inhibit the growth of breast cancer cells T.W. Moody a,*, G. Czerwinski b, N.I. Tarasova b, C.J. Michejda b a b
Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute, Rockville, MD 20850, USA Molecular Aspects of Drug Design Section, Macromolecular Structure Laboratory, Advanced BioScience Laboratories, National Cancer Institute, Frederick, MD 21702, USA Received 30 October 2001; accepted 20 December 2001
Abstract The effects of vasoactive intestinal peptide (VIP)-ellipticine (E) derivatives were investigated on breast cancer cells. VIP-ALALA-E and VIP-LALA-E inhibited 125I-VIP binding to MCF-7 cells with an IC50 values of 1 and 0.2 AM respectively. VIP-ALALA-E and VIP-LALA-E caused elevation of cAMP in MCF-7 cells with ED50 values of 1 and 0.1 AM. VIP-LALA-E caused increased c-fos mRNA in MCF-7 cells. Radiolabeled VIP-LALA-E was internalized at 37 jC and delivered the cytotoxic E into MCF-7 cells. VIP-LALA-E inhibited the clonal growth of MCF-7 cells, decreased cell viability based on trypan blue exclusion and reduced 35S-methionine uptake. These results indicate that VIP-E derivatives function as breast cancer VPAC1 receptor agonists which inhibit MCF-7 cellular viability. D 2002 Elsevier Science Inc. All rights reserved. Keywords: VIP-ellipticine derivatives; VIP agonists; VPAC1 receptor; Breast cancer
Introduction Vasoactive intestinal peptide (VIP) is a 28 amino acid peptide which functions as a growth factor in the central nervous system and periphery [1–3]. It is structurally related to pituitary adenylate cyclase activating polypeptide (PACAP) a 27 amino acid peptide [4]. VIP and PACAP bind with high affinity to VPAC1 and VPAC2 receptors which contain 459 and 430 amino acids respectively [5,6]. PACAP but not VIP binds with high affinity to PAC1 receptors which contain 495 amino acids [7,8].
*
Corresponding author. Tel.: +1-301-402-3128; fax: +1-301-402-4422. E-mail address: [email protected] (T.W. Moody). 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 7 4 1 - 1
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VPAC1 receptors interact with a stimulatory guanine nucleotide binding protein (Gs) resulting in adenylyl cyclase activation [9]. Addition of VIP or PACAP to breast cancer MCF-7 cells results in cAMP elevation. The increase in cAMP causes protein kinase A activation. Protein kinase A can phosphorylate protein substrates such as CREB leading to increased nuclear oncogene expression [10]. VIP causes increased c-fos and c-myc expression [9] in MCF-7 cells. VIP stimulates the proliferation of MCF-7 cells, whereas the VIP receptor antagonist VIPhybrid inhibits proliferation [11]. Recently we found that VIPhybrid enhances the ability of taxol or doxorubicin to kill MCF-7 cells [11]. Alternatively cytotoxic drugs may be coupled to a receptor agonist and internalized leading to breast cancer cell death [12]. Here tetra- and pentapeptide ellipticine (E) derivatives were attached to the C-terminal of VIP. The VIP-E derivatives bound to VPAC1 receptors on MCF-7 cells and transiently increased the cAMP and c-fos mRNA. The VIP-E derivatives, however, were cytotoxic for breast cancer cells. Methods Cell lines Breast cancer cells (MCF-7 and T47D) were cultured in DMEM containing 10% fetal bovine serum (Life Technologies, Rockville, MD). The cells were mycoplasm-free and were used when they were in exponential growth phase. Peptide Synthesis VIP-E derivatives (9-methoxy-1-chloro-5,11-dimethyl-6H-pyrido[4,3–h]carbazole) was synthesized as described previously [13]. The fmoc-pentapeptide (ALALA) or fmoc-tetrapeptide (LALA) was synthesized. The N-terminal was deprotected with 20% piperidine I DMF and coupled to E. After 2 hr, the product (LALA-E or ALALA-E) was purified by HPLC. The LALA-E or ALALA-E was then coupled to VIP-COOH with carbodiimide. Peptide purity was greater 98% based on HPLC, amino acid and mass spectroscopy analysis. The biological activity of VIP-ALALA-E and VIP-LALA-E was compared to that of VIP-G (Bachem, San Diego, CA); VIP has an amidated C-terminal whereas in VIP-G, an additional amino acid is added to the C-terminal, which has a free carboxyl group. Receptor binding For receptor binding studies, breast cancer cells in 24 well plates were washed 4 times with DMEM. Then 250 Al of the receptor binding medium (DMEM containing 2% BSA and 2 mg/ml bacitracin) was added with 100,000 cpm 125I-VIP (NEN, Boston, MA; 2200 Ci/mmol) in the absence or presence of competitor [9]. After incubation at 37 jC for 30 minutes, the plates were rinsed 3 times with receptor binding medium. The cells containing bound peptide were solubilized in 0.2 N NaOH and counted in a gamma counter. VIP-LALA-E was reacted with 125I using the chloramine T procedure and the 125I-VIP-LALA-E purified by HPLC [9]. The ability of 125I-VIP-LALA-E to internalize into MCF-7 cells was investigated.
