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, cell migration, immune system stimulation or suppression, stem cell (hematopoietic) expansion, angiogenesis (new blood vessel generation from existing ones), actions of estrogen and androgen, adipose differentiation, hypothalamus actions, and actions of other hormonal systems beside the expression of genes of P450 family and some others[1,2,3,4,5,6,7,8,9,10]. 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.

 

 

AhR-Mode-of-Action.gif

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[11,12]. 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[13,14]. The biological potency was estimated as 5 times greater than that of b-nephthoflavone (BNF), one of potent artificial ligands for the AhR. The estimated Ki value (the smaller the value is, 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).


 

 

                           ITE-Model-Space-Filling.gif

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 endogenous or physiological ligand), 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[15]. 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 until the next round of AhR activation is needed.


 

 

                 Interconversion-ITE-and-ITC.gif

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 multiple therapeutic capabilities. More specifically, ITE cancer therapeutic efficacy and specificity, together with possible low side effect(s) of the ITE therapy, will be presented.

 

4-1. ITE Has Multiple Capabilitis

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 mostly by probing with its artificial ligands. Analyzing data on AhR functions studied with its artificial ligands in literature together with data generated by using ITE directly, it is evident that ITE may well be able to:

  • Block angiogenesis (new blood vessel generation)[7]

  • Inhibit cell division[16,17,18]

  • Promote apoptosis (programed cell death)[19,20]

  • Induce cell differentiation[2,3,21]

  • Prohibit cell migration[4,21,22]

  • Curb estrogen actions[8,23]

  • Obstruct androgen actions[24,25]

  • Stimulate or suppress immune system[26,27,28]

  • Repress the transformation from normal to fat cells[29,30,31]

  • Control food intake and energy balance[32,33]

 

These capabilities are extremely valuable in developing ITE into an efficacious and sustainable therapeutic agent in the intervention of several disorders, such as cancer, obesity, and diseases caused by imbalanced actions of immune systems.

1.) 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.

2.) Once cancer cell division is blocked by ITE, the cancer may stop growing. Similarly, if primitive fat cells (the precursor cells destined to become fat cells) are prevented from dividing, the organism may stop becoming larger. The ability to inhibit cell division means that ITE may bring cancer and obesity under tight control.

3.) When cancer or fat cells are ordered to die through the action of the hormone ITE together with its receptor (the Ah receptor), the development of cancer or obesity could be halted.

4.) If cancer cells are coaxed into differentiating, they will no longer be cancer cells. The cells will be inactive and die away eventually. This could help cure the cancer. In the case of obesity, the precursor cells (called preadipocytes or primitive fat cells) for fat cells keep multiplying actively and become fat cells. ITE could be used to convert these cells into inactive and dying ones.

5.) Cancer cell migration (metastasis) is one of the deadliest events to cancer patients in cancer development. ITE's capability of blocking cell migration is therefore extremely valuable in an efficacious and sustainable cancer therapy.

6.) Some breast and prostate cancers are dependent on estrogen and androgen, respectively, for progression. Together with the other capabilities in fighting cancer, ITE has thus the added power over therapy for estrogen-dependent breast cancers and androgen-dependent prostate cancers.

7.) The Ah receptor has been discovered to be deeply involved in immune system homeostasis. Whether it is involved in stimulation or suppression of the system is most probably determined by the physiological and pathological context. If ITE could stimulate immune system of a cancer patient to combat cancer and clear up individual cancer cells while attacking the cancer directly with the other capabilities, cancer eradication will finally be a possibility. Without the help from the immune system, cancer elimination would be very difficult. Similarly, if it can stimulate the immune systems of AIDS patients, ITE could then help them effectively fight HIV virus. On the other hand, if it can be used to suppress otherwise overly active immune system, ITE could then be used to treat conditions caused by autoimmunity. There is a recent example of this application[34], actually.

8.) ITE may be able to inhibit the transformation from a normal cell to a fat cell and affect functions of a region named hypothalamus in the brain to control appetite and energy balance. Both of these capabilities would have great importance in treating obesity. More importantly, they represent independent strategies in therapeutic intervention of this disorder.

