Plant Medicines in Cancer Treatment
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Review of Literature
Plants as therapeutic agents
Plant medicines are the most widely used medicines in the world today. The use of herbs and plants as the first medicine is a universal phenomenon. Every culture on earth, through written or oral tradition, has relied on the vast variety of natural chemistry found in healing plants for their therapeutic properties (Serrentino 1991). Plants with therapeutic potential may be defined as any plant that can be put to culinary or medicinal use. Recent researches found that food and their constituents act in a manner similar to modern drugs without the dreaded side effects (Serrentino 1991). Sometimes plant medicine is viewed as complementary medicine, working closely with allopathic drugs. Nearly 5.1 billion people worldwide employ natural plant-based remedies as their primary medicines for both acute and chronic health problems, from treating common cold to controlling blood pressure and cholesterol (Stockwell, 1988).
Most of the drugs were substances with a particular therapeutic action extracted from plants. Some medicines, such as the cancer drug Taxol from Taxus brevifolia and the anti-malarial quinine from Cinchona pubescens are manufactured from the plants. Other medicinal agents such as pseudoephedrine originally derived from ephedra species and methylsalicylate, derived from gaultheria procumbens are now synthesized. Plant medicines remain indispensable to modern pharmacology and clinical practice. Much of the current drug discovery and development process are plant-based, and new medicines derived from plants are inevitable.
A food can be regarded as a "functional food" if it is demonstrated to affect one or more target functions in the body beyond adequate nutrition and improves health/well-being or reduces the risk of diseases (Tsao and Akhtar, 2005). On this basis, a functional food can be a natural food, a food to which a positive component has been added, or from which a deleterious component has been removed or a food where the nature of one or more components has been modified (Tsao and Akhtar, 2005). While searching for new sources of functional food, attention has been paid to vegetables from the Cruciferae family, which more often used in the human diets. The cruciferous vegetables may thus become a potential source of a nutritious food or food ingredients. Recent research showed that cruciferous vegetables contain an appropriate amount of bioactive compounds such as GLs, ITCs, tocopherols, L-ascorbic acid, vitamin B, reduced glutathione, inositol phosphates and polyphenolic compounds [Nakamura et al, 2001; Zielinski and Kozlowska, 2003; Zielinski et al, 2005; Takaya et al, 2003].
The family Cruciferae (Brassicaceae) is an economically important family with about 350 genera and 3000 species that includes several edible plants. Despite the great diversity among the crucifers, members of only a few genera are eaten. The most commonly eaten cruciferous vegetables belong to the genus Brassica that includes broccoli, cabbage, cauliflower, kale and Brussels sprouts. Other cruciferous vegetables used in the human diet such as radish, water cress, wasabi, horseradish, garden cress, Italian cress, Swiss chard and crambe belong to another genera of the family such as Raphanus, Nasturtium, Wasabia, Armoracia, Lepidium, Eruca, Beta and Crambe respectively.
Cruciferous vegetables are important dietary constituents in many parts of the world and appear to account for about 10 - 15% of total vegetable intake, reaching almost 25% in countries with a high consumption (Bosetti et al, 2002; Chiu et al, 2003). However, regional pattern of crucifer consumption varies substantially in different parts of the world. The highest intake of cruciferous vegetable was reported to that of people in China, who consumed more than 100 g per day, representing about one-fourth of their total vegetable intake (Chiu et al, 2003). Other Asians and some Middle Eastern populations in Japan, Singapore, Thailand and Kuwait also have a relatively high intake of cruciferous vegetables, ranging from 40 - 80 g per day (Bosetti et al, 2002; Seow et al, 2002; Shannon et al, 2002; Memon et al, 2002). However, the only study carried out in India (Rajkumar et al, 2003) showed a lower daily intake of cruciferous plants, of about 17 g per day. In North America, the daily estimated consumption was in the range of 16 - 40 g per day (Lin et al, 1998) and in South America, it was about 3 - 15 g per day (Atalah et al, 2001). The daily intake of cruciferous vegetables was reported to be about 5 - 30 g per day in Europe (Bosetti et al, 2002), 50 g per day in Australia (Nagle et al, 2003) and 15 g per day in South Africa (Steyn et al, 2003) respectively.
R. sativus is believed to have originated in southern Asia and was cultivated in Egypt. The first cultivated R. sativus was black variety and later on white and red R. sativus were developed. It was highly esteemed in ancient Greece, and the Greek physician Androcydes ordered his patients to eat R. sativus as a preservative against intoxication. The Japanese white R. sativus, also named daikon, is the vegetable for which the literature reports the highest per capita consumption, quoted at 55 g per day in Japan (Talalay and Fahey, 2001). In addition to this, Japanese also consumes R. sativus sprouts under the name of "Kaiware Daikon".
Varieties of R. sativus
There are six main varieties of R. sativus such as Daikons, Red Globe, White Globe, Black, White Icicles and California Mammoth White
- Daikons (R. sativus L)
This variety is native to Asia. They are large and carrot-shaped, have a white flesh that is juicy and a bit hotter than a red radish, but milder than black.
- Red Globe (R. sativus var. red)
This variety is the most popular in the United States. It is small, round or oval shaped, referred to as "button" red radishes and have a solid crisp flesh.
- White Globe (R. sativus var.white)
This variety is small and oval shaped, referred to as "hailstone" or "white button". They have white flesh and milder than the red variety.
- Black (R. sativus var. niger)
This variety is thought to be native to Egypt and Asia. They are turnip-like in size and shape. They are quite pungent and drier than other varieties of radishes.
- White Icicles (R. sativus L var. thin)
This variety is long and tapered. They have a white flesh that is milder than the red variety.
- California Mammoth White (R. sativus L var. large)
A larger variety than the white icicle, these varieties have oblong- shaped roots and their flesh is slightly pungent.
Nutritive value of R. sativus
R. sativus root and its leafy part are ideal vegetables as they provide an excellent source of vitamin C. Leafy part contains almost six times the vitamin C content of its root and also a good source of calcium and iron. R. sativus is also a good source of potassium and folic acid. It is very low in fats. Approximately, 100 g of raw vegetable provides roughly 20 Kcal, coming largely from carbohydrates (Table 2.1). Thus R. sativus is a dietary food that is relatively filling for its caloric value. Some sources list R. sativus as being rich in dietary fiber, whereas other sources differ in respect of its roughage content (USDA Nutrient Database, 1999; Duke and Ayensu, 1985).
Health benefits of R. sativus (Traditional usage of R. sativus)
According to Hakeem Hashmi, an eminent Unani physician from India, R. sativus is unparallel in curing any kind of ailments. All the parts of R. sativus including its seed, stem, root and leaves are used in food and medicine. R. sativus is a unique vegetable having a hot and cold effect on the body simultaneously. R. sativus, like other members of the cruciferous family (cabbage, kale, broccoli, Brussels sprouts) contains cancer-protective properties.
- Liver and gall bladder disorders
Throughout the history, R. sativus root and seeds have been effective when used as medicinal food for liver disorders. They contain sulfur-based compounds such as GLs and ITCs that increase the flow of bile and help to maintain healthy gallbladder and liver (Chevallier, 1996). They are useful in treating jaundice and also an excellent remedy for gall bladder stone.
