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Bioconjugate Chem. 2000, 11, 278−281
TECHNICAL NOTES Synthesis of Biotinylated Glyfoline for Immunoelectron Microscopic Localization Tsann-Long Su,*,† Chin-Tarng Lin,‡ Ching-Huang Chen,† and Hue-Ming Huang‡ Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, and Institute of Pathology, College of Medicine, National Taiwan University, Taipei 100, Taiwan
Antineoplastic glyfoline (1) has potent antitumor efficacy against various murine and human solid tumors. To elucidate the actual mechanism of action, we synthesized biotinylated glyfoline (B-Gly) and used it for the visualization of glyfoline-binding sites in nasopharyngeal carcinoma (NPC) cells. Under the electron microscope (EM), after cells were incubated for 6-36 h, the reaction products of anti-B-Gly were seen on some areas of the external cell surface and on the outer and inner membranes of the mitochondria. Pure EM morphology of NPC cells after glyfoline treatment revealed the similar morphological change of mitochondria. These findings indicate that the binding site of glyfoline in NPC is the inner membrane of the mitochondria, suggesting that B-Gly can be used as a marker for glyfoline localization.
The natural antineoplastic glyfoline (1, Figure 1), a 9-acridone alkaloid isolated from Glycosmis citrifolia, was previously revealed to have significant antitumor activity both in vitro and in vivo against several murine solid tumors (1). Glyfoline does not interact with DNA or with topoisomerases I and II, but is capable of partially inhibiting nucleoside transport across the cell membrane (2). Recently, we found most human tumor cells such as leukemia K-256 and nasopharyngeal carcinoma (NPC) tumor cells showed apoptotic change when they were treated with glyfoline (3). Moreover, we found that glyfoline may damage tumor cells at the G2/M phase. Hence, the action mechanism of glyfoline is novel and is quite different from other antitumor drugs currently being used in clinical settings. It is, therefore, of great interest to study the possible action mechanism of glyfoline and its binding site(s) in the glyfoline-treated tumor cells. NPC cell lines developed in our laboratory were used for identification of glyfoline cytotoxic effect and action mechanism. NPC cells showed apoptosis after treatment with 8.8 µM (2 × IC50) glyfoline (3). Apoptotic changes were judged by DNA fragmentation, TUNEL, and cytofluorometric analyses. However, no apoptotic gene expression, including ICE-R,β, Bcl-2, Bax, or c-myc genes, were involved, nor did cytokines such as TNF-R appear to induce abnormal expression. Although G2/M phase arrest was identified by FACScan analysis and cytochrome c was found to be released from the mitochondria into the cytosol in NPC cells (4), the actual target site for glyfoline action remained undetermined. Glyfoline is a fluorescent compound. When NPC cells * To whom correspondence should be addressed. Phone: 8862-2789-9045. Fax: 886-2-2782-7685. E-mail: tlsu@ ibms.sinica.edu.tw). † Institute of Biomedical Sciences. ‡ Institute of Pathology.
Figure 1. Chemical structure of glyfoline and biotinylated glyfoline.
were treated with glyfoline, the fluorescent glyfoline was observed surrounding the cell membrane at first and then penetrating into the cytoplasm of the cell under light fluorescent microscope. However, the fluorescence of the agent disappeared within a few minutes. Therefore, in the present experiment, we synthesize biotinylated glyfoline (B-Gly, 2, Figure 1) and use it for the localization of the glyfoline target site in NPC cells using light and immunoelectron microscopes. The synthetic route to B-Gly (2) with a spacer between biotin and glyfoline is illustrated in Scheme 1 (5, 6). By following the procedure developed by Mouton et al. (7), the known hydroxysuccinimido biotinate (3) (7) was treated with 4-aminobutyric acid in dry DMSO containing triethylamine to give 4-(biotinylamido)butyric acid (4), which was then reacted with N-hydroxysuccinimide and DCC in dry DMSO at room temperature for 2 days to yield N-hydroxysuccinimido 4-biotinylamido-butyrate (5). Treatment of potassium phthalimide (6) with 1,4-
10.1021/bc990105v CCC: $19.00 © 2000 American Chemical Society Published on Web 12/28/1999
Technical Notes
Bioconjugate Chem., Vol. 11, No. 2, 2000 279
Scheme 1 Chemical Synthesis of Biotinylated Glyfoline (2)
