Accepted Manuscript Review Chemopreventive and Therapeutic Effects of Nimbolide in Cancer: the Underlying Mechanisms Lakshmi Narendra Bodduluru, Eshvendar Reddy Kasala, Nagaraju Thota, Chandana C Barua, Ramakrishna Sistla PII: DOI: Reference:

S0887-2333(14)00074-5 http://dx.doi.org/10.1016/j.tiv.2014.04.011 TIV 3304

To appear in:

Toxicology in Vitro

Received Date: Accepted Date:

6 December 2013 14 April 2014

Please cite this article as: Bodduluru, L.N., Kasala, E.R., Thota, N., Barua, C.C., Sistla, R., Chemopreventive and Therapeutic Effects of Nimbolide in Cancer: the Underlying Mechanisms, Toxicology in Vitro (2014), doi: http:// dx.doi.org/10.1016/j.tiv.2014.04.011

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CHEMOPREVENTIVE AND THERAPEUTIC EFFECTS OF NIMBOLIDE IN CANCER: THE UNDERLYING MECHANISMS

a

a

a

b

Lakshmi Narendra Bodduluru , Eshvendar Reddy Kasala , Nagaraju Thota , Chandana C Barua , Ramakrishna c,*

Sistla a

Department of Pharmacology & Toxicology, National Institute of Pharmaceutical Education and Research

(NIPER), Guwahati-781032, Assam, India. Email: [email protected] ; [email protected] ; [email protected] b

Department of Pharmacology and Toxicology, College of Veterinary Science, Assam Agricultural University,

Guwahati-781032, Assam, India. Email: [email protected] c

Medicinal Chemistry & Pharmacology Division, Indian Institute of Chemical Technology (IICT), Hyderabad-

500007, Andhra Pradesh, India. Email: [email protected]

* Correspondence author Dr. Sistla Ramakrishna, Principal Scientist, Council of Scientific and Industrial Research- Indian Institute of Chemical Technology (CSIR-IICT). Telephone: +91 40 27193753 E-mail address: [email protected]

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ABSTRACT Cancer chemoprevention is a strategy taken to block, reverse or retard the multistep process of carcinogenesis, including the blockage of its vital morphogenetic milestones viz. normal-preneoplasia-neoplasia-metastasis. Naturally occurring phytochemicals are becoming increasingly popular over synthetic drugs for several reasons, including safety, efficacy, and easy availability. Nimbolide, a triterpene derived from the leaves and flowers of neem, is widely used in traditional medical practices for treating various human ailments. The neem limonoid exhibits multiple pharmacological effects among which its anticancer activity is the most promising. The preclinical and mechanistic studies carried over the decades have shown that nimbolide inhibits tumorigenesis and metastasis without any toxicity and unwanted side effects. Nimbolide exhibits anticancer activity through selective modulation of multiple cell signaling pathways linked to inflammation, survival, growth, invasion, angiogenesis and metastasis. The present review highlights the current knowledge on molecular targets that contribute to the observed anticancer activity of nimbolide related to (i) inhibition of carcinogenic activation and induction of antioxidant and carcinogen detoxification enzymes, (ii) induction of growth arrest and apoptosis; and (iii) suppression of proinflammatory signaling pathways related to cancer progression. Keywords: Cancer, Nimbolide, Chemoprevention, Apoptosis, NF-κB.

Highlights
Cancer is a complex disease with dysregulation of multiple cell signaling pathways.
Chemoprevention is a strategy to block, reverse or retard carcinogenesis.
Nimbolide has shown significant anticancer activity in vitro and in vivo.
Nimbolide acts by inducing apoptosis and inhibiting tumor cell proliferation.


Nimbolide suppresses the NF-κB, Wnt, PI3K-Akt, MAPK and JAK-STAT signaling pathways.

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1. INTRODUCTION Cancer is a leading cause of death in economically developed countries and the second leading cause of death in developing countries (Jemal et al., 2011). The global burden of cancer continues to increase largely because of aging and growth of world population as well as increasing adoption of cancer-associated lifestyle choices like smoking, sedentary habits and westernized diets. As per Globocan 2012 report, an estimated 14.1million people were diagnosed with cancer across the world and 8.2 million people died from the disease (Globocan 2012). It is also estimated that if this current trend continues, there would be 22 million new cases and around 13.2 million deaths worldwide occurring each year by 2030 (Ferlay et al., 2010). Cancer development is a multistep process in which a cell acquires essential alterations that dictate the progressive transformation of normal cells into cancer cells. The cellular alterations include evading apoptosis, self-sufficiency in growth signals, limitless replicating potential, evading growth suppressors, sustained angiogenesis, tissue invasion and metastasis (Hanahan and Weinberg, 2011; Singh, 2013). In spite of significant progress in understanding the biology of cancer and development of anticancer therapies, the number of deaths caused by the dreadful disease remains unabated. The main cause for this disappointment is, even though it is well understood that cancer is a hyperproliferative disorder mediated through dysregulation of multiple genes and cell signaling pathways, most cancer drug developments remain focused on modulation of a single gene product or cell signaling pathway (Paul et al., 2011). Chemotherapy and specific targeted drugs have been developed to disrupt the gene products or pathways. However, problems such as ineffective targeting and drug resistance have plagued these agents. Therefore, the current paradigm in cancer chemotherapy is to use a combination of several drugs or a drug that modulates multiple targets (Hasima and Aggarwal, 2012). Over the decades, phytochemicals have gained considerable attention from researchers and clinicians because of their safety, efficacy, and immediate availability (Gupta et al., 2013). This mounting interest has led to the development of several clinically available anticancer drugs. These include Vinca alkaloid’s vinblastine and vincristine, paclitaxel (taxol), the epipodophyllotoxin derivative etoposide and the camptothecin derivatives topotecan and irinotecan. Phytochemicals derived from medicinal plants have the ability to target multiple signaling pathways, exhibit minimal (or no) toxicity, and thus are ideal as alternatives and complementary forms for cancer treatment. Nimbolide is one such compound that has the potential to modulate multiple signaling pathways in cancer cells. Neem (Azadirachta indica L), is a traditional medicinal plant of the Meliaceae family widely distributed in Asia, Africa and other tropical parts of the world. Neem is extensively used in traditional medical practices (Ayurveda, Unani and Homoeopathy) for treating various human ailments. All parts of the neem tree offer tremendous potential for medicinal, agricultural and industrial exploitation and have been evaluated for antiinflammatory, antipyretic, antihistamine, antifungal, antitubercular, antiprotozoal, vasodilatory, antimalarial, diuretic, spermicidal, antiarthritic, antiprotozoal, insect repellent, antifeedant and antihormonal activities (Biswas et al., 2002). Limonoids, the modified triterpenes formed as secondary metabolites by plants in the Meliaceae and Rutaceae families, have attracted considerable research attention as promising candidates for cancer chemoprevention. Nimbolide (5, 7, 4′-trihydroxy-3′, 5′-diprenylflavanone), a tetranortriterpenoid with α, βunsaturated ketone system and a γ-lactone ring (Fig.1) was first derived from the leaves and flowers of neem. This isoprenoid has been shown to exhibit numerous biological activities, including anti-feedant (Suresh et al., 2002), anti-malarial (Rochanakij et al., 1985), anti-microbial (Biswas et al., 2002), anti-HIV (Udeinya et al., 2004) and anticancer activities (Paul et al., 2011). Nimbolide exhibits anticancer activity in a wide variety of cancer cells.

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Literature evidence reveals that α, β-unsaturated ketone structural element is responsible for the anticancer activity of nimbolide (Sastry et al., 2006). This review focuses on the anticancer biology of nimbolide with main emphasis on the in vitro and in vivo studies and on the underlying molecular mechanisms.

