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Cancer Biology & Therapy 12:6, 467-476; September 15, 2011; 2011 Landes Bioscience

Anticancer biology of Azadirachta indica L (neem)

A mini review
Rajkumar Paul,1,* Murari Prasad2 and Nand K. Sah3

School of Biotechnology; Rajiv Gandhi Technological University; 2Environmental Chemistry Division; Advanced Material Processing Research Institute (CSIR); Bhopal, India; 3 Department of Botany; TNB College; Bhagalpur, India

Keywords: neem, Azadirachta indica, herbal medicine, nimbolide, azadirachtin, anticancer drug Arya-veppu (Malyalam), Vaypum (Tamil), Bevu (Kannad) and Nimtree, Vepu, Vempu, Vepa (Telugu). In east Africa it is also known as Mwarobaini (Swahili) which literally means the tree of the 40 as it is considered as a treatment for 40 different diseases. The whole plant is full of domestic, industrial and pharmaceutical values (Tables 1 and 2). The preparations using foliar extracts of this plant are applied as a panacea for skin problems, such as, eczema, ringworm, acne and cancer.1 A large number of unique chemical compounds have been puried from this plant that are being used effectively as antiseptic, antiviral, antipyretic, anti-inammatory, anti-ulcer, anti-malarial, antifungal and anticancer agents (Tables 4 and 5). Some of the most studied compounds include nimbin, azadirachtin, nimbidiol, quercetin and nimbidin, among others (Tables 3 and 4).2,3 Because of its tremendous therapeutic signicance (Fig. 5), neem is also known as a village pharmacy, particularly in India. This unique plant, has been closer to human culture and civilization since time immemorial. Cancer continues to be an enigmatic challenge for cancer biologists and medical practitioners. Several tantalizing claims for discovering a sure cure for cancer have been made by scientists from time to time; yet a dependable cure against most cancers remains a challenge even today. One of the primary reasons for this is the multiple pathways of survival adopted by cancer cells. Blocking a few pathways of their survival does not ensure their targeted elimination. That is why researchers are now trying to target them through multiple pathways with minimum possible side effects and discomfort to patients. In recent years, certain herbal products and ethnomedicines (Table 1) have drawn keen attention of researchers and medical practitioners primarily because of convincing anticancer properties with negligible unpleasant side effects and discomfort to patients.4,5 During the last decade, the anticancer properties of neem have been investigated in detail from several scientic angles. The active ingredients obtained from the plant have been demonstrated unequivocally to induce apoptosis in several types of tumor cells and to organize and gear up the immune system to take on the cancer cells through cross priming. Regular use of neem and its preparations have been found to prevent onset of cancer through multiple mechanisms including production of substantial levels of antioxidants and carcinogen-detoxifying enzymes. Against a wide variety of human cancer cell lines and animal models for human cancers including colon, stomach, Ehrlichs carcinoma, lung, liver, skin, oral, prostate and breast

Neem (Azadirachta indica), a member of the Meliaceae family, is a fast growing tropical evergreen tree with a highly branched and stout, solid stem. Because of its tremendous therapeutic, domestic, agricultural and ethnomedicinal significance, and its proximity with human culture and civilization, neem has been called the wonder tree and natures drug store. All parts of this tree, particularly the leaves, bark, seed-oil and their purified products are widely used for treatment of cancer. Over 60 different types of biochemicals including terpenoids and steroids have been purified from this plant. Pre-clinical research work done during the last decade has fine-tuned our understanding of the anticancer properties of the crude and purified products from this plant. The anticancer properties of the plant have been studied largely in terms of its preventive, protective, tumor-suppressive, immunomodulatory and apoptotic effects against various types of cancer and their molecular mechanisms. This review aims at scanning scattered literature on the anticancer biology of A. indica, related toxicity problems and future perspectives. The cogent data on the anticancer biology of products from A. indica deserve multi-institutional clinical trials as early as possible. The prospects of relatively cheaper cancer drugs could then be brighter, particularly for the under-privileged cancer patients of the world.

