- Email: [email protected]
Telomerase inhibition and telomere erosion: a two-pronged strategy in cancer therapy
Update
328
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NB-DNJ on epididymal histology and function have yet to be examined. The sulfoglycolipid seminolipid (sulfogalactosylalkylacylglycerol) is a well-recognized component of the plasma membrane of spermatozoa and has been shown to be essential for spermatogenesis [24] and play a significant role in the recognition of the zona pellucida (the membrane surrounding a mammalian oocyte) by sperm [25]. Therefore, an agent that alters the biosynthesis of glycosphingolipids would be expected to affect the formation of the cell membrane. The extensive damage observed in the nucleus, mitochondrial sheath and acrosome of sperm following treatment with NB-DNJ might also be linked to altered glycosphingolipids associated with these organelles, but this remains to be established. It will be interesting to determine whether, at doses used to induce infertility, NB-DNJ also causes significant changes in other tissues, such as the brain, that are rich in sulfoglucolipids. To be developed as a male contraceptive any compound will need to have negligible risks associated with its use (no toxicity), while having very high efficacy. Whether, after safety evaluation, this particular compound succeeds or not, the report from Platt’s group provides the foundation for developing new male contraceptives. This represents a new avenue of research in the field of male contraceptive development. References 1 Anderson, R.A. et al. (2002) Male contraception. Endocr. Rev. 23, 735 – 762 2 Turner, T.T. (1991) Spermatozoa are exposed to a complex microenvironment as they traverse the epididymis. Ann. New York Acad. Sci. 637, 364 – 383 3 Zirkin, B.R. et al. (1989) Maintenance of advanced spermatogenic cells in the adult rat testis: quantitative relationship to testosterone concentration within the testis. Endocrinology 124, 3043 – 3049 4 Lobl, T.J. et al. (1983) Contraceptive efficacy of testosterone-estradiol implants in male rhesus monkeys. Contraception 27, 383 – 389 5 van Houten, M.E. et al. (2000) Differences in reproductive endocrinology between Asian men and Caucasian men. Asian J. Androl. 2, 13–20 6 Ewing, L.L. et al. (1977) Synergistic interaction of testosterone and oestradiol inhibits spermatogenesis in rats. Nature 269, 409 – 411 7 Kamischke, A. et al. (2001) Intramuscular testosterone undecanoate and norethisterone enanthate in a clinical trial for male contraception. J. Clin. Endocrinol. Metab. 86, 303 – 309 8 McLachlan, R.I. et al. (2002) Effects of testosterone plus medroxyprogesterone acetate on semen quality, reproductive hormones, and germ cell populations in normal young men. J. Clin. Endocrinol. Metab. 87, 546–556
Vol.24 No.7 July 2003
9 Ramachandra, S.G. et al. (2002) Effect of chronic administration of 7alpha-methyl-19-nortestosterone on serum testosterone, number of spermatozoa and fertility in adult male bonnet monkeys (Macaca radiata). Reproduction 124, 301 – 309 10 Jackson, H. et al. (1979) Contraception for the male: problems with progress. Clin. Obstet. Gynaecol. 6, 129 – 155 11 Liu, G.Z. et al. (1985) Trials of gossypol as a male contraceptive. In Gossypol: A Potential Contraceptive for Men (Segal, S., ed.), pp. 9 – 16, Plenum Publishers 12 Zavos, P.M. et al. (1996) The inhibitory effects of gossypol on human sperm motility characteristics: possible modes of reversibility of those effects. Tohoku J. Exp. Med. 179, 167– 175 13 Liu, G.Z. et al. (1988) Effects of K salt or a potassium blocker on gossypol-related hypokalemia. Contraception 37, 111 – 117 14 Shi, Y.L. et al. (2003) Ion-channels in human sperm membrane and contraceptive mechanisms of male antifertility compounds derived from Chinese traditional medicine. Acta Pharmacol. Sin. 24, 22 – 30 15 Cheng, C.Y. et al. (2002) Indazole carboxylic acids in male contraception. Contraception 65, 265– 268 16 Cooper, T.G. (2002) The epididymis as a target for male contraception. In The Epididymis: From Molecules to Clinical Practice (Robaire, B. and Hinton, B.T., eds), pp. 483 – 502, Kluwer Academic Plenum Publishers 17 Frayne, J. et al. (1999) The potential use of sperm antigens as targets for immunocontraception; past, present and future. J. Reprod. Immunol. 43, 1 – 33 18 Ren, D. et al. (2001) A sperm ion channel required for sperm motility and male fertility. Nature 413, 603 – 609 19 van der Spoel, A.C. et al. (2002) Reversible infertility in male mice after oral administration of alkylated imino sugars: a nonhormonal approach to male contraception. Proc. Natl. Acad. Sci. U. S. A. 99, 17173 – 17178 20 Butters, T.D. et al. (2003) Therapeutic applications of imino sugars in lysosomal storage disorders. Curr. Top. Med. Chem. 3, 561 – 574 21 Platt, F.M. et al. (2001) Inhibition of substrate synthesis as a strategy for glycolipid lysosomal storage disease therapy. J. Inherit. Metab. Dis. 24, 275 – 290 22 Cox, T. et al. (2000) Novel oral treatment of Gaucher’s disease with Nbutyldeoxynojirimycin (OGT 918) to decrease substrate biosynthesis. Lancet 355, 1481– 1485 23 Trasler, J. et al. (1998) Characterization of the testis and epididymis in mouse models of human Tay Sachs and Sandhoff diseases and partial determination of accumulated gangliosides. Endocrinology 139, 3280– 3288 24 Fujimoto, H.K. et al. (2000) Requirement of seminolipid in spermatogenesis revealed by UDP-galactose: Ceramide galactosyltransferase-deficient mice. J. Biol. Chem. 275, 22623 – 22626 25 White, D. et al. (2000) Role of sperm sulfogalactosylglycerolipid in mouse sperm-zona pellucida binding. Biol. Reprod. 63, 147 – 155 0165-6147/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0165-6147(03)00141-X