- Kidney disorders
R. sativus root, seeds and leaves are diuretic in nature and increase the urine output. Their diuretic properties help to flush out the toxins accumulated in the kidneys and protect them from infections and inflammatory conditions. It is an old belief that R. sativus can aid in the treatment as well as prevention of kidney stones (Chopra et al, 1986).
- Respiratory disorders
R. sativus is an anti-congestive and relieves congestion of the respiratory system. It has found to be beneficial in problems associated with bronchitis (Bown, 1995) and asthma (Duke and Ayensu, 1985).
- Skin disorders
R. sativus helps to cure skin disorders such as leucoderma, rashes, cracks, etc and also refreshes the skin by maintaining the moisture content of the skin (Duke and Ayensu, 1985).
- Digestive disorders
R. sativus root, seeds and leaves are rich in roughage (indigestible carbohydrates) which facilitates digestion, retain water and relieve constipation (Chopra et al, 1986). They also soothe the digestive system and stimulate appetite (Chevallier, 1996)
- Nervous and vascular disorders
R. sativus decreases nervous tensions and is also useful in enhancing blood circulation. It is a remedy for insomnia, hypochondria and irritative conditions of the central nervous system (Panda, 1999).
- Other benefits
R. sativus is germicidal and suppresses phlegm. It is a good appetizer, mouth fresher, laxative, regulates metabolism, remedy for headache, acidity, piles, nausea, obesity, sore throat, whooping cough, dyspepsia, etc (Nadkarni, 1976; Kapoor, 1990).
Chemical constituents of R. sativus
GLs are an important and unique class of secondary plant metabolites found in the seeds, roots and leaves of R. sativus (Daxenbichler et al, 1991; Blazevic and Mastelic, 2009). GLSs include several naturally occurring thioglucosides with a common structure (Figure 2.2) characterized by side chains (R) with varying aliphatic, aromatic and heteroaromatic carbon skeletons, all presumably derived from amino acids by a chain-lengthening process and hydroxylation or oxidation (Larsen, 1981).
In the intact cell, GLs are separated from thioglucosidase (EC 188.8.131.52), an enzyme generally known as myrosinase. When the plant cell structure is damaged, myrosinase catalyzes the hydrolysis of GLs to yield D-glucose, sulfate and a series of compounds including isothiocyanates, thiocyanates and nitriles, depending on both the substrate and the reaction conditions, especially the pH (Figure 2.2). GLs are also hydrolyzed by thioglucosidase activity of the intestinal microflora (Jeffery and Jarrell, 2001).
4-(methylthio)-3-butenyl glucosinolate (glucoraphasatin), 4-(methylsulfinyl) butyl glucosinolate (glucoraphanin) and 4- (methylsulfinyl)-3-butenyl glucosinolate (glucoraphenin) are the most predominant GLs in the root and seeds of R. sativus (Daxenbichler et al, 1991; Carlson et al, 1985). These GLs on hydrolysis by myrosinase yield MTBITC, sulforaphane and sulforaphene respectively. GLs are not uniformly distributed and are highest in the distal end of the root, decreasing in upper root sections with the lowest level in vegetative tops (Esaki and Onozaki, 1980).
Apart from GLs and their breakdown products, R. sativus also contains polyphenolics such as phenolic acid, flavonoids and anthocyanins. Several polyphenolic compounds including sinapic acid esters and kaempferol were isolated from R. sativus sprouts (Takaya et al, 2003). Twelve acylated anthocyanins (pelargonidin) were isolated from R. sativus red variety (Otsuki et al, 2002). Phytochemical screening showed the presence of other phytochemicals such as triterpenes, alkaloids, saponins and coumarins in R. sativus seeds (Mohamed et al, 2008).
The myrosinase - catalyzed hydrolysis of glucosinolates. (Adapted from Rusk et al, 2000)
Novel classes of plant defensins (small basic cysteine rich peptides) such as Raphanus sativus antifungal peptide 1 and 2 (RsAFP1 and RsAFP2) were isolated from the seeds of R. sativus (Terras et al, 1992a). RsAFP1 and RsAFP2 are highly basic oligomeric proteins composed of small (5 KDa) polypeptides that are rich in cysteine. Both RsAFP1 and RsAFP2 have a broad spectrum antifungal activity and show a high degree of specificity to filamentous fungi (Terras et al, 1992b). They are active against both phytopathogenic fungi such as Fusarium culmorum and Botrytis cinerea (Terras et al, 1992b), human pathogenic fungi such as Candida albicans (Aerts et al, 2007) and occasionally possess antibacterial activity. However, they are non-toxic to humans and plant cells. R. sativus 2S storage albumins were identified as second novel class of antifungal protein (Terras et al, 1992a). They also inhibit the growth of different plant pathogenic fungi and certain bacteria (Terras et al, 1992a).
At least eight distinguishable isoperoxidases were isolated and purified to apparent homogeneity from Korean R sativus roots. Among them are two cationic isoperoxidases such as C1 and C3 and four anionic isoperoxidases such as A1, A2, A3n and A3 (Lee and Kim, 1994). Plant peroxidases play an important role in several physiological functions such as removal of peroxide, oxidation of indole-3-acetic acid and toxic reductants, wound healing and cell wall biosynthesis (Hammerschmidt et al, 1982). Further, peroxidase represents an important component of an early response in plants to pathogen attack and plays a key role in the biosynthesis of lignin, which limits the extent of pathogen spread (Bruce and West, 1989). The products of this enzyme in the presence of a hydrogen donor and hydrogen peroxide have antimicrobial activity and even antiviral activity (Van Loon and Callow, 1983). Recently, a novel heme peroxidase intrinsically resistant to H2O2 was isolated from R. sativus (Japanese daikon), which showed relatively stronger oxidative stability than that of reference horse radish peroxidase (HRPA2) (Rodríguez et al, 2008).
Biological activities of R. sativus
Evidence from numerous investigations reveals that the biological and pharmacological functions of R. sativus are mainly due to its GLs and its breakdown products - ITCs (Esaki and Onozaki, 1982; Nakamura et al 2001; Barillari et al, 2006; Papi et al, 2008). These compounds provide to R. sativus its characteristic odor and flavor as well as most of their biological properties. GLs and/or ITCs have long been known for their fungicidal, bacteriocidal, nematocidal and allelopathic properties (Brown et al, 1991) and have attracted intense research interest because of their cancer chemoprotective attributes (Fahey et al, 2001; Verhoeven et al, 1997). Polyphenolics, alkaloids, saponins, isoperoxidases and antifungal peptides are also accountable for significant part of the health benefits of R. sativus. These constituents are reported to exhibit several biological effects, including radical scavenging activity (Takaya et al, 2003), gut stimulatory, uterotonic and spasmogenic effects (Gilani and Ghayur, 2004; Ghayur and Gilani, 2005), anti-hyperlipidemic activity (Wang et al, 2002) and anti-atherogenic effects (Suh et al, 2006) and would perhaps work synergistically with GLSs and ITCs of R. sativus.
