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).