Chemoprevention- A Promising Approach for Overcoming Cancer Burden The idea of interrupting the process of carcinogenesis using either natural or synthetic external substances was introduced in the 1970s by Dr. Michael Sporn, who coined the term, ‘‘chemoprevention’’. Chemoprevention is defined as the use of either natural or synthetic substances or their combination to block, reverse or to retard the process of carcinogenesis (Sporn et al., 1976). Primary chemoprevention refers to the use of an agent that prevents carcinogenesis in healthy individuals who are at high risk. Secondary chemoprevention refers to preventing the full transition to malignancy in a patient who already has developed a pre-malignant lesion. Tertiary chemoprevention refers to the use of an agent that prevents a second primary cancer or metastasis in a patient who had a first malignancy that has been treated (Tsao et al., 2004). Chemopreventive agents are subdivided into two main categories: (i) blocking agents, which inhibit the initiation step by preventing carcinogen activation and (ii) suppressing agents, which inhibit malignant cell proliferation during promotion and progression steps of carcinogenesis (Wattenberg, 1985; Surh, 2003).

2. ANTICANCER ACTIVITY OF NIMBOLIDE 2.1 Chemopreventive Effects of Nimbolide and Neem Chemopreventive agents retard carcinogenesis by a variety of mechanisms directed at all major stages of carcinogenesis (Fig.2) (Wattenberg, 1997). The possible methods of intervening carcinogenesis include, modulation of carcinogen biotransformation, scavenging free radicals and altering the expression of genes involved in cell signaling, particularly those regulating cell proliferations, apoptosis and differentiation (Hursting et al., 1999). Induction of phase II detoxification enzymes such as glutathione S-transferase (GST) or NAD(P)H quinone oxidoreductase (QR) is one of the major mechanisms of protection against initiation of carcinogenesis (Talalay, 2000). Neem preparations were reported to induce the activity of antioxidant and detoxification enzymes, reduce the activities of cytochrome P450 (CYP)-dependent monooxygenases, inhibit cellular proliferation, induce apoptosis and possess cancer chemopreventive potential against chemically-induced carcinogenesis models (Subapriya et al., 2005; Vinothini et al., 2009). In other studies, nimbolide inhibited the development of 7,12dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinomas by influencing multiple mechanisms, including prevention of procarcinogen activation and oxidative DNA damage, upregulation of antioxidant and carcinogen detoxification enzymes (glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GR), gamma glutamyl transpeptidase (GGT), NAD(P)H dehydrogenase quinone 1(NQO1), manganesesuperoxide dismutase (Mn-SOD), catalase (CAT), GST, QR), induction of apoptosis, inhibition of tumour cells proliferation, invasion, angiogenesis and metastasis (Sritanaudomchai et al., 2005; Priyadarsini et al., 2009; Kumar et al., 2010; Gupta et al., 2013). The chemopreventive activity of neem extracts and nimbolide was reported in many preclinical studies and the details are summarized in Table 1.

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2.2 In vitro Cytotoxicity of Nimbolide The cytotoxicity of nimbolide has been extensively studied over the last years in a large variety of cancer cell lines. Cohen et al., examined nimbolide for cytotoxicity against N1E-155 murine neuroblastoma and 143B TKhuman osteosarcoma cell lines, and found nimbolide to be more potent antiproliferative agent compared to azadirachtin (Cohen et al., 1996). Roy et al., investigated the inhibitory effect of nimbolide on the growth of leukemic (HL-60, U937 and THP-1) and melanoma (B16) cell lines and observed that, all the cell lines were sensitive to the cytotoxic effects of nimbolide (Roy et al., 2007). Sastry et al., tested the in vitro cytotoxicity of nimbolide against a panel of human cancer cell lines and reported IC50 values ranging from 4.17 to 15.56 with an average of 8.31µM (Sastry et al., 2006). Nimbolide demonstrated superior cytotoxicity compared to positive control cisplatin against HL-60, SMMC7721, A549, MCF-7, and SW-480 cell lines (Chen et al., 2011). Furthermore, nimbolide exerts antiproliferative and apoptotic inducing effects on BeWo, MCF-7, MDA-MB-231, HeLa, HT-29 and PC-3 cancer cell lines (Roy et al., 2006; Kumar et al., 2009; Priyadarsini et al., 2010; Elumalai et al., 2012). In addition, nimbolide abrogates canonical nuclear factor-kappa B (NF-κB) to induce apoptosis in HepG2 and WiDr cancer cell lines (Babykutty et al., 2012; Kavitha et al., 2012). In another study Gupta et al., reported that nimbolide selectively sensitized human colon cancer cells to tumor necrosis factor-related apoptosisinducing ligand (TRAIL) through reactive oxygen species (ROS) and extracellular-signal-regulated kinase (ERK)dependent upregulation of death receptors (DRs), p53 and Bcl-2-associated X protein (Bax) (Gupta et al., 2011). These results indicate that nimbolide exerts its effects selectively in the cancer cells. Table 2 summarizes the cytotoxic activity of nimbolide on cancer cell lines.

2.3 Anticancer Activity of Nimbolide in Combination with Chemotherapeutic and TRAIL-based regimens Besides anticancer properties as single agent, nimbolide has also been reported to possess additive or synergistic tumor killing activity in combination with different cytotoxic stimuli such as tumor necrosis factor alpha (TNF-α), TRAIL and chemotherapeutic drugs. Nimbolide was found to enhance the cytotoxic and apoptotic inducing effect of cytokine (TNF-α) and chemotherapeutic drugs (5-fluorouracil, thalidomide) in KBM-5 cells through suppression of inhibitor of kappa B (IκB)-kinase (IKK)-induced NF-κB pathway (Gupta et al., 2010). Nimbolide exerts its NF-κB inhibitory effect by specifically targeting the cysteine (Cys179) in the IKK-β activation loop. The above finding was supported by the evidence that, addition of reducing agent and/or mutation of cysteine residue (Cys179) of IKK-β to alanine abolished the inhibitory effect of nimbolide on IKK activation. In addition, combined treatment with nimbolide and TRAIL acted in concert to induce apoptosis via ROS and ERK-mediated up-regulation of DR5 and DR4, down-regulation of cell survival proteins, and up-regulation of pro-apoptotic protein’s p53 and Bax (Gupta et al., 2011). The upregulation of DR5 and DR4 was not restricted to colon cancer cells but also occurred in chronic myeloid leukemia (KBM-5), multiple myeloma (U266), embryonic kidney carcinoma (A293), pancreatic cancer (AsPC-1) and breast cancer cells (MCF-7, MDA-MB-231). Gene silencing of the receptors reduced the effect of limonoid on TRAIL-induced apoptosis. In addition, gene silencing of ERK1&2 abolished the enhancement of TRAIL-induced apoptosis and upregulation of DR5 and DR4 (Gupta et al., 2011). Importantly, the combination treatment with nimbolide and TRAIL synergized to induce apoptosis in different tumor cell lines, but not in normal breast cells indicating some tumor specificity (Gupta et al., 2011). These reports suggest that using nimbolide as sensitizer in TRAIL-based combination regimens may be a novel strategy to enhance the efficacy of anticancer therapy.

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2.4 Studies in tumor bearing animal models In addition to its antitumor activity in a variety of cancer cell lines, nimbolide also suppressed tumor growth in vivo in a number of animal studies. In animal models nimbolide at doses of 10 and 100 µg/kg, (intragastric) was shown to exhibit chemopreventive activity in DMBA-induced buccal pouch carcinogenesis by modulating the levels of phase I and phase II xenobiotic metabolizing enzymes (XMEs), antioxidants, invasion and angiogenesis (Priyadarsini et al., 2009). In another hamster model of oral carcinogenesis, the limonoid was shown to exhibit antiproliferative and apoptosis-inducing activities (Kumar et al., 2010). In a xenograft mouse model of colon cancer, administration of nimbolide at doses of 5 and 20mg/kg intraperitoneally inhibited the survival and growth of colorectal carcinoma (CRC) xenografts (Gupta et al., 2013). Nimbolide suppressed the growth of CRC xenografts through modulation of expression of NF-κB regulated gene products linked to survival, proliferation, invasion and angiogenesis. Moreover, pharmacokinetic studies in mice bearing CRC xenografts demonstrated that nimbolide was well absorbed and distributed with the highest concentrations found within the tumor. More specifically, concentration levels of 222ng/ml and 409ng/ml were detected in the plasma of mice treated with nimbolide at 5mg/kg and 20mg/kg of body weight respectively. Similarly, the concentration levels of 345ng/g and 868ng/g of tumor tissue were obtained from the mice treated with nimbolide at 5mg/kg and 20mg/kg of body weight respectively. Overall, the bioavailability of nimbolide and its efficacy in inhibiting the growth of the CRC xenograft at relatively lower concentrations strengthens its therapeutic value.

