THE SCIENTIFIC FOUNDATION

1. THE ARYL HYDROCARBON RECEPTOR

The aryl hydrocarbon (Ah) receptor (AhR) is a ligand inducible transcription factor, member of a so-called basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) superfamily. Upon binding to its ligand, the receptor mediates and interacts with a series of biological processes including cell division, apoptosis (programmed cell death), cell differentiation, actions of estrogen and androgen, adipose differentiation, hypothalamus actions, immune system homeostasis, vascular development and remodeling, and actions of other hormonal systems beside the expression of genes of P450 family and some others^[1-7]. The genomic action of AhR is depicted in Fig. 1. Once bound to its ligand, the receptor participates in biological processes through translocation from cytoplasm into nucleus, heterodimerization with another factor named Ah receptor nuclear translocator (Arnt), attachment of the heterodimer to the regulatory region termed Ah response element (AhRE) of genes under AhR regulation, and then either enhancement or inhibition of transcription of those genes.

Fig. 1. The genomic action of the aryl hydrocarbon (Ah) receptor (AhR).

2. A PHYSIOLOGICAL LIGAND FOR THE RECEPTOR

The receptor system has been studied so far with its artificial ligands (exogenous chemicals that happen to have binding affinities to the receptor). While studies with those AhR artificial ligands greatly advanced our understanding toward the receptor system, thorough elucidation of the physiological roles the system plays and the potential therapeutic benefits the system may offer are impossible without the identification of AhR physiological ligand. To identify the physiological ligand for the receptor, we purified ~20 mg of an endogenous AhR ligand from lungs of ~70 adult pigs. We then unequivocally identified its previously unknown structure by means of UV, FT-IR, and mass spectroscopy; extensive nuclear magnetic resonance (NMR) spectroscopic studies; micro-scale chemical reactions, and thorough theoretical (quantum mechanical) calculations^[8]. The structure (Fig. 2, 3A), 2-(1’H-indole-3’-carbonyl)-thiazole-4-carboxylic acid methyl ester (short for ITE), was confirmed further by chemical synthesis^[9]. The biological potency was estimated as 5 times greater than that of b-nephthoflavone (BNF), one of the potent artificial ligands for the AhR. The estimated Ki value (the smaller the value, the higher the affinity for receptor binding) for ITE is 3 nM vs. 2 and 0.5 nM for, respectively, BNF and TCDD (another potent AhR artificial ligand).

Fig. 2. The space-filling structure of ITE, a physiological ligand or natural hormone for the aryl hydrocarbon (Ah) receptor (AhR).

3. THE INTERCONVERSION BETWEEN ITE AND ITC

To investigate the biological production and metabolism of ITE (the AhR physiological ligand or natural hormone), we identified its precursor (or prehormone). The precursor is a carboxylate, 2-(1’H-indole-3’-carbonyl)-thiazole-4-carboxylate (short for ITC) (Fig. 3), which is unable to bind to the Ah receptor and thus inactive in the system. We also discovered an enzymatic system in animal tissue to convert ITC to ITE and an inhibitor to the conversion system. ITE was then found to be able to undergo hydrolysis into ITC catalyzed by an esterase. There is a report in literature that an esterase is inducible by an AhR artificial ligand^[10]. Therefore, it is highly possible that ITE could induce one or more esterase(s) hydrolyzing itself into ITC to establish a feedback control loop. Those discoveries lead to our current understanding toward the system as follows (Fig. 3B). When AhR activation is called for, the expression or activity of the enzyme(s) converting ITC to ITE will be enhanced and/or the inhibitor will be removed or inactivated to make ITE (undetectable in major adult organs including the blood) from ITC (circulating in blood). While performing other biological duties, the ITE activated receptor would also enhance the expression of one or more esterase(s) to hydrolyze ITE into ITC (inactive prehormone) to halt the action of ITE and its receptor^{[11, 12]}.