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Introduction Neem (Azadirachta indica) is a tropical evergreen, profusely branched tree with oblique leaves and stout trunk of timber values with insect-repellant properties. Earlier this plant was also known as Melia azadirachta (L) and Antelaea azadirachta (L). It is a native of Myanmar (Burma) and India. But, it may be found in Bangladesh, Sri Lanka and African countries, as well. It is about 20 ft tall and belongs to Meliaceae family (Fig. 1AD). The neem tree is known by a variety of names, such as, Indian lilac (English), Azadirakhta (Persian), Margosa and neeb (Arabic), Tamar (Burmese), Kohomba (Sinhala), Pokok semambu (Malaysia), Dogon yaro (some Nigerian languages), neem (Hindi and Bangla), nimba (Sanskrit and Marathi),
*Correspondence to: Rajkumar Paul; Email: Submitted: 05/20/11; Accepted: 06/09/11 DOI: 10.4161/cbt.12.6.16850

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Figure 1. (A) A neem tree,52 (B) an inflorescence of neem,53 (C) fruits of neem54 and (D) seeds of neem.55

cancers; neem products have been demonstrated to produce impressive anticancer results. This review aims at providing an in-depth analysis of the scattered research work on the anticancer therapeutic potential of neem vis--vis its toxicological problems and future perspectives. Anti-Carcinogenic and Anti-Mutagenic Effect of Neem That neem extracts possess potent ability to remove cancerous phenotype (tumor commonly termed as nasoor), has long been known to people in Asia, particularly in India. During the last two decades, researchers in India and abroad have gathered convincing data to suggest that the onset of cancerous phenotype due to certain mutagens and pro-carcinogens may be treated effectively by extracts obtained from various parts of the neem tree. The chemopreventive effects of dietary doses of aqueous neem leaf extract was studied on in vivo murine system against 3H-B- -P (Benz- -pyrene)-induced initiation of cancer measured in terms of 3H-B- -P-DNA adduct.6 Their results indicated that neem leaf extract reduced the metabolic activation of 3H-B- -P with

consequent decrease in the level of 3H-B- -P-DNA adduct formation. These molecular and biochemical modulations observed at the initiation phase of carcinogenesis highlight the chemopreventive signicance of A. indica extracts. In another interesting study, Chaimuangraj et al. observed that dietary feeding of the neem leaf extract (20, 100, 250 mg/Kg body weight) signicantly inhibited the azoxymethane-induced aberrant crypt foci (ACF) and also signicantly decreased the proliferating cell nuclear antigen (PCNA) labeling indices (p < 0.0006) in the colon epithelium in rats. The preventive action of neem owers has been demonstrated by several research groups against the neoplastic developments due to chemicals including DMBA and B P.8-10 These results indicate that dietary use of extracts from various parts of A. indica may play a promising role in future drug discovery and development programs as far as chemoprevention of cancer is concerned. Most of the ethnomedicinal and early studies on neem with respect to its anticancer properties suffered from lack of credible mechanistic principles. Serious attempts at unraveling the possible scientic interpretations of the chemopreventive/anticancer effects of neem extracts have been made in recent years. O6-alkylguanines are potently mutagenic and


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Table 1. Ethnomedicinal and ritualistic significance of neem (A. indica) S. no Ethnomedicinal/Ritualistic uses On the eve of Hindi New Years Day in the month of Chaitra (MarchApril) a paste from the neem leaf or its aqueous lysate is orally taken. This is also known as Gudipadwa in some parts of India. Use of neem boughs with green leaves is prevalent during Nagpanchmi, a festival for worshipping deadly snakes. On the eve of the festival neem leaves are consumed as a precaution against the poisonous snakes. It is assumed that oral consumption of neem may help to overcome problems associated with snake bite. Neem twigs are hung outside the labor room during and after the birth of a child for some days in order to avoid the evil spirits that may otherwise jeopardize the life of the new born. Soft leaves of neem are used as bed cover for patients suffering from small pox. Powdered neem leaves are used externally for healing wounds. Earlier, neem leaves were burnt into powdered form, mixed with mustard oil and applied on open wounds to speed healing. Scientific significance This date signifies the end of winter and beginning of spring followed by summer. During this seasonal change health problems aggravate. Oral consumption of foliar paste of neem may take care of these problems. Remarks The ancient sages in India enshrined these rituals in the culture and civilization of this region only because of its medicinal efficacy. This ritual has been prevalent in India, particularly in the eastern states since time immemorial. The antidotes against snake venom may be worked out. The ancient sages understood the significance of neem as an antibiotic. So, it was enforced into the social custom as a ritual during child birth. It is not understood how neem acts as an antiviral.

Certain ingredients of neem may be acting as an antidote for snake venom.

