Cancer Chemopreventive Potential of Apples, Apple Juice, and Apple Components

1608 Review
Cancer Chemopreventive Potential of Apples,

Apple Juice, and Apple Components
Author Clarissa Gerhauser
Affiliation Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany
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Key words Abstract AIS: àlcohol-insoluble substance'
●
" apples ! AP-1: activator protein 1
●
" Malus sp.
Apples (Malus sp., Rosaceae) are a rich source of Apaf-1: apoptotic protease activating
●
" Rosaceae
nutrient as well as non-nutrient components factor 1
●
" cancer chemoprevention
and contain high levels of polyphenols and other APE: apple polyphenol extract
●
" oligomeric procyanidins
●
" polyphenols phytochemicals. Main structural classes of apple B(a)P: benzo[a]pyrene
constituents include hydroxycinnamic acids, di- bw: body weight
hydrochalcones, flavonols (quercetin glycosides), CI: confidence interval
catechins and oligomeric procyanidins, as well as COBRA: combined bisulfite restriction
triterpenoids in apple peel and anthocyanins in analysis
red apples. Several lines of evidence suggest that Cox-1: cyclooxygenase-1
apples and apple products possess a wide range Cyp1A: cytochrome P450 1A
of biological activities which may contribute to DEP-1: density-enhanced protein-tyrosine
health beneficial effects against cardiovascular phosphatase-1
disease, asthma and pulmonary dysfunction, dia- DMBA: 7,12-dimethylbenz[a]anthracene
betes, obesity, and cancer (reviewed by Boyer and DMH: 1,2-dimethylhydrazine
Dedication Liu, Nutr J 2004). The present review will sum- DNMT: DNA methyltransferase
In memory of marize the current knowledge on potential can- DPPH: 1,1-diphenyl-2-picrylhydrazyl
Prof. B. L. Pool-Zobel cer preventive effects of apples, apple juice and EGCG: (–)-epigallocatechin 3-gallate
apple extracts (jointly designated as apple prod- EGF: epidermal growth factor
received May 25, 2008
ucts). In brief, apple extracts and components, es- EGFR: epidermal growth factor receptor
revised July 17, 2008
pecially oligomeric procyanidins, have been FCS: fetal calf serum
accepted July 31, 2008
shown to influence multiple mechanisms rele- Fr.P: procyanidin-enriched fraction P
Bibliography vant for cancer prevention in in vitro studies. GJIC: gap-junctional intracellular
DOI 10.1055/s-0028-1088300 These include antimutagenic activity, modula- communication
Planta Med 2008; 74: 1608–
tion of carcinogen metabolism, antioxidant activ- GSK3β: glycogen synthase kinase 3β
1624
ity, anti-inflammatory mechanisms, modulation GST: glutathione S-transferase
© Georg Thieme Verlag KG
Stuttgart · New York of signal transduction pathways, antiproliferative HDAC: histone deacetylase
Published online October 14, and apoptosis-inducing activity, as well as novel IL-2Rα: interleukin-2 receptor α-chain
2008 mechanisms on epigenetic events and innate im- IQ: 2-amino-3-methylimidazo
ISSN 0032-0943 munity. Apple products have been shown to pre- [4,5-f]quinoline
vent skin, mammary and colon carcinogenesis in MAP-kinase: mitogen-activated protein kinase
Correspondence
Dr. Clarissa Gerhäuser animal models. Epidemiological observations in- NSP: non-starch polysaccharides
Deutsches Krebsforschungs- dicate that regular consumption of one or more NHS: Nurses Health Study
zentrum (DKFZ) apples a day may reduce the risk for lung and co- ODC: ornithine decarboylase
Toxikologie und lon cancer. OPC: oligomeric procyanidins
Krebsrisikofaktoren OR: odds ratio
Im Neuenheimer Feld 280
Abbreviations ORAC: oxygen radical absorbance
9120 Heidelberg
! capacity
Germany
Tel.: +49-6221-42-3306 AC: aberrant crypts PARP: poly(ADP-ribose)polymerase
Fax: +49-6221-42-3359 ACF: aberrant crypt foci PBMC: peripheral blood mononuclear
[email protected] AE02/-03/-04: apple juice polyphenol extract cells
Gerhauser C. Cancer Chemopreventive Potential … Planta Med 2008; 74: 1608 – 1624
Review 1609
PGs: prostaglandins SCFA: short-chain fatty acids

PTM: permeability transition pore TNF-α: tumor necrosis factor-α
PKC: protein kinase C TPA: 12-O-tetradecanolyphorbol 13-acetate
ROS: reactive oxygen species TRAIL: TNF-related apoptosis-inducing ligand
RR: relative risk TSG: tumor suppressor gene
Introduction (quercetin glycosides), catechins and oligomeric procyanidins,

! as well as anthocyanins in red apples (selected chemical struc-
During the past years, we have made tremendous progress in our tures in ●
" Fig. 1) [13], [14], [15], [16], [17], [18]. Cider apples are
understanding of the carcinogenic process at the cellular and particularly rich in polyphenols [19], [20], [21]. Consequently,
molecular level. This has led to the development of a promising freshly squeezed apple juice from cider apples has highest levels
new approach to cancer prevention, termed “chemoprevention” of the four major classes of apple constituents, whereas com-
[1], which aims to block, inhibit or reverse the development and mercially available clear juice (often made from concentrates)
progression of precancerous cells through use of non-cytotoxic is very poor in polyphenols (summarized from [12] in ● " Table 2).
nutrients and/or pharmacological agents [2]. Carcinogenesis is Apples and apple juice are good sources of oligomeric procyani-
generally a slow process and often takes decades from tumor in- dins (OPC) composed of (epi)catechin units [22], [23], [24], [25],
itiation to diagnosis, offering a considerable time frame for che- [26], which have recently gained interest because of potential
mopreventive approaches. Accordingly, the validation and uti- health promoting effects (review in [27]). In apples, 63 – 77 % of
lization of dietary components, natural products, or their syn- all polyphenols were attributed to OPC [13]. Similar to the differ-
thetic analogs as potential cancer chemopreventive agents in ences in polyphenol levels in apple juices, polyphenolic extracts
the form of functional foods or nutraceuticals has become an im- prepared by enrichment on adsorber resins from cloudy apple
portant issue in current health- and cancer-related research. juice contained higher amounts of OPC (48 – 61 %) than extracts
Apples (Malus sp., Rosaceae) and apple juice are the most con- from clear juice (28 – 49 %) [28].
sumed fruit and fruit juice in Germany, with an average annual In addition to polyphenols, apple peel contains considerable
per capita consumption of 18.4 kg and 12.8 L, respectively [3], amounts of lipophilic triterpenoids, which are concentrated in
[4]. Several lines of evidence suggest that apples and apple prod- the cuticular wax layer. The most abundant triterpene, ursolic
ucts possess a wide range of biological activities which may con- acid, was isolated in amounts up to 50 mg per medium size fruit
tribute to health beneficial effects against cardiovascular dis- [29]. A series of thirteen triterpenes with antiproliferative activ-
ease, asthma and pulmonary dysfunction, diabetes, obesity and ity was isolated from apple peel by extraction with organic sol-
cancer [5]. This review will summarize the present knowledge vents [30].
on potential cancer preventive effects of apples, apple juice and
apple extracts (jointly designated as apple products). After a
short summary of nutrient and phytochemical composition, the Absorption, Bioavailability and Metabolism of
manuscript gives an overview of results from bio-availability Apple Juice Constituents
studies and in vitro investigations of apple polyphenols, triterpe- !
noids and apple pectin on cancer preventive mechanisms. Stud- Apple components may influence multiple mechanisms contri-
ies with apple products in animal models of skin, mammary and buting to cancer prevention as outlined below. However, to do
colon carcinogenesis and in xenograft models for tumor growth so in vivo they must be absorbed and achieve effective concen-
and invasion are described, followed by a brief compilation of trations at the target site in the correct metabolic form [31]. Sev-
animal studies with apple-derived dietary fiber and apple pec- eral earlier reviews have summarized evidence on absorption,
tin. Subsequently, short-term human intervention studies on bioavailability and metabolism of polyphenolic compounds, in-
the modulation of antioxidant parameters with apples and apple cluding all major classes of apple and apple juice constituents
juice are summarized. Finally, the current evidence on links be- [32], [33], [34]. Boyer and Liu summarized data related to bio-
tween apple consumption and human cancer occurrence based availability and metabolism of apple components, covering
on epidemiological studies is presented.
Table 1 Average nutrient content in apples and apple juice (per 100 g fresh
Apples and Apple Juice: Nutrient and weight) [6]
Phytochemical Composition Apples Apple juice
!
Water (g) 85.3 88.1
Apples and apple juice contain nutrient as well as non-nutrient
Energy (kcal/kJ) 54/227 48/203
components, including dietary fiber, minerals, and vitamins, as
Protein (g) 0.3 0.07
summarized in ● " Table 1. With the exception of protein levels,
Fat (g) 0.6 n. a.
fiber, and natural vitamin C contents, the average nutrient com- Carbohydrates (g) 11.4 11.1
position of apples and apple juice is quite similar [6]. Apples are Fibre (g) 2.0 0.77
a rich source of phenolic constituents, which are distributed in ● Pectin (g) 0.5 0.032
the peel, core and pulp [7], [8]. Content and composition of phe- Potassium (mg) 144 116
nolic compounds vary strongly in dependence of the apple vari- Calcium (mg) 7.0 4.2
ety, area of cultivation, and time and year of harvest [9], [10], Magnesium (mg) 6.0 6.9
[11], [12]. The total polyphenol content of apples represents Phosphorus (mg) 12.0 7.0
about 0.01 to 1 % of the fresh weight. Main structural classes in- Vitamin C (mg) 12.0 1.4
clude hydroxycinnamic acids, dihydrochalcones, flavonols Organic fruit acids (g) 0.5 0.74
1610 Review
Fig. 1 Chemical structures of selected typical ap-

