Can indole-based extracts prevent colorectal cancer via early ...

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Jan 30, 2010 - Antitumor activity of novel indirubin derivatives in rat tumor model. Clin Cancer Res. 2007; 13:253-9. 25. Benkendorff K, McIver CM, Abbott CA.
Commentary

Cancer Biology & Therapy 9:5, 380-382; March 1, 2010; © 2010 Landes Bioscience

Can indole-based extracts prevent colorectal cancer via early apoptotic pathways? Shusuke Toden,* Benjamin L. Scherer and Julie M. Clarke Preventative Health National Research Flagship; Adelaide, Australia; CSIRO Food and Nutritional Sciences; Adelaide, SA Australia

Key words: apoptosis, colorectal cancer, tyrian purple, azoxymethane Abbreviations: AOM, azoxymethane; AARGC, acute apoptotic response to genotoxic carcinogen Submitted: 01/30/10 Accepted: 02/01/10 Previously published online: www.landesbioscience.com/journals/cbt/ article/11353 *Correspondence to: Shusuke Toden; Email: [email protected] Commentary to: Westley CB, McIver CM, Abbott CA, Leu RKL, Benkendorff K. Enhanced acute apoptotic response to azoxymethane-induced DNA damage in the rodent colonic epithelium by Tyrian purple precursors: A potential colorectal cancer chemopreventative. Cancer Biol Ther 2010; This issue.

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Colorectal cancer (CRC) is the second most common cause of cancer-related death in the western world1 and is increasing in incidence in many Asian countries, coinciding with a shift to western style diets.2,3 Diet and lifestyle are the most important aetiological factors for CRC accounting for up to 70% of cases while fewer than 20% of cases are attributable directly to genetic causes.4,5 Research has focused on chemopreventive agents that reduce the incidence of CRC by inducing programmed cell death (apoptosis) of damaged colonocytes. Apoptosis, characterised by the deletion of single cells without disturbing the tissue architecture or function,6 defends against tumorigenesis by removing cells that have become genomically unstable or have undergone irreversible genotoxic damage.6 Apoptosis occurs via a series of molecular events including APC/WNT signals, p53 gene mutations and chromosomal instability.7 These events are frequently altered in tumor cells8 allowing them to avoid apoptotic surveillance and thereby enabling the development and progression of cancer. Azoxymethane (AOM) is a genotoxic carcinogen widely used in rodent models to evaluate the effects of dietary and pharmaceutical agents on the development of CRC. Treatment with AOM induces DNA adducts in colonocytes, which, if not repaired or the cell deleted, have the potential to lead to DNA mutations and tumorigenesis.9 AOM-induced tumors in rodents are often mutated at the β-catenin and K-ras genes, which is similar to human CRC.10 However, unlike human tumors, AOM-induced tumors are rarely mutated at the APC gene and are not mutated

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within the p53 gene.11 Although the model has limitations, it is still widely recognised and used to examine the effects of potential dietary chemopreventative agents. To evaluate the effects of dietary components on CRC development in rodents, AOM is typically injected in two doses to rodents that have been pre-fed test and control diets. Following 26–40 w of further feeding of experimental diets the animals are killed and their large bowel scored for colonic tumor size, number and frequency.12 However, in order to investigate the mechanisms of early stage carcinogenesis the acute apoptotic response to genotoxic carcinogen (AARGC) AOM rodent model has been developed.13-15 In this model, the rodents are pre-fed the test diets, given a single injection of AOM and killed at different time points after carcinogen treatment to enable the determination of the levels of cancer biomarkers including DNA adducts, apoptosis and cellular proliferation of colonic epithelial cells. To date, the AARGC rodent model has been mainly used to examine the effects of dietary components such as starches and oils.15-17 In particular, resistant starches which escape digestion in the small intestine have been shown to enhance apoptosis following colonic DNA damage.17 Resistant starch is fermented extensively by the large bowel microflora producing short chain fatty acids, in particular butyrate, which may protect against CRC.18,19 A similar rodent study has demonstrated the potential protective effects of fish oil showing significantly higher colonic apoptotic levels together with reduced epithelial adduct formation compared to those animals fed on a corn oil diet.13