Fig. 3. The structure of ITE (A.) and the regulated interconversion between ITE and ITC, a precursor and also a major metabolite of ITE (B.)

4. THE THERAPEUTIC POTENTIALS OF ITE

With the system of ITE and its receptor described, we will analyze the therapeutic potentials of ITE starting from its in vivo antiangiogenic property. Evidence of other therapeutic capabilities, the cancer therapeutic efficacy, the cancer therapeutic specificity, and possible low side effect(s) of ITE will be presented.

4-1. ITE is Antiangiogenic in vivo

While we devoted most of our time and energy to the purification, identification, and confirmation of ITE and to the investigation of its biological production and metabolism, we conducted certain experiments to explore its therapeutic potentials. We discovered that ITE strongly inhibits angiogenesis (generation of new blood vessels from the existing ones) induced by both bFGF (basic Fibroblast Growth Factor) (Fig. 4) and tumor cells (data not shown) in a mouse corneal grafting model. With a concentration of 100 times lower (100 mg/kg bodyweight) than that used in Fig. 4, ITE can still effectively block the process (data not shown). The property alone is very important in combating cancer, obesity, and blinding retinopathy. Cancer cannot grow beyond 1 to 2 mm in diameter without newly formed blood vessels to supply nutrients with oxygen and remove wastes. Similar to cancer, adipose tissue expansion requires also new blood vessel generation to sustain the process. The major cause of blinding retinopathy is the excessive generation of new but low quality blood vessels eventually leading to blindness. ITE does not seem to stop here, however.

Fig. 4. While i.p. injection of vehicle (DMSO:propylene glycol, 1:1, v/v) did not affect the bFGF induced angiogenesis (A1 to A3), ITE in the vehicle (10 mg/kg bodyweight) inhibited strongly the angiogenesis process (A4 to A6). The quantification (B.) confirms the visual impression. The inhibition is still very effective at a much lower concentration (100 mg/kg bodyweight, data not shown).

4-2. ITE Has other Capabilities

In addition to the antiangiogenic property, ITE may well have other capabilities. The Ah receptor (AhR) happens to be able to bind, with different affinities, to several groups of exogenous chemicals (thus artificial ligands) such as polycyclic aromatic hydrocarbons exemplified by 3-methylchoranthrene (3-MC) and halogenated aromatic hydrocarbons typified by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Known functions of the AhR thus far have been learned by probing with its artificial ligands. Analyzing data on AhR functions studied with its artificial ligands in literature, it is evident that ITE may well be able to also inhibit cell division^[13-15], induce cell differentiation^{[3, 7]}, promote apoptosis^{[16, 17]}, and antagonize estrogen^{[4, 18]} and androgen^{[19, 20]} signaling systems for efficient cancer intervention. This multiple mechanism based combating capability makes the ITE unique, especially in therapeutic intervention of estrogen-dependent breast cancer and androgen-dependent prostate cancer. For the obesity therapy, besides choking fat tissues to death, ITE will be efficacious not only because it can inhibit cell division and promote apoptosis but also because it can block the transformation of a normal cell to fat cells^[21-23] and can control food intake and energy balance^{[24, 25]}.

4-3. Evidence Supporting the Efficacy of ITE Cancer Therapy

Even though most of the artificial ligands for AhR are environmental toxins and thus cannot be used as therapeutic agents, for the purpose of understanding AhR functions, TCDD, 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF), and 8-methyl-1,3,6-trichlorodibenzofuran (8-MCDF) were used to discover that the Ah receptor was able to inhibit the growth of carcinogen induced rat mammary tumor^{[26, 27]} and human breast tumor cell (MCF-7) xenograft^[28]. Fig. 5 represents this type of studies^[27].