ple juice components belonging to the structural
classes of hydroxycinnamic acids, dihydrochal-
cones, flavan-3-ols (catechins and procyanidins),
flavonols (quercetin-glycosides) and triterpenoids.
Table 2 Polyphenol content of apples and apple juice (mg/kg fresh weight or mg/L) (adapted from [12], [13])
Apples a Fresh juice b Commercial juice b
(n = 8) from dessert from cider clear (n = 3) cloudy

apples (n = 4) apples (n = 7) (n = 21)
Total polyphenols 662 –2119 154 – 178 261 – 970 110 – 173 152 – 459
● Hydroxycinnamic acids 45 – 384 57 – 68 134 – 593 69 – 122 74 – 259
● Dihydrochalcones 20 – 155 10 – 35 34 – 171 9 – 54 14 – 87
● Flavan-3-ols: Mono- and dimers 116 – 411 50 – 95 70 – 393 14 – 32 46 – 124
Oligomeric procyanidins 388 – 1622 n. d. n. d. n. d. n. d.
● Flavonols (quercetin-glycosides) 34 – 83 0.4 – 4 0.4 – 27 4–7 2 – 14
● Anthocyanins (in red apples) 0– 37 n. d. n. d. n. d. n. d.
a
From [13]. Values were multiplied × 10 to adjust for mg/kg.
b
From [12].
n. d. not determined
studies performed until 2003 [5]. They concluded that flavonoid will be further conjugated. Flavonoid glycosides may be absor-
aglycones apparently pass through epithelial cells where they bed intact in low levels. Mostly, they will be absorbed after hy-
Review 1611
drolysis by small intestinal hydrolases such as β-glucosidases or passage [47]. This observation was confirmed by Kahle et al.,
lactase phloridzin hydrolase, and also conjugated [5], [35], [36]. who detected about 90 % of the ingested OPC in ileostomy bags
According to a comprehensive review by Manach et al. on 97 bio- with a maximum 2 h after consumption of 1 L cloudy apple juice
availability studies with polyphenols in humans [34], metabo- [38]. Still, the mean degree of OPC polymerization was reduced
lites present in blood result from digestive and hepatic activity. from 5.7 (juice) to 3.4 within 2 h and further declined with
Plasma concentrations of total metabolites range from 0 to 4 μM time. Interestingly, polyphenols present in ileostomy bags still
after an intake of 50 mg aglycone equivalents, with relative uri- demonstrated antioxidant activity against peroxyl radicals and
nary excretion up to 43 % of the ingested dose, depending on the potently scavenged DPPH radicals after passage through the gas-
polyphenol. Overall, gallic acid and isoflavones (which are not trointestinal tract [48]. In vitro incubations with human colonic
present in apples) are the best absorbed polyphenols, followed microflora suggest that OPC will be catabolized into low molec-
by catechins and quercetin glucosides, but with different kinet- ular weight phenolic acids when they reach the colon [49]. Bio-
ics. The least well absorbed apple polyphenols are the procyani- logical properties of these metabolites should therefore be con-
dins and anthocyanins (if present). It was concluded that data sidered.
are still too limited for assessment of hydroxycinnamic acids Overall, these studies suggest that low molecular weight constit-
and other polyphenols. Most of the studies were performed uents in apple juice are likely to be absorbed and metabolized.
with purified compounds, and more research needs to address OPC are stable in the stomach and will reach the colon, were
questions of bioavailability from whole foods such as apples, they may exert a local effect before they are degraded by the mi-
which includes effects of food matrix, processing, digestion, and croflora.
interactions between different food components [5].
Kahle et al. performed an apple juice intervention study with
ileostomy patients [37], [38]. Colonic degradation is minimal in Bioactivities Indicative of Cancer Chemopreventive
these patients, therefore they represent an interesting study col- Potential: In Vitro Investigations
lective to investigate which portion of ingested polyphenols is !
absorbed and how much would reach the colon after ingestion. Carcinogenesis evolves through a network of events with multi-
Eleven volunteers drank 1 L of cloudy apple juice after an over- ple pathways which virtually all can be modulated exogenously
night fast. Ileostomy bags were collected immediately before [50]. Cancer preventive strategies include mechanisms to trap
and 1 – 8 h after apple juice consumption. Most of the ingested and remove carcinogens from the organism, thus minimizing
polyphenols were absorbed from or metabolized in the small in- their contact with DNA and reducing their genotoxic and promu-
testine. Maximum total recovery in the bags was after 2 h. Only tagenic action. Another approach is to prevent the activation of
0 – 33 % of the consumed hydroxycinnamic acids, and 10 % procarcinogens to ultimate carcinogens and to enhance their de-
(∼11 mg) of chlorogenic acid in particular, were found in ileosto- toxification through modulation of xenobiotic metabolism (anti-
my bags. In an earlier study conducted by Olthof et al., in which a initiating mechanisms). Further, chemopreventive agents may
dose of 1000 mg purified chlorogenic acid (representative of target cellular alterations associated with tumor promotion and
chlorogenic acid intake in coffee drinkers) was applied to ileos- progression by antioxidant and anti-inflammatory activity, in-
tomy patients, about 67 % were excreted in ileostomy bags with- hibition of signaling pathways which result in enhanced cell
in 24 h [39]. These differences may be due to the 10-fold higher proliferation, cell growth inhibitory mechanisms, and induction
absolute amount applied in this earlier study. The same group of programmed cell death. Recent research also indicates epige-
reported that in humans with intact colon, 50 % of ingested netic mechanisms and modulation of immune functions as nov-
chlorogenic acid was extensively metabolized to hippuric acid el targets of chemoprevention. Apple components have been
(N-benzoylglycine) by colonic microorganisms [40]. Similar re- shown to influence all of these mechanisms as summarized in
sults were observed in rats [41]. ●" Fig. 2.
In Kahle's ileostomy study with apple juice, recovery of querce-

