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Ascorbic Acid in Cancer Chemoprevention: Translational Perspectives and Efficacy Mohammad F. Ullah1,*, Showket H. Bhat1, Eram Hussain1, Faisel Abu-Duhier1, Aamir Ahmad2 and S.M. Hadi3 1

Prince Fahad Research Chair, Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, University of Tabuk, Tabuk-71491, KSA; 2Department of Pathology, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201, USA; 3Department of Biochemistry, Faculty of Life Sciences, AMU, Aligarh202002 (U.P.), India Abstract: Chemoprevention, which is referred to as the use of nontoxic natural or synthetic chemicals to intervene in multistage carcinogenesis has since decades attracted a considerable interest in plant-derived chemical constituents often termed as “phytochemicals” or sometimes as “Nutraceuticals” in case they are derived from dietary sources. A comprehensive search of the literature show that such an interest in natural product pharmacology has surged in the last 25 years and particularly risen at exponential rates since the last one decade. Phytochemicals such as curcumin (from spice turmeric), resveratrol (from red wine) and genistein (from soy) share the major efforts as indicated by overwhelming publications, despite skepticism concerning their bioavailability. Ascorbic acid (AA), the popular anti-oxidant in fruits and vegetables, has even a longer historical perspective than these dietary agents as for more than 35 years; there had been lingering questions about the efficacy of AA in cancer therapy. The footprints of AA from “scurvy” to “cancer” though complex seems to carry potential provided the puzzle could be set right. The use of AA in cancer treatment has been debated extensively as evident from the literature but surprisingly the complementing early phase bench work on the mechanistic studies for anticancer action was rather retarded. Proposed mechanisms of action for AA in the prevention and treatment of cancer includes antioxidant as well as pro-oxidant properties, stimulation of the immune system, altering carcinogen metabolism, enhancement of collagen synthesis necessary for tumor encapsulation and interference with cancer cell signaling. The observation that the intravenous administration of AA enhances its bioavailability to the extent of deriving pharmacological benefits against cancer has in recent years partially supported the clinical plausibility (efficacy) of AA towards realizing its translational advantage. Here, we provide an overview of AA with regard to its potential in the management of cancer disease.

Keywords: Ascorbic acid, chemoprevention, phytochemicals. 1. INTRODUCTION Cancer is responsible for approximately 13% of deaths worldwide [1] and remains a growing health problem around the world particularly with the steady rise in life expectancy. Cancer development is a dynamic, long-term and multistage process that involves many complex factors in its initiation, promotion, and progression. During this process, accumulation of genetic and epigenetic alterations leads to the progressive transformation of a normal cell into a localized tumor mass which later metastasize to near and distant tissues and organs. Cancer cells acquire immunity against physiologically imposed restrictions to growth and division by their ability to posses: (i) self-sufficiency in growth signals, (ii) insensitivity to anti-growth signals, (iii) evasion of programmed cell death (apoptosis), (iv) limitless replicative potential, (v) sustained angiogenesis, and (vi) tissue invasion and metastasis [2]. Cancer being a multifactorial disease, it is *Address correspondence to this author at the Prince Fahad Research Chair, Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, University of Tabuk, Tabuk-71491, KSA; Cell #: +966568958324; E-mail: [email protected] 1-5/12 $58.00+.00

suggested that diet and its constituents unlike drugs posses pleotropic action mechanism and thus has the advantage of simultaneously influencing multiple pathways that are altered in cancer. The concept that cancer can be prevented, or its onset postponed, by certain diet-derived substances, has in the recent years promoted considerable interest. It has been estimated that more than two-third of human cancers could be prevented through appropriate lifestyle modification including dietary habits. Epidemiologic and laboratory studies indicate that a high consumption of antioxidant-rich fruit and vegetables can reduce the risk of cancer [3, 4]. Ascorbic acid (AA) Fig. (1) is considered to be one of the most active anti-oxidative components of fruits and vegetables [5] and has since long been shown to exert chemopreventive effects against cancer [6, 7]. Unlike most other chemotherapeutic drugs, higher doses of intravenous/oral administration of AA are well tolerated and clinically safe in cancer patients [5]. Although partially discredited in the past due to few null clinical outcomes [8, 9], relatively recent studies [10] related to the bioavailability of pharmacologically active doses of ascorbic acid in physiological system

© 2012 Bentham Science Publishers

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has enhanced its clinical plausibility, thus warranting the molecule to be revisited for its chemopreventive efficacy.

Fig. (1). Chemical structure of ascorbic acid.

2. TRACES FROM SCURVY TO CANCER The pharmacological value of AA was virtually introduced in the mid- 18th century when Dr. James Lind (a Scottish army physician) first demonstrated that the juice of fresh citrus cures scurvy [11]. The molecule has thus the commendation of being associated with the first clinical trial in the history of medicine. However, it was in late 1920s that the AA was actually isolated and introduced by Albert SzentGyorgyi as an active principle of citrus juice responsible for its observed effects in the prevention and treatment of scurvy [12]. The pathology of scurvy has been described as a generalized structural breakdown of the intercellular matrix associated with undifferentiated cell proliferation [13]. In 1954, W.J. McCormick (a Canadian physician) proposed that cancer is a collagen disease implicated to an AA deficiency [14]. His observations that the generalized stromal changes of scurvy are identical with the local stromal changes observed in the immediate vicinity of invading neoplastic cells provided the first evidence to link cancer with AA [15]. It was believed that the nutrient known to be capable of preventing such generalized changes in scurvy presumably shall have similar effects in cancer. As reviewed by Cameron et al. [16], the host resistance to cancer has an important component where regulation of stromal activity leads to the encapsulation of neoplastic cells in a dense, impenetrable barrier of fibrous tissue. Such a collagenous barrier is scanty and illdefined in highly anaplastic invasive tumors, moderate in amount in tumors of moderate rapidity of growth and very abundant in slow growing tumors. It was also realized that in terminal human cancer (stage of invasiveness), the associated pre-mortal features of anemia, cachexia, extreme lassitude, hemorrhages, ulceration, susceptibility to infections, and abnormally low tissue, plasma, and leukocyte ascorbate levels, are virtually identical with the pre-mortal features of advanced human scurvy.

