review article attenuation of carcinogenesis and the

Review ArticleAttenuation of Carcinogenesis and the MechanismUnderlying by the Influence of Indole-3-carbinol and ItsMetabolite 331015840-Diindolylmethane A Therapeutic Marvel

V L Maruthanila J Poornima and S Mirunalini

Department of Biochemistry and Biotechnology Faculty of Science Annamalai UniversityAnnamalainagar Tamilnadu 608 002 India

Correspondence should be addressed to S Mirunalini mirunasankargmailcom

Received 3 February 2014 Revised 7 April 2014 Accepted 19 April 2014 Published 8 May 2014

Academic Editor Masahiro Oike

Copyright copy 2014 V L Maruthanila et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Rising evidence provides credible support towards the potential role of bioactive products derived from cruciferous vegetablessuch as broccoli cauliflower kale cabbage brussels sprouts turnips kohlrabi bok choy and radishes Many epidemiologicalstudies point out that Brassica vegetable protects humans against cancer since they are rich sources of glucosinolates in addition topossessing a high content of flavonoids vitamins andmineral nutrients Indole-3-carbinol (I3C) belongs to the class of compoundscalled indole glucosinolate obtained from cruciferous vegetables and is well-known for tits anticancer properties In particularI3C and its dimeric product 331015840-diindolylmethane (DIM) have been generally investigated for their value against a number ofhuman cancers in vitro as well as in vivo This paper reviews an in-depth study of the anticancer activity and the miscellaneousmechanisms underlying the anticarcinogenicity thereby broadening its therapeutic marvel

1 Introduction

Plant materials present in the human diet contain a largenumber of naturally occurring compounds thatmay be usefulto protect the body against cancer development Recentlymany candidates have been assayed to identify the presenceof anticarcinogens in their diet [1] Recognition of diet as aprimary causative factor to overcome cancer risk has directedmuch research attention towards the chemoprotective role ofcertain compounds in foods [2] Technological progress inmanipulating plant metabolism and metabolites combinedwith the explosive growth of the ldquofunctional foodrdquo industryhas led to many attempts to enhance the concentration ofthese health-promoting compounds in specific plant-basedfoods [2]

The anticancer properties of cruciferous vegetables areprimarily documented by the Roman statesman Cato andElder (234-149 BC) who in his study of drug wrote ldquoif a can-cerous ulcer appears on the breasts apply a crushed cabbageleaf and it will make it well [3]rdquo It is now well set up that

cruciferous vegetables contain a forerunner phytochemicalopenly glucosinolate that undergoes hydrolysis by the plantenzyme myrosinase yielding a bioactive compound knownas I3C [3] It is rapidly converted to many condensationproducts as it is chemically unbalanced in aqueous andgastric acidic environment The dietary indoles I3C andDIM occur naturally as glucosinolate conjugates in Brassicavegetables and are released upon hydrolysis [3] The cancer-protecting properties of Brassica (ie broccoli cauliflowerkale cabbage brussels sprouts bok choy radishes turnipsand kohlrabi) utilization are most likely mediated throughldquobioactive compoundsrdquo that induce a variety of physiologicalprocess including direct or indirect antioxidant action detox-ifying enzymes inducing apoptosis and cell cycle regulation[4]

DIM is readily detected in the livers and feces of rodentsfed I3C whereas the parent I3C compound has not beendetected in tissues of these rodents [5] Thus the naturaleffects of I3C are attributable to DIM which show evidenceof antitumorigenic activities in vivo and in vitro by reducing

Hindawi Publishing CorporationAdvances in Pharmacological SciencesVolume 2014 Article ID 832161 7 pageshttpdxdoiorg1011552014832161

2 Advances in Pharmacological Sciences

the growth of prostate colon and breast cancer cells [6 7]I3C and its derivatives also suppress cell propagation andinduce apoptosis in colon cancer cells [8] as well as in othertypes of cancer cells including prostate [9] breast [7] bladder[10] pancreas [11] and hepatoma [12] This review mainlyfocuses on the role of I3C and DIM against various types ofmalignancy and underlying mechanisms

2 Cruciferous Vegetables andTheir Derivatives

Glucosinolates are a class of organic compounds that givepungent smell and piquant taste in the cruciferous vegetablesand some condiments such as wasabi andmustardThemainfunction of glucosinolates in plants is that it acts as naturalpesticides and accelerates the resistance against herbivores[13]

The central carbon of all glucosinolates is bound 6 to athioglucose group to a sulfate group via the nitrogenmolecule[22] The central carbon of each glucosinolate is boundto a side group and this makes each glucosinolate unique[22] Glucosinolates are classified as aliphatic (eg alkenylalkyl hydroxyalkenyl or w-methylthioalkyl) aromatic (egbenzyl substituted benzyl) or heterocyclic with the R-groupof the aliphatic glucosinolates being derived from alaninemethionine leucine or valine and those of aromatic andheterocyclic glucosinolates being derived from phenylala-nine or tyrosine or tryptophan [14] (Figure 1(a)) Althoughglucosinolates are related to inhibition of carcinogenesisit is actually their hydrolysis products not the glucosino-lates themselves that are biologically active Hydrolysis ofglucosinolates is catalyzed by the enzyme myrosinase alsoknown as 120573-thioglucoside glucohydrolase [13] In a low pHenvironment I3C is converted into polymeric products andDIM is the main one DIM is a major in vivo acid catalyzedcondensation product of I3C [23] (Figures 1(b) and 1(c))Theyare no more natural products from DIM Maciejewska et alsynthesized a series ofDIMderivatives bearing fluoro bromoiodo and nitro substituents in indole or benzene rings andtested for cytotoxicity against human melanoma cell linesafter characterizing their structure [23] These derivativeswere found to cause 50 inhibition of the viability of ME18and ME18R cell lines at concentrations ranging between97 and 173mM [23] These finding clearly suggested thatsynthetic analog of DIM has potential role in cancer therapyin the near future once their systemic toxicity studies havebeen determined [23]