In summary, experimental studies together with theoretical analysis on the hormone ITE do suggest that it can fight cancer and obesity with multiple combating strategies. This multiple mechanism based combating capability makes the ITE unique, especially in therapeutic intervention of cancers. The multiplicity of ITE combating capabilities makes its efficacy sustainable and the sustainability of ITE potency makes cancer eradication a final possibility. 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 from a normal cell to fat cells and can control food intake and energy balance. The role of the Ah receptor plays in immune systems will certainly help to expand effectively the therapeutic spectrum of ITE.

 

4-2. Evidence Supporting the Efficacy of ITE Cancer Therapy

Even though most of the artificial ligands for AhR are environmental toxins[1,2] and thus cannot be used as therapeutic agents, for the purpose of understanding functions of liganded AhR, its artificial ligands such as TCDD, 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF), 8-methyl-1,3,6-trichlorodibenzofuran (8-MCDF), and those derived from indole or tryptophan were used to reveal that the liganded AhR was able to inhibit the metastasis of prostate tumors in a strain of transgenic mice[22] and the growth of carcinogen induced rat mammary tumors[35,36,37], human breast tumor cell xenografts[38,39], and tumors caused by gene mutations[40].

 

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

An important issue in cancer therapy is that it is highly desirable for a therapeutic agent specifically working in cancer cells instead of normal cells to enhance its potency and reduce side effects. This type of specificity can be achieved if there are more target molecules the agent binds in cancer cells than in normal cells. The target molecule for ITE is the Ah receptor. Supporting the possible specificity of the ITE therapy, the Ah receptor was reported to be highly concentrated in pancreatic cancer tissues from patients but very diluted in all normal pancreatic tissues examined[41]. Similarly, the enhanced AhR expression is also documented with cancers of prostate[42,43], lung[44], ovary (personal communication, Dr. Jing Zheng, Dept. of Ob/Gyn, University of Wisconsin-Madison), and stomach[45]. This means that the therapeutic specificity of ITE could be achieved in these reported cases of cancers at least.

 

4-4. Possibility of Low Side Effect for the ITE Therapy

Side effects are constant problems for most of therapies. Lower therapeutic side effect(s) not only improves quality of life but also enhances efficacy of a therapy through raising and extending the dosing levels, dosing frequencies, and dosing durations. With the grand therapeutic opportunity of ITE outlined, we will analyze why we think the ITE hormone therapy may well have low side effect/s. Aspects of metabolism of exogenous chemicals vs. a natural hormone, probability of "off-target" binding and interaction of exogenous chemicals vs. a natural hormone, and reported studies in ITE biology and toxicology will be analyzed.

 

4-4-1. 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. 4, for an example) and proteins causing a lot of adverse reactions including even 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.

 

 

Unsafe-Metabolism.gif

 

Fig. 4. 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.

 

4-4-2. ITE should have 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, but not limited to, 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 ITE 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.

 

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

Adverse reactions from animals dosed with ITE at the tested doses have not been observed from a reported study of ITE biology and toxicity together with a toxic AhR artificial ligand, TCDD[46]. Another independent report has reached similar conclusions[34].

 

 

5. CITIED LITERATURES

1. Poland A, Knutson JC. 2,3,7,8-tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Annu. Rev. Pharmacol. Toxicol. 1982;22:517-554.

2. Poellinger L. Mechanistic aspects--the dioxin (aryl hydrocarbon) receptor. Food Addit Contam. 2000;17(4):261-6.

3. Puga A, Tomlinson CR, Xia Y. Ah receptor signals cross-talk with multiple developmental pathways. Biochem Pharmacol. 2005;69(2):199-207.

4. Hsu EL, Yoon D, Choi HH, et al. A proposed mechanism for the protective effect of dioxin against breast cancer. Toxicol. Sci. 2007;98(2):436-444.

5. Stevens EA, Mezrich JD, Bradfield CA. The aryl hydrocarbon receptor: a perspective on potential roles in the immune system. Immunology. 2009;127(3):299-311.

6. Boitano AE, Wang J, Romeo R, et al. Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells. Science. 2010;329(5997):1345-1348.

7. DeLuca HF, Clagett-Dame M, Song J, Helfand S, Akhtar N. United States Patent: 7419992 - Use of aryl hydrocarbon receptor ligand as a therapeutic intervention in angiogenesis-implicated disorders. 2008. Available at: http://patft.uspto.gov/ [Accessed October 7, 2009].