Damage to proteins, lipids and DNA by reactive oxygen species (ROS) and reactive nitrogen species (RNS) can lead to a variety of chronic diseases such as cancer, cardiovascular, inflammatory and age-related neurodegenerative diseases (Borek, 1997; Richardson, 1993). ROS/RNS can damage cell membranes, disrupt enzymes, reduce immunity (Ahsan et al, 2003) and induce mutations (Loft and Poulsen, 1996). ROS/RNS are by-products of normal aerobic metabolism and could occur during mitochondrial/microsomal electron transport chain, phagocytic activity or generated from oxidase enzymes and transition metal ions (Nohl et al, 2003; Aruoma et al, 1989). Other sources of ROS/RNS are environmental factors such as pollution, sun damage, cigarette smoke or even some kinds of the foods (Schroder and Krutmann, 2004). These reactive species and the resulting oxidative damages are usually counteracted by the antioxidant defense mechanisms (Bagchi and Puri, 1998). Recent studies evidence that plant-based diets, particularly those rich in vegetables and fruits, provide a considerable amount of antioxidant phytochemicals such as vitamins C and E, glutathione, polyphenolics, sulfur containing compounds and pigments, which offer protection against cellular damage (Dimitrios, 2006).
Ascorbic acid is found to be the most effective antioxidant in inhibiting lipid peroxidation initiated by a peroxyl radical initiator among several types of antioxidants including a-tocopherol (Fei et al, 1989). Ascorbic acid is also capable of scavenging hydrogen peroxide, singlet oxygen, superoxide and hydroxyl radicals efficiently (Fei et al, 1989). It is also involved in the regeneration and recycling of tocopherols and ß-carotene (Niki et al, 1995). Numerous studies have shown that ascorbic acid is effective in lowering the risk of developing cancers (Block, 1991) and cardiovascular diseases (Trout, 1991). In spite of the overwhelming evidence on the health benefits, however, there are reports that demonstrated the pro-oxidant activity of ascorbic acid (Podmore, 1998). Tocopherols are essential vitamins with their major role as antioxidants in protecting polyunsaturated fatty acids (PUFAs) and other components of cell membranes and low-density lipoprotein (LDL) from oxidation, thereby preventing the onset of heart diseases (Rimm et al, 1993).
Polyphenolics is an extremely comprehensive phrase that covers many different subgroups of phenols and phenolic acids. These compounds are most commonly present in fruits and vegetables. They are essential to the physiology of plants, being involved in diverse functions such as lignification, pigmentation, pollination, allelopathy, pathogen/predator resistance and growth (Haslam, 1996). Polyphenolics include single-ring structure such as hydroxybenzoic acids and hydroxycinnamic acids and multi-ring structure such as flavonoids, which can be further classified into anthocyanins, flavan-3-ols, flavones, flavanones and flavonols. Some of the flavonoids such as flavan-3-ols can be found in their dimeric, trimeric and polymeric forms. Most of the polyphenolics are often associated or conjugated with sugar moieties that further complicate the polyphenolic profile of vegetables. Polyphenolics are especially important as antioxidants, because they have high redox potentials, which permit them to act as reducing agents, hydrogen donors, singlet oxygen quenchers and metal chelator (Kahkonen et al, 1999) and alleviate free radical mediated cellular injury (Shahidi and Wanasundara, 1992).
The antioxidant ability of individual polyphenolics may differ, but, as a group, they are one of the strongest groups of antioxidants. The antioxidant activity of a polyphenolic compound is chiefly determined by its structure, in particular the electron delocalization over an aromatic nucleus (Tsao and Akhtar, 2005). When these compounds react with a free radical, delocalization of the gained electron over the phenolic antioxidant and the stabilization of the aromatic nucleus by the resonance effect take place that prevent the continuation of the free radical-mediated chain reaction (Tsao and Akhtar, 2005).
GLs are a group of sulfur-containing compounds found in the cruciferous plants such as R. sativus, broccoli, cabbage, mustard, wasabi etc. These compounds are found to be strong antioxidants, which are indeed through activation of detoxification enzyme mechanisms for the efficient removal of xenobiotics, rather than through direct radical scavenging capability (Zhang and Talalay, 1998). This property of GLs and its hydrolysis products - ITCs is considered as one of the major contributors to its anti-cancer activity (Zhang and Talalay, 1998).
Antioxidant activity of R. sativus
R. sativus is one of the major sources of dietary phenolic acids and flavonoids, which are mostly present as sugar conjugates (Takaya et al, 2003). The major phenolic acids found in R. sativus sprout are sinapic acid and ferulic acid, which are present in conjugated form as 1-sinapoyl-1-ß-D-glucopyranoside, ß-D-(3-sinapoyl) frucofuranosyl -a-D-(6-sinapoyl) glucopyranoside and 1-feruloyl-ß-D-glucopyranoside (Takaya et al, 2003). The major flavonoids present in R. sativus sprouts is kaempferol that occurs in a conjugated form as kaempferol-3,7-O- a-L-dirhamnopyranoside and kaempferol-3-O- a-L-rhamnopyranosyl-(1-4)- ß-D-glucopyranoside (Takaya et al, 2003).
Lugasi et al (1998) demonstrated the strong antioxidant property of squeezed juice extracted from a black R. sativus root through its ability to donate electrons, chelate metal ions and scavenge free radicals in a H2O2/·OH-luminol system. Since HPLC analysis revealed the presence of a considerable amount of GLs degradation products and polyphenols in the squeezed juice of black R. sativus, antioxidant activity of black R. sativus root could be attributed to these compounds.
Takaya et al (2003) tested methanolic extracts from 11 different plants including Daikon R. sativus sprouts for their ability to scavenge free radicals. Daikon R. sativus sprouts proved to be the most potent, almost 1.8 times more effective than Vitamin C.
Souri et al (2004) studied the antioxidant activity of 26 commonly used vegetables in Iranian diet and found that methanolic extract of R. sativus leaf significantly inhibited the peroxidation of linoleic acid as compared to standard antioxidant such as a-tocopherol and quercetin.
Katsuzaki et al (2004) found that hot water extract of Daikon R. sativus extract showed more significant antioxidant activity than the extract obtained at an ambient temperature. L-tryptophan was isolated and identified as the compound responsible for the antioxidant activity. They also found that L-tryptophan changed to 5-hydroxy tryptophan (5-HTP), a precursor to serotonin in the rat liver microsome model system. A plant-based 5-HTP supplement is popular for its anti-depressant, appetite suppressant and sleep aiding properties.
Lugasi et al (2005) further demonstrated that squeezed juice from black R. sativus significantly alleviated the free radical reaction in rats with hyperlipidaemia by decreasing the lipid peroxidation reactions and by improving the antioxidant status.
Recent study also showed that R. sativus extract reduced the extent of lipid peroxidation in a dose dependent manner in rat liver homogenate treated with cumene hydroperoxide by increasing the levels of reduced glutathione and thereby protecting the liver from the toxin induced oxidative damages (Chaturvedi, 2008).
Salah-Abbes et al (2008a) showed the protective effect of Tunisian R. sativus root extract against toxicity induced by zearalenone in mice by virtue of its ability to alleviate oxidative stress through stimulation and improvement of the antioxidant status.
Polyphenolics in R. sativus may act in a synergistic or additive manner with GLs and/or ITCs and exert their antioxidant activity through inhibition of lipid peroxidation, enhancing the cellular antioxidant enzymes and increasing the glutathione in the cells. Apart from these phytochemicals, R. sativus also contain several classes of peroxidases that could play a significant role in the elimination of toxic peroxides and thus reduce the impact of free radical mediated cellular injury (Wang et al, 2002).