tin glycosides in the ileostomy bags was extremely low [37], Antimutagenic potential
[38]. Only two of five derivatives present in the juice, i. e., quer- Some dietary fibers can act as scavengers of exogenous and en-
cetin 3-O-arabinoside and quercetin 3-O-rhamnoside, were de- dogenous mutagens [51]. Apple pectin and pectin from other
tected in the bags, and recovery was only 6 % and 10 % of the in- sources were effective against the direct-acting standard muta-
gested dose. This was in line with earlier studies by Walle et al. gen 1-nitropyrene, which represents the predominant nitrated
Their data from an ileostomy study suggested that quercetin polyaromatic hydrocarbon emitted in diesel exhaust [52]. In the
mono- and diglucosides provided by an onion meal were effi- classical Ames test, preincubation of Salmonella strains with ap-
ciently hydrolyzed in the small intestine by β-glucosidases to ple pectin before addition of the mutagen dose-dependently de-
quercetin which was then absorbed [42]. Interestingly, 23 % - creased the mutagenic activity. Pectins are characterized by a
81 % of 14C-labeled quercetin applied p. o. or i. v. to healthy volun- rhamnogalacturonan backbone structure with neutral side
teers was exhaled as 14CO2, indicating a complex metabolism of chains. Removal of the side chains by hydrolysis removed the an-
quercetin in humans [43]. timutagenic potential, indicating that side chains are a prerequi-
The metabolic fate of OPC is of particular interest due to high site for antimutagenic polysaccharides. Polymers with acidic
abundance in apple juice and other dietary sources [44], [45]. side chains were equally effective, whereas linear neutral poly-
Based on in vitro incubations to mimic gastric passage, Spencer mers or highly branched compounds were inactive [52].
et al. suspected that cleavage of higher OPC to mixtures of mono- Besides direct scavenging of mutagens, dietary fibers are sup-
mers and dimers in the stomach may enhance their absorption posed to act as antimutagens in the intestinal tract by increasing
in the small intestine [46]. However, Rios et al. demonstrated in fecal mass through their water-binding capacity, therefore dilut-
a human intervention study that OPC from cocoa, which are ing mutagen concentrations and increasing fecal transit time.
chemically very similar to apple OPC, were stable during gastric Ferguson et al. and Kestell et al. compared antimutagenic prop-
1612 Review
●
Fig. 2 Schematic presentation of colon carcinogenesis (left) and mechanisms relevant for the cancer preventive potential of apple constituents (right). OH ,
hydroxyl radicals; ROO , peroxyl radicals; O2 –, superoxide anion radicals; TSG, tumor supressor gene; HDAC, histone deacetylase.
● ●
erties of resistant starch vs. non-starch polysaccharides (NSP) gens capable of reacting with DNA and thus initiating carcino-
against the food-derived heterocyclic aromatic amine IQ (2-ami- genesis [55]. Phase 2 enzymes such as glutathione S-transferases
no-3-methylimidazo[4,5-f]quinoline). Apple pectin (10 % in the (GST) and sulfotransferases conjugate activated phase 1 metabo-
diet) was tested as an example of soluble dietary fiber of the lites and xenobiotics to endogenous ligands like glutathione,
NSP group. Both types of fiber significantly enhanced fecal bulk glucuronic, acetic, or sulfuric acid and enhance excretion and de-
and transit times. However, whereas resistant starch significant- toxification in form of these conjugates. Reduction of elevated
ly elevated IQ carcinogen bioavailability, non-starch polysac- phase 1 enzyme activities to physiological levels and enhancing
charides including apple pectin reduced IQ bioavailability and excretion of carcinogens via upregulation of phase 2 enzymes is
enhanced fecal excretion, thereby reducing the risk of mutage- considered a logical strategy in chemoprevention.
nicity. These differences were associated with distinct effects Pohl et al. investigated the effect of apple juice extracts on Cyp1A
on the expression of enzymes involved in the metabolism of IQ expression and activity in the Caco-2 colon cancer cell line and
[53], [54]. demonstrated a strong reduction at the mRNA, protein and ac-
tivity level [56]. Zessner et al. identified the flavonoid quercetin
Modulation of phase 1 and phase 2 carcinogen as the most potent inhibitor of Cyp1A activity from apple juice
metabolism extracts, with inhibitory potential in the nM range. The aglycone
Enzymes of the phase 1 of drug metabolism (cytochromes P450) was orders of magnitude more potent than its glycosides, but
activate xenobiotics by addition of functional groups which ren- overall, these were still more potent than hydroxycinnamates
der these compounds more water-soluble. Phase 1 functionali- and dihydrochalcones ([57], [58] and unpublished results).
zation may be required to efficiently detoxify carcinogens. How- Quercetin or 4-week intervention with quercetin-rich fruit juice
ever, carcinogens similar to benzo[α]pyrene [B(a)P], a planar reduced DNA adduct formation with B(a)P-diol epoxide, an ac-
polycyclic aromatic hydrocarbon, are capable of inducing the active metabolite of B(a)P formed via Cyp1A activity, in human
tivity of phase 1 enzymes such as cytochrome P450 1A (Cyp1A). lymphocytes in vitro and ex vivo [59], [60]. Besides quercetin,
This may further increase the risk to produce ultimate carcino- fractions of apple juice extracts containing OPC demonstrated
Review 1613
potent Cyp1A-inhibitory activity when enzyme activity was tes- colon cancer cells [74], and prevented Cr(VI)-induced lipid per-
ted in a cell-free assay with crude cell homogenates as enzyme oxidation, DNA damage and NF-κB activation in human lung ep-
source [58]. This may however be due to unspecific protein bind- ithelial A549 cells [75].
ing effects. It has been suggested that H2O2 and other non-genotoxic factors
Veeriah et al. focused on the induction of phase 2 enzymes by may contribute to tumor promotion by inhibition of gap-junc-
apple juice constituents. Using a microarray-based approach, tional intracellular communication (GJIC) [76]. Lee et al. repor-
treatment of the HT29 adenocarcinoma cell line with a mixture ted that apple extracts significantly prevented H2O2-mediated
of apple juice polyphenols for 24 h resulted in 1.6- to 2.1-fold in- inhibition of GJIC measured with WB-F344 rat liver epithelial
duction of mRNA levels of GSTP1, GSTT2 and MGST2, as well as cells by a scrape loading/dye transfer technique [77]. Treatment
of sulfotransferases CHST5, CHST6, and CHST7. At the same time, with 500 μM H2O2 reduced the number of communicating cells
mRNA expression of epoxide hydrolase, which contributes to the by about 90 %. Simultaneous treatment with increasing concen-
metabolic activation of B(a)P and other carcinogens, was signifi- trations of apple extract equivalent to 15 – 25 mg/mL fresh ap-
cantly reduced by 50 % [61]. Similarly, treatment of the preneo- ples increased cell-cell communication to control levels. Similar
plastic colon adenoma cell line LT97 with an apple juice extract effects were obtained with apple-derived flavonols (quercetin),
induced mRNA expression (measured by microarray analyses flavan-3-ols [(–)-epicatechin, procyanidin B2], and vitamin C,
and confirmed by quantitative RT-PCR) of selected phase 2 en- whereas chlorogenic acid and phloretin were inactive.
zymes (GSTP1, GSTT2, GSTA4, UGT1A1, UGT2B7) and total en-
zyme activities, indicating potential protection of the cells Anti-inflammatory mechanisms
against toxicological insults [62]. Using induction of NAD(P)H: Prostaglandins (PGs) are hormone-like endogenous mediators of
quinone oxidoreductase in Hepa1c1c7 mouse hepatoma cells as inflammation and are formed from arachidonic acid by cyclooxy-
a simple colorimetric test system to detect inducers of Phase 2 genase-1 (Cox-1) and the inducible form Cox-2, which is often
enzymes, we recently identified apple aroma compounds as elevated in tumor tissue. Excessive production of PGs is thought
novel inducers of phase 2 enzymes in polyphenolic apple juice to be a causative factor of cellular injury and may ultimately lead
extracts ([58] and manuscript in preparation]. to carcinogenesis by inhibition of apoptosis (programmed cell
death) as well as stimulation of cell proliferation, formation of
Antioxidant activities new blood vessels (angiogenesis) and tumor invasiveness [78].
Manifestation of oxidative stress by infections, immune diseases For activity-guided fractionation of apple juice extracts we used
and chronic inflammation has been associated with carcinogen- sheep seminal vesicle microsomes as a source of Cox-1 to detect
esis [63]. Overproduction of reactive oxygen species (ROS) may anti-inflammatory activity. Inhibition of Cox-1 activity was
lead to the formation of highly reactive oxidation products, acti- highest with all fractions containing (–)-epicatechin, which
vation of carcinogens, formation of oxidized DNA bases and DNA dose-dependently inhibited Cox-1 activity by 50 % at a concen-
strand breaks. These then cause mistakes during DNA replica- tration of 2.2 μg/mL (7.5 μM). OPC may also contribute to the
tion and genetic alterations, increased transformation frequen- anti-inflammatory potential [58].
cies, induced transcription of redox-regulated proteins and ulti- The transcription factor NF-κB plays an important role in the in-
mately result in enhanced cell proliferation and tumor promo- duction of proinflammatory enzymes including Cox-2 and the in-
tion/progression [63]. ducible nitric oxide synthase. NF-κB is inducible by the proinflam-
Eberhardt et al. demonstrated that radical scavenging activity of matory cytokine tumor necrosis factor-α (TNF-α), bacterial lipo-
fresh apples was mainly attributed to the phytochemical content polysaccharides, tumor promoter 12-O-tetradecanolyphorbol 13-
rather than to that of vitamin C [64]. Interestingly, several acetate (TPA) and other factors [79]. In a study by Davis et al., NF-
groups reported that highest antioxidant activity was associated κB was induced in human umbilical vein endothelial cells trans-
with apple peel rather than pulp, and identified quercetin-glyco- fected with a NF-κB-driven reporter construct. Pretreatment with
sides as active principle, which are mainly found in the skin [65], apple polyphenol extracts for 24 h significantly reduced TNF-α-
[66], [67], [68], [69]. A comprehensive comparison of radical mediated expression of the reporter gene [80]. Similarly, reduc-
scavenging activity of apple extracts, fractions and subfractions tion of nuclear NF-κB was observed when MCF-7 cells were pre-
with their phytochemical composition revealed that all major treated with apple extracts for 2 h and stimulated with TNF-α for
classes of apple phytochemicals contribute to antioxidant activ- 30 min [81]. Inhibition of proteasomal activity was identified as
ity against peroxyl radicals measured in the ORAC assay, where- the underlying mechanisms, which is important to release NF-κB
as DPPH (1,1-diphenyl-2-picrylhydrazyl) and superoxide anion from a complex with its inhibitor IκB in the cytosol.
radicals were potently scavenged by more lipophilic fractions
containing quercetin-glycosides and OPC [58]. Several other Inhibition of signaling pathways
studies demonstrated potent in vitro peroxyl radical scavenging Uncontrolled cell proliferation often involves disorganization of
potential and consequent inhibition of lipid peroxidation. Apple signaling pathways. Binding of growth factors (e. g., epidermal
extracts and individual polyphenols inhibited copper-induced growth factor EGF) to their receptors located in the cell mem-
oxidation of human low density lipoproteins, lipid peroxidation brane (e. g., epidermal growth factor receptor EGFR) stimulates