3 Bioavailability of I3C and DIM

Reed et al [24] in his extensive article of pharmacokineticstudies reported that women received oral doses of 400 600800 1000 and 1200mg I3C and these serial plasma sampleswere analyzed by high-performance liquid chromatography-mass spectrometrymethod for the detection and quantitationof the I3C and DIM I3C itself is not detectable in plasma[24] The only detectable I3C-derived product is DIM Highinitial value plasma DIM for all subjects decreased to near

N

OSO3

GIc

SR

(minus)

(a)

NH

OH

(b)

NH

NH

(c)

Figure 1 Structures of (a) glucosinolates (b) I3C and (c) DIM

or below the limit of quantitation within the 12 h samplingperiod [24] Physiologically based pharmacokinetic (PBPK)model is developed using plasma and tissue (brain heartliver kidneys and lungs) concentration data for DIM tocompare the pharmacokinetic properties and biodistributionof pure crystalline and a novel formulation (BioResponse-DIM (BR-DIM)) of DIM after oral administration to mice[25] I3C is hard to develop as a drug because it is highlyunstable and can transform into many other derivativessuch as DIM 5611121718-hexahydrocyclonona[12-b45-b101584078-b10158401015840] triindole (CTr) indolo [32-b] carbazole (ICZ) N-methoxyindole-3-carbinol (NI3C) two tetramers one linear(LTET) and one cyclic (CTET) and 1-(3-hydroxymethyl)indolylmethane (HI-IM) Anderton et al reported oral dosesof I3C to female CD-1 mice and studied the disposition ofI3C and its acid condensation productsDIM ICZ LTr(1) andHI-IM in blood liver kidney lung heart and brain I3C wasrapidly absorbed distributed and eliminated from plasmaand tissues falling below the limit of detection by 1 h [5] Themajor acid condensation product of I3C DIM has provenvery stable in acidic conditions during prolonged exposureto high temperature and humidity [4] Another report BR-DIM has proven very stable and it is not converted into otherforms [4] Based on these outcomes authors suggested thatone could exploit further development of DIM as a potentialtherapeutic agent

4 Metabolism and Distribution

Under the acidic conditions of the gastric tract I3C under-goes condensation to form several oligomeric productsparticularly DIM [23]The tissue distribution of I3C has beendetermined in mice by radio-labelled I3C [26] Both I3Cand DIM have been detected in kidneys lungs heart liverplasma and brain samples of mice treated with 250mgkg ofI3C as early as 15mins after administration [26]These resultssuggest that I3C is rapidly absorbed and spread to a numberof well-perfused tissues where it is transformed to DIM toperform its anticancer actions [26]

Advances in Pharmacological Sciences 3

5 Epidemiological Studies

For more than 25 years the interdependence between nutri-tion and the development and progression of cancer havebeen recognized [27] The key challenge is to identify thespecific components responsible for contributing to thisrelationship [27] In the United States cancers of the lungscolon rectum breast and prostate account for almost half ofthe total cancer incidence [27] Cohen et al [28] examined theassociation of fruit and vegetable intake and prostate cancerrisk among newly diagnosedmen residing in the SeattleWAarea With each 10 g of cruciferous vegetables consumed perday one could expect an 8 decrease in risk for colorectalcancer

Several recent case-control studies in the US Swedenand China found that measures of cruciferous vegetableintake are significantly lower inwomen diagnosedwith breastcancer than in cancer-free control groups [29] High intakeof cruciferous vegetables has been associated with lowerrisk of lung and colorectal cancer in some epidemiologicalstudies but there is evidence that genetic polymorphismsmay influence the effectiveness of cruciferous vegetables onhuman cancer risk [30] Epidemiological studies show thatconsumption of large quantities of fruits and vegetables par-ticularly cruciferous vegetables is associated with a reducedoccurrence of cancer [30]

6 Experimental Studies

Studies in various experimental models have shown thatI3C can alter the metabolism of carcinogens and provideprotection against chemically induced carcinogenesis [31]When administered before or at the same time as the car-cinogen oral I3C has been originated to inhibit the spreadingout of cancer in a variety of animal models and tissuesincluding cancers of the mammary gland (breast) [7] colon[8] stomach [31] lung [26] and liver [32]) However anumber of studies have found that I3C actually promotedor enhanced the development of cancer when administeredchronically after the carcinogen [32]

The cancer promoting effects of I3C is first reported ina trout model of liver cancer [32] However I3C also hasbeen found to promote cancer of the thyroid liver colonand uterus in rats [33] Although the long-term effects of I3Csupplementation on cancer risk in humans are not knownbut the contradictory results of animal studies have ledsome to caution against the widespread use of I3C and DIMsupplements in humans until their potential risks and benefitsare better understood [33]