Fig. 5. AhR artificial ligands, 6-MCDF (left) and 8-MCDF(right), inhibit the growth of rat mammary tumor induced by 7,12-dimethylbenz[a]anthracene. Animals were injected i.p. weekly with corn oil as vehicle control or compounds dissolved in the vehicle^[27] (reproduced with permission from Dr. Stephen Safe of Texas A & M University, College Station, TX 77843).

4-4. Evidence Supporting the Specificity of ITE Cancer Therapy

Supporting the possible specificity of the ITE therapy, the Ah receptor was reportedly to be highly expressed in pancreatic cancer tissues from 14 out of 15 patients but faintly expressed in all normal pancreatic tissues examined^[29]. Furthermore, the AhR artificial ligands were shown to be able to inhibit the in vitro growth of pancreatic cancer cells expressing the Ah receptor^[29]. Similarly, the enhanced AhR expression is also documented with prostate cancer^[30].

4-5. Possibility of Low Side-effect for ITE Therapy

With the grand therapeutic opportunity of ITE outlined, we will analyze why we think the ITE hormone therapy may well have low side effect. Aspects of metabolism of exogenous chemicals vs. a natural hormone, probability of “off-target” binding and interaction of exogenous chemicals vs. a natural hormone, preliminary observation of ITE dosed animals, and a reported study in ITE biology and toxicology will be analyzed.

4-5-1. ITE has a natural way of metabolism

In our body, the metabolism of exogenous chemicals, including those AhR artificial ligands, presents quite a challenge. In an effort to get rid of those exogenous chemicals, many chemically active intermediates or radicals will be produced unavoidably during the metabolic elimination of those chemicals. Those radicals or chemically active intermediates will assault many cellular substances including nucleic acids (Fig. 6, for an example) and proteins causing a lot of adverse reactions including the induction of cancers while a simple enzymatic action, without producing any chemically active intermediate, will convert the lipophilic ITE into a polar ITC circulating naturally in the blood (Fig. 3). ITC could then be easily excreted, most probably, through the urinary system when its level goes higher. It is, therefore, very clear that it may have a completely different consequence of administering the natural hormone ITE from that of exogenous chemicals including AhR artificial ligands. Therefore, the concern that most of AhR artificial ligands happen to be environmental toxicants and thus main functions of the Ah receptor seem to “mediate” toxicological responses has also been addressed.

Fig. 6. Some chemically active intermediates from metabolic elimination of exogenous chemicals, including AhR artificial ligands, will attack a base, the genetic signal, on the DNA chain thus destroying or disrupting the flow of genetic information.

4-5-2. ITE should have very low chance of “off-target” action

One of the important reasons for current therapeutic agents to be high in side effects is that since they are designed by humans, not the nature, they tend to have very high chance to bind to and interact with other molecules (including proteins and receptors) than their expected targets in the body. These “off-target” bindings and interactions account for significant opportunities for side effects. On the other hand, the binding of the natural hormone to its receptor (the Ah receptor) is very specific and precise since it is designed and manufactured by the nature. The hormone ITE, other than those human designed chemicals, will then have very low chance of binding to and interact with other proteins or molecules to provoke “off-target” problems.

4-5-3. No adverse effect of ITE has been observed so far

At least visually, we have not observed any adverse reaction from mice or rats when we were conducting animal experiments with ITE at a concentration of as high as 10 mg/kg bodyweight. The visual impression was supported by a reported study of ITE biology and toxicity together with a toxic AhR artificial ligand, TCDD^[31].

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5. CITIED LITERATURES

1. Schmidt, J.V. and Bradfield, C.A. 1996. Ah receptor signaling pathways. Annu Rev Cell Dev Biol. 12:55-89.

2. Whitlock, J.P.J. 1999. Induction of cytochrome P4501A1. Ann. Rev. Pharmacol. Toxicol. 39:103-125.

3. Poellinger, L. 2000. Mechanistic aspects-the dioxin (aryl hydrocarbon) receptor. Food Add. Contam. 17(4):261-266.

4. Safe, S., and McDougal, A. 2002. Mechanism of action and development of selective aryl hydrocarbon receptor modulators for treatment of hormone-dependent cancers. Internatl. J. Oncol. 20(6):1123-1128.