Many times, particularly in rural India, child birth takes place in a clean room in the residential place. Presence of neem twigs with leaves may help avoid viral/bacterial infections to mother and the child. Small pox used to be an epidemic. Now it has been controlled to a large extent. During those days, neem leaves were used as an antiviral agent. The antibiotic nature of neem is exploited still in certain parts of India, particularly by rural populations.

pro-carcinogenic. O6-methylguanine-DNA methyltransferase (MGMT) is an enzyme that detoxies O6-alkylguanines and thus tries to maintain genomic integrity of the cells. Recently ethanolic and aqueous extracts of neem have been demonstrated to enhance the activity of MGMT leading to time-dependent demethylation of O6-methylguanine and consequent prevention of its cytotoxic lesions.11 A facilitated elimination of these lesions by increasing the activity of MGMT is likely to be a benecial chemoprevention strategy. More recently, development of DMBA-induced hamster buccal pouch (HBP) carcinoma has been observed to be inhibited by nimbolide and azadirachtin in a concentration-dependent manner through multiple mechanisms including prevention of activation of procarcinogen, oxidative DNA damage and upregulation of antioxidant and carcinogen detoxifying enzymes.12 Effect of Neem on Drug Metabolizing Enzymes In certain cases, neem preparations have also been observed to potentiate the antitumor activities of certain drugs besides affording protection against life threatening side effects of these chemotherapeutic agents. For example, pre-treatment of Swiss mice with NLP not only suppressed leucopenia and neutropenia but also potentiated the antitumor activities of cyclophosphamide concomitantly. In an interesting observation HPLC-generated neem leaf fractions have been shown as positive modulators of the phase-I and phase-II xenobiotic-metabolizing enzymes, lipid and protein oxidation and anti-oxidant defense enzymes leading to attenuation of the DMBA-induced HBP carcinoma in the Swiss mice.13

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S. no 1 Parts of neem tree Bark 2 Twig 3 4 5 6 7 8 Leaf Flower Fruit Seed Oil Gum

This practice has been long in use in India.

Table 2. Parts of neem (A. indica) tree with medicinal uses Treatable ailments

Analgesic, alternative and curative of fever Cough, asthma, piles, phantom tumor, intestinal worms, spermatorrhoea, obstinate urinary disorder, diabetes Leprosy, eye problem, epistaxis, intestinal worms, anorexia, biliousness, skin ulcers, cancer Bile suppression, elimination of intestinal worms, phlegm Piles, intestinal worms, urinary disorder, epistaxis, phlegm, eye problem, diabetes, wounds, leprosy Leprosy, intestinal worms, cancer Leprosy, intestinal worms Scabies, wounds, ulcers, skin diseases

Cisplatin (cis) and 5-uorouracil (5-FU) are established chemotherapeutic agents against certain forms of cancer. But, they also cause terrible side effects including programmed cell death of circulating blood cells. In order to reduce these side effects granulocyte colony stimulating factor (GCSF) is often administered while treatment with cis and 5-FU. Recently pretreatment with neem leaf preparation (NLP) has been found to protect circulating blood of cis and 5-FU treated Swiss mice signicantly. NLP could, therefore, be a better substitute than GCSF which is expensive and is also known to promote angiogenesis and tumor development in the experimental animals.14

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Table 3. Medicinal properties of neem (A. indica) tree preparations S. no 1 2 3 4 5 6 7 8 9 10 11 Therapeutic uses Immunostimulant activity Hypoglycaemic activity Antiulcer effect Antifertility effect Antimalarial activity Antifungal activity Antibacterial activity Antiviral activity Anticancer activity Antioxidant activity Effect on central nervous system Types of preparations Aqueous extracts of neem bark and leaf Aqueous extracts of neem leaves Aqueous extracts of neem leaf and bark Seed oil Seed and leaf extracts Extracts of neem leaf, neem oil seed kernels Aqueous leaf extract Aqueous leaf extract Aqueous leaf extract Leaf and seed extract Leaf extract References 45 46, 56 57 4749, 58, 59 6062 6364 6365 24, 27, 3132, 66, 67 24, 27, 31, 32 1213, 15, 16 68