3. MECHANISM OF ACTION OF NIMBOLIDE Understanding the molecular mechanism of action of drugs is essential to predict the potential therapeutic and side effects of the substances. For this purpose, the molecular and cellular mechanism by which nimbolide induces cytotoxicity has been under investigation. Accumulating evidence suggests that the antitumor effect of nimbolide is attributed to its ability to target a plethora of signaling pathways governing apoptosis, cell cycle progression, cell proliferation and survival. The diverse molecular targets influenced by nimbolide includes the transcription factors, growth factors and their receptors, cytokines, enzymes, and genes regulating cell proliferation and apoptosis. This section is aimed at providing an overview of the causative relationship between individual signaling targets and various nimbolide induced cellular responses.

3.1 Induction of Apoptosis Cancer development is associated with increased proliferation and decreased apoptosis (Hanahan and Weinberg, 2000). Cancer cells evade apoptosis by downregulation of DRs, overexpression of antiapoptotic proteins, reduced expression of proapoptotic proteins and caspases, and constitutive activation of pro-survival and anti-apoptotic transcription factor NF-κB (Reed, 1999; Vermeulen et al., 2005; Plati et al., 2008). Evasion of apoptosis creates a permissive environment for genomic instability with accumulation of mutations that increase cell survival, block differentiation, promote angiogenesis, invasion and metastasis, eventually culminating in neoplastic transformation (Lowe and Lin, 2000). Induction of apoptosis is currently recognized as one of the active strategies to arrest proliferation of cancer cells (Sun et al., 2004). Nimbolide has been reported to induce apoptosis by disruption of mitochondrial outer membrane potential (MOMP) (Priyadarsini et al., 2010; Elumalai et al., 2012). The disruption of MOMP constitutes a central coordinating event in nimbolide-induced cell death resulting in activation of the caspase cascade and apoptosis. There is evidence that production of ROS initiated by nimbolide is involved in mitochondrial membrane permeabilization and cell death induction. Generation of ROS was detected in cancer cell

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lines of different origin, and this was reversed by addition of reduced glutathione suggesting ROS involvement in the cytotoxicity of nimbolide (Kumar et al., 2009; Priyadarsini et al., 2010; Gupta et al., 2011). In addition, incubation with glutathione or N-acetyl cysteine (NAC) prior to administration of nimbolide rescued cells from undergoing apoptosis. This provides an inference that nimbolide transduces the apoptosis signal via generation of ROS. The ROS generation was linked to activation of proapoptotic p38 and SAPK/JNK kinases indicating that ROS acts upstream of the MAPKs in the signal pathway of nimbolide (Gupta et al., 2011). This prooxidant behavior of nimbolide is similar to that exhibited by several antioxidant phytochemicals such as curcumin, resveratrol and tea polyphenols in cancer cell lines and can be attributed to the redox status, free radical source, partial pressure of oxygen and ability to participate in a fenton type chemical reaction (Mohan et al., 2007; Madan et al., 2008; Thayyullathil et al., 2008). Furthermore, nimbolide has been reported to modulate expression levels of multiple B-cell lymphoma 2 (Bcl-2) family proteins. Nimbolide treatment causes upregulation of the pro-apoptotic Bcl-2 family proteins- Bax, Bcl-2-associated death promoter (Bad), BH3 interacting-domain (Bid), cytochrome-c (Cyt-c), second mitochondria-derived activator of caspase (Smac)/direct inhibitor of apoptosis-binding protein with low pI (DIABLO) and apoptotic protease activating factor 1 (Apaf-1), while the expression of anti-apoptotic proteins Bcl-2, myeloid cell leukemia-1 (Mcl-1), B-cell lymphoma-extra-large (Bcl-xL), X-linked inhibitor of apoptosis protein (xIAP)-1&2 were downregulated (Kumar et al., 2009; Gupta et al., 2010; Gupta et al., 2011; Elumalai et al., 2012; Kavitha et al., 2012; Raja Singh et al., 2013). Hence the ratio of pro-apoptotic to antiapoptotic proteins was altered in favor of apoptosis. The aforementioned studies demonstrate that nimbolide is a potent pro-apoptotic agent in several cell lines, and its activity is mediated through different mechanisms namely permeabilization of outer mitochondrial membrane, generation of ROS, and modulation of Bcl-2 family proteins leading to a strong inhibition of clonal expansion of initiated cells.

3.2 Inhibition of Tumor Cell Proliferation In multicellular organisms, cellular homeostasis is maintained through a balance in cell proliferation and apoptosis. The cell cycle is strictly regulated in normal cells by checkpoints, sequential activation and inactivation of cyclin-dependent kinases (CDKs) that drive cell cycle progression through phosphorylation and dephosphorylation of several regulatory proteins. Defects in cell cycle regulation and mutations in genes controlling the cell cycle are common phenomena in cancer and agents that suppress proliferation of tumor cells have therapeutic value. Like chemotherapeutic agents, several cancer chemopreventive agents like nimbolide, curcumin, resveratrol and epigallocatechingallate (EGCG) have been described that can suppress the proliferation of tumor cells. Flow cytometric analysis of U937 cells exposed to nimbolide revealed cell cycle disruption by decreasing the number of cells in G0/G1 phase with an initial accumulation in S and G2/M phases. Upon increasing the dose and duration of exposure, the S and G2/M fraction decreased with a reciprocal increase of cells in the subG1 fraction (Roy et al., 2007). In a study, Roy et al., reported that HT-29 cells exposed to nimbolide showed G2/M arrest accompanied by upregulation of p21, cyclin D2, chk2 and downregulation of cyclin A, cyclin D1, cdk2 and Rad17. Similar to U937cells, HT-29 cells exposed to higher doses of nimbolide for a longer period exhibited apoptotic features characterized by increasing number of cells in sub-G1 phase, appearance of annexin-V positive cells and expression of phosphotidylserine in the outer leaflet of mitochondria (Roy et al., 2006). Similarly, nimbolide suppressed the viability of HeLa cells in a dose-dependent manner by inducing cell cycle arrest at G0/G1 phase accompanied by p53-dependent p21 accumulation and down-regulation of the cell cycle regulatory protein’s cyclin B, cyclin D1 and proliferating cell nuclear antigen (PCNA) (Priyadarsini et al., 2010). Nimbolide demonstrated its growth inhibitory effects on human choriocarcinoma (BeWo) cells by downregulation of PCNA, a

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cofactor for DNA polymerase-δ that plays a central role in the cell cycle besides blocking apoptosis by inhibition of GADD45 and MyD118 (Kumar et al., 2009). The neem triterpene downregulated the expression of proteins involved in cell survival and proliferation (cyclin D1, c-Myc and Ki67) and this may contribute to the growth inhibitory effects on colon cancer cells (Gupta et al., 2013). Nimbolide arrested cells at S-phase through modulation of the cell cycle regulatory protein’s cyclin A/cyclin D1 and exerts its growth inhibitory effects on WiDr colon cancer cells (Babykutty et al., 2012). In animal models, nimbolide inhibited the development of HBP carcinomas by downregulation of cyclin D1, GST-P and PCNA, enhanced expression of p21waf1 and p53, suggesting regulative effects on cell cycle progression (Kumar et al., 2010). Overall, these results indicate that nimbolide inhibits tumor cell proliferation and exerts its growth inhibitory effects through alterations of cyclins, cdks, PCNA and p53 levels.