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Commentary

Commentary

In this issue of Cancer Biology & Therapy, Westley et al. have used the AARGC model to show that oral administration of muricid extract (ME), containing a mixture of tyrindoleninone and 6-bromoisatin, increased the distal colonic apoptotic index in a dose-dependent manner.20 This study is important as the first in vivo investigation of the potential anticancer effects of indole-based natural products such as tyrian purple. Tyrian purple is generated from secretions of the hypobrancial gland of Muricidae, a predatory marine gastropod,21 and can also be found in muricid egg masses providing protection against microbial infection during larval development.22 It is unknown whether this anti-bacterial effect of ME is related to its pro-apoptotic effect. Previous studies have demonstrated that indole-based compounds induce apoptosis in cancer cell lines.23-25 Extracts from the egg masses of the Australian mollusc D. orbita, mainly 6-bromoisatin and tyrindoleninone, induced apoptosis and necrosis in Jurkat T lymphoma cells and significantly reduced proliferation in a range of solid tumor and lymphoma cell lines.25 The possible protective mechanisms of these indole-based compounds include increased apoptosis via activation of effector caspase-3 and -7 and inhibition of antiapoptotic gene products via suppression of TNF induced NFκB activation.26 Tyrian purple components of bromine substituted indigo and indirubin isomers, derived from the Mollusk Hexaplex trunculus, have also been shown to inhibit neuronal apoptosis by selectively inhibiting glycogen synthase kinase-3.28 Some indole-based compounds such as indirubins and isatins display anti-proliferative activities against colon cancer cell lines as well as being inducers of apoptosis23,29 while others do not induce apoptosis.28 Further investigation is required to determine the specific pro-apoptotic mechanisms of action of the ME derivatives tyrindolenione and 6-bromoisatin. In this study the ME were administered by oral gavage in an oil solution to reduce the rate of degradation in the stomach. In vitro studies showed that the primary bioactive tyrindoleninone would have been completely degraded to 6-bromoisatin, a compound known to induce apoptosis

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in cultured tumor cells, and tyriverdin, within 1 h in the stomach environment.23 Considering that the gastric emptying rate of a mouse gavaged with a lipid-containing solution is likely to be about 13–15 min,30 it is unclear what proportion of specific bioactives escaped gastric oxidation and were responsible for the pro-apoptotic effect. The authors suggested that the effectiveness of these bioactives might be further improved by enteric coating to decrease the impact of gastric oxidation. Ideally future in vivo studies would also enable identification the bioactives, and would include pharmacological measures demonstrating bioactive absorption . The impact of these indole-based compounds on colonocyte proliferation remains unclear. While Westley et al. showed no evidence for altered proliferation rates using PCNA labelling, as the authors have noted PCNA immuno-staining does not detect early changes in proliferation status which occur acutely after a genotoxic event. Use of Ki67 immunostaining is recommended under these circumstances.31 In summary, the work of Westley et al. provides interesting pre-clinical experimental evidence that indole-based extracts induce apoptosis in colonoctyes of genotoxin-treated mice supporting the suggestion that ME may provide protection against early steps in CRC initiation. We eagerly await definitive preclinical demonstration of the impact of these extracts on colorectal tumor incidence and burden and the identification of the active compounds responsible for these effects. References 1. Stewart BW, Kleihues P. World cancer report. Lyon: IARC, 2003. 2. Key TJ, Allen NE, Spencer EA, Travis RC. The effect of diet on risk of cancer. Lancet 2002; 360:861-8. 3. Sung JJ, Lau JY, Goh KL, Leung WK. Increasing incidence of colorectal cancer in Asia: implications for screening. Lancet Oncol 2005; 6:871-6. 4. Doll R, Peto R. The causes of cancer: quantitative estimates of avoidable risks of cancer in the United States today. J Natl Cancer Inst 1981; 66:1191-308. 5. Johnson IT, Lund EK. Review article: nutrition, obesity and colorectal cancer. Aliment Pharmacol Ther 2007; 26:161-81. 6. Bellamy CO, Malcomson RD, Harrison DJ, Wyllie AH. Cell death in health and disease: the biology and regulation of apoptosis. Semin Cancer Biol 1995; 6:3-16. 7. Watson AJ. Apoptosis and colorectal cancer. Gut 2004; 53:1701-9. 8. Kasibhatla S, Tseng B. Why target apoptosis in cancer treatment? Mol Cancer Ther 2003; 2:573-80.