Skin tumor induction is often associated with upregulation of certain marker enzymes, such as alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase. Upon treatment with aqueous A. indica leaf extract (AAILE) there was signicant decrease in the activity of these marker enzymes. The AAILE treatment reduced oxidative stress by decreasing lipid peroxidation levels and by enhancing the reduced glutathione contents and activities of various antioxidant enzymes.15,16 Hanachi et al. demonstrated that oral administration of 5% (w/v) of A. indica extract suppressed the diethylnitrosamine and acetylaminouorene induced pre-neoplastic nodules in the male Sprague drawly rats apparently by increasing the distribution of antioxidant elements and GST activity. Their results suggest that neem extract protects against onset of hepatocarcinoma with negligible side complications on normal cells. In another interesting piece of research performed on 7-wk-old Swiss albino mice,18 80% ethanolic extract of the leaves of A. indica has been shown to enhance the phase-II hepatic enzymes, such as, Glutathione S Transferase and DT-diaphorase. These enzymes, which are known to be involved in detoxication of certain chemical carcinogens, were studied vis--vis B P and DMBA (7,12 dimethyl benz anthracene)-induced onset of carcinogenesis. The neem extract was observed not only to induce the phase-II enzymes but also to enhance the activity of the hepatic antioxidant enzymes including, glutathione reductase, glutathione peroxidase and superoxide dismutase. Similar results on the anti-oxidant activity of the aqueous foliar extract and ethanolic extracts of ower and stem bark of Siamese neem has been made by Sithisarn et al. Neem may, therefore, be used as a potent preventive and adjuvant therapy against cancer. Neem Arrests Tumor Cell Growth and Proliferation There is interesting and compelling evidence to suggest that neem may be used as a tumor suppressor. Researchers in India, Europe and Japan have found that polysaccharides and limonoids present in the neem bark, leaves and seed oil reduced tumors and cancers, and showed efcacy against lymphocytic leukemia. In Japan, hot water extracts from neem bark showed remarkable effectiveness against several types of tumors. Some

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of these extracts were equal to or better than the standard anticancer agents, particularly against solid tumors. For example, ethanolic extracts (80%) of neem leaf, when administered at doses of 250500 mg per kilogram, suppressed the average number of papilomas as well as overall tumor burden induced by B P and DMBA signicantly (p < 0.005 and p < 0.001) in the 7-wk-old Swiss albino mice model.18 In another interesting molecular study, a Japanese group has demonstrated that nimbolide, a triterpenoid present in certain edible parts of A. indica, arrested the HT-29 (human colon carcinoma cells) in G2 /M and G0 /G1 stages apparently through upregulation of p21 which is a well known down-stream effector of the p53 gene. p53 is a very important anticancer protein (product of p53 gene) that regulates a large number of genes that are involved in tumorigenesis. Nimbolide has also been shown to upregulate cyclin D2 and Chk2; and to suppress the expression of cyclin A, cyclin E, Cdk2 and Rad17 at the same time.20 Extending these studies on the U937, HL-60, THP1 and B16 cell lines, Kumar and authors21 have demonstrated that nimbolide exerted antiproliferative and apoptotic effects apparently through the suppression of bcl-2/bax ratio and through the mediation of increased expression of Apaf-1 and caspase-3. These observations suggest that neem ingredients act through molecular pathways to register anticancer effects. Subapriya et al.22 also investigated molecular pathways of anticancer effects of the ethanolic neem leaf extract (ENLE) on DMBA-induced carcinogenesis in buccal pouch of hamster. Their observations suggest involvement of the PCNA (Proliferating cell nuclear antigen), mutant p53 and bcl2 genes. These pro-cancer genes are upregulated during DMBA-induced carcinogenesis. The use of ENLE has been observed by them to suppress these genes as well as DMBA-induced cell proliferation and differentiation. In another interesting study by Bieberich et al, neem seed oil has been found to suppress breast tumor development in the ex vivo experimental models of female mice. They have observed that 50 l of neem seed oil inoculated at a dilution of 1:25 every week around the ex vivo developing breast cancer (MCF-7 cell line) for about 50 d reduced tumor burden by 50%. These observations provide emphatic evidence to help evolve working strategies to make use of antitumor efcacy of neem extracts on human beings.


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Table 4. Triterpenoids and steroids isolated from neem (A. indica) S. no 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Name of constituents Nimbin Nimbinin Nimbidic acid (Salannic acid) Salannin Deacetyl Nimbin Nimbolide Meliantriol Azadirone Epoxyazadiradione Azadiradione Gedunin 7-Deacetyl Gedunin Meldenin Salannin-lactone Nimbin-lactone Azadirachtin Vepinin Nimbolin A Nimbolin B Vilasinin Nimbidinin Nomolin Chemical formula C30H36O9 C28H34O6 C26H34O7 C34H44O9 C28H34O8 C27H30O7 C30H50O5 C28H36O4 C28H34O6 C28H34O5 C28H34O7 C26H34O6 C28H38O5 C34H44O10 C30H36O10 C35H44O16 C28H36O5 C39H46O8 C39H46O10 C26H34O6 C26H36O5 Melting point (C) 205 202 228 167 208 245 176 192 202 205 218 259 240 244 184 155 180 243 282 255 166 177 Isolated from Oil, trunk and root bark Oil, trunk and root bark Oil Oil Seeds and bark Leaves Oil Oil Oil Oil Oil Oil Oil Oil Oil Seeds Oil Trunk wood Trunk wood Oil Leaves Fruits Fruits

