NF-κB; CDK4; CDK6

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.[1]

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Nimbolide or an ethanol soluble fraction of A. indica leaves (Azt) that contains nimbolide as the principal cytotoxic agent is highly cytotoxic against glioblastoma multiforme in vitro. Azt caused cell-cycle arrest, most prominently at the G1-S stage in glioblastoma multiforme cells expressing EGFRvIII, an oncogene present in about 20% to 25% of glioblastoma multiformes. Azt/nimbolide directly inhibited CDK4/CDK6 kinase activity leading to hypophosphorylation of the retinoblastoma protein, cell-cycle arrest at G1-S, and cell death. Independent of retinoblastoma hypophosphorylation, Azt also significantly reduced proliferative and survival advantage of glioblastoma multiforme cells in vitro by downregulating Bcl2 and blocking growth factor-induced phosphorylation of Akt, extracellular signal-regulated kinase 1/2, and STAT3. These effects were specific because Azt did not affect mTOR or other cell-cycle regulators.[2]

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The present study was aimed to investigate the effect of nimbolide on IGF signalling and cell cycle arrest in MCF-7 and MDA-MB-231 breast cancer cell lines. The protein expression of IGF signalling molecules and cell cycle protein levels was assessed by western blot analysis. In order to study the interaction of nimbolide on IGF-1 signalling pathway, IGF-I and phosphoinositide 3-kinase (PI3K) inhibitor (LY294002) were used to treat MCF-7 and MDA-MB-231 cells. Further, the cell cycle arrest was analysed by flow cytometry. The protein expression of IGF signalling molecules was significantly decreased in nimbolide-treated breast cancer cells. PI3K inhibitor and IGF-I with nimbolide treatment notably inhibited phosphorylated Akt. The cell cycle arrest was observed at the G0/G1 phase, and accumulation of apoptotic cells was observed in nimbolide-treated breast cancer cell lines. Nimbolide also increased the protein expression of p21 and decreased the cyclins in both the cell lines. Nimbolide decreases the proliferation of breast cancer cells by modulating the IGF signalling molecules, which could be very useful for the breast cancer treatment.[3]

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Nimbolide inhibited proliferation, induced apoptosis, and suppressed NF-κB activation and NF-κB-regulated tumorigenic proteins in colorectal cancer cells. The suppression of NF-κB activation by nimbolide was caused by sequential inhibition of IκB kinase (IKK) activation, IκBα phosphorylation, and p65 nuclear translocation. Furthermore, the effect of nimbolide on IKK activity was found to be direct. [5]

Nimbolide inhibits GBM tumor growth in vivo[2]
So far we have shown that nimbolide markedly reduces GBM cell viability in vitro. To verify, whether nimbolide can repress tumor growth in vivo, we took three different approaches. All in vivo approaches were in compliance with institutional and IACUC guidelines. In the first approach, we created flank xenografts using U87EGFRvIII cells and injected Azt or EtoH (control) directly into the tumor bed. In the second approach, we injected nimbolide or DMSO (control) through the tail vein (i.v.) after creating flank xenografts of U87EGFRvIII cells. In the third approach, U87EGFRvIII were seeded orthotopically in the cortex of Nu/Nu mice and nimbolide or DMSO (control) was injected (i.v.). In the first model, tumors were allowed to grow for seven days following which 62.5 μl of 95% EtOH or Azt (0.34 μg/g body weight) was injected into the tumor bed of the left and right flank respectively, every other day for eight days. Other than the tumor burden, mice injected with EtOH or Azt were healthy, and did not show lethargy, locomotion or behavioral abnormalities. EtOH exposure caused skin reddening (the reddish area next to the tumor in Fig 6A). Azt, which is extracted with ethanol from leaves is dark green in color and when injected into tumors, gave a dark green hue to tumors (Fig 6A), but did not cause tumor necrosis as judged by histological analysis of tumor sections (Fig 6E, G). However, Azt significantly (p = 0.00013) reduced growth of GBM tumors (Fig 6 A-C). Following three injections, the volume of Azt-injected tumors were 50% less than control tumors, while at the end of the study, Azt-treated tumors were less than 10% of the volume of control tumors (Fig 6C).[2]