5. Safe, S., and Wormke, M. 2003. Inhibitory aryl hydrocarbon receptor-estrogen receptor a cross-talk and mechanisms of action. Chem. Res. Toxicol. 16(7):807-816.

6. Walisser, J.A., Bunger, M.K., Glover, E., and Bradfield, C.A. 2004. Gestational exposure of Ahr and Arnt hypomorphs to dioxin rescues vascular development. Proc. Natl. Acad. Sci. USA. 101(47):16677-16682.

7. Puga, A., Tomlinson, C.R., and Xia, Y. 2005. Ah receptor signals cross-talk with multiple developmental pathways. Biochem. Pharmacol. 69(2):199-207.

8. Song, J., Clagett-Dame, M., Peterson, R.E., Hahn, M.E., Westler, W.M., Sicinski, R.R., and DeLuca, H.F. 2002. A ligand fro the aryl hydrocarbon receptor isolated from lung. Proc. Natl. Acad. Sci. USA. 99(23):14694-14699.

9. Grzywacz, P., Sicinski, R.R., and DeLuca, H.F. 2003. A concise synthesis of an AHR endogenous ligand with the indolecarbonylthiazole skeleton. Heterocycles. 60(5):1219-1224.

10. Korza, G., and Ozols, J. 1988. Complete covalent structure of 60-kDa esterase isolated from 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced rabbit liver microsomes. J. Biol. Chem. 263(7):3486-3495.

11. Song, J., Clagett-Dame, M., Hahn, M.K., Peterson, R.E., and DeLuca, H.F. 2008. The production and metabolism of ITE, an aryl hydrocarbon (Ah) receptor physiological ligand. Manuscript Prepared.

12. Song, J., Barycki, R., Clagett-Dame, M., and DeLuca, H.F. 2008 An enzymatic system converting ITC to ITE, an Ah receptor physiological ligand. Manuscript in Preparation.

13. Elizondo, G., Fernandez-Salguero, P., Sheikh, M.S., Kim, G.Y., Fornace, A.J., Lee, K.S., and Gonzalez, F.J. 2000. Altered cell cycle control at the G(2)/M phases in aryl hydrocarbon receptor-null embryo fibroblast. Mol. Pharmacol. 57(5):1056-1063.

14. Puga, A., Xia, Y., and Elferink, C. 2002. Role of the aryl hydrocarbon receptor in cell cycle regulation. Chemico-Biol. Interact. 141(1-2):117-130.

15. Marlowe, J.L., Knudsen, E.S., Schwemberger, S., and Puga, A. 2004. The aryl hydrocarbon receptor displaces p300 from E2F-dependent promoters and represses S phase-specific gene expression. J. Biol. Chem. 279(28):29013-29022.

16. Kramer, H.J., Podobinska, M., Bartsch, A., Battmann, A., Thoma, W., Bernd, A., Kummer, W., Irlinger, B., Steglich, W., and Mayser, P. 2005. Malassezin, a novel agonist of the aryl hydrocarbon receptor from the yeast Malassezia furfur, induces apoptosis in primary human melanocytes. Chembiochem. 6(5):860-865.

17. Park, K.T., Mitchell, K.A., Huang, G.M., and Elferink, C.J. 2005. The aryl hydrocarbon receptor predisposes hepatocytes to Fas-mediated apoptosis. Mol. Pharmacol. 67(3):612-622.

18. Oenga, G.N., Spink, D.C., and Carpenter, D.O. 2004. TCDD and PCBs inhibit breast cancer cell proliferation in vitro. Toxicol. in vitro. 18(6):811-819.