dibromobutane (7) in dry DMF (30 mL) afforded N-(4bromobutyl)phthalimide (8), which was reacted with glyfoline (1) in MeCN/K2CO3 to give 6-O-4-(phthalimidoN-yl)butylglyfoline (9). The N-protecting function of 9 was removed by treating with hydrazine in an ice-bath to afford 6-O-(4-aminobutyl)glyfoline (10), which was then condensed with compound 5 to give the desired B-gly (2) in good yield. The chemical properties of the highly specific and very strong binding of biotin to avidin (9) provided a technology for the identification of drug’s target site(s) in cells even when the compound is present at very low concentrations. Biotin-peptide conjugates were widely used as tools for detecting monoclonal antibody receptors. For example, a polypeptide hormone covalently linked with biotin was incubated with cells and later retrieved by adsorption on immobilized avidin for studies of hormone catabolism and for isolation of hormone-receptor complexes (10). However, conjugation of biotin to the chemical agent may change or inhibit the activity of this chemical agent at certain degree. Hoffman et al. (11) reported that there is a dramatic effect on the ability of succinylavidin to bind biotinylated insulin. The attachment of the bulky insulin molecule to biotin appears to exert a steric impediment to avidin binding. This suggests that avidin weakens the ability of biotinylinsulin to interact with insulin receptors and that the interference is inversely proportional to the distance between the biotin and insulin. During the course of our study, we also found that there is also an effect of avidin binding to the B-Gly after the B-Gly binds to the cytoplasm of NPC cells. Therefore, we used antibody against biotin to replace the avidin. NPC cells were treated with B-Gly and used antibody to detect biotin under the light microscope. We observed that the conjugate was found to bind to the cell surface
membrane and bind diffusely in the cytoplasm after the first hour of incubation. Within 6 h, the conjugate gradually concentrated in many vesicles. These findings indicate that B-Gly binds to the cell membrane, then enters into the cytoplasm, probably by passive diffusion. After 6-24 h, this phenomenon became more prominent, indicating that a certain specific organelle in the cytoplasm is the target site for glyfoline binding. However, the light microscopic observation cannot clarify the definite target organelle. The fact that under the electron microscope, in certain tumor cells, electron dense reaction product of anti-BGly was seen concentrated in the mitochondria (Figure 2), especially in certain cristae and in the inner membrane, suggests that B-Gly may first bind to the mitochondrial outer membrane and then move to the inner membrane. The inner membranes of the mitochondria, especially the cristae, were bound by B-Gly. Gradual degeneration and formation of myelinated bodies with local disappearance of cristae and loss of the dense granule (calcium apatite) in the matrix of the mitochondria were apparent, suggesting that B-Gly not only binds to the inner membrane of mitochondria, but also causes membrane disintegration that may result in leakage of cytochrome c and matrix substance into the cytosol. This increase in the cytosolic fraction then results in activation of the caspase system to induce apoptosis (13-16). Additionally, the increase of calcium ion concentration in the cytoplasm may help to activate endonuclease, causing DNA condensation and fragmentation and cellular apoptosis. Furthermore, since B-Gly binds to the inner membrane and causes degeneration of the mitochondrial inner membrane, it may induce a decrease in ATP production and activate reactive oxygen species production (17), resulting in a hypoxic state of treated cells and subsequent membrane damage. However, it is
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search grants (NSC 86-2314-B-001 to T.L.S.; NSC 862314-002-281 to C.T.L.) from National Science Council, Taipei, Taiwan. LITERATURE CITED
Figure 2. Immunoelectron microscopic localization of B-Gly in NPC cells. NPC-TW06 cells (12) were incubated with B-Gly for 48 h and subjected to tissue preparation. Briefly, the NPCs were grown on the Teflon-coated coverslips and fixed in 3% formaldehyde and 1% glutaraldehyde mixture for 15 min and incubated with peroxidase labeled goat antibodies against biotin and then with peroxidase substrate (diaminobenzidine), followed by fixation in 1% OsO4 and routine electron microscopic procedure. Reaction product of anti-B-Gly is seen on the inner, some outer and cristal membranes of many mitochondria (large arrowheads). Many mitochondria reveal focal degeneration of cristae with dilatation of matrix chambers, which contain some myelinated bodies stained with or without reaction product (small arrowheads). The ground substance and other organelles in the cytoplasm show no specific reaction product.
