Research Article Mrunmayee P.Toraskar et al. / Journal of Pharmacy Research 2009, 2(10),1638-1641 ISSN: 0974-6943 Available online through http://jprsolutions.info Isothiocyanate: cancer chemopreventive agent

Mrunmayee P.Toraskar*, Vithal M. Kulkarni , Shweta T.Dhanashire, Vilasarao J.Kadam Department of Pharmaceutical chemistry, Bharati Vidyapeeth’s College of Pharmacy, Sector 08, CBD Belapur, Navi-Mumbai – 400614, India.. Received on: 20-05-2009; Accepted on:15-07-2009 ABSTRACT Isothiocyanates- plant derived dietary compounds, are promising chemopreventive agents generally non-toxic substances that interfere with the process of cancer development. Several natural and synthetic isothiocyanates have demonstrated cancer-preventive properties in animals treated with chemical carcinogens, including polycyclic aromatic hydrocarbon nitrosamines. Some of these compounds, the isothiocyanates – particularly allyl isothiocyanate and phenethyl isothiocyanate, have anticancer activity. Epidemiologic and clinical studies indicate a positive correlation between the general consumption of cruciferous vegetables and the decreased incidence of some cancers including non-Hodgkin’s lymphoma, liver, prostate, cervical, ovarian, lung, gastrointestinal tract. Natural isothiocyanates such as sulforaphane and phenethyl isothiocyanate possess strong antitumor activities both in vitro and in vivo. It exerts strong anticarcinogenic effects in a number of animal models of cancer, presumably by modulation of xenobiotic-metabolizing enzymes, such as by inhibition of cytochrome P- 450 and/or by induction of phase II detoxifying enzymes.

Keywords: Isothiocyanates, Cancer chemopreventive agent, Apoptosis

INTRODUCTION Chemopreventive isothiocyanates Cancer chemoprevention is regarded as the use of synthetic zymatically and enzymatically by myrosinase (ß-thioglucoside or natural agents to inhibit, delay or reverse the process of carcino- glucohydrolase) during food preparation, cooking and chewing. genesis through intervention before the appearance of invasive dis- Antineoplastic effect of cruciferous vegetables is attributed to ease. Isothiocyanates, plant-derived dietary compounds, are isothiocyanates, which occur as thioglucoside conjugates chemopreventive agents. Suppression of cell proliferation and in- (glucosinolates) in a variety of edible plants, including broccoli, wa- duction of differentiation and apoptosis during the promotion and tercress and cabbage. (4) progression stages are ideal preventive devices1. Vegetables are a good source of trace non-nutrient com- pounds that contribute to the prevention of cancer. Some of these MECHANISM OF ACTION OF ISOTHIOCYANATES compounds, the isothiocyanates – particularly allyl isothiocyanate and phenethyl isothiocyanate, have both anti-carcinogenic and anti- cancer activity 2. Glucosinolates and myrosinase are compartmentalised in plants Isothiocyanates acts as chemopreventive by inducing apoptosis pro- but interact when tissue damage occurs. Myrosinase activity of in- cess i.e cell death The mechanism by which Phenyl ethyl testinal bacteria may also contribute to the degradation of isothiocyanates inhibits chemically induced cancer involves inhibi- glucosinolates in vivo. Two of the most prevalent glucosinolates are tion of carcinogen activation due to inhibition of cytochrome P-450 sinigrin and gluconasturtin. Myrosinase-catalysed hydrolysis of sini- dependent mono-oxygenases and increased carcinogen inactivation grin and gluconasturtin cleaves the thioglucoside linkage; yielding due to induction of phase II enzymes, including glutathione trans- 4 glucose and an unstable iohydroximate-O-sulphonate aglycone that ferase .Cytotoxicity was associated with an initial decrease in GSH spontaneously rearranges to form allyl isothiocyanate and phenethyl and GSSG, with a concomitant formation of the GSH adduct S-(N- isothiocyanate (PEITC), respectively, other products, nitriles and iso- phenethylthiocarbamoyl) glutathione