[Show abstract][Hide abstract] ABSTRACT: Many viral proteins are responsible for causing induction of apoptosis in the target cells. Hemagglutinin neuraminidase (HN), a multifunctional protein of Newcastle disease virus (NDV), is one of such proteins. The present study was undertaken to determine the apoptotic potential of the HN gene in cultured human cervical cancer cell line (HeLa cell) and to elucidate the molecular mechanisms involved. The results of the study indicate that HN protein causes apoptosis in HeLa cells, as observed by the translocation of Phosphatidylserine, activation of caspases, cleavage of PARP and DNA fragmentation. Further, we report that expression of HN protein up-regulates SAPK/JNK pathway leading to transactivation of c-Jun which in turn activates apoptosis signaling. The results of our study provide an insight into the mechanism through which HN induces apoptosis.
Full-text · Article · Aug 2015 · Applied biochemistry and biotechnology
[Show abstract][Hide abstract] ABSTRACT: Nuclear factor kappa-B (NF-κB), a key anti-apoptotic factor, plays a critical role in tumor cell growth, metastasis and angiogenesis. The transcriptional activity of NF-κB is normally suppressed in the cytoplasm due to its association with a natural inhibitor molecule IκB. Phosphorylation of the IκB at Ser 32 and Ser 36 by the IκB kinase complex (IKK) marks the degradation of the molecule by the 26S proteasome. As NF-κB is constitutively activated in the most of the tumor cells, inhibition of the activities of IKK may significantly sensitize the tumor cells to apoptosis. In the present study, we investigated the effect of IκB kinase specific blocker PS1145 on DMBA induced skin tumor of male Wistar rats. We examined the apoptotic effect of PS1145 on DMBA induced tumor by various histopathological and molecular techniques. Our results demonstrated the significant expression of major pro-apoptotic genes like caspase 2, 3, 8, 9 and p53 in PS1145 treated tumor bearing group at mRNA levels as well as significant (P<0.05) down regulation in the expression levels of NF-κB and VEGF, the major pro-inflammatory and pro-angiogenic factors, respectively. The histopathological examination showed that the tumor progression, mitotic, AgNOR and PCNA indices were significantly reduced in PS1145 treatment groups as compared to PBS control on day 28 of post-treatment. Further, significant increase in TUNEL positive nuclei and observation of peculiar apoptotic nuclei in transmission electron microscopy were seen in PS1145 treatment group. We conclude that intravenous application of PS1145 promotes direct apoptosis in DMBA induced skin tumor in male Wistar rats by blocking NF-κB and VEGF activities.
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Full-text · Article · Aug 2015 · Cell Biology International
[Show abstract][Hide abstract] ABSTRACT: The use of viruses for treatment of cancer overcomes the bottlenecks of chemotherapy and radiotherapy. Several viruses and their proteins have been evaluated for oncolytic effect. The VP3 protein (apoptin) of chicken anemia virus is one such protein with an inherent ability to lyse cancer and transformed cells while leaving normal cells unharmed. In the present study, the apoptosis inducing potential of VP3 protein of CAV was evaluated in human cervical cancer cell line (HeLa). It was found that in VP3-induced apoptosis, caspase-dependent intrinsic pathway plays an important role with the cleavage of poly (ADP-ribose) polymerase (PARP) and there was no evidence of involvement of death receptor-mediated extrinsic pathway. The results of this study provide intuitive information and strengthen the candidacy of apoptin as a viral oncotherapeutic agent.