3.3 Suppression of NF-κB Activation Cancer cells evade apoptosis through constitutive activation of NF-κB, a ubiquitous and evolutionary conserved prosurvival and antiapoptotic transcription factor that is activated in response to a number of carcinogens and inflammatory stimuli, including cytokines (TNF-α), interleukin-1, growth factors, cigarette smoke, environmental pollutants, ionizing radiation and oxidative stress. NF-κB plays a critical role in tumor cell survival, proliferation, differentiation, inflammation, immune response, apoptosis, invasion and metastasis (Dreesen and Brivanlou, 2007). In resting state, NF-κB exists in the cytoplasm as an inactive heterotrimer consisting of p50, p65 and IκB proteins (IκBα, IκBβ and IκBε). In response to activation signals, the IκB is phosphorylated at serine residues 32 and 36, ubiquitinated at lysine residues 21 and 22 by E3 ubiquitin ligase complex and targeted for degradation by the 26S proteasome. IκB degradation allows the translocation of NF-κB heterodimer (p65, p50) to the nucleus where it binds to a specific DNA sequence resulting in transcription of target genes that play a pivotal role in oncogenic progression (Shen and Tergaonkar, 2009; Chaturvedi et al., 2011). The carcinogens (cigarette smoke, phorbol 12-myristate 13-acetate (PMA), okadaic acid (OA)) and inflammatory agents like lipopolysaccharide (LPS) are potent activators of NF-κB, and nimbolide suppressed the activation of NF-κB induced by these agents in a dose-dependent manner. Nimbolide not only suppressed the inducible form of NF-κB, but also inhibited constitutively activated NF-κB in multiple myeloma cells (U266, MM.1S and RPMI-8226). The limonoid inhibited NF-κB pathway through direct interaction with cys179 of IKK-β, leading to suppression of IκBα phosphorylation and subsequent proteasome degradation, inhibition of p65 nuclear translocation, and down-regulation of NF-κB regulated gene products (Gupta et al., 2010). Nimbolide attenuated the canonical NF-κB signaling pathway in hepG2 cells by downregulating the expression of NF-κB-p50 & p65, IκBα and IKKβ. Further, nimbolide attenuated the TNF-α induced activation of NF-κB, which provides substantial evidence that nimbolide can block both the inducible as well as constitutive activation of NF-κB (Gupta et al., 2010; Kavitha et al., 2012). NF-κB, in addition to influencing tumor cell proliferation and survival, also promotes tumor invasion, metastasis and angiogenesis, which are major causes of cancer related morbidity and mortality. Matrix metalloproteinases (MMPs) are a group of enzymes which play an important role in the tumor invasion during metastasis. These are secreted as inactive zymogens and require activation to exert efficient degradation of the extra cellular matrix (ECM). Nimbolide abrogates PMA-induced phosphorylation of ERK1/2 and suppresses PMAstimulated expression of MMP-2/9 which suggests its role in preventing metastasis and tumor invasion (Gupta et al., 2011; Gupta et al., 2013). In addition, nimbolide also inhibits the transcription of VEGF-A subsequently leading to stunted in vitro tube formation of HUVECs (Babykutty et al., 2012; Mahapatra et al., 2012). All

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together, the above reports indicate that nimbolide retards tumor cell migration, invasion and angiogenesis by downregulating MMP-2/9, VEGF-A expression via inhibiting ERK1/2, reducing the nuclear translocation and DNA-binding activity of NF-κB in cancer cells. 3.4 Other Signaling Pathways Modulated by Nimbolide Nimbolide was reported to have an additional effect on signaling pathways other than those discussed in the preceding sections. NF-κB has been recognized to enhance its carcinogenic potential by coordinately activating the canonical Wnt/ β-catenin signaling pathway. Du and Geller reported that there exists a functional cross-talk between NF-κB and Wnt/ β-catenin signaling pathway and the activation of NF-κB is recognized to synergistically induce activation of canonical Wnt/ β-catenin in various tumors (Du and Geller, 2010). The functional cross-talk between NF-κB and Wnt/ β-catenin signaling pathway is mediated through GSK-3β, a key component of the Wnt signaling. Activation of GSK-3β leads to accumulation of free β-catenin in the cytosol which eventually translocate to the nucleus trans-activating various target genes implicated in tumorigenesis. Nimbolide downregulates the expression of GSK-3β, β-catenin in HepG2 cells and hampers the activation of the canonical Wnt/β-catenin signaling (Kavitha et al., 2012). In another study, nimbolide repressed cell proliferation by inhibiting IGF1/IGF1RP13K/Akt pathway and induced apoptosis through activation of both extrinsic and intrinsic apoptotic pathways in prostate cancer cells (Raja Singh et al., 2013). The signaling pathways frequently dysregulated in glioblastoma include the EGFR/EGFRvIII driven PI3K-Akt pathway, Ras-MAP kinase pathway, JAK-STAT pathway and the RB pathway. The recent findings of Karkare et al., demonstrated that nimbolide suppresses glioblastoma viability and restrains tumor growth by inhibiting CDK4/6 activity, leading to RB hypophosphorylation and cell cycle arrest and by inhibiting growth factor pathways hyperactivated in glioblastoma including the PI3K-Akt, MAP kinase and JAK-STAT pathway (Karkare et al., 2013). The overview of all the signaling pathways modulated by nimbolide is shown in (Fig.3).

4. CONCLUSION Cancer is a group of over 200 neoplastic diseases caused by dysregulation of multiple cell signaling pathways. To effectively halt the tumor progression, combinations of several drugs or a drug that modulates multiple targets would be ideal, as targeting or inhibiting a single gene product or cell signaling pathway is unlikely to provide a significant benefit. Consequently, chemopreventive agents which modulate the multiple molecular and cellular pathways are now the attractive objects in cancer research. Nimbolide is one such compound that displays those traits in cell-based as well as in animal models and appears to be a fascinating molecule for chemoprevention/ chemotherapy. Nimbolide was demonstrated to modulate the expression and/ or activity of transcription factors involved in critical pathways of carcinogenesis, including carcinogen activation and detoxification, growth arrest and apoptosis, and proinflammatory mediated signaling pathway (Fig.4). Overall the scientific reports from the cell-based and in vivo studies support the apoptosis inducing, antiproliferative, antiinvasive, antimetastatic and antiangiogenic activities of nimbolide. These studies also demonstrate that nimbolide’s effect against several cancers is not through any single mechanism; however, like curcumin, EGCG and resveratrol, nimbolide’s activity involves multiple cellular and molecular targets. Till to date, only little information is available regarding its bioavailability, pharmacokinetics and pharmacodynamics in relation to its anticancer effects. Therefore, extensive preclinical and clinical research in this field will help to establish the safety and efficacy of nimbolide as a chemopreventive/ therapeutic agent against several human cancers.