Ullah et al.

stomach cancer risk. [23]. Further epidemiological evidence of a dietary link to pancreatic cancer reported consistent inverse relationships between AA and the incidence of pancreatic cancer [24]. Another study examining the relationship of dietary and supplemental factors with esophageal cancer reported that the high index of AA intake was associated with decreased risks of esophageal cancer [25]. A systematic study of AA blood levels in patients with lung cancer and an evaluation of their modifications when the patients were orally treated with daily high doses of ascorbic acid (5g/day) have shown hypovitaminosis C subclinic conditions (to lower level of physiologic range) and administration of periodic haematic dosages of AA have shown a rapid increase of its blood concentration (over 1500 micrograms, the higher level of normal range). The study proposed that such high haematic levels of AA remain generally constant for some time and appear beneficial in increasing the defense reactions of the cancer patient [26]. A study associated with South Australian Central Cancer Registry examined the dietary habits and incidence of colorectal cancer and found AA to be protective [27]. Other studies have reported reduction of colonic polyp (precursor to colorectal cancer) with AA intake [28]. Several studies have established a trend of inverse relationship between cervical neoplasia as well as invasiveness and dietary AA [29-31]. Breast cancer patients when compared to a matched group of controls demonstrated significantly lower levels of ascorbate in plasma [32]. Consistent with this finding an association between dietary antioxidant vitamin intake and the risk of breast cancer in a prospective study in Iowa showed that women who reported consuming at least 500 mg AA daily had a non-significant relative risk of developing breast cancer compared with women who did not supplement with AA [33]. 4. SOURCES, BIOSYNTHESIS, METABOLISM AND BIOAVAILABILITY OF ASCORBIC ACID Sources AA is one of the most important water-soluble vitamins, naturally present in fruits and vegetables. Fruits rich in AA include orange, lemons, arbutus, avocado, grapefruit, watermelon, papaya, strawberries, cantaloupe, mango, pineapple, raspberries and cherries whereas common vegetable sources are green leafy vegetables, tomatoes, broccoli, green and red peppers, cauliflower and cabbage [5, 34]. The AA content of some dietary sources has been summarized in (Table 1) [35]. Biosynthesis

3. EPIDEMIOLOGICAL EVIDENCE: INVERSE RELATIONSHIP OF ASCORBIC ACID AND CANCER Epidemiological studies provide strong evidence of a protective effect of AA for cancers such as of esophagus, lung, pancreas, stomach, colorectal, breast and cervix [17, 18]. An inverse relation between plasma AA and cancer mortality has been reported [19]. One of the most consistent epidemiological findings on AA has been an association with high intake of AA or AA rich foods and reduced risk of stomach cancer [20-22]. A study analyzing the existing epidemiological data in literature showed that 9 of 10 casecontrol studies and 10 of 11 non-controlled studies yielded a significant inverse relationship between AA intake and

AA is synthesized by both plants as well as animals although it remains a dietary requirement for humans since they lack the last enzyme in the biosynthetic pathway (Lgulonolactone oxidase) and thus cannot synthesize it [36]. As Plant-derived ascorbate is the major source of AA in the human diet, we describe here the biosynthetic pathway of AA in plants, the Smirnoff-Wheeler pathway Fig. (2). The Dmannose/ L-galactose (Man/L-gal) pathway was proposed by Wheeler et al. in 1998 [37]. In a sequence of enzyme catalyzed reactions, L-galactose (L-Gal) is rapidly converted to ascorbate by L-galactose dehydrogenase (L-GalDH), which oxidizes C1 of L-Gal to L-galactono-1,4-lactone (L-GalL). All eight steps of the D-mannose/ L-galactose (Smirnoff–

Ascorbic Acid in Cancer Chemoprevention

Table 1.

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L-Ascorbic acid content of selected fruits and vegetables. reproduced from [35] by permission from john wiley and sons

Source

mg (100g)-1


molg-1 Fresh Weight

Acerola (west Indian cherry)

1300

73.00

Apple

2-10

0.11-0.56

Apricot

7-10

0.39-0.56

Avocado

15-20

0.84-1.12

Banana

10-30

0.56-1.68

Blackberry

15

0.84

Broccoli

113

6.35

Broccoli (cooked)

90

5.05

Brussels sprouts

87-109

4.94-6.12

Cabbage (raw)

46-47

2.64

Cauliflower

64-78

3.63-4.38

Cauliflower(cooked)

55

3.09

Carrot

6

0.34

Cranberry

12

0.67

Cherry

5-8

0.28-0.45

Blackcurrant

200-210

11.2-11.8

Redcurrant

40

2.25

Damson

3

0.17

Gooseberry

40

2.25

Gourd

8

0.45

Passion fruit

25

1.40

Grapefruit

40

1.18

Guava

230-300

13.1-16.8

Horseradish

120

6.74

Kale

186

1.01

Kale (cooked)

62

3.48

Kiwi

60

3.41

Lemon

50

2.84

Lettuce

15

0.85

Lime

25

1.40

Loganberry

30

1.68

Lychee

45

2.55

Melon

10-35

0.57-1.97

Orange

50

2.84

Orange (juice)

50

2.84

Tangerine

30

1.68

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(Table 1) contd….

Source

mg (100g)-1


molg-1 Fresh Weight

Peach

7-31

0.39-1.76

Peach (canned)

6

0.34

Pepper (green)

128

0.72

Plum

3

0.17

Pea

25

1.40

Pear

3-4

0.17-0.23

Pineapple

12-25

0.68-1.40

Pineapple (canned)

12

0.68

Pomegranate

6

0.34

Potato (new)

30

1.68

Potato (Oct, Nov)

20

1.14

Potato (Dec)

15

0.85

Potato (Jan, Feb)

10

0.57

Potato (Mar, May)

8

0.45

Potato (boiled)

16

0.90

Quince

15

0.84

Raspberry

25

1.40

Rosehip

1000

5.62

Spinach

51

2.86

Spinach (cooked)

28

1.57

Strawberry

59-60

3.37

Tomato

20-25

1.14-1.40

Tomato (juice)

