review-anticancer effect of emblica
TRANSCRIPT
Amla as an antineoplastic agent
Preclinical studies have shown that the aqueous extract of amla causes a concentration-
dependent cytotoxic effect on L 929 cells in vitro and that the IC50 was observed to be
16.5mg/ml (Jose et al., 2001). The extract also caused apoptosis in Dalton’s lymphoma
ascites and CeHa cell lines (Rajeshkumar et al., 2003). Khan et al. (2002) studied the
antiproliferative activity of the extract in the human tumor cell lines of different
histological orgins (human erythromyeloid K562, B-lymphoid Raji, T-lymphoid Jurkat,
erythroleukemic HEL) and observed it to be effective.
Recently, Ngamkitidechakul et al. (2010) have observed that the aqueous extract of amla,
which contains tannins(43%), uronic acid (11%), and gallic acid (21%), inhibitedthe
growth of A549 (lung), HepG2 (liver), HeLa (cervical),MDA-MB-231 (breast), SK-OV3
(ovarian), and SW620(colorectal) cells in vitro. However, at the same concentrationthe
extract did not cause similar level of cytotoxicityin the MRC5, normal lung fibroblast,
suggesting it to besafe for normal cells (Ngamkitidechakul et al., 2010). Theextract also
induced apoptosis in HeLa, A549, MDA-MB-231, and SK-OV3 cells (Ngamkitidechakul
et al., 2010).
An amla extract possesses antiproliferative activity inMCF7 and MDA-MB-231 breast
cancer cell lines andalso induces an increase in ERamRNA in these cells(Lambertini et
al., 2004). The extract was devoid ofcytotoxic effects on the normal Chinese hamster
ovarycell line, suggesting it to be selectively cytotoxic to onlyneoplastic cells (Sumantran
et al., 2007). Administeringthe extract to Dalton’s lymphoma-bearing mice caused
areduction in ascitic volume (when the tumor cells wereinoculated in the peritoneum) and
solid tumor growth(when inoculated subcutaneously). The amla extractsignificantly
reduced the solid tumors and prolongedsurvival time. At a molecular level, the extract
wasobserved to inhibit the cell cycle-regulating enzyme,Cdc25 phosphatase, in a dose-
dependent manner and theIC50 was observed to be 5 mg/ml (Jose et al., 2001).
Studies have also shown that some of the compoundspresent in amla are effective in
inhibiting the proliferationof neoplastic cells in vitro and also in tumor-bearinganimals.
The hydrolyzable tannins of amla are also reportedto possess selective cytotoxicity to the
human oralsquamous cell carcinoma and salivary gland tumor celllines, while they were
nontoxic to the normal humangingival fibroblasts. The dimeric compounds, oenotheinB,
woodfordin C, and woodfordin D, were more effectivethan the monomeric compounds,
while the macrocyclicellagitannin oligomers were more effective than gallicacid and
epigallocatechin gallate. These compounds alsoinduced apoptosis in the neoplastic cells
and mechanisticstudies showed that the effect was mediated by theprooxidant actions, but
not through the generation ofhydrogen peroxide (Sakagami et al., 2000).
Zhang et al. (2004) evaluated the antiproliferative effectsof 18 phytochemicals of amla
(norsesquiterpenoids,phenolic compounds, and proanthocyanidin polymers)in B16F10,
HeLa, and MK-1 cells in vitro. Among thenorsesquiterpenoids, it was observed that the
glycosidephyllaemblicins B and C were highly potent in all thethree cells [B16F10 (GI50
at 2.0, 3.5 mg/ml, respectively),HeLa (GI50 at 3.0, 12.0 mg/ml, respectively), and MK-
1(GI50 at 7.0 mg/ml for both compounds)]. However, withrespect to the phenolic
compounds, all showed inhibitoryactivity against the three tumor cell lines (at
aconcentration of <68 mg/ml), and were more effectiveagainst B16F10 than against HeLa
and MK-1 cells. Thehighest activity was observed with corilagin, geraniin,elaeocarpusin,
and prodelphinidins B1 and B2 againstB16F10 (Zhang et al., 2004).
