Target cell induced activation of NK cells in vitro: cytokine production and enhancement of cytotoxic function

This study examines the effect of fixed AK-5 tumour cells on rat NK cells. Co-culture of NK cells with fixed tumour cells augmented the cytotoxicity of NK cells against NK-sensitive targets, YAC-1 and AK-5, and induced the secretion of IFN-gamma by NK cells. Antibody against IFN-gamma suppressed the anti-tumour activity of NK cells, whereas the addition of T cells during co-culture enhanced this activity. However, macrophages and B cells had no significant effect when present during co-culture with NK cells. All the inducible cytotoxicity was contained within the NK (CD161+) and NKT (CD3+, CD161+) subsets of lymphocytes. However, in the presence of T cells, the cytolytic potential of NKT cells was higher than that of NK cells alone. The augmentation of cytotoxic activity of NK cells by AK-5 cells in presence of T cells was dependent on IL-2 and IFN-gamma secretion. NK cell activation was blocked by specific antibodies to IL-2 and IFN-gamma in the presence of T cells. Interaction between fixed AK-5 cells with NK and T cell populations induced the expression of Fas-L and perforin in NK cells. These data demonstrate that fixed AK-5 cells initiated cytokine synthesis by NK cells, and the enhanced cytotoxic activity in the presence of T cells was induced as a consequence of the products secreted by activated T lymphocytes. The present observations reflect the possible interactions taking place in vivo after the transplantation of AK-5 tumour in animals. They also suggest direct activation of NK cells after their interaction with the tumour cells.     

Lipoxygenase inhibition by novel fatty acid ester from Annona squamosa seeds

Studies on the seeds of Annona squamosa yielded a novel lipoxygenase inhibitor fatty acid ester, (+) - annonlipoxy (1). Compound 1 was screened for its enzyme inhibitory activity against lipoxygenase (E.C.1.14.18.1), exhibiting activity with IC(50) 69.05 +/- 5.06 microm. Baicalein (IC(50) 22.6 +/- 0.5 microm) was used as a positive control. Crude extracts of Annona squamosa fruit pulp and seeds were screened for its enzyme inhibitory activity against lipoxygenase and acetylcholinesterase. The crude ethanolic extract of fruit pulp and seeds of Annona squamosa also exhibited lipoxygenase activity with 22.2 and 26.7% inhibition, while the pet.ether extract of seeds of A. squamosa exhibited 52.7% inhibition at a concentration of 40 microg/200 ml. The crude ethanolic extract of seeds of Annona squamosa was also bioassayed for acetylcholinesterase inhibition and it was found inactive.     

Induction of apoptosis in AK-5 cells by rotenone involves participation of caspases.

AK-5 tumour cells undergo apoptosis after treatment with rotenone an electron transport inhibitor and oligomycin which inhibits mitochondrial ATPases. Apoptotic process involves the induction of caspases 2 and 3, whereas caspase 1 does not seem to be participating in rotenone/oligomycin induced apoptosis. DEVD which is a specific inhibitor of caspase 3, inhibited apoptosis in AK-5 cells. We have also observed a significant lowering of intracellular pH in AK-5 cells which are induced into the apoptotic process by rotenone. These results suggest an important role for mitochondrial electron transport in the induction of apoptosis in AK-5 tumour cells.     

Target-cell-induced anergy in natural killer cells: suppression of cytotoxic function

 Our earlier studies have demonstrated that natural killer (NK) cells are the effectors that participate during the spontaneous regression of AK-5 tumour in syngeneic hosts. We have shown that the tumour cells are killed by necrosis and apoptosis. In this study, we have examined the induction of functional anergy in NK cells following coculture with fixed AK-5 tumour cells at high ratio. NK cells, upon coculture with fixed AK-5 cells (1:1 ratio), showed loss of cytotoxic function against both AK-5 (antibody-dependent cell cytotoxicity) as well as YAC-1 targets. The response of these cells to the activation by recombinant interleukin-2 and recombinant interferon γ was poor. Induction of tumour necrosis factor α (TNFα) secretion was observed after coculture of NK cells with fixed AK-5 cells. The cocultured cell supernatant inhibited the cytotoxic activity of NK cells, which was partially restored with anti-TNFα antibody. In addition, NK cells, after treatment with fixed tumour cells showed overexpression of the Fas receptor. We have also observed induction of apoptosis in cocultured NK cells. These studies suggest that the fixed tumour cells (antigen) at high ratio are able to suppress NK cell function as well as induce death in NK cells. Received: 16 September 1999 / Accepted: 13 January 2000     