induced in rat liver microsomes by ascorbic acid and FeSO4, and signal transduction cascades. Growth-stimulating signals are
peroxidation of linoleic acid in a micellar system [18], [70], [71]. transduced to the nucleus via phosphorylation/activation steps
In cell culture, apple juice extracts and polyphenols from apple mediated by protein kinase cascades (e. g., Ras/Raf/MAP kinase
pomace also reduced oxidative DNA damage induced by mena- cascade), resulting in activation or repression of gene transcrip-
dione or H2O2 treatment in colon cancer cell lines, and reduced tion and consequently up- or down-regulated protein expres-
tert-butylhydroperoxide-induced intracellular ROS measured sion. Consequently, overexpression of hormone/growth factors
by 2′,7′-dichlorofluorescin oxidation [72], [73]. Apple polyphe- and their receptors might present a growth advantage to preneo-
nol extracts protected from H2O2-induced cytotoxicity in Caco-2 plastic cells [82], [83], [84].
1614 Review
Kern et al. [85] and Friedrich et al. [86] investigated the potential all, the adenoma cell line was more sensitive to the antiprolifer-
of apple juice extracts and apple juice polyphenols to influence ative action of the apple extracts than the HT29 cell line. The
EGF signaling. Apple juice extract effectively inhibited protein growth inhibitory potential increased with incubation time
tyrosine kinase activity of EGFR and suppressed EGFR autophos- with both cell lines. As anticipated by the higher content in fla-
phorylation and the subsequent MAP kinase cascade. Procyani- vonols, AE03 was more growth inhibitory than AE02 and AE04
din dimers B1 and B2 as well as two quercetin glycosides pos- [94]. When a native extract was compared with a composed
sessed substantial EGFR-inhibitory properties [85], [86]. Apple mixture of low molecular weight apple polyphenols (including
juice polyphenols also blocked the signaling cascade leading to flavan-3-ol mono- and dimers, but no OPC), the native extract
the induction of ornithine decarboxylase (ODC), which is essen- was about twice as potent as the mixture in inhibiting HT29
tial for cellular proliferation by formation of polyamines, but is cell growth. This indicated that OPC contribute to a substantial
often overexpressed in tumor cells [87]. An OPC-rich fraction P part to the antiproliferative activity of apple extracts [61].
(Fr. P) potently inhibited protein kinase C (PKC) activity by 70 % Anaerobic fermentation of the three apple extracts with human
in human colon cancer-derived SW620 cells. This was associated fecal slurries for 24 h resulted in the generation of mM concen-
with downregulation of polyamine biosynthesis and activation tration of short-chain fatty acids (SCFA) (also compare [95]).
of apoptosis [87]. Inhibition of cytosolic PKC activity in a cell- SCFA production from AE02 was in the order of acetate (max.
free system, but not in intact HT29 cells was also shown by 29 mM) > propionate (max. 6 mM) > butyrate (max. 5 mM). Al-
Kern et al. [88]. though SCFA and particularly butyrate have been associated
Kern et al. also investigated the effect of apple juice extracts on with inhibition of cell proliferation by induction of cell differen-
key elements of the Wnt signaling pathway, which is often acti- tiation and apoptosis [96], the antiproliferative potential of all
vated in colon carcinogenesis through mutation of the tumor three fermented extracts was considerably reduced in both cell
suppressor gene Apc (adenomatous polyposis coli). Apc protein lines after 24 – 48 h of incubation, and fermentation led to an al-
forms a complex with GSK3β (glycogen synthase kinase 3β) and most complete degradation of apple polyphenols. Low amounts
other factors and regulates levels of β-catenin, which otherwise of two metabolites, phloroglucinol and 3,4-dihydroxyphenyl-
accumulates, translocates to the nucleus and activates transcrip- propionic acid, were detected [94].
tion of growth promoting proteins. Mutation of Apc or inhibition Butyrate-mediated effects on cell proliferation have been associ-
of GSK3β by Wnt signaling disrupts this regulatory process [82]. ated with histone hyperacetylation due to inhibition of histone
Apple juice polyphenols inhibited GSK3β kinase activity in a deacetylase (HDAC) [97]. Waldecker et al. compared the poten-
cell-free system as well as in intact HT29 cells, but induced tial of apple juice extracts AJE03B and AJE04 ( = AE03 and AE04),
GSK3β protein expression measured by Western blotting. Over- fermented in the presence or absence of apple pectin, or pectin
all, the extract did not influence downstream signaling parame- alone, to inhibit proliferation and nuclear HDAC activity in HeLa
ters regulated by the Wnt signaling pathway [89]. Mad 38, HT29 and Caco-2 cells [95]. HeLa Mad 38 cells are stably
transfected with an HDAC inhibition-inducible reporter con-
Inhibition of cell proliferation by native and fermented struct. Significant induction of reporter gene activity was ob-
apple juice extracts served with fermentation supernatants of all samples. Fermen-
Multiple studies have demonstrated cell growth inhibitory po- tation supernatants of the extract + pectin combinations were
tential of apple juice components in cultured cancer cell lines. most active, although the butyrate content was lower than in
These results should be interpreted with some caution. Similar the fermented pectin sample. This indicated that additional
to green tea polyphenols [90], [91], apple polyphenols have HDAC inhibitors may have been formed during fermentation of
been shown to artefactually induce formation of hydrogen per- the apple juice extracts. Similar conclusions were drawn when
oxide in cell culture medium which could account for some or HDAC activity was directly measured with nuclear extracts of
all of the reported effects in cell culture. H2O2 formation by apple all three cell lines treated with increasing concentrations of fer-
phenolics was first described by Lapidot et al. in 2002 [92]. The mentation supernatants [95].
effect was particularly high in serum-free incubations, since se-
rum was able to decompose H2O2 due to residual enzyme activi- Induction of programmed cell death (apoptosis)
ty [92]. Fridrich et al. confirmed H2O2 production by apple juice Programmed cell death or apoptosis is a physiological process
extracts in cell culture media [86]. Janzowski et al. recently re- involved in the maintenance of multi-cellular organisms and
vealed that H2O2 formation only occurred in bicarbonate-buf- plays an important role in development, metamorphosis, hor-
fered solutions (C. Janzowski, personal communication). This monal atrophy and chemical-induced cell death [98]. Generally,
observation may explain why H2O2 was not detected in a study apoptosis can be induced by two major pathways: the extrinsic,
on polyphenolic apple extracts by Liu and Sun, who added death receptor-mediated pathway and the intrinsic, mitochon-
10 mM HEPES to cell culture media [93]. Overall, these findings drial-mediated activation [99]. Stimulation of the death receptor
should be taken into consideration when polyphenol extracts pathway leads to receptor aggregation, which then initiates re-
from apples or other sources are investigated in cell culture. cruitment and activation of initiator caspase-8. Caspase-8 acti-
Veeriah et al. compared antiproliferative activity of three apple vation subsequently triggers apoptosis by cleavage of down-
extracts in HT29 and the adenoma cell line LT97 [61], [94]. Ex- stream effector caspases. The mitochondrial pathway of cell
tracts were prepared either from apple juice (AE02 and AE04) death is mediated by Bcl-2 family proteins, which are a group of
or apple pomace after enzyme treatment to release cell wall anti- (e. g., Bcl-2, Bcl-xL) and proapoptotic proteins (e. g., Bax,
bound compounds (AE03), respectively, and had distinct poly- Bak). Bcl-2 family proteins regulate the passage of small mole-
phenol profiles (compare [28], AE02 = AS02, AE03 = AS03B and cules like cytochrome c through the mitochondrial permeability
AE04 = AS04). The pomace extract AE03 contained only 1/8 of transition pore (PTM). Release of cytochrome c then activates
the hydroxycinnamic acids and less total polyphenols, but about Apaf-1 (apoptotic protease activating factor 1), allowing it to as-
10-fold higher flavonol levels than the two juice extracts. Over- semble the multiprotein caspase-activating complex àpopto-
Review 1615
some' and to bind to and activate procaspase-9 and the down- Several investigators analyzed the cell growth inhibitory poten-
stream effector caspase cascade. Caspases are proteolytic en- tial of apple-derived OPC in various cancer cell lines. Raul and
zymes that are synthesized as enzymatically inert zymogens colleagues utilized the human cell line SW620 derived from a
and act in a self-amplification cascade. Initiator caspases-8 and lymph node metastasis of a colon adenocarcinoma patient to
-9 are characterized by longer pro-domains that mediate trans- identify links between polyamine metabolism and inhibition of
duction of death signals and assembly of activating complexes. cell proliferation by OPC. Fraction P (see Inhibition of signaling
The major effector caspases-3, -6 and -7 execute apoptosis by pathways) at 50 μg/mL accumulated SW620 cells in G2/M phase
cleavage of key cellular proteins that cause the typical morpho- of the cell cycle after 24 and 48 h of incubation, increased the
logical changes observed in cells undergoing apoptosis. Cleavage sub-G1 fraction indicative of apoptosis induction after 72 h, and
of the DNA repair-associated enzyme poly(ADP-ribose)polymer- activated caspase-3 activity [87]. Modulation of enzymes of the
ase (PARP) is accepted as a major marker of apoptosis induction. polyamine pathway led to an accumulation of N1-acetyl-poly-
The 26S proteasome system is a large protease complex that also amines. Cotreatment with an inhibitor of polyamine oxidase,
plays an important role in the regulation of cell growth and cell which metabolizes N1-acetyl-polyamines to polyamines, sensi-
death [100]. The proteasome controls the turnover of a variety of tized cells to Fr. P-induced cell growth inhibition [104]. The au-
intracellular regulatory proteins involved in cell cycle and apop- thors observed a significant reduction in total cellular polyamine
tosis. Short-term exposure to proteasome inhibitors protects pool, up-regulation of TRAIL (TNF-related apoptosis-inducing li-
cells from toxic stimuli; long-term exposure however is toxic to gand)-responsive death receptors DR4/DR5, and inhibition of
nearly all cells and is associated with induction of apoptosis. nuclear HDAC activity [105]. This was of interest as SW620 cells
Based on earlier results with (–)-epigallocatechin 3-gallate are usually TRAIL resistant. In contrast, apoptosis induction by
(EGCG) from green tea (review in [101]), Chen et al. investigated Fr. P alone involved depolarization of the mitochondrial mem-
the influence of apples and other fruit and vegetables on the 26S brane potential and induction of the intrinsic mitochondrial-
proteasome as a mechanism to inhibit cell proliferation [102]. In mediated apoptosis pathway [105]. Similarly, Miura et al. repor-
a cell-free system, an apple “extract” consisting of freshly pre- ted an increase in mitochondrial membrane permeability, re-
pared, centrifuged, and sterile filtered apple juice, inhibited chy- lease of cytochrome c, and activation of caspase-9 and -3 by ap-
motrypsin-like activity of the proteasome when added at 1 – 10 % ple procyanidins in B16 mouse melanoma cells and BALB-
(v/v) concentrations to the incubation mixture. Grape “extract” MC.E12 mouse mammary tumor cells [106]. Procyanidin di- and
