Anju Shrivastava

INHIBITION OF TUMOR NECROSIS FACTOR (TNF-ALPHA )-MEDIATED APOPTOSIS BY HEPATITIS C VIRUS CORE PROTEIN

Hepatitis C virus (HCV) putative core protein has displayed many intriguing biological properties. Since tumor necrosis factor (TNF) plays an important role in controlling viral infection, in this study the effect of the core protein was investigated on the TNF-α induced apoptosis of human breast carcinoma cells (MCF7). HCV core protein when expressed inhibited TNF-α-induced apoptotic cell death unlike the control MCF7 cells, as determined by cell viability and DNA fragmentation analysis. Additionally, HCV core protein blocked the TNF-induced proteolytic cleavage of the death substrate poly(ADP-ribose) polymerase from its native 116-kDa protein to the characteristic 85-kDa polypeptide. Results from this study suggest that the HCV core protein plays a role in the inhibition of TNF-α-mediated cell death. Thus, the ability of core protein to inhibit the TNF-mediated apoptotic signaling pathway may provide a selective advantage for HCV replication, allowing for evasion of host antiviral defense mechanisms.

VEGI, a new member of the TNF family activates Nuclear Factor-κB and c-Jun N-terminal kinase and modulates cell growth

Recently a new member of the human tumor necrosis factor (TNF) family named as VEGI was reported. However, very little is known about the biological activities displayed by this cytokine. In this report, we show that in myeloid cells VEGI activated the transcription factor κB (NF-κB) as determined by the electrophoretic mobility shift assay, induced degradation of IκBα, and nuclear translocation of p65 subunit of NF-κB. VEGI also activated NF-κB-dependent reporter gene expression. In addition, VEGI activated c-Jun N-terminal kinase. When examined for growth modulatory effects, VEGI inhibited the proliferation of breast carcinoma (MCF-7), epithelial (HeLa), and myeloid (U-937 and ML-1a) tumor cells; and activated caspase-3 leading to PARP cleavage. VEGI-induced cytotoxicity was potentiated by inhibitors of protein synthesis. VEGI also induced proliferation of normal human foreskin fibroblast cells. The activity of VEGI could neither be neutralized by antibodies against TNF, nor could it compete with TNF binding, indicating that the activity of VEGI is not due to TNF and it binds to a distinct receptor. These results suggest that VEGI, a new member of the TNF family, has a signaling pathway similar to TNF and is most likely a multifunctional cytokine.

Cisplatin primes murine peritoneal macrophages for enhanced expression of nitric oxide, proinflammatory cytokines, TLRs, transcription factors and activation of MAP kinases upon co-incubation with L929 cells.

Cisplatin, a chemotherapeutic drug, may also act as a biological response modifier. Cisplatin (10mug/ml) treatment of macrophages for 24h activates them to produce enhanced amounts of nitric oxide (NO), ROI, proinflammatory cytokines and exhibit increased tumoricidal activity, which may or may not be contact mediated. In the present investigation, we report that the treatment of macrophages with cisplatin for a short period of 2h is sufficient to make them more receptive to interaction with tumor cells. Macrophages pretreated with cisplatin for 2h, and co-incubated with L929 cells, produced enhanced NO, TNF-alpha, IL-1beta, IL-12 and IFN-gamma. Production of NO, TNF-alpha, IL-1beta, IL-12 and IFN-gamma was maximum at 24h of co-incubation. Enhanced transcription of iNOS, TNF-alpha, IL-1beta, IL-12 and IFN-gamma genes in cisplatin-pretreated macrophages were observed between 12 and 24h of co-incubation with L929 cells. Cisplatin-treated macrophages on co-incubation with L929 cells also expressed enhanced transcription of Toll-like receptor (TLR)-2 and TLR-4 genes and their proteins. It is observed that cisplatin-pretreated macrophages on co-incubation with L929 cells showed activation of mitogen-activated protein (MAP) kinases and NF-kappaB. Pharmacological inhibitors like PD98059, SB202190 and wortmannin strongly inhibited the production of NO and proinflammatory cytokines suggesting the probable role of p42/44, p38 MAPK and PI3K in the above process. The c-Jun amino terminal kinase (JNK) inhibitor SP600125 was less effective in inhibiting the production of NO and proinflammatory cytokines. The data thus suggests that pretreatment of macrophages with cisplatin makes them biologically more responsive to interaction with L929 cells and become activated.