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I-VIP-LALA-E was incubated with MCF-7 cells at 4 jC for 2 hr in the presence or absence of 1 AM VIP. One of the plates was incubated at 37 jC for 5 min. The plates were washed 3 times with receptor binding buffer to remove free peptide. The cells were treated with 150 mM NaCl/100 mM acetic acid for 5 min at 4 jC. The supernatant, which contained radiolabeled peptide bound to the cell surface, was counted. The pellet was dissolved in 100 mM NaOH and contained radiolabeled peptide which was internalized. Cyclic AMP
For the cAMP assays, MCF-7 cells in 24 well plates were rinsed 2 times in DMEM. Then the cells were incubated in 250 Al of DMEM containing 2% BSA and 100 AM IBMX (Sigma Chemical, St. Louis, MO). After 20 minutes at 37 jC, VIP-like peptides were added. After 5 minutes at 37 jC, the plates were placed on ice and 250 Al of cold ethanol added. The cell extracts were mixed and frozen at 80 jC until assay. The samples were thawed and assayed for cAMP by radioimmunoassay (New England Nuclear, Boston, MA) as described previously [14]. C-fos mRNA For the mRNA experiments, MCF-7 cells were cultured in 15 cm dishes in DMEM containing 0.5% fetal bovine serum. After 4 hours, the cells were treated with stimuli such as 1 AM VIP-LALA-E for 60 minutes. Total RNA was isolated using the guanidinium isothiocyanate method. Ten Ag of denatured RNA was separated in a 0.66 M formaldehyde 1% agarose gel. The gel was treated with ethidium bromide to assess RNA integrity. The RNA was blotted onto a nytran membrane overnight and the membrane hybridized with cDNA probes labeled with 32P-dCTP using a random priming kit (Boehringer Mannheim, Kansas City, MO). The membrane was exposed to Kodak XAR-2 film at 80 jC for 1 day and the autoradiogram developed. The autoradiograms were analyzed using a Molecular Dynamics densitometer. Proliferation The ability of VIP-E derivatives to alter breast cancer growth was investigated in vitro using a clonogenic growth assay [15]. 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, Rockford, ME), VIP analogs and 5 104 single viable cells. For each cell line and peptide concentration, triplicate wells were plated. After 2 weeks, 1 ml of 0.1% p-iodonitrotetrazolium violet was added and after 16 hours at 37 jC the plates were screened for colony formation; the number of colonies larger than 50 Am in diameter were counted using an Omnicon image analysis system. For the cytotoxicity assays, MCF-7 cells (103) were dispersed in a 96 well plate in SIT medium in the presence of increasing concentrations VIP-LALA-E. After 4 days, 0.4% trypan blue was added and the cytotoxicity determined. The number of trypan blue negative cells was divided by the total number and the percentage of viable cells calculated. For the 35S-methionine assays, MCF-7 cells were placed in 24 well plates containing DMEM with 10% FBS and VIP-E derivatives. After 16 hr, 106 cpm of 35S-methionine (New England Nuclear, Boston, MA) was added for 2 hr. The cells were
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washed 3 times in SIT medium and the pellet dissolved and counted in a Beckman h counter after addition of Aquasol.
Results VIP derivatives bind with high affinity to breast cancer cells The specificity of 125I-VIP binding was investigated (Fig. 1). VIP-LALA-E had little effect of I-VIP binding at 0.01 AM whereas almost all specific binding was inhibited at 10 AM. 125I-VIP binding was half maximally (IC50) inhibited using 0.15 AM VIP-LALA-E. VIP-G and VIP-ALALA-E inhibited specific binding of 125I-VIP to MCF-7 cells in a dose-dependent manner with IC50 values of 0.2 and 1 AM respectively. VIP, which has an amidated C-terminal, had an IC50 value of 0.01 AM (data not shown). These results indicate that the order of peptide potency to bind to MCF-7 cells is VIP-LALA-E = VIP-G > VIP-ALALA-E. The internalization of 125I-VIP-LALA-E was investigated. Fig. 2 shows that most of the 125I-VIPLALA-E bound specifically to the cell surface at 4 jC, whereas it was internalized at 37 jC. Similarly, 125 I-VIP was not internalized at 4 jC but was internalized at 37 jC using T47D cells (data not shown). 125
VIP derivatives function as VPAC1 receptor agonists The ability of the VIP derivatives to alter cAMP was investigated. Table 1 shows that 5 min after addition to MCF-7 cells, VIP-LALA-E increased the cAMP weakly, moderately and strongly at 0.01, 0.1 and 1 AM respectively. The half maximal concentration for VIP-LALA-E to elevate cAMP (ED50) was 0.2 AM. Similarly VIP-G and VIP-ALALA-E had ED50 values of 0.1 and 2 AM
Fig. 1. VIP derivative binding. The % specific binding of 125I-VIP was determined as a function of VIP-LALA-E(.), VIP-G (E) and VIP-ALALA-E (o) concentration. The mean value F S.D. of 4 determinations each repeated in quadruplicate is indicated.