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C28H34O6 C28H34O5 205 Nimolicin 17-Hydroxy-azadiradione 17-Epi-azadiradione 1-Methoxy-1,2-dihydroepoxy-azadiradione 1, 2-Diepoxy-azadiradione 7-Acetylneotrichilenone 7-Deacetyl-7-benzoylazadiradione 7-Deacetyl-7-benzoylepoxy-azadiradione 7-Deacetyl-7-benzoylgedunin Nimbinene 6-Deacetyl nimbinene Nimbandiol 6-o-Acetyl-nimbandiol 3-Deacetylsalannin Salannol 1,3-Diacetyl-vilasinin Nimocinol (6 -Hydroxy-azadirone) -Sitosterol -Sitosterol--D-glucoside Cycloeucalenol 24-Methylene-cycloartanol 4,14 -Dimethyl 5-ergosta-8,24 (28)-dien-3-ol 4 -Methyl-5-ergosta-8,24 (28)-dien-3-ol Neem leaf glycoprotein C28H34O6 C28H34O5 C29H38O7 C28H34O7 C28H36O5 C33H36O5 C33H36O6 C33H36O7 C28H34O7 C26H32O6 C26H32O7 C28H34O8 C32H42O8 C32H44O8 C30H40O7 C28H36O5 C29H50O C35H60O6 C30H50O C31H52O C30H50O C29H48O 17-Hydroxy-azadiradione C28H34O6 205 235 110 208 Amorphous Amorphous 278 134 141 121 178 214 208 157 130 140 283 138 122 -

Fruits Fruits Fruits

Seeds Seeds Seeds Seeds Seeds Seeds Oil, leaves and bark Oil, leaves and bark Oil, leaves and bark Oil Oil Oil Oil Leaves Blossom, leaves and wood oil Blossom, leaves and heart wood Wood oil Wood oil Heart wood Heart wood Leaf

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Table 5. Non-terpenoid and non-steroid constituents of neem (A. indica) S. no 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Name of constituents Kaemferol Quercetin Myricetin Sugiol Nimbiol Glucoside of Quercetin Glucoside of Kaemferol Melicitrin Quercetin-3-galactoside (Hyperin) Kaemferol-3-glucoside (Astragalin) Quercitrin Rutin Isorhamnetin Rhamnoside of Quercetin 5-Hydroxy-methyl furfural Chemical formula C15H10O6 C15H10O7 C15H10O8 C20H28O2 C18H24O2 C21H20O12 C21H20O11 C20H18O12 C21H20O12 C21H20O11 C21H20O11 C27H30O16 C16H12O7 C21H20O11 C6H6O3 Melting point (C) 276 313 357 292 250 237 178 250 214 305 245 Isolated from Blossoms Blossoms and leaves Blossoms Trunk bark Trunk bark Leaves Leaves Blossoms Blossoms and leaves Blossoms Leaves Leaves Leaves Leaves Fruit

Neem Induces Apoptosis in Cancer Cells Apoptosis or programmed cell death is a genetically driven process that has been kept conserved largely among the metazoans since millions of years. This is such a ne natural method of surgery that requires biochemical blades led by a host of caspase enzymes to wipe out the unwanted, redundant, wayward, incorrigible and irreparable cells of the body without shedding even a single drop of blood and without any material loss to the body as a whole. The medical practitioners have, therefore, accepted elimination of cancer cells through orchestration of apoptosis as a therapy of choice. Neem extracts and its puried products have been examined for induction of apoptosis among the cancer cells. Following DNA fragmentation and cell viability assays, an ethanolic extract of neem has been shown to induce apoptosis in prostate cancer cells (PC-3) in a dose-dependent manner.21 These observations are commensurate with the ndings that treatment with neem extract (1) suppressed the level of expression of bcl-2 protein, which is a strong pro-survival factor in cancer cells and (2) at the same time enhanced the level of expression of pro-apoptotic Bax protein. The neem extract could thus be a potentially effective treatment against prostate cancer. Working on the choriocarcinoma (BeWo) cells, Kumar et al.24 have shown that nimbolide, which is an active anticancer principle of the neem leaf and owers, induces apoptosis through engagement of the mitochondrial pathway. The involvement of this pathway is based on the observation that nimbolide mediates upregulation of Apaf1 and caspase3 and decrease in the bcl2/bax ratio. Treatment of U937, HL-60, THP1 and B16 cells with nimbolide has been observed to disrupt cell cycle check points (G2 /M, G0 /G1 and G/S) in correlation to induction of apoptosis.25 The molecular connection that exists between cell cycle disruption and apoptosis is not clearly shown by them in these cells. Sometimes, cell cycle disruption leading to arrest of cells at the cell cycle check points is attributed to immunity against apoptosis. This is true at least