Caspase-2/NEDD-2 protease mediates execution of apoptosis in AK-5 tumor cells

AK-5 tumour cells have been shown to undergo apoptosis in vitro and in vivo. The efficient killing of tumour cells by necrosis and apoptosis leads to spontaneous regression of the tumour. To investigate a possible involvement of caspase-2/Nedd-2 protease in AK-5 apoptosis, we introduced Nedd-2 gene in antisense orientation and showed inhibition of tumour cell apoptosis. Similarly introduction of the bcl-2 gene in tumour cells also inhibited the apoptotic programme. NK cells which have previously been shown to be the effector cells also fail to induce apoptosis in Nedd-2 antisense and bcl-2 transfected clones whereas NK mediated cytotoxic activity is not altered in the transfectants. These results suggest participation of Nedd-2 protease in the induction of apoptosis in AK-5 cells leading to tumour regression. This revised version was published online in June 2006 with corrections to the Cover Date.     

Suppression of tumourogenicity and overexpression of cell adhesion molecules in AK-5 cells transfected with wild-type p53 gene

We have previously shown overexpression of p53 and rearrangement of the p53 gene in AK-5 tumour. In order to study the role of p53 in AK-5 tumourogenicity, we introduced wild-type p53 in AK-5 cells. We have shown suppression of tumourogenicity in AK-5 cells after transfixion with wt p53. In one of the transfected clones 3B4, there was complete loss of tumour forming ability. Clone 3B4 also showed cellular aggregation which correlated well with the higher expression of cell adhesion molecules like fibronectin and hyaluronectin. These observations demonstrate tumour suppression in AK-5 after introduction of wt p53.     

In vitro antibacterial activity in seed extracts of Manilkara zapota, Anona squamosa, and Tamarindus indica

Extracts prepared from seeds of Manilkara zapota, Anona squamosa, and Tamarindus indica were screened for their antibacterial activity by disc diffusion and broth dilution methods. Acetone and methanol extracts of T. indica seeds were found active against both gram-positive and gram-negative organisms. MIC values of potent extracts against susceptible organisms ranged from 53-380 μg/mL. Methanol extract of T. indica and acetone extract of M. zapota seeds were found to be bactericidal.     

Differential cytotoxic effects ofAnnona squamosa seed extracts on human tumour cell lines: Role of reactive oxygen species and glutathione

Annonaceous acetogenins are a new class of compounds that have been reported to have potent pesticidal, parasiticidal, anti-microbial, cell growth inhibitory activities. In this study, organic and aqueous extracts from the defatted seeds ofAnnona squamosa (custard apple) were tested on different human tumour cell lines for antitumoural activity. While organic and aqueous extracts induced apoptosis in MCF-7 and K-562 cells, they failed to do so in COLO-205 cells. Treatment of MCF-7 and K-562 cells with organic and aqueous extracts resulted in nuclear condensation, DNA fragmentation, induction of reactive oxygen species (ROS) generation and reduced intracellular glutathione levels. In addition downregulation of Bcl-2 and PS externalization by Annexin-V staining suggested induction of apoptosis in MCF-7 and K-562 cells by both the extracts through oxidative stress. On the contrary, COLO-205 cells showed only PS externalization but no change in ROS and glutathione levels. These observations suggest that the induction of apoptosis byA. squamosa extracts can be selective for certain types of cancerous cells     

Partial purification and characterization of a novel antifungal compound against Aspergillus spp. from Synechocystis sp. PCC 6803

The antifungal compound AK-3 is purified and characterized from intracellular metabolites of the unicellular cyanobacteria, Synechocystis sp. PCC 6803. AK-3 clearly had antifungal effects upon growth of the fungi Aspergillus spp. From 2 g of dry cell powder, 4.8 mg of AK-3 was obtained with a yield of 0.24%. Its structure was elucidated by NMR, UV spectra, amino acid analysis by chemical degradation, and MS measurements, including MALDI-TOF and MS-MS techniques. AK-3 contains a high level of antifungal activity compared to itraconazole and is seemingly a new compound based on its amino acid composition. Extraction was performed with an acetic acid/water mixture (pH 3.0) while detection at UV 214 nm found AK-3 did not react with ninhydrin. These results suggest that this compound is a cyclic peptide with high hydrophilicity and a phenolic ring.     