Identification and characterization of peptide: N- glycanase from Dictyostelium discoideum

BackgroundPeptide: N- glycanase (PNGase) enzyme cleaves oligosaccharides from the misfolded glycoproteins and prepares them for degradation. This enzyme plays a role in the endoplasmic reticulum associated degradation (ERAD) pathway in yeast and mice but its biological importance and role in multicellular development remain largely unknown.ResultsIn this study, the PNGase from the cellular slime mold, Dictyostelium discoideum (Dd PNGase) was identified based on the presence of a common TG (transglutaminase) core domain and its sequence homology with the known PNGases. The domain architecture and the sequence comparison validated the presence of probable functional domains in Dd PNGase. The tertiary structure matched with the mouse PNGase. Here we show that Dd PNGase is an essential protein, required for aggregation during multicellular development and a knockout strain of it results in small sized aggregates, all of which did not form fruiting bodies. The in situ hybridization and RT-PCR results show higher level of expression during the aggregate stage. The expression gets restricted to the prestalk region during later developmental stages. Dd PNGase is a functional peptide:N-glycanase enzyme possessing deglycosylation activity, but does not possess any significant transamidation activity.ConclusionsWe have identified and characterized a novel PNGase from D. discoideum and confirmed its deglycosylation activity. The results emphasize the importance of PNGase in aggregation during multicellular development of this organism.

Cisplatin-stimulated murine bone marrow-derived macrophages secrete oncostatin M.

Cisplatin (CP), a widely used anticancer drug activates cells of the immune system to a tumoricidal state, and thus functions as a potent biological response modifier. Expression of oncostatin M (OSM), a novel cytokine having a growth regulatory effect, was studied in bone marrow‐derived macrophages treated with cisplatin. Supematants from CP‐stimulated macrophages were found to be cytostatic for OSM‐sensitive A375 melanoma cells. Immunoblot analysis with anti‐OSM antibody revealed that expression of OSM in macrophages upon CP stimulation is a rapid process and within 30 min of CP treatment, a significant amount of OSM is secreted into the culture supernatant. These results therefore indicate that CP can stimulate murine bone marrow‐derived macrophages to produce OSM which can be implicated as one of the cytostatic/cytocidal factors in the antitumour action of cisplatin‐stimulated macrophages.

Protein kinase C: a potential pathway of macrophage activation with cisplatin

Cisplatin (CP) has been reported to activate murine macrophages to tumoricidal state, however, its mechanism of action is not known. In the present study it is reported that the production of: (a) interleukin-1 (IL-1); (b) tumor necrosis factor (TNF); (c) nitric oxide (NO); and (d) macrophage-mediated cytotoxicity by cisplatin-treated bone marrow-derived macrophages were inhibited by PKC inhibitors H-7 and chelerythrine chloride. Also, it was observed that treatment of macrophages with CP resulted in the translocation of PKC from the cytosol to the membrane fraction. These findings suggest the involvement of PKC in the activation of bone marrow-derived macrophages with cisplatin.