not known what enzyme or protein at the inner membrane is the major target site for B-Gly. Apparently, the main target site of B-Gly is the mitochondria. This phenomenon is similar to that seen in fluorescent Rhodamine 123 (18). For comparison, NPC cells were treated with glyfoline and prepared for routine electron microscopic observation. Results also showed marked alternation of mitochondrial cristal and inner membrane (data not shown) similar to the B-Gly-treated cells. This finding suggested that B-Gly can be used as a marker for glyfoline localization. In summary, the above new findings suggest that the target site of glyfoline may be the mitochondrial inner membrane. The binding of B-Gly to the mitochondria may be resulted in the release of cytochrome c into the cytosol. The damaged mitochondria may also release calcium into the cytosol, produce free radicals, and deprive tumor cell from ATP, any of which would accelerate cellular apoptosis. The mode of action of this agent is quite different from currently available anticancer drugs for chemotherapy, indicating that glyfoline may be a good candidate for antitumor chemotherapy. Currently, we are studying whether glyfoline itself also targets to the mitochondria in NPC cells and releases cytochrome c that may induce apoptosis in the tumor cells. The present studies will facilitate to understand a clear action mechanism of glyfoline. ACKNOWLEDGMENT
This work was supported in part by a program project grant (1997) from the Institute of Biomedical Sciences, Academia Sinica, Taipei, to T.L.S. and C.T.L. and re-
(1) Su, T.-L., Kohler, B., Chou, T. C., Chun, M. W., and Watanabe, K. A. (1992) Synthesis of the Acridone Alkaloids Glyfoline and Congeners. Structure-Activity Relationship Studies of Cytotoxicity Acridones. J. Med. Chem. 35, 27032710. (2) Su, T.-L., and Chou, T.-C. (1994) Experimental Antitumor Activity of Acridone Alkaloids, Acronycine and Glyfoline. Chin. Pharm. 46, 371-391. (3) Su, T.-L., Yang, D.-M., Huang, H.-M., Yang, W.-K., Lin, C.T., and Whang-Peng, J. (1997) Effects of antitumor agent, glyfoline on human cancer cells in vitro. Proc. Am. Assoc. Cancer Res. 38, 219. (4) Huang, H.-M., Su, T.-L., Wu, H.-C., and Lin, C.-T. Unpublished data. (5) Melting points were determined on a Fargo Melting Apparatus and were uncorrected. Column chromatography was carried out on silica gel G60 (70-230 mesh, ASTM, Merck). Thin-layer chromatography was performed on silica gel 60F254 (Merck) with short-wavelength UV light for visualization. Element analyses were determined on a Heraeus NCH-Rapid Apparatus. 1H NMR spectra were recorded on a Brucker AMX-400 spectrometer with Me4Si as the internal standard. Chemical shifts are reported in parts per million (δ), and the signals are described as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad), and brs (broad singlet). Values reported for coupling constants are first order. (6) Melting point, 1H NMR spectra and elemental analysis of compounds described in the text are follows. Compound 4: mp 220-221 °C. 1H NMR (DMSO-d6): δ 1.31, 1.50, 2.06, 2.35 (each 2H, brs, 4 × CH2), 1.62 (4H, brs, 2 × CH2), 2.52, 2.60, and 2.82 (each 1H, s, CH), 3.05 (2H, brs, CH2), 3.10 (1H, s, CH), 4.32, and 12.1 (1H, br exchangeable, COOH). Anal. calcd for C14H23N3O4S: C, 51.04; H, 7.04; N, 12.76. Found: C, 51.23; H, 6.95; N, 12.65. C. Compound 5: mp 119-121 °C. 1H NMR (DMSO-d6): δ 1.29 (2H, br CH2), 1.49 and 1.58 (4H, m, 2 × CH2), 1.73 and 2.05 (each 2H, br, 2 × CH2), 2.57 (1H, s, CH), 2.80 (4H, s, 2 × CH2), 2.81 (1H, s, CH), 3.02 (4H, br, 2 × CH2), 4.12 and 4.29 (each 1H, s, CH), 6.34, 6.42, and 7.85 (each 1H, br, exchangeable, 3 × NH). Anal. calcd for C18H26N4O6S: C, 50.69; H, 6.14; N, 13.14. Found: C, 50.75; H, 6.08; N, 13.26. Compound 8: mp 73-74 °C. 1H NMR (DMSO-d6): δ 1.86 (4H, brs 2 × CH2), 3.45 and 3.78 (each 2H, brs, NCH2 and BrCH2), 7.71 and 7.85 (each 2H, brs, 4 × ArH). Anal. calcd for C12H12BrNO2: C, 51.08; H, 4.29; N, 4.96. Found: C, 51.20; H, 4.35; N, 5.05. Compound 9: mp 135136 °C. 1H NMR (DMSO-d6): δ 1.98 (4H, brs, 2 × CH2), 3.46, 3.80 (6H, s, 2 × OMe), 3.87 and 3.90 (each 3H, s, 2 × Me), 4.16 (3H, s, N.