Pyrogallol, a catechin compound of amla, is also reportedto possess a potent
antiproliferative effect on human lungcancer cell lines and, to a lesser degree, on the
humanbronchial epithelium cell line. Detailed studies with thehuman lung cancer cell
lines H441 (lung adenocarcinoma)and H520 (lung squamous cell carcinoma) haveshown
that pyrogallol inhibited the growth of these cells,triggered apoptosis by increasing Bax
and concomitantlydecreasing Bcl-2, arrested the cells in the G2/M phase byaffecting the
cyclin B1, Cdc25C and increasing thephosphorylation of Cdc2 (Thr14). The in-vitro
observationsalso extended into in-vivo studies with xenograftnude mice (Yang et al.,
2009).
Gallic acid, another chief constituent of amla, is alsoshown to cause a concentration- and
time-dependentinhibition of proliferation, and to induce apoptosis inBEL-7404 cells
(Zhong et al., 2009). Gallic acid is alsoshown to cause apoptosis in human non-small-cell
lungcancer NCI-H460 cells (Ji et al., 2009), A375.S2 humanmelanoma cells (Ji et al.,
2009), human bladder transitionalcarcinoma cell line (TSGH-8301 cell) (Lo et al.,2010)
and HeLa cervical cancer cells (You et al., 2010).Consuming gallic acid (0.3–1% in
drinking water)inhibited the growth of prostate cancer and retardedthe progression to
advanced-stage adenocarcinoma inmice with transgenic adenocarcinoma of the prostate
bysuppressing cell cycle progression and cell proliferationand, concomitantly, increasing
apoptosis (Raina et al.,2008). Gallic acid also suppressed lung xenograft tumorgrowth (Ji
et al., 2009). Some of the other phytochemicalssuch as quercetin and kampferol also
possess antineoplasticeffects in the various cultured cell lines (Table 2)and their presence
may have also resulted in the observedantineoplastic effect.
Chemomodulatory effects
Chemotherapy is known to possess deleterious effectson normal cells. At times, the
effects can be extremelysevere and can compel the physician to discontinue orreduce the
dose of treatment. This will affect cancercontrol and ultimately the survival of the patient.
Inaddition, the development of drug resistance is anothermajor problem in the treatment
of cancer as chemoresistancecan lead to unabated proliferation of the defianttumor cells
and the administered antineoplastic agent cancause nonspecific toxicity to the normal
cells. Accordingly,an agent that can selectively protect the normalcells against the
deleterious effects of chemotherapy(chemoprotective agents), or can sensitize the
tumorcells to anticancer drugs (chemosensetizers), is anattractive proposition in cancer
treatment and the goalof researchers (Coleman et al., 1988).
The aqueous extract of amla has been observed to beeffective at reducing
cyclophosphamide-induced suppressionof humoral immunity and to restore the levels
ofglutathione and the antioxidant enzymes in the kidneysand liver of mice (Haque et al.,
2001). Amla is reportedto decrease cyclophosphamide-induced DNA damage asmeasured
by a reduction in both micronuclei and chromosomalaberration in the bone marrow cells
of mice (Sharmaet al., 2000a). Amla reduced the levels of cytochrome (Cyt)P450,
increased the levels of the antioxidant glutathione,antioxidant enzymes [glutathione
peroxidase (GPx), glutathionereductase], and increased the detoxificationenzyme
glutathione-S-transferase (GST), which therebycontributed to these observations (Sharma
et al., 2000a).
In-vitro studies have shown that amla effectivelysuppressed the proliferation of the
human hepatocellularcarcinoma (HepG2) and lung carcinoma (A549) cells andsynergized
the cytotoxic effects of doxorubicin andcisplatin, two important clinically used
antineoplasticdrugs (Pinmai et al., 2008). The ethanolic extract of amlaalso protected the
cardiac myoblasts H9c2 cells againstdoxorubicin-induced toxicity (Wattanapitayakul et
al.,2005). Together these observations suggest that it isquite possible that amla prevents
doxorubicin-inducedcardiotoxicity to the normal cardiac myoblasts and,concomitantly,
sensitizes the antineoplastic effects oncancer cells. However, detailed studies are required
forthis hypothesis to be validated, especially in the relevantanimal models of study.
Amla as a radioprotective agent
Since the discovery of the deleterious effects of ionizingradiation, studies have been
focused on developingchemical radioprotectors that have the ability to decreasethe ill
effects of radiation on normal tissues (Arora et al.,2005). The thiol compound amifostine
is credited withbeing the only radioprotector to have been approved bythe Food and Drug
Administration to reduce theincidence and severity of xerostomia in head and neckcancer
patients undergoing radiotherapy (Arora et al.,2005). Unfortunately, the application of
this drug has sofar been less than hoped for, owing to its untowardtoxicity often being
evidenced at the optimal radioprotectivedoses (Arora et al., 2005).