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5. Conflict of interest: Authors declare no conflict of interest. 6. Acknowledgements: We would like to thank the Directors of National Institute of Pharmaceutical Education and Research (NIPER)-Guwahati and Indian institute of chemical technology (IICT)-Hyderabad for their support and encouragement. 7. References Arora, N., Koul, A., Bansal, M.P., 2011. Chemopreventive activity of Azadirachta indica on two-stage skin carcinogenesis in murine model. Phytotherapy Research 25, 408-416. Babykutty, S., Ps, P., Rj, N., Kumar, M.A., Nair, M.S., Srinivas, P., Gopala, S., 2012. Nimbolide retards tumor cell migration, invasion, and angiogenesis by downregulating MMP-2/9 expression via inhibiting ERK1/2 and reducing DNA-binding activity of NF-KB in colon cancer cells. Molecular Carcinogenesis 51, 475-490. Balasenthil, S., Arivazhagan, S., Ramachandran, C.R., Ramachandran, V., Nagini, S., 1999. Chemopreventive potential of neem (Azadirachta indica) on 7, 12-dimethylbenz [a] anthracene (DMBA) induced hamster buccal pouch carcinogenesis. Journal of ethnopharmacology 67, 189-195. Baral, R., Chattopadhyay, U., 2004. Neem (Azadirachta indica) leaf mediated immune activation causes prophylactic growth inhibition of murine Ehrlich carcinoma and B16 melanoma. International immunopharmacology 4, 355-366. Bharati, S., Rishi, P., Koul, A., 2012. Azadirachta indica exhibits chemopreventive action against hepatic cancer: Studies on associated histopathological and ultrastructural changes. Microscopy Research and Technique 75, 586-595. Biswas, K., Chattopadhyay, I., Banerjee, R.K., Bandyopadhyay, U., 2002. Biological activities and medicinal properties of neem (Azadirachta indica). Current science 82, 1336-1345. Chaturvedi, M.M., Sung, B., Yadav, V.R., Kannappan, R., Aggarwal, B.B., 2011. NF-KB addiction and its role in cancer: 'one size does not fit all'. Oncogene 30, 1615-1630. Chen, J., Chen, J., Sun, Y., Yan, Y., Kong, L., Li, Y., Qiu, M., 2011. Cytotoxic triterpenoids from Azadirachta indica. Planta medica 77, 1844-1847. Cohen, E., Quistad, G.B., Casida, J.E., 1996. Cytotoxicity of nimbolide, epoxyazadiradione and other limonoids from neem insecticide. Life sciences 58, 1075-1081. Dasgupta, T., Banerjee, S., Yadava, P.K., Rao, A.R., 2004. Chemopreventive potential of Azadirachta indica (Neem) leaf extract in murine carcinogenesis model systems. Journal of ethnopharmacology 92, 23-36. Dreesen, O., Brivanlou, A.H., 2007. Signaling pathways in cancer and embryonic stem cells. Stem cell reviews 3, 7-17. Du, Q., Geller, D.A., 2010. Cross-regulation between Wnt and NF-KB signaling pathways. Forum on immunopathological diseases and therapeutics 1, 155-181. Elumalai, P., Gunadharini, D.N., Senthilkumar, K., Banudevi, S., Arunkumar, R., Benson, C.S., Sharmila, G., Arunakaran, J., 2012. Induction of apoptosis in human breast cancer cells by nimbolide through extrinsic and intrinsic pathway. Toxicology letters 215,131-42. Ferlay, J., Shin, H.R., Bray, F., Forman, D., Mathers, C., Parkin, D.M., 2010. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. International journal of cancer 127, 2893-2917. Gangar, S.C., Sandhir, R., Rai, D.V., Koul, A., 2006. Modulatory effects of Azadirachta indica on benzo (a) pyrene-induced forestomach tumorigenesis in mice. World Journal of Gastroenterology 12, 2749-2755. GLOBOCAN 2012: Estimated http://globocan.iarc.fr

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cancer

incidence,

mortality

and

prevalence

worldwide

in

2012.

Gupta, S., Prasad, S., Sethumadhavan, D.R., Nair, M.S., Mo, Y.-Y., Aggarwal, B.B., 2013. Nimbolide, a Limonoid Triterpene, Inhibits Growth of Human Colorectal Cancer Xenografts by Suppressing the Proinflammatory Microenvironment. Clinical Cancer Research 19, 4465-4476. Gupta, S.C., Prasad, S., Reuter, S., Kannappan, R., Yadav, V.R., Ravindran, J., Hema, P.S., Chaturvedi, M.M., Nair, M., Aggarwal, B.B., 2010. Modification of cysteine 179 of IkBa kinase by nimbolide leads to downregulation of NF-kB-regulated cell survival and proliferative proteins and sensitization of tumor cells to chemotherapeutic agents. Journal of Biological Chemistry 285, 35406-35417. Hanahan, D., Weinberg, R.A., 2000. The hallmarks of cancer. Cell 100, 57-70. Hanahan, D., Weinberg, R.A., 2011. Hallmarks of cancer: the next generation. Cell 144, 646-674. Hasima, N., Aggarwal, B.B., 2012. Cancer-linked targets modulated by curcumin. International journal of biochemistry and molecular biology 3, 328-351. Hursting, S.D., Slaga, T.J., Fischer, S.M., DiGiovanni, J., Phang, J.M., 1999. Mechanism-based cancer prevention approaches: targets, examples, and the use of transgenic mice. Journal of the National Cancer Institute 91, 215225. Jemal, A., Bray, F., Center, M.M., Ferlay, J., Ward, E., Forman, D., 2011. Global cancer statistics. CA: a cancer journal for clinicians 61, 69-90. Karkare, S., Chhipa, R.R., Liu, X., Henry, H., Gasilina, A., Nassar, N., Pauletti, G.M., Ghosh, P.K., Dasgupta, B., Roychoudhury, J., 2013. Direct inhibition of Retinoblastoma phosphorylation by Nimbolide causes cell cycle arrest and suppresses glioblastoma growth. Clinical Cancer Research, doi: 10.1158/1078-0432.CCR-13-0762. Kavitha, K., Vidya Priyadarsini, R., Anitha, P., Ramalingam, K., Sakthivel, R., Purushothaman, G., Singh, A.K., Karunagaran, D., Nagini, S., 2012. Nimbolide, a neem limonoid abrogates canonical NF-KB and Wnt signaling to induce caspase-dependent apoptosis in human hepatocarcinoma (HepG2) cells. European journal of pharmacology 681, 6-14. Kigodi, P.G.K., Blaskó, G.b., Thebtaranonth, Y., Pezzuto, J.M., Cordell, G.A., 1989. Spectroscopic and biological investigation of nimbolide and 28-deoxonimbolide from Azadirachta indica. Journal of natural products 52, 1246-1251. Koul, A., Mukherjee, N., Gangar, S.C., 2006. Inhibitory effects of Azadirachta indica on DMBA-induced skin carcinogenesis in Balb/c mice. Molecular and cellular biochemistry 283, 47-55. Kumar, G.H., Mohan, K.V.P.C., Rao, A.J., Nagini, S., 2009. Nimbolide a limonoid from Azadirachta indica inhibits proliferation and induces apoptosis of human choriocarcinoma (BeWo) cells. Investigational new drugs 27, 246-252. Kumar, G.H., Priyadarsini, R.V., Vinothini, G., Letchoumy, P.V., Nagini, S., 2010. The neem limonoids azadirachtin and nimbolide inhibit cell proliferation and induce apoptosis in an animal model of oral oncogenesis. Investigational new drugs 28, 392-401. Lowe, S.W., Lin, A.W., 2000. Apoptosis in cancer. Carcinogenesis 21, 485-495. Madan, E., Prasad, S., Roy, P., George, J., Shukla, Y., 2008. Regulation of apoptosis by resveratrol through JAK/STAT and mitochondria mediated pathway in human epidermoid carcinoma A431 cells. Biochemical and biophysical research communications 377, 1232-1237. Mahapatra, S., Young, C.Y.F., Kohli, M., Karnes, R.J., Klee, E.W., Holmes, M.W., Tindall, D.J., Donkena, K.V., 2012. Antiangiogenic Effects and Therapeutic Targets of Azadirachta indica Leaf Extract in Endothelial Cells. Evidence-Based Complementary and Alternative Medicine. doi:10.1155/2012/303019. Manoharan, S., Rajmani Ramachandran, C., Ramachandran, V., Nagini, S., 1996. Inhibition of 4-nitroquinoline-1oxide-induced oral carcinogenesis by plant products. Journal of clinical biochemistry and nutrition 21, 141149.