16

0.90

Watercress

68-79

3.82-4.44

Wheeler) pathway, starting from the central metabolite fructose 6-P, have been confirmed by genetic analysis [38]. Metabolism & Bioavailability AA present in diet is readily available and easily absorbed by active transport in the intestine [39]. The major metabolites of AA in human are dehydroascorbic acid, 2,3diketogulonic acid, xylose, xylonate, lyxonate and oxalate [40]. In the context of bioavailability of ingested ascorbic acid in humans, the average daily intake level that is sufficient to meet the nutritional requirement of ascorbic acid or recommended dietary allowances (RDA) for adults (>19 yr) are 90 mg/day for men and 75 mg/day for women [41]. AA concentrations in plasma are tightly controlled as a function of dose [42]. The bioavailability of AA is complete for 200 mg as a single dose and decreases above 500 mg and higher, due to urinary excretion [43]. Plasma is completely saturated at doses of 400 mg daily and higher, producing a steady-state

plasma concentration of approximately 80 μM. AA concentration in relation to dose has been measured in circulating neutrophils, lymphocytes and monocytes. These cells contain 1–4 mM concentrations of AA and saturate at doses between 100 and 200 mg daily [43, 44]. The cellular uptake of AA requires its oxidation to dehydroascorbic acid (DHA) which is transported by facilitative diffusion via glucose transporters (GLUT) into the cell where DHA is reduced intracellularly to AA [45, 46]. Unlike DHA, the transport of ascorbic acid occurs by sodium-dependent transporters SVCT1 and SVCT 2 [47]. SVCT1 (distributed along transporting epithelial systems such as intestine, kidney and liver) and SVCT2 (widely expressed) mediates the intestinal and renal reabsorption of AA. Although AA transporters are widely distributed, several tissues utilize the transport of DHA by the GLUTs [48]. As mentioned above, DHA is then rapidly reduced on the internal side of the plasma membrane, which prevents its efflux. It needs a mention that plasma levels of AA are tightly controlled and are around 50 μM [49]. How-

Ascorbic Acid in Cancer Chemoprevention

ever, Padayatty et al. have shown that intravenous administration of AA bypasses such tight control and results in concentrations as much as 70 folds higher than those achieved by maximum oral consumption [10]. Thus high plasma and tissue concentrations of AA can be achieved by intravenous route of administration and thus could be bioavailable in concentrations relevant to exert pharmacological effects, a major determinant of its translational value.

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of interference, whether initiation, promotion or progression in multistage cancer disease. In regard to the initiation stage of carcinogenesis, properties of ascorbic acid related to its anti-oxidant chemistry and xenobiotic metabolism are believed to be important, whereas pro-oxidant action and induction of apoptosis are involved in anti-promotion activity. The anti-progression actions include suppression of growth factor signaling pathways, suppression of cancer cell growth, tumor mass encapsulation related properties and inhibition of angiogenesis and metastasis [18]. However, in the context of the conceptualization of AA as an anti-cancer drug, there is a lag in investigations related to mechanistic studies and therefore a balancing approach needs to be implemented whereby both the characterization of anti-cancer action mechanisms and well targeted controlled clinical trials are considered equally significant. 5.1. Neutralization of Cellular Oxidative Insults by Ascorbic Acid As An Efficient Electron Donor

Fig. (2). The mannose/L-galactose ascorbate biosynthesis pathway. Enzymes: 1, phosphomannose isomerase; 2, phosphomannose mutase; 3, GDP-mannose pyrophosphorylase; 4, GDP-mannose-3,5epimerase; 5, GDP-L-galactose phosphorylase/guanylytransferase; 6, L-galactose 1-P phosphatase ; 7, L-galactose dehydrogenase; 8, L-galactono-1, 4-lactone dehydrogenase. Reprinted by permission from Macmillan Publishers Ltd: Wheeler GL, Jones MA, Smirnoff N. Nature, 1998; 393: 365-9.

5. MECHANISTIC VIEWPOINT OF CHEMOPREVENTIVE ACTION OF ASCORBIC ACID AGAINST CANCER Several studies during the last few decades have suggested that AA exhibits anticancer activities. Multiple biochemical and molecular mechanisms seem to contribute to the observed effects of AA against cancer cells. Presumably the action mechanism by which it manifests its anticancer properties are of pleiotropic nature in which more than one mechanism seems to be implicated depending upon the stage

Evidence in literature indicates that a large proportion of cancers could be prevented through modifiable lifestyle related risk factors such as smoking, obesity, physical activity, and diet [50]. It has been noted that these lifestyle-related factors might relate to the process of carcinogenesis through oxidative stress that occurs as a result of damage induced by reactive oxygen and nitrogen species (RONS), which produce potentially mutagenic DNA damage and impair the ability of repair mechanisms due to oxidative inactivation of enzymes [51, 52]. Certain physiological and biochemical actions of AA are due to its action as an efficient electron donor, for example; ascorbate promotes hydroxylation reactions, in part by donating electrons to metal ion cofactors of enzymes hydroxylase and oxygenase as it contributes to balance the active center of metal ions in a reduced state for optimal activity of enzymes [53]. AA has strong anti-oxidant tendency as it donates its electrons and thereby prevents other molecular targets of ROS/RNS from being oxidized. When AA donates electrons, species formed after the loss of one electron is a free radical; ascorbyl radical which when compared to other free radicals is relatively stable with a half-life of 10-5 seconds and has a low reactivity due to resonance stabilization of the unpaired electron and readily dismutates to ascorbate and dehydroascorbic acid (DHA) [54]. Ascorbyl radical with the loss of another electron is further converted to dehydroascorbic acid which is either reverted to AA by reduction or further metabolized [55]. AA has the advantage of an ideal antioxidant as both ascorbate and the ascorbyl radical, the latter formed by one electron oxidation of ascorbate have low reduction potentials [56] and can thus react with most other biologically relevant radicals and oxidants. Oxidative processes can affect DNA integrity which may trigger a cascade of alterations including strand breaks, mutations in DNA molecule or breakdown of protective mechanisms due to oxidative inactivation of DNA repair enzymes, thereby contributing to the carcinogenic events [57]. It has been estimated that one human cell is exposed to approximately 1.5
10 5 oxidative hits a day from hydroxyl radicals and other such reactive species [58]. The hydroxyl radical is known to react with all components of the DNA molecule: damaging both the purine and pyrimidine bases and also the deoxyribose backbone [59]. DNA can also be

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damaged by reactive nitrogen species, some of which can be derived from nitrosamines [60, 61]. AA as an antioxidant is capable of neutralizing multiple reactive oxygen species (ROS) including hydroxyl, alkoxyl, peroxyl, superoxide anion and hydroperoxyl radicals as well as reactive nitrogen species (RNS) such as nitrogen dioxide, nitroxide, peroxynitrite at very low concentrations [62]. In addition to scavenging reactive oxygen species and reactive nitrogen species, AA can regenerate other small molecule antioxidants, such as -tocopherol, glutathione (GSH), urate, and -carotene, from their respective radical species [63]. Moreover, AA has also been reported to reduce in vivo oxidative damage as indicated by a reduction in markers of oxidative damage of DNA, lipid, and proteins [62]. The reactive species which are the target of the anti-oxidant action of AA has been listed in (Table 2) [54, 62, 64-71]. Table 2.