With regard to the radioprotective effects of amla, studieshave shown that administering
(50, 100, 200, 400, and800 mg/kg b.wt./day) amla once daily for 7 consecutivedays
before exposure to sublethal dose of g-radiation(9Gy) protected mice against the
radiation-inducedsickness and mortality (Singh et al., 2005). Among allthe doses studied,
the optimal effect was observed at100 mg/kg b.w. as it delayed the radiation-induced
lethalityand caused a survival of 87.5% when compared withplacebo-treated irradiated
cohorts in which no survivorswere observed (Singh et al., 2005).
Administration of amla (100 mg/kg b.wt.) ameliorated theradiation (5Gy)-induced
gastrointestinal damage asevaluated by the histopathological studies, by quantifyingthe
crypt cell population, mitotic figures, and villuslength at all the assay points (12 h–30
days). Reports alsosuggest that amla ameliorated the radiation-inducedhemopoietic
damage (Hari Kumar et al., 2004). Feedingmice with 2.5 g/kg b.wt. of amla for 10
consecutive daysbefore exposure to a single dose of 7Gy of radiationincreased the total
leukocyte count, bone marrow viability,and levels of hemoglobin. However, treatment
withamla after exposure to irradiation (continuously for another15 days) was not as
effective when compared withadministeration before radiation, suggesting it to be ofuse
only when exposure to radiation is planned (HariKumar et al., 2004).
Mechanistic studies have shown that feeding amlaenhanced the activity of the various
antioxidant enzymes(catalase, superoxide dismutase, and GPx), the phase IIdetoxifying
enzyme, GST, and the antioxidant thiol,glutathione, in the blood, with a concomitant
decrease inthe levels of lipid peroxides (Hari Kumar et al., 2004).Similar results were
also observed by Jindal et al. (2009) inmice intestine and together both these studies
confirmthat amla significantly reduces the deleterious effects ofradiation at least in part
through its antioxidant andinhibition of lipid peroxidation activities. The
phytochemicalsellagic acid, gallic acid, and quercetin (Fig. 2)present in amla also possess
radioprotective effects andare shown in Table 3.
Amla as a chemopreventive agent
Cancer chemoprevention has traditionally been definedas a dietary or therapeutic
approach for the prevention,delay, or reversal of carcinogenesis with nontoxic
agents(Bonte, 1993; Pastorino, 1994; Sporn and Suh, 2002).
Epidemiological studies have provided convincing evidencethat natural dietary
compounds can modify theprocess of carcinogenesis, which includes the threedecisive
steps: namely initiation, promotion, and progression,in several types of human cancer
(Sporn and Suh,2002). Experimental studies have also validated theefficacy of a number
of bioactive dietary components,supporting the acceptance of natural dietary
compoundsas chemopreventive agents in the near future. Amla isreported to be effective
in stopping initiation, promotion,and progression of cancer and the ability of amla to
renderchemopreventive effects is discussed in the followingsections.
Sancheti et al. (2005) investigated the chemopreventiveeffects of amla in two-stage
carcinogenesis {[7,12-dimethylbenz(a)anthracene] (DMBA)-induced and crotonoil
promoted} in mice by considering the delay intumorigenesis, cumulative number of
papillomas, tumorincidence, tumor yield, and tumor burden as the endpoints. The
researchers observed that feeding amla for7 consecutive days before and after DMBA
application wasless effective than when administered during the promotion(starting from
the time of croton oil treatment andcontinued till the end of experiment for 16 weeks).
However, the best effect was observed when amla was fedthroughout the experimental
period, that is, before andafter DMBA application and during the promotional
stage.These observations may be because of the various protectivemechanisms that were
operating. When amla is administeredbefore DMBA treatment, there will be an increase
inthe levels of antioxidant and phase II enzymes, with aconcomitant decrease in the phase
I detoxifying enzymes,which cumulatively may prevent/reduce the process
ofcarcinogenesis. However, when administered during thepromotion, amla may trigger
the selective apoptosis of themutated and preneoplastic cells and decrease the
carcinogenesis(explained later). The phytochemicals, such asellagic acid, gallic acid, and
quercetin, present in amla alsopossess chemopreventive effects and may have
beenresponsible for the beneficial effects (Table 4).