Telomerase inhibition and telomere erosion: a two-pronged strategy in cancer therapy Fred O. Odago and Stanton L. Gerson Division of Hematology/Oncology and the Comprehensive Cancer Center at Case Western Reserve University and University Hospitals of Cleveland, 10900 Euclid Ave., BRB 3-West, Cleveland, OH 44106-4937, USA
Inhibition of telomerase activity in cancerous cells is a very potent factor in the abrogation of cellular Corresponding author: Stanton L. Gerson ([email protected]). http://tips.trends.com
immortalization. However, targeting telomerase alone is not sufficient to elicit cell death because tumor cells can acquire the ability to lengthen their telomeres independently of telomerase activity, in a process
Update
TRENDS in Pharmacological Sciences
referred to as alternative lengthening of telomeres. In this article, we suggest that a biphasic approach, by both eroding intrinsic levels of telomeres and inhibiting telomerase activity, would result in a synergistic beneficial effect that could be used in cancer therapy. Research in cancer, at present, is focused mostly on identifying therapeutic agents that can limit the exponential proliferation of transformed cells. The identification of a single therapeutic target that will act as a panacea in cancer treatment is compounded by the fact that the transformation of normal cells into malignancies is a multi-step process, which thus necessitates targeting the various acquired capabilities of these cells [1]. Recently, it has become apparent that targeting telomerase, a ribonucleoprotein responsible for adding repetitive units of TTAGGG to the ends of telomeres (structures at the end of chromosomes that protect them from end-to-end fusion), could be a potent factor in the control of invasive cancerous cells [2]. The normal addition of these nucleotide repeats averts a crisis in which increased shortening and eventual loss of telomeres results in the cell entering senescence (Fig. 1). Loss of telomeres also results in end-to-end chromosomal fusions, yielding karyotypic disarray, which leads eventually to the death of the cell [3]. Thus, limiting the levels of telomerase within tumorigenic cells to allow the cells to enter senescence and eventually undergo cell death would be a favorable anticancer strategy. Telomerase as a target of cancer control is favored further by observations that levels of the catalytic subunit of this enzyme are particularly elevated in most tumorigenic cells compared with such levels in normal cells [4]. Strategies that silence telomerase gene expression or inhibit telomerase function Various strategies have been shown to affect the intrinsic levels of telomerase as a result of either a direct or an Normal somatic cell chromosome
(a) (b)
GGTTAGGGTTAG CAAUCCCAAUC
Cell senesces and eventually dies
(c)
Cancerous cell chromosome
GTTAGGGTTAGGGTTAG CAAUCCCAAUC
TRENDS in Pharmacological Sciences
Fig. 1. Involvement of telomerase in cancer. (a) The conversion of normal dividing cells into senescent cells involves the loss of telomeric ends (dark blue) in a process known as telomeric arm erosion. The telomerase enzyme (red arrow) is used to lengthen the telomeres (inset), and prevent cells from entering senescence and undergoing end-to-end chromosomal fusions. (b) The conversion of normal somatic cells to cancerous cells involves the upregulation of telomerase expression resulting in increased telomere lengths and a decreased likelihood of senescence. (c) Cancerous cells can be directed to senesce and eventually die by the use of paclitaxel, a putative telomere eroding agent. Telomerase inhibitors (shown in Table 1) enhance the effect of paclitaxel in a synergistic manner. http://tips.trends.com
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indirect action (Table 1). These strategies include: (1) chemoprevention [5 – 7]; (2) anti-retroviral attack using a chain-terminating nucleoside triphosphate analog known to inhibit reverse transcriptases [e.g. azidothymidine (AZT)] [8,9]; (3) an antisense strategy using oligonucleotides [2,10,11]; and (4) the use of G-quadraplex ligands [12]. Chemoprevention is the use of agents, either pharmacological or natural, that inhibit the development of tumors by either preventing the DNA damage that initiates carcinogenesis or arresting or reversing the progression of pre-malignant cells in which such DNA damage has already occurred [5]. An example of a pharmacological agent is oltipraz, which is a potent inducer of the enzymes involved in the detoxification of carcinogens such as aflatoxin. Oltipraz has been shown to have a marked inhibitory effect on carcinogenesis within the liver and to indirectly prevent the accumulation of telomerase [6]. Other chemopreventive agents include natural diet constituents such as curcuminin (present in the spice turmeric), genistein (present in soya) and catechins (present in tea). These agents have been shown to have tumor-suppressing properties in transgenic mice studies and have resulted in the downregulation of intrinsic levels of telomerase [7]. Another method of inhibiting telomerase is by enhancing the folding of telomeric DNA (guanine repeats) into a four-stranded quadraplex structure held together by Hoogsten hydrogen-bonded arrays of guanine bases. The