7 Therapeutic Action of Indole Glucosinolates

71 Apoptosis Apoptosis or programmed cell death is ahighly regulated process that involves activation of a series ofmolecular events leading to cell death that is characterizedby cellular morphological change chromatin condensationand apoptotic bodieswhich are associatedwithDNAcleavageinto ladders [34] The nuclear factor kappa B (NF-120581B)signaling plays critical roles in regulating cell proliferation

survival tumor invasion metastasis drug resistance andstress response [35] We confirmed that NF-120581B activityis significantly upregulated by docetaxel gemcitabine oroxaliplatin treatment and that the NF-120581B inducing activityof these agents was completely abrogated in cells pretreatedwith DIM [36] We found that DIM or the formulatedBR-DIM treatment could restrict its nuclear localizationand inactivate NF-120581B DNA-binding activity in prostate [14]breast [15] and pancreatic cancer cells [36] resulting inthe inhibition of transcriptional downregulation of severalNF-120581B downstream genes causing inhibition of cell growthand inducing apoptotic cell death Collectively these resultsclearly suggest that DIM pretreatment which inactivates NF-120581B activity along with other cellular effects of DIM maycontribute to enhanced cell growth inhibition and apoptosiswith suboptimal doses of cytotoxic chemotherapeutic agentswith minimal side effects (Figure 2)

I3C trigger the stress-induced MAP-kinases p38 and C-jun N-terminal kinase (JNK) in prostate cancer cells and toinhibit constitutively active STAT3 a transcription factor inpancreatic cancer cells [18] Irrespective of the cell type I3Csuppressed NF-120581B activation induced by various agents [18]NF-120581B inhibition correlated with suppression of inhibitor ofkappa B kinase (IKK) and I120581B120572 phosphorylation ubiquitina-tion degradation with p65 phosphorylation nuclear translo-cation and acetylation [20] I3C also downregulated NF-120581B120572 regulated reporter gene transcription and gene productsinvolved in cell proliferation antiapoptosis and invasion[20] This led to the potentiation of apoptosis induced bycytokines and chemotherapeutic agents [20] Collectively theconcerted effects on those proapoptotic components underliethe ability of I3CDIM to induce mitochondrial dependentapoptosis in tumor cells [20]

72 Regulation of Redox Status Reactive oxygen species(ROS) including H

2O2can cause different combinations of

apoptosis necrosis and autophagy in a cell line dependentand stimulus-dependent manner [37] The capacity of I3Cto form adducts with electrophiles or free radicals appearstoo autonomous on their chemical reactivity hence thescavenging ability of I3C is compatible with the adductformation [38] Arnao et al [38] investigate the ability of I3Cto trap a metastable synthetic-free radical and inhibition ofcarcinogenesis This induction may be produced by I3C itselfandor I3C derived polymerization products such as DIMand others [39]

According to Benabadji et al [40] they reported thatDIM and 6-methoxy-DIM in DPPH model their IC

50 were

50 and 40 smaller than that of vitamin E due to theirhydrogen-donating ability with the presence of two NndashHgroups as an H-donating group necessary to react with freeradical and slightly less potent than the standard phenolicantioxidant BHA in 120573-carotene model with IC

504 and 9

smaller for DIM and 6-methoxy-DIM

73 Anti-Inflammatory Effect The effect of DIM on inflam-matory responses and itsmolecularmechanisms ofDIMhavebeen examined using lipopolysaccharide (LPS) stimulated


Anticarcinogenic activity of Indole

glucosinolates

Prostrate cancer Breast cancer Cervical cancer

Skin cancer Endometrial cancer

Anti-inflammatory effects

Free radical production

Angiogenesis and metastasis

DNA repair in normal cells

DNA-carcinogen adduct formation

Programmed cell death

-Increasing

-Decreasing

Figure 2 Sources of anticancer effect of indole glucosinolates (I3C and DIM) and its mode of action

RAW2647 murine macrophages [16] DIM inhibits LPS-induced increases in protein levels of inducible nitric oxidesynthase (iNOS) which are accompanied by decreased iNOSmRNA levels and transcriptional activity [16] In additionDIM suppresses LPS-induced NF-120581B transcriptional andDNA-binding activity translocation of p65 (RelA) to thenucleus and degradation of inhibitor of kappa B alpha(I120581B120572) [16] Also the results of RT-PCR analysis exposed thatLPS augmented the steady-state levels of proinflammatorycytokines such as TNF-120572 IL-1120573 PLA2 and interleukin-6 (IL-6) transcripts which are substantially suppressed by DIMtreatment [16] DIM pretreatment significantly repressedLPS-induced phosphorylation of SAPKJNK whereas thephosphorylation of other MAPK family proteins (p38 orERK-12) is unaltered by DIM pretreatment [41]

74 Cell Cycle Arrest Cell cycle arrest is defined as the haltof the cell cycle I3C is reported to inhibit cyclin dependentkinase 2 (CDK2) kinase activities in MCF-7 cells throughselective alterations in cyclin E composition size distributionand subcellular localization of the CDK2 protein complex[42] Cell-cycle arrest involves the upregulation of the CDKinhibitors p21 WAF1 and p27 KIP1 and the concurrentdownregulation of cyclin D1 cyclin E and CDKs 2 4 and6 attributable to the effect of I3C and DIM on regulatingSP1-promoter binding activity [19] Inhibition of CDK46cyclin D1 and CDK2 cyclin E activities led to decreased Rbphosphorylations which cause the Rb protein to bind to theE2F transcription factor [43] This E2F sequestration blocksthe transcription of S phase genes resulting in G