was about equally active, whereas green tea was more potent trimers induced DNA laddering as a sign of apoptosis in KATO III
with up to 96 % inhibition at the same test concentrations. In a human stomach cancer cells, although at extremely high con-
cellular system with intact leukemic Jurkat T cells, 5 % green tea, centrations of up to 5 mg/mL [107]. Since DNA fragmentation
apple or grape “extract” all resulted in the accumulation of ubi- was caspase-independent and prevented by co-treatment with
quitinated proteins (as a sign of intracellular proteasome inhibi- the antioxidant N-acetylcysteine, these results may be caused
tion) concomitant with activation of effector caspase-3/-7 and by artifactual H2O2 formation as outlined above.
induction of PARP cleavage. These results indicate that inhibi- He and Liu reported that in addition to polyphenols [64], triter-
tion of chymotrypsin-like activity of the proteasome can be re- penoids isolated from apple peel inhibited proliferation of
garded as an interesting novel mechanism of apple and grape HepG2 human hepatoma cells, MCF-7 human breast cancer
“extract” contributing to apoptosis induction [102]. cells, and Caco-2 human colon cancer cells and may contribute
Kern et al. analyzed the potential of apple juice polyphenol ex- to the anticancer activity of apples [30]. Overall, 2α-hydroxyur-
tract AE02 (see Inhibition of cell proliferation) to induce apoptosis solic acid had the highest anti-proliferative activity and was
in HT29 cells [88]. The AE02 extract potently induced caspase-3 more active than the most abundant ursolic acid (● " Fig. 1). Of
activity and DNA fragmentation measured by an ELISA assay, al- note, ursolic acid and other pentacyclic triterpene acids have
though at relatively high concentrations. Since these experi- been associated with cancer preventive mechanisms at all stages
ments were performed under serum-free conditions without ad- of tumorigenesis and may have antimetastatic potential by in-
dition of catalase, formation of H2O2 may be responsible for part hibition of angiogenesis and tumor invasion [108].
of these results. Apoptosis induction by AE02 was also detected
by PARP cleavage under normal cell culture conditions in the Recent novel mechanisms of apple polyphenols
presence of 10 % fetal calf serum (FCS). PAPR cleavage was dose- Density-enhanced protein-tyrosine phosphatase-1 (DEP-1) has
dependent and increased up to 72 h [88]. Quercetin and phlore- been recognized as a candidate tumor suppressor protein in co-
tin dose-dependently induced both caspase-3 activity and DNA lon epithelium and reduces cell proliferation and cell migration
cleavage under serum-free conditions, whereas phloridzin [109]. Apple juice extract induced DEP-1 mRNA and protein ex-
(phloretin-2'-glucoside), which is present in apples and apple pression in LT97 colon adenoma cells, whereas protein expres-
juice/extract (●" Fig. 1), was basically inactive. The apoptosis in- sion was reduced in HT29 and Caco-2 cells. In contrast, butyrate
ducing potential of the aglycone phloretin in HT29 cells was fur- and green tea extract stimulated DEP-1 expression in all three
ther investigated by Park et al. [103]. Under serum-deprived cell lines. It was postulated that DEP-1-mediated regulation of
conditions (1 % FCS), phloretin at 100 μM induced both the cell proliferation might represent a hitherto unrecognized
death-receptor as well as the mitochondrial pathway of apopto- mechanism of cell growth inhibition by dietary nutrients [109].
sis induction, detected by activation of the initiator caspases-8 Epigenetic events such as the methylation of CpG rich sequences
and -9 and the effector caspases-3 and -7 as well as by PARP in gene promoter regions increase with increasing stage of ma-
cleavage. Activation of caspase-9 was accompanied by release lignancy in various human cancers, and often result in silencing
of cytochrome c and the mitochondrial protein Smac/Diablo of tumor suppressor genes [110], [111]. Promoter hypermethyla-
from the mitochondria to the cytoplasm, and upregulation of tion has been identified as a very early event in carcinogenesis.
proapoptotic Bax levels [103]. Consequently, development of agents or food components that
prevent or reverse the hypermethylation-induced inactivation
1616 Review
of tumor suppressor genes is an attractive approach for cancer ficacy can only be demonstrated in animal models or human in-
prevention. EGCG from green tea and genistein from soybean tervention studies with tumor incidence and multiplicity (e. g.,
have been demonstrated to inhibit DNA methyltransferases number of tumors per animal) as endpoints. When considering
DNMT in vitro. This was associated with the reactivation of application of a compound or product for prevention of cancer
methylation-silenced genes such as p16INK4a[112]. Fini et al. re- in humans, toxicological and safety issues also have to be con-
cently discovered that treatment of the human colon cancer cell sidered.
line RKO with an apple polyphenol extract resulted in demethyl- One study has addressed toxicology and safety of a polyphenol-
ation of the DNA repair gene hMLH1, analyzed by methylation rich extract from unripe apples (Applephenon®) which is sold in
specific PCR. This was confirmed at the mRNA and protein level. Japan as a food additive and nutritional supplement. The prod-
Similarly, reversal of methylation in promoter regions of the tu- uct contains high levels of OPC (64 %, dimers to 15-mers), 12 %
mor supresssor genes p14ARF and p16INK4a was demonstrated by flavan-3-ol monomers, 7 % flavonoids, and 18 % non-flavonoids
COBRA (combined bisulfite restriction analysis). Re-expression [118]. One gram of extract was reported to contain polyphenols
of mRNA of both genes was detected in RKO and SW48 cells. equivalent to approximately four apples [119]. In the Ames mu-
Post-translational inhibition of expression of the two main DNA tagenicity test, only one out of five bacterial strains showed a
methyltransferases, DNMT-1 and DNMT-3b, was postulated as slight increase in revertants indicative of mutagenic potential.
an underlying mechanism for the inhibitory effect of the apple No signs of mutagenicity were detected in the chromosomal
polyphenol extract [113]. aberration test in Chinese hamster lung cell culture and the mi-
γδT cells are innate immune cells that participate in host respon- cronucleus test in Sprague-Dawley rats. Also, no signs of toxicity
ses against many pathogens and cancers. Recent evidence has at a dose of 2000 mg/kg body weight (bw) were observed in an
accumulated that dietary factors may strengthen the immune acute and subchronic toxicity test. The extract was therefore re-
system by activation of these cells, and that this may contribute garded as safe [120].
to cancer preventive potential [114]. Human γδT cells can be As a first indication of cancer chemopreventive efficacy in vivo,
categorized into two distinct populations, Vδ1 and Vδ2 T cells, apple products have been tested in experimental animal models
based on the expression of cell surface receptors. Vδ2 T cells con- for chemically- or genetically-induced tumors of the skin, breast,
stitute the majority of γδT cells in peripheral blood and the lym- and colon, as well as in xenograft models for solid tumors and
phatic system and are potent antimicrobial and antitumor effec- melanoma.
tor cells, whereas Vδ1 T cells are located preferentially in skin ep- Oral administration of aqueous apple peel extracts (derived from
ithelium and in the intestine and have immune regulatory func- 1 mL water/g apple peel, given ad libitum) significantly reduced
tions [115]. Holderness et al. screened a library of >100.000 nat- the number of 7,12-dimethylbenz[a]anthracene (DMBA)-initi-
ural products including nutritional supplements to identify nov- ated and TPA-induced mouse skin papillomas by 55 %. The effect
el agonist of γδT cells, based on up-regulation of IL-2Rα (interleu- was explained by antioxidant properties which may block ROS-
kin-2 receptor α-chain) as a marker of γδT cell activation. Treat- mediated signal transduction pathways via MAP-kinase cascade
ment of bovine and human peripheral blood mononuclear cells and transcription factor AP-1 [121]. Liu et al. investigated the po-
(PBMC) with a water-soluble extract from peels of unripe apples tential of a polyphenol-enriched apple extract on DMBA-in-
at a concentration of 10 μg/mL induced IL-2Rα immune-positiv- duced mammary carcinogenesis in rats. Application by gavage
ity. The activity was linked to a polymeric fraction of condensed of 9 – 54.4 mg extract (equivalent to 3.3 – 20 g apples) per kg bw
tannins (= oligo- and polymeric proanthocyanidins). In human two weeks prior to and for 24 weeks after carcinogen treatment
PBMC, both Vδ1 and Vδ2 T cells were equally activated by the lowered the number of tumor-bearing animals dose-dependent-
tannin fraction, in addition to non-γδT cells including natural ly by 17 %, 39 %, and 44 %. Tumor numbers per animal were also
killer cells, natural killer T-cells and αβT cells, but not B cells. In- reduced by 25 %, 25 %, and 61 % after 24 weeks [122]. In a rat xen-
terestingly, immune-modulatory response was not limited to ograft model for tumor invasion and metastases with AH109A
lymphocytes in vitro. Akiama et al. demonstrated that mucosal rat ascites hepatoma cells, a commercially available apple poly-
γδT cells in the small intestine of mice expanded in response to phenol extract from unripe apples was tested. Intervention with
polyphenols from unripe apples. In this study, the results were the extract (added to the chow at 0.3 and 1 % concentrations) for
discussed in relation to prevention of food allergies [116]. 21 days after tumor cell inoculation significantly reduced the
(Over)-activation of immune cells may not always improve weight of solid tumors by 64 % and 58 %. In addition, numbers of
health. As an example, over-activated γδT cells have been associ- lung and lymphatic node metastases were strongly reduced
ated with inflammatory or celiac bowel disease [114]. In line from 17 per 10 rats to 1 per 10 rats in the two extract groups
with these observations, it was shown recently that the apple [123]. Apple polyphenols and OPC (both applied at 1 % in drink-
tannin fraction increased expression of the cell surface receptor ing water) also inhibited the growth of transplanted B16 mouse
CD11b, which is involved in leukocyte adhesion and migration melanoma cells in vivo, and increased the survival rate of the
[117]. This may be related with a pro-inflammatory rather than host mice transplanted with B16 cells [106].
an anti-inflammatory response to the extract. Overall, the phys- Several groups investigated the potential of various apple products
iological consequences of these novel observations need further [clear and cloudy apple juice, apple polyphenol extract (APE), OPC,
investigation. apple pectin] to prevent colon carcinogenesis (● " Table 3). Barth et
al. compared the effects of clear and cloudy apple juice in a rat
model for chemically-induced colon carcinogenesis using 1,2-di-
Apples and Cancer Prevention: In Vivo methylhydrazine (DMH) as a carcinogen [124]. After intervention
Investigations in Animal Models for eight weeks, cloudy apple juice was more potent in inhibiting
! carcinogen-induced epithelial cell proliferation and DNA damage
In vitro studies on molecular mechanisms will provide a hint to than clear apple juice. Also, cloudy apple juice reduced the number
potential cancer preventive effects in vivo. Chemopreventive ef- of aberrant crypt foci (ACF) as a pre-neoplastic marker for colon
Review 1617
Table 3 Summary of colon cancer preventive effects of apple products in animal models
Model (carcinogen) a Intervention, dose Duration b Endpoint, resultsc Ref.