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Researchers next examined if systemic administration of nimbolide will be sufficiently bioavailable to have an effect on the growth of flank GBM xenografts. Indeed, compared to DMSO treatment (control), nimbolide (10μg/Kg body wt) had a significant (p < 0.0001) inhibitory effect on growth of GBM tumors (Fig 7 A, B). When the study ended (day 11) we observed a three to four fold reduction of tumor volume in nimbolide-treated animals. Many preclinical drugs in trial fail because of their inability to cross the blood brain barrier at quantities sufficient to have an effect on intracranial tumors. Therefore, we examined whether nimbolide had any effect on orthotopic GBM xenografts. We used three groups of animals – in the first group, we injected U87EGFRvIII cells labeled with lentiviral luciferase, treated mice with DMSO or nimbolide (i.v) every other day (n = 4/subgroup) and measured tumor growth on day 14 (Fig 7C). In the second group, we injected U87EGFRvIII cells labeled DsRed, treated mice with DMSO or nimbolide (i.v) every other day (n = 3/subgroup), euthanized mice on day 14 and monitored tumor growth by measuring tumor area in 2 mm thick coronal brain sections (Fig 7D, E). In the third group, we injected unlabeled U87EGFRvIII cells, treated mice with DMSO or nimbolide (IV) every other day (n = 5/subgroup) and followed survival (Fig 7F). All animals ultimately died; however as revealed by bioluminescence assay (Fig 7C) and fluorescence microscopy of brain sections (Fig 7D), nimbolide reduced tumor growth and prolonged survival of mice (Fig 7E; Log Rank p value 0.017). By measuring tumor area (total area within yellow contour line in D), we found that nimbolide-treated tumors were about 40% smaller relative to DMSO-treated tumors (p value 0.018). Extensive proliferation in GBMs causes metabolic stress leading to necrosis. Nimbolide-injected tumors were not only smaller but also did not show necrosis at the same time point when control tumors showed widespread necrosis (asterisks in Fig 7D). Future studies will be required to determine if nimbolide could be used at higher doses to increase brain bioavailability and if structural modifications of nimbolide could make it more stable, bioavailable and permissive through the brain tissue. The collective results from our three different in vivo approaches raise the possibility that either application of nimbolide to the tumor bed post-surgery through wafers and/or systemic administration of a safe, optimized dose of nimbolide could be therapeutically effective in GBM.[2]

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In vivo, nimbolide (at 5 and 20 mg/kg body weight), injected intraperitoneally after tumor inoculation, significantly decreased the volume of colorectal cancer xenografts. The limonoid-treated xenografts exhibited significant downregulation in the expression of proteins involved in tumor cell survival (Bcl-2, Bcl-xL, c-IAP-1, survivin, and Mcl-1), proliferation (c-Myc and cyclin D1), invasion (MMP-9, ICAM-1), metastasis (CXCR4), and angiogenesis (VEGF). The limonoid was found to be bioavailable in the blood plasma and tumor tissues of treated mice. Conclusions: Our studies provide evidence that nimbolide can suppress the growth of human colorectal cancer through modulation of the proinflammatory microenvironment.[5]

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Nimbolide plays an important role in treating human diseases. In these years, the anticancer property of nimbolide has been paid more and more attention. However, the role of nimbolide in non-small cell lung cancer (NSCLC) remains unclear. In this study, we found that nimbolide treatment suppressed the invasion and migration of NSCLC cells, in a dose-dependent manner. Moreover, nimbolide treatment dose-dependently inhibited ERK1/2 activation, decreased Snail and MMP-3 expression, and increased E-cadherin expression. Further, we found that nimbolide treatment upregulated DUSP4 expression. DUSP4 knockdown attenuated nimbolide-mediated inhibition of cell invasion, migration and ERK1/2 activation. We also found that DUSP4 knockdown suppressed the effect of nimbolide on MMP-3, Snail and E-cadherin expression. Taken together, our study demonstrates that nimbolide treatment can upregulate the expression of DUSP4, thus inhibiting ERK1/2 activation. Inhibition of ERK1/2 pathway by nimbolide decreases MMP-3 and Snail expression, and increases E-cadherin expression, which finally inhibits NSCLC cell invasion and migration. Therefore, nimbolide may act as a novel drug to inhibit NSCLC invasion and metastasis through manipulation of ERK1/2 signaling and DUSP4 expression.[4]

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ELISA assay[4]
Cells were incubated with or without nimbolide for 12 h. Cell suspension was centrifugated at 1000 rpm for 10 min at 4 °C. ELISA assay was performed with MMP-3 ELISA Kit, according to the manufacturer’s instruction. Briefly, each well of the 96-well plate was precoated with anti-MMP-3 antibody, and 50 μl of cell suspension or reference standard solution was added. After incubation with the secondary antibody for 2 h, HRP solution was added. Finally, stabilized chromogen was added and OD values were detected by Bio-Rad Model 680 Microplate Reader at 595 nm. The concentration of MMP-3 was determined through comparing with the reference standard.