1arrest

also the involvement of p53 and cell cycle arrest in the I3C-mediated effect has been studied in various cancer cells [44]

Treatment of I3C betters the expression of p53 (Ser 15)and CDKIs such as p21 and p27 while cyclin D1 expressionis suppressed and cyclin E is not altered [45] Collectively thedata for the cell cycle analysis the Plk-1 assay and the western

blot of p53 and CDKIs explain that I3C augments theexpression of p53 and CDKIs and that I3C induced cell cyclearrest at G

0G1in A549 cells [45] In addition cotreatment

with I3C and wortmannin prevents both phosphorylation ofp53 at Ser 15 and p21 expression Hence it is clear that A549cell arrest by I3C is involved in the PI3 K and p53 signalpathways [45]

75 Angiogenesis Angiogenesis is the physiological processthrough which new blood vessels form from preexistingvessels This is distinct from vasculogenesis which is thede novo formation of endothelial cells from mesoderm cellprecursors [46] Dysregulated angiogenesis that consists ofthe unbalanced production of pro- and antiangiogenic factorsis linked to a number of pathological situations [21] Forexample the overexpression of angiogenic factors includingvascular endothelial growth factor (VEGF) IL-6 and matrixmetalloproteinases (MMP-9) is closely associated with thedevelopment of cancers and metastasis [21] The effect ofI3C on LPS-activated macrophage-induced tube formationand its associated factors in endothelial EAhy926 cells areinvestigated [21] LPS significantly enhanced the capillary-like structure of endothelial cells (ECs) cocultured withmacrophages but no such effect was observed in single-cultured ECs [21] I3C on the other hand suppressed suchenhancement in concert with decreased secretions of VEGFNO IL-6 and MMPs [21] The results obtained from culti-vating ECs with conditioned medium (CM) collected frommacrophages suggested that both ECs andmacrophages wereinactivated by I3C [21]

76 Detoxification Detoxification is the physiological ormedicinal removal of toxic substances from living organisms[47] Both the Phase I and Phase II detoxification centersin the liver and the intestinal epithelial cells can be accel-erated by some minor exogenous agent like I3C [47] Many


Table 1 Modulation of genes involved in various mechanisms by the influence of I3C and DIM

Mechanisms involved Upregulated genes (uarr) modulated by I3Cand DIM

Downregulated genes (darr) modulated byI3C and DIM References

Apoptosis JNKSAPK and Bax Bcl-xl Bcl-2 surviving and NF-120581B [14ndash16]Xenobiotic metabolism CYP CYP 1A1 CYP 1A2 and CYP 1B1 mdash [17]Antioxidant GSH and GST mdash [4]Transcription factor Nrf2 and ATF3 NF-120581B and STAT3 [4 18]Cell cycle p21 WAF1 and p27 KIP1 Cyclin D1 E CDK2 and CDK6 [19]Inflammation NAG-1 NF-120581B and MMP-9 [16 20]Angiogenesis VEGF IL-6 and MMP-9 mdash [21]

researchers indicate that the ability of cruciferous vegetablesto motivate Phase I and Phase II detoxification particularlytheir I3C content is a primary factor in which these nutrientsare related to reduce cancer risk in humans [48] Animalsare exposed to or injected with carcinogens the animalsreceiving the cruciferous vegetables or the I3C in their foodsupply have a significantly lower tumor incidence than theanimals fed the same diet but without cruciferous vegetablesor I3C fortification [48] Table 1 showed the genes modulatedby I3C and DIM in the above mechanisms

8 Anticancer Effects of I3C and DIM

DIM inhibits proliferation of human breast cancer cellsat concentrations achievable through oral supplementationwith I3C (10ndash50120583M) [49] Recently a report by Fan etal [50] demonstrated that DIM protects cancer cells andnormal epithelial cells against ROS in a breast cancer type 1susceptibility protein (BRCA1-)dependentmannerMoreoverI3C inhibits DMBA initiated and TPA promoted mouseskin tumour formation [51] I3C also exhibits inhibitory andpreventive effects on prostate tumors inmice [9]Other inves-tigators reported that this activity of I3C is associated withits action as a nonspecific inducer of powerful cytochromeenzymes responsible for ldquoPhase Irdquo detoxificationmetabolismthe efficiency of I3C to inhibit prostate tumor growth [52]The anticancer efficacy of I3C is well proven includingthe reduction of cervical intraepithelial neoplasia (CIN)and its progression to cervical cancer [53] It is perceivedthat the diminished phosphatase and tensin homolog pro-tein (PTEN) expression is observed during the progressionfrom low-grade to high-grade cervical dysplasia in humansand in a mouse model for cervical cancer the K14HPV16transgenic mice promoted with estrogen [53] PTEN couldimpede tumor progression by inhibiting proliferation and byincreasing tumor cell apoptosis [53] This is supported bythe previous findings that I3C decreased proliferating cellnuclear antigen- (PCNA-)positive cells and increased TdT-mediated dUTPnick-end labeling- (TUNEL-)positive cells inabnormal cervical epithelium of HPV16 mice [54]