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in cases where cell cycle arrest has been demonstrated to accrue through p53 and its downstream attendant p21 gene.26 Subapriya and coworkers22,27 have found convincing results on the apoptosis-inducing ability of ENLE on DMBA-induced cancer cells/tissues in the HBP model. They have shown that ENLE enhanced the expression of pro-apoptotic genes, such as, bim, caspase-8 and caspase-3, and at the same time suppressed bcl2, PCNA and mutant p53 in the DMBA-induced cancer cells (Fig. 2). These observations are correlated reasonably well to elimination of the neoplasm. It is, thus obvious that crude extracts and puried drugs from neem possess credible ability to induce programmed cell death among cancer/tumor cells. This therapy of cancer may be considered for human patients, as well. The Role of Neem in Bioimmunotherapy of Cancer The innate and the acquired immune systems keep vigil on the overall defense of the body. Gearing up the immune system against targeted attack on health risks has been contemplated as a dependable therapy known as bioimmunotherapy. Cancer biologists and medical practitioners have accepted bioimmunotherapy as one of the dependable therapeutic regimens against cancer. Several drugs of immunological origin are in great demand. For example, interferons (IFN) are well known therapeutic cytokines that are used as anticancer agents against many types of cancer cells in vitro and in vivo.28 It is used individually and in combination with other anticancer agents including ionizing radiations. Subversion of the bodys immune and genetic system is often a prelude to initiation of cancer. Some recent inputs to cancer research demonstrate that neem exerts anticancer effects against various types of cancers efciently by gearing up the bodys immune response. Neem modulated active specic immunotherapy intends to boost the hosts antitumor immune response by proper presentation of tumor-associated antigen (TAA) or its derivatives to B and T cells, where the role of antigen presenting cells (APC) is predominant. Closely in line with this view,


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Figure 2. Functional mechanisms of neem. 24,27,31-32

Baral et al.29 demonstrated that neem leaf preparation (NLP) acted as an adjuvant in the course of generation of appreciable level of anti-serum in C57BL/6 mice against B16MelSAg (vaccine). This anti-serum must show anticancer property if it is to be dependable. Vaccination of the mice with B16MelSAg+NLP

indeed prevented the growth of B16 melanoma tumor more efciently than B16MelSAg and NLP alone. This result was adequately supported by the observation that anti-serum (raised against B16MelSAg+NLP) when injected subcutaneously after mixing with B16Mel tumor in the syngenic C57BL/6 mice