Chemical species scavenged by ascorbate. Reproduced from [62] by permission from Federation of American Societies for Experimental Biology

Reactive Oxygen Species

Reference

Hydroxyl radical (•OH)

[54]

•

Alkoxyl radicals (RO )

[54]

Peroxyl radicals (RO2•)

[54] •-

Superoxide anion/hydroperoxyl radical (O2 /HO2•)

[54]

Hypochlorous acid (HOCl)

[64]

Ozone (O3)

[65]

Single oxygen (1O2)

[66]

Reactive Nitrogen Species Nitrogen dioxide (NO2•)

[67]

Dinitrogen trioxide/dinitrogen tetroxide (N2O3/N 2O4)

[68]

Nitroxide (NO)

[69]

Peroxynitrite/peroxynitrous acid (ONOO /ONOOH)

-

[70]

Antioxidant-Derived Radicals -Tocopheroxyl radical (-TO•)

[54]

Thiyl/sulphenyl radicals (RS•/RSO•)

[54]

•

[54]

Urate radical (UH ) -Carotene radical cation (-C ) •+

[71]

5.2. Pro-Oxidant Action Mechanism of Ascorbic Acid Specific to Tumor Microenvironment It is understood that ROS toxicity induced by certain chemotherapeutic agents can be an effective means of selectively eradicating malignant cells [72]. In normal cells, there exists a balance between the free radical generation and the antioxidant defense [73]. However, it has been clearly

documented that tumor cells are under persistent oxidative stress and have an altered antioxidant system [72] and thus further ROS stress in these malignant cells reaching a threshold level could result in apoptosis [74]. These observations further suggest that neoplastic cells may be more vulnerable to oxidative stress because they function with a heightened basal level of ROS due to increased rate of growth and metabolism [75]. Thus, in cancer cells, an enhanced exposure to ROS, generated through the redox activity in the intracellular and extracellular milieu can overwhelm the cells antioxidant capacity, leading to irreversible damage and apoptosis. Studies with ROS producing systems such as hypoxanthine/xanthine oxidase have shown that their administration leads to cytotoxic effects which are limited to cancer cells with low or no toxicity to normal cells [76]. Chen et al., [77] carried out a study which observed that ascorbate at pharmacologic concentrations was prooxidant, generating hydrogen peroxide-dependent cytotoxicity towards a variety of cancer cells in vitro without adversely affecting normal cells. The study suggested that AA in pharmacologic concentrations may act as a prodrug leading to the formation of ascorbate radical (Asc•
) and hydrogen peroxide in extracellular space. Certain properties of naturally occurring anti-oxidants including AA, such as cleavage of DNA, generation of reactive oxygen species in the presence of transition metal ions [78] and induction of apoptosis are similar to those of several known anti-cancer drugs [79]. The apoptosis-inducing activity of AA has been ascribed to its pro-oxidant action and is inhibited by catalase, antioxidants like N-acetylcysteine and GSH, Ca2+ and Fe3+ depletion and stimulated by H2O2 and Cu2+ [80]. In an earlier study site-specific DNA cleavage by AA in the presence of Cu (II) has been described [81]. Copper ions are known to interact with both DNA phosphates and bases through coordination binding [82]. Accordingly, Hadi and co-workers [83] put forward several lines of evidence to propose that the selective toxicity of AA against cancer cells may be explained by the observation that copper levels are elevated in cancer cells [74] and that the AA is able to mobilize nuclear copper [84] leading to DNA damage. Thus the copperdependent redox activity is an important determinant of prooxidant activity of AA. Among oxygen radicals, the hydroxyl radical is most electrophilic with high reactivity and therefore possesses a small diffusion radius. Thus, in order to cleave DNA, it must be produced in the close vicinity of cellular DNA [85]. The location of the redox-active metal is of utmost importance because the hydroxyl radical, due to its extreme reactivity, interacts exclusively in the vicinity of the bound metal. This is also in concurrence with the observation that the AA induces cell death, nuclear fragmentation and internucleosomal DNA cleavage in human myelogenous leukemia cell lines [86]. Such internucleosomal DNA “laddering” often used as an indicator of apoptosis may reflect, at least partially, DNA fragmentation by nonenzymatic processes by metal-chelating agents that promote the redox activity of endogenous copper ions, resulting in the production of hydroxyl radicals at the site [87]. It may be noted that although antioxidant action mechanism of AA is of critical importance in prevention of carcinogenic insults, transition to pro-oxidant action may be an important pathway through which preneoplatic cells and neoplastic cells can be selectively killed while normal cells survive Fig. (3).

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Fig. (3). Possible mechanism for chemopreventive effects of dietary antioxidants. Reprinted by permission from American Chemical Society: Qian YP, Cai YJ, Fan GJ et al., J Med Chem 2009; 52: 1963-74.

exhibit a general state of chronic inflammation which is related to the tumor itself and the interaction of host factors with the tumor. Elevation in the level of classical inflammatory markers such as fibrinogen [94, 95], CRP [96, 97] and IL-6 [98] have been well-documented in cancer patients, with numerous studies demonstrating that elevation is associated with poor survival. Mayland et al. demonstrated that, in 50 patients with advanced malignancies of various types, a correlation between high CRP levels and AA deficiency existed [99]. In an interventional study, Block et al. [100] examined 396 healthy nonsmokers randomized to receive 1000 mg/day AA, 800 IU/day vitamin E, or placebo, for 2 months. A statistically significant decrease in plasma CRP levels was found only in the group receiving AA. A recent study showed that AA at pharmacologic concentration had the potential to influence the expressions of angiogenic and angiostatic chemokine genes in hepatocellular carcinoma (HCC) cell lines [101]. AA has also been shown to inactivate pro-inflammatory nuclear factor-kB in endothelial cells during the inflammation process [102]. In consideration of the above findings it is required that studies are undertaken to further investigate the possibility that AA potentially increases immune function in cancer patients and such immuno-modulatory action might be an important mechanism for its anti-cancer properties.