9 Drug Resistance

While a number of therapeutic options are available forthe cure of various cancers a major clinical problem

is the development of drug resistance [55] Intrinsic andacquired resistances are the two broad classifications ofresistance to anticancer drugs Natural compounds such asindoles which induce apoptosis in human cancer cells with-out causing unwanted toxicity in normal cells can be usefulin combination with conventional chemotherapeutic agentsfor the treatment of human malignancies with diminishedtoxicity and higher efficacy [55]

10 Conclusion

As seen throughout the review cruciferous vegetables impartseveral mechanisms of action to develop its outsized numberof function The experimental studies as described abovesuggest that I3C and DIM the components of cruciferousvegetables have therapeutic potential both for preventionand for treatment of cancer Moreover the future research canfocus on the advantageous effect of I3C and DIM in variousclinical studies to overcome the cancer epidemic among thepopulation by the application of diverse technologies

Conflict of Interests

The authors declare that they have no conflict of interests

References

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[2] J W Finley ldquoProposed criteria for assessing the efficacy ofcancer reduction by plant foods enriched in carotenoidsglucosinolates polyphenols and selenocompoundsrdquo Annals ofBotany vol 95 no 7 pp 1075ndash1096 2005

[3] C A de Kruif J W Marsman J C Venekamp et al ldquoStructureelucidation of acid reaction products of indole-3-carbinoldetection in vivo and enzyme induction in vitrordquo Chemico-Biological Interactions vol 80 no 3 pp 303ndash315 1991

[4] S Banerjee D Kong Z Wang B Bao G G Hillman and FH Sarkar ldquoAttenuation of multi-targeted proliferation-linkedsignaling by 331015840-diindolylmethane (DIM) from bench toclinicrdquoMutation Research vol 728 no 1-2 pp 47ndash66 2011

[5] M J Anderton M M Manson R D Verschoyle et alldquoPharmacokinetics and tissue disposition of indole-3-carbinoland its acid condensation products after oral administration to


micerdquo Clinical Cancer Research vol 10 no 15 pp 5233ndash52412004

[6] D J Kim D H Shin B Ahn et al ldquoChemoprevention of coloncancer by Korean food plant componentsrdquo Mutation Researchvol 523-524 pp 99ndash107 2003

[7] K M W Rahman and F H Sarkar ldquoInhibition of nucleartranslocation of nuclear factor-120581B contributes to 331015840-Diindolylmethane-induced apoptosis in breast cancer cellsrdquoCancer Research vol 65 no 1 pp 364ndash371 2005

[8] S Chintharlapalli R Smith III I Samudio W Zhang andS Safe ldquo11-Bis(31015840-indolyl)-1-(p-substitutedphenyl)methanesinduce peroxisome proliferator-activated receptor 120574-mediatedgrowth inhibition transactivation and differeatiation markersin colon cancer cellsrdquo Cancer Research vol 64 no 17 pp5994ndash6001 2004

[9] M Nachshon-Kedmi S Yannai A Haj and F A FaresldquoIndole-3-carbinol and 331015840-diindolylmethane induce apoptosisin human prostate cancer cellsrdquo Food and Chemical Toxicologyvol 41 no 6 pp 745ndash752 2003

[10] W Kassouf S Chintharlapalli M Abdelrahim G Nelkin SSafe and A M Kamat ldquoInhibition of bladder tumor growthby 11-bis(31015840-indolyl)-1-(p- substitutedphenyl)methanes a newclass of peroxisome proliferator-activated receptor 120574 agonistsrdquoCancer Research vol 66 no 1 pp 412ndash418 2006

[11] M Abdelrahim K Newman K Vanderlaag I Samudio and SSafe ldquo331015840-Diindolylmethane (DIM) and its derivatives induceapoptosis in pancreatic cancer cells through endoplasmic retic-ulum stress-dependent upregulation of DR5rdquo Carcinogenesisvol 27 no 4 pp 717ndash728 2006

[12] Y Gong G L Firestone and L F Bjeldanes ldquo331015840-Diindolyl-methane is a novel topoisomerase II120572 catalytic inhibitor thatinduces S-phase retardation and mitotic delay in human hep-atoma HepG2 cellsrdquoMolecular Pharmacology vol 69 no 4 pp1320ndash1327 2006

[13] A-S Keck and J W Finley ldquoCruciferous vegetables cancerprotective mechanisms of glucosinolate hydrolysis productsand seleniumrdquo Integrative Cancer Therapies vol 3 no 1 pp 5ndash12 2004

[14] Y Li S R Chinni and F H Sarkar ldquoSelective growth regulatoryand pro-apoptotic effects of DIM is mediated by Akt andNF-kappaB pathways in prostate cancer cellsrdquo Frontiers inBioscience vol 10 no 1 pp 236ndash243 2005

[15] KMW Rahman S Banerjee S Ali et al ldquo331015840-diindolylmeth-ane enhances taxotere-induced apoptosis in hormone-refractory prostate cancer cells through survivin down-regulationrdquo Cancer Research vol 69 no 10 pp 4468ndash44752009

[16] H J Cho M R Seon Y M Lee et al ldquo331015840-Diindolylmethanesuppresses the inflammatory response to lipopolysaccharide inmurine macrophagesrdquo The Journal of Nutrition vol 138 no 1pp 17ndash23 2008