F344 rats (DMH) ● clear apple juice (ad lib.) (37.9 mg 7 w (b-p) ● genetic damage 21 % ↓ , [124]
(male, 118 g bw) polyphenols d and 22.6 mg pectin/kg proliferation 45 % ↓
bw/day)
● cloudy apple juice (ad lib.) (40.7 mg 7 w (b-p) ● genetic damage 72 % ↓ *,
polyphenols d and 91.4 mg pectin/kg proliferation 72 % ↓ *, ACF/colon 19 %
bw/day) ↓ , AC/colon 29 % ↓
F344 rats (DMH) ● cloudy apple juice (ad lib.) (39.3 mg 7 w (b-p) ● genetic damage 77 % ↓ *, [126]
(male, 100 g bw) polyphenols d and 88 mg pectin/kg proliferation 74 % ↓ *,
bw/day) ACF/colon 15 % ↓,
large ACF/colon 35 % ↓
● APE (39.5 mg polyphenols d/kg bw/ 7 w (b-p) ● genetic damage 20 % ↓ ,
day) proliferation 34 % ↓
● cloud fraction (dissolved at 0.75 g/L) 7 w (b-p) ● genetic damage 32 % ↓ ,
proliferation 45 % ↓
● APE and cloud fraction combined 7 w (b-p) ● genetic damage 53 % ↓ ,
proliferation 34 % ↓
Wistar rats (AOM) ● procyanidin fraction P with 78.4 % 6 w (p) ● ACF/colon 50 % ↓* [87]
(male, 230 – 245 g bw) OPC (0.01 % in drinking water)
Donryu rats (AOM) ● apple pectin (20 % in the diet) 32 w (b-p) ● carcinoma incidence 75 % ↓*, [127]
(male, 200 g bw, 4 w) carcinoma multiplicity 72 % ↓*
● body weight (at 30 w) 15 % ↓ *,
fecal weight 48 %ñ*