5.3. Ascorbic Acid Potentiates the Host-Immune Response

5.4. Interference with Carcinogen Metabolism As An Anti-Induction Mechanism of Ascorbic Acid

The immune system plays an extraordinary role in the prevention of cancer. First, the immune system can protect the host from virus-induced tumors by eliminating or suppressing viral infections. Second, the timely elimination of pathogens and early resolution of inflammation can prevent the establishment of an inflammatory environment conducive to tumorigenesis. Third, the immune system can specifically identify and eliminate tumor cells on the basis of their expression of tumor-specific antigens or molecules induced by cellular stress. The third process is referred to as tumor immune surveillance, whereby the immune system identifies cancerous and/or precancerous cells and eliminates them before they can cause harm [88]. Lymphocytes are most numerous in the stroma of slow-growing tumors and scanty or virtually absent around rapidly growing lesions. It is believed that AA may act as immune stimulator thereby enhancing the host resistance to tumor [16]. The concentration of AA in phagocytes and lymphocytes is far higher than in plasma, indicating that it may have functional roles in these immune system cells. Studies have supported this idea as it has been demonstrated that AA affects random migration and chemotaxis of phagocytes [89], transformation of influenza virusinfected lymphocytes [90], production of interferon [91], and replication of viruses [92]. In addition the in vivo effect of AA on human natural killer (NK) cell activity measured using K562 tumor cells as targets show a biphasic response: a transient slight suppression between 1 to 2 hours (20% of control) which was followed by a significant enhancement (an over-shoot) at 8 hours that was further increased at 24 hours [93]. The study concluded that AA is a potent immunomodulator and its effect in enhancement of NK cytotoxicity may explain one mechanism by which AA exerts its probable anti-cancer activity. Cancer patients are known to

The cytochrome P450 (CYP)-dependent monooxygenase system is predominantly involved in detoxification mechanisms catalyzing oxidative metabolism of xenobiotics including a wide variety of drugs and potential carcinogens. In the initial phase of detoxification, many compounds are first converted to polar metabolites by CYP, which facilitates their elimination in the second phase. However, in the process some compounds may also be inadvertently bio-activated by CYP to reactive intermediates that show detrimental biological effects [103]. For example, carcinogenic polyaromatic hydrocarbons (PAH) such as cigarette tobacco constituent benzo(a)pyrene undergo metabolic activation by CYP1A1 and CYP 1B1to form B[a]P-7,8-oxide which further undergoes epoxide hydrolase-mediated metabolism to the proximate carcinogen B[a]P-7,8-dihydro-7,8-diol. Further activation of dihydrodiol metabolite by CYP 1A1/1B1 results in the formation of the ultimate carcinogen 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P [104]. Similarly, N-nitrosamines are mutagenic and carcinogenic compounds widely present in the environment. The majority of N-nitrosamines tested has been shown to cause cancer at different organs in a variety of animal species [105] and may be causative agents in human cancer. N-Nitrosamines require metabolic activation to exert their carcinogenic effects. CYP2E1 is the most active species known in the metabolism of N-nitrosodimethylamine (NDMA) and N-nitrosopyrroli dine (NPYR) [106]. CYP2A contribute to the preferential activation of N-nitrosopiperidine (NPIP) [107] while Nnitrosodibutylamine (NDBA) is mainly activated by CYP1A1 [108]. One of the studies examining the protective role of AA against such genotoxic responses demonstrated the ascorbic acid to be protective towards the Nnitrosamines-induced DNA damage in HepG2 cells. A con-

1764 Current Drug Targets, 2012, Vol. 13, No. 14

centration of 10
M AA led to a maximum reduction in NDBA (94%), NPYR (81%), NPIP (80%) and NDMA (61%)-induced DNA damage [109]. It was thus suggested that AA may exert its protective effect towards Nnitrosamines by interfering with the enzyme systems catalyzing the metabolic activation of N-nitrosamines and thereby blocking the production of genotoxic intermediates. Further the authors reported that the protective effect of AA (5–10
M) towards NDMA-induced DNA damage could be related in part to the reduction of human CYP2E1 activity (32%), while the reduction of DNA damage induced by NPYR and NPIP may be also attributed in part to the inhibition of human CYP2E1 (32%) and CYP2A6 (82%) activities by AA which is involved in their metabolism. It is also known that CYP1 family of enzymes (CYP1A1, CYP1A2, and CYP1B1) are important in the metabolism of several xenobiotics such as PAH and heterocyclic amines and the expression of these enzymes is inducible by PAHs such as 2,3,7,8-tetrachloridibenxo-p-dioxin (TCDD) [110]. TCDD is known to induce a variety of toxic effects, including hepatotoxicity, and cancers [111] and has been classified as a group I carcinogen by the International Agency for Research on Cancer [112]. In a recent study by Chang et al., [113], CYP1A1 enzyme activity in HepG2 cells treated with AA in the presence or absence of TCDD was measured and the data showed that AA prevented the TCDD-induced expression of CYP1A1 mRNA and further reduced TCDD-inducible EROD activities in a dose-dependent manner. Thus, inhibition of human cytochrome P450s (CYPs) activities may be a feasible mechanism by which AA exert its protective effect. In addition other related mechanisms such as enhancement of detoxification pathways that convert these reactive compounds to less toxic and more easily excreted products also exist [114]. Indeed the ability to induce phase II enzymes, NAD(P)H:quinone oxidoreductase activity [115] and UDPglucuronyl transferase [116] has also been reported for AA. 5.5. Stabilization of Intercellular Matrix & “Walling off” Effect by Ascorbic Acid AA is essential for the structural integrity of the intercellular matrix as it is closely related to the replenishment of principal matrix structures. Robert McCormick hypothesized that cancer metastases spread through weakened collagen and that metastases could be blocked by AA, which makes collagen stronger [15]. AA plays an important role in the synthesis of collagen and thus in the stability of the microenvironment of both the normal and neoplastic cells. Collagen formation is thus an important factor in the encapsulation of tumors to inhibit or slow down the invading metastatic stage by contributing to the development of a barrier or walling off the tumor [53]. Ascorbate deficiency significantly reduces hydroxylation of proline and lysine to hydroxyproline and hydroxylysine respectively, thereby interfering with the required collagen cross-linking. Additionally the strength of the intercellular matrix also depends upon the abundance of certain long-chain mucopolysaccharide polymers, the glycosaminoglycans and proteoglycans [117, 118]. Normally the intercellular matrix is maintained in steady-state equilibrium with the homeostatic balance between the formation of new macromolecules (polymerization) and decay (depolymerization) of the pre-existing ones. In cancer the matrix de-