[17] M Yoshida S Katashima J Ando et al ldquoDietary indole-3-carbinol promotes endometrial adenocarcinoma developmentin rats initiated with N-ethyl-N1015840 -nitro-N-nitrosoguanidinewith induction of cytochromeP450s in the liver and consequentmodulation of estrogenmetabolismrdquoCarcinogenesis vol 25 no11 pp 2257ndash2264 2004

[18] J P Lian B Word S Taylor G J Hammons and B DLyn-Cook ldquoModulation of the constitutive activated STAT3transcription factor in pancreatic cancer prevention effects ofindole-3-carbinol (I3C) and genisteinrdquoAnticancer Research vol24 no 1 pp 133ndash137 2004

[19] C Hong G L Firestone and L F Bjeldanes ldquoBcl-2 family-mediated apoptotic effects of 331015840-diindolylmethane (DIM) inhuman breast cancer cellsrdquo Biochemical Pharmacology vol 63no 6 pp 1085ndash1097 2002

[20] Y Takada M Andreeff and B B Aggarwal ldquoIndole-3-carbinolsuppresses NF-120581B and I120581B120572 kinase activation causing inhi-bition of expression of NF-120581B-regulated antiapoptotic andmetastatic gene products and enhancement of apoptosis inmyeloid and leukemia cellsrdquo Blood vol 106 no 2 pp 641ndash6492005

[21] K Kunimasa T Kobayashi K Kaji and T Ohta ldquoAntiangio-genic effects of indole-3-carbinol and 331015840-diindolylmethane areassociated with their differential regulation of ERK12 and Aktin tube-formingHUVECrdquoThe Journal of Nutrition vol 140 no1 pp 1ndash6 2010

[22] J D Hayes M O Kelleher and I M Eggleston ldquoThe cancerchemopreventive actions of phytochemicals derived from glu-cosinolatesrdquo European Journal of Nutrition vol 47 no 2 pp 73ndash88 2008

[23] DMaciejewskaM Rasztawicka IWolska E Anuszewska andB Gruber ldquoNovel 331015840-diindolylmethane derivatives Synthesisand cytotoxicity structural characterization in solid staterdquoEuropean Journal of Medicinal Chemistry vol 44 no 10 pp4136ndash4147 2009

[24] G A Reed D W Arneson W C Putnam et al ldquoSingle-dose and multiple-dose administration of indole-3-carbinolto women pharmacokinetics based on 331015840-diindolylmethanerdquoCancer Epidemiology Biomarkers and Prevention vol 15 no 12pp 2477ndash2481 2006

[25] M J Anderton M M Manson R Verschoyle et alldquoPhysiological modeling of formulated and crystalline 331015840 -diindolylmethane pharmacokinetics following oral administra-tion in micerdquo Drug Metabolism and Disposition vol 32 no 6pp 632ndash638 2004

[26] F Kassie L B Anderson R Scherber et al ldquoIndole-3-carbinolinhibits 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone plusbenzo(a)pyrene-induced lung tumorigenesis in AJ mice andmodulates carcinogen-induced alterations in protein levelsrdquoCancer Research vol 67 no 13 pp 6502ndash6511 2007

[27] GMurillo and R GMehta ldquoCruciferous vegetables and cancerpreventionrdquo Nutrition and Cancer vol 41 no 1-2 pp 17ndash282001

[28] J H Cohen A R Kristal and J L Stanford ldquoFruit and vegetableintakes and prostate cancer riskrdquo Journal of the National CancerInstitute vol 92 no 1 pp 61ndash68 2000

[29] C B Ambrosone S E McCann J L Freudenheim J RMarshall Y Zhang and P G Shields ldquoBreast cancer risk in pre-menopausal women is inversely associatedwith consumption ofbroccoli a source of isothiocyanates but is notmodified byGSTgenotyperdquoThe Journal of Nutrition vol 134 no 5 pp 1134ndash11382004

[30] J V Higdon B Delage D E Williams and R H DashwoodldquoCruciferous vegetables and human cancer risk epidemiologicevidence andmechanistic basisrdquo Pharmacological Research vol55 no 3 pp 224ndash236 2007

[31] LWWattenberg andW D Loub ldquoInhibition of polycyclic aro-matic hydrocarbon-induced neoplasia by naturally occurringindolesrdquo Cancer Research vol 38 no 5 pp 1410ndash1413 1978

[32] S Hendrich and L F Bjeldanes ldquoEffects of dietary cabbageBrussels sprouts Illicium verum Schizandra chinensis andalfalfa on the benzo[a]pyrene metabolic system in mouse liverrdquoFood and Chemical Toxicology vol 21 no 4 pp 479ndash486 1983


[33] B M Lee and K-K Park ldquoBeneficial and adverse effects ofchemopreventive agentsrdquo Mutation Research vol 523-524 pp265ndash278 2003

[34] Y-C Chen S-Y Lin-Shiau and J-K Lin ldquoInvolvement of p53and HSP70 proteins in attenuation of UVC-induced apoptosisby thermal stress in hepatocellular carcinoma cellsrdquoPhotochem-istry and Photobiology vol 70 no 1 pp 78ndash86 1999

[35] M Karin and F R Greten ldquoNF-120581B linking inflammationand immunity to cancer development and progressionrdquo NatureReviews Immunology Cancer Cell vol 5 no 10 pp 749ndash7592005