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Fig. 2. Internalization. The % of 125I-VIP-LALA-E bound specifically to the MCF-7 cell surface (m) or internalized (n) was determined at 4 jC or 37 jC. The mean value F S.D. of 3 determinations each repeated in quadruplicate is indicated.
respectively. The order of peptide potency to elevate MCF-7 cAMP was VIP-LALA-E = VIP-G > VIP-ALALA-E. The ability of VIP-E derivatives to cause increased nuclear oncogene expression was investigated. Fig. 3 shows that 1 AM VIP-ALALA-E or VIP-LALA-E caused increased c-fos
Table 1 VIP derivatives and cAMP Addition
cAMP, fmol
None VIP-G 0.01 AM VIP-G 0.1 AM VIP-G 1 AM VIP-LALA-E 0.01 AM VIP-LALA-E 0.1 AM VIP-LALA-E 1 AM VIP-ALALA-E 0.01 AM VIP-ALALA-E 0.1 AM VIP-ALALA-E 1 AM
7.0 F 0.7 13.2 F 1.7* 26.5 F 6.1* 47.9 F 2.6* 7.9 F 1.0 22.5 F 1.2* 31.1 F 4.9* 8.7 F 0.7 11.5 F 1.8* 18.9 F 4.3*
The mean value F S.D. of 4 determinations is indicated using MCF-7 cells. This experiment is representative of 2 others; p < 0;05, *; using student’s t-test. The structures of the peptides are shown below: VIP-G HSDAVFTDNYTRLRKQMAVKKYLNSILN-G VIP-LALA-E HSDAVFTDNYTRLRKQMAVKKYLNSILN-LALA-E VIP-ALALA-E HSDAVFTDNYTRLRKQMAVKKYLNSILN-ALALA-E.
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Fig. 3. VIP-E derivatives and c-fos mRNA. Left: One hour after addition of VIP-ALALA-E and VIP-LALA-E to MCF-7 cells increased c-fos mRNA. Right: As a control, RNA was assessed by determining the 18S and 28S rRNA. This experiment is representative of 2 others.
mRNA 1 hr after addition to MCF-cells. Similar results were obtained using 1 AM VIP-G (data not shown). VIP-E derivatives inhibit breast cancer cellular proliferation The effects of the VIP analogs on the growth of MCF-7 cells was investigated. Table 2 shows that 1 but not 0.1 AM VIP-LALA-E and 1 but not 0.1 AM VIP-ALALA-E significantly reduced colony formation of MCF-7 cells. In contrast, VIP-G had little effect on colony formation. The ability of 1 AM VIP-LALA-E to inhibit breast cancer colony formation was reversed by 10 nM VIP (data not shown). These results indicate that VIP-LALA-E and VIP-ALALA-E inhibit MCF-7 cellular proliferation.
Table 2 Effect of VIP analogs on breast cancer growth Addition
MCF-7 colony number
None VIP-LALA-E, 0.1 AM VIP-LALA-E, 1 AM VIP-ALALA-E, 0.1 AM VIP-ALALA-E, 1 AM VIP-G, 0.1 AM VIP-G, 1 AM
85 F 23 135 F 18 23 F 9* 93 F 24 48 F 11* 92 F 12 115 F 15
The mean value F S.D. of 3 determinations is shown; p < 0.05, * using student’s t-test. This experiment is representative of 2 others.
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Table 3 Cytotoxicity of VIP analogs Addition
% MCF-y viable cells
None VIP-LALA-E, 0.1 AM VIP-LALA-E, 1 AM VIP-G, 0.1 AM VIP-G, 1 AM
100 75 28 108 110
F F F F F
5 5 9* 8 9
The mean value F S.D. of 8 determinations is shown; p < 0.05, *. This experiment is representative of 2 others.
Because VIP-LALA-E but not VIP-G inhibited MCF-7 colony formation it effects on cellular survival were investigated. Table 3 shows that 1 but not 0.1 AM VIP-LALA-E significantly decreased survival of MCF-7 cells. In contrast, 0.1 or 1 AM VIP-G had no effect on MCF-7 cellular survival. Similarly, 0.1 AM VIP-LALA-E and 1 AM VIP-LALA-E as well as 1 AM VIP-ALALA-E significantly inhibited 35 S-methionine uptake into MCF-7 cells (Fig. 4). In contrast, 35S-methionine uptake was unaffected by 0.01 AM VIP-LALA-E, 0.01 and 0.1 AM VIP-ALALA-E or 0.01, 0.1 and 1 AM VIP-G. These results indicate that VIP-LALA-E and VIP-ALALA-E but not VIP-G decrease MCF-7 cell viability.
Fig. 4. 35S-methionine uptake. The 35S-methionine taken up by MCF-7 cells (cpm) was determined as a function of VIPLALA-E (n), VIP-ALALA-E (m) and VIP-G (5). The mean value F S.D. of 4 determinations is indicated. This
experiment is representative of 2 others.