Ullah et al.

polymerization in the immediate vicinity of proliferating invasive cells has been observed. Such an enhanced degradation is due to the secretion of lysosomal glycosidases by the neoplastic cells while AA has an inhibitory activity against these enzymes [16]. Indeed the level of AA in cancer is low and thus the natural restraint on the activity of these enzymes is evaded. The intercellular matrix which is the target of hydrolytic enzymes secreted by cancer cells is required to facilitate cell to cell communication and it has been observed that the inhibition of cell-to-cell communication is strongly related to the advanced carcinogenic process like the tumor promotion [119, 120]. For example, the inhibition of gap junction intercellular communication is linked to the carcinogenic process as the impairment results in cellular proliferation [121]. AA has been shown to have a protective action against the H2O2-induced inhibition of gap junction intercellular communication [122, 123]. 5.6. Ascorbic Acid Targets the Cancer Cell Survival Signaling Pathways Initiation and progression of neoplastic growth involves significant alterations of signal transduction through inhibition or activation of constitutive factors of the signaling pathways. AA derivatives; fatty acid esters of AA such as ascorbyl palmitate and ascorbyl stearate have attracted considerable interest as anticancer compounds [124]. Naidu and coworkers have demonstrated that ascorbyl stearate inhibited cell proliferation by interfering with cell cycle progression and induced apoptosis by modulation of insulin-like growth factor 1-receptor expression in human brain tumor glioblastoma (T98G) cells [125]. The AKT family members, AKT 1, 2, and 3, have been shown to be over expressed and constitutively activated in human tumors such as breast, pancreatic, ovarian, prostatic and gastric carcinomas [126, 127]. Inhibitory effects of ascorbyl stearate was also reported in a panel of human ovarian and pancreatic cancer cells suggesting that the anti-proliferative effect and induction of apoptosis in these cells were mediated through cell cycle arrest and modulation of the IGF-I receptor and PI3K/AKT2 survival pathways [128]. In the same study the authors reported an anti-tumorigenic effect of Asc-S in vivo using a human ovarian carcinoma xenograft nude mouse (C57BL/6) model. In another study high concentrations of AA were found to induce apoptosis of human gastric cancer cells by p38-MAP kinase-dependent up-regulation of transferrin receptor [129]. It was shown that the use of p38 mitogen activated protein kinase (MAPK) inhibitor countered the upregulation of TfR and reduced the apoptotic potential of AA. As observed in various malignancies, solid human tumors contain regions of very low oxygen concentrations creating hypoxia. Thus in order to survive, cancer cells must therefore adapt themselves to hypoxia, and this process is mainly achieved by the activation of the transcription factor hypoxia-inducible factor 1 (HIF-1) [130, 131]. Since the activity of HIF-1 is an essential requirement for solid tumor progression, its inhibition represents a very attractive target for cancer therapy [132, 133]. Under hypoxic conditions, the activity of HIF-1 hydroxylases decreases, resulting in a decreased rate of HIF-1
degradation and the transcriptional activation of HIF-1 target genes which are known to encode angiogenic factors, glycolytic enzymes, survival factors and invasion factors. In this

Ascorbic Acid in Cancer Chemoprevention

context it has been demonstrated that the in vitro activity of prolyl hydroxylases is enhanced in the presence of ascorbate and loading cells with ascorbate results in the inhibition of HIF-1 activation by hypoxia [134, 135]. 6. ASCORBIC ACID: EVIDENCE OF TRANSLATIONAL EFFICACY AGAINST CANCER The evidence based on the observations that AA has significant anti-cancer properties demonstrated against in vitro and pre-clinical cancer models as well against human cancers presents a viable potential towards the feasible utility of its translational value. However, the underlying principles of its therapeutic action needs to be further examined at molecular and genetic levels as the possibility of customized action specific to individual patients might exist and perhaps might explain the large variations in the results of various clinical trials and individual patient responses reported in case studies. Such understanding can guide decisions about which cancer patients might potentially benefit from pharmacologic ascorbate therapy. Below we provide a series of evidence in literature that demands an attention towards ascorbic acid as a clinically potential molecule in cancer chemoprevention: i)

Earlier studies by Cameron and Pauling reported clinical benefits and improved survival using both oral and intravenously administered AA in the treatment of terminal cancer [136, 137]. Later, in two double blind, placebo-controlled trials, investigators at the Mayo clinic found that a high-dose oral administration of AA had no effect on cancer survival [8, 9]. These trials were considered definitive possibly because the difference in the in vivo levels of AA achieved between the oral and intravenous administration was not adequately appreciated. Plasma levels of ascorbic acid are tightly controlled and are around 50 μM [49]. However, Padayatty et al. have shown that intravenous administration of ascorbic acid bypasses such tight control and results in concentrations as much as 70 folds higher than those achieved by maximum oral consumption [10]. Thus concentrations of ascorbic acid achieved through oral administration might have preventive role for cancer risk but the therapeutic intervention may require high pharmacological doses achievable only via intravenous administration.

ii) In Japan two uncontrolled trials conducted at two different hospitals during the 1970s also confirmed the increase in survival time of terminal cancer patients supplemented with ascorbate. At the Fukuoka Torikai Hospital, the average survival time after being labeled “terminal” was 43 days for 44 patients supplemented with low levels of ascorbate (less than 4 grams daily), while 246 days for 55 patients supplemented with higher dosages of ascorbate (greater than 5 grams daily — averaging 29 grams daily) and starting at the time of “terminal” diagnosis [138]. At the Kamioka Kozan Hospital where 19 terminally-ill control patients survived an average of 48 days compared to six patients on high levels of AA who lived an average of 115 days i.e. 2.4 times longer than the control group [139, 18].