[36] S Banerjee ZWang D Kong and F H Sarkar ldquo331015840-diindolyl-methane enhances chemosensitivity of multiple chemothera-peutic agents in pancreatic cancerrdquoCancer Research vol 69 no13 pp 5592ndash5600 2009

[37] Y Chen E McMillan-Ward J Kong S J Israels and SB Gibson ldquoOxidative stress induces autophagic cell deathindependent of apoptosis in transformed and cancer cellsrdquo CellDeath and Differentiation vol 15 no 1 pp 171ndash182 2008

[38] M B Arnao J Sanchez-Bravo and M Acosta ldquoIndole-3-carbinol as a scavenger of free radicalsrdquo Biochemistry andMolecular Biology International vol 39 no 6 pp 1125ndash11341996

[39] S Sun J HanWM Ralph et al ldquoEndoplasmic reticulum stressas a correlate of cytotoxicity in human tumor cells exposed todiindolylmethane in vitrordquo Cell Stress and Chaperones vol 9pp 76ndash87 2004

[40] S H Benabadji R Wen J-B Zheng X-C Dong and S-GYuan ldquoAnticarcinogenic and antioxidant activity of diindolyl-methane derivativesrdquoActa Pharmacologica Sinica vol 25 no 5pp 666ndash671 2004

[41] M Karin ldquoInflammation-activated protein kinases as targetsfor drug developmentrdquo Proceedings of the American ThoracicSociety vol 2 no 4 pp 386ndash390 2005

[42] H H Garcia G A Brar D H H Nguyen L F Bjeldanesand G L Firestone ldquoIndole-3-carbinol (I3C) inhibits cyclin-dependent kinase-2 function in human breast cancer cells byregulating the size distribution associated cyclin E forms andsubcellular localization of the CDK2 protein complexrdquo TheJournal of Biological Chemistry vol 280 no 10 pp 8756ndash87642005

[43] S Safe S Papineni and S Chintharlapalli ldquoCancer chemother-apy with indole-3-carbinol bis(31015840-indolyl)methane and syn-thetic analogsrdquoCancer Letters vol 269 no 2 pp 326ndash338 2008

[44] C-Y Chen Y-L Hsu Y-C Tsai and P-L Kuo ldquoKotomolideA arrests cell cycle progression and induces apoptosis throughthe induction of ATMp53 and the initiation of mitochondrialsystem in human non-small cell lung cancer A549 cellsrdquo Foodand Chemical Toxicology vol 46 no 7 pp 2476ndash2484 2008

[45] G L Firestone and L F Bjeldanes ldquoIndole-3-carbinol and 3-31015840-diindolylmethane antiproliferative signaling pathways controlcell-cycle gene transcription in human breast cancer cells byregulating promoter-Sp1 transcription factor interactionsrdquo TheJournal of Nutrition vol 133 no 7 pp 2448Sndash2455S 2003

[46] W Risau and I Flamme ldquoVasculogenesisrdquo Annual Review ofCell and Developmental Biology vol 11 pp 73ndash91 1995

[47] W D Loub LWWattenberg and DW Davis ldquoAryl hydrocar-bon hydroxylase induction in rat tissues by naturally occurringindoles of cruciferous plantsrdquo Journal of the National CancerInstitute vol 54 no 4 pp 985ndash988 1975

[48] R McDanell A E M McLean and A B Hanley ldquoDifferentialinduction of mixed-function oxidase (MFO) activity in rat liver

and intestine by diets containing processed cabbage correlationwith cabbage levels of glucosinolates and glucosinolate hydrol-ysis productsrdquo Food and Chemical Toxicology vol 25 no 5 pp363ndash368 1987

[49] I Chen A McDougal F Wang and S Safe ldquoAryl hydrocarbonreceptor-mediated antiestrogenic and antitumorigenic activityof diindolylmethanerdquo Carcinogenesis vol 19 no 9 pp 1631ndash1639 1998

[50] S Fan Q Meng T Saha F H Sarkar and E M Rosen ldquoLowconcentrations of diindolylmethane a metabolite of indole-3-carbinol protect against oxidative stress in a BRCA1-dependentmannerrdquo Cancer Research vol 69 no 15 pp 6083ndash6091 2009

[51] B Srivastava and Y Shukla ldquoAntitumour promoting activity ofindole-3-carbinol inmouse skin carcinogenesisrdquoCancer Lettersvol 134 no 1 pp 91ndash95 1998


[53] D-Z Chen M Qi K J Auborn and T H Carter ldquoIndole-3-carbinol and diindolylmethane induce apoptosis of humancervical cancer cells and in murine HPV16-transgenic preneo-plastic cervical epitheliumrdquoThe Journal of Nutrition vol 131 no12 pp 3294ndash3302 2001

[54] B Davidson I Goldberg and J Kopolovic ldquoAngiogenesisin uterine cervical intraepithelial neoplasia and squamouscell carcinoma An immunohistochemical studyrdquo InternationalJournal of Gynecological Pathology vol 16 no 4 pp 335ndash3381997

[55] G Szakacs J K Paterson J A Ludwig C Booth-Genthe andM M Gottesman ldquoTargeting multidrug resistance in cancerrdquoNature Reviews Drug Discovery vol 5 no 3 pp 219ndash234 2006