Infectious diseases are the world's leading cause of untimely death, killing approximately 50,000 people every year. Bacteria have a remarkable ability to develop resistance to most pharmaceutical antibiotics. An increase in such antibiotic-resistant bacteria are menacing the human population with a recurrence of infectious diseases that were once thought to be under control, at least in developed countries (Pinner et al, 1996). These antibiotic-resistant bacteria have also caused unique problems in treating infections in patients with cancer and AIDS (Dennesen et al, 1998). Since tenacious and virulent bacteria develop immunity to solitary antibiotics at an alarming speed, there is an imperative need for a holistic targeted approach to search for novel antimicrobials from natural sources, especially from plant kingdom.
Long before mankind ascertained the existence of microbes, the fact that certain plants had therapeutic potential was very well accepted.
Since ancient times, man has used plants as the widespread remedial tool to treat common infectious diseases. Some of these traditional medicines are still included as part of the habitual treatment of various maladies. Bearberry (Arctostaphylos uva-ursi) and cranberry juice (Vaccinium macrocarpon) are employed to treat urinary tract infections, while species such as lemon balm (Melissa officinalis), garlic (Allium sativum) and tee tree (Melaleuca alternifolia) are described as broad-spectrum antimicrobial agents (Heinrich et al, 2004).
Plant based antimicrobials represent a vast unexploited source for medicines, which need to be explored further. They have an immense therapeutic potential as they are effectual in the treatment of infectious diseases while concomitantly alleviating many of the side effects that are frequently connected with synthetic antimicrobials (Cowan, 1999). Plant based anti-infective agents generally have manifold effects on the body and often act beyond the symptomatic treatment of the infectious diseases. Plants have a virtually unlimited capacity to produce secondary metabolites, especially for their defense against predation by microorganisms, insects and herbivores. Many of these secondary metabolites give plants their characteristic odors and also responsible for plant pigments. Antimicrobial phytochemicals are divided into several categories based on their structural similarity as follows:
These are the simplest bioactive phytochemicals consisting of a single substituted phenolic ring. Cinnamic acid and caffeic acids are the common representatives of this group. Phenolic acids are reported to be effective against viruses (Wild, 1994), bacteria (Brantner et al, 1996) and fungi (Duke, 1985). The number and site of the hydroxyl group on the phenol structure are considered to be related to their relative toxicity to microorganisms. Phenolic acids which are in the higher oxidized state are often more inhibitory towards microorganisms than the one with the lower oxidation state (Scalbert, 1991). Thus the mechanisms thought to be responsible for the antimicrobial activity of phenolic acid could include enzyme inhibition by the oxidized compound through interaction with - SH groups or through nonspecific interaction with the microbial proteins (Mason and Wasserman, 1987).
They are aromatic compounds with two ketone substitutions in the phenolic ring. They are ubiquitous in nature and show general antimicrobial properties (Duke, 1997). They are extremely active as they can switch between hydroquinone and quinone through oxidation/reduction reactions. Quinones bind with proteins irreversibly, leading to inactivation of proteins and loss of function (Stern et al, 1996). They may also make substrates unavailable to the microbes.
They are phenolic structures containing hydroxyl groups. They are ubiquitous and are commonly found in fruits, vegetables, nuts, tea, wine, honey, etc. They are known to be effective antimicrobial compounds against a wide variety of microorganisms (Cushnie and Lamb, 2005). Catechins are the most extensively researched flavonoids for their possible antimicrobial activity due to their occurrence in green tea (Toda et al, 1989). Flavonoids have the ability to complex with extracellular proteins as well as with bacterial cell walls, rendering them inactive (Cushnie and Lamb, 2005). More lipophilic flavonoids may also have the ability to disrupt microbial membrane (Tsuchiya et al, 1996).
Terpenoids and essential oils
Essential oils are secondary metabolites that are highly supplemented in compounds based on an isoprene structure (Cowan, 1999). They are called as terpenes and usually occur as di, tri, tetra, hemi and sesquiterpenes. When the compounds contain extra elements such as oxygen, they are called as terpenoids. Camphor, farnesol, artemisin and capsaicin are the common examples of terpenoids. Terpenes and terpenoids are active against an array of bacteria (Habtemariam et al, 1993) and fungi (Rana et al, 1997). Previous research showed that terpenoids present in the essential oils of plants could be useful in the control of Listeria monocytogenes (Aureli et al, 1992). The mechanism action of terpenes is not yet established precisely, but is speculated to be due to the disruption of bacterial cell membrane by the lipophilic terpenoids (Mendoza et al, 1997).
Alkaloids constitute large groups of compounds containing a nitrogen atom in a heterocyclic ring, with a broad range of biological activities. The first medically functional alkaloid was morphine isolated from Papaver somniferum (Fessenden and Fessenden, 1982). Alkaloids are generally found to have potent antimicrobial activity (Ghoshal et al, 1996). Solamargine, a glycoalkaloid from the berries of Solanum khasianum reported to be useful against HIV infection and intestinal infections associated with AIDS (McMahon et al, 1995). Berberine is an important and frequently studied member of the alkaloid group. It is potentially efficient against trypanosomes (Freiburghaus et al, 1996) and plasmodial infections (Wright et al, 1992). The mode of action responsible for the antimicrobial activity of alkaloids may be attributed to their ability to intercalate with DNA and arresting the metabolic activity of the bacterial cells (Phillipson and O'Neill, 1987).
Sulfur-containing compounds encompass a wide array of compounds and usually found in the plants as glucosides (glucosinolates, alliin, etc). These glucosides, during the rupturing of the plant cell wall, are hydrolyzed into volatile sulfur compounds such as ITCs, allicin, allyl sulfide, diallyl disulfate, etc. Biological activity of sulfur-containing compounds is considered to be chiefly due to glucoside degradation products, as intact glucosides usually display much fewer biological activities than their subsequent hydrolysis products (Donkin et al, 1995).
The mechanism of action responsible for the antimicrobial activity of sulfur-containing compounds varies. Antimicrobial activity of ITCs, degradation products of GLs, is thought to be related to its NCS group, in which the central carbon atom is highly electrophilic, which could interact irreversibly with the nucleophilic targets of a microbial cell wall. Allicin, degradation product of alliin, does not initiate leakage of cellular content, fusion or aggregation of membrane, but can permeate into cells through phospholipid bilayer and interact with the -SH groups in enzymes and proteins thereby modifying their activities, which is regarded as the mechanisms responsible for the antimicrobial activity of allicin (Rabinkov et al, 1998).
Antimicrobial activity of cruciferous vegetables
6-methylsulfinyl hexyl ITC, a volatile fraction extracted from Wasabi japonica is another ITC with considerable antibacterial activity, especially against Escherichia coli and Staphylococcus aureus (Ono et al, 1998). Similarly, sulphoraphane, an ITC from broccoli displayed a significant bactericidal effect against intracellular Helicobacter pylori in a human epithelial cell line (Haristoy et al, 2005).