-Me), 4.22 (2H, brs, N.-CH2), 6.96 and 8.08 (each 1H, d, J ) 8.0 Hz), 7.74 and 7.78 (each 2H, brs, 4 × ArH), 14.0 (1H, s, OH). Anal. calcd for C30H30N2O9: C, 64.05; H, 5.38; N, 4.98. Found: C, 64.17; H, 5.43; N, 5.06. Compound 10: mp 175-176 °C. 1H NMR (DMSO-d6): δ 1.82 and 1.89 (each 2H, m, 2 × CH2), 2.89 (2 H, brs, OMe), 3.69, 3.78, 3.81 and 3.84 (each 3H, s, 3 × OMe), 4.22 (3H, s, NMe), 4.40 (2H, s, J ) 4.8 Hz, NCH2), 7.22 and 7.95 (each 1H, d, J ) 8.0 Hz, Ar-H), 8.12 (2H, br, exchangeable, NH2), 14.11 (1H, s, exchangeable, OH). Anal. calcd for C22H28N2O7: C, 61.10; H, 6.53; N, 6.48. Found: C, 6.21; H, 6.48; N, 6.53. Compound 2: mp 127-128 6 °C. 1H NMR (DMSO-d6): δ 1.30 (2H, m, CH2) 1.5-1.52 (4H, m, 2 × CH2), 1.65 (6H, m, 3 × CH2), 1.82 (2H, m, CH2), 2.07 (4H, m, 2 × CH2), 2.57 (1H, d, J ) 12.3 Hz, CH), 2.81 (1H, dd, J ) 5.91 Hz, CH2) 3.69, 3.75, 3.81, and 3.82 (each 3H, s, 4 × OMe), 4.11 (3H, s, NMe), 4.18 (1H, m, CH), 4.19 (2H, t, J ) 5.82 Hz, CH2), 4.29 (1H, t, J ) 4.80 Hz, CH), 7.19 and 7.94 (each 1H, J ) 8.0 Hz, 2 × ArH), 7.77 and 7.78 (each 1H, exchangeable 2 × NH), 14.0 (1H, s, exchangeable, OH). Anal. calcd for C39H49N5O10S: C, 56.09; H, 6.80; N, 9.09. Found: C, 56.15; H, 6.94; N, 8.91.
Technical Notes (7) Mouton, C. A., Pang, D., Natraj, C. D., and Shafer, J. A. (1982) A Reagent for Covalently Attaching Biotin to Proteins via a Cleavable Connector Arm. Arch. Biochem. Biophys. 218, 101-108. (8) Lin, C.-T., Chen, C.-C., How, S.-W., Huang, W.-M., and Peck, K. (1992) Localization of HPV-16 DNA Sequence in CaSKi Cells by Electron Microscopic Hybridocytochemistry. J. Histochem. Cytochem. 40, 467-478. (9) Green, N. M. (1975) Avidin. Adv. Protein Chem. 29, 85133. (10) Hoffman, K., Finn, F. M., Friccsn, H. J. Diaconesou, C., and Zahn, H. (1997) Biotinylinsulins as Potential Tools for Receptor Studies. Proc. Natl. Acad. Sci. U.S.A. 74, 26972700. (11) Hofmann, K., Judith, A., Montibeller, J. A., and Finn, F. M. (1982) Avidin Binding of Carboxysubstituted Biotin and Analogues. Biochemistry 21, 978-984. (12) Lin, C. T., Chen, W. Y., Chen, W., Huang, H. W., Wu, H. C., Hsu, M. M., Chuang, S. M., and Wang, C. C (1993) Charaterization of seven newly established nasopharyngeal carcinoma cell lines. Lab. Invest. 68, 716-723.
Bioconjugate Chem., Vol. 11, No. 2, 2000 281 (13) Cai, J., Yang, J., and Jones, D. P. (1998) Mitochondrial Control of Apoptosis: the Role of Cytochrome C. Biochim. Biophys. Acta 1366, 139-149. (14) Allen, R. T., Cluck, M. W., and Agrawal, D. K. (1998) Mechanisms Controlling Cellular Suicide: Role of Bcl-2 and Caspases. Cell. Mol. Life Sci. 54, 427-445. (15) Kroemer, G., Dalloprta B., and Resche-Rigon M. (1998) The Mitochondrial Death/Life Regulator in Apoptosis and Necrosis. Annu. Rev. Physiol. 60, 619-642. (16) Cecconi, F., Alvarez-Bolado, G., Meyer, B. I., Roth, K. A., and Gruss, P. (1998) Apaf1 (CED-4 homolog) Regulates Programmed Cell Death in Mammalian Development. Cell 94, 727-737. (17) Skulachev V. P. (1998) Cytochrome C in the Apoptotic and Antioxidant Cascades. FEBS Lett. 423, 275-280. (18) Bernal, S. D., Lampidis, T. J., Summerhayes, I. C., and Chen, L. B. (1982) Rhodamine-123 Selectively Reduces Clonal Growth of Carcinoma Cells In Vitro. Science 218, 117-119.
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