C57BL/6 APC Min/+ mice ● cloudy apple juice (ad lib.) 10 w ● small intestinal adenoma 38 % ↓* [128]
(male, 7 w) (14 mg polyphenols d/kg bw/day)
● APE (0.2 % in drinking water) 10 w ● small intestinal adenoma 40 % ↓*,
(70 mg polyphenols d/kg bw/day) hematocrit ñ*, spleen weights ↓ *
C57BL/6 APC Min/+ mice ● dehydrated apple pomace 8w ● small intestinal adenoma 132 %, [131]
(female, 4 w) (20 % in diet) small intestinal polyp burden
111 % ↑ *,
colon polyp diameter 40 % ↑*,
colon polyp burden 150 % ↑ *
a
DMH, 1,2-dimethylhydrazine; AOM, azoxymethane; bw, body weight; w, age in weeks.
b
w, weeks; dietary intervention before (b), during (d), post (p) carcinogen treatment.
c
inhibition ( ↓), induction (↑), *significant results. ACF, aberrant crypt foci; AC, aberrant crypts. Endpoints without effect of intervention are not shown.
d
OPC content was not considered.
carcinogenesis, whereas clear apple juice was ineffective. These re- tive effect. Rather, a transient effect on fecal bacterial enzyme ac-
sults may be explained by a higher content in procyanidins in tivities was discussed [127].
cloudy apple juice than in clear juice, as shown by Oszmianski et The C57BL/6-ApcMin (ApcMin) mouse strain commonly used in
al. [125] and Hümmer et al. [28], who recently also determined chemoprevention studies is a genetically engineered model that
that the cloud fraction responsible for the turbidity of cloudy apple develops multiple intestinal neoplasia. Ten weeks of interven-
juice contained up to 60% of oligomeric procyanidins (personal tion with both cloudy apple juice and a polyphenol-enriched ap-
communication). Results obtained with cloudy apple juice were ple juice extract (1 : 1 mix of AE02 and AE03 at 0.2 % in drinking
reproduced in a second investigation by Barth et al. [126]. In this water) in male ApcMin mice significantly lowered the number of
study, neither isolated APE nor the cloud fraction alone or in com- adenomas in the small intestine by 38 % and 40 %. Extract treat-
bination significantly reduced the number of ACF. The potentially ment also improved hematocrit values, which are reduced in
important role of OPC for colon cancer prevention was demonstra- ApcMin mice as a sign of intestinal bleeding. Also, spleen weights,
ted by Gosse et al. [87], [104], [105]. In the AOM (azoxymethane)- which were about 5-fold increased in ApcMin compared to wild-
induced rat model, a procyanidin-enriched fraction P from apples type mice, were significantly reduced by extract intervention
at a very low dose (0.01 % in drinking water) significantly reduced [128], [129]. In a recent study by Mandir et al., the influence of
the number of ACF/colon by 50 % [87]. In an earlier study by Ohka- dehydrated apple pomace was investigated in the ApcMin mouse
mi et al., addition of 20 % apple pectin to the diet significantly re- model. Apple pomace is a waste product of apple juice produc-
duced the incidence and multiplicity of AOM-induced colonic ade- tion and a good source of highly fermentable non-starch poly-
nomas and carcinomas in rats [127]. Concomitantly, body weights saccharides (23.9 g/100 g). At a dose of 20 % in the diet, apple po-
were significantly reduced by apple pectin intervention, although mace significantly enhanced the number of small intestinal pol-
the animals consumed the same amount of food/day as the control yps and polyp burden in female ApcMin mice. Also, colonic ade-
group (not adjusted for differences in available energy). Since noma size and burden was significantly enhanced. This was sur-
treatment with citrus pectin similarly reduced body weight in- prising, but is consistent with some earlier reports on fermenta-
crease, but not tumor numbers, the authors argued that reduction ble resistant carbohydrates. The results were explained by in-
of bw alone was not sufficient to explain the strong cancer preven- creased formation of short-chain fatty acids (SCFA), which may
1618 Review
stimulate intestinal cell growth [130]. However, other studies short chain fatty acids were significantly enhanced by all apple
have demonstrated a protective effect, e. g., with rye bran, indi- diets in comparison with the controls (190 μmol/cecum =
cating that the role of dietary fiber in colon cancer prevention is 84 mM) with highest concentrations of 560 μmol/cecum (=
still an area of controversial debate ([96], [131] and references 122 mM) in the combination group. Overall, the authors conclu-
cited therein). ded that the effect of apple pectin and the polyphenol-rich frac-
tion on large intestine fermentations and lipid metabolism was
enhanced when both fractions were fed in combination, sug-
In Vivo Effects of Apple-Derived Dietary Fiber in gesting interactions between fiber and polyphenols of apple
Animal Models [139].
!
Several short-term dietary intervention studies in rodents have
been performed to investigate the influence of apple-derived di- Human Short-Term Intervention Studies:
etary fiber and cell wall components on intestinal fermentation Modulation of Antioxidant Status and Markers of
products, fecal steroids and serum lipids. This was of interest Oxidative Stress
since, e. g., the ratio of the secondary bile acids lithocholic acid !
to deoxycholic acid, formed by bacterial metabolism from pri- In vitro, apple polyphenols have been identified as potent radi-
mary bile acids synthesized in the liver, is regarded as a risk in- cal-scavengers and antioxidants (see above). But are these
dex for colorectal cancer [132]. Shimizu et al. examined the ef- mechanisms effectively translated to the in vivo situation? Al-
fect of a commercially available apple pulp powder low in fiber though animal studies have indicated cancer preventive efficacy
(32.8 %) and rich in carbohydrates (59.8 %, dextrin) on fecal ste- of apple products, extrapolation of the results to the human sit-
roid profiles in rats [133]. The apple powder was added at a con- uation is difficult. In humans, exposure to low doses of endoge-
centration of 15.2 % to the diet and fed for three weeks. The inter- nous or exogenous carcinogens and tumor promoters may occur
vention significantly reduced serum triacylglycerol and serum constantly and life-long. In addition, genetic polymorphisms,
and liver phospholipid levels in comparison with a cellulose con- variations in DNA methylation and epigenomic events may in-
trol group. Excretion of total and several major bile acids was sig- fluence the response of humans to carcinogens and protective
nificantly increased, whereas the ratio between secondary bile agents [31]. Consequently, proof of cancer preventive efficacy in
acids and total bile acids was significantly reduced by the apple humans requires very large and long-lasting controlled clinical
pulp powder [133]. trials.
So-called “enzymatic liquefaction” of apple pomace with cellu- Short-term human intervention studies can provide an indica-
lases and pectinases to produce pomace liquefaction juices (B- tion of potential health-promoting or cancer preventive activity
juices) may increase the extraction of valuable apple compo- based on the modulation of biomarkers. Several recent studies
nents including dietary fiber and polyphenols [134]. B-juices have focused on the modulation of antioxidant status and mark-
are rich in colloids composed of galacturonic acid (49 – ers of oxidative stress by consumption of apple and apple juice.
64 mol %), arabinose (14 – 23 mol %) and galactose (6 – 15 mol %), In a study by Ko et al., improved antioxidant capacity vs. hydrox-
with minor amounts of rhamnose, xylose, and glucose [135]. yl radicals was detected in serum of 10 healthy male volunteers
Sembries et al. compared the influence of colloids from B-juices 30 min after consumption of 150 mL apple juice. Apple juice was
and an àlcohol-insoluble substance' (AIS) on SCFA profiles and compared with a variety of fruit juices. Orange juice provided
intestinal microbiota in rats. Similar to the results with apple the best antioxidant effect, whereas pear juice was inactive
pectin [127] (see above), animals fed for 6 weeks with 5 % B-juice [140]. Similarly, one serving of 1 L of apple juice caused a signifi-
colloids gained about 25 % less weight than control rats, al- cant increase of serum DPPH radical scavenging activity in 12
though food consumption was not significantly different. Both healthy subjects 1 h after juice ingestion [141]. In a study con-
interventions significantly lowered luminal pH values and in- ducted by Maffei et al., 6 healthy, non-smoking male volunteers
creased the weight of cecal contents. Apple colloids enhanced consumed a homogenate obtained from 600 g unpeeled apples.
total SCFA, acetate, and propionate concentrations in cecum Results indicated a significant inhibition of H2O2-induced micro-
and distal colon, whereas AIS also increased butyrate levels. nuclei frequency in lymphocytes collected at 3 h after apple con-
This was attributed to cellulose present only in AIS, but not apple sumption, compared with samples at 0 h. Values gradually re-
colloids. AIS intervention enhanced the numbers of cecal micro- turned to baseline starting from 6 to 24 h [142]. In line with
biota of the Eubacterium rectale cluster. In contrast, apple col- these observations, Briviba et al. reported that consumption of
loids increased fecal concentrations of Bacteriodaceae [136]. 1 kg organically or conventionally grown apples once by 10
Similar to the results of Shimizu et al. [133], apple colloids and healthy male volunteers did not change antioxidant capacity in
AIS also increased excretion of primary bile acids in feces up to plasma, endogenous DNA strand breaks, and protection from
30 % and 88 %, whereas concentrations of secondary bile acids H2O2-induced DNA damage in lymphocytes when measured
were reduced [137]. Analogous effects were obtained when dilu- 24 h after consumption. However, lymphocyte DNA had lower
ted B-juice was applied to rats directly as a drink, suggesting that levels of so called Endo-III sensitive sites (specific for oxidized
administration of extraction juices enriched in phytochemicals pyrimidines) and was protected from hydroxyl radicals. The
and dietary fiber resulted in beneficial nutritional effects [138]. type of apple production had no influence on polyphenol levels
These are promising results, but long-term cancer preventive and any biological effect measured in this study [143]. Mayer et
effects of B-juice colloids need to be investigated. al. used a high-throughput fluorescence screening method to
The effect of apple components on cecal fermentations and lipid measure antioxidative capacity in human serum [144]. Two flu-
metabolism was also tested by Aprikian et al. [139]. Rats were orophores were developed as hydrophilic and hydrophobic oxi-
fed with diets supplemented with 5 % apple pectin, 10 % high- dation markers for the aqueous and lipid phase of serum. Forty-
polyphenol freeze-dried apple, or both, for three weeks. Cecal seven healthy human volunteers consumed 1 kg of apples daily
Review 1619
for four days, providing 2.7 g total phenolics/kg fresh apples. Ap- not. Case-control studies acquire data on food consumption etc.
ple consumption increased the anti-oxdidant capacity in the retrospectively and are more prone to errors. The results are ex-
aqueous phase, but not in the lipophilic phase 3 h after the first pressed as an odds ratio (OR), defined as the ratio of the odds of
apple consumption. The effect was only transient and did not in- an event (here: apple-eating) occurring in the group of cancer
crease with longer apple intake for four days [144]. Consumption cases, to the odds of it occurring in the control group. OR = 1.0
of a blueberry/apple juice mixture (1 L daily, providing an extra would indicate that apple consumption is equal in both groups,
18 mg quercetin) for four weeks significantly increased the total whereas an OR < 1.0 would demonstrate that the control group is
antioxidant capacity in plasma of 8 female volunteers. Also, more likely to eat apples than the group of cases. This observa-