Recently shin et al (2004) studied the bactericidal activity of Korean and Japanese wasabi roots, stems and leaves against Helicobacter pylori and found that the leaves exhibited more potent bactericidal activity than the roots, even though AITC level of the leaves was lesser than that of the roots. These results suggest that certain components other than AITC in wasabi are effective in inhibiting the growth of Helicobacter pylori.
Antimicrobial activity of R. sativus
The health benefits of R. sativus have been promoted for centuries, but few studies have been conducted to prove its medicinal and pharmaceutical value. There have been very few studies of the antibacterial activity of R. sativus. Abdou et al (1972) described the antibacterial activity of an aqueous extract of R. sativus root against Escherichia coli, Pseudomonas pyocyaneus, Salmonella typhi and Bacillus subtilis. However, R. sativus when extracted with isopropyl alcohol and ethanol inhibited only E. coli and showed no inhibitory activity towards the remaining bacteria. Similarly, when extracted with ether, petroleum ether and chloroform, R. sativus failed to reduce the growth of any of the bacteria studied.
Esaki and Onozaki (1982) identified the pungent principle of R. sativus root (MTBITC) as antimicrobial to Escherichia coli, Staphylococcus aureus, Saccharomyces cerevisiae, and Aspergillus oryzae. Further research revealed that 2-thioxo-3-pyrrolidinecarbaldehyde, produced by the degradation of MTBITC could be the actual component responsible for the antimicrobial activity of R. sativus. Khan et al (1985) reported the antibacterial activity of the roots, flowers and pods against bacteria such as Staphylococcus aureus and Bacillus subtilis.
Recently, Rani et al (2008) demonstrated the antimicrobial activity of crude water extract, supernatant and methanolic extract of R. sativus seeds against a variety of bacteria and fungi. All the extracts exhibited significant antibacterial activity against Hafnia alvei, Enterobacter agglomerans, Lactobacillus and Bacillus thuringiensis, whereas fungal species such as Penicillium lilacinum, Paecilomyces variotii, Spadicoides stoveri, Penicillium funiculosum displayed variable degrees of inhibition.
Chemoprevention of cancer
Cancer, presently the second leading cause of death may outrank cardiovascular diseases both in the developed and developing countries in a few decades (Oliveria et al, 1997). Cancer is a dynamic process that entails many intricate factors (Nowell, 1986). Hanahan and Weinberg, (2000) implied that cancer is a sign of significant alterations in cellular physiology, which result in unrepressed malignant growth. Carcinogenesis can be regarded as a gradual accumulation of genetic and biochemical changes occur through three stages (initiation, promotion and progression) that represent the progressive transformation of normal cells into highly aggressive malignant cells (Pitot and Dragan, 1994). Initiation is defined as a mutagenic event resulting from exposure and interaction of carcinogens with cellular constituents, especially DNA. Promotion is characterized by persistence and replication of a clone of abnormal cells that ultimately grow into a definable focus of preneoplastic cells. Progression is considered as a final stage of cancer development that alters the preneoplastic cells into an invasive and metastatic cell population.
The recent development in understanding the carcinogenic process at the cellular and molecular level has enhanced the probability that cancer prevention, either primary or secondary will rely increasingly on interventions collectively termed "chemoprevention". Chemoprevention is described as a pharmacological approach intends to arrest or reverse the development and progression of precancerous cells through the use of nutrients and/or pharmacological agents during the stage between tumor initiation and progression (Kelloff et al, 1994). Since carcinogenesis is a multistage process often having a latency of many years or decades, there are a considerable opportunities for intervention with innovative approaches and potential to interrupt this process at different steps during the initiation, promotion or progression (Kelloff et al, 1996; Greenwald, 2001). Molecular advances have led to the detection of genetic lesions and cellular components that may be involved in the initiation and progression of malignancies and thus constitute probable targets for chemoprevention. The emerging field of cancer prevention by chemopreventive agents offers substantial potential for reducing the incidence and mortality of cancer. Because of its promising effects, chemoprevention has been largely identified as a powerful treatment strategy by clinicians. Chemopreventive agents may exert their effect either by blocking or metabolizing carcinogens or by inhibiting the growth of cancerous cells. The most significant beneficial effects of chemopreventive agents are their non-toxic nature and negligible chemoresistance. Further, they display selective activity by initiating apoptotic pathways to impede cancer in abnormal cancer cells and at the same time induce detoxifying enzymes, which rendering them non-toxic to normal cells.
Phytochemicals as Chemopreventive agents
The majority of human cancers are induced by environmental aspects such as biological, chemical and radioactive factors existing in the milieu. It has been projected that more than two-thirds of human cancers could be averted by lifestyle modifications including dietary changes (Surh, 2003). Epidemiological studies have strongly indicated that certain daily-consumed dietary phytochemicals could have cancer protective effects against several forms of human cancers (Block et al, 1992). Because of the likely protection following long-term administration to humans, diet has been regarded as a potential source of chemopreventive agents. Indeed, a number of natural compounds with inhibitory effects on cancer formation have been identified from diet or source of diet.
Numerous in vitro and in vivo studies evaluated the chemopreventive effects of phytochemicals and elucidated their mode of cancer prevention. These studies have eventually resulted in the discovery of several classes of phytochemicals that possess cancer preventive effects such as ITCs from cruciferous vegetables, catechins from green tea, resveratrol from grape seeds and red wine, curcuminoids from turmeric, procyanidins from fruits and nuts, isoflavones from soyabean and antioxidant vitamins in various foods (Surh, 2003; Greenwald, 1996; Block, 1991). Multiple cellular and molecular mechanisms appear to account for the overall chemopreventive effects of these dietary phytochemicals. These include induction of the cellular defense system such as activation of detoxifying and antioxidant enzymes; inhibition of cell proliferation and cell cycle progression; induction of differentiation and apoptosis; modulation of expression of genes related to apoptotic pathways and inhibition of angiogenesis and metastasis by modulating cellular signaling pathways (Surh, 1999; Chen and Kong, 2005). These signal transduction pathways are now recognized as the potential molecular targets for chemoprevention by dietary phytochemicals (Hu and Kong, 2004).
Chemopreventive efficacy of cruciferous vegetables
Vegetables of the Brassicaceae family, particularly those belonging to Brassica genus (broccoli, cabbage, cauliflower, radish, mustard, etc) have received much attention because they are reported to possess significant anticancer activity (Zhang and Talalay, 1994; Verhoeven et al, 1997). GLs and their hydrolysis products (ITCs) are reported to be responsible for the chemopreventive activity of cruciferous vegetables. A modulation of detoxification enzymes (inhibition of phase I enzymes and induction of phase II enzymes) has been proposed as the probable mechanism pertinent to the chemopreventive properties of ITCs (Thornalley, 2002; Zhang, 2004). These compounds have also been shown to possess anti-proliferative and apoptosis inducing activities in several cancer cell lines in vitro (Wu et al, 2005). Apart from GLS and ITCs, cruciferous vegetables contain other potentially protective constituents such as phenolic acid, flavonoids, alkaloids, vitamins, fibers and pigments whose chemopreventive effects have been reported by numerous investigations (Yang et al, 1997; Bhatt and Pezzuto, 2002; Ferguson and Harris, 1996; Albanes et al, 1995).