Recently, Ngamkitidechakul et al. (2010) have alsoobserved that the aqueous extract of
amla containingtannins (43%), uronic acid (11%), and gallic acid (21%)was effective in
delaying and reducing DMBA-inducedand (12-otetradecanoylphorbol-13-acetate)-
promoted skincarcinogenesis in mice. The topical application of theextract (1, 2, or 4mg
in 0.1 ml acetone) 1 h before each(12-otetradecanoylphorbol-13-acetate) application until
thetermination of the experiment caused a concentrationdependentdecrease in the
appearance and incidenceof skin papillomas (Ngamkitidechakul et al., 2010).
Theseresults clearly suggest the effectiveness of amla whenapplied topically and also its
possible use as a skin careproduct.
In Ayurveda amla is considered to be a hepatoprotectiveagent and scientific studies have
validated this traditionalbelief. Studies have shown that amla protects againstchemical-
induced carcinogenesis and oxidative stress.With regard to chemoprevention, studies by
Rajeshkumaret al. (2003) have shown that feeding amla decreased theN-
nitrosodiethylamine-induced liver tumors in rats. Amladecreased the levels of serum g-
glutamyl transpeptidase,alkaline phosphatase, glutamate pyruvate transaminase,and
bilirubin (Rajeshkumar et al., 2003). Similar observationswere also made when the
chemopreventive effectsof amla were studied against diethylnitrosoamineinducedand 2-
acetylaminoflourine-promoted hepatocarcinogenesisin rats (Sultana et al., 2008).
Prophylactic treatment with amla for 7 consecutive daysbefore the single administration
of thioacetamide reversesthe thioacetamide-induced oxidative stress andearly
promotional events of primary hepato-carcinogenesisin rats. Amla inhibited the serum
levels of SGOT,SGPT, and GGT; decreased levels of lipid peroxide, inhibitedaberrant
synthesis of DNA; decreased the activitiesof GST, GR, G6PD, and ornithine
decarboxylase;and concomitantly increased the glutathione content andGPx activity in
the liver (Sultana et al., 2004).Studies have also shown that administering amla
reducesthe cytotoxic effects of the proven carcinogens such as3,4-benzo(a)pyrene (Nandi
et al., 1997), benzo[a]pyrene(Sharma et al., 2000a), DMBA (Banu et al., 2004)
byreducing the mutagenesis, oxidative stress, lipid peroxides,phase I enzymes
[cytochrome (Cyt) P450 and Cytb5], and concomitantly increasing the
antioxidants(glutathione) and enzymes (GPx, glutathione reductase,and phase II
detoxifying enzyme GST (Nandi et al., 1997;Sharma et al., 2000a; Banu et al., 2004).
In addition to these observations, amla has been scientificallystudied for its protective
role against country liquor(Gulati et al., 1995), ethanol (Pramyothin et al., 2006; Reddyet
al., 2009), carbon tetrachloride (Sultana et al., 2005; Leeet al., 2006; Mir et al., 2007),
ochratoxin (Verma andChakraborty, 2008), hexachlorocyclohexane (Anilakumaret al.,
2007), paracetamol (Gulati et al., 1995), and theantituberculosis drugs (rifampicin,
isoniazid, and pyrazinamide)(Tasduq et al., 2005; Panchabhai et al., 2008)-
inducedoxidative stress and damage to the liver. Most of theseagents are known to be
hepatotoxins and to initiate andpromote carcinogenesis. By preventing oxidative stress
andthe resulting damage, amla protects against both hepatotoxicityand possible
carcinogenesis.
Mechanisms of action (Fig. 3)
Amla is a free radical scavenger
Excess generation of free radicals, the reactive oxygenspecies [ROS superoxide anion
radical (O2K– ), hydroxylradical (OHK) and hydrogen peroxide (H2O2)], and thereactive
nitrogen species [RNS nitric oxide (NO),peroxynitrite (ONOO– )], respectively, causes
oxidativestress and nitrosative stress. The free radicals that aregenerated are highly
reactive and cause damage to themembrane lipids, proteins, and DNA (Devasagayam et
al.,2004). Accordingly, their prevention is important inpreventing cell damage,
mutagenesis, and carcinogenesis.In-vitro studies have shown that amla scavenges 2,2-
diphenyl-1-picrylhydrazyl radicals (Naik et al., 2005;Hazra et al., 2010), superoxide
anions (Naik et al., 2005;Hazra et al., 2010), hydroxyl radical (Hazra et al., 2010),nitric
oxide (Hazra et al., 2010), hydrogen peroxide (Hazraet al., 2010), peroxynitrite (Hazra et
al., 2010), singletoxygen (Hazra et al., 2010), and hypochlorous acid (Hazraet al., 2010).