8. Safe S, McDougal A. Mechanism of action and development of selective aryl hydrocarbon receptor modulators for treatment of hormone-dependent cancers (Review). Int J Oncol. 2002;20(6):1123-8.

9. Lindén J, Lensu S, Tuomisto J, Pohjanvirta R. Dioxins, the aryl hydrocarbon receptor and the central regulation of energy balance. Front Neuroendocrinol. 2010. Available at: http://www.ncbi.nlm.nih.gov/pubmed/20624415 [Accessed October 19, 2010].

10. Fetissov SO, Huang P, Zhang Q, et al. Expression of hypothalamic neuropeptides after acute TCDD treatment and distribution of Ah receptor repressor. Regul. Pept. 2004;119(1-2):113-124.

11. Song J, Clagett-Dame M, Peterson RE, et al. A ligand for the aryl hydrocarbon receptor isolated from lung. Proc Natl Acad Sci U S A. 2002;99(23):14694-9.

12. DeLuca HF, Jiasheng Song, Clagett-Dame M, et al. United States Patent: 6916834 - Preparations and use of an Ah receptor ligand, 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester. 2005. Available at: http://patft.uspto.gov/ [Accessed October 7, 2009].

13. Grzywacz P, Sicinski RR, DeLuca HF. A Concise Synthesis of an AHR Endogenous Ligand with the Indolecarbonylthiazole Skeleton. HETEROCYCLES. 2003;60(5):1219.

14. DeLuca HF, Grzywacz PK, Sicinski RR. United States Patent: 7002019 - Synthesis of indole thiazole compounds as ligands for the Ah receptor. 2006. Available at: http://patft.uspto.gov/ [Accessed October 27, 2010].

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

16. Elizondo G, Fernandez-Salguero P, Sheikh MS, et al. Altered cell cycle control at the G(2)/M phases in aryl hydrocarbon receptor-null embryo fibroblast. Mol Pharmacol. 2000;57(5):1056-63.

17. Puga A, Marlowe J, Barnes S, et al. Role of the aryl hydrocarbon receptor in cell cycle regulation. Toxicology. 2002;181-182:171-7.

18. Marlowe JL, Knudsen ES, Schwemberger S, Puga A. The aryl hydrocarbon receptor displaces p300 from E2F-dependent promoters and represses S phase-specific gene expression. J Biol Chem. 2004;279(28):29013-22.

19. Krämer H, Podobinska M, Bartsch A, et al. Malassezin, a novel agonist of the aryl hydrocarbon receptor from the yeast Malassezia furfur, induces apoptosis in primary human melanocytes. Chembiochem. 2005;6(5):860-865.

20. Park K, Mitchell KA, Huang G, Elferink CJ. The aryl hydrocarbon receptor predisposes hepatocytes to Fas-mediated apoptosis. Mol. Pharmacol. 2005;67(3):612-622.

21. Hall JM, Barhoover MA, Kazmin D, et al. Activation of the Aryl-Hydrocarbon Receptor Inhibits Invasive and Metastatic Features of Human Breast Cancer Cells and Promotes Breast Cancer Cell Differentiation. Mol Endocrinol. 2009. Available at: http://www.ncbi.nlm.nih.gov/pubmed/20032195 [Accessed January 27, 2010].

22. Fritz WA, Lin T, Safe S, Moore RW, Peterson RE. The selective aryl hydrocarbon receptor modulator 6-methyl-1,3,8-trichlorodibenzofuran inhibits prostate tumor metastasis in TRAMP mice. Biochem. Pharmacol. 2009;77(7):1151-1160.

23. Oenga GN, Spink DC, Carpenter DO. TCDD and PCBs inhibit breast cancer cell proliferation in vitro. Toxicol In Vitro. 2004;18(6):811-9.

24. Jana NR, Sarkar S, Ishizuka M, et al. Cross-talk between 2,3,7,8-tetrachlorodibenzo-p-dioxin and testosterone signal transduction pathways in LNCaP prostate cancer cells. Biochem Biophys Res Commun. 1999;256(3):462-8.

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

26. Veldhoen M, Hirota K, Westendorf AM, et al. The aryl hydrocarbon receptor links TH17-cell-mediated autoimmunity to environmental toxins. Nature. 2008;453(7191):106-109.