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9. Hirose Y, Yoshimi N, Makita H, Hara A, Tanaka T, Mori H, et al. Early alterations of apoptosis and cell proliferation in azoxymethane-initiated rat colonic epithelium. Jpn J Cancer Res 1996; 87:575-82. 10. Dashwood RH, Suzui M, Nakagama H, Sugimura T, Nagao M. High frequency of beta-catenin (ctnnb1) mutations in the colon tumors induced by two heterocyclic amines in the F344 rat. Cancer Res 1998; 58:1127-9. 11. De Filippo C, Caderni G, Bazzicalupo M, Briani C, Giannini A, Fazi M, et al. Mutations of the Apc gene in experimental colorectal carcinogenesis induced by azoxymethane in F344 rats. Br J Cancer 1998; 77:2148-51. 12. Reddy BS. The fourth DeWitt S. Goodman lecture. Novel approaches to the prevention of colon cancer by nutritional manipulation and chemoprevention. Cancer Epidemiol Biomarkers Prev 2000; 9:239-47. 13. Hong MY, Chapkin RS, Morris JS, Wang N, Carroll RJ, Turner ND, et al. Anatomical site-specific response to DNA damage is related to later tumor development in the rat azoxymethane colon carcinogenesis model. Carcinogenesis 2001; 22:1831-5. 14. Hu Y, Martin J, Le Leu R, Young GP. The colonic response to genotoxic carcinogens in the rat: regulation by dietary fibre. Carcinogenesis 2002; 23:1131-7. 15. Hong MY, Chapkin RS, Wild CP, Morris JS, Wang N, Carroll RJ, et al. Relationship between DNA adduct levels, repair enzyme and apoptosis as a function of DNA methylation by azoxymethane. Cell Growth Differ 1999; 10:749-58. 16. Le Leu RK, Hu Y, Young GP. Effects of resistant starch and nonstarch polysaccharides on colonic luminal environment and genotoxin-induced apoptosis in the rat. Carcinogenesis 2002; 23:713-9. 17. Le Leu RK, Brown IL, Hu Y, Young GP. Effect of resistant starch on genotoxin-induced apoptosis, colonic epithelium and lumenal contents in rats. Carcinogenesis 2003; 24:1347-52. 18. Whitehead RH, Young GP, Bhathal PS. Effects of short chain fatty acids on a new human colon carcinoma cell line (LIM1215). Gut 1986; 27:1457-63. 19. Topping DL, Clifton PM. Short-chain fatty acids and human colonic function: roles of resistant starch and nonstarch polysaccharides. Physiol Rev 2001; 81:1031-64. 20. Westley CB, McIver CM, Abbott CA, Le Leu RK, Benkendorff K. Enhanced acute apoptotic response to azoxymethane-induced DNA damage in the rodent colonic epithelium by Tyrian purple precursors: a potential colorectal cancer chemopreventative. Cancer Biol Ther 2010; 9:371-78. 21. Cooksey CJ. Tyrian purple: 6,6'-dibromoindigo and related compounds. Molecules 2001; 6:736-69. 22. Benkendorff K, Bremner J, Davis A. Tyrian purple precursors in the ess masses of the Australian muricid, Dicathais orbitia: a possible deffensive role. J Chem Ecol 2000; 26:1037-50. 23. Vine KL, Locke JM, Ranson M, Benkendorff K, Pyne SG, Bremner JB. In vitro cytotoxicity evaluation of some substituted isatin derivatives. Bioorg Med Chem 2007; 15:931-8. 24. Kim SA, Kim YC, Kim SW, Lee SH, Min JJ, Ahn SG, et al. Antitumor activity of novel indirubin derivatives in rat tumor model. Clin Cancer Res 2007; 13:253-9. 25. Benkendorff K, McIver CM, Abbott CA. Bioactivity of the murex homeopathic remedy and of extracts from an australian muricid mollusc against human cancer cells. Evid Based Complement Alternat Med 2009; In press. 26. Sethi G, Ahn KS, Sandur SK, Lin X, Chaturvedi MM, Aggarwal BB, et al. Indirubin enhances tumor necrosis factor-induced apoptosis through modulation of nuclear factor-kappaB signaling pathway. J Biol Chem 2006; 281:23425-35.

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27. Kim DH, Lee J, Kim KN, Kim HJ, Jeung HC, Chung HC, et al. Anti-tumor activity of N-hydroxy7-(2-naphthylthio) heptanomide, a novel histone deacetylase inhibitor. Biochem Biophys Res Commun 2007; 356:233-8. 28. Meijer L, Skaltsounis AL, Magiatis P, Polychrono poulos P, Knockaert M, Leost M, et al. GSK-3selective inhibitors derived from Tyrian purple indirubins. Chem Biol 2003; 10:1255-66.

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29. Lee JW, Moon MJ, Min HY, Chung HJ, Park EJ, Park EJ, et al. Induction of apoptosis by a novel indirubin-5-nitro-3'-monoxime, a CDK inhibitor, in human lung cancer cells. Bioorg Med Chem Lett 2005; 15:3948-52. 30. Symonds EL, Butler R, Omari TI. A mouse model for assessing the impact of ingested nutrients on gastric emptying rate. Clin Exp Pharmacol Physiol 2007; 34:132-3.

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31. Hayat MA. Microscopy, immunohistochemistry and antigen retrieval. Methods for light and electron microscopy. In: Kuo J, ed. Principles and Techniques of Electron Microscopy, NY: Kluwer Academic/ Plenium Publishers 2001; 233-47.

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