Induction of nitric oxide production by natural killer cells: its role in tumor cell death.

It has been recognized that natural killer (NK) cells destroy AK-5 tumor cells, largely by cytolysis and apoptosis. The objective of this study was to elucidate the existence and the role of nitric oxide (NO) during this killing. The target cell killing ability of NK cells was associated with an increased production of NO with higher expression of inducible nitric oxide synthase. In part, the production of NO was confirmed by significant increase in cell lysis in the presence of l-arginine and attenuation of cell lysis, DNA fragmentation, and apoptosis by N(omega)-nitro-l-arginine methyl ester (L-NAME). An increased oxidation of intracellularly trapped dichlorofluorescein was observed in NK cells, which was effectively prevented by L-NAME. Exposure of AK-5 cells to chemically generated NO also induced DNA fragmentation in AK-5 cells. Further evidence for the involvement of NO in apoptosis was provided by the inhibition of specific cleavage of PARP and activation of CPP32 by L-NAME. Increased production of NO with simultaneous enhancement of the cytotoxic activity of NK cells from sc tumor-transplanted animals has been implicated in tumor regression when compared to the ip tumor-bearing animals. Overall, these observations suggest an important role for NO during NK cell-mediated apoptosis and lysis of AK-5 cells.     

Involvement of 5-lipoxygenase in programmed cell death of cancer cells

We investigated the involvement of 5-lipoxygenase activity in the early phases of programmed cell death (PCD) induced by H2O2 or retinoids in different human tumour cells (erythroleukaemia, neuroblastoma, melanoma). Apoptotic cells showed enhanced 5-lipoxygenase activity which was paralleled by decreased superoxide dismutase activity and increased light emission. Ultraweak luminescence, mainly due to membrane lipid peroxidation by lipoxygenase activation, increased in all cell lines tested within 10-15 min after induction of PCD, in a concentration and time-dependent manner. At the same time, we observed a significant increase in the intracellular steady state level of the 5-lipoxygenase metabolite leukotriene B4. Furthermore, 5-lipoxygenase metabolite 5-hydroxyeicosatetraenoic acid was able to induce PCD in all cell lines tested. Conversely, the general lipoxygenase inhibitor nordihydroguaiaretic acid and the selective 5-lipoxygenase inhibitor caffeic acid protected the different tumour cells from H2O2-induced PCD to a similar extent. These results show the activation of the 5-lipoxygenase pathway in PCD of three different cancer cell lines.     

Antitumor activity of curcumin is mediated through the induction of apoptosis in AK-5 tumor cells

Curcumin, the yellow pigment of turmeric (Curcuma longa), used commonly as a spice, has been shown to possess anticarcinogenic activity. Curcumin inhibited AK-5 tumor growth and induced apoptosis in AK-5 cells. Curcumin induced apoptosis is mediated through the activation of caspase-3, which is specifically inhibited by the tetrapeptide Ac-DEVD-CHO. In addition, curcumin induced tumor cell death is caused through the generation of reactive oxygen intermediates which is inhibited by N-acetyl-L-cysteine. Our studies suggest that the apoptotic process induced by curcumin is the mechanism mediating AK-5 tumor cell death.     

Evaluation of mosquitocidal activity of Annona squamosa leaves against filarial vector mosquito,Culex quinquefasciatus Say

Methanolic extract of leaves of A. squamosa was tested for mosquitocidal effect against C. quinquefasciatus. A liquid mosquito insecticide formulation was prepared with the extract (1, 3 and 5 %w/w) using deodorized kerosene as solvent and investigated for its knock-down and 24 hr mortality. The extract formulation produced dose dependent activity, exhibited significantly shorter knock down KD50 and KD90 values and produced significant mortality. The results suggest the potential mosquitocidal effect of A. squamosa on C. quinquefasciatus.     

Adenylate kinase 2, a mitochondrial enzyme

The subcellular compartmentalization of the isozymes of ATP: AMP phosphotransferase (adenylate kinase) was analyzed in HeLa cells, RAG cells, and RAG-human hybrids that express human AK-2. In HeLa cells and in the hybrids, human AK-2 was present in a mitochondrial fraction prepared from cell extracts and in mitochondria purified by density gradient centrifugation. Human AK-1 was, as expected, distributed in the soluble cytoplasmic fraction of the cells. The rodent isozymes which are homologous to human AK-1 and AK-2 have been determined.This work was supported by NIH Grants HD 04807-07 and HD 06285-04.     