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Discussion Previously we found that the growth of breast cancer cells is inhibited by VIPhybrid, a VPAC1 receptor antagonist [9]. High concentrations of VPAC1 but not VPAC2 receptor mRNA were present in MCF-7 cells and VIPhybrid inhibited 125I-VIP binding to MCF-7 cells with high affinity (IC50 = 0.5 AM). VIPhybrid blocked the ability of VIP to cause cAMP elevation and nuclear oncogene expression. Also VIPhybrid inhibited the basal growth of breast cancer cells and potentiated the cytotoxicity of chemotherapeutic agents [11]. Here the ability of VIP-chemotherapeutic derivatives to kill breast cancer cells was investigated. The N-terminal of VIP is essential for biological activity whereas the C-terminal is amidated [16]. The C-terminal of VIP, however, can be extended and VIP-G binds with only 3-fold lower affinity than does VIP [16]. Also, VIP-aba-G-G-(D)A-G was labeled with Tc-99m with retention of biological activity [17]. VIP-LALA-E, VIP-G and VIP-ALALA-E inhibited 125I-VIP binding to MCF-7 cells with IC50 values of 0.2, 0.2 and 1 AM. The VIP-LALA-E and VIP-ALALA-E functioned as a VIP receptor agonists in that they elevated cAMP. Also, VIP-LALA-E and VIP-ALALA-E increased c-fos mRNA. These results indicate that VIP-LALA-E, VIP-G and VIP-ALALA-E function as VPAC1 receptor agonists. The VIP-LALA-E was internalized at 37 jC by MCF-7 cells. The VPAC1 receptor-VIP-LALA-E complex may undergo endocytosis to endosomes. Subsequently the ligand may be delivered to lysosomes and metabolized by endogenous proteases. The ellipticine derivative may be released into the cytosol and kill breast cancer cells. In this regard, 1 AM VIP-ALALA-E or VIP-LALA-E decreased MCF-7 colony formation. In contrast, VIP-G which lacks the cytotoxic moiety, did not decrease MCF-7 colony formation. Similarly, 1 AM VIP-LALA-E but not VIP-G decreased MCF-7 cell viability based on trypan blue exclusion and 35S-methionine uptake. These results indicate that VIP-ALALA-E and VIP-LALA-E but not VIP-G are cytotoxic for MCF-7 cells. Previously, TGFa-PE40 conjugates were utilized to bind to EGF receptors and kill breast cancer cells [12]. TGFa-PE40 bound with high affinity to EGF receptors and was internalized into endosomes. The TGFa-PE40 was transferred to the Golgi apparatus and then the endoplasmic reticulum prior to release of the toxic PE40 into the cytosol. A key factor was the high density of EGF receptors (100,000/cell). Similarly approximately 100,000 125I-VIP binding sites are present on breast cancer cells [9,18–20]. For CCK receptors, CCK-ALALA-E CCK receptor complex was sorted in cellular endosomes, however, the ligand was delivered to lysozomes and the receptor recycled to the cell surface after 1–2 hr [21]. Because the toxicity of the ellipticine derivatives is high, it was calculated that after one cycle of internalization the concentration of cytotoxic drug in the cytosol was 0.1 AM [22,23]. Similarly the VPAC1 receptor may deliver high concentration of ellipticine derivatives into MCF-7 cells. It remains to be determined if the VPAC1 receptor is recycled to the cell surface after internalization.
Conclusion VIP-E derivatives bind with high affinity to breast cancer cells and function as VPAC1 receptor agonists. While the VIP-E derivatives are cytotoxic for breast cancer cells in vitro, it remains to be determined if they will be useful therapeutic agents in vivo.
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Acknowledgements The authors thank M. Casibang and J. Leyton for technical assistance.
References [1] Said SI, Mutt V. Polypeptide with broad biological activity: Isolation from the small intestine. Science 1970;69: 1217 – 8. [2] Gozes I, McCune SL, Jacobson L, Warren D, Moody T, Fridkin M, Brenneman DJ. A hybrid antagonist to vasoactive intestinal peptide. Effects on cellular function in the central nervous system. J Pharm Exp Ther 1991;257: 959 – 66. [3] Moody TW. Peptides and growth factors in non-small cell lung cancer. Peptides 1996;17:545 – 55. [4] Arimura A. Pituitary adenylate cyclase activating polypeptide (PACAP): Discovery and current status of research. Regul Peptides 1992;37:287 – 303. [5] Ishihara R, Shigemoto R, Mori K, Takahashi K, Nagata S. Functional expression and tissue distribution of a novel receptor for vasoactive intestinal polypeptide. Neuron 1992;8:811 – 9. [6] Harmar T, Lutz EM. Multiple receptors for PACAP and VIP. Trends in Pharmacol Sci 1993;15:97 – 9. [7] Pisegna JR, Wank SA. Multiple cloning and functional expression of the pituitary adenylate cyclase activating polypeptide type I receptor. Proc Natl Acad Sci U S A 1993;90:6345 – 9. [8] Spengler D, Waeber C, Pantaloni C, Holsboer F, Bockaert J, Seeberg PH, Journot L. Differential signal transduction by five splice variants of the PACAP receptor. Nature 1993;365:170 – 5. [9] Zia H, Hida T, Jakowlew S, Birrer M, Gozes Y, Reubi JC, Fridkin M, Gozes I, Moody TW. Breast cancer growth is inhibited by VIPhybrid, a synthetic VIP receptor antagonist. Cancer Res 1996;56:3486 – 9. [10] Whitmarsh AJ, Davies RJ. Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J Mol Med 1996;74:589 – 607. [11] Moody TW, Leyton J, Chan D, Brenneman DC, Fridkin M, Gelber E, Levy A, Gozes I. VIP receptor antagonists and chemotherapeutic drugs inhibit the growth of breast cancer cells. Br Cancer Res Treatment 2001;68:55 – 64. [12] Pastan I, Pai LH, Brinkman U, Fitzgerald DJ. Recombinant immunotoxins. Br Cancer Res Treatment 1996;38:3 – 9. [13] Czerwinski GC, Tarasova NI, Michejda C. Cytotoxic agents directed to peptide hormone receptors. Defining the requirements for a successful drug. Proc Natl Acad Sci U S A 1998;95:11520 – 5. [14] Korman LY, Carney DN, Citron M, Moody TW. Secretin/VIP stimulated secretion of bombesin/gastrin releasing peptide from human small cell carcinoma of the lung. Cancer Res. 1986;46:1214 – 8. [15] Mahmoud S, Staley J, Taylor J, Bogden A, Moreau JP, Coy D, Avis I, Cuttitta F, Mulshine JL, Moody TW. (Psi13,14) bombesin analogues inhibit the growth of small cell lung cancer in vitro and in vivo. Cancer Res 1991;51: 1298 – 802. [16] Fahrenkrug J. Glycine-extended processing intermediate of proVIP: A new bioactive form of VIP in the rat. Biomed Res 1992;13(Sup. 2):19 – 23. [17] Thakur ML, Marcus CS, Saeed S, Pallela V, Minami C, Diggles L, Pham HL, Ahdoot R, Kalinowski EA, Moody T. Imaging tumors in humans with Tc-99 m-VIP. Ann NY Acad Sci 2000;921:37 – 44. [18] Reubi JC, Laderach U, Waser B, Gebbers JD, Robberecht P, Laissue JA. Vasoactive intestinal peptide/pituitary adenylate cyclase activating polypeptide receptor subtypes in human tumors and their tissues of origin. Cancer Res 2000;60: 3105 – 12. [19] Gespach C, Bawab W, De Cremoux P, Calvo F. Pharmacology, molecular identification and functional characteristics of vasoactive intestinal peptide receptors in human breast cancer cells. Cancer Res 1988;48:5079 – 88. [20] Waschek J, Richards ML, Bravo DT. Differential expression of VIP/PACAP receptor genes in breast, intestinal and pancreatic cell lines. Cancer Lett 1995;92:143 – 9. [21] Tarasova NI, Stauber RH, Choi JK, Hudson EA, Czerwinski G, Miller JL, Pavlakis GN, Michejda CJ, Wank SA. Visualization of G protein-coupled receptor trafficking with the aid of green fluorescent protein. Endocytosis and recycling of cholecystokinin receptor type A. J Biol Chem 1997;272:14817 – 24.
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[22] Rivalle C, Wendling R, Tambourin P, Lhoste JM, Bisafni E, Cherman JC. Antitumor amino-substituted pyrido (3V,4V-4,5) pyrrolo (2,3-g)isoquinolines and pyrido (4,3-h)carbazole derivatives: Synthesis and evaluation of compounds resulting from new side chains and heterocyclic modifications. J Med Chem 1983;26:181 – 5. [23] Fitzpatrick JJ, Garnett MC. Studies in the mechanism of action of an MTX-HSA-MoAb conjugate. Anticancer Drug Design 1995;10:1 – 9.
VIP-ellipticine derivatives inhibit the growth of breast cancer cells T.W. Moody a,*, G. Czerwinski b, N.I. Tarasova b, C.J. Michejda b a b
Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute, Rockville, MD 20850, USA Molecular Aspects of Drug Design Section, Macromolecular Structure Laboratory, Advanced BioScience Laboratories, National Cancer Institute, Frederick, MD 21702, USA Received 30 October 2001; accepted 20 December 2001
Abstract The effects of vasoactive intestinal peptide (VIP)-ellipticine (E) derivatives were investigated on breast cancer cells. VIP-ALALA-E and VIP-LALA-E inhibited 125I-VIP binding to MCF-7 cells with an IC50 values of 1 and 0.2 AM respectively. VIP-ALALA-E and VIP-LALA-E caused elevation of cAMP in MCF-7 cells with ED50 values of 1 and 0.1 AM. VIP-LALA-E caused increased c-fos mRNA in MCF-7 cells. Radiolabeled VIP-LALA-E was internalized at 37 jC and delivered the cytotoxic E into MCF-7 cells. VIP-LALA-E inhibited the clonal growth of MCF-7 cells, decreased cell viability based on trypan blue exclusion and reduced 35S-methionine uptake. These results indicate that VIP-E derivatives function as breast cancer VPAC1 receptor agonists which inhibit MCF-7 cellular viability. D 2002 Elsevier Science Inc. All rights reserved. Keywords: VIP-ellipticine derivatives; VIP agonists; VPAC1 receptor; Breast cancer
Introduction Vasoactive intestinal peptide (VIP) is a 28 amino acid peptide which functions as a growth factor in the central nervous system and periphery [1–3]. It is structurally related to pituitary adenylate cyclase activating polypeptide (PACAP) a 27 amino acid peptide [4]. VIP and PACAP bind with high affinity to VPAC1 and VPAC2 receptors which contain 459 and 430 amino acids respectively [5,6]. PACAP but not VIP binds with high affinity to PAC1 receptors which contain 495 amino acids [7,8].