The phytochemicals, such as gallic acid,ellagic acids, emblicanin A, and emblicanin B,
are alsoreported to possess free-radical-scavenging effects in the2,2-diphenyl-1-
picrylhydrazyl assay and efficacy was asfollows: A emblicanin greater than B emblicanin
greaterthan gallic acid greater than ellagic acid greater thanascorbic acid (Pozharitskaya
et al., 2007).
Studies have also shown that the methanol extract ofamla and its various fractions
(hexane, ethyl acetate, andwater fractions) possess NO scavenging effects. Theisolated
compounds, such as gallic acid, methyl gallate,corilagin, furosin, and geraniin, which
were isolated fromthe ethyl acetate fraction that possessed the best NOscavengingeffect,
were also effective. Gallic acid wasfound to be a major compound in the ethyl
acetateextract and geraniin showed highest NO-scavengingactivity among the isolated
compounds (Kumaran andKarunakaran, 2006).
Amla decreases phase I enzymes
Phase I drug-metabolizing enzymes, especially the CYPP450 mixed-function oxidases,
which are involved in thebiotransformation of xenobiotics, can transform a
nontoxicchemical (procarcinogen) into a harmful toxic substance(ultimate carcinogen),
which can induce damage tothe nucleic acids and other macromolecules (Percival,1997).
Studies have also shown that administering theethanolic extract of amla reduced the
hepatic levels ofthe activating enzymes, Cyt P450 and Cyt b5, which areimportant in
converting the procarcinogen DMBA intoultimate carcinogen (Banu et al., 2004). In
addition, theinhibition of microsomal-activating enzymes, includingCyt P450, was also
responsible for the antimutageniceffects of amla against 2-aminofluorene (Arora et
al.,2003), aflatoxin B1, and benzo[a]pyrene-induced mutagenesisin the Ames test
(Sharma et al., 2000b).
Amla increases glutathione S-transferase, a phase II enzyme
The reactive species formed by the phase I enzymes areoften detoxified by phase II drug-
metabolizing enzymes.In the reaction, the hydrophobic intermediates generatedby the
phase I enzymes are converted to a water-solublegroup, thus decreasing their reactive
nature, and allowingsubsequent excretion (Jana and Mandlekar, 2009).A properly
functioning and balanced phase II systemwould detoxify the metabolically activated
carcinogen,thereby preventing mutagenesis and carcinogenesis.Agents preferentially
activating phase II over phase Ienzymes can be more beneficial as
chemopreventiveagents (Percival, 1997; Jana and Mandlekar, 2009).Studies have shown
that amla increases the level of GSTand thereby reduces the toxic effects of N-
nitrosodiethylamine(Jeena et al., 1999; Rajeshkumar et al., 2003),benzo[a]pyrene
(Sharma et al., 2000a), cyclophosphamide(Sharma et al., 2000a), thioacetamide (Sultana
et al.,2004), CCl4 (Sultana et al., 2005), ionizing radiation (HariKumar et al., 2004),
hexachlorocyclohexane (Anilakumaret al., 2007), arsenic (Panchabhai et al., 2008),
ethanol(Reddy et al., 2009), and ochratoxin (Sultana et al., 2004).Molecular studies have
also shown that amla increasedGSTP1 expression (Niture et al., 2006), thereby
validatingthe biochemical observation.
Amla decreases ornithine decarboxylase
Ornithine decarboxylase (ODC), the rate-limiting enzymein polyamine synthesis, is
important in polyaminesynthesis. High levels of ODC are an adverse prognosticfactor as
it is observed to be important in tumor proliferation,progression, and metastasis and for
the survivalof cancer patients (Manni et al., 2002).
Studies have shown that administering amla inhibitedthioacetamide-induced hyper-
proliferation in rat liverby decreasing the levels of ODC activity and
thymidineincorporation in DNA (Sultana et al., 2004). These observationsclearly indicate
the inhibitory effects of amla onODC and DNA replication, steps that are important
intumor cell proliferation.
Amla increases the antioxidant enzymes
The antioxidant enzymes, superoxide dismutase, GPx,and catalase, cooperate or, in a
synergistic method, workto protect cells against oxidative stress. The
superoxidedismutase catalyses the dismutation of superoxideradicals, a major form of
ROS, into hydrogen peroxide,which is acted on by the GPx and catalase to give
water.When an appropriate balance exists between these threeenzymes, oxidative stress is
reduced and the cells areprotected from the cytotoxic and mutagenic effects of theROS
(Devasagayam et al., 2004).