formation of this quadraplex structure at the 30 end of telomeric DNA effectively hinders telomerase from adding further repeats. The use of G-quadraplex ligands, small molecules that stabilize the secondary structure comprising the guanine-rich repetitive DNA, is of particular importance in strategies aimed at prolonging the effects of telomerase inhibition [12]. Using an antisense strategy, oligonucleotides complementary to the telomerase RNA component (hTR) can be expressed in cancerous cells that exhibit elevated levels of telomerase. Exogenously added oligomers complementary to the template region of hTR, such as 20 -O-MeRNA and peptide nucleic acid (PNA) (a DNA mimic with a neutral amide backbone), have been shown to inhibit telomerase activity by preventing access of the RNA template to telomeric DNA sequences. This has resulted in an enhanced shortening of telomeres, eventually resulting in cell death by apoptosis after an extended period of treatment [2]. 20 -O-alkyl-derivatives and PNA oligomers bind tighter to complementary RNA sequences than do analogous DNA oligomers, resulting in an improved antisense selectivity, and have also been shown to have enhanced efficacy and pharmacological properties, compared with analogous DNA oligomers that have neither a peptide backbone nor been substituted at the 20 position [10]. In studies using immortal human cells and PNA oligomers that were complementary to telomerase RNA, telomerase activity was effectively inhibited, telomeres were markedly shortened and, after a lag period, cell proliferation was arrested. These observations served as markers of a suppressed cellular ‘immortality’ [11].
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Table 1. Strategies used to inhibit telomerase activity Strategy
Agents
Mode of action
Refs
Chemoprevention
Oltipraz
[6]
G-quadraplex ligands
Natural diet constituents such as genistein, catechins and curcuminin Acridine compounds
Antisense strategy
20 O-MeRNAa and PNAb
Anti-retroviral attack
Azidothymidine
Induces the synthesis of detoxification enzymes for carcinogens, thus lowering carcinogen-induced telomerase activity Interfere with cellular processes involved in tumor promotion and progression, including telomerase Enhanced folding of telomeric DNA (guanine repeats) into a four-stranded quadraplex structure that hinders telomerase from adding further repeats Prevent access of telomerase RNA template to telomeric DNA sequences by complementary sequence binding to the template Chain-terminating nucleoside triphosphate analog that prevents lengthening of telomeric DNA sequences
[7] [12]
[2,9,10,13]c
[8,9]
RNA with a methyl group attached to the 20 position. Peptide nucleic acid, a mimic of DNA with a peptide backbone. c http://www.geron.com/. a
b
Another strategy that could be used to downregulate telomerase is the administration of AZT. AZT is a nucleoside triphosphate analog that can act on the RNA template of telomerase as a chain terminator, thus preventing extension of telomeric DNA sequences. Previous studies have shown that telomerase activity in the ciliated protozoan Tetrahymena thermophila could be inhibited in vitro by AZT, and that this nucleoside analog also suppressed developmentally regulated de novo telomere addition [8]. The potential of both antisense- and AZT-induced inhibition of telomerase as potent factors in limiting exponential cell (tumor) growth has been demonstrated recently [9]. In these studies, using telomerase-positive human pharynx FaDu tumor cells, treatment with antisense to the hTR resulted in inhibition of telomerase activity, shortened telomere length and reduced cell growth rate. Similar results were observed using the chain terminator AZT. However, telomerase inhibition in these experiments resulted in cytotoxicity only after depletion of pre-existing telomeres had occurred over a significant period of time [9], consistent with results that show dependence of cellular arrest on a lag period [11]. Synergy in cancer therapy: simultaneous erosion of telomeres and telomerase inhibition It is evident that inhibiting only the function of telomerase or the expression of telomerase [2,5– 11] is not sufficient to control exponential cellular proliferation. Cells might acquire a telomerase-independent mode of telomere lengthening termed alternative lengthening of telomeres (ALT) within the lag period that is necessary to result in cytotoxicity [13]. Thus, the requirement for residual telomere depletion has limited the therapeutic efficacy of telomerase inhibitors. Future research should therefore focus on a biphasic approach to target exponential cell growth by both eroding intrinsic levels of telomeres and preventing de novo telomere regeneration by telomerase. The first evidence of such a synergistic effect and the great promise held by such an approach in limiting cellular proliferation and tumor growth has been demonstrated recently [9]. Treatment of mice containing implanted FaDu pharyngial cancer cells with paclitaxel, an anti-proliferative agent http://tips.trends.com