Anchorage independent growth[2]
For Anchorage independent growth, 2 × 104 GBM cells were mixed with 0.7% top agar and layered on top of 1% bottom agar made in 2X DMEM with 20% FCS and antibiotics. Cells were fed with medium containing EtOH, DMSO (control) or Azt, nimbolide (Purchased from Bio Vision) every third day and allowed to grow for two weeks. Colonies were stained with crystal violet and imaged. Colony quantitation was done using Image J software.[2]

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Western blot analysis[3]
The cells (1 × 106 per plate) were cultured in 100-mm culture plates containing growth medium, and after 24h the cells (70–80% confluent) were washed twice with serum-free medium and starved by incubating them in 5 ml of serum-free medium. After starvation, the cells were treated with dimethyl sulfoxide (vehicle), 2 and 4 μM of nimbolide for MCF-7 cells and 4 and 6 μM of nimbolide for MDA-MB-231 cells. After 24h treatment period, the cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing 1X protease and phosphatase inhibitor cocktail, and the protein concentrations were determined by Lowry's method.22 The cell lysates (50 µg) were electrophoresed in 12% sodium dodecyl sulfate (SDS) polyacrylamide gel and then transferred into PVDF membranes. The membranes were incubated with primary antibodies against IGF signalling molecules, PCNA, c-Myc, p21, cyclins and β-actin in tris-buffered saline. After washing, the membranes were incubated with HRP-conjugated antimouse IgG (1:5000) and goat antirabbit IgG (1:5000). The protein bands were detected using chemiluminescence system (ECL kit) and quantified.

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Growth factor (IGF-I), PI3K inhibitor (LY294002) and nimbolide treatment[3]
The cells (1 × 106 per plate) were cultured in 100-mm culture plates containing growth medium and after 24 hours, the cells (70–80% confluent) were washed twice with serum-free medium and starved by incubating them in 5 ml of serum-free medium. The cells were pretreated with or without IGF-I (50 ng ml−1),23 LY294002 (50 μM) in cell culture medium for 1 h. After pretreatment, 2 and 4 μM of nimbolide for MCF-7 cells and 4 and 6 μM of nimbolide for MDA-MB-231 cells were given. After 24h, the cells were lysed with RIPA buffer containing protease and phosphatase inhibitor, and the protein was quantified. pAkt was analysed by western blot analysis.

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Cell cycle analyses by flow cytometer[3]
The cells (1 × 106 per plate) were cultured in 100-mm culture plates containing growth medium. After starvation, 2 and 4 μM of nimbolide for MCF-7 cells and 4 and 6 μM of nimbolide for MDA-MB-231 cells were given. After 24h, the cells were harvested with 0.25% trypsin and centrifuged at 3000xg for 5 min. Then, the cells were washed with PBS. After centrifugation, the cells were fixed in 100% ice-cold methanol overnight at −20 °C. The cells were then incubated in 50 µg ml−1 of propidium iodide in PBS and 1 mg m−1 of ribonuclease in PBS for 30 min. Cell cycle analyses were performed on a FACSCalibur, and the data were analysed using CellQuest Pro software.