Preclinical studies have conclusively shown that amlaameliorates the oxidative and
xenobiotic-induced stress,mutagenesis, and carcinogenesis by increasing the
antioxidantenzymes. Reports suggest that amla increasesthe antioxidant enzymes and
prevents benzo[a]pyrene(Sharma et al., 2000a), cyclophosphamide (Sharma et al.,2000a),
DMBA (Banu et al., 2004), g-radiation (Hari Kumaret al., 2004; Jindal et al., 2009),
hexachlorocyclohexane(Anilakumar et al., 2007), and ethanol (Pramyothin et al.,2006)-
induced toxic effects.
Amla decreases lipid peroxidation
Lipid peroxidation is one of the most evaluated consequencesof free radicals on
membrane structure. Thepolyunsaturated fatty acids are vulnerable to peroxidativeattack
and this can cause loss of fluidity, decreasedmembrane potential, increased permeability
for protonsand calcium ions and eventually loss of cell membranes,and result in
pathological and toxicological processes(Devasagayam et al., 2004). The major aldehydic
endproduct of lipid peroxidation is malondialdehyde and ismutagenic in the bacterial and
mammalian systems ofstudies.
Multiple studies have shown that amla possesses inhibitoryeffects on lipid peroxidation
induced by various inducers.In-vitro studies have shown that amla prevents
radiationinducedlipid peroxidation (Naik et al., 2005) and this effectalso extends to
animal studies (Hari Kumar et al., 2004;Jindal et al., 2009). Amla inhibits cadmium
(Khandelwalet al., 2002), carbon tetra chloride (Sultana et al., 2005),arsenic (Panchabhai
et al., 2008), ethanol (Reddy et al.,2009), ochratoxin (Chakraborty and Verma, 2010),
Nnitrosodiethylamine(Rajeshkumar et al., 2003), and thioacetamide(Anilakumar et al.,
2007)-induced lipid peroxidation.By inhibiting lipid peroxidation amla may
contributetoward the observed beneficial effects, at least in part.Amla possess anti-
inflammatory effects
Chronic inflammation has been proved to cause freeradicals and the resulting oxidative
and nitrosative stressis known to directly or indirectly contribute toward malignantcell
transformation by inducing genomic instability,alterations in epigenetic events,
inappropriate geneexpression, enhanced proliferation of mutated cells, resistanceto
apoptosis, tumor neovascularization, and metastasis(Kundu and Surh, 2005).
Experiments have shown that the aqueous fraction ofmethanol extract of the leaves
possesses anti-inflammatoryeffects in carrageenan-induced and dextran-induced rathind
paw edema. Mechanistically, it was observed that theextract inhibited migration of
human polymorphonuclearcells and exerted its anti-inflammatory effects (Asmawiet al.,
1993). Studies have also shown that amla extract andthe phytochemical pyrogallol also
possess anti-inflammatoryeffects and inhibited the Pseudomonas aeruginosa
laboratorystrain PAO1-dependent expression of the neutrophilchemokines IL-8, GRO-a,
GRO-g, of the adhesionmolecule, ICAM-1, and of the pro-inflammatory cytokine,IL-6
(Nicolis et al., 2008). Recently, Muthuraman et al.(2010) have also observed that the
phenolic compoundsfrom amla possess anti-inflammatory effects in thecarrageenan and
cotton pellet-induced acute and chronicinflammatory response in animal models of study.
Theeffect was significant at high doses and was comparable tothe positive control,
diclofenac (Muthuraman et al., 2010).
Antimutagenic effects
The initial step in the process of carcinogenesis is inductionof mutation in the oncogenes
or tumor-suppressorgenes of the genome of a somatic cell. Therefore, itsprevention is of
great importance (Weisburger, 2001).Multiple studies carried out in the last two decades
haveconclusively shown that amla prevents DNA damageagainst different carcinogens
and mutagens. Using thestandard Ames test, Sharma et al. (2000b) observed forthe first
time that the aqueous extract of amla inhibitedaflatoxin B1 and benzo[a]pyrene-induced
mutagenesis inthe Salmonella typhimurium strains TA 98 and TA 100.Amla is also
reported to increase the levels and activitiesof O6-methylguanine-DNA
methyltransferase, an enzymeimportant for removing the highly mutagenicadducts
formed by alkylating agents in human lymphocytes(Niture et al., 2006). Amla was also
effective inpreventing the radiation-induced damage in the plasmidDNA assay (Naik et
al., 2005), suggesting its effectivenessagainst different classes of mutagens.