inside cells which was then ex- cyanates, may also be formed3. Cruciferous vegetables such as broc- ported from cell.Then S-(N-phenethyl thiocarbamoyl) glutathione coli, cabbage and kale, are rich sources of sulfur containing com- spontaneously fragmented to GSH and phenethyl isothiocyanate,GSH pounds called glucosinolates. Glucosinolates are degraded non-en- oxidized to GSSG and glutathionyl-protein disulphides and phenethyl isothiocyanate hydrolyzed to phenylethy lamine5.Induction of apoptosis by dietary isothiocyanates due to activation of the stress- *Corresponding author. activated protein kinase JNK1( Junction N-terminal Kinase) with cleav- Tel.: + 91-022 27662973 age of p22 BID protein to p15, p13 and p11 fragments. Telefax: +91- E-mail: [email protected] Journal of Pharmacy Research Vol.2.Issue 10.October 2009 1638-1641 Mrunmayee P.Toraskar et al. / Journal of Pharmacy Research 2009, 2(10),1638-1641 Mechanism of induction of tumour cell apoptosis by dietary EXAMPLES OF ISOTHIOCYANATE ( 6) isothiocyanates(5) 1. Benzyl Isothiocyanate The mechanism of induction of apoptosis involves the entry of the isothiocyanate into cells and formation of the GSH conjugate, S-(N- CH2NCS alkyl or arylalkyl thiocarbamoyl) glutathione. Concurrently, there is a slow inactivation of phenyl ethyl isothiocyanates by spontaneous hydrolysis then expulsion of the GSH conjugate via the MRP protein channel, with a consequent depletion of cellular GSH.There is a tem- 2. Phenethyl Isothiocyanate porary, reversible modification of cellular protein thiols – protein thiocarbamoylation. N=C=S Event 3 triggers recruitment of adaptor protein FADD to apoptosis-mediating cell death receptors (tumour necrosis receptor 1 and/or the fas receptor) and activates caspase-8. After this there is cleavage of mitochondrial protein BID, then release of cytochrome c 3. Phenyl Methyl Isothiocyanate from mitochondria and activation of caspase-3. Activation of the stress activation protein kinase JNK (which induces increased expression of the fas receptor ligand – further potentiating apoptosis) via activa- tion of the mitogen activated kinase MEKK1. N=C=S All these events induces apoptosis, DNA fragmentation and cell death. Where the isothiocyanate (RN=C=S) is administered as the corre- sponding thiol conjugate (RNH(C=S)-SR’), this sequence of events is preceded by fragmentation of the thiol conjugate to the free 4. Phenylhexyl Isothiocyanate isothiocyanate (CH2)6NCS

5. Sulphoraphane O

S NCS H3C

ferases (GSTs), and further metabolized to mercapturic acids. These isothiocyanate metabolites can be measured in the urine, and are highly correlated with dietary intake of cruciferous vegetables .

BIOLOGICAL ACTIVITIES (2,8) Effects on Biotransformation Enzymes Involved in Carcinogen Me- tabolism

Biotransformation enzymes play important roles in the metabo- lism and elimination of a variety of chemicals, including drugs, toxins and carcinogens. In general, phase -I biotransformation enzymes cata- lyze reactions that increase the reactivity of hydrophobic (fat-soluble) compounds, preparing them for reactions catalyzed by phase -II Metabolism and Bioavailability: biotransformation enzymes. Myrosinase a class of enzymes that catalyzes the hydrolysis (break- down) of glucosinolates, is physically separated from glucosinolates Inhibition of Phase - I Biotransformation Enzymes when plant cells are intact.8When cruciferous vegetables are chopped Some procarcinogens (carcinogen precursors) require or chewed, myrosinase can interact with glucosinolates and release biotransformation by phase-I enzymes, such as those of the cyto- isothiocyanates from their precursors.During metabolism, chrome P450 (CYP) family, in order to become active carcinogens that isothiocyanates are conjugated (bound) to glutathione, an activity are capable of binding DNA and inducing mutations. Inhibition of that is promoted by a family of enzymes called glutathione-S-trans- specific CYP enzymes involved in carcinogen