No preview · Article · Mar 2015 · Applied biochemistry and biotechnology
[Show abstract][Hide abstract] ABSTRACT: The viral gene oncotherapy in combination with cytokines emerges as an exciting strategy for cancer therapy due to its minimal side effects and tumor specificity. HN is the surface protein of NDV which is involved in virus infectivity and is known to kill many cancerous cell types. TNF-a, a multifactorial cytokine has direct anti-tumor activity by activating the extrinsic pathways of apoptosis. In the present study, HN gene of NDV and TNF-a of human were cloned at multiple cloning sites (MCS) 1 and 2 of bicistronic expression vector pVIVO2. Expression pattern of recombinant clone was checked on transcriptional and translational level by RT-PCR, Immunofluorescence assay and flow cytometry. On flow cytometric analysis HN gene expression was found to be 28.30 1.21; 5.22 0.60%, and TNF-a gene expression was found to be 15.44 0.42; 6.51 0.757%, in HeLa cells transfected with pVIVO.nd.hn.hu.tnf and pVIVO2 empty vector control, respectively. These assays confirm that HN and TNF-a act synergistically in the induction of apoptosis in HeLa cells.
Full-text · Article · Oct 2014 · Animal Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Development and study of dog mammary tumour xenograft in immunosuppressed Swiss Albino Mice adds a new dimension in cancer research as dog tumors have many similarities with human tumors regarding progression, histopathology, molecular mechanism, immune response and therapy. Failure of the immune system to recognize and eliminate cancer cells leads to cancer progression and the fight between immune cells and cancer cells has a great role in understanding the mechanism of cancer progression and elimination. Rejection and acceptance of tumour xenograft depends on efficiency of CD4+, CD8+ and NK cell populations. In the present investigation, dog mammary tumor xenograft in cyclosporine-A and γ-irradiated, immunosuppressed Swiss Albino mice was developed and the immune cell status of graft accepted and rejected mice was assessed. It was observed that all the major immune cells (CD4+, CD8+ and NK cells) play an equal role in tumour rejection.
Full-text · Article · Oct 2014 · Indian journal of experimental biology
[Show abstract][Hide abstract] ABSTRACT: The Non-Structural protein 1 of Canine Parvovirus – 2 (CPV2.NS1) plays a major role in viral cytotoxicity and pathogenicity. CPV2.NS1 has been proven to cause apoptosis in HeLa cells in vitro in our laboratory. Here we report that CPV2.NS1 has no toxic side effects on healthy cells but regresses skin tumors in Wistar rats. Histopathological examination of tumor tissue from CPV2.NS1 treated group revealed infiltration of mononuclear and polymorphonuclear cells with increased extra cellular matrix, indicating signs of regression. Tumor regression was also evidenced by significant decrease in mitotic index, AgNOR count and PCNA index, and increase in TUNEL positive apoptotic cells, in CPV2.NS1 treated group. Further, CPV2.NS1 induced anti-tumor immune response through significant increase in CD8+ and NK cell population in CPV2.NS1 treated group. These findings suggest that CPV2.NS1 can be a possible therapeutic candidate as an alternative to chemotherapy for treatment of cancer.
Full-text · Article · Aug 2014 · Research in Veterinary Science
[Show abstract][Hide abstract] ABSTRACT: The use of chemotherapy and/or radiotherapy for treatment of cancer is limited due to genotoxic side effects on healthy
cells, involvement of anti-apoptotic signal transduction pathways that prevent cell death, and requirement of functional p53
for induction of apoptosis in cancerous cells. Efforts are beings made worldwide to develop new anticancer therapies as an
alternative to chemotherapy. And viral gene therapy is one of the most potent therapeutics that is being ventured worldwide.
Canine parvovirus-2 (CPV-2) is one of those viruses that have an inherent oncolytic property. The non-structural protein-1
(NS1 protein) of CPV-2 plays a major role in parvoviral cytotoxicity and pathogenicity in permissive cells. The oncolytic
potential of CPV2-NS1 has been established in vitro. Prior to taking up the in vivo studies, the present study was undertaken
to clone Canine Parvovirus NS1 gene and reporter gene GFP in eukaryotic expression vector pVIVO2-mcs, and to
characterize the double construct in mammalian cells. The genes were successfully cloned in pVIVO2-mcs and
characterized for their expression as demonstrated by fluorescence microscopy and immunofluorescence staining. This
characterized double gene construct will further be used to evaluate the oncolytic potential of CPV-2 NS1 in experimentally
induced in vivo tumour model.