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Mohan, K.V.P., Gunasekaran, P., Varalakshmi, E., Hara, Y., Nagini, S., 2007. In vitro evaluation of the anticancer effect of lactoferrin and tea polyphenol combination on oral carcinoma cells. Cell Biology International 31, 599-608. Paul, R., Prasad, M., Sah, N.K., 2011. Anticancer biology of Azadirachta indica L (neem): a mini review. Cancer biology & therapy 12, 467-476. Plati, J., Bucur, O., Khosravi-Far, R., 2008. Dysregulation of apoptotic signaling in cancer: molecular mechanisms and therapeutic opportunities. Journal of cellular biochemistry 104, 1124-1149. Priyadarsini, R.V., Manikandan, P., Kumar, G.H., Nagini, S., 2009. The neem limonoids azadirachtin and nimbolide inhibit hamster cheek pouch carcinogenesis by modulating xenobiotic-metabolizing enzymes, DNA damage, antioxidants, invasion and angiogenesis. Free radical research 43, 492-504. Priyadarsini, R.V., Murugan, R.S., Sripriya, P., Karunagaran, D., Nagini, S., 2010. The neem limonoids azadirachtin and nimbolide induce cell cycle arrest and mitochondria-mediated apoptosis in human cervical cancer (HeLa) cells. Free radical research 44, 624-634. Raja Singh, P., Arunkumar, R., Sivakamasundari, V., Sharmila, G., Elumalai, P., Suganthapriya, E., Brindha Mercy, A., Senthilkumar, K., Arunakaran, J., 2013. Anti-proliferative and apoptosis inducing effect of nimbolide by altering molecules involved in apoptosis and IGF signalling via PI3K/Akt in prostate cancer (PC3) cell line. Cell biochemistry and function. doi: 10.1002/cbf.2993. Reed, J.C., 1999. Mechanisms of apoptosis avoidance in cancer. Current opinion in oncology 11, 68-75. Rochanakij, S., Thebtaranonth, Y., Yuthavong, Y., 1985. Nimbolide, a constituent of Azadirachta indica, inhibits Plasmodium falciparum in culture. Southeast Asian journal of tropical medicine and public health 16, 66-72. Roy, M.K., Kobori, M., Takenaka, M., Nakahara, K., Shinmoto, H., Isobe, S., Tsushida, T., 2007. Antiproliferative effect on human cancer cell lines after treatment with nimbolide extracted from an edible part of the neem tree (Azadirachta indica). Phytotherapy Research 21, 245-250. Roy, M.K., Kobori, M., Takenaka, M., Nakahara, K., Shinmoto, H., Tsushida, T., 2006. Inhibition of colon cancer (HT-29) cell proliferation by a triterpenoid isolated from Azadirachta indica is accompanied by cell cycle arrest and up-regulation of p21. Planta medica 72, 917-923. Sastry, B.S., Suresh Babu, K., Hari Babu, T., Chandrasekhar, S., Srinivas, P.V., Saxena, A.K., Madhusudana Rao, J., 2006. Synthesis and biological activity of amide derivatives of nimbolide. Bioorganic & medicinal chemistry letters 16, 4391-4394. Shen, H.-M., Tergaonkar, V., 2009. NFKB signaling in carcinogenesis and as a potential molecular target for cancer therapy. Apoptosis 14, 348-363. Singh, S.R., 2013. Cancer Stem Cells: Recent Developments and Future Prospects. Cancer Letters 338, 1-2. Sporn, M.B., Dunlop, N.M., Newton, D.L., Smith, J.M., 1976. Prevention of chemical carcinogenesis by vitamin A and its synthetic analogs (retinoids). Federation proceedings 35, 1332-1338. Sritanaudomchai, H., Kusamran, T., Kuakulkiat, W., Bunyapraphatsara, N., Hiransalee, A., Tepsuwan, A., Kursamran, W., 2005. Quinone reductase inducers in Azadirachta indica A. Juss flowers, and their mechanisms of action. Asian Pacific Journal of Cancer Prevention 6, 263-269. Subapriya, R., Kumaraguruparan, R., Nagini, S., 2006. Expression of PCNA, cytokeratin, Bcl-2 and p53 during chemoprevention of hamster buccal pouch carcinogenesis by ethanolic neem (Azadirachta indica) leaf extract. Clinical biochemistry 39, 1080-1087. Subapriya, R., Nagini, S., 2003. Ethanolic neem leaf extract protects against N-methyl-N'-nitro-Nnitrosoguanidine-induced gastric carcinogenesis in Wistar rats. Asian Pacific Journal of Cancer Prevention 4, 215-224.

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Subapriya, R., Velmurugan, B., Nagini, S., 2005. Modulation of xenobiotic-metabolizing enzymes by ethanolic neem leaf extract during hamster buccal pouch carcinogenesis. Journal of experimental and clinical cancer research 24, 223-230. Subash C. Gupta, S.P., Simone Reuter, Ramaswamy Kannappan, Vivek R. Yadav, Jayaraj Ravindran,, Padmanabhan S. Hema, M.M.C., Mangalam Nair, and Bharat B. Aggarwal., 2011. Nimbolide Sensitizes Human Colon Cancer Cells to TRAIL through Reactive Oxygen Species- and ERK-dependent Upregulation of Death Receptors, p53, and Bax. The journal of biological chemistry 286, 1134-1146. Sun, S.-Y., Hail, N., Lotan, R., 2004. Apoptosis as a novel target for cancer chemoprevention. Journal of the National Cancer Institute 96, 662-672. Suresh, G., Gopalakrishnan, G., Wesley, S.D., Pradeep Singh, N., Malathi, R., Rajan, S., 2002. Insect antifeedant activity of tetranortriterpenoids from the Rutales. A perusal of structural relations. Journal of agricultural and food chemistry 50, 4484-4490. Surh, Y.-J., 2003. Cancer chemoprevention with dietary phytochemicals. Nature Reviews Cancer 3, 768-780. Talalay, P., 2000. Chemoprotection against cancer by induction of phase 2 enzymes. Biofactors 12, 5-11. Tepsuwan, A., Kupradinun, P., Kusamran, W.R., 2002. Chemopreventive potential of neem flowers on carcinogeninduced rat mammary and liver carcinogenesis. Asian Pacific Journal of Cancer Prevention 3, 231-238. Thayyullathil, F., Chathoth, S., Hago, A., Patel, M., Galadari, S., 2008. Rapid reactive oxygen species (ROS) generation induced by curcumin leads to caspase-dependent and-independent apoptosis in L929 cells. Free Radical Biology and Medicine 45, 1403-1412. Tsao, A.S., Kim, E.S., Hong, W.K., 2004. Chemoprevention of cancer. CA: a cancer journal for clinicians 54, 150180. Udeinya, I., Mbah, A., Chijioke, C., Shu, E., 2004. An antimalarial extract from neem leaves is antiretroviral. Transactions of the Royal Society of Tropical Medicine and Hygiene 98, 435-437. Vermeulen, K., Van Bockstaele, D.R., Berneman, Z.N., 2005. Apoptosis: mechanisms and relevance in cancer. Annals of hematology 84, 627-639. Vinothini, G., Manikandan, P., Anandan, R., Nagini, S., 2009. Chemoprevention of rat mammary carcinogenesis by Azadirachta indica leaf fractions: Modulation of hormone status, xenobiotic-metabolizing enzymes, oxidative stress, cell proliferation and apoptosis. Food and Chemical Toxicology 47, 1852-1863. Wattenberg, L.W., 1985. Chemoprevention of cancer. Cancer Research 45, 1-8. Wattenberg, L.W., 1997. An overview of chemoprevention: current status and future prospects. Proceedings of the Society for Experimental Biology and Medicine 216, 133-141.

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Figures (Preference of color: In web only)

Fig.1 Structure of nimbolide (4α,5α,6α,7α,15β,17α)-7,15:21,23-Diepoxy-6-hydroxy-4,8-dimethyl-1-oxo-18,24dinor-11,12-secochola-2,13,20,22-tetraene-4,11-dicarboxylic Αcid γ-lactone Methyl Ester). (CAS Number: 2599037-8; Molecular Formula: C27H30O7; Molecular Weight: 466.52)

Fig.2 Schematic representation of multistep-carcinogenesis and its intervention by nimbolide. Carcinogenesis is initiated with the transformation of the normal cell into a cancer cell (initiation). These cancer cells undergo tumour promotion into preneoplastic cells, which progress to neoplastic cells. Nimbolide prevents carcinogenesis by inhibiting metabolic activation of the procarcinogen and/or alternatively stimulate the detoxification of

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carcinogens, suppresses the promotion and progression by inducing growth arrest and apoptosis, inhibition of inflammation-promoted cell proliferation, angiogenesis, invasion and metastasis.

Fig.3 Schematic representation of the molecular mechanisms for the anticancer activity of nimbolide. Nimbolide is known to exert anticancer effects by inducing apoptosis through activation of death receptor (extrinsic) and mitochondrial (intrinsic) pathways. It also induces cell cycle arrest through altering the levels of cyclin and CDKs. Nimbolide enhances the levels of antioxidant and detoxification enzymes through Nrf2 activation. In addition, nimbolide restrains the nuclear translocation and DNA binding of NF-κB via interaction with IKK and suppressed the expression of NF-κB regulated genes involved in cell proliferation, migration, invasion, metastasis and angiogenesis. The inhibition of NF-κB pathway in turn inhibits the disassociation of GSK-3β from the multienzyme complex thereby restraining Wnt/β-catenin signaling. Nimbolide also suppresses tumor growth and survival by modulating growth factor pathways namely the PI3K-Akt, MAPK and JAK-STAT.