activation inhibits the Journal of Pharmacy Research Vol.2.Issue 10.October 2009 1638-1641 Mrunmayee P.Toraskar et al. / Journal of Pharmacy Research 2009, 2(10),1638-1641 Clinically, it is usually diagnosed in patients over 50 years old. Thus, development of cancer in animal models.Isothiocyanates,including there could be opportunity for intervention using cancer PEITC and BITC(Benzyl isothiocyanates), have been found to inhibit chemopreventive compounds that prevent or slow the progression carcinogen activation by CYP enzymes. of this disease. Recent studies have indicated that using dietary Induction of Phase - II Biotransformation Enzymes chemopreventive compounds, such as isothiocyanates, might be a Many isothiocyanates, particularly SFN (Sulphoraphane), promising strategy to decrease the incidence of prostate cancer. Some are potent inducers of phase -II enzymes in cultured human cells. isothiocyanates derived from cruciferous vegetables, such as Phase -II enzymes, including GSTs, UDP-glucuronosyl transferases sulforaphane (SFN), are highly effective in preventing or reducing (UGTs), quinone reductase and glutamate cysteine ligase, plays im- the risk of cancer11. portant roles in protecting cells from DNA damage by carcinogens and reactive oxygen species Curcumin (from turmeric) is one of the most well known naturally Anti-inflammatory Activity occurring compounds. It has cancer chemopreventive potential Inflammation promotes cellular proliferation and inhibits against many types of cancerous cells, including prostate cancer. It is apoptosis, increasing the risk of developing cancer. Isothiocyanates a potent nuclear factor-kappaB (NF-kB) inhibitor as well as an inducer’s have been found to decrease the secretion of inflammatory signaling of apoptosis12 molecules by white blood cells and to decrease DNA binding of NF- kappaB, a pro-inflammatory transcription factor. 4. Glioblastoma Cancer Preservation Of Normal Cell Cycle Regulation Dietary Iberin isothiocyanate: After cell divides it passes through a sequence of stages Glioblastoma is the most common malignant tumor of the adult central known as the cell cycle before dividing again.Following cell damage nervous system and is hallmarked by high proliferation of tumor cells the cell cycle can be transiently arrested to allow for DNA repair or with increased cellularity and necrosis2. Recent studies indicate that activation of pathways leading to cell death (apoptosis) if the damage natural isothiocyanates such as sulforaphane and phenethyl cannot be repaired. Defective cell cycle regulation may result in the isothiocyanate possess strong antitumor activities both in vitro and propagation of mutations that contribute to the development of can- in vivo13 cer. A number of isothiocyanates, including AITC, BITC, PEITC and SFN, have been found to induce cell cycle arrest in cultured cells. 5. Breast Cancer Risk In Premenopausal Women ROLE OF ISOTHIOCYANATES IN VARIOUS CANCER TYPES Isothiocyanates, formed from glucosinolates in cruciferous veg- 1.Lung Cancer etables, could also be responsible for inverse associations between Phenethyl isothiocyanate and benzyl isothiocyanate: - cruciferous vegetable consumption and breast cancer risk13. These compounds inhibited the metabolic activation of nicotine-de- The anti carcinogenic effects of cruciferous vegetables may derive rived nitrosamino ketone (NNK), a powerful pulmonary carcinogen from the glucosinolates they contain; these are degraded into indoles found in cigarette smoke, capable of inducing lung tumours in ani- and isothiocyanates. Indoles were studied in relation to breast cancer mals whatever the route of administration. With regard to partly because of their effects on estrogen metabolism and isothiocyanates, much research, in particular by Hecht and colleagues, isothiocyanates are chemopreventive agents in animal models, per- has focused on two compounds: phenethyl isothiocyanate and ben- haps due in part to their potent effects on