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reduced the tumor burden quite signicantly as compared with parallel controls. In an interesting study, Sarkar et al.30 have shown that antiserum raised against NLP in the Swiss mice may react with carcinoembryonic antigen (CEA) as well as with a peptide derived from it. NLP may thus be considered for use not only as a potential immune adjuvant but also as an effective antigen for inducing active immunity against at least certain tumors. These results may help evolve formal strategies for treatment of tumors, particularly CEA positive ones, albeit a convincing explanation of this observation is yet to come. Recent work done by Bose and Baral31 shows that NLP enhances NK-cell mediated cytotoxicity of human cancer cells (K562) possibly through CD40-CD40L dependent endogenous production of IL-12 and activation of p38 MAPK pathway. It has been suggested that these pathways critically control perforin to eliminate tumor cells. An active ingredient (Neem Leaf Glycoprotein, NLGP) recently isolated from neem leaf preparation has been investigated for its immunomodulatory role in the restoration of the impaired chemotactic movement of peripheral blood mononuclear cells (PBMC) toward the head and neck squamous cell carcinoma to exert antitumor activities.32 Their study is actually based on an earlier nding that NLGP exerts immunomodulation-mediated restriction of murine tumors in vivo. They have observed that NLGP enhanced expression of IFN which in turn downregulated CXCR3B (splice variant of CXCR3, responsible for lymphocyte apoptosis) in the in vitro human HNSCC-PBMC. As a result, the ratio of CXCR3A/ CXCR3B increased and restored chemotaxis of PBMCs toward the tumor. Activation of the immune system by NLGP was also studied in terms of upregulation of early markers, CD69 and CD45RO and downregulation of CD45RA and CD62L (L-selectin) on the lymphocyte, monocyte and dendritic cells.33 NLGP has been observed to prompt the type I response through activation of T cells that begin secretion of greater amount of signature T-helper (Th) 1 cytokines, such as IFN. NLGP also gears up type II response measured in terms of IL4 secretion but to a comparatively less amount than type I. These workers have further shown that NK-cells and cytotoxic T-cells isolated from the immunosuppressed patients of HNSCC (head and neck squamous cell carcinoma) may be activated effectively by NLGP to kill K562 (erythroleukemia) cells and KB (oral cancer) cells, respectively. NLGP-induced activation of CTL response was found dependent on IFN-mediated upregulation of perforin/ granzyme B in the killer cells, but independent of this in the NK cells.34 A similar preparation from neem (NLP) was observed to replenish the pool of WBC, which was reduced by cyclophosphamide (CYP) in mice.35 This result was in good correlation to signicant inhibition of CYP-induced tumor growth and consequent survival rate of mice following pre-treatment with NLP. In another interesting study, pretreatment with neem leaf preparation (NLP) suppressed murine Enrlich carcinoma and B16 melanoma in the Swiss and C57BL/6 mice. These observations correlated well to activation of NK and NK-T cells, as well as enhanced secretion of TNF and IFN.36 TNF is a controversial cytokine that may act as antitumor and protumor depending upon several factors including the type of cell line, external

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and internal stimuli, etc. However, a large body of modern literature on cancer shows that TNF is a strong survival factor for cancer, because it activates NFB (a strong transcription factor) that may induce innumerous genes that support survival of cancer.37 Using BALB/-c mice as an experimental model it has been observed that there was a signicant enhancement in the peritoneal macrophage activity and expression of activation marker CD44 following regular subcutaneous administration of aqueous neem extract. This extract was also observed to enhance the CD4, CD8 and CD25 positive sub-population of lymphocytes. At the same time, metastatic activity of sarcoma L-1 and lymphosarcoma RAW cells was attenuated by this extract.38 Nitric oxide (NO), a key tumoricidal agent is known to regulate T-cell proliferation, cytokine production, cell signaling and apoptosis.39 Several laboratories have demonstrated that the T-cell derived cytokines IFN, along with IL-2 and/or TNF, are macrophage activators and serve as a potent inducer of NO.40 NLGP along with tumor antigen vaccination showed NO production and upregulation of IL-12 and excess IFN.41 Neem leaf preparation enhances tumor antigen presentation of the macrophages42 and dendritic cells to the T and B cells for induction of antitumor immunity by allowing generation of immune effector/memory response.43 That NLGP can enable maturation of the myeloid derived DCs and can optimize the antitumor T-cell functions is also known. Thus, it can be used as a candidate vaccine tool for antigen specic cancer immunotherapy.44 Toxicological Risk Factors Involved in the Use of Neem Extracts

In spite of its versatile qualities neem deserves use with care. Indiscriminate use of its extracts, particularly when taken in overdoses, may cause unpleasant side effects. Some people are excessively allergic to neem products which may cause itching, swelling of mouth and throat, wheezing and sometimes breathing difculties. Use of neem or its products may also be a reason for damage to liver and kidney that may result in jaundice and in low or no urine production, respectively. Although neem has some hematostimulatory effect as observed in mice, it may also destroy red blood corpuscles.45 Excessive use of neem may bring about neurotoxicity. One of the important concerns about its use is its ability to interfere with the normal reproductive systems fostering infertility.46-49 DNA methylation is a major epigenetic regulatory mechanism. A statistically insignicant reduction in methylated deoxycytidine has been observed in the infertile male group treated with neem than in the untreated ones.50 Neem has been in use since the dawn of civilization, yet no severe harmful effect has been reported. Since ancient times, therefore, neem tree is kept at bay from the regular dwelling places. Conclusion Neem (Azadirachta indica) has been an ancient source of herbal panacea against a variety of human health problems. Researchers have been trying to purify the active ingredients from this plant and to work out their mechanistic, therapeutic and clinical