Full-text · Article · Jan 2014 · Indian Journal of Biotechnology
[Show abstract][Hide abstract] ABSTRACT: The canine parvovirus type 2 (CPV-2) causes an acute disease in dogs. It has been found to induce cell cycle arrest and DNA damage leading to cellular lysis. In this paper, we evaluated the apoptotic potential of the "new CPV-2a" in MDCK cells and elucidated the mechanism of the induction of apoptosis. The exposure of MDCK cells to the virus was found to trigger apoptotic response. Apoptosis was confirmed by phosphatidylserine translocation, DNA fragmentation assays, and cell cycle analysis. Activation of caspases-3, -8, -9, and -12 and decrease in mitochondrial potential in CPV-2a-infected MDCK cells suggested that the CPV-2a-induced apoptosis is caspase dependent involving extrinsic, intrinsic, and endoplasmic reticulum pathways. Increase in p53 and Bax/Bcl2 ratio was also observed in CPV-2a-infected cells.
Full-text · Article · Oct 2013 · Applied biochemistry and biotechnology
[Show abstract][Hide abstract] ABSTRACT: In the present study, Indian Newcastle disease virus (NDV)
isolates UP/3 and UP/4 collected from suspected field samples
were characterized to be velogenic by pathogenic and
phylogenetic analysis. Phylogenetic analysis based on the
nucleotide sequences of the F gene indicated that UP/3 and UP/4
isolates belong to genotype VII and genotype IV, respectively.
These isolates possessed the amino acid sequence 112R/K-R-QK/
R-R-F117 in the F0 cleavage site, which is typical of velogenic
NDV pathotype. All the NDV strains analyzed have shown to
have non-synonymous to synonymous base substitution rate in the
F gene ranging between 0.074-0.197, demonstrating the presence
of purifying selection. HN gene of the isolates was found to have
an open reading frame encoding 571 amino acids. The deduced
amino acid sequences of the HN glycoprotein revealed that the
cysteine residues essential for intra-molecular disulphide bonds to
stabilize the HN molecules, antigenic sites and key residues for
receptor binding were all conserved in Indian isolates
Full-text · Article · Aug 2013 · Indian Journal of Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Apoptosis is programmed cell death that normally occurs during development and aging in multicellular animals. Apoptosis also occurs as a defense mechanism against disease or harmful external agents. It can be initiated by a variety of stimuli including viruses and viral proteins. Canine parvovirus type 2 (CPV-2) that causes acute disease in dogs has been found to induce cell cycle arrest and DNA damage leading to cellular lysis. Though non structural protein 1 (NS1) of many parvoviruses has been found to be apoptotic, no report on the apoptotic potential of NS1 of CPV-2 (CPV-2.NS1) exists. In this study, we evaluated the apoptotic potential of CPV-2.NS1 in HeLa cells. CPV-2.NS1 has been found to induce apoptosis which was evident through characteristic DNA fragmentation, increase in hypodiploid cell count, phosphatidyl serine translocation and activation of caspase-3. Increase in caspase-3 activity and no change in p53 activity with time in CPV-2.NS1 expressing HeLa cells showed the induction of apoptosis to be caspase dependent and p53 independent.
[Show abstract][Hide abstract] ABSTRACT: Viral gene oncotherapy is emerging as a biotherapeutic cancer treatment modality based on targeted killing of cancer cells by viral genes. Newcastle disease virus (NDV) has the property to cause selective oncolysis of tumor cells sparing normal cells. NDV has a single stranded negative sense RNA genome, which is 15,186 nucleotide long and consists of six genes, which codes for eight proteins. NDV like other paramyxoviruses has the ability to generate multiple proteins from the P gene. P protein is encoded by an unedited transcript of the P gene, whereas the V and W protein are the results of RNA editing event in which one and two G residues are inserted at a conserved editing site within the P gene mRNA resulting in V and W transcripts, respectively. Although NDV is known to cause oncolysis by triggering apoptosis, the role of different viral proteins in selective oncolysis is still unclear. P gene edited products are known for its anti-apoptotic property in homologous host. In the present study, NDV P gene and its RNA edited products were amplified, cloned, sequenced and in vitro expression was done in HeLa cells. Further constructs were assayed for their apoptosis inducing ability in HeLa cells. Preliminary study suggested that P, V and W proteins are not apoptotic to HeLa cells.