Abbreviations: Akt/PKB, Protein kinase-B; AP-1, Activator protein 1; Apaf-1, Apoptotic protease activating factor 1; APC, Adenomatous polyposis coli; Bad, Bcl2 -associated death promoter; Bax, Bcl2 -associated X protein; Bcl2, B cell lymphoma 2; Bcl-xL, B-cell lymphomaextra-large; Bid, BH3 interacting-domain; C/EBP, CCAAT-enhancer-binding proteins; CDKs, Cyclin dependent kinases; Chk2, Checkpoint kinase 2; COX-2, Cyclooxygenase-2; DIABLO, Direct inhibitor of apoptosis-binding protein with low pI; DR, Death receptor; EGFR, Epidermal growth factor receptor; ERK, Extracellular-signal-regulated kinase; ERα, Estrogen receptor alpha; FADD, Fas-associated death domain; FasL, Fatty acid synthase ligand; GADD45, Growth arrest and DNA damage 45; GSK-3, Glycogen synthase kinase 3; GST-P, Glutathione S-transferase pi; IAP, Inhibitor of apoptosis protein; ICAM, Intercellular adhesion molecule; IGF-1, Insulin-like growth factor 1; IκB, Inhibitor of kappa B; IKK, IκB kinase; JAK, Janus kinase; JNK, c-jun N-terminal kinase; KEAP, Kelch-like erythroid-cell-derived protein with CNC homology (ECH)-associated protein; MAPKs, Mitogen-activated protein kinases; MCL-1, Myeloid cell leukemia 1; MMPs, Matrix metalloproteinases; NF-κB, Nuclear factor kappa B; NL, Nimbolide; Nrf-2, Nuclear factor (erythroid-derived 2)-like 2; Ras, rat sarcoma; Raf, Rapidly accelerated fibrosarcoma; PARP, Poly (ADP-ribose) polymerase; PDGF, Platelet-derived growth factor; PI3K, Phosphoinositide 3kinase; ROS, Reactive oxygen species; Smac, Second mitochondria-derived activator of caspases; TNF-α, Tumor necrosis factor alpha; STAT, Signal transducer and activator o f transcription; TRAIL, Tumor necrosis factor apoptosis inducing ligand; TRK, Tyrosine kinase; VEGF, Vascular endothelial growth factor; VEGF-A, Vascular endothelial growth factor-A.

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Fig.4 Molecular targets of nimbolide. The diverse molecular targets influenced by nimbolide include the transcription factors, growth factors and their receptors, cytokines, protein kinases, enzymes and genes regulating cell proliferation and apoptosis. Arrow headlines ( ) indicate that these molecules are up-regulated by nimbolide, whereas blunt-headlines ( ) indicate a decrease or inhibition. Abbreviations: AP-1, Activator protein 1; Apaf-1, Apoptotic protease activating factor 1; Bad, Bcl-2-associated death promoter; Bax, Bcl-2associated X protein; Bcl2, B cell lymphoma 2; Bcl-xL, B-cell lymphoma-extra-large; Bid, BH3 interacting-domain; CAT, Catalase; CDKs, Cyclin dependent kinases; C/EBP, CCAAT-enhancer-binding proteins; Chk2, Checkpoint kinase 2; COX-2, Cyclooxygenase-2; CYP, Cytochrome P450; CD, Cluster of differentiation; Cyt-c, Cytochrome-C; ERα, Estrogen receptor alpha; ERK, Extracellular-signal-regulated kinase; FADD, Fas-associated death domain; FasL, Fatty acid synthase ligand; GPx, Glutathione peroxidase; GR, Glutathione reductase ; GST, Glutathione-S-transferase; GSK-3, Glycogen synthase kinase 3; GST-P, Glutathione S-Transferase pi; HDAC, Histone deacetylases; HO-1, Heme oxygenase-1; ICAM, Intercellular adhesion molecule; IGF-1, Insulin-like growth factor 1; IKK, IκB kinase β; IL-6, Interleukin -6; JNK, C-jun N-terminal kinase; NF-κB, Nuclear factor kappa B; Nrf-2, Nuclear factor (erythroid-derived 2)-like 2; NK, Natural killer cells; MCL-1, Myeloid cell leukemia 1; MMPs, Matrix metalloproteinases; NQO, NAD(P)H:quinone oxidoreductase; PCNA, Proliferating cell nuclear antigen; MAPKs, Mitogen-activated protein kinases; QR, NADPH:quinone oxidoreductase; Rb, Retinoblastoma; Smac, Second mitochondriaderived activator of caspases; SOD, Superoxide dismutase; STAT, Signal transducer and activator of transcription; TRAIL, Tumor necrosis factor apoptosis inducing ligand; VEGF, Vascular endothelial growth factor. xIAP, X-linked inhibitor of apoptosis protein.

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Fig.2 Schematic representation of multistep-carcinogenesis and its intervention by nimbolide. Carcinogenesis is initiated with the transformation of the normal cell into a cancer cell (initiation). These cancer cells undergo tumour promotion into preneoplastic cells, which progress to neoplastic cells. Nimbolide prevents carcinogenesis by inhibiting metabolic activation of the procarcinogen and/or alternatively stimulate the detoxification of carcinogens, suppresses the promotion and progression by inducing growth arrest and apoptosis, inhibition of inflammation-promoted cell proliferation, angiogenesis, invasion and metastasis.

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Fig.3 Schematic representation of the molecular mechanisms for the anticancer activity of nimbolide. Nimbolide is known to exert anticancer effects by inducing apoptosis through activation of death receptor (extrinsic) and mitochondrial (intrinsic) pathways. It also induces cell cycle arrest through altering the levels of cyclin and CDKs. Nimbolide enhances the levels of antioxidant and detoxification enzymes through Nrf2 activation. In addition, nimbolide restrains the nuclear translocation and DNA binding of NF-κB via interaction with IKK and suppressed the expression of NF-κB regulated genes involved in cell proliferation, migration, invasion, metastasis and angiogenesis. The inhibition of NF-κB pathway in turn inhibits the disassociation of GSK-3β from the multienzyme complex thereby restraining Wnt/β-catenin signaling. Nimbolide also suppresses tumor growth and survival by modulating growth factor pathways namely the PI3K-Akt, MAPK and JAK-STAT.

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Fig.4 Molecular targets of nimbolide. The diverse molecular targets influenced by nimbolide include the transcription factors, growth factors and their receptors, cytokines, protein kinases, enzymes and genes regulating cell proliferation and apoptosis. Arrow headlines ( ) indicate that these molecules are up-regulated by nimbolide, whereas blunt-headlines ( ) indicate a decrease or inhibition.

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Table.1 Chemopreventive and chemotherapeutic effects of nimbolide and neem extracts in animal models.

COMPOUND/ EXTRACT

CANCER

CARCINOGEN/ CELL LINE

ANIMAL

MECHANISM OF ACTION

REFERENCES

(Dose) Modulation of lipid peroxidation,

ENLE Gastric

MNNG

Wistar rats

Skin

DMBA & TPA

Male LACA mice

(200 mg/kg)

AAILE (300 mg/kg)

Enhancing antioxidant enzymes (GSH, GPx, GST, SOD, and CAT) in stomach, liver and blood.

Induces apoptosis by induction of Bax, caspase-3 & 9, and inhibition of Bcl2.

(Subapriya and Nagini, 2003)

(Arora et al., 2011)

Modulation of phase-II detoxification enzymes. ENLE Fore stomach

B(a)P

(250 & 500 mg/kg )

Swiss albino mice

Elevation of antioxidant enzymes

(Dasgupta et al., 2004)

Inhibits lipid peroxidation and LDH- induced damages. Modulation of phase-II detoxification enzymes.