*
Corresponding author. Tel.: +1-301-402-3128; fax: +1-301-402-4422. E-mail address: [email protected] (T.W. Moody). 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 7 4 1 - 1
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VPAC1 receptors interact with a stimulatory guanine nucleotide binding protein (Gs) resulting in adenylyl cyclase activation [9]. Addition of VIP or PACAP to breast cancer MCF-7 cells results in cAMP elevation. The increase in cAMP causes protein kinase A activation. Protein kinase A can phosphorylate protein substrates such as CREB leading to increased nuclear oncogene expression [10]. VIP causes increased c-fos and c-myc expression [9] in MCF-7 cells. VIP stimulates the proliferation of MCF-7 cells, whereas the VIP receptor antagonist VIPhybrid inhibits proliferation [11]. Recently we found that VIPhybrid enhances the ability of taxol or doxorubicin to kill MCF-7 cells [11]. Alternatively cytotoxic drugs may be coupled to a receptor agonist and internalized leading to breast cancer cell death [12]. Here tetra- and pentapeptide ellipticine (E) derivatives were attached to the C-terminal of VIP. The VIP-E derivatives bound to VPAC1 receptors on MCF-7 cells and transiently increased the cAMP and c-fos mRNA. The VIP-E derivatives, however, were cytotoxic for breast cancer cells. Methods Cell lines Breast cancer cells (MCF-7 and T47D) were cultured in DMEM containing 10% fetal bovine serum (Life Technologies, Rockville, MD). The cells were mycoplasm-free and were used when they were in exponential growth phase. Peptide Synthesis VIP-E derivatives (9-methoxy-1-chloro-5,11-dimethyl-6H-pyrido[4,3–h]carbazole) was synthesized as described previously [13]. The fmoc-pentapeptide (ALALA) or fmoc-tetrapeptide (LALA) was synthesized. The N-terminal was deprotected with 20% piperidine I DMF and coupled to E. After 2 hr, the product (LALA-E or ALALA-E) was purified by HPLC. The LALA-E or ALALA-E was then coupled to VIP-COOH with carbodiimide. Peptide purity was greater 98% based on HPLC, amino acid and mass spectroscopy analysis. The biological activity of VIP-ALALA-E and VIP-LALA-E was compared to that of VIP-G (Bachem, San Diego, CA); VIP has an amidated C-terminal whereas in VIP-G, an additional amino acid is added to the C-terminal, which has a free carboxyl group. Receptor binding For receptor binding studies, breast cancer cells in 24 well plates were washed 4 times with DMEM. Then 250 Al of the receptor binding medium (DMEM containing 2% BSA and 2 mg/ml bacitracin) was added with 100,000 cpm 125I-VIP (NEN, Boston, MA; 2200 Ci/mmol) in the absence or presence of competitor [9]. After incubation at 37 jC for 30 minutes, the plates were rinsed 3 times with receptor binding medium. The cells containing bound peptide were solubilized in 0.2 N NaOH and counted in a gamma counter. VIP-LALA-E was reacted with 125I using the chloramine T procedure and the 125I-VIP-LALA-E purified by HPLC [9]. The ability of 125I-VIP-LALA-E to internalize into MCF-7 cells was investigated.
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I-VIP-LALA-E was incubated with MCF-7 cells at 4 jC for 2 hr in the presence or absence of 1 AM VIP. One of the plates was incubated at 37 jC for 5 min. The plates were washed 3 times with receptor binding buffer to remove free peptide. The cells were treated with 150 mM NaCl/100 mM acetic acid for 5 min at 4 jC. The supernatant, which contained radiolabeled peptide bound to the cell surface, was counted. The pellet was dissolved in 100 mM NaOH and contained radiolabeled peptide which was internalized. Cyclic AMP
For the cAMP assays, MCF-7 cells in 24 well plates were rinsed 2 times in DMEM. Then the cells were incubated in 250 Al of DMEM containing 2% BSA and 100 AM IBMX (Sigma Chemical, St. Louis, MO). After 20 minutes at 37 jC, VIP-like peptides were added. After 5 minutes at 37 jC, the plates were placed on ice and 250 Al of cold ethanol added. The cell extracts were mixed and frozen at 80 jC until assay. The samples were thawed and assayed for cAMP by radioimmunoassay (New England Nuclear, Boston, MA) as described previously [14]. C-fos mRNA For the mRNA experiments, MCF-7 cells were cultured in 15 cm dishes in DMEM containing 0.5% fetal bovine serum. After 4 hours, the cells were treated with stimuli such as 1 AM VIP-LALA-E for 60 minutes. Total RNA was isolated using the guanidinium isothiocyanate method. Ten Ag of denatured RNA was separated in a 0.66 M formaldehyde 1% agarose gel. The gel was treated with ethidium bromide to assess RNA integrity. The RNA was blotted onto a nytran membrane overnight and the membrane hybridized with cDNA probes labeled with 32P-dCTP using a random priming kit (Boehringer Mannheim, Kansas City, MO). The membrane was exposed to Kodak XAR-2 film at 80 jC for 1 day and the autoradiogram developed. The autoradiograms were analyzed using a Molecular Dynamics densitometer. Proliferation The ability of VIP-E derivatives to alter breast cancer growth was investigated in vitro using a clonogenic growth assay [15]. 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, Rockford, ME), VIP analogs and 5 104 single viable cells. For each cell line and peptide concentration, triplicate wells were plated. After 2 weeks, 1 ml of 0.1% p-iodonitrotetrazolium violet was added and after 16 hours at 37 jC the plates were screened for colony formation; the number of colonies larger than 50 Am in diameter were counted using an Omnicon image analysis system. For the cytotoxicity assays, MCF-7 cells (103) were dispersed in a 96 well plate in SIT medium in the presence of increasing concentrations VIP-LALA-E. After 4 days, 0.4% trypan blue was added and the cytotoxicity determined. The number of trypan blue negative cells was divided by the total number and the percentage of viable cells calculated. For the 35S-methionine assays, MCF-7 cells were placed in 24 well plates containing DMEM with 10% FBS and VIP-E derivatives. After 16 hr, 106 cpm of 35S-methionine (New England Nuclear, Boston, MA) was added for 2 hr. The cells were
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washed 3 times in SIT medium and the pellet dissolved and counted in a Beckman h counter after addition of Aquasol.