Earlier studies demonstrated the inhibitory effects of naturally occurring ITCs on chemically induced carcinogenesis in a variety of experimental animal models (Verhoeven et al, 1997; Hecht, 1999; Conaway et al, 2002). In line with these findings, several case-controlled epidemiological studies observed an inverse relationship between high intake of cruciferous vegetables and risks of many types of cancer (Lin et al, 1998; London et al, 2000; Spitz et al, 2000; Zhao et al, 2001; Kristal and Lampe, 2002).
Sulforaphane (SUL) is one of the most studied ITCs of cruciferous vegetable. Several lines of evidence, both in vitro and in vivo, demonstrated the chemopreventive potential of sulforaphane (Zhang et al, 1992b; Zhang et al, 1994; Chung et al, 2000; Wu et al, 2005; Agudo et al, 2004; Singh AV et al, 2004; Singh SV et al, 2004; Choi and Singh, 2005). Besides sulforaphane, phenethyl isothiocyanate (PEITC), benzyl isothiocyanate (BITC), 4-(methylthio) butyl isothiocyanate (MTBuITC), 4-(methylthio)-3-butenyl isothiocyanate (MTBITC) and indole-3-carbinol are the other classes of ITCs showing promising chemopreventive activity in both in vitro and in vivo studies.
Molecular and cellular targets of ITCs for chemoprevention
It has been ascertained that ITCs can inhibit tumor formation through multiple mechanisms, including: (i) shielding DNA from oxidative damage by modulating detoxifying enzymes; (ii) alleviating oxidative stress by inducing and maintaining cellular antioxidants; (iii) suppressing cell proliferation by blocking cell cycle progression and/or initiating apoptotic pathway, thus impeding or eliminating clonal expression of initiated, transformed and/or neoplastic cells. Further, initiation of cellular differentiation and anti-inflammatory/anti-infective property could also contribute to the overall chemopreventive effects of ITCs. Besides, ITCs have the ability to modulate several cellular and molecular targets implicated in cancer development.
Modulation of phase I and phase II detoxification enzymes by ITCs
Most of the dietary and environmental carcinogens metabolize through an oxidative pathway called Phase I metabolism, which converts the procarcinogens into highly reactive intermediates. This physiological event is catalyzed by an enzyme called cytochrome P450 (CYP). CYPs occur in many isoforms and play significant roles in the activation of carcinogens to electrophiles which bind to DNA and generate DNA adduct. The DNA adduct formation is considered to be a crucial step for cancer initiation by carcinogens. Inhibition of CYPs involved in the generation of DNA adducts often results in the suppression of cancer formation.
Numerous evidence shows that ITCs may inhibit DNA adduct formation and chemical tumorigenesis through modification of the level of certain CYP isoforms in rodent models by way of competitive or mechanism based irreversible inhibition (Yang et al, 1994; Zhang et al, 1994; Nakajima et al, 2001) or through downregulation of CYPs in target tissues (Hecht, 2000; Conaway et al, 2002). However, substantial variations occur in the inhibition of different CYPs by ITCs.
Nakajima et al (2001) showed that PEITC selectively inhibited and inactivated human CYPs expressed from baculovirus-infected insect cells. PEITC strongly inhibited CYP 1A2 and to a lesser extent, CYP 2A6 by competitive inhibition, while CYP 2B6 and CYP 2C9 was inhibited by PEITC through noncompetitive inhibition and CYP 2E1 via a mechanism based irreversible inhibition (Nakajima et al, 2001). Unlike PEITC, BITC inhibited and inactivated CYPs both from rat and human mostly by a mechanism based irreversible inhibition, which occurs chiefly through protein modification (Goosen et al, 2000). However, the efficiency of inactivating CYPs by SUL and AITC appeared to be less prominent when compared with the ability of PEITC and BITC in inhibiting specific CYPs (Conaway et al, 1996).
On the other hand, inhibitory effect of ITCs on the activation of carcinogen may be carcinogen-specific. BITC was shown to inhibit 7, 12 - dimethylbenz(a)anthracene (DMBA) induced mammary tumors in rat (Wattenberg, 1977) and benzo[a]pyrene (BaP) induced pulmonary neoplasia in A/J mice (Wattenberg, 1987). However, it showed no effect on mouse lung carcinoma initiated by the tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) or by N-nitrosodiethylamine (DEN) (Morse et al, 1990), as well as on esophageal tumor induced by N-nitrosomethylbenzylamine (NMBA) in rats (Wilkinson et al, 1995).
On the contrary, PEITC encompasses a broad inhibitory activity against neoplasia induced by N-nitrosamines and has been shown by multiple studies both in mice and rats (Morse et al, 1989; Hecht et al, 1996; Hecht et al, 1999). But, PEITC showed an insignificant inhibitory effect on BaP-induced lung DNA adduct formation and lung carcinogenesis in A/J mice (Adam-Rodwell et al, 1993).
SUL significantly inhibited mammary tumor induced by DMBA in rats, but was ineffective as an inhibitor of BaP-induced lung tumor in mice (Zhang et al, 1994). Similarly, SUL had no effect on NNK-induced lung neoplasia in mice (Chung et al, 1997).
In contrast to the diverse result on phase I enzymes, many ITCs are strong inducers of phase II enzymes. These enzymes represent an essential part of cellular defense against reactive carcinogens or oxidants through elimination of highly reactive intermediate as water soluble products in the bile or urine. Several studies identified ITCs as major inducers of phase II carcinogen-detoxifying enzymes including quinone reductase-1 (QR-1) (Brooks et al, 2001; Kirlin et al, 1999), glutathione-s-transferase (GST) (Brooks et al, 2001), UDP-glucuronosyl transferase (UGT) (Petri et al, 2003) and ? -glutamylcysteine synthetase (GCS) (Bonnesen et al, 2001; Sharf et al, 2003).
Induction of carcinogen-detoxifying enzymes by ITCs appears to occur at the transcriptional level and may be mediated through the antioxidant responsive element (ARE). Nuclear factor E2-related factor 2 (Nrf2), a member of the Cap 'n' Collar (CNC) family of leucine zipper (bZIP) transcriptional factors binds to the ARE and mediates the transcriptional activation of an array of detoxification and antioxidant enzymes (Itoh et al, 1997).
A number of upstream signaling pathways leading to the transcriptional activation of Nrf2 and ARE are illustrated in Figure 2.3. The precise mechanisms through which ITCs elicit the Nrf2 transactivation is rather unclear. Nrf2 is normally sequestered in the cytoplasm as an inactive complex by Keap1 protein, which is anchored to the cytoskeleton proteins (Itoh et al, 1999). Upon stimulation by ARE-mediated inducers including ITCs, Nrf2 is dissociated from Keap1 protein, apparently through the cleavage of disulfide bond between Nrf2 and Keap1 (Dinkova-Kostova et al, 2002). Nrf2 then translocates to the nucleus and binds to the ARE through a heterodimeric combination with
Model of the signaling pathways involved in activation of Nrf2 and ARE. Nrf2 is anchored to Keap1 in the cytosol. Upon activation of upstream proteins kinases (MAPKs, PI3K, PKC and PERK), and/or direct effect on Keap1, Nrf2 is released from Keap1 and translocates into the nucleus, where Nrf2 binds to the ARE with association of small Maf inside the nucleus. Isothiocyanates may also directly cause the cleavage of disulfide bond between Nrf2 and Keap1 (Keum et al, 2004).
other factors such as Maf proteins and activates expression of detoxifying enzymes.