19. Jana, N.R., Sarkar, S., Ishizuka, M., Yonemoto, J., Tohyama, C., and Sone, H. 1999. Cross-talk between 2,3,7,8-tetrachlorodibenzo-p-dioxin and testosterone signal transduction pathways in LNCaP prostate cancer cells Biochem. Biophy. Res. Comm. 256(3):462-468.

20. Morrow, D., Qin, C.H., Smith, R., and Safe, S. 2004. Aryl hydrocarbon receptor-mediated inhibition of LNCaP prostate cancer cell growth and hormone-induced transactivation. J. Ster. Biochem. Mol. Biol. 88(1):27-36.

21. Shimba, S., Wada, T., and Tezuka, M. 2001. Arylhydrocarbon receptor (AhR) is involved in negative regulation of adipose differentiation in 3T3-L1 cells: AhR inhibits adipose differentiation independently of dioxin. . J. Cell Sci. 114(15):2809-2817.

22. Hanlon, P.R., Ganem, L.G., Cho, Y.C. Yamamoto, M., and Jefcoate, C.R. 2003. AhR- and ERK-dependent pathways function synergistically to mediate 2,3,7,8-tetrachlorodibenzo-p-dioxin suppression of peroxisome proliferator-activated receptor-gamma 1 expression and subsequent adipocyte differentiation. . Toxicol. Appl. Pharmacol. 189(1):11-27.

23. Hanlon, P.R., Cimafranca, M.A., Liu, X.Q., Cho, Y.C., and Jefcoate, C.R. 2005. Microarray analysis of early adipogenesis in C(3)H10T1/2 cells: Cooperative inhibitory effects of growth factors and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. 207(1):39-58.

24. Fetissov, S.O., Huang, P., Zhang, Q., Mimura, J., Fujii-Kuriyama, Y., Rannug, A., Hokfelt, T., and Ceccatelli, S. 2004. Expression of hypothalamic neuropeptides after acute TCDD treatment and distribution of Ah receptor repressor. Reg. Peptides. 119(1-2):113-124.

25. Yang, C., Boucher, F., Tremblay, A., and Michaud, J.L. 2004. Regulatory interaction between arylhydrocarbon receptor and SIM1, two basic helix-loop-helix PAS proteins involved in the control of food intake. J. Biol. Chem. 279(10):9306-9312.

26. Holcomb, M., and Safe, S. 1994. Inhibition of 7,12-dimethylbenz[a]anthrancene-induced rat mammary tumor growth by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Cancer Let. 82:43-47.

27. McDougal, A., Wilson, C., and Safe, S. 1997. Inhibition of 7,12-dimethylbenz[a]anthracene-induced rat mammary tumor growth by aryl hydrocarbon receptor agonists. Cancer Let. 120:53-63.

28. Gierthy, J.F., Bennett, J.A., Bradly, L.M., and Cutler, D.S. 1993. Correlation of in vitro and in vivo growth suppression of MCF-7 human breast cancer by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Cancer Res. 53:3149-3153.

29. Koliopanos, A., Kleeff, J., Xiao, Y., Safe, S., Zimmermann, A., Buchler, M. W., and Friess, H. 2002. Increased arylhydrocarbon receptor expression offers a potential therapeutic target for pancreatic cancer. Oncogene. 21:6059-6070.

30. Kashani, M., Steiner, G., Haitel, A., Schaufler, K., Thalhammer, T., Amann, G., Kramer, G., Marberger, M., and Scholler, A. 1998. Expression of the aryl hydrocarbon receptor (AhR) and the aryl hydrocarbon receptor nuclear translocator (ARNT) in fetal, benign hyperplastic, and malignant prostate. Prostate. 37(2):98-108.

31. Henry, E.C., Bemis, J.C., Henry, O., Kende, A.S., and Gasiewicz, T.A. 2006. A potential endogenous ligand for the aryl hydrocarbon receptor has potent agonist activity in vitro and in vivo. Arch Biochem Biophys. 450(1):67-77.

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