Full-text · Article · Feb 2013 · Indian journal of experimental biology
[Show abstract][Hide abstract] ABSTRACT: The canine Parvovirus 2, non-structural 1 (NS1) is a novel candidate tumor suppressor gene. To confirm the expression of the NS1 in HeLa cells after transfection there was a need to raise antiserum against CPV2- NS1. Therefore, this study was carried out to express and purify the recombinant NS1 (rNS1), and characterize the polyclonal serum. CPV2-NS1, complete coding sequence (CDS) was amplified, cloned in pET32a+ and expressed in BL21 (DE3) (pLysS). SDS-PAGE analysis revealed that the expression of the recombinant protein was maximum when induced with 1.5 mM IPTG. The 6 x His tagged fusion protein was purified on Ni-NTA resin under denaturing conditions and confirmed by western blot using CPV2 specific antiserum. The rabbits were immunized with the purified rNS1 to raise anti-NS1 polyclonal antiserum. The polyclonal serum was tested for specificity and used for confirming the expression of NS1 in HeLa transfected with pcDNA.cpv2.ns1 by indirect fluorescent antibody test (IFAT), flow cytometry and western blot. The polyclonal antiserum against NS1 could be very useful to establish functional in vitro assays to explore role of NS1 in cancer therapeutics.
Full-text · Article · Sep 2012 · Indian journal of experimental biology
[Show abstract][Hide abstract] ABSTRACT: Cancer is one of the killer diseases in humans and needs alternate curative measures despite recent improvement in modern treatment modalities. Oncolytic virotherapy seems to be a promising nonconventional way to treat cancers. Newcastle disease virus (NDV), a poultry virus, is nonpathogenic to human and domestic animals and has a long history of being used in oncotherapy research in several preclinical studies. The ability of NDV to successfully infect and destroy cancer cells is dependent on the strain and the pathotype of the virus. Adaptation of viruses to heterologous hosts without losing its replicative and oncolytic potential is prerequisite for use as cancer virotherapeutics. In the present study, velogenic NDV was adapted for replication in HeLa cells, and its cytotoxic potential was evaluated by observing morphological, biochemical, and nuclear landmarks of apoptosis. Our results indicated that the NDV-induced apoptosis in HeLa cells was dependent on upregulation of TNF-related apoptosis-inducing ligand (TRAIL) and caspases activation. Different determinants of apoptosis evaluated in the present study indicated that this strain could be a promising candidate for cancer therapy in future.
Full-text · Article · May 2012 · Applied biochemistry and biotechnology
[Show abstract][Hide abstract] ABSTRACT: In the present study recombinant VP3 (rVP3) was expressed in E. coli BL21 (DE3) (pLysS) and its polyclonal antibodies were characterized. SDS-PAGE analysis revealed that the expression of recombinant protein was maximum when induced with 1.5 mM IPTG for 6 h at 37 degrees C. The 6xHis-tagged fusion protein was purified on Ni-NTA and confirmed by Western blot using CAV specific antiserum. Rabbits were immunized with purified rVP3 to raise anti-VP3 polyclonal antibodies. Polyclonal serum was tested for specificity and used for confirming expression of VP3 in HeLa cells transfected with pcDNA.cav.vp3 by indirect fluorescent antibody test (IFAT), flow cytometry and Western blot. Available purified rVP3 and polyclonal antibodies against VP3 may be useful to understand its functions which may lead to application of VP3 in cancer therapeutics.
Full-text · Article · May 2012 · Indian journal of experimental biology