Involvement of adenosine kinase in the phosphorylation of formycin B in CHO cells

In Chinese hamster ovary cells, [3H]formycin B is metabolized into formycin B-5'-monophosphate, formycin A-5'-monophosphate and higher phosphorylated derivatives of formycin A which are incorporated into RNA. Mutants of CHO cells independently selected for resistance to various adenosine analogs viz. toyocamycin, tubercidin, 6-methylmercaptopurine riboside, which contain no detectable activity of adenosine kinase (AK) in cell extracts, all exhibited between 2- to 3-fold increased resistance to formycin B. Formycin B-resistant mutants of CHO cells are also affected in AK, as indicated by the absence of AK activity in cell extracts. Both types of AK- mutants showed reduced uptake and phosphorylation of [3H]formycin B in comparison to the parental (AK+) cells. In addition, toxicity of formycin B towards CHO cells was reduced in presence of adenosine in a concentration dependent manner. These observations strongly indicate that in CHO cells, formycin B is phosphorylated via AK and that like other nucleoside analogs its phosphorylation may be essential for the drugs cellular toxicity.     

Three Novel Chemical Compounds of Annonaceous Acetogenins from the Seeds of Annona squamosa

Seven compounds (A1--A7) were isolated from the seeds of Annona squamosa L. Three of them, A2, A3, and A5, are new compounds of annonaeeous aeetogenins, and they were name neo-desacetyluvaricin (A2), neo-annonin B (A3) and neo-reticulatacin A (A5), respectively.     

一些孟加拉当地产水果的抗氧化活性

研究了A.heterophyllus,A.squamosa,T.bellirica,S.samarangense,A.carambolaand O.europ水果的抗氧化活性。首先将这些成熟的水果切成小片,然后用日光晒干,最后用研磨器磨成粉末。这些水果粉末用99·99%的乙醇进行乙醇提取。这些提取物的活性通过其清除稳定的1,1-diphenyl-2-picrylhydrazyl(DPPH)自由基的能力进行测定。实验结果表明,所有A.heterophyllus,A.squamosa,T.bellirica,S.samarangense,A.carambolaand O.europa这些水果的乙醇提取物都具有抗氧化活性,这些水果的乙醇提取物的的IC50值分别是410,250,34,200,30和76μg/mL。其中A.carambola具有最高的抗氧化活性,其次是T.bellirica,O.europa,S.samarangense,A.squamosa和A.heterophyllus。结果表明A.carambola,T.bellirica和O.europa对人体健康是非常有益的。     

PAF-acether in AK-5 tumour cells

PAF-Acether fraction derived from stimulated AK-5 tumour cells, aggregated human platelets. The platelet aggregating ability increased linearly with increasing concentration of the stimulant, calcium ionophore A23187, and reached a maximum at 6 microM in 25 minutes. This factor had biological and chemical properties identical to authentic PAF-acether. Our results demonstrate that, although PAF-acether is produced mainly from pro-inflammatory cells, it appears to be produced even in tumour cells.     

Extracts of the Brown Seaweed <Emphasis Type="Italic">Ascophyllum nodosum</Emphasis> Induce Gibberellic Acid (GA<Subscript>3</Subscript>)-independent Amylase Activity in Barley

Extracts of the brown seaweed Ascophyllum nodosum have been used as a biostimulant to promote growth and productivity in a number of agricultural production systems. Although the extracts have been shown to improve seedling emergence and vigor in a variety of plants, including barley, the mechanism(s) of this growth-promoting effect is(are) largely unknown. In our study, A. nodosum extract induced amylase activity in barley seed-halves; a significant difference in amylase activity was observed in seeds without an embryo. The addition of activated charcoal to the treatment media negated the bioactivity of the extracts suggesting the organic nature of bioactive compounds in A. nodosum extracts. The extracts induced amylase activity in a gibberellic acid (GA)-deficient barley mutant (grd2). LC-MS-MS analysis failed to detect the presence of GA₃ in the extracts. ABA supplementation of the medium caused a significant reduction of amylase activity in GA-treated seeds compared with those treated with the A. nodosum extract. Taken together, our results suggest that the organic components of A. nodosum extract induce amylase activity independent of GA₃ and might act in concert with GA-dependent amylase production leading to enhanced germination and seedling vigor in barley. Being derived from a renewable resource, the bioactive compounds from A. nodosum could be used to improve crop productivity in sustainable agricultural systems.