Nimbolide sensitizes human colon cancer cells to TRAIL through reactive oxygen species- and ERK-dependent up-regulation of death receptors, p53, and Bax

TNF-related apoptosis-inducing ligand (TRAIL) shows promise as a cancer treatment, but acquired tumor resistance to TRAIL is a roadblock. Here we investigated whether nimbolide, a limonoid, could sensitize human colon cancer cells to TRAIL. As indicated by assays that measure esterase activity, sub-G(1) fractions, mitochondrial activity, and activation of caspases, nimbolide potentiated the effect of TRAIL. This limonoid also enhanced expression of death receptors (DRs) DR5 and DR4 in cancer cells. Gene silencing of the receptors reduced the effect of limonoid on TRAIL-induced apoptosis. Using pharmacological inhibitors, we found that activation of ERK and p38 MAPK was required for DR up-regulation by nimbolide. Gene silencing of ERK abolished the enhancement of TRAIL-induced apoptosis. Moreover, our studies indicate that the limonoid induced reactive oxygen species production, which was required for ERK activation, up-regulation of DRs, and sensitization to TRAIL; these effects were mimicked by H(2)O(2). In addition, nimbolide down-regulated cell survival proteins, including I-FLICE, cIAP-1, cIAP-2, Bcl-2, Bcl-xL, survivin, and X-linked inhibitor of apoptosis protein, and up-regulated the pro-apoptotic proteins p53 and Bax. Interestingly, p53 and Bax up-regulation by nimbolide was required for sensitization to TRAIL but not for DR up-regulation. Overall, our results indicate that nimbolide can sensitize colon cancer cells to TRAIL-induced apoptosis through three distinct mechanisms: reactive oxygen species- and ERK-mediated up-regulation of DR5 and DR4, down-regulation of cell survival proteins, and up-regulation of p53 and Bax.     

The neem limonoids azadirachtin and nimbolide induce cell cycle arrest and mitochondria-mediated apoptosis in human cervical cancer (HeLa) cells

Abstract

Limonoids from the neem tree (Azadirachta indica) have attracted considerable research attention in recent years owing to their potent antioxidant and anti-proliferative effects. The present study was designed to investigate the cellular and molecular mechanisms by which azadirachtin and nimbolide exert cytotoxic effects in the human cervical cancer (HeLa) cell line. Both azadirachtin and nimbolide significantly 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 proteins cyclin B, cyclin D1 and PCNA. Characteristic changes in nuclear morphology, presence of a subdiploid peak and annexin-V staining pointed to apoptosis as the mode of cell death. Increased generation of reactive oxygen species with decline in the mitochondrial transmembrane potential and release of cytochrome c confirmed that the neem limonoids transduced the apoptotic signal via the mitochondrial pathway. Altered expression of the Bcl-2 family of proteins, inhibition of NF-κB activation and over-expression of caspases and survivin provide compelling evidence that azadirachtin and nimbolide induce a shift of balance toward a pro-apoptotic phenotype. Antioxidants such as azadirachtin and nimbolide that can simultaneously arrest the cell cycle and target multiple molecules involved in mitochondrial apoptosis offer immense potential as anti-cancer therapeutic drugs.     

Nimbolide inhibits IGF‐I‐mediated PI3K/Akt and MAPK signalling in human breast cancer cell lines (MCF‐7 and MDA‐MB‐231)

The insulin‐like growth factor I (IGF‐I) signalling pathway contributes a major role on various cancer cell proliferation, survival and cell cycle. 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. Copyright © 2014 John Wiley & Sons, Ltd.     

Biosynthesis of nimbolide from [2-14C,(4R)4-3H1] mevalonic acid lactone in the leaves of Azadirachta indica

Nimbolide was biosynthesized from [2-¹⁴C, (4R)4-³H₁]mevalonic acid lactone in the leaves of Azadirachta indica. The nimbolide had a ³H:¹⁴C ratio of 3:5 which gives support to the suggestion of the involvement of a triterpenoid intermediate with a double bond at the Δ⁸⁽⁹)-position in the biosynthesis of nimbolide.     