27. Quintana FJ, Basso AS, Iglesias AH, et al. Control of T(reg) and T(H)17 cell differentiation by the aryl hydrocarbon receptor. Nature. 2008;453(7191):65-71.

28. Stockinger B. Beyond toxicity: aryl hydrocarbon receptor-mediated functions in the immune system. J. Biol. 2009;8(7):61.

29. Shimba S, Wada T, Tezuka M. 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. 2001;114(Pt 15):2809-2817.

30. Hanlon PR, Ganem LG, Cho YC, Yamamoto M, Jefcoate CR. AhR- and ERK-dependent pathways function synergistically to mediate 2,3,7,8-tetrachlorodibenzo-p-dioxin suppression of peroxisome proliferator-activated receptor-gamma1 expression and subsequent adipocyte differentiation. Toxicol. Appl. Pharmacol. 2003;189(1):11-27.

31. Hanlon PR, Cimafranca MA, Liu X, Cho YC, Jefcoate CR. Microarray analysis of early adipogenesis in C3H10T1/2 cells: cooperative inhibitory effects of growth factors and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. 2005;207(1):39-58.

32. Fetissov SO, Huang P, Zhang Q, et al. Expression of hypothalamic neuropeptides after acute TCDD treatment and distribution of Ah receptor repressor. Regul. Pept. 2004;119(1-2):113-124.

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

34. Quintana FJ, Murugaiyan G, Farez MF, et al. An endogenous aryl hydrocarbon receptor ligand acts on dendritic cells and T cells to suppress experimental autoimmune encephalomyelitis. Proc. Natl. Acad. Sci. U.S.A. 2010;107(48):20768-20773.

35. McDougal A, Wilson C, Safe S. Inhibition of 7,12-dimethylbenz[a]anthracene-induced rat mammary tumor growth by aryl hydrocarbon receptor agonists. Cancer Lett. 1997;120(1):53-63.

36. Holcomb M, Safe S. Inhibition of 7,12-dimethylbenzanthracene-induced rat mammary tumor growth by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Cancer Lett. 1994;82(1):43-7.

37. McDougal A, Wormke M, Calvin J, Safe S. Tamoxifen-induced antitumorigenic/antiestrogenic action synergized by a selective aryl hydrocarbon receptor modulator. Cancer Res. 2001;61(10):3902-3907.

38. Gierthy JF, Bennett JA, Bradley LM, Cutler DS. 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. 1993;53(13):3149-3153.

39. Zhang S, Lei P, Liu X, et al. The aryl hydrocarbon receptor as a target for estrogen receptor-negative breast cancer chemotherapy. Endocr. Relat. Cancer. 2009;16(3):835-844.

40. Kawajiri K, Kobayashi Y, Ohtake F, et al. Aryl hydrocarbon receptor suppresses intestinal carcinogenesis in ApcMin/+ mice with natural ligands. Proc. Natl. Acad. Sci. U.S.A. 2009;106(32):13481-13486.

41. Koliopanos A, Kleeff J, Xiao Y, et al. Increased arylhydrocarbon receptor expression offers a potential therapeutic target for pancreatic cancer. Oncogene. 2002;21(39):6059-6070.

42. Kashani M, Steiner G, Haitel A, et al. Expression of the aryl hydrocarbon receptor (AhR) and the aryl hydrocarbon receptor nuclear translocator (ARNT) in fetal, benign hyperplastic, and malignant prostate. Prostate. 1998;37(2):98-108.

43. Gluschnaider U, Hidas G, Cojocaru G, et al. beta-TrCP inhibition reduces prostate cancer cell growth via upregulation of the aryl hydrocarbon receptor. PLoS ONE. 2010;5(2):e9060.

44. Lin P, Chang H, Tsai W, et al. Overexpression of aryl hydrocarbon receptor in human lung carcinomas. Toxicol Pathol. 2003;31(1):22-30.

45. Peng T, Chen J, Mao W, et al. Potential therapeutic significance of increased expression of aryl hydrocarbon receptor in human gastric cancer. World J. Gastroenterol. 2009;15(14):1719-1729.

46. Henry EC, Bemis JC, Henry O, Kende AS, Gasiewicz TA. A potential endogenous ligand for the aryl hydrocarbon receptor has potent agonist activity in vitro and in vivo. Arch. Biochem. Biophys. 2006;450(1):67-77.

 

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