Results VIP derivatives bind with high affinity to breast cancer cells The specificity of 125I-VIP binding was investigated (Fig. 1). VIP-LALA-E had little effect of I-VIP binding at 0.01 AM whereas almost all specific binding was inhibited at 10 AM. 125I-VIP binding was half maximally (IC50) inhibited using 0.15 AM VIP-LALA-E. VIP-G and VIP-ALALA-E inhibited specific binding of 125I-VIP to MCF-7 cells in a dose-dependent manner with IC50 values of 0.2 and 1 AM respectively. VIP, which has an amidated C-terminal, had an IC50 value of 0.01 AM (data not shown). These results indicate that the order of peptide potency to bind to MCF-7 cells is VIP-LALA-E = VIP-G > VIP-ALALA-E. The internalization of 125I-VIP-LALA-E was investigated. Fig. 2 shows that most of the 125I-VIPLALA-E bound specifically to the cell surface at 4 jC, whereas it was internalized at 37 jC. Similarly, 125 I-VIP was not internalized at 4 jC but was internalized at 37 jC using T47D cells (data not shown). 125
VIP derivatives function as VPAC1 receptor agonists The ability of the VIP derivatives to alter cAMP was investigated. Table 1 shows that 5 min after addition to MCF-7 cells, VIP-LALA-E increased the cAMP weakly, moderately and strongly at 0.01, 0.1 and 1 AM respectively. The half maximal concentration for VIP-LALA-E to elevate cAMP (ED50) was 0.2 AM. Similarly VIP-G and VIP-ALALA-E had ED50 values of 0.1 and 2 AM
Fig. 1. VIP derivative binding. The % specific binding of 125I-VIP was determined as a function of VIP-LALA-E(.), VIP-G (E) and VIP-ALALA-E (o) concentration. The mean value F S.D. of 4 determinations each repeated in quadruplicate is indicated.
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Fig. 2. Internalization. The % of 125I-VIP-LALA-E bound specifically to the MCF-7 cell surface (m) or internalized (n) was determined at 4 jC or 37 jC. The mean value F S.D. of 3 determinations each repeated in quadruplicate is indicated.
respectively. The order of peptide potency to elevate MCF-7 cAMP was VIP-LALA-E = VIP-G > VIP-ALALA-E. The ability of VIP-E derivatives to cause increased nuclear oncogene expression was investigated. Fig. 3 shows that 1 AM VIP-ALALA-E or VIP-LALA-E caused increased c-fos
Table 1 VIP derivatives and cAMP Addition
cAMP, fmol
None VIP-G 0.01 AM VIP-G 0.1 AM VIP-G 1 AM VIP-LALA-E 0.01 AM VIP-LALA-E 0.1 AM VIP-LALA-E 1 AM VIP-ALALA-E 0.01 AM VIP-ALALA-E 0.1 AM VIP-ALALA-E 1 AM
7.0 F 0.7 13.2 F 1.7* 26.5 F 6.1* 47.9 F 2.6* 7.9 F 1.0 22.5 F 1.2* 31.1 F 4.9* 8.7 F 0.7 11.5 F 1.8* 18.9 F 4.3*
The mean value F S.D. of 4 determinations is indicated using MCF-7 cells. This experiment is representative of 2 others; p < 0;05, *; using student’s t-test. The structures of the peptides are shown below: VIP-G HSDAVFTDNYTRLRKQMAVKKYLNSILN-G VIP-LALA-E HSDAVFTDNYTRLRKQMAVKKYLNSILN-LALA-E VIP-ALALA-E HSDAVFTDNYTRLRKQMAVKKYLNSILN-ALALA-E.
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Fig. 3. VIP-E derivatives and c-fos mRNA. Left: One hour after addition of VIP-ALALA-E and VIP-LALA-E to MCF-7 cells increased c-fos mRNA. Right: As a control, RNA was assessed by determining the 18S and 28S rRNA. This experiment is representative of 2 others.
mRNA 1 hr after addition to MCF-cells. Similar results were obtained using 1 AM VIP-G (data not shown). VIP-E derivatives inhibit breast cancer cellular proliferation The effects of the VIP analogs on the growth of MCF-7 cells was investigated. Table 2 shows that 1 but not 0.1 AM VIP-LALA-E and 1 but not 0.1 AM VIP-ALALA-E significantly reduced colony formation of MCF-7 cells. In contrast, VIP-G had little effect on colony formation. The ability of 1 AM VIP-LALA-E to inhibit breast cancer colony formation was reversed by 10 nM VIP (data not shown). These results indicate that VIP-LALA-E and VIP-ALALA-E inhibit MCF-7 cellular proliferation.
Table 2 Effect of VIP analogs on breast cancer growth Addition
MCF-7 colony number
None VIP-LALA-E, 0.1 AM VIP-LALA-E, 1 AM VIP-ALALA-E, 0.1 AM VIP-ALALA-E, 1 AM VIP-G, 0.1 AM VIP-G, 1 AM
85 F 23 135 F 18 23 F 9* 93 F 24 48 F 11* 92 F 12 115 F 15
The mean value F S.D. of 3 determinations is shown; p < 0.05, * using student’s t-test. This experiment is representative of 2 others.