In addition, studies with experimental animals have shownthat amla prevents cadmium
(Khandelwal et al., 2002), lead(Dhir et al., 1990), aluminium (Dhir et al., 1990),
nickel(Dhir et al., 1991), cesium chloride (Ghosh et al., 1992),arsenic (Biswas et al.,
1999), chromium (Sai Ram et al.,2003), 3,4-benzo(a)pyrene (Nandi et al., 1997),
benzo[a]-pyrene (Sharma et al., 2000a), DMBA (Nandi et al., 1997),and
cyclophosphamide (Sharma et al., 2000a)-inducedDNA damage. Together these
observations clearly suggestthe effectiveness of amla in preventing mutagenesis andDNA
damage, which would inhibit/reduce the incidenceand process of carcinogenesis, at least
in part.
Amla possesses immunomodulatory effects
Immune activation is an effective protective approachagainst emerging infectious
diseases and certain cancers.Immunostimulants enhance the overall immunity of thehost,
present a nonspecific immune response againstmicrobial pathogens and increase humoral
and cellularimmune responses, by either enhancing cytokine secretion,or by directly
stimulating B-lymphocytes or T-lymphocytes(Spelman et al., 2006). In Ayurveda, amla is
considered tobe an immunostimulatory agent and scientific studies havevalidated this
(Warrier et al., 1996; Kulkarni, 1997; Khan,2009; Krishnaveni and Mirunalini, 2010).
Studies have shown that amla enhances natural killer(NK) cell activity and antibody-
dependent cellularcytotoxicity in BALB/c mice bearing Dalton’s lymphomaascites
tumor. Amla increases the life span of tumorbearinganimals and this was because of the
increase inthe activation of splenic NK cell activity and antibodydependent cellular
cytotoxicity. However, the increase insurvival was completely abrogated when the NK
cell andkiller cell activities were depleted, either by cyclophosphamideor anti-asialo-
GM1 antibody treatment, validatingthat the observed effects were because of its
immunomodulatoryeffects (Suresh and Vasudevan, 1994).
Amla and its phytochemicals modulate the levels of proteins important in cell cycle
progression
Cancer is frequently considered to be a disease of the cellcycle and a convincing body of
data has proved that thedisruption of the normal regulation of cell-cycle progressionand
division are important events in cancer development(Hanahan and Weinberg, 2000;
Kastan andBartek, 2004). The progression of the cell cycle is atightly regulated and
highly ordered process involvingmultiple checkpoints that assess extracellular
growthsignals, cell size, and DNA integrity (Kastan and Bartek,2004). The cyclin-
dependent kinases (CDKs) and theirrespective partners (cyclin) are responsible for
theprogression of the cell cycle, whereas the CDK inhibitorsact as brakes to stop cell
cycle progression (Hartwell andWeinert, 1989). The genesis of cancer is principally
becauseof the derailed expression or activation of positiveregulators and functional
suppression of negative regulators(Hartwell and Weinert, 1989; Kastan and Bartek,
2004).
Studies by Jose et al. (2001) have shown for the first timethat amla extract caused a dose-
dependent inhibition ofthe cell cycle-regulating enzyme Cdc25 phosphatasein vitro, with
an IC50 of 5 mg/ml (Jose et al., 2001). Thephytochemical pentagalloylglucose is shown
to cause G1arrest in human Jurkat T cells by elevating p27Kip1 andp21Cip1/WAF1
proteins (Chen and Lin, 2004). Gallicacid induces cell cycle arrest by decreasing CDKs
andcyclins. It phosporylates Cip1/p21 and cell division cycle2 (Cdc2), Cdc25A, and
Cdc25C in DU145 cells (Sunet al., 2004). It also induces G2/M phase cell cycle arrestby
regulating 14-3-3b release from Cdc25C; activation ofchk2; decreasing CDK1, cyclin B1,
and Cdc25C; increasingphosphorylation of p-Cdc2 (Tyr-15), Cip1/p21 andCdc25C in
human bladder transitional carcinoma cells(TSGH-8301cells) (Ou et al., 2010). Gallic
acid feedingalso reduces Cdc2, CDK2, CDK4, CDK6, cyclin B1, and Ein the prostatic
tissue of mice with transgenic adenocarcinomaof the mouse prostate (Raina et al., 2008).