quercetin plasma levels increased significantly from 1.5 ng/mL tion would suggest (but not prove) that regular apple consump-
plasma (5.0 nM) before intervention to 3.1 ng/mL (10.6 nM) at tion may be linked to a reduced risk of getting cancer.
the end of the study [59]. This study was followed by a larger Epidemiological evidence accumulated over the past years
scale study with 168 subjects, who consumed a blueberry/apple points to the cancer preventive potential of apples especially for
juice mixture (1 L daily, providing an extra 97 mg quercetin) for lung and colorectal cancer. The results of several cohort and
four weeks. In this follow-up study, analysis of effects of 34 ge- case-control studies are summarized in ● " Table 4. In the Nurses'
netic polymorphisms on lymphocytic DNA damage was inclu- Health Study (NHS), a large prospective cohort study conducted
ded. Plasma concentrations of quercetin and ascorbic acid, and in the USA, a significant reduced risk for lung cancer was ob-
antioxidant capacity were significantly increased. Lymphocytic served among the women, while no effect was seen among men
DNA was protected against ex vivo H2O2-induced oxidative DNA in the Health Professionals' Follow-up Study [149]. The Zutphen
damage, whereas levels of ex vivo induced B(a)P-diol epoxide- Elderly Study aimed at identifying risk factors for chronic disea-
DNA adducts were 28 % increased upon intervention. Six genetic ses in elderly men in The Netherlands. Uptake of apples was non-
polymorphisms significantly influenced the outcome of the in- significantly related with reduced lung cancer risk, whereas tea
tervention [NQO1*2 → quercetin levels; Cat1 → vitamin C levels; consumption as the major source (87 %) of catechins had no pro-
GSTT1 deletion → plasma antioxidant capacity and levels of in- tective effect [150]. Also, in a large Finnish cohort study, the risk
duced oxidative damage in lymphocytes; Cyp1B1*5 and COMT1 for lung cancer was significantly reduced by 60 % in men who ate
→ B(a)P-diole epoxide-DNA adduct levels] [60]. most apples (>47 g/d) compared to those who did not eat apples
Overall, these studies suggest that apple or apple juice consump- at all [151]. Finally, statistically significant inverse associations
tion results in a brief, transient increase in plasma antioxidant between lung cancer risk and onions and apples as the main
capacity 0.5 to 3 h after consumption. Eventually, longer contin- food source of the flavonoid quercetin were found in a case-con-
uous exposure for 2 – 4 weeks is required to obtain a sustained trol study conducted in Hawaii. No significant differences were
increase. Based on reports of Lotito and Frei, these results have observed when apple consumption was related to the most com-
to be interpreted with caution. In a series of in vitro, ex vivo and mon lung cancer cell types, i. e. squamous cell carcinoma and
in vivo studies they demonstrated that the increase in human adenocarcinoma [152].
plasma antioxidant capacity after apple consumption is not In addition to the consistent association with lung cancer pre-
caused by apple-derived antioxidants, but most likely due to a vention, recent publications also indicate preventive effects of
metabolic effect of fructose, which is provided by apple products apple consumption on colorectal carcinogenesis. In the NHS,
in large quantities, on urate, an important endogenous antioxi- those 20 % of women who consumed the most apples had a sig-
dant in plasma [145], [146], [147]. nificantly reduced risk of developing colorectal adenomas in
comparison to the 20 % with the lowest intake [153]. In a case-
control study conducted in Uruguay, apple consumption was as-
Apple Consumption and Cancer Incidence in sociated with a significant, dose-dependent reduction in colo-
Humans: Epidemiological Evidence rectal cancer risk in men and women [154]. In a South-Korean
! case-control study, fruit consumption (apples combined with
Observations of the eating behavior of the general public in ret- banana, pear and watermelon) lowered the risk for colon cancer
ro- or prospective epidemiological studies allow to draw conclu- in men, but not in women [155]. A meta-analysis of multicenter
sions on relations between consumption of specific food items case control studies conducted in Italy revealed that consump-
and cancer risk [148]. Best evidence comes from large scale pro- tion of ≥1 apple/day in comparison with ≤ 1 apple/day signifi-
spective cohort studies, which select a collective of healthy peo- cantly reduced the odds ratio (OR) for colorectal cancer as well
ple and regularly interview for dietary habits and cancer inci- as for cancers of the oral cavity (OR 0.79, 95 % CI 0.62 – 1.0), lar-
dence over a long period of time. These studies permit epidemi- ynx (OR 0.58, 95 % CI 0.44 – 0.76), breast (OR 0.82, 95 % CI 0.73 –
ologists to calculate a relative risk (RR), that is the ratio of the 0.92) and ovary (OR 0.85, 95 % CI 0.72 – 1.0) [156]. A recent case-
probability of cancer occurring in the exposed (here: apple-eat- control study from Scotland did not find a statistically significant
ing) group versus the non-exposed group (here: no or very low association between apple consumption and colon cancer risk
apple consumption). The collective can be divided in a number [157]. High apple consumption (> 94 g/day) was associated with
of subgroups with increasing exposure (mostly 3, tertiles; 4, a reduced renal cancer risk. The reduction was particularly
quartiles; 5 quintiles), and the RR is calculated for each sub- strong for the 10 % of people who ate the most apples and for
group. This allows evaluation of dose-response relationships. non-smokers, whereas no effect was seen in smokers [158].
RR values can be > 1.0 within a certain confidence interval (CI),
indicating an increased risk, or < 1.0, indicating protection. Only
studies with a CI not including 1.0 are considered as statistically Summary and Conclusions
significant. !
Case-control studies compare a group of patients who have a Apples are a rich source of phytochemicals (polyphenols, triter-
disease with a group of patients (or healthy controls) who do penoids) and dietary fiber, which have been associated with can-
1620 Review
Table 4 Epidemiological cohort and case-control studies on apple consumption and cancer risk
Type of cancer Type Study No. of Years of Effect of apple consumption Ref.
of study population cancer follow-up
cases
Lung cancer
Nurses' Health Study cohort 77283 women 519 12 y For increases of one serving of [149]
(NHS) apples and pears, RR = 0.63; 95 %
CI = 0.43 – 0.91
Health Professionals' cohort 47778 men 274 10 y no effect [149]
Follow-up Study
(HPFS)
Zuthphen Elderly cohort 728 men 42 10 y Catechins from apples account for [150]
Study 8 % of total catechin intake. RR for
7.5 mg increase in catechin from
apples = 0.67, 95 % CI = 0.38 – 1.17
Finnish study cohort 5218 men 169 max. 30 y RR for highest (> 47 g/d) vs. lowest [151]
(0 g/d) quartile: 0.4 (95 % CI = 0.22 –
0.74)
Hawaii study case-control 582 cases + 582 controls OR for highest (> 49.7 g/d) vs. [152]
lowest (< 2.3 g/d) quartile: 0.6,
95 % CI = 0.4 – 1.0 (P for trend 0.03)
Colorectal cancer
Nurses' Health Study cohort 34467 women 1 720 18 y OR for adenoma prevalence [153]
(NHS) comparing highest vs. lowest
quintile: 0.83, 95 % CI = 0.7 – 0.98
(P for trend 0.05)
Uruguay case-control 160 cases + 287 hospital controls OR for highest vs. lowest tertile: [154]
0.4, 95 % CI = 0.25 – 0.66
(P for trend <0.001)
South-Korea case-control 162 cases + 2 576 hospital controls OR for highest vs. lowest quartile [155]
(combined with banana, pear and
watermelon): 0.36, 95 % CI = 0.16 –
0.84 (only in men)
Italy (meta-analysis) case-control 1953 cases + 4154 hospital controls OR for ≥ 1 apple/d vs. <1 apple/d: [156]
0.8, 95 % CI = 0.71 – 0.9
Scotland case-control 1456 cases + 1456 population-based controls OR for highest vs. lowest quartiles: [157]
0.94, 95 % CI = 0.62 – 1.5
(P for trend 0.9)
Renal cancer
Sweden case-control 379 cases + 353 population-based controls OR for highest (> 94 g/d) vs. [158]
lowest (< 15 g/d) quartile:
0.65, 95 % CI = 0.43 – 0.98
(P for trend 0.02).
Top 10 % of consumption:
OR = 0.36, 95 % CI = 0.14 – 0.93
Non-smokers only: 175 cases + 191 controls OR for highest (> 94 g/d) vs. lowest
(< 15 g/d) quartile: 0.5, 95 %
CI = 0.28 – 0.88 (P for trend 0.01).
n. s. for smokers
Data were corrected for potential confounding factors, such as age, gender, smoking habits, alcohol consumption, medical treatments, and vitamin
supplementation as indicated in the original references. RR, relative risk; OR, odds ratio; 95 % CI, confidence interval. Only results with a 95 % CI not including 1.0 are
considered as statistically significant. n. s. not significant.
cer preventive mechanisms in in vitro studies. These include and modulation of immune functions as novel targets of apple
anti-mutagenic effects, enhanced detoxification through modu- products.
lation of xenobiotic metabolism, antioxidant effects (demonstra- Bioavailability studies have indicated that low molecular weight
ted in vitro and in vivo), anti-inflammatory activity by inhibition polyphenols of apples may be absorbed after hydrolysis and fur-
of Cox activity and NF-κB, inhibition of signaling pathways, in- ther conjugated. Overall, plasma levels appear to be low. Transi-
cluding the EGF/EGFR-mediated activation of the MAP kinase ent anti-oxidant activity 0.5 to 3 h after apple and apple juice
pathway, PKC and polyamine metabolism, and GSK3β involved consumption has been observed in human short-term interven-
in Wnt-signaling, cell growth inhibitory mechanisms, and in- tion studies. Application for 2 – 4 weeks may be required for sus-
duction of programmed cell death by activation of both the tained antioxidant effects. Some studies indicate that these anti-
death receptor and the mitochondrial pathway. Recent studies oxidant activities are more likely due to metabolic effects of
have identified chymotrypsin-like activity of the 26S protea- fructose than to polyphenolic antioxidants [147]. OPC, which
some, tumor suppressor protein DEP-1, epigenetic mechanisms, are the most abundant polyphenols in apples, are poorly absor-
Review 1621
bed, pass the stomach and will reach the colon, where they may 7 Thielen C, Will F, Zacharlas J, Dietrich H, Jacob H. Polyphenols in apples:
Distribution of polyphenols in apple tissue and comparison of fruit
exert local antioxidant, anti-proliferative, or immune modulat-
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Some studies indicate that combination of polyphenols and ap-
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the population, including children. Almost all studies summar- 19 Sanoner P, Guyot S, Marnet N, Molle D, Drilleau JP. Polyphenol profiles
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20 Alonso-Salces RM, Barranco A, Abad B, Berrueta LA, Gallo B, Vicente F.
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Acknowledgement
zones of a French cider apple variety (Malus domestica var. Kermer-
!
rien). J Agric Food Chem 1998; 46: 1698 – 705
I would like to thank the German Federal Ministry of Education 22 Guyot S, Marnet N, Drilleau J. Thiolysis-HPLC characterization of apple
and Research (BMBF) for financial support of our studies with procyanidins covering a large range of polymerization states. J Agric
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Cancer Chemopreventive Potential of Apples, Apple Juice, and Apple Components

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Cancer Chemopreventive Potential of Apples,

Author Clarissa Gerhauser

PGs: prostaglandins SCFA: short-chain fatty acids

Introduction (quercetin glycosides), catechins and oligomeric procyanidins,

Fig. 1 Chemical structures of selected typical ap-

Apples a Fresh juice b Commercial juice b

(n = 8) from dessert from cider clear (n = 3) cloudy

In Kahle's ileostomy study with apple juice, recovery of querce-

Model (carcinogen) a Intervention, dose Duration b Endpoint, resultsc Ref.

fecal weight 48 %ñ*

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