that has been shown to target tubulin and also result in telomere erosion, resulted in a 2.5-fold increase (from less than 12% to 30%) in the number of apoptotic cells per tumor, compared with saline-treated controls. A reduction in tumor size by , 67% was also observed ðP , 0:05Þ: In groups treated with both paclitaxel and AZT, there was a sixfold increase in the number of apoptotic cells (from 12% to 72%) and an 80% reduction in tumor size ðP , 0:05Þ [9]. Animal survival also improved. The development of strategies using the antisense model and AZT to limit the function of telomerase are still in their infancy for several reasons, including the need to optimize drug delivery, the duration of action and the dosing regimen. Geron Corporation, the pharmaceutical company that first cloned the RNA template of telomerase, has developed an antisense agent (GRN163) that is currently in preclinical trials and has so far shown no toxicity at therapeutically effective doses (http://www. geron.com/). However, scientists still need to continue to identify ways to enhance selectivity with respect to telomerase inhibition and telomere erosion in transformed cells while protecting progenitor cells from the side-effects of such inhibition. It will be important to characterize telomerase activity in various tumors in various cell lines to produce a ‘data-bank’ that could be useful in the development of treatments of telomerasepositive cancers. Acknowledgements This work was supported by Public Health Service Grant RO1CA86357
References 1 Hanahan, D. et al. (1997) The hallmarks of cancer. Cell 100, 57 – 70 2 Corey, D.R. et al. (1999) Inhibition of human telomerase in immortal human cells leads to progressive telomere shortening and cell death. Proc. Natl. Acad. Sci. U. S. A. 96, 14276 – 14281 3 Counter, C.M. et al. (1992) Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J. 5, 1921– 1929 4 Meyerson, M. et al. (1997) hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell 90, 785 – 795 5 Glover, K.Y. and Papadimitrakopoulou, V.A. (2003) Chemoprevention of head and neck cancer. Curr. Oncol. Reports 5, 152– 157 6 Kensler, T.W. et al. (2002) Strategies for chemoprevention of liver cancer. Eur. J. Cancer Prev. (Suppl. 2), S58– S64
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7 Gescher, A.J. et al. (2001) Cancer chemoprevention by dietary constituents: a tale of failure and promise. Lancet Oncol. 2, 371 – 379 8 Strahl, C. and Blackburn, E.H. (1994) The effects of nucleoside analogs on telomerase and telomeres in tetrahymena. Nucleic Acids Res. 22, 893 – 900 9 Mo, Y. et al. (2003) Simultaneous targeting of telomeres and telomerase as a cancer therapeutic approach. Cancer Res. 63, 579– 585 10 Corey, D.R. (2002) Telomerase inhibition, oligonucleotides, and clinical trials. Oncogene 21, 631 – 637 11 Shammas, M.A. et al. (1999) Telomerase inhibition by peptide nucleic
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acids reverses ‘immortality’ of transformed human cells. Oncogene 18, 6191– 6200 12 Haider, S.M. et al. (2003) Structure of a G-quadraplex – ligand complex. Cancer Res. 326, 117– 125 13 Bryan, T.M. et al. (1997) Evidence for an alternative mechanism for maintaining telomere length in human tumors and tumor-derived cell lines. Nat. Med. 3, 1271– 1274 0165-6147/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0165-6147(03)00165-2
Pharmacological treatment of headache using traditional Persian medicine Ali Gorji Institut fu¨r Physiologie, Universita¨t Mu¨nster, Robert-Koch-Strasse 27a, 48149 Mu¨nster, Germany
Naturally occurring substances derived from plants currently have, and will continue to have, a relevant place in drug discovery. The medieval Persian physicians have provided long lists of plants that they used to treat cephalalgia. Some of these substances are employed in clinical practice today; however, still more of these naturally occurring substances could be of use in modern medicine. The use of medicinal plants for the treatment of headache in Persia can be traced back to the 6th century BC [1]. However, most of the documents that outline the treatment of headache are derived from the medieval period. Some of these texts, such as Qanoon-fel-teb (The Canon) by Ebn-e-Sina (980– 1037) and Raˆzi’s (860 – 940) Ketab-alhawi (Continents), became reference sources for medical studies in many European universities from the 13th to the 18th centuries (Fig. 1). Medieval Persian physicians described the treatment of headache using many substances with variable modes of action (Table 1). They attributed the therapeutic actions of plants to a specific analgesic, sedative or prophylactic drug property of variable strength [1,2]. In the medical texts of medieval Persia, the physicians classified cephalalgia as simple and non-recurrent (seda), recurrent bilateral (bayzeh) or recurrent unilateral (shaqhiqheh) headache. This classification system was important in the design of the treatment plan, which included the prescription of medicinal herbs [1,2]. Despite progress in the development of therapy in recent years, effective and potent drugs are still required for the treatment of headache. The search for new pharmacologically active analgesics obtained from plants has led to the discovery of some clinically useful drugs that, during the past two centuries, have played a major role in the treatment of human diseases. However, most medicinal plants prescribed by Persian physicians remain Corresponding author: Ali Gorji ([email protected]). http://tips.trends.com
Fig. 1. During the renaissance, Persian medical knowledge and theories influenced contemporary medicine. At this time, higher education reached the peaks of prosperity in Persian history, and many painters and poets described the importance of this period. ‘Celebration of learning’, painted by Mahmoud Farshchian, is one of these works. Reprinted with the permission from the Saad’abad cultural complex and the Farshchian Museum, Tehran, Iran.