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iii) Rozanova et al., [140] conducted a study where they conjugated AA with extracts of medicinal herbs for treatment against cancer cell lines. This study showed that this conjugation stimulated apoptosis and disrupted cell cycle. In addition, 20–40% of cells underwent apoptosis within 24 h of treatment. The result suggested that AA can act as a catalyst in the treatment of cancer. The earlier attempt of this kind was made in 1983 when Kimoto et al., showed enhancement of anti-tumor activity of ascorbate by a copper:glycylglycyl complex [141]. iv) Aidoo et al., [142] have investigated the anti-mutagenic effects of AA on the frequency of 6-thioguanineresistant (6-TGr) T-lymphocytes produced in Fischer 344 rats dosed with the direct-acting alkylating agent, N-ethyl-N-nitrosourea (ENU). The frequency of 6-TGr T-lymphocytes from the spleen measured five weeks after ENU treatment indicated that ENU produced a substantial mutagenic response. Pretreatment and/or posttreatment of rats with AA administered in the drinking water appeared to inhibit the response. In order to evaluate the time effects of the AA supplement on ENU mutagenicity, rats were exposed to the mutagen together with ascorbic acid, which was given continuously for the entire duration of the experiment. At specific times after ENU treatment, the frequency of 6-TGr T-cells was determined in lymphocytes isolated from the spleen and the thymus. Time-dependent increases in the frequency of 6-TGr T-cells were observed with ENU treatment; AA significantly reduced the ENU-mediated mutagenic responses, most dramatically in the spleen (P < 0.0001 at weeks 6 and 8) and to a lesser extent in the thymus (P < 0.01 at week 6 and P < 0.006 at week 8). The authors proposed it as an explanation for epidemiological data that link AA ingestion with decreased cancer risk. v) Levine and coworkers have demonstrated that pharmacologic AA concentrations achievable through intravenous administration were cytotoxic to many types of cancer cells in vitro and significantly impeded tumor progression in vivo without toxicity to normal tissues [143]. A recent study tested 10 cancer cell lines with AA and the results showed that pharmacologic ascorbic acid induced cytotoxicity in all tested cancer cells, with IC50 less than 4 mM, a concentration easily achievable in humans. Treatment in mouse pancreatic cancer xenografts showed that intraperitoneal ascorbic acid at 4 g/kg daily reduced tumor volume by 42% [144]. vi) Several laboratories have shown the tumoricidal effect of methylglyoxal on cancer-bearing animals [145]. In a study the effect of methylglyoxal alone and in combination with creatine and AA was demonstrated on cancerbearing animals by measuring the increase in life span and tumor cell growth inhibition. The results indicated that anticancer effect of methylglyoxal was significantly augmented by AA and further enhanced by AA and creatine in combination. Nearly 80% of the animals treated with methylglyoxal plus AA and creatine were completely cured and devoid of any malignant cells within the peritoneal cavity [146]. Further as reviewed by Verrax and Calderon [131], in (Table 3), AA has

1766 Current Drug Targets, 2012, Vol. 13, No. 14

Table 3.

Ullah et al.

Influence of ascorbic acid on the efficacy of different therapeutic drugs. Reproduced from [131] by permission from Elsevier Ltd

Treatment

Influence of Ascorbic Acid

Reference

5-Fluorouracil

a b

[147] [151]

Bleomycin

a

[147]

Doxorubicin

a

[149]

Paclitaxel

a

[149]

a

Cisplatin

 a

[149] [152]

Cyclophosphamide

b

[151]

Procarbazin

b

[151]

b [151]

Asparaginase Vinblastine

b

[151]

Adriamycin

b

[151]

Gemcitabin



b

[148]

a

Vincristin

 a

[153] [154]

X-rays

a b

[147] [150]

Trisenox

a a c d

[155] [156] [157] [158]

Methotrexate

a

[147]

TRAIL ligand



d

[159]

Bortezomib



a

[160]

a

In vitro results In vitro results in combination with menadione In vitro results d In vitro results in cells loaded with ascorbic acid. b c

been reported to increase the efficacy of several chemotherapeutic drugs either in vitro or in vivo and also augment the treatment potential of radiotherapy [147160]. vii) As mentioned earlier AA is also involved in immune cell functions and immune responses against cancer cells. In a study using AA depleted animal model (the mice lacking L-gulono- -lactone oxidase (Gulo), the enzyme required for the biosynthesis of ascorbic acid), the effects of ascorbic acid on NK cell cytotoxicity against ovarian cancer cells, MOSECs (murine ovarian surface epithelial cells) was examined. The Gulo(
/
) mice depleted of AA survived for a shorter time than the normal control or Gulo(
/
) mice supplemented with

AA after tumor challenge regardless of treatment with IL-2. In addition, the CD69 and NKG2D expression was clearly reduced in NK cells isolated from mice depleted of AA as compared to that in the normal control and the mice supplemented with AA. It was also observed that IFN-  secretion by NK cells isolated from Gulo(
/
) mice depleted of AA was decreased after NK cells were co-cultured with MOSECs [161]. The results suggested that ascorbic acid at the normal plasma concentration has an essential role in maintaining the NK cytotoxicity against cancer cells and thus aid in immune surveillance. viii) Wright et al., [162] studied the single nucleotide polymorphisms (SNPs) in SLC23A1 and SLC23A2 – genes that encode key AA transport proteins and its affect on