Nimbolide ameliorates the streptozotocin-induced diabetic retinopathy in rats through the inhibition of TLR4/NF-κB signaling pathway

BackgroundDiabetic retinopathy (DR) is a common problem in the diabetic patients due to the high blood glucose level. DR affects more number of diabetic patients worldwide with irreversible vision loss.ObjectiveThe current investigation was focused to reveal the therapeutic actions of nimbolide against the streptozotocin (STZ)-provoked DR in rats through inhibition of TLR4/NF-κB pathway.MethodologyDR was provoked to the rats through administering a single dose of STZ (60 mg/kg) intraperitoneally. The DR rats were then supplemented with the 50 mg/kg of nimbolide for 60 days. The bodyweight and blood glucose level was measured using standard methods. The lipid profiles (cholesterol, TG, LDL, and HDL), inflammatory markers, and antioxidants level was detected using respective kits. The level of MCP-1, VEGF, and MMP-9 was quantified using kits. The morphometric analysis of retinal tissues were done. The mRNA expressions of target genes were studied using RT-PCR assay.ResultsNimbolide treatment effective decreased the food intake and blood glucose, and improved the bodyweight of STZ-provoked animals. The levels of pro-inflammatory mediators, cholesterol, TG, LDL, and HDL, MCP-1, VEGF, and MMP-9 was remarkably suppressed by the nimbolide treatment. Nimbolide also improved the antioxidants, retinal thickness and cell numbers. The TLR4/NF-κB pathway was appreciably inhibited by the nimbolide.ConclusionOverall, our findings demonstrated that the nimbolide attenuated the STZ-provoked DR in rats through inhibiting the TLR4/NF-κB pathway.     

Nimbolide ameliorates the streptozotocin-induced diabetic retinopathy in rats through the inhibition of TLR4/NF-κB signaling pathway

BackgroundDiabetic retinopathy (DR) is a common problem in the diabetic patients due to the high blood glucose level. DR affects more number of diabetic patients worldwide with irreversible vision loss.ObjectiveThe current investigation was focused to reveal the therapeutic actions of nimbolide against the streptozotocin (STZ)-provoked DR in rats through inhibition of TLR4/NF-κB pathway.MethodologyDR was provoked to the rats through administering a single dose of STZ (60 mg/kg) intraperitoneally. The DR rats were then supplemented with the 50 mg/kg of nimbolide for 60 days. The bodyweight and blood glucose level was measured using standard methods. The lipid profiles (cholesterol, TG, LDL, and HDL), inflammatory markers, and antioxidants level was detected using respective kits. The level of MCP-1, VEGF, and MMP-9 was quantified using kits. The morphometric analysis of retinal tissues were done. The mRNA expressions of target genes were studied using RT-PCR assay.ResultsNimbolide treatment effective decreased the food intake and blood glucose, and improved the bodyweight of STZ-provoked animals. The levels of pro-inflammatory mediators, cholesterol, TG, LDL, and HDL, MCP-1, VEGF, and MMP-9 was remarkably suppressed by the nimbolide treatment. Nimbolide also improved the antioxidants, retinal thickness and cell numbers. The TLR4/NF-κB pathway was appreciably inhibited by the nimbolide.ConclusionOverall, our findings demonstrated that the nimbolide attenuated the STZ-provoked DR in rats through inhibiting the TLR4/NF-κB pathway.     

Nimbolide attenuate the lipid accumulation,oxidative stress and antioxidant in primary hepatocytes

Nimbolide is a bioactive compound found in Azadirachta indica. This work was devised to investigate the potential effects of nimbolide on intracellular lipid deposition and its associated redox modulation in primary hepatocytes (Heps). Lipid accumulation was induced in Heps by supplementing 1 mM oleic acid for 24 h which was marked by significant accumulation of lipids. The results demonstrated that nimbolide can decrease intracellular cholesterol, free fatty acids and triglycerides. Nimbolide may also improve hepatocytes function through its antioxidant effects by inhibiting oxidative DNA damage and lipid peroxidation by curtailing the reactive oxygen species levels. Further it also restore the mitochondrial potential, improving the endogenous antioxidant levels such as GSH and antioxidant enzyme activities. Nimbolide increased (P?<?0.05) liver X receptor-α (LXRα), peroxisome proliferator-activated receptor-γ (PPARγ) and sterol regulatory element-binding protein-1c (SREBP1c) gene expression in Heps. The biological significance of nimbolide may involve hypolipidemic effect, lipid peroxidation inhibition, DNA damage inhibition, ROS inhibition, restoring mitochondrial function, increases in GSH and SOD & CAT activities, and direct regulation of LXRα, PPARγ and SREBP1c gene expression. Nimbolide may be used as effective lipid lowering compound and lipid deposition-induced Heps changes.     