ITCs may also regulate Nrf2-mediated gene transcription by different mechanisms. There is also a report indicating that ITCs possess pro-oxidant properties and could generate oxidative stress by themselves in the cells (Zhang, 2004). However, the amount of oxidative stress produced by ITCs seem to be dose-dependent, mild at low concentrations with signal strength ample enough to trigger the cellular defense system leading to the synchronized activation of Nrf2 signaling pathway (Nakamura et al, 2000). This oxidative stress generated by low dose of ITCs is supposed to be at a sub-toxic level that would not cause any adverse effects such as DNA damage, mutation or tissue degeneration as usually caused by carcinogens.
Inhibition of cell proliferation by ITCs
Till the date, most of the studies have been focused on the ability of ITCs to block chemical carcinogenesis through inhibition of carcinogen-activating enzymes and induction of carcinogen-detoxifying enzymes. ITCs may also be important as cancer therapeutic agents. Recent studies have shown that ITCs have the ability to induce growth inhibition rapidly and are also known to induce both cell cycle arrest and apoptosis in a number of human cancer cell lines (Gamet-Payrastre et al, 2000; Xu and Thornalley, 2000, Chiao et al, 2002; Fimognari et al, 2002). Studies have also demonstrated that ITCs appear to be selective in inhibiting the cell growth and found to be more toxic to malignant cells rather than to normal cells (Gamet-Payrastre et al, 2000; Xiao et al, 2003).
Induction of cell cycle arrest by ITCs
Cell cycle depends on DNA replication (S phase) and segregation of chromosomes to the daughter cells (M phase), which are spaced by intervals of growth and reorganization (G1 and G2 phases). The systematic progression of the eukaryotic cell cycle is controlled by a series of proteins called cyclins, which exert their effects by binding to and activating a series of specific cyclin-dependent kinases (CDKs). This process is further modulated by inhibitory proteins such as p21/WAF-1, p16/INK41 and p27/Kip-1, collectively termed as CDK inhibitors (CDIs) (Weinstein, 2000).
Cell cycle arrest occurs in response to cellular stress such as DNA damage through activation of cellular checkpoints such as G1/S or G2/M phase until errors in DNA are rectified. However, apoptosis (programmed cell death) is initiated, if the DNA damage is extensive and beyond any repair. Thus cell cycle arrest and apoptosis are considered as closely coupled protective cellular mechanisms. While tumor cells are regarded as a clone of transformed cells, induction of cell cycle arrest or apoptosis is considered as a potential chemopreventive approach (Keum et al, 2004).
In addition to its modulatory effects on carcinogen metabolism, induction of cell cycle arrest in several cancer cell lines was also identified as a potential mechanism underlying the chemopreventive activities of ITCs. Hasegawa et al (1993) first observed the significant accumulation of cells at G2/M phase after the treatment of HeLa cells with ITCs. Similar effects were consequently, seen in a wide variety of cells. However, molecular targets of ITCs vary and indeed molecular mechanisms precisely responsible for the cell cycle arrest by ITCs are still far from clear.
SUL caused significant accumulation of Jurkat cells (Fimognari et al, 2002) and HT29 cells (Gamet-Payrastre et al, 2000) at G2/M phase, which was associated with an increase in cyclin B1 level. However, the same SUL treatment of LNCaP cells arrested cells in G1 phase with a simultaneous decrease in cyclin D1 expression (Chiao et al, 2002). On the contrary, AITC-induced G2/M phase arrest in LNCaP cells was accompanied by a prominent decrease in cyclin B1 level, but the comparable effect was not noticed in PC-3 cells (Xiao et al, 2003). Similarly, AITC arrested HL60 cells in G1 phase, in contrast to G2/M phase in other cells, whereas BITC arrested the cells in both G1 and G2/M phases (Zhang et al, 2003). From these results, it can be implied that ITCs certainly have cell cycle-arresting activity, but ITCs may arrest different cells at different phases of the cell cycle which primarily depends on the type of ITC and cell lines. Furthermore, ITCs have the ability to induce cell cycle arrest as early as 3 h after the cell treatment (Zhang et al, 2003).
Induction of apoptosis by ITCs
Apoptosis (programmed cell death) is a highly regulated protective mechanism through which damaged or superfluous cells are eliminated from the system. Apoptosis is recognized to be vital for normal development, turnover and replacement of cells such as skin cells. Apoptosis can be initiated either at the cell surface (death receptor or extrinsic pathway) or from internal events within the cell (mitochondrial or intrinsic pathway). Both pathways lead to activation of caspases which are responsible for the execution of cell death by cleaving cellular substrates. Extrinsic pathway depends on ligand activated recruitment of adaptor proteins by the death receptor and consequent activation of caspase-8. The intrinsic pathway entails the release of proapoptotic molecules from mitochondria to the cytosol such as cytochrome c, which then activate caspase cascade. The chief regulators of this pathway are members of the Bcl-2 family proteins (Figure 2.4).
Additionally, apoptosis of individual cells serves as a defense mechanism against cancer development in an organism by removing genetically damaged or redundant cells that have been inappropriately stimulated to divide by a mitotic stimulus. In fact, apoptosis is deranged in cancer cells, which display reduced propensity towards apoptotic stimuli. Most of the chemopreventive agents have been shown to demonstrate their inhibitory effect through the induction of apoptosis.
Role of caspases in apoptosis
Caspases (cysteine-aspartic acid proteases) belong to cysteine proteases family and serve as the major effectors of apoptosis. The activation of caspases leads to distinctive morphological changes of the cell such as shrinkage, chromatin condensation, DNA fragmentation and plasma membrane blebbing (Degterev et al, 2003). Induction to commit suicide is needed for proper development of organisms, to get rid of cells that pose a threat to the organism (e.g. cell infected with virus or cancer cells), and to remove cells with damaged DNA. Cells undergoing apoptosis are eventually removed by phagocytosis.
There are two types of caspases: initiator (apical) caspases and effector (executioner) caspases, both of which are synthesized as inactive proenzymes. Initiator caspases are the first to be activated and include caspase - 2, 8, 9 and 10. These in turn cleave inactive forms of effector caspases (3, 6 and 7), thereby activating them. Effector caspases consecutively cleave, degrade or activate other cellular proteins within the cell triggering the apoptotic process (Boatright and Salvesen, 2003). The initiation of this cascade reaction is regulated by caspase inhibitors (Concha and Abdel-Meguid, 2002).
Caspase activation can be mediated by intrinsic factors such as Bcl-2 on the mitochondrial membrane. Bcl-2 is normally found associated with Apaf-1. Damage causes Bcl-2 to disassociate from Apaf-1 leading to the release of cytochrome c into the cytosol. New complex forms which is comprised of cytochrome c, Apaf-1, and caspase-9 (the apoptosome). Caspases-9 is cleaved and activates other caspases leading to an expanding cascade of proteolytic activity within the cell.
Schematic representation of the apoptotic pathways involving p53, Bcl-2 family and caspases.