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Table 3 Cytotoxicity of VIP analogs Addition
% MCF-y viable cells
None VIP-LALA-E, 0.1 AM VIP-LALA-E, 1 AM VIP-G, 0.1 AM VIP-G, 1 AM
100 75 28 108 110
F F F F F
5 5 9* 8 9
The mean value F S.D. of 8 determinations is shown; p < 0.05, *. This experiment is representative of 2 others.
Because VIP-LALA-E but not VIP-G inhibited MCF-7 colony formation it effects on cellular survival were investigated. Table 3 shows that 1 but not 0.1 AM VIP-LALA-E significantly decreased survival of MCF-7 cells. In contrast, 0.1 or 1 AM VIP-G had no effect on MCF-7 cellular survival. Similarly, 0.1 AM VIP-LALA-E and 1 AM VIP-LALA-E as well as 1 AM VIP-ALALA-E significantly inhibited 35 S-methionine uptake into MCF-7 cells (Fig. 4). In contrast, 35S-methionine uptake was unaffected by 0.01 AM VIP-LALA-E, 0.01 and 0.1 AM VIP-ALALA-E or 0.01, 0.1 and 1 AM VIP-G. These results indicate that VIP-LALA-E and VIP-ALALA-E but not VIP-G decrease MCF-7 cell viability.
Fig. 4. 35S-methionine uptake. The 35S-methionine taken up by MCF-7 cells (cpm) was determined as a function of VIPLALA-E (n), VIP-ALALA-E (m) and VIP-G (5). The mean value F S.D. of 4 determinations is indicated. This
experiment is representative of 2 others.
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Discussion Previously we found that the growth of breast cancer cells is inhibited by VIPhybrid, a VPAC1 receptor antagonist [9]. High concentrations of VPAC1 but not VPAC2 receptor mRNA were present in MCF-7 cells and VIPhybrid inhibited 125I-VIP binding to MCF-7 cells with high affinity (IC50 = 0.5 AM). VIPhybrid blocked the ability of VIP to cause cAMP elevation and nuclear oncogene expression. Also VIPhybrid inhibited the basal growth of breast cancer cells and potentiated the cytotoxicity of chemotherapeutic agents [11]. Here the ability of VIP-chemotherapeutic derivatives to kill breast cancer cells was investigated. The N-terminal of VIP is essential for biological activity whereas the C-terminal is amidated [16]. The C-terminal of VIP, however, can be extended and VIP-G binds with only 3-fold lower affinity than does VIP [16]. Also, VIP-aba-G-G-(D)A-G was labeled with Tc-99m with retention of biological activity [17]. VIP-LALA-E, VIP-G and VIP-ALALA-E inhibited 125I-VIP binding to MCF-7 cells with IC50 values of 0.2, 0.2 and 1 AM. The VIP-LALA-E and VIP-ALALA-E functioned as a VIP receptor agonists in that they elevated cAMP. Also, VIP-LALA-E and VIP-ALALA-E increased c-fos mRNA. These results indicate that VIP-LALA-E, VIP-G and VIP-ALALA-E function as VPAC1 receptor agonists. The VIP-LALA-E was internalized at 37 jC by MCF-7 cells. The VPAC1 receptor-VIP-LALA-E complex may undergo endocytosis to endosomes. Subsequently the ligand may be delivered to lysosomes and metabolized by endogenous proteases. The ellipticine derivative may be released into the cytosol and kill breast cancer cells. In this regard, 1 AM VIP-ALALA-E or VIP-LALA-E decreased MCF-7 colony formation. In contrast, VIP-G which lacks the cytotoxic moiety, did not decrease MCF-7 colony formation. Similarly, 1 AM VIP-LALA-E but not VIP-G decreased MCF-7 cell viability based on trypan blue exclusion and 35S-methionine uptake. These results indicate that VIP-ALALA-E and VIP-LALA-E but not VIP-G are cytotoxic for MCF-7 cells. Previously, TGFa-PE40 conjugates were utilized to bind to EGF receptors and kill breast cancer cells [12]. TGFa-PE40 bound with high affinity to EGF receptors and was internalized into endosomes. The TGFa-PE40 was transferred to the Golgi apparatus and then the endoplasmic reticulum prior to release of the toxic PE40 into the cytosol. A key factor was the high density of EGF receptors (100,000/cell). Similarly approximately 100,000 125I-VIP binding sites are present on breast cancer cells [9,18–20]. For CCK receptors, CCK-ALALA-E CCK receptor complex was sorted in cellular endosomes, however, the ligand was delivered to lysozomes and the receptor recycled to the cell surface after 1–2 hr [21]. Because the toxicity of the ellipticine derivatives is high, it was calculated that after one cycle of internalization the concentration of cytotoxic drug in the cytosol was 0.1 AM [22,23]. Similarly the VPAC1 receptor may deliver high concentration of ellipticine derivatives into MCF-7 cells. It remains to be determined if the VPAC1 receptor is recycled to the cell surface after internalization.
Conclusion VIP-E derivatives bind with high affinity to breast cancer cells and function as VPAC1 receptor agonists. While the VIP-E derivatives are cytotoxic for breast cancer cells in vitro, it remains to be determined if they will be useful therapeutic agents in vivo.
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Acknowledgements The authors thank M. Casibang and J. Leyton for technical assistance.
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