Ascorbic Acid in Cancer Chemoprevention

gastric cancer risk in 279 incident cases and 414 ageand gender-matched controls drawn from a populationbased case–control study in Poland. Compared to subjects who were homozygous for the common G allele of the SLC23A2, SNP rs12479919, carriers of the AA genotype had a 41% lower risk of gastric cancer [odds ratio (OR) = 0.59, 95% confidence interval (CI): 0.36– 0.95; P trend = 0.06]. A haplotype that contained the common allele of the rs6139591, rs2681116 and rs14147458 SNPs in SLC23A2 was also significantly inversely associated with gastric malignancy. Ascorbic acid is highly concentrated in gastric mucosa and gastric juice and individuals with chronic gastritis or overt cancer have substantially lower concentrations than their healthier counterparts, suggesting a particularly important role for this micronutrient in the aetiology of gastric cancer [163]. ix) Several case studies have been reported over the last few decades that show remarkable chemopreventive properties of AA against cancer: 1) Cameron et al., reported a case study where response of a patient with histologically proven reticulum cell sarcoma to no treatment other than large doses of AA was described. At the time of first diagnosis, the disease was widely disseminated, and a very dramatic regression of all parameters of disease activity was induced by the continuous administration of large doses of AA. Reduction in dosage some months later coincided with reactivation of the disease process. The reinstitution of regular high-dose AA therapy induced a second complete remission [164]; 2) Riordan et al., reported the translational utility of high dose i.v. ascorbic acid in treating patients with adenocarcinoma of the kidney [165]; 3) Padayatty et al., [166] reported three cases describing the regression of a pulmonary metastatic renal cancer, a primary bladder tumour with multiple satellite tumours and a diffuse large B-cell lymphoma in patients receiving high-dose intravenous AA therapy. x) In 1997, expert panels at the World Cancer Research Fund and the American Institute for Cancer Research proposed that AA can reduce the risk of several types of cancer including stomach, mouth, pharynx, esophagus, lung, pancreas and cervical cancers [50]. AA was also selected as a model nutrient by NIH scientists to develop strategies to optimize nutrition as a new way to promote health and prevent disease by involving quantitative strategies that could be translated for use by the general public [167]. xi) An in vivo study was carried out by Yeom et al. [168] to test the carcinostatic effects of AA in mice with sarcoma cells. The survival rate was increased by 20% in the group that received high-dose concentrations of AA, compared to the control. These results suggested that the high concentrations of AA can inhibit the angiogenesis in cancer cells. xii) Verrax and Calderon [169] observed that pharmacologic concentrations of ascorbate killed various cancer cell lines very efficiently (EC50 ranging from 3 to 7 mM). In agreement with other reports previously published, in vivo results showed that both intravenous and intraperi-

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toneal administration of ascorbate induced pharmacologic concentrations (up to 20 mM) in blood. In contrast, the concentrations reached orally remained physiological. According to pharmacokinetic data, parenteral administration of ascorbate decreased the growth rate of a murine hepatoma, whereas oral administration of the same dosage did not. It was also reported that pharmacologic concentrations of ascorbate did not interfere with but rather reinforced the activity of important chemotherapeutic drugs. xiii) A recent Phase I clinical trial reported by Monti et al., [170] provided an initial safety evaluation of AA added to gemcitabine and erlotinib in patients with stage IV pancreatic cancer. In the nine patients who completed the study, AA concentrations were reached safely and with minimal associated adverse events that could be attributed to AA. Peak AA concentrations were achieved as high as 30 millimoles/L in the highest dose group. These concentrations were similar to reported concentrations in patients who received AA intravenously without concomitant chemotherapy [171]. The usual plasma AA concentrations in people are 0.010–0.080 millimoles/L and are dependent on dietary and supplement intake. Even with massive oral supplementation of gram quantities daily, taken every few hours, plasma AA concentrations in people do not exceed 0.25 millimoles/L [10]. As the authors suggested, the data from this trial indicated that pharmacologic ascorbic acid concentrations were achievable in patients who received intravenous AA in combination with gemcitabine and erlotinib ; and further warrant Phase II trial to assess the combination therapy for progression free and overall survival. Indeed there are studies which have previously indicated that AA may also be efficient in treating cancers when used in combination with other active chemotherapeutic drugs. A recent single-arm, multicenter phase 2 study demonstrated that oral consumption of 1 gm of AA in combination with bortezomib and oral melphalan (0.1 mg/kg) is an effective and well tolerated frontline treatment regime for patients with newly diagnosed multiple myeloma [172]. xiv) Reactive oxygen/nitrogen species generated by antineoplastic agents are prime suspects for the toxic sideeffects of acute or chronic chemotherapy. In a recent clinical study, Banu and co-workers reported that coadministration of Vitamin C and E restored the antioxidant status in patients suffering from breast-cancer and undergone chemotherapy with standard anti-cancer regimen including fluorouracil, doxorubicin and cyclophosphamide [173]. The study reflects the potential of ascorbic acid as adjuvant in cancer treatment to ameliorate the chemotherapy associated oxidative damages to normal cells for a better post-treatment management of cancer patients. CONCLUSION Among the highly sought for alternative medical treatments AA is one of the most popular drug used by non mainstream physicians orally and parenterally for many decades as a therapeutic agent to treat diverse conditions including

1768 Current Drug Targets, 2012, Vol. 13, No. 14

infections, autoimmune diseases and cancer. A partial validation of the possible role of AA in prevention or regression of cancer as a lead drug or adjuvant to standard chemotherapeutic regimen has emerged with the feasibility of high bioavailable levels achieved through pharmacologically active doses. The observation that ascorbic acid acts as a pro-drug targeting the cancer cells while sparing the normal ones provides the molecule one of the desired characteristic of an ideal anti-cancer drug. AA is well tolerated except for few conditions such as pre existing renal disease, glucose 6-phosphate dehydrogenase deficiency and oxalate nephrolithiasis where patients may respond aggressively against the high dose IV AA and thus require careful screening of the patients. Although the studies supporting the role of AA in cancer chemoprevention might appear inconclusive at the current stage but are definitely optimistic. It is thus suggested that public health campaigns should be directed towards increasing the consumption of fruits, as well as vegetables, rich in AA. Furthermore, controlled and large clinical trials must be carried out in concurrence with the laboratory studies to investigate the precise action mechanism of AA in cancer chemoprevention and expand the possibility of its translational utility.

Ullah et al. [12]

[13] [14] [15] [16] [17] [18] [19]

[20]

[21]

[22]

CONFLICT OF INTEREST

[23]

The authors declare no conflict of interest. The authors alone are responsible for the content and writing of this paper.

[24] [25] [26]

ACKNOWLEDGEMENTS Declared none. [27]

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Revised: September 18, 2012

PMID: 23140287

1771

Accepted: November 03, 2012