This eventually results in the digestion of structural proteins in the cytoplasm, chromosomal DNA degradation and phagocytosis of the cell. External signals can also affect the caspase activation cascade. TNF and Fas receptors on the cell surface can be triggered upon ligand binding (TNF, Fas, certain toxins and chemicals) to cleave caspase-8 which then goes on to initiate increased proteolysis within the cell and its ultimate removal by phagocytosis (Denault and Salvesen, 2002).
Role of Bcl-2 family members in apoptosis
Of late, the Bcl-2 gene family has been recognized to play critical roles in the regulation of apoptosis (Cory and Adams, 2002). Study of the mechanism of apoptosis by Bcl-2-related genes presents new possibilities for prevention and treatment of several human diseases including cancer (Green, 1998; Reed, 1995). Some of the proteins within this family including Bcl-2 and Bcl-XL inhibit apoptosis and, whereas others such as Bad, Bax, Bik, Bid and Bak, promote apoptosis (Cory and Adams, 2002). They form homo-oligomers and hetero-oligomers, which act directly at the outer mitochondrial membrane. Indeed, the ratio between these two subsets determines, in part, the susceptibility of cells to apoptotic stimuli (Oltvai et al, 1993).
Role of p53 in apoptosis
p53 acts as a transcriptional activator by triggering the transcription of proteins involved in DNA repair (Lohrum and Vousden, 1999). If the damage is beyond repair, p53 activates the apoptotic pathway which is considered as a last resort to avoid proliferation of cells containing mutated DNA (Jin and Levine, 2001). Thus normal p53 function has been demonstrated to be crucial in the induction of apoptosis in human following DNA damage. This result was further supported by the findings that p53 is the most commonly mutated tumor suppressor gene and found in about 50 - 55% of all human cancers (Malkin, 2001). Lack of p53 function may contribute to the complex network of molecular events leading to tumor formation, as this may allow mutated cells to escape apoptosis. Further, loss of p53 may prompt preneoplastic cells to accumulate additional mutation by obstructing the normal apoptotic response to genotoxic damage (Bode and Dong, 2004).
In normal cells, the p53 protein is latent and highly unstable with a half-life measured in minutes. It is maintained at low levels by targeted degradation mediated by its negative regulator, Mdm2 (Alarcon-Vargas and Ronai, 2002). During DNA damage and/or other stress signals, the half-life increases significantly leading to accumulation of p53 and transcription of target genes such as p21WAF1/CIP1 and Bax (El-Diery, 1998). The outcome of this increased transcription depends on the type of cell but usually manifested as a very prolonged G1 arrest or apoptosis (El-Diery, 1998). There are several potential mediators of p53-induced apoptosis. The Bax is an apoptosis-inducing member of the Bcl-2 protein family, whose transcription is directly activated by p53-binding sites in the regulatory region of the gene (Thornborrow et al, 2002). Furthermore, p53 also participates in the initiation of apoptosis by acting directly at mitochondria. Localization of p53 to the mitochondria occurs in response to apoptotic signals and precedes cytochrome c release and procaspase-3 activation (Haupt et al, 2003).
Mechanism of apoptosis induction by ITCs
A large number of ITCs have been reported to induce apoptosis in a variety of cultured human and animal cell lines as well as animal tissues and cancer cell xenografts in vivo (Singh AV et al, 2004; Srivastava et al, 2003; Bonnesen et al, 2001; Zhang et al, 2003; Chiao et al, 2002; Gamet-Payrastre et al, 2000; Xiao et al, 2003; Xu et al, 2000; Huang et al, 1998; Fimognari et al, 2002; D'Agostini et al, 2001). Moreover, treatment of cells with ITCs leads to the activation of caspases involved in multiple apoptotic pathways. However, the precise intracellular signaling pathways initiated by ITCs leading to apoptosis are likely to be complex and are only partly understood. There are substantial differences among cell lines with respect to the potential targets for apoptosis that are modulated by various ITCs. Furthermore, most of the previous work has been focused on the mitochondrial pathway.
Chen et al (1998) reported that ITC-induced apoptosis in HeLa and HL60 cells was mediated by c-Jun NH2-terminal kinases (JNK), whose activation may result from oxidative stress induced by ITCs. Nevertheless, Rose et al (2003) showed that treatment of HepG2 cells with ROS scavengers did not impede PEITC-induced apoptosis. In fact, another study indicated that PEITC inhibits a phosphatase that inactivates JNK (Chen et al, 2002). Besides, JNK activation could also occur from inhibition of Bcl-2 as over-expression of Bcl-2 was found to suppress PEITC induced JNK activation in HeLa cells (Chen et al, 1998). Down-regulation of Bcl-2 was reported in various cancer cell lines treated with ITCs (Xiao et al, 2003; Fimognari et al, 2002; Singh AV et al, 2004; Srivastava et al, 2003).
ITCs may also induce apoptosis through modulation of other cellular targets. Increased expression of pro-apoptotic Bax was detected in prostate, colon and leukemia cells treated with SUL (Singh AV et al, 2004; Gamet-Payrastre et al, 2000; Fimognari et al, 2002), but not in AITC-treated prostate cancer cell lines (Srivastava et al, 2003; Xiao et al, 2003). Interestingly, increased expression of Bax, which is downstream of p53, was accompanied by an increase in p53 expression in Jurkat cells treated with SUL (Fimognari et al, 2002), but not in SUL-treated HT29 cells (Gamet-Payrastre et al, 2000). Likewise, anti-apoptotic Bcl-XL was significantly downregulated by AITC in LNCaP cells but not by AITC or SUL in PC-3 cells (Xiao et al, 2003; Singh AV et al, 2004). Even though, Huang et al (1998) reported that upregulation of p53 is needed for PEITC-induced apoptosis, AITC, PEITC and SUL could be able to induce apoptosis in p53 deficient PC-3 cell lines (Singh AV et al, 2004; Xiao et al, 2003; Xiao et al, 2002), suggesting that ITCs could initiate apoptosis through p53 independent pathway. These results were confirmed by a recent study done by Pappa et al (2006) that different ITCs have a distinct profile of cell growth inhibition, potential to induce p53 independent apoptosis and have the ability to modulate Bcl-2 family protein expression in the human colon cancer cell lines.
Chemopreventive efficacy of R. sativus
Recent studies substantiate the beneficial role of R. sativus in the prevention of human cancers. Papi et al (2008) reported that MTBITC extracted from Japanese R. sativus sprout showed promising anti-proliferative activity in a dose-dependent manner and induced apoptosis in the colon cancer cell lines (LoVo, HCT116 and HT29) through increased expression of Bax and caspases-9 and decreased expression of Bcl-2 protein along with the cleavage of PARP-1. Further, it was demonstrated that MTBITC displayed interesting antioxidant/radical scavenging property, associated with negligible toxicity on normal human lymphocytes.
Barillari et al (2008) employed standardized Kaiware Daikon extract (KDE) containing 10.5% glucoraphasatin and 3.8% glucoraphenin in combination with myrosinase as a natural chemopreventive agent against the colon cancer cell lines (LoVo, HCT116 and HT29) in comparison with pure MTBITC and sulforaphene. They found out that KDE significantly reduced the cell growth in a dose-dependent manner, surpassing the pure MTBITC and sulforaphane at the same dose, with no significant toxicity o
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