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aspects on the reliable systems. Over 60 various products from neem are available in the puried form. These products vis-vis the crude extracts of neem are being tested expediently on suitable in vitro and in vivo systems to establish their anticancer and immunomodulation effects. There are compelling experimental evidences to suggest that neem products e.g., Azadirachtin A, Nimbolide, Nimbidin etc., possess convincing anticancer properties. Molecular mechanisms of their action have been investigated. Due to these reasons, it is imperative that suitable clinical trials should be conducted to pave the way to bring these products into the market as certied anticancer drugs. The scientic data show that suitable combinations of drugs tempered with radiotherapy and bioimmunotherapy may be of great promise in nding a sure cure for cancer patients. Neem products may cause some unpleasant side effects, particularly if taken in overdoses. It is, therefore, advisable that these products be used under the supervision and prescription of qualied medical practitioners and physicians. Future Perspectives The world market for alternative medicine has increased to about 10.895 billion USD as per a recent report by the World
1. Ganguli S. Neem: A therapeutic for all seasons. Curr Sci 2002; 82:1304. 2. 3. 4. Biswas K, Chattopadhyay I, Banerjee RK, Bandyopadhyay U. Biological activities and medicinal properties of Neem (Azadirachta indica). Curr Sci 2002; 82:1336-45. 5. Thomas R, Sah NK, Sharma PB. Therapeutic biology of Jatropha curcas. Curr Pharm Biotechnol 2008; 9:315-24; PMID: 18691091; DOI: 10.2174/138920108785161505. 6. Gangar SC, Sandhir R, Rai DV, Koul A. Modulatory effects of Azadirachta indica on benzo(a)pyreneinduced forestomach tumorigenesis in mice. World J Gastroenterol 2006; 12:2749-55; PMID: 16718763. 7. Chaimuangraj S, Arakaki J, Suzui M, Morioka T, Kinjo T, Kaneshiro T, et al. Antioxidative and Modifying Effects of a Tropical Plant Azadirachta indica (Neem) on Azoxymethane-induced Preneoplastic Lesions in the Rat Colon. Asian Pac J Cancer Prev 2006; 7:467-71; PMID: 17059347. 8. Sritanaudomchai H, Kusamran T, Kuakulkiat W, Bunyapraphatsara N, Hiransalee A, Tepsuwan A, et al. Quinone reductase inducers in Azadirachta indica A. Juss flowers, and their mechanisms of action. Asian Pac J Cancer Prev 2005; 6:263-9; PMID: 16235984. 9. Tepsuwan A, Kupradinun P, Kusamran WR. Chemopreventive potential of neem flowers on carcinogen-induced rat mammary and liver carcinogenesis. Asian Pac J Cancer Prev 2002; 3:231-8; PMID: 12718580. 10. Aruna K, Sivaramakrishnan VM. Plant products as protective agents against cancer. Indian J Exp Biol 1990; 28:1008-11; PMID: 2283166. 11. Niture SK, Rao US, Srivenugopal KS. Chemopreventive strategies targeting the MGMT protein: Augmented expression in human lymphocytes and tumor cells by ethanolic and aqueous extracts of several Indian medicinal plants. Int J Oncol 2006; 29:1269-78; PMID: 17016661.

Health Organization (WHO). The global market for herbal medicine alone is pegged at $5 trillion with an annual growth rate of about 11 per cent.51 The rapidly growing market of the herbal/alternative medicine shows its world-wide popularity and acceptance. A. indica (Neem) is a discovery of the pristine civilizations that has withstood trial of time and wisdom over the generations for treatment of various types of wounds (nassor) in the body, particularly those with poor prognosis and diagnosis. Convincing pre-clinical observations on the efcacy of its various extracts and puried products against most types of cancer have been made on suitable systems. There is no doubt that the products of this plant enshrine bright prospects as a reliable cure for cancer, but its protocol needs ne tuning for use in the clinical trials. One of the important reasons is its nominal side effects. A multi-institutional trial may be set up to spruce up chances for its speedy acceptance in the clinics, particularly where cancer medicines are scarcely affordable. However, this suggestion may not nd warm favor from companies that invest sumptuously to produce costly cancer medicines.
Disclosure of Potential Conicts of Interest

No potential conicts of interest were disclosed.

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Cancer Biology & Therapy Volume 12 Issue 6