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NB-DNJ on epididymal histology and function have yet to be examined. The sulfoglycolipid seminolipid (sulfogalactosylalkylacylglycerol) is a well-recognized component of the plasma membrane of spermatozoa and has been shown to be essential for spermatogenesis [24] and play a significant role in the recognition of the zona pellucida (the membrane surrounding a mammalian oocyte) by sperm [25]. Therefore, an agent that alters the biosynthesis of glycosphingolipids would be expected to affect the formation of the cell membrane. The extensive damage observed in the nucleus, mitochondrial sheath and acrosome of sperm following treatment with NB-DNJ might also be linked to altered glycosphingolipids associated with these organelles, but this remains to be established. It will be interesting to determine whether, at doses used to induce infertility, NB-DNJ also causes significant changes in other tissues, such as the brain, that are rich in sulfoglucolipids. To be developed as a male contraceptive any compound will need to have negligible risks associated with its use (no toxicity), while having very high efficacy. Whether, after safety evaluation, this particular compound succeeds or not, the report from Platt’s group provides the foundation for developing new male contraceptives. This represents a new avenue of research in the field of male contraceptive development. References 1 Anderson, R.A. et al. (2002) Male contraception. Endocr. Rev. 23, 735 – 762 2 Turner, T.T. (1991) Spermatozoa are exposed to a complex microenvironment as they traverse the epididymis. Ann. New York Acad. Sci. 637, 364 – 383 3 Zirkin, B.R. et al. (1989) Maintenance of advanced spermatogenic cells in the adult rat testis: quantitative relationship to testosterone concentration within the testis. Endocrinology 124, 3043 – 3049 4 Lobl, T.J. et al. (1983) Contraceptive efficacy of testosterone-estradiol implants in male rhesus monkeys. Contraception 27, 383 – 389 5 van Houten, M.E. et al. (2000) Differences in reproductive endocrinology between Asian men and Caucasian men. Asian J. Androl. 2, 13–20 6 Ewing, L.L. et al. (1977) Synergistic interaction of testosterone and oestradiol inhibits spermatogenesis in rats. Nature 269, 409 – 411 7 Kamischke, A. et al. (2001) Intramuscular testosterone undecanoate and norethisterone enanthate in a clinical trial for male contraception. J. Clin. Endocrinol. Metab. 86, 303 – 309 8 McLachlan, R.I. et al. (2002) Effects of testosterone plus medroxyprogesterone acetate on semen quality, reproductive hormones, and germ cell populations in normal young men. J. Clin. Endocrinol. Metab. 87, 546–556
Vol.24 No.7 July 2003
9 Ramachandra, S.G. et al. (2002) Effect of chronic administration of 7alpha-methyl-19-nortestosterone on serum testosterone, number of spermatozoa and fertility in adult male bonnet monkeys (Macaca radiata). Reproduction 124, 301 – 309 10 Jackson, H. et al. (1979) Contraception for the male: problems with progress. Clin. Obstet. Gynaecol. 6, 129 – 155 11 Liu, G.Z. et al. (1985) Trials of gossypol as a male contraceptive. In Gossypol: A Potential Contraceptive for Men (Segal, S., ed.), pp. 9 – 16, Plenum Publishers 12 Zavos, P.M. et al. (1996) The inhibitory effects of gossypol on human sperm motility characteristics: possible modes of reversibility of those effects. Tohoku J. Exp. Med. 179, 167– 175 13 Liu, G.Z. et al. (1988) Effects of K salt or a potassium blocker on gossypol-related hypokalemia. Contraception 37, 111 – 117 14 Shi, Y.L. et al. (2003) Ion-channels in human sperm membrane and contraceptive mechanisms of male antifertility compounds derived from Chinese traditional medicine. Acta Pharmacol. Sin. 24, 22 – 30 15 Cheng, C.Y. et al. (2002) Indazole carboxylic acids in male contraception. Contraception 65, 265– 268 16 Cooper, T.G. (2002) The epididymis as a target for male contraception. In The Epididymis: From Molecules to Clinical Practice (Robaire, B. and Hinton, B.T., eds), pp. 483 – 502, Kluwer Academic Plenum Publishers 17 Frayne, J. et al. (1999) The potential use of sperm antigens as targets for immunocontraception; past, present and future. J. Reprod. Immunol. 43, 1 – 33 18 Ren, D. et al. (2001) A sperm ion channel required for sperm motility and male fertility. Nature 413, 603 – 609 19 van der Spoel, A.C. et al. (2002) Reversible infertility in male mice after oral administration of alkylated imino sugars: a nonhormonal approach to male contraception. Proc. Natl. Acad. Sci. U. S. A. 99, 17173 – 17178 20 Butters, T.D. et al. (2003) Therapeutic applications of imino sugars in lysosomal storage disorders. Curr. Top. Med. Chem. 3, 561 – 574 21 Platt, F.M. et al. (2001) Inhibition of substrate synthesis as a strategy for glycolipid lysosomal storage disease therapy. J. Inherit. Metab. Dis. 24, 275 – 290 22 Cox, T. et al. (2000) Novel oral treatment of Gaucher’s disease with Nbutyldeoxynojirimycin (OGT 918) to decrease substrate biosynthesis. Lancet 355, 1481– 1485 23 Trasler, J. et al. (1998) Characterization of the testis and epididymis in mouse models of human Tay Sachs and Sandhoff diseases and partial determination of accumulated gangliosides. Endocrinology 139, 3280– 3288 24 Fujimoto, H.K. et al. (2000) Requirement of seminolipid in spermatogenesis revealed by UDP-galactose: Ceramide galactosyltransferase-deficient mice. J. Biol. Chem. 275, 22623 – 22626 25 White, D. et al. (2000) Role of sperm sulfogalactosylglycerolipid in mouse sperm-zona pellucida binding. Biol. Reprod. 63, 147 – 155 0165-6147/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0165-6147(03)00141-X