Activation of P388D1 macrophage cell line by chemotherapeutic drugs

It has been found that certain antineoplastic drugs impart their function with a distinct duality. Besides being tumoricidal, they are capable of acting as immunopotentiator. This led us to investigate the effect of cytosine arabinoside (CA), vincristine sulphate (VS), cyclophosphamide (CS), mitomycin C (MMC), hydroxy urea (HU) and lipopolysaccharide (LPS) on a macrophage cell line P388D1. Supernatants collected from P388D1 cells treated with CA, VS, CS, MMC, HU or LPS demonstrated enhanced production of tumor necrosis factor (TNF) confirmed by bioassay on L929 tumor target cells and increased interleukin-1 (IL-1) production by standard thymocyte proliferation bioassay. Also, supernatants showed increased amounts of nitric oxide and lysozyme using Griess reaction and reduction in turbidity of Micrococcus lysodeikticus, respectively. The above findings demonstrate that these drugs may be used not only as chemotherapeutic agents but also as macrophage-activating agents.

Natural Compounds: Role in Reversal of Epigenetic Changes

The hallmarks of carcinogenesis are characterized by alterations in the expression of multiple genes that occur via genetic and epigenetic alterations, leading to genome rearrangements and instability. The reversible process of epigenetic regulation, which includes changes in DNA methylation, histone modifications, and alteration in microRNA (miRNA) expression that alter phenotype without any change in the DNA sequence, is recognized as a key mechanism in cancer cell metabolism. Recent advancements in the rapidly evolving field of cancer epigenetics have shown the anticarcinogenic potential of natural compounds targeting epigenetic mechanism as a common molecular approach for cancer treatment. This review summarizes the potential of natural chemopreventive agents to reverse cancer-related epigenetic aberrations by regulating the activity of histone deacetylases, histone acetyltransferases, DNA methyltransferase I, and miRNAs. Furthermore, there is impetus for determining novel and effective chemopreventive strategies, either alone or in combination with other anticancer agents that exhibit similar properties, for improving the therapeutic aspects of cancer.

Involvement of ras and map kinase (ERK‐1) in cisplatin‐induced activation of murine bone marrow‐derived macrophages

Cisplatin (cis‐diamminedichloroplatinum II), a potent antitumor compound, stimulates immune responses by activating monocytes/macrophages and other cells of the immune system. However, the mechanism by which cisplatin activates these cells is poorly characterised. Our earlier findings indicate that cisplatin treatment stimulates rapid tyrosine phosphorylation in a number of cellular proteins in murine macrophages. This initial tyrosine phosphorylation is an important regulatory mechanism and is followed by activation of several other proteins. In the present study, we report the involvement of other key molecules and the role of tyrosine phosphorylation in their activation in the signaling cascade of cisplatin. We observed the involvement of Ras (a low molecular weight GTP‐binding protein) and ERK‐1 (a MAP kinase) in this signaling cascade. Cisplatin treatment results in an increase in the expression of both Ras and ERK‐1 in a dose‐dependent manner, which was dependent upon tyrosine phosphorylation. Genistein a PTK inhibitor inhibited the cisplatin induced expression of Ras and ERK‐1. These findings indicate that Ras and ERK‐1 are important signaling molecules involved in the tumoricidal activation of macrophages with cisplatin and is dependent on initial tyrosine phosphorylation.

Synthesis, characterization and antitumour studies on N-salicyl-N′-thiobenzohydrazide and its copper(II) complex

SummaryThe new potential tetradentate ligand N-salicyl-N′-thiobenzohydrazide (H2SBTH) and its Cu complex, [Cu(SBTH)], have been prepared and characterized by physico-chemical studies. In vivo antitumour activity of [Cu(SBTH)] has been tested against breast tumour in C3H/J strain mice; LD50 values were also calculated. The cytotoxicity and antitumour effect of [Cu(SBTH)] is a maximum at 100 mg kg-1 body weight injected intraperitoneally in mice carrying breast tumour. In vitro results of the ligand and the complex on P-815 (murine mastocytoma) and K-562 (human erythroleukemia cells) indicate that these compounds show significant inhibition on 3H-thymidine and 3H-uridine incorporation in DNA and RNA in these tumour cells. Light microscopic study of the treated tumour mass demonstrated that certain cellular degradation, such as disappearance of mitotic figures, loss in cellular compactness, distortion of nucleus and disruption of cytoplasmic boundaries, take place in complex-treated mice with tumours.