IL-2- and IFN gamma-enhanced natural cytotoxic activity: analysis of the role of different lymphoid subsets and implications for activation routes

To elucidate the immunoregulatory mechanisms by which human recombinant interleukin 2 (r.IL-2) and human recombinant interferon (IFN)-gamma influence natural cytotoxic activity of human peripheral blood lymphocytes (PBL), experiments were performed in which we studied: the kinetics of augmentation of natural cytotoxic activity by IL-2 and IFN-gamma; the phenotypes of the natural cytotoxic precursor cells acted upon by IL-2 and IFN-gamma; the role of IL-2-induced IFN-gamma in the natural cytotoxic activity enhancing effects of IL-2 and (d) the surface phenotypes of the natural cytotoxic cells activated by IL-2 or IFN-gamma. Three phenotypical distinct precursor cells can be identified. The phenotypes of the precursor cells sensitive to r.IFN-gamma are T3-, Leu 7+or-, FcR-gamma+. The phenotypes of the precursor cells sensitive to r.IL-2 are T3-, Leu 7-, FcR-gamma+or-. It appears that during prolonged culture relatively more of the FcR-gamma- precursor cells acquire natural cytotoxic activity upon stimulation with r.IL-2. Antibodies to IFN-gamma, known to neutralize both recombinant and natural IFN-gamma, completely inhibited the IFN-gamma-induced augmentation of natural cytotoxic activity but only slightly affected the IL-2-mediated augmentation. This indicates that only part of the augmenting effect of r.IL-2 is mediated through IL-2-induced IFN-gamma. This implies that r.IL-2 augments natural cytotoxic activity predominantly through an IFN-gamma-independent pathway. The phenotype of the effector cells expressing natural cytotoxic activity after a 4-day culture period with IL-2 or IFN-gamma was analyzed by depletion experiments. Virtually all the cells that exerted the IFN-gamma-enhanced natural cytotoxic activity are FcR-gamma+, whereas a portion of the cells that exerted the IFN-gamma-enhanced natural cytotoxic activity are FCR-gamma-. Both natural killer cells and lymphokine-activated killer cells contribute to natural cytotoxic activity. The relative contributions of both activities to the natural cytotoxic activity augmented by IL-2, respectively, IFN-gamma, is discussed.     

Neuromelanogenic and Cytotoxic Properties of Canine Brainstem Peroxidase

We have isolated a heme protein from canine midbrains that possesses potent peroxidase activity. This enzyme catalyzes the oxidation of dopamine to neuromelanin in the presence of H2O2. We have further shown that the isolated peroxidase possesses potent cytotoxic activity in the presence of superoxide or H2O2 and Cl-. The enzyme possesses an endogenous NAD(P)H oxidase activity that can promote the cytotoxic activity by virtue of its production of superoxide. Other enzymes such as dihydroorotate dehydrogenase and galactose oxidase, which produce O2- and H2O2, respectively, are also effective in promoting the cytotoxic activity of the brainstem peroxidase. Although rat erythrocytes were routinely used as the target cell, other cell types, including rat hepatoma and mouse neuroblastoma cells, are also susceptible to the toxic action of the peroxidase. The cytotoxic action of the brainstem peroxidase is dramatically enhanced by kainic acid and is significantly enhanced by Mn2+, whereas dopamine was found to be a potent inhibitor of the cytotoxic activity. Based on these findings, we postulate a central role for the brainstem peroxidase in dopamine metabolism as well as in the biochemical and anatomical changes associated with Parkinson's disease.     