Telomerase inhibition and telomere erosion: a two-pronged strategy in cancer therapy Fred O. Odago and Stanton L. Gerson Division of Hematology/Oncology and the Comprehensive Cancer Center at Case Western Reserve University and University Hospitals of Cleveland, 10900 Euclid Ave., BRB 3-West, Cleveland, OH 44106-4937, USA
Inhibition of telomerase activity in cancerous cells is a very potent factor in the abrogation of cellular Corresponding author: Stanton L. Gerson ([email protected]). http://tips.trends.com
immortalization. However, targeting telomerase alone is not sufficient to elicit cell death because tumor cells can acquire the ability to lengthen their telomeres independently of telomerase activity, in a process
Update
TRENDS in Pharmacological Sciences
referred to as alternative lengthening of telomeres. In this article, we suggest that a biphasic approach, by both eroding intrinsic levels of telomeres and inhibiting telomerase activity, would result in a synergistic beneficial effect that could be used in cancer therapy. Research in cancer, at present, is focused mostly on identifying therapeutic agents that can limit the exponential proliferation of transformed cells. The identification of a single therapeutic target that will act as a panacea in cancer treatment is compounded by the fact that the transformation of normal cells into malignancies is a multi-step process, which thus necessitates targeting the various acquired capabilities of these cells [1]. Recently, it has become apparent that targeting telomerase, a ribonucleoprotein responsible for adding repetitive units of TTAGGG to the ends of telomeres (structures at the end of chromosomes that protect them from end-to-end fusion), could be a potent factor in the control of invasive cancerous cells [2]. The normal addition of these nucleotide repeats averts a crisis in which increased shortening and eventual loss of telomeres results in the cell entering senescence (Fig. 1). Loss of telomeres also results in end-to-end chromosomal fusions, yielding karyotypic disarray, which leads eventually to the death of the cell [3]. Thus, limiting the levels of telomerase within tumorigenic cells to allow the cells to enter senescence and eventually undergo cell death would be a favorable anticancer strategy. Telomerase as a target of cancer control is favored further by observations that levels of the catalytic subunit of this enzyme are particularly elevated in most tumorigenic cells compared with such levels in normal cells [4]. Strategies that silence telomerase gene expression or inhibit telomerase function Various strategies have been shown to affect the intrinsic levels of telomerase as a result of either a direct or an Normal somatic cell chromosome
(a) (b)
GGTTAGGGTTAG CAAUCCCAAUC
Cell senesces and eventually dies
(c)
Cancerous cell chromosome
GTTAGGGTTAGGGTTAG CAAUCCCAAUC
TRENDS in Pharmacological Sciences
Fig. 1. Involvement of telomerase in cancer. (a) The conversion of normal dividing cells into senescent cells involves the loss of telomeric ends (dark blue) in a process known as telomeric arm erosion. The telomerase enzyme (red arrow) is used to lengthen the telomeres (inset), and prevent cells from entering senescence and undergoing end-to-end chromosomal fusions. (b) The conversion of normal somatic cells to cancerous cells involves the upregulation of telomerase expression resulting in increased telomere lengths and a decreased likelihood of senescence. (c) Cancerous cells can be directed to senesce and eventually die by the use of paclitaxel, a putative telomere eroding agent. Telomerase inhibitors (shown in Table 1) enhance the effect of paclitaxel in a synergistic manner. http://tips.trends.com
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indirect action (Table 1). These strategies include: (1) chemoprevention [5 – 7]; (2) anti-retroviral attack using a chain-terminating nucleoside triphosphate analog known to inhibit reverse transcriptases [e.g. azidothymidine (AZT)] [8,9]; (3) an antisense strategy using oligonucleotides [2,10,11]; and (4) the use of G-quadraplex ligands [12]. Chemoprevention is the use of agents, either pharmacological or natural, that inhibit the development of tumors by either preventing the DNA damage that initiates carcinogenesis or arresting or reversing the progression of pre-malignant cells in which such DNA damage has already occurred [5]. An example of a pharmacological agent is oltipraz, which is a potent inducer of the enzymes involved in the detoxification of carcinogens such as aflatoxin. Oltipraz has been shown to have a marked inhibitory effect on carcinogenesis within the liver and to indirectly prevent the accumulation of telomerase [6]. Other chemopreventive agents include natural diet constituents such as curcuminin (present in the spice turmeric), genistein (present in soya) and catechins (present in tea). These agents have been shown to have tumor-suppressing properties in transgenic mice studies and have resulted in the downregulation of intrinsic levels of telomerase [7]. Another method of inhibiting telomerase is by enhancing the folding of telomeric DNA (guanine repeats) into a four-stranded quadraplex structure held together by Hoogsten hydrogen-bonded arrays of guanine bases. The formation of this quadraplex structure at the 30 end of telomeric