inhibition of phase -I and zyl isothiocyanate9. induction of phase -II enzymes14 2. Prostate, Liver, Ovarian, And Colon Cancer Sulphoraphane :- Food Sources Cruciferous Vegetables Isothiocyanates are components of certain plants and vegetables Cruciferous vegetables, such as bok choi, broccoli, Brussels sprouts, that have selective biological activities and functions against car- cabbage, cauliflower, kale, kohlrabi, mustard, radish, rutabaga, turnip cinogenesis. Sulforaphane, a potent cancer preventive agent, is a and watercress contain a series of relatively unique secondary me- dietary isothiocyanate compound found as a precursor glucosinolate tabolites of amino acids, called glucosinolates. Sinigrin, the predomi- in cruciferous vegetables such as cauliflower, broccoli, and Brussels nant aliphatic glucosinolate in cruciferous vegetables, is hydrolyzed sprouts9. Epidemiologic and clinical studies reviewed by Murillo and to yield allyl isothiocyanate (AITC), which, after absorption and me- colleagues indicate a positive correlation between the general con- tabolism in humans, is excreted in the urine as an N-acetylcysteine sumption of cruciferous vegetables and the decreased incidence of (NAC) conjugate15.Unlike some other phytochemicals, glucosinolates some cancers including non-Hodgkin’s lymphoma, liver, prostate, are present in relatively high concentrations in commonly consumed cervical, ovarian, lung, a gastrointestinal tract. Oral administration of portions of cruciferous vegetables. For example ½ cup of raw broccoli sulforaphane inhibited or retarded experimental multistage carcino- might provide more than 25 mg of total glucosinolates. Total genesis models including cancers of the breast, colon, stomach, and glucosinolate contents of selected cruciferous vegetables are pre- lung10. sented in Table 1. Some cruciferous vegetables are better sources of 3. Prostate Cancer specific glucosinolates (and isothiocyanates) than others. Vegetables Phenylethyl isothiocyanate and curcumin: of relatively good sources of some isothiocyanates currently under Prostate cancer is one of the most prevalent malignancies and the study for their cancer-preventive properties 8 are listed in Table 2 second leading cause of cancer deaths in men in the United States. Journal of Pharmacy Research Vol.2.Issue 10.October 2009 1638-1641 Mrunmayee P.Toraskar et al. / Journal of Pharmacy Research 2009, 2(10),1638-1641 Table 1. Glucosinolate Content of Selected Cruciferous Vegetables From these studies it was confirmed that isothiocyanate acts as can- Food (raw) Serving Total Glucosinolates (mg) cer chemoprevenive agent. Brussels sprouts ½ cup (44 g) 104 Garden cress ½ cup (25 g) 98 CONCLUSION Mustard greens ½ cup, chopped (28 g) 79 By consuming cruciferous vegetables containing isothiocyanates we Turnip ½ cup, cubes (65 g) 60 may prevent cancer or may prolong it. Intervention with chemo pre- Cabbage, Savoy ½ cup, chopped (45 g) 35 ventive agent like isothiocyanate at an earliest stage in cancer is more Kale 1 cup, chopped (67 g) 67 Watercress 1 cup, chopped (34 g) 32 attractive than attempting to eradicate fully developed tumours with Cabbage, red ½ cup, chopped (45 g) 29 chemotherapeutic drugs. Broccoli ½ cup, chopped (44 g) 27 Horseradish 1 tablespoon (15 g) 24 REFERENCES: Cauliflower ½ cup, chopped (50 g) 22 1. S.V. Bhide & G. B. Maru, Chemoprevention of cancer, Omega Scien- tific publisher, 1998, 12,13,120,121 Table 2. Food Sources of Selected Isothiocyanates and Their 2. Unmesh Jadhav1, Ravesanker Ezhilarasan, Dietary Isothiocyanate Glucosinolate Precursors Iberin Inhibits Growth and Induces Apoptosis in Human Glioblastoma Cells, J Pharmacol Sci 103, 247 – 251 (2007 Isothio- Glucosinolate Food Sources 3. http://www2.warwick.ac.uk/fac/med/research/csri/proteindamage/ cyanate (precursor) researchinterest /isothiocyanates Allyl Sinigrin Broccoli, Brussels sprouts, cabbage, 4. Dong Xiao, Yan Zeng,. Caspase-Dependent Apoptosis Induction by Isothiocyanate (AITC) horseradish, mustard, radish Phenethyl Isothiocyanate, a Cruciferous Vegetable-Derived Cancer Benzyl Glucotropaeolin Cabbage, garden cress, Indian cress Chemopreventive Agent, Is Mediated by Bak and Bax, clin cancer Isothiocyanate (BITC) res.2005:1197) April1, 2005 Phenethyl- Gluconasturtiin Watercress 5. Xu, K. and Thornalley, P.J. (2001) Involvement of GSH metabolism Isothiocyanate (PEITC) in the cytotoxicity of the phenethyl isothiocyanate and its cysteine Sulforaphane (SFN) Glucoraphanin Broccoli, Brussels sprouts, cabbage conjugate to human leukaemia cells in vitro. Biochem. Pharmacol. 