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

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Autoimmune Diseases


Anesthesiology Research and Practice

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MEDIATORSINFLAMMATION

of


the growth of prostate colon and breast cancer cells [6 7]I3C and its derivatives also suppress cell propagation andinduce apoptosis in colon cancer cells [8] as well as in othertypes of cancer cells including prostate [9] breast [7] bladder[10] pancreas [11] and hepatoma [12] This review mainlyfocuses on the role of I3C and DIM against various types ofmalignancy and underlying mechanisms

2 Cruciferous Vegetables andTheir Derivatives

Glucosinolates are a class of organic compounds that givepungent smell and piquant taste in the cruciferous vegetablesand some condiments such as wasabi andmustardThemainfunction of glucosinolates in plants is that it acts as naturalpesticides and accelerates the resistance against herbivores[13]

The central carbon of all glucosinolates is bound 6 to athioglucose group to a sulfate group via the nitrogenmolecule[22] The central carbon of each glucosinolate is boundto a side group and this makes each glucosinolate unique[22] Glucosinolates are classified as aliphatic (eg alkenylalkyl hydroxyalkenyl or w-methylthioalkyl) aromatic (egbenzyl substituted benzyl) or heterocyclic with the R-groupof the aliphatic glucosinolates being derived from alaninemethionine leucine or valine and those of aromatic andheterocyclic glucosinolates being derived from phenylala-nine or tyrosine or tryptophan [14] (Figure 1(a)) Althoughglucosinolates are related to inhibition of carcinogenesisit is actually their hydrolysis products not the glucosino-lates themselves that are biologically active Hydrolysis ofglucosinolates is catalyzed by the enzyme myrosinase alsoknown as 120573-thioglucoside glucohydrolase [13] In a low pHenvironment I3C is converted into polymeric products andDIM is the main one DIM is a major in vivo acid catalyzedcondensation product of I3C [23] (Figures 1(b) and 1(c))Theyare no more natural products from DIM Maciejewska et alsynthesized a series ofDIMderivatives bearing fluoro bromoiodo and nitro substituents in indole or benzene rings andtested for cytotoxicity against human melanoma cell linesafter characterizing their structure [23] These derivativeswere found to cause 50 inhibition of the viability of ME18and ME18R cell lines at concentrations ranging between97 and 173mM [23] These finding clearly suggested thatsynthetic analog of DIM has potential role in cancer therapyin the near future once their systemic toxicity studies havebeen determined [23]

3 Bioavailability of I3C and DIM

Reed et al [24] in his extensive article of pharmacokineticstudies reported that women received oral doses of 400 600800 1000 and 1200mg I3C and these serial plasma sampleswere analyzed by high-performance liquid chromatography-mass spectrometrymethod for the detection and quantitationof the I3C and DIM I3C itself is not detectable in plasma[24] The only detectable I3C-derived product is DIM Highinitial value plasma DIM for all subjects decreased to near

N

OSO3

GIc

SR

(minus)

(a)

NH

OH

(b)

NH

NH

(c)

Figure 1 Structures of (a) glucosinolates (b) I3C and (c) DIM

or below the limit of quantitation within the 12 h samplingperiod [24] Physiologically based pharmacokinetic (PBPK)model is developed using plasma and tissue (brain heartliver kidneys and lungs) concentration data for DIM tocompare the pharmacokinetic properties and biodistributionof pure crystalline and a novel formulation (BioResponse-DIM (BR-DIM)) of DIM after oral administration to mice[25] I3C is hard to develop as a drug because it is highlyunstable and can transform into many other derivativessuch as DIM 5611121718-hexahydrocyclonona[12-b45-b101584078-b10158401015840] triindole (CTr) indolo [32-b] carbazole (ICZ) N-methoxyindole-3-carbinol (NI3C) two tetramers one linear(LTET) and one cyclic (CTET) and 1-(3-hydroxymethyl)indolylmethane (HI-IM) Anderton et al reported oral dosesof I3C to female CD-1 mice and studied the disposition ofI3C and its acid condensation productsDIM ICZ LTr(1) andHI-IM in blood liver kidney lung heart and brain I3C wasrapidly absorbed distributed and eliminated from plasmaand tissues falling below the limit of detection by 1 h [5] Themajor acid condensation product of I3C DIM has provenvery stable in acidic conditions during prolonged exposureto high temperature and humidity [4] Another report BR-DIM has proven very stable and it is not converted into otherforms [4] Based on these outcomes authors suggested thatone could exploit further development of DIM as a potentialtherapeutic agent

4 Metabolism and Distribution

Under the acidic conditions of the gastric tract I3C under-goes condensation to form several oligomeric productsparticularly DIM [23]The tissue distribution of I3C has beendetermined in mice by radio-labelled I3C [26] Both I3Cand DIM have been detected in kidneys lungs heart liverplasma and brain samples of mice treated with 250mgkg ofI3C as early as 15mins after administration [26]These resultssuggest that I3C is rapidly absorbed and spread to a numberof well-perfused tissues where it is transformed to DIM toperform its anticancer actions [26]
















50 were


504 and 9





glucosinolates









-Increasing

-Decreasing




1arrest
















9 Drug Resistance



10 Conclusion




References
































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of
















50 were


504 and 9





glucosinolates









-Increasing

-Decreasing




1arrest
















9 Drug Resistance



10 Conclusion




References
































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



glucosinolates









-Increasing

-Decreasing




1arrest
















9 Drug Resistance



10 Conclusion




References
































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of









9 Drug Resistance



10 Conclusion




References
































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

review article attenuation of carcinogenesis and the

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