Amla and some of its constituents cause apoptosis and cytotoxicity of neoplastic cells
Apoptosis, a process by which the cell is committed todeath by not initiating an
inflammatory response, isvital in regulating tissue homeostasis (Sun et al., 2004;Ghobrial
et al., 2005). A large body of evidence has provedthat the processes of neoplastic
transformation, progression,and metastasis involve alterations of the normalapoptotic
pathway and that the number of cell deaths isvery low in these cells (Sun et al., 2004;
Ghobrial et al.,2005). Therefore, the induction of apoptosis is arguablythe most potent
defence against cancer as it effectivelyeliminates the mutated and severely damaged
cells.Accordingly, agents that can eliminate mutated, preneoplastic,and neoplastic cells
by sparing the normal cellsare supposed to be an effective chemopreventive agentand to
offer therapeutic advantage in the elimination ofcancer cells (Sun et al., 2004; Ghobrial et
al., 2005).
The ability of the extract of amla and some of itsphytochemicals to induce apoptosis in
cancer cells contributesto the understanding of its anticancer andchemopreventive
potential. Studies have shown thatthe aqueous extract of amla induces apoptosis
andinhibits the growth of HeLa, MDA-MB-231, and SKOV3without affecting the normal
lung fibroblast, MRC5(Ngamkitidechakul et al., 2010). The hydrolyzable tanninspossess
selective cytotoxicity to the human oral squamouscell carcinoma and salivary gland
tumor cell lines, whereasthey were nontoxic to the normal human gingival
fibroblasts(Sakagami et al., 2000). Studies have also shown thatquercetin (Son et al.,
2004), gallic acid (Isuzugawa et al.,2001), ellagic acid (Losso et al., 2004), and pyrogallol
(Yanget al., 2009) also possess cytotoxic and apoptogenic effectson the neoplastic and
transformed cells, but not in normalcells. Together, these observations clearly suggest
that thepresence of these compounds in amla resulted in the eliminationof the mutated
and neoplastic cells and resultedin the desired effects in both antineoplastic effects
andchemoprevention.
Amla and some of its constituents prevent Metastasis
Cancer cells differ from normal cells; the most importantbeing the loss of differentiation,
self-sufficiency in growthsignals, limitless replicative potential, decreased
drugsensitivity, increased invasiveness, and metastasis (Hanahanand Weinberg, 2000).
Metastasis, the process by whichsome of the neoplastic cells spread from the primary
siteto distant tissue, is the life-threatening aspect of cancer.It is the hallmark of cancer
and is responsible for thefailure of treatment and death. The process of tumormetastasis is
extremely complex and involves myriad biochemicalinteractions operating concurrently
or sequentially.The important steps in the process of metastasisare (i) invasion and
migration, (ii) intravasation, (iii)circulation, (iv) extravasation, and (v)
colonization,proliferation, and angiogenesis (Chiang and Massague´,2008; Leber and
Efferth, 2009). Cell invasion is one ofthe fundamental processes required during tumor
progressionand metastasis and matrix metalloproteinases(MMPs), a group of enzymes
that regulate cell-matrixcomposition, are important in this process (Chiang andMassague
´, 2008; Leber and Efferth, 2009).
Recent studies have suggested that the aqueous extractof amla was effective in preventing
the invasion of MDAMB-231 cells in the in-vitro matrigel invasion
assay(Ngamkitidechakul et al., 2010). The amla phytochemical,kaempferol, inhibited the
expression of stromelysin 1(MMP-3) in the MDA-MB-231 breast cancer cell
line(Phromnoi et al., 2009). The polyphenol gallic acid is alsoreported to possess
inhibitory effects on gastric adenocarcinomacell migration, decreased expression of
MMP-2/9 in vitro (Ho et al., 2010), and metastasis of P815mastocytoma cells to the liver
of DBA/2 mice (Ohno et al.,2001). The flavanol, quercetin, decreased the expressionof
gelatinases A and B (MMP-2 and MMP-9) in thehuman metastatic prostate PC-3 cells
(Vijayababu et al.,2006) and stromelysin 1 (MMP-3) in the MDA-MB-231breast cancer
cell line (Phromnoi et al., 2009) andinhibited the lung metastasis of murine colon 26-L5
carcinomacells (Ogasawara et al., 2007) and B16-BL6 murinemelanoma metastasis in
mice (Piantelli et al., 2006).