DNA effectively hinders telomerase from adding further repeats. The use of G-quadraplex ligands, small molecules that stabilize the secondary structure comprising the guanine-rich repetitive DNA, is of particular importance in strategies aimed at prolonging the effects of telomerase inhibition [12]. Using an antisense strategy, oligonucleotides complementary to the telomerase RNA component (hTR) can be expressed in cancerous cells that exhibit elevated levels of telomerase. Exogenously added oligomers complementary to the template region of hTR, such as 20 -O-MeRNA and peptide nucleic acid (PNA) (a DNA mimic with a neutral amide backbone), have been shown to inhibit telomerase activity by preventing access of the RNA template to telomeric DNA sequences. This has resulted in an enhanced shortening of telomeres, eventually resulting in cell death by apoptosis after an extended period of treatment [2]. 20 -O-alkyl-derivatives and PNA oligomers bind tighter to complementary RNA sequences than do analogous DNA oligomers, resulting in an improved antisense selectivity, and have also been shown to have enhanced efficacy and pharmacological properties, compared with analogous DNA oligomers that have neither a peptide backbone nor been substituted at the 20 position [10]. In studies using immortal human cells and PNA oligomers that were complementary to telomerase RNA, telomerase activity was effectively inhibited, telomeres were markedly shortened and, after a lag period, cell proliferation was arrested. These observations served as markers of a suppressed cellular ‘immortality’ [11].
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Table 1. Strategies used to inhibit telomerase activity Strategy
Agents
Mode of action
Refs
Chemoprevention
Oltipraz
[6]
G-quadraplex ligands
Natural diet constituents such as genistein, catechins and curcuminin Acridine compounds
Antisense strategy
20 O-MeRNAa and PNAb
Anti-retroviral attack
Azidothymidine
Induces the synthesis of detoxification enzymes for carcinogens, thus lowering carcinogen-induced telomerase activity Interfere with cellular processes involved in tumor promotion and progression, including telomerase Enhanced folding of telomeric DNA (guanine repeats) into a four-stranded quadraplex structure that hinders telomerase from adding further repeats Prevent access of telomerase RNA template to telomeric DNA sequences by complementary sequence binding to the template Chain-terminating nucleoside triphosphate analog that prevents lengthening of telomeric DNA sequences
[7] [12]
[2,9,10,13]c
[8,9]
RNA with a methyl group attached to the 20 position. Peptide nucleic acid, a mimic of DNA with a peptide backbone. c http://www.geron.com/. a
b
Another strategy that could be used to downregulate telomerase is the administration of AZT. AZT is a nucleoside triphosphate analog that can act on the RNA template of telomerase as a chain terminator, thus preventing extension of telomeric DNA sequences. Previous studies have shown that telomerase activity in the ciliated protozoan Tetrahymena thermophila could be inhibited in vitro by AZT, and that this nucleoside analog also suppressed developmentally regulated de novo telomere addition [8]. The potential of both antisense- and AZT-induced inhibition of telomerase as potent factors in limiting exponential cell (tumor) growth has been demonstrated recently [9]. In these studies, using telomerase-positive human pharynx FaDu tumor cells, treatment with antisense to the hTR resulted in inhibition of telomerase activity, shortened telomere length and reduced cell growth rate. Similar results were observed using the chain terminator AZT. However, telomerase inhibition in these experiments resulted in cytotoxicity only after depletion of pre-existing telomeres had occurred over a significant period of time [9], consistent with results that show dependence of cellular arrest on a lag period [11]. Synergy in cancer therapy: simultaneous erosion of telomeres and telomerase inhibition It is evident that inhibiting only the function of telomerase or the expression of telomerase [2,5– 11] is not sufficient to control exponential cellular proliferation. Cells might acquire a telomerase-independent mode of telomere lengthening termed alternative lengthening of telomeres (ALT) within the lag period that is necessary to result in cytotoxicity [13]. Thus, the requirement for residual telomere depletion has limited the therapeutic efficacy of telomerase inhibitors. Future research should therefore focus on a biphasic approach to target exponential cell growth by both eroding intrinsic levels of telomeres and preventing de novo telomere regeneration by telomerase. The first evidence of such a synergistic effect and the great promise held by such an approach in limiting cellular proliferation and tumor growth has been demonstrated recently [9]. Treatment of mice containing implanted FaDu pharyngial cancer cells with paclitaxel, an anti-proliferative agent http://tips.trends.com