ENLE Skin

DMBA

(250 & 500 mg/kg )

Swiss albino mice

Elevation of antioxidant enzymes

(Dasgupta et al., 2004)

Inhibits lipid peroxidation and LDH- induced damages.

AAILE Oral

NQO

Male Wistar rats

Antioxidant and anti-inflammatory activity.

(Manoharan et al., 1996)

Buccal pouch

DMBA

Male Syrian hamsters

Modulation of lipid peroxidation, antioxidant and detoxification systems (GSH, GPx, GGT & GST).

(Balasenthil et al., 1999)

Skin

DMBA

Male Balb/c mice

Modulatory effects on lipid peroxidation and antioxidant defense system (GPx, GR).

Fore stomach

B(a)P

Balb/c mice

Modulates phase I (CYP450, cytochrome b5), phase II and antioxidant enzymes.

(Gangar et al., 2006)

Swiss & C57BL/6 mice

Stimulates cell mediated (CD4+, CD8+) and innate immune functions (NK cells, macrophages).

(Baral and Chattopadhyay, 2004)

(1 g/kg)

AAILE (100 mg/kg)

AAILE (400 mg/kg)

AAILE (100 mg/kg)

(Koul et al., 2006)

Xenograft NLP (1 unit/mice)

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Undifferentiate

EAC

d carcinoma

Melanoma

B16 Modulates hormone (estradiol) and receptor status (ERα), xenobiotic metabolizing enzymes (XMEs) (CYP1A1, CYP1B1) and lipid & protein oxidation.

Ethyl acetate & methanolic neem leaf fractions

Breast

DMBA

Rat

Upregulates antioxidant enzymes (SOD, CAT, GPx & GSH).

(Vinothini et al., 2009)

Inhibits ROS-induced damage, cell proliferation.

(1& 10 mg/kg)

Induce apoptosis.

Neem flowers (10 & 12.5% (w/w) in diet)

Breast

DMBA

Modulates phase I (CYP1A1, 1B1, 2C11, 2E1) and phase II enzyme expression in hepatocytes. Liver

AFB1

Buccal pouch

DMBA

ENLE (200 mg/kg)

ENLE

(Subapriya et al., 2005)

(Bharati et al., 2012)

Male Balb/c mice

Alters the activity of carcinogen biotransformation enzymes.

DMBA

Syrian hamsters

Inhibits cell proliferation, Induces apoptosis.

DMBA

Syrian hamsters

Buccal pouch

(Subapriya et al., 2006)

Modulates carcinogen metabolism by decreasing phase-I enzymes (CYP450, b5 ) and increasing phase-II enzymes (GST, DT-diaphorase and NADPH-diaphorase).

NDEA

(Kumar et al., 2010)

Modulates phase I and phase II XMEs, Buccal pouch

(10 & 100 µg/kg)

Nimbolide

Xenograft Colon

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Induces differentiation and apoptosis.

Liver

Nimbolide & Azadirachtin

(5 & 20 mg/kg)

Inhibits cell proliferation,

DMBA

(100 µg/g BW)

Nimbolide & Azadirachtin

Syrian hamster

Buccal pouch

AAILE

(Tepsuwan et al., 2002)

Male Wistar rats

Syrian hamsters

(200 mg/kg)

(10 & 100 µg/kg)

Female SD rats

Nude mice (HCT-116)

Block tumour invasion, angiogenesis and inhibit HDAC.

Suppresses NF-κB activation and modulates the expression of NF-κB regulated gene products linked to survival, proliferation, invasion and angiogenesis.

(Priyadarsini et al., 2010)

(Gupta et al., 2013)

Ethanol soluble fraction of Azadirachta indica leaves

Induce cell cycle arrest and apoptosis,

Xenograft Brain

Nude mice (U87EGFRvIII)

Inhibit PI3K-Akt, MEK-Erk1/2 and JAK-STAT pathways.

(Karkare et al., 2013)

(0.34 µg/g) Nimbolide Brain

Nude mice

(10 µg/kg)

(U87EGFRvIII)

Nimbolide

Xenograft Brain

(200µg/kg )

Induce cell cycle arrest and apoptosis,

Xenograft

(Karkare et al., 2013)

Induce cell cycle arrest and apoptosis, Nude mice

(U87EGFRvIII)

Inhibit PI3K-Akt, MEK-Erk1/2 and JAK-STAT pathways.

Inhibit PI3K-Akt, MEK-Erk1/2 and JAK-STAT pathways.

(Karkare et al., 2013)

Abbreviations: AAILE, Aqueous Azadirachta indica leaf extract; AFB1, Aflatoxin B1; B(a)P, Benzo[a]pyrene; DMBA 7,12-Dimethylbenz(a)anthracene; ENLE, Ethanolic neem leaf extract; EAC, Ehrlich ascites carcinoma; MNNG; Methylnitronitrosoguanidine; NDEA, N-nitrosodiethylamine; NQO, 4Nitroquinoline 1-oxide; NLP, Neem leaf preparation; TPA, 12-O-Tetradecanoylphorbol-13-acetate.

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Table.2 In vitro cytotoxicity of nimbolide against tumor cell lines. Cell lines

Histotype

Origin

IC50

References

A549

Lung carcinoma

Human

1.9 µM

(Chen et al., 2011)

B16

Melanoma

Mouse

1.74 µM

(Roy et al., 2007)

BC-1

Lymphoma

Human

0.39µg/ml

(Kigodi et al., 1989)

BeWo

Choriocarcinoma

Human

1.19 µM

(Kumar et al., 2009)

COL-2

Colon carcinoma

Human

0.41µg/ml

(Kigodi et al., 1989)

HT29

Colon carcinoma

Human

6.54 µM

(Roy et al., 2006; Sastry et al., 2006) (Chen et al., 2011; HL-60

Leukemia promyelocytic

Human

0.8 µM Roy et al., 2007)

HOP-62

Lung carcinoma

Human

10.37 µM

(Sastry et al., 2006)

HeLa

Cervical carcinoma

Human

5 µM

(Priyadarsini et al., 2010)

Hepa 1c1c7

Hepatoma

Mouse

0.42µg/ml

(Sritanaudomchai et al., 2005)

HepG2

Hepatic carcinoma

Human

5 µM

(Kavitha et al., 2012)

HT-1080

Fibro sarcoma

Human

0.31µg/ml

(Kigodi et al., 1989)

KB

Nasopharyngeal

Human

0.25µg/ml

(Kigodi et al., 1989)

MDA-MB-231

Breast carcinoma

Human

4 µM

(Elumalai et al., 2012)

MCF-7

Breast carcinoma

Human

4.5 µM

(Chen et al., 2011; Elumalai et al., 2012) N1E-115

Neuroblastoma

Mouse

3.2 µM

(Cohen et al., 1996)

OVCAR-5

Ovarian carcinoma

Human

4.17 µM

(Sastry et al., 2006)

P-388

Leukemia lymphocytic

Mouse

0.065µg/ml

(Kigodi et al., 1989)

PC3

Prostate carcinoma

Human

2 µM

(Raja Singh et al., 2013; Sastry et al., 2006) RAW 264.7

Macrophage

Mouse

5 µM

(Cohen et al., 1996)

SK-LU-1

Lung carcinoma

Human

0.42µg/ml

(Kigodi et al., 1989)

SK-MEL-2

Melanoma

Human

0.53µg/ml

(Kigodi et al., 1989)

SMMC-7721

Hepatocellular carcinoma

Human

2.2 µM

(Chen et al., 2011)

SW-480

Colon carcinoma

Human

2.3 µM

(Chen et al., 2011)

SW-620

Colon carcinoma

Human

8.25 µM

(Sastry et al., 2006)

THP-1

Leukemia monocytic

Human

1.42 µM

(Roy et al., 2007)

U937

Lymphoma histiocytic

Human

1.24 µM

(Roy et al., 2007)

U87EGFRvIII

Glioblastoma

Human

3 µM

(Karkare et al., 2013)

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WiDr

Colon carcinoma

Human

2.5 µM

(Babykutty et al., 2012)

143B-TK

Osteosarcoma

Human

4.3 µM

(Cohen et al., 1996)

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Conflict of interest: Authors declare no conflict of interest in the present work.

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