61, 165-177. (8,10,16) RESULT AND DISCUSSION 6. Susan J.Thomson,Kristin K.Brown,Juliet M.Phenethyl Isothiocyanate Sulforaphane is an isothiocyanate that has been isolated from SAGA Triggers Apoptosis in Jurkat CellsMade Resistant by the broccoli. Laurence Gamet-Payrastre, Pengfei and Li, SolangeLumeau Overexpression of Bcl-2,Cancer Research 2006;66(13).July,2006. etal investigated the effect of sulforaphane on the growth and viabil- 7. Rong Yu, Sandhya Mandlekar,,Chemopreventive Isothiocyanates Induce Apoptosis and Caspase-3-like Protease Activity, Cancer Re- ity of HT29 cells during their exponentially growing phase. This com- search 58, 402-408. February I. 1998 pound has been shown to block the formation of tumors initiated by 8. Jane Higdon, Ph.D, Linus Pauling Institute,Oregon,state chemicals in the rat. It is a very potent monofunctional inducer of University,September 9,2005 phase II enzymes in both cultured cells and mouse tissues; and it has 9. G.J.Caine& G.Y.H.Lip, Dietary prevention of carcinogenesis in smok- ers, Journal Of Internal Medicine2002; 252:577-578. recently been shown to inhibit at least one cytochrome P-450 (CYP2E1) 10. Laurence Gamet-Payrastre, Sulforaphane, a Naturally Occurring involved in the activation of a variety of carcinogens.10 Isothiocyanate, Induces Cell Cycle Arrest and Apoptosis in HT29 Review article by Kuper et al. regarding tobacco use and car- Human Colon Cancer Cells, Cancer Research 60, 1426–1433, March cinogenesis. Studies show that vegetable consumption decreases 1, 2000 11. Jung-Hwan Kim1, Changjiang Xu, Young-Sam, Inhibition of EGFR the risk of lung cancer. In studies to date, much work has been fo- signaling in human prostate cancer PC-3 cells by combination, treat- cused on isothiocyanates. These compounds inhibited the metabolic ment with b-phenylethyl isothiocyanate and curcumin, Carcinogen- activation of nicotine-derived nitrosaminoketone (NNK), a powerful esis vol.27 no.3 pp.475–48 pulmonary carcinogen found in cigarette smoke, capable of inducing 12. Changjiang Xu, Guoxiang Shen, ERK and JNK signaling pathways are involved in the regulation of activator protein 1 and cell death elic- lung tumors in animals whatever the route of administration. It has ited by three isothiocyanates in human prostate cancer PC-3 cells, shown that isothiocyanates are effective inhibitors of tumour devel- Carcinogenesis vol.27 no.3 pp.437–445 opment in carcinogen-treated rodents. Isothiocyanate and benzyl 13. Nhu-An Pham,1,2 James W., The dietary isothiocyanate sulforaphane isothiocyanate in combination may be chemo preventive for lung targets pathways of apoptosis, cell cycle arrest, and oxidative stress in human pancreatic cancer cells and inhibits tumor growth in severe cancer in smokers. Indeed, a mixture of these agents given in the diet combined immunodeficient mice, Mol Cancer Ther 2004;3(10). Oc- inhibits lung-tumour induction in mice treated with NNK and benzo tober 2004 [a] pyrene. 14. Christine B. Ambrosone, Susan E. McCann, Breast Cancer Risk in Limited data from clinical trials suggests that glucosinolate- Premenopausal Women Is Inversely Associated with Consumption of Broccoli, a Source of Isothiocyanates, but Is Not Modified by GST rich foods increases phase II enzyme activity in humans. 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Journal of Pharmacy Research Vol.2.Issue 10.October 2009 1638-1641