that has been shown to target tubulin and also result in telomere erosion, resulted in a 2.5-fold increase (from less than 12% to 30%) in the number of apoptotic cells per tumor, compared with saline-treated controls. A reduction in tumor size by , 67% was also observed ðP , 0:05Þ: In groups treated with both paclitaxel and AZT, there was a sixfold increase in the number of apoptotic cells (from 12% to 72%) and an 80% reduction in tumor size ðP , 0:05Þ [9]. Animal survival also improved. The development of strategies using the antisense model and AZT to limit the function of telomerase are still in their infancy for several reasons, including the need to optimize drug delivery, the duration of action and the dosing regimen. Geron Corporation, the pharmaceutical company that first cloned the RNA template of telomerase, has developed an antisense agent (GRN163) that is currently in preclinical trials and has so far shown no toxicity at therapeutically effective doses (http://www. geron.com/). However, scientists still need to continue to identify ways to enhance selectivity with respect to telomerase inhibition and telomere erosion in transformed cells while protecting progenitor cells from the side-effects of such inhibition. It will be important to characterize telomerase activity in various tumors in various cell lines to produce a ‘data-bank’ that could be useful in the development of treatments of telomerasepositive cancers. Acknowledgements This work was supported by Public Health Service Grant RO1CA86357
References 1 Hanahan, D. et al. (1997) The hallmarks of cancer. Cell 100, 57 – 70 2 Corey, D.R. et al. (1999) Inhibition of human telomerase in immortal human cells leads to progressive telomere shortening and cell death. Proc. Natl. Acad. Sci. U. S. A. 96, 14276 – 14281 3 Counter, C.M. et al. (1992) Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J. 5, 1921– 1929 4 Meyerson, M. et al. (1997) hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell 90, 785 – 795 5 Glover, K.Y. and Papadimitrakopoulou, V.A. (2003) Chemoprevention of head and neck cancer. Curr. Oncol. Reports 5, 152– 157 6 Kensler, T.W. et al. (2002) Strategies for chemoprevention of liver cancer. Eur. J. Cancer Prev. (Suppl. 2), S58– S64
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7 Gescher, A.J. et al. (2001) Cancer chemoprevention by dietary constituents: a tale of failure and promise. Lancet Oncol. 2, 371 – 379 8 Strahl, C. and Blackburn, E.H. (1994) The effects of nucleoside analogs on telomerase and telomeres in tetrahymena. Nucleic Acids Res. 22, 893 – 900 9 Mo, Y. et al. (2003) Simultaneous targeting of telomeres and telomerase as a cancer therapeutic approach. Cancer Res. 63, 579– 585 10 Corey, D.R. (2002) Telomerase inhibition, oligonucleotides, and clinical trials. Oncogene 21, 631 – 637 11 Shammas, M.A. et al. (1999) Telomerase inhibition by peptide nucleic
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acids reverses ‘immortality’ of transformed human cells. Oncogene 18, 6191– 6200 12 Haider, S.M. et al. (2003) Structure of a G-quadraplex – ligand complex. Cancer Res. 326, 117– 125 13 Bryan, T.M. et al. (1997) Evidence for an alternative mechanism for maintaining telomere length in human tumors and tumor-derived cell lines. Nat. Med. 3, 1271– 1274 0165-6147/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0165-6147(03)00165-2
Pharmacological treatment of headache using traditional Persian medicine Ali Gorji Institut fu¨r Physiologie, Universita¨t Mu¨nster, Robert-Koch-Strasse 27a, 48149 Mu¨nster, Germany
Naturally occurring substances derived from plants currently have, and will continue to have, a relevant place in drug discovery. The medieval Persian physicians have provided long lists of plants that they used to treat cephalalgia. Some of these substances are employed in clinical practice today; however, still more of these naturally occurring substances could be of use in modern medicine. The use of medicinal plants for the treatment of headache in Persia can be traced back to the 6th century BC [1]. However, most of the documents that outline the treatment of headache are derived from the medieval period. Some of these texts, such as Qanoon-fel-teb (The Canon) by Ebn-e-Sina (980– 1037) and Raˆzi’s (860 – 940) Ketab-alhawi (Continents), became reference sources for medical studies in many European universities from the 13th to the 18th centuries (Fig. 1). Medieval Persian physicians described the treatment of headache using many substances with variable modes of action (Table 1). They attributed the therapeutic actions of plants to a specific analgesic, sedative or prophylactic drug property of variable strength [1,2]. In the medical texts of medieval Persia, the physicians classified cephalalgia as simple and non-recurrent (seda), recurrent bilateral (bayzeh) or recurrent unilateral (shaqhiqheh) headache. This classification system was important in the design of the treatment plan, which included the prescription of medicinal herbs [1,2]. Despite progress in the development of therapy in recent years, effective and potent drugs are still required for the treatment of headache. The search for new pharmacologically active analgesics obtained from plants has led to the discovery of some clinically useful drugs that, during the past two centuries, have played a major role in the treatment of human diseases. However, most medicinal plants prescribed by Persian physicians remain Corresponding author: Ali Gorji ([email protected]). http://tips.trends.com
Fig. 1. During the renaissance, Persian medical knowledge and theories influenced contemporary medicine. At this time, higher education reached the peaks of prosperity in Persian history, and many painters and poets described the importance of this period. ‘Celebration of learning’, painted by Mahmoud Farshchian, is one of these works. Reprinted with the permission from the Saad’abad cultural complex and the Farshchian Museum, Tehran, Iran.