In vivo Experiments[2]
Animals were monitored daily by animal care personnel. For flank in vivo xenografts experiments, 1.5 × 106 U87EGFRvIII cells were injected into the flanks of nu/nu mice. For glioma initiation experiment, cell were pretreated with EtOH (control) or Azt for 1h and injected into the flanks. Mice did not receive any further injections of EtOH or Azt. For glioma progression experiment, similar numbers of untreated cells were injected and tumors were allowed to grow for seven days. Following this, 62.5 ul of 95% ethanol or Azt (0.34 μg/g body weight) was injected directly into the tumor bed of the left and right flank respectively, every other day for eight days. To examine the effect of systemic effect of nimbolide, we injected 1.5 × 106 U87EGFRvIII cells into the flanks of nu/nu mice, allowed tumor seeding for three days and treated animals with nimbolide (@10μg/Kg body wt, IV) or vehicle (DMSO) every day for seven days. Tumor growth was monitored by measuring tumor volume every other day by using the formula π/6 × A × B2 (A = larger diameter; B = smaller diameter). Tumors were dissected out and imaged using an iPAD 3. For intracranial xenograft experiments, U87EGFRvIII cells were infected with LeGo-Cer2 lentivirus expressing firefly luciferase or lentivirus expressing DsRed fluorescent reporter. 1 × 105 cells, unlabeled or labeled cells (depending on experimental group – see results) were injected into the cerebral cortex of male nu/nu mice (2mm caudal to bregma; 2 mm right of midline; 3 mm deep) using a stereotactic device. Tumor seeding was allowed for three days following which mice received nimbolide (@ 200μg/Kg body wt, IP) or vehicle (DMSO). All animals were imaged twice – once, three days after tumor seeding before start of treatment to ensure they had similar starting signals and next, on day 14. Five minutes before imaging, mice were anesthetized and injected (IP) with luciferin (150 mg/Kg) and imaged using IVIS (Xenogen). All mice were euthanized following observation of lethargy and/or neurological symptoms. For DsRed-labeled tumors, 2 mm thick coronal brain sections were dissected and tumor area was measured using the Image J software.

[1]. Chemopreventive and therapeutic effects of nimbolide in cancer: the underlying mechanisms. Toxicol In Vitro. 2014 Aug;28(5):1026-35.

[2]. Direct inhibition of retinoblastoma phosphorylation by nimbolide causes cell-cycle arrest and suppresses glioblastoma growth. Clin Cancer Res. 2014 Jan 1;20(1):199-212.

[3]. lumalai P, Arunkumar R, Benson CS, Sharmila G, Arunakaran J. Nimbolide inhibits IGF-I-mediated PI3K/Akt and MAPK signalling in human breast cancer cell lines (MCF-7 and MDA-MB-231). Cell Biochem Funct. 2014 Jul;32(5):476-84.

[4]. Lin H, Qiu S, Xie L, Liu C, Sun S. Nimbolide suppresses non-small cell lung cancer cell invasion and migration via manipulation of DUSP4 expression and ERK1/2 signaling. Biomed Pharmacother. 2017 Aug;92:340-346.

[5]. Gupta SC, Prasad S, Sethumadhavan DR, Nair MS, Mo YY, Aggarwal BB. Nimbolide, a limonoid triterpene, inhibits growth of human colorectal cancer xenografts by suppressing the proinflammatory microenvironment. Clin Cancer Res. 2013 Aug 15;19(16):4465-76.

methyl 2-[(1R,2S,4R,6R,9R,10S,11R,15R,18R)-6-(furan-3-yl)-7,9,11,15-tetramethyl-12,16-dioxo-3,17-dioxapentacyclo[9.6.1.02,9.04,8.015,18]octadeca-7,13-dien-10-yl]acetate has been reported in Pyrola and Azadirachta indica with data available.

C27H30O7

466.52300

466.199

C, 69.51; H, 6.48; O, 24.01

25990-37-8

25990-37-8

12313376

White to off-white solid powder

1.3±0.1 g/cm3

608.6±55.0 °C at 760 mmHg

321.9±31.5 °C

0.0±1.7 mmHg at 25°C

1.597

2.66

0

7

4

34

1040

9

CC1=C2C(C[C@H]1C3=COC=C3)O[C@@H]4[C@H]5C6[C@@](C)(C=CC(=O)[C@]6(C)[C@@H](CC(=O)OC)[C@]24C)C(=O)O5

JZIQWNPPBKFOPT-LSYMHUITSA-N

InChI=1S/C27H30O7/c1-13-15(14-7-9-32-12-14)10-16-20(13)27(4)17(11-19(29)31-5)26(3)18(28)6-8-25(2)22(26)21(23(27)33-16)34-24(25)30/h6-9,12,15-17,21-23H,10-11H2,1-5H3/t15-,16-,17-,21-,22+,23-,25-,26+,27-/m1/s1

methyl 2-[(1R,2S,4R,6R,9R,10S,11R,15R,18R)-6-(furan-3-yl)-7,9,11,15-tetramethyl-12,16-dioxo-3,17-dioxapentacyclo[9.6.1.02,9.04,8.015,18]octadeca-7,13-dien-10-yl]acetate

Nimbolide; 25990-37-8; Nimbolide?; NSC-309909; CHEMBL426690; N993G4LGD6; SCHEMBL2146198; DTXSID20948894;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~107.18 mM)

Solubility in Formulation 1: ≥ 1 mg/mL (2.14 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1 mg/mL (2.14 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)