Nimbolide prevents myocardial damage by regulating cardiac biomarkers,antioxidant level,and apoptosis signaling against doxorubicin‐induced cardiotoxicity in rats

The current work planned to assess the protecting properties of nimbolide against doxorubicin (DOX)‐treated myocardial damage. Myocardial damage was produced with 2.5 mg/kg of DOX given on alternative days (14 days). Thiobarbituric acid reactive substances (TBARS) levels of a lipid peroxidative marker were elevated, whereas reduced body weight, heart weight, blood pressure indices and reduced levels of antioxidants like glutathione‐S‐transferase, superoxide dismutase, catalase, glutathione peroxidase, glutathione, and glutathione reductase were observed in the heart tissue of DOX‐treated animals. DOX‐treated animals showed augmented levels of cardiac markers likes monocyte chemotactic protein‐1, interferon‐gamma, aspartate transferase, creatine kinase, lactate dehydrogenase, creatine kinase‐muscle/brain, heart‐type fatty acid‐binding protein, glycogen phosphorylase isoenzyme BB, transforming growth factor‐β, brain natriuretic peptide, myoglobin, and cTnI in serum. Histopathological assessment confirmed the DOX‐induced cardiotoxicity. Furthermore, DOX‐induced rats showed augmented inflammatory mediators (nuclear factor‐κB [NF‐kB], tumor necrosis factor‐α [TNF‐α], and interleukin‐1β [IL‐1β]) and increased PI3K/Akt signaling proteins (PI3K, p‐Bad/Bad, caspase‐3, and p‐Akt), whereas decreased oxidative markers (HO‐1 and NQO‐1) and p‐PTEN were observed. Nimbolide‐supplemented rats showed reduced activity/levels of cardiac markers and TBARS levels in serum and heart tissue. Levels of enzymatic and nonenzymatic antioxidants were augmented in the heart tissue of nimbolide‐supplemented rats. Nimbolide influence decreased apoptosis, inflammation, and enhanced antioxidant markers through the modulation of p‐Bad/Bad, caspase‐3, PI3K, p‐Akt, TNF‐α, NF‐kB, IL‐1β, HO‐1, NQO‐1, and p‐PTEN markers. The histopathological explanations were observed to be in line with biochemical analysis. Therefore, the finding of current work was that nimbolide has a defensive effect on the myocardium against DOX‐induced cardiac tissue damage.     

Cytotoxic activity toward KB cells of 2-substituted naphtho[2,3-b]furan-4,9-diones and their related compounds

We investigated the cytotoxic activity of 2-substituted naphtho[2,3-b]furan-4,9-diones. We have previously synthesized 33 types of 2-substituted and related compounds, and the cytotoxic activity of these compounds was then examined by a KB cell culture assay. 2-(3-Furanoyl)benzoic acids and 1,4-naphthoquinones had no activity. 2-Acetyl-4,9-dimethoxynaphtho[2,3-b]furan 4 showed low activity. However, parent naphtho[2,3-b]furan-4,9-dione 2 and most 2-substituted derivatives exhibited cytotoxic activity. The parent structure was therefore for cytotoxicity. 2-Formylnaphtho[2,3-b]furan-4,9-dione 11 had particularly potent activity (ED50=0.09 microg/ml).     

Mechanism of target cell lysis by cytotoxic T lymphocytes (CTL) employing RSV-induced H-2 congenic and recombinant mouse tumor cells: demonstration of soluble mediators released from H-2-restricted CTL clones

The secretion and the specificity of cytotoxic mediators from H-2-restricted cytotoxic T lymphocytes (CTL) were examined using non-virus-producing target tumor cells induced by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) in B10 congenic and recombinant mice. By using rat concanavalin A supernatant, two H-2-restricted CTL clones were established from cytotoxic effector cells of B10.A(5R) mice primed with SR-RSV-induced syngeneic tumor Cell-free supernatants from the H-2-restricted CTL clones cocultured with syngeneic tumor cells had selectively high cytotoxic activity for syngeneic and H-2-compatible tumor cells, but not for H-2-incompatible tumor cells. YAC-1 cells, and B10.A(5R) blasts as defined in the 5-hr 51Cr-release assay. The cytotoxic activity was detected in the cell-free supernatants from the CTL clones cocultured with the CTL-sensitive syngeneic and H-2-compatible tumor cells, but not with the CTL-insensitive tumor cells and YAC-1 cells. The cytotoxic activity of the cell-free supernatant could be adsorbed by the syngeneic tumor cells, but not by YAC-1 and L(s) cells. Thus, the H-2-restricted CTL clones against SR-RSV-induced tumor cells were capable of releasing cytotoxic mediators by coculturing with syngeneic or H-2-compatible tumor cells, and the cytotoxic mediators showed a certain H-2-restricted manner in killing the target cells. These results suggest that the lysis of RSV-induced tumor cells by H-2-restricted CTL can at least in part be mediated by cytotoxic factors.     

Generation of natural killer-like cytotoxicity from human thymocytes with interleukin-2

Human thymocytes cultured in the presence of an interleukin-2 (IL-2) source (the supernatant of the MLA 144 cell line) that contains no lectin or interferon (IFN) activity become cytotoxic to K562 target cells. Inclusion of allogeneic lymphoblastoid cells in these cultures results in the earlier detection of cytotoxic activity. Addition of IFN to the thymocyte cultures has no effect on the development of cytotoxic activity. In contrast, the cytotoxic activity of IL-2 activated thymocytes can be augmented by subsequent exposure to IFN. The specificity and cell surface phenotype of the cytotoxic thymocytes are similar to that of peripheral blood natural killer cells.     

Specific cytotoxic activity of a virus-induced murine lymphoma

The NS8 lymphoma induced with Radiation Leukemia Virus (RadLV) presents in vitro a specific cytotoxic activity. This line was established from a lymphoma induced by injection of lymph node cells from mice bearing the MCA-fibrosarcoma T2 to congenic mice. These cells present a cytotoxic activity limited to the T2 cells. They develop no significant cytotoxic activity against other syngeneic or allogeneic tumoral lines.     

Antitumor action of seminal ribonuclease, its dimeric structure, and its resistance to the cytosolic ribonuclease inhibitor

Bovine seminal RNase (BS-RNase) is a homodimeric enzyme with a cytotoxic activity selective for tumor cells. In this study, the relationships of its cytotoxic activity to its dimeric structure and its resistance to the cytosolic RNase inhibitor (cRI) are investigated systematically by site-directed mutagenesis. The results show that (1) the dimericity of BS-RNase is essential for its full cytotoxic action; (2) the role of the dimeric structure in the antitumor activity is that of making the enzyme insensitive to the cytosolic RNase inhibitor; (3) a RNase may not be completely insensitive to cRI to exploit a full cytotoxic potential.     

Nigexine, a phospholipase A2 from cobra venom with cytotoxic properties not related to esterase activity. Purification, amino acid sequence, and biological properties

The venoms of the Naja species are known to be cytotoxic. This toxicity has been attributed to the presence of small nonenzymatic polypeptides of 60 amino acid residues, designated as cardiotoxins or cytotoxins. We investigated the cytotoxic potency of Naja nigricollis venom fractions and isolated another type of cytotoxic component which is even more potent than cardiotoxins. This cytotoxic compound, which was designated as nigexine, was purified to homogeneity and its amino acid sequence was determined. Nigexine is a basic phospholipase A2 consisting of a single chain of 118 amino acids. A detailed investigation of the cytotoxic effects on epithelial FL cells, C-13T neuroblastoma cells, and promyelocytic leukemia HL 60 cells revealed that nigexine not only altered cell viability but also prevented cell proliferation. This is a property that was specific to nigexine since other phospholipases A2 from various sources had no detectable cytotoxic activity. The cytotoxic activity of nigexine was not dependent on the presence of divalent cations, unlike its enzymatic activity. In particular, the cytotoxic activity of nigexine was identical in the presence or absence of either 2 mM Ca2+ or Sr2+, or 6 mM EDTA. We also present evidence based on chemical modifications that cytotoxic activity was not correlated with enzymatic activity. Thus, modification with parabromophenacyl bromide totally abolished the enzymatic activity of nigexine, which nevertheless retained 6-20% of the cytotoxicity of native nigexine. Conversely, treatment with cyanogen bromide gave a compound that retained 7% of the enzymatic activity